CiteULike: jyuh's Wagner

An analytical workflow for investigating cytokine profiles.

JC Siebert — 2008-08-20T14:50:54-00:00

Cytometry. Part A : the journal of the International Society for Analytical Cytology, Vol. 73, No. 4. (April 2008), pp. 289-298.

Understanding cytokine profiles of disease states has provided researchers with great insight into immunologic signaling associated with disease onset and progression, affording opportunities for advancement in diagnostics and therapeutic intervention. Multiparameter flow cytometric assays support identification of specific cytokine secreting subpopulations. Bead-based assays provide simultaneous measurement for the production of ever-growing numbers of cytokines. These technologies demand appropriate analytical techniques to extract relevant information efficiently. We illustrate the power of an analytical workflow to reveal significant alterations in T-cell cytokine expression patterns in type 1 diabetes (T1D) and breast cancer. This workflow consists of population-level analysis, followed by donor-level analysis, data transformation such as stratification or normalization, and a return to population-level analysis. In the T1D study, T-cell cytokine production was measured with a cytokine bead array. In the breast cancer study, intracellular cytokine staining measured T cell responses to stimulation with a variety of antigens. Summary statistics from each study were loaded into a relational database, together with associated experimental metadata and clinical parameters. Visual and statistical results were generated with custom Java software. In the T1D study, donor-level analysis led to the stratification of donors based on unstimulated cytokine expression. The resulting cohorts showed statistically significant differences in poststimulation production of IL-10, IL-1 beta, IL-8, and TNF beta. In the breast cancer study, the differing magnitude of cytokine responses required data normalization to support statistical comparisons. Once normalized, data showed a statistically significant decrease in the expression of IFN gamma on CD4+ and CD8+ T cells when stimulated with tumor-associated antigens (TAAs) when compared with an infectious disease antigen stimulus, and a statistically significant increase in expression of IL-2 on CD8+ T cells. In conclusion, the analytical workflow described herein yielded statistically supported and biologically relevant findings that were otherwise unapparent.

Cloning and functional characterization of human SMCT2 (SLC5A12) and expression pattern of the transporter in kidney.

E Gopal — 2008-08-16T06:28:29-00:00

Biochimica et biophysica acta, Vol. 1768, No. 11. (November 2007), pp. 2690-2697.

Recently, we cloned two Na(+)-coupled lactate transporters from mouse kidney, a high-affinity transporter (SMCT1 or slc5a8) and a low-affinity transporter (SMCT2 or slc5a12). Here we report on the cloning and functional characterization of human SMCT2 (SLC5A12) and compare the immunolocalization patterns of slc5a12 and slc5a8 in mouse kidney. The human SMCT2 cDNA codes for a protein consisting of 618 amino acids. When expressed in mammalian cells or Xenopus oocytes, human SMCT2 mediates Na(+) -coupled transport of lactate, pyruvate and nicotinate. The affinities of the transporter for these substrates are lower than those reported for human SMCT1. Several non-steroidal anti-inflammatory drugs inhibit human SMCT2-mediated nicotinate transport, suggesting that NSAIDs interact with the transporter as they do with human SMCT1. Immunofluorescence microscopy of mouse kidney sections with an antibody specific for SMCT2 shows that the transporter is expressed predominantly in the cortex. Similar studies with an anti-SMCT1 antibody demonstrate that SMCT1 is also expressed mostly in the cortex. Dual-labeling of SMCT1 and SMCT2 with 4F2hc (CD98), a marker for basolateral membrane of proximal tubular cells in the S1 and S2 segments of the nephron, shows that both SMCT1 and SMCT2 are expressed in the apical membrane of the tubular cells. These studies also show that while SMCT2 is broadly expressed along the entire length of the proximal tubule (S1/S2/S3 segments), the expression of SMCT1 is mostly limited to the S3 segment. These studies suggest that the low-affinity transporter SMCT2 initiates lactate absorption in the early parts of the proximal tubule followed by the participation of the high-affinity transporter SMCT1 in the latter parts of the proximal tubule.

probeCheck - a central resource for evaluating oligonucleotide probe coverage and specificity.

Alexander Loy — 2008-08-13T10:07:37-00:00

Environmental microbiology (21 July 2008)

The web server probeCheck, freely accessible at http://www.microbial-ecology.net/probecheck, provides a pivotal forum for rapid specificity and coverage evaluations of probes and primers against selected databases of phylogenetic and functional marker genes. Currently, 24 widely used sequence collections including the Ribosomal Database Project (RDP) II, Greengenes, SILVA and the Functional Gene Pipeline/Repository can be queried. For this purpose, probeCheck integrates a new online version of the popular ARB probe match tool with free energy (DeltaG) calculations for each perfectly matched and mismatched probe-target hybrid, allowing assessment of the theoretical binding stabilities of oligo-target and non-target hybrids. For each output sequence, the accession number, the GenBank taxonomy and a link to the respective entry at GenBank, EMBL and, if applicable, the query database are displayed. Filtering options allow customizing results on the output page. In addition, probeCheck is linked with probe match tools of RDP II and Greengenes, NCBI blast, the Oligonucleotide Properties Calculator, the two-state folding tool of the DINAMelt server and the rRNA-targeted probe database probeBase. Taken together, these features provide a multifunctional platform with maximal flexibility for the user in the choice of databases and options for the evaluation of published and newly developed probes and primers.

EGFR signalling as a negative regulator of Notch1 gene transcription and function in proliferating keratinocytes and cancer

Vihren Kolev — 2008-08-02T07:28:43-00:00

Nature Cell Biology, Vol. 10, No. 8. (06 July 2008), pp. 902-911.

Which hospitals have significantly better or worse than expected mortality rates for acute myocardial infarction patients? Improved risk adjustment with present-at-admission diagnoses.

GJ Stukenborg — 2008-07-25T04:39:10-00:00

Circulation, Vol. 116, No. 25. (18 December 2007), pp. 2960-2968.

BACKGROUND: Public reports that compare hospital mortality rates for patients with acute myocardial infarction are commonly used strategies for improving the quality of care delivered to these patients. Fair comparisons of hospital mortality rates require thorough adjustments for differences among patients in baseline mortality risk. This study examines the effect on hospital mortality rate comparisons of improved risk adjustment methods using diagnoses reported as present-at-admission. METHODS AND RESULTS: Logistic regression models and related methods originally used by California to compare hospital mortality rates for patients with acute myocardial infarction are replicated. These results are contrasted with results obtained for the same hospitals by patient-level mortality risk adjustment models using present-at-admission diagnoses, using 3 statistical methods of identifying hospitals with higher or lower than expected mortality: indirect standardization, adjusted odds ratios, and hierarchical models. Models using present-at-admission diagnoses identified substantially fewer hospitals as outliers than did California model A for each of the 3 statistical methods considered. CONCLUSIONS: Large improvements in statistical performance can be achieved with the use of present-at-admission diagnoses to characterize baseline mortality risk. These improvements are important because models with better statistical performance identify different hospitals as having better or worse than expected mortality.

Present-at-admission diagnoses improved mortality risk adjustment among acute myocardial infarction patients.

GJ Stukenborg — 2008-07-25T04:37:05-00:00

Journal of clinical epidemiology, Vol. 60, No. 2. (February 2007), pp. 142-154.

OBJECTIVE: Hospital mortality outcomes for acute myocardial infarction (AMI) patients are a focus of quality improvement programs conducted by government agencies. AMI mortality risk-adjustment models using administrative data typically adjust for baseline differences in mortality risk with a limited set of common and definite comorbidities. In this study, we present an AMI mortality risk-adjustment model that adjusts for comorbid disease and for AMI severity using information from secondary diagnoses reported as present at admission for California hospital patients. STUDY DESIGN AND SETTING: AMI patients were selected from California hospital administrative data for 1996 through 1999 according to criteria used by the California Hospital Outcomes Project Report on Heart Attack Outcomes, a state-mandated public report that compares hospital mortality outcomes. We compared results for the new model to two mortality risk-adjustment models used to assess hospital AMI mortality outcomes by the state of California, and to two other models used in prior research. RESULTS: The model using present-at-admission diagnoses obtained substantially better discrimination between predicted survival and inpatient death than the other models we considered. CONCLUSION: AMI mortality risk-adjustment methods can be meaningfully improved using present-at-admission diagnoses to identify comorbid disease and conditions related closely to AMI.

The Systems Biology Research Tool: evolvable open-source software

Jeremiah Wright — 2008-06-30T10:00:02-00:00

BMC Systems Biology, Vol. 2, No. 1. (2008)

BACKGROUND:Research in the field of systems biology requires software for a variety of purposes. Software must be used to store, retrieve, analyze, and sometimes even to collect the data obtained from system-level (often high-throughput) experiments. Software must also be used to implement mathematical models and algorithms required for simulation and theoretical predictions on the system-level.RESULTS:We introduce a free, easy-to-use, open-source, integrated software platform called the Systems Biology Research Tool (SBRT) to facilitate the computational aspects of systems biology. The SBRT currently performs 35 methods for analyzing stoichiometric networks and 16 methods from fields such as graph theory, geometry, algebra, and combinatorics. New computational techniques can be added to the SBRT via process plug-ins, providing a high degree of evolvability and a unifying framework for software development in systems biology.CONCLUSIONS:The Systems Biology Research Tool represents a technological advance for systems biology. This software can be used to make sophisticated computational techniques accessible to everyone (including those with no programming ability), to facilitate cooperation among researchers, and to expedite progress in the field of systems biology.

Effect of time of day of dialysis shift on serum biochemical parameters in patients on chronic hemodialysis.

J Mattana — 2008-07-03T12:56:10-00:00

American journal of nephrology, Vol. 15, No. 3. (1995), pp. 208-216.

It is unknown whether predialysis serum biochemical parameters may differ among chronic hemodialysis patients depending on the shift during which they are dialyzed. We studied 115 patients on chronic hemodialysis in our institution for 3 consecutive months and compared clinical and biochemical parameters based on the shift during which they were dialyzed. Predialysis serum potassium was found to be progressively higher for patients dialyzed on later as compared with earlier dialysis shifts, and phosphate was significantly higher for patients dialyzed during the evening shift as well. Regression analysis suggested that higher of potassium and phosphate levels were related to the time of day these sessions and not to patient age, amount of dialysis given or diet. By contrast, serum albumin, creatinine, sodium, and chloride levels were found to differ depending on dialysis shift, though these differences appeared to be accounted for by patient age. We concluded that the time of day of the beginning of the dialysis shift appears to mildly influence the levels of serum predialysis biochemical parameters which are important in monitoring patients on chronic hemodialysis, in particular potassium and phosphate. Further insight into the mechanism of this observed effect might improve our ability to interpret and treat derangements of these serum biochemical parameters in patients on chronic hemodialysis.

Methyl balance and transmethylation fluxes in humans.

SH Mudd — 2008-05-14T15:59:03-00:00

The American journal of clinical nutrition, Vol. 85, No. 1. (January 2007), pp. 19-25.

Various questions have been raised about labile methyl balance and total transmethylation fluxes, and further discussion has been encouraged. This report reviews and discusses some of the relevant evidence now available. The fact that, if needed, labile methyl balance is maintained by methylneogenesis appears to be established, but several aspects of transmethylation remain uncertain: definitive measurements of the rate of total transmethylation in humans of both sexes on various diets and at various ages; the extent to which synthesis of phosphatidylcholine has been underestimated; and the relative contributions of the 2 pathways for the formation of sarcosine (ie, N-methylglycine). The available evidence indicates that the quantitatively most important pathways for S-adenosylmethionine-dependent transmethylation in mammals are the syntheses of creatine by guanidinoacetate methyltransferase, of phosphatidylcholine by phosphatidylethanolamine methyltransferase, and of sarcosine by glycine N-methyltransferase. Data presented in this report show that S-adenosylmethionine and methionine accumulate abnormally in the plasma of humans with glycine N-methyltransferase deficiency but not of those with guanidinoacetate N-methyltransferase deficiency or in the plasma or livers of mice devoid of phosphatidylethanolamine N-methyltransferase activity. The absence of such accumulations in the latter 2 conditions may be due to removal of S-adenosylmethionine by synthesis of sarcosine. Steps that may help clarify the remaining issues include the determination of the relative rates of synthesis of sarcosine, creatine, and phosphatidylcholine by rapid measurement of the rates of radiolabel incorporation into these compounds from L-[methyl-3H]methionine administered intraportally to an experimental animal; clarification of the intracellular hepatic isotope enrichment value during stable-isotope infusion studies to enhance the certainty of methyl flux estimates during such studies; and definitive measurement of the dietary betaine intake from various diets.

Loss of the glycine N-methyltransferase gene leads to steatosis and hepatocellular carcinoma in mice.

ML Martínez-Chantar — 2008-05-14T15:55:30-00:00

Hepatology (Baltimore, Md.), Vol. 47, No. 4. (April 2008), pp. 1191-1199.

Glycine N-methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMT-knockout (GNMT-KO) mice. GNMT-KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT-KO mice coincidently with the suppression of the Ras inhibitors Ras-association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1-3 and cytokine-inducible SH2-protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT-KO mice. Conclusion: These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice.

Mobilization of osmotically inactive Na+ by growth and by dietary salt restriction in rats.

M Schafflhuber — 2008-05-12T02:37:06-00:00

American journal of physiology. Renal physiology, Vol. 292, No. 5. (May 2007)

The idea that an osmotically inactive Na(+) storage pool exists that can be varied to accommodate states of Na(+) retention and/or Na(+) loss is controversial. We speculated that considerable amounts of osmotically inactive Na(+) are lost with growth and that additional dietary salt excess or salt deficit alters the polyanionic character of extracellular glycosaminoglycans in osmotically inactive Na(+) reservoirs. Six-week-old Sprague-Dawley rats were fed low-salt (0.1%; LS) or high-salt (8%; HS) diets for 1 or 4 wk. At their death, we separated the tissues and determined their Na(+), K(+), and water content. Three weeks of growth reduced the total body Na(+) content relative to dry weight (rTBNa(+)) by 23%. This "growth-programmed" Na(+) loss originated from the bone and the completely skinned and bone-removed carcasses. The Na(+) loss was osmotically inactive (45-50%) or osmotically active (50-55%). In rats aged 10 wk, compared with HS, 4 wk of LS reduced rTBNa(+) by 9%. This dietary-induced Na(+) loss was osmotically inactive ( approximately 50%) and originated largely from the skin, while approximately 50% was osmotically active. LS for 1 wk did not reduce skin Na(+) content. The mobilization of osmotically inactive skin Na(+) with long-term salt deprivation was associated with decreased negatively charged skin glycosaminoglycan content and thereby a decreased water-free Na(+) binding capacity in the extracellular matrix. Our data not only serve to explain discrepant results in salt balance studies but also show that glycosaminoglycans may provide an actively regulated interstitial cation exchange mechanism that participates in volume and blood pressure homeostasis.

Large-scale chemical dissection of mitochondrial function.

Bridget K Wagner — 2008-03-06T11:51:11-00:00

Nat Biotechnol (24 February 2008)

Mitochondrial oxidative phosphorylation (OXPHOS) is under the control of both mitochondrial (mtDNA) and nuclear genomes and is central to energy homeostasis. To investigate how its function and regulation are integrated within cells, we systematically combined four cell-based assays of OXPHOS physiology with multiplexed measurements of nuclear and mtDNA gene expression across 2,490 small-molecule perturbations in cultured muscle. Mining the resulting compendium revealed, first, that protein synthesis inhibitors can decouple coordination of nuclear and mtDNA transcription; second, that a subset of HMG-CoA reductase inhibitors, combined with propranolol, can cause mitochondrial toxicity, yielding potential clues about the etiology of statin myopathy; and, third, that structurally diverse microtubule inhibitors stimulate OXPHOS transcription while suppressing reactive oxygen species, via a transcriptional mechanism involving PGC-1alpha and ERRalpha, and thus may be useful in treating age-associated degenerative disorders. Our screening compendium can be used as a discovery tool both for understanding mitochondrial biology and toxicity and for identifying novel therapeutics.

High-throughput mammalian two-hybrid screening for protein-protein interactions using transfected cell arrays

Andrea Fiebitz — 2008-02-08T01:10:49-00:00

BMC Genomics, Vol. 9 (06 February 2008), 68.

Endocannabinoids acting at vascular CB1 receptors mediate the vasodilated state in advanced liver cirrhosis.

S Bátkai — 2008-04-20T00:46:28-00:00

Nature medicine, Vol. 7, No. 7. (July 2001), pp. 827-832.

Advanced cirrhosis is associated with generalized vasodilation of unknown origin, which contributes to mortality. Cirrhotic patients are endotoxemic, and activation of vascular cannabinoid CB1 receptors has been implicated in endotoxin-induced hypotension. Here we show that rats with biliary cirrhosis have low blood pressure, which is elevated by the CB1 receptor antagonist SR141716A. The low blood pressure of rats with CCl4-induced cirrhosis was similarly reversed by SR141716A, which also reduced the elevated mesenteric blood flow and portal pressure. Monocytes from cirrhotic but not control patients or rats elicited SR141716A-sensitive hypotension in normal recipient rats and showed significantly elevated levels of anandamide. Compared with non-cirrhotic controls, in cirrhotic human livers there was a three-fold increase in CB1 receptors on isolated vascular endothelial cells. These results implicate anandamide and vascular CB1 receptors in the vasodilated state in advanced cirrhosis and indicate a novel approach for its management.

Classification of chronic kidney disease by estimated glomerular filtration rate

Marsik — 2008-03-16T14:23:10-00:00

European Journal of Clinical Investigation, Vol. 38, No. 4. (April 2008), pp. 253-259.

SCH 503034, a novel hepatitis C virus protease inhibitor, plus pegylated interferon alpha-2b for genotype 1 nonresponders.

C Sarrazin — 2008-04-02T03:29:01-00:00

Gastroenterology, Vol. 132, No. 4. (April 2007), pp. 1270-1278.

BACKGROUND & AIMS: SCH 503034 is a novel and potent oral hepatitis C virus (HCV) protease inhibitor. In this phase Ib study, we assessed safety parameters and virologic response of combination of SCH 503034 plus pegylated (PEG) interferon (IFN) alpha-2b in patients with HCV genotype 1 infections who were previously nonresponders to PEG-IFN-alpha-2b +/- ribavirin therapy. METHODS: This was a multicenter, open-label, 2-dose level, 3-way crossover, randomized (to crossover sequence) study carried out in 3 medical centers in Europe. Adult patients received SCH 503034 200 mg (n = 14) or 400 mg (n = 12) 3 times daily orally and PEG-IFN-alpha-2b 1.5 microg/kg subcutaneously once each week. Patients received SCH 503034 as monotherapy for 1 week, PEG-IFN-alpha-2b as monotherapy for 2 weeks, and combination therapy for 2 weeks with washout periods between each treatment period. RESULTS: Combination therapy with SCH 503034 and PEG-IFN-alpha-2b was well tolerated, with no clinically significant changes in safety parameters. Mean maximum log(10) changes in HCV RNA were -2.45 +/- 0.22 and -2.88 +/- 0.22 for PEG-IFN-alpha-2b plus 200 mg and 400 mg SCH 503034, respectively, compared with -1.08 +/- 0.22 and -1.61 +/- 0.21 for SCH 503034 200 mg and 400 mg, respectively, and -1.08 +/- 0.22 and -1.26 +/- 0.20 for PEG-IFN-alpha-2b alone in the 200 mg and 400 mg SCH 503034 groups, respectively. CONCLUSIONS: SCH 503034 plus PEG-IFN-alpha-2b was well tolerated in patients with HCV genotype 1 nonresponders to PEG-IFN-alpha-2b +/- ribavirin. These preliminary results of antiviral activity of the combination suggest a potential new therapeutic option for this hard-to-treat, nonresponder patient population.

Gene expression-based screening identifies microtubule inhibitors as inducers of PGC-1alpha and oxidative phosphorylation

Zoltan Arany — 2008-03-17T23:10:36-00:00

Proceedings of the National Academy of Sciences (17 March 2008), 0800979105.

The transcriptional coactivator PGC-1alpha is a potent regulator of several metabolic pathways, including, in particular, the activation of oxidative phosphorylation and mitochondrial biogenesis. Recent evidence suggests that increasing PGC-1alpha activity may have beneficial effects in various conditions, including muscular dystrophy, diabetes, and neurodegenerative diseases. We describe here a high-throughput screen to identify small molecules that induce PGC-1alpha expression in skeletal muscle cells. A number of drug classes are identified, including glucocorticoids, microtubule inhibitors, and protein synthesis inhibitors. These drugs induce PGC-1alpha mRNA, and the expression of a number of genes known to be regulated by PGC-1alpha. No induction of these target genes is seen in PGC-1alpha / cells, demonstrating that the drugs act through PGC-1alpha. These data demonstrate the feasibility of high-throughput screening for inducers of PGC-1alpha. Moreover, the data identify microtubule inhibitors and protein synthesis inhibitors as modulators of PGC-1alpha and oxidative phosphorylation. 10.1073/pnas.0800979105

Disorders of renal magnesium handling explain renal magnesium transport.

CA Wagner — 2008-03-23T13:51:04-00:00

J Nephrol, Vol. 20, No. 5. (t 2007), pp. 507-510.

Magnesium is essential for bone stability, neuronal excitability, muscular relaxation and many other metabolic functions. Despite its fundamental biological importance, mechanisms controlling systemic magnesium homeostasis are only partially understood. The kidney plays a central role in maintaining magnesium balance as evident from several rare inherited disorders of renal magnesium transport. Recent studies shed new light on molecular mechanisms of renal magnesium handling and its control. Mutations in the claudin 16 (paracellin) paracellular protein in the thick ascending limb (TAL) of Henle's loop and in the transient receptor potential cation channel, subfamily 6, member 6 (TRPM6) magnesium channel expressed in distal tubules found in patients with renal magnesium wasting and hypomagnesemia underscore the importance of these transport proteins. A study by Hou et al (J Biol Chem 2007; 282: 17114-22) demonstrates a pathomechanism for claudin 16 mutations that gives interesting insights into the function of the TAL. Moreover, Groenestege and colleagues report (J Clin Invest 2007; 117: 2260-7) the identification of the epidermal growth factor (EGF) as a hormonal regulator of TRPM6 activity, and thereby explain how mutations in EGF can cause familial hypomagnesemia. Interestingly, cetuximab, a drug used in treatment of certain cancers, acts an inhibitor of the EGF receptor and causes hypomagnesemia which may be due to the inhibition of EGF signaling.

Effect of immunisation against angiotensin II with CYT006-AngQb on ambulatory blood pressure: a double-blind, randomised, placebo-controlled phase IIa study

Alain Tissot — 2008-03-12T02:17:12-00:00

The Lancet, Vol. 371, No. 9615., pp. 821-827.

SummaryBackground Hypertension can be controlled adequately with existing drugs such as angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers. Nevertheless, treatment success is often restricted by patients not adhering to treatment. Immunisation against angiotensin II could solve this problem. We investigated the safety and efficacy of CYT006-AngQb--a vaccine based on a virus-like particle--that targets angiotensin II to reduce ambulatory blood pressure.Methods In this multicentre, double-blind, randomised, placebo-controlled phase IIa trial, 72 patients with mild-to-moderate hypertension were randomly assigned with a computer-generated randomisation list to receive subcutaneous injections of either 100 [mu]g CYT006-AngQb (n=24), 300 [mu]g CYT006-AngQb (24), or placebo (24), at weeks 0, 4, and 12. 24-h ambulatory blood pressure was measured before treatment and at week 14. The primary outcomes were safety and tolerability. Analyses were done by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00500786.Findings Two patients in the 100 [mu]g group, three in the 300 [mu]g group, and none in the placebo group discontinued study treatment. All patients were included in safety analyses; efficacy analyses did not include the five dropouts, for whom no data were available at week 14. Five serious adverse events were reported (two in the 100 [mu]g group, two in the 300 [mu]g group, and one in the placebo group); none were deemed to be treatment related. Most side-effects were mild, transient reactions at the injection site. Mild, transient influenza-like symptoms were seen in three patients in the 100 [mu]g group, seven in the 300 [mu]g group, and none in the placebo group. In the 300 [mu]g group, there was a reduction from baseline in mean ambulatory daytime blood pressure at week 14 by -9[middle dot]0/-4[middle dot]0 mm[punctuation space]Hg compared with placebo (p=0[middle dot]015 for systolic and 0[middle dot]064 for diastolic). The 300 [mu]g dose reduced the early morning blood-pressure surge compared with placebo (change at 0800 h -25/-13 mm[punctuation space]Hg; p<0[middle dot]0001 for systolic, p=0[middle dot]0035 for diastolic).Interpretation Immunisation with CYT006-AngQb was associated with no serious adverse events; most observed adverse events were consistent with local or systemic responses similar to those seen with other vaccines. The 300 [mu]g dose reduced blood pressure in patients with mild-to-moderate hypertension during the daytime, especially in the early morning.Funding Cytos Biotechnology AG.

Potential role of retinoids in the therapy of renal disease.

J Wagner — 2008-03-07T03:52:37-00:00

Nephrol Dial Transplant, Vol. 16, No. 3. (March 2001), pp. 441-444.

Genome-wide gene expression profiling reveals renal genes regulated during metabolic acidosis

Marta Nowik — 2008-02-22T04:22:21-00:00

Physiol. Genomics, Vol. 32, No. 3. (19 February 2008), pp. 322-334.

Production and excretion of acids are balanced to maintain systemic acid-base homeostasis. During metabolic acidosis (MA) excess acid accumulates and is removed from the body, a process achieved, at least in part, by increasing renal acid excretion. This acid-secretory process requires the concerted regulation of metabolic and transport pathways, which are only partially understood. Chronic MA causes also morphological remodeling of the kidney. Therefore, we characterized transcriptional changes in mammalian kidney during MA to gain insights into adaptive pathways. Total kidney RNA from control and 2- and 7-days NH4Cl treated mice was subjected to microarray gene profiling. We identified 4,075 transcripts significantly (P < 0.05) regulated after 2 and/or 7 days of treatment. Microarray results were confirmed by qRT-PCR. Analysis of candidate genes revealed that a large group of regulated transcripts was represented by different solute carrier transporters, genes involved in cell growth, proliferation, apoptosis, water homeostasis, and ammoniagenesis. Pathway analysis revealed that oxidative phosphorylation was the most affected pathway. Interestingly, the majority of acutely regulated genes after 2 days, returned to normal values after 7 days suggesting that adaptation had occurred. Besides these temporal changes, we detected also differential regulation of selected genes (SNAT3, PEPCK, PDG) between early and late proximal tubule. In conclusion, the mammalian kidney responds to MA by temporally and spatially altering the expression of a large number of genes. Our analysis suggests that many of these genes may participate in various processes leading to adaptation and restoration of normal systemic acid-base and electrolyte homeostasis. 10.1152/physiolgenomics.00160.2007

Function of connexins in the renal circulation

C Wagner — 2008-02-19T09:22:30-00:00

Kidney Int, Vol. 73, No. 5. (12 December 2007), pp. 547-555.

The H-Invitational Database (H-InvDB), a comprehensive annotation resource for human genes and transcripts.

2008-01-28T03:45:18-00:00

Nucleic Acids Res, Vol. 36, No. Database issue. (January 2008)

Here we report the new features and improvements in our latest release of the H-Invitational Database (H-InvDB; http://www.h-invitational.jp/), a comprehensive annotation resource for human genes and transcripts. H-InvDB, originally developed as an integrated database of the human transcriptome based on extensive annotation of large sets of full-length cDNA (FLcDNA) clones, now provides annotation for 120 558 human mRNAs extracted from the International Nucleotide Sequence Databases (INSD), in addition to 54 978 human FLcDNAs, in the latest release H-InvDB_4.6. We mapped those human transcripts onto the human genome sequences (NCBI build 36.1) and determined 34 699 human gene clusters, which could define 34 057 (98.1%) protein-coding and 642 (1.9%) non-protein-coding loci; 858 (2.5%) transcribed loci overlapped with predicted pseudogenes. For all these transcripts and genes, we provide comprehensive annotation including gene structures, gene functions, alternative splicing variants, functional non-protein-coding RNAs, functional domains, predicted sub cellular localizations, metabolic pathways, predictions of protein 3D structure, mapping of SNPs and microsatellite repeat motifs, co-localization with orphan diseases, gene expression profiles, orthologous genes, protein-protein interactions (PPI) and annotation for gene families. The current H-InvDB annotation resources consist of two main views: Transcript view and Locus view and eight sub-databases: the DiseaseInfo Viewer, H-ANGEL, the Clustering Viewer, G-integra, the TOPO Viewer, Evola, the PPI view and the Gene family/group.

Genome-scale RNAi profiling of cell division in human tissue culture cells

Ralf Kittler — 2007-12-03T17:43:20-00:00

Nature Cell Biology, Vol. 9, No. 12. (11 November 2007), pp. 1401-1412.

The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

DS Gerhard — 2007-01-18T23:32:12-00:00

Genome Res, Vol. 14, No. 10B. (October 2004), pp. 2121-2127.

The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

A central role for the JNK pathway in mediating the antagonistic activity of pro-inflammatory cytokines against transforming growth factor-beta-driven SMAD3/4-specific gene expression.

F Verrecchia — 2008-01-14T15:01:18-00:00

J Biol Chem, Vol. 278, No. 3. (17 January 2003), pp. 1585-1593.

We have focused our attention on the molecular events underlying the antagonistic activities of pro-inflammatory cytokines against transforming growth factor-beta (TGF-beta)/SMAD signaling. Using jnk1/2-knockout (jnk(-/-)) and I kappa B kinase-gamma/nemo(-/-) fibroblasts, we have determined the specific roles played by the JNK/AP-1 and NF-kappa B/Rel pathways in this phenomenon. We demonstrate that, in a cellular context devoid of JNK activity (i.e. jnk(-/-) fibroblasts), interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) did not inhibit the formation of SMAD-DNA complexes and the resulting SMAD-driven transcription in response to TGF-beta. On the other hand, lack of NF-kappa B activity in nemo(-/-) fibroblasts did not affect the antagonistic effect of pro-inflammatory cytokines against TGF-beta. In the latter cell type, overexpression of antisense c-jun mRNA or of a dominant-negative form of MKK4 blocked the inhibitory activity of TNF-alpha, similar to what was observed in normal human dermal fibroblasts. Among JNK substrates, c-Jun and JunB (but not activating transcription factor-2) antagonized TGF-beta/SMAD signaling in a JNK-dependent manner. Overexpression of JNK1 in jnk(-/-) fibroblasts restored the ability of cytokines and Jun proteins to interfere with SMAD signaling. In junAA mouse embryo fibroblasts, in which c-Jun can no longer be phosphorylated by JNK, JunB substituted for c-Jun in mediating the cytokine effect against SMAD-driven transcription in a JNK-dependent manner. These results suggest a critical role for JNK-mediated c-Jun and JunB phosphorylation in transmitting the inhibitory effect of pro-inflammatory cytokines against TGF-beta-induced SMAD signaling. In addition, we demonstrate that such a JNK-dependent regulatory mechanism underlies the antagonistic activity of TNF-alpha against TGF-beta-induced up-regulation of type I and III collagens in fibroblasts.

Glycogen Synthase Kinase-3alpha Reduces Cardiac Growth and Pressure Overload-induced Cardiac Hypertrophy by Inhibition of Extracellular Signal-regulated Kinases

Peiyong Zhai — 2007-12-29T14:50:13-00:00

J. Biol. Chem., Vol. 282, No. 45. (9 November 2007), pp. 33181-33191.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase having multiple functions and consisting of two isoforms, GSK-3alpha and GSK-3[beta]. Pressure overload increases expression of GSK-3alpha but not GSK-3[beta]. Despite our wealth of knowledge about GSK-3[beta], the function of GSK-3alpha in the heart is not well understood. To address this issue, we made cardiac-specific GSK-3alpha transgenic mice (Tg). Left ventricular weight and cardiac myocyte size were significantly smaller in Tg than in non-Tg (NTg) mice, indicating that GSK-3alpha inhibits cardiac growth. After 4 weeks of aortic banding (transverse aortic constriction (TAC)), increases in left ventricular weight and myocyte size were significantly smaller in Tg than in NTg, indicating that GSK-3alpha inhibits cardiac hypertrophy. More severe cardiac dysfunction developed in Tg after TAC. Increases in fibrosis and apoptosis were greater in Tg than in NTg after TAC. Among signaling molecules screened, ERK phosphorylation was decreased in Tg. Adenovirus-mediated overexpression of GSK-3alpha, but not GSK-3[beta], inhibited ERK in cultured cardiac myocytes. Knockdown of GSK-3alpha increased ERK phosphorylation, an effect that was inhibited by PD98059, rottlerin, and protein kinase Cepsilon (PKCepsilon) inhibitor peptide, suggesting that GSK-3alpha inhibits ERK through PKC-MEK-dependent mechanisms. Knockdown of GSK-3alpha increased protein content and reduced apoptosis, effects that were abolished by PD98059, indicating that inhibition of ERK plays a major role in the modulation of cardiac growth and apoptosis by GSK-3alpha. In conclusion, up-regulation of GSK-3alpha inhibits cardiac growth and pressure overload-induced cardiac hypertrophy but increases fibrosis and apoptosis in the heart. The anti-hypertrophic and pro-apoptotic effect of GSK-3alpha is mediated through inhibition of ERK. 10.1074/jbc.M705133200

Database resources of the National Center for Biotechnology Information.

David L Wheeler — 2007-12-02T21:06:38-00:00

Nucleic Acids Res (27 November 2007)

In addition to maintaining the GenBank(R) nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides analysis and retrieval resources for the data in GenBank and other biological data available through NCBI's web site. NCBI resources include Entrez, the Entrez Programming Utilities, My NCBI, PubMed, PubMed Central, Entrez Gene, the NCBI Taxonomy Browser, BLAST, BLAST Link, Electronic PCR, OrfFinder, Spidey, Splign, RefSeq, UniGene, HomoloGene, ProtEST, dbMHC, dbSNP, Cancer Chromosomes, Entrez Genome, Genome Project and related tools, the Trace, Assembly, and Short Read Archives, the Map Viewer, Model Maker, Evidence Viewer, Clusters of Orthologous Groups, Influenza Viral Resources, HIV-1/Human Protein Interaction Database, Gene Expression Omnibus, Entrez Probe, GENSAT, Database of Genotype and Phenotype, Online Mendelian Inheritance in Man, Online Mendelian Inheritance in Animals, the Molecular Modeling Database, the Conserved Domain Database, the Conserved Domain Architecture Retrieval Tool and the PubChem suite of small molecule databases. Augmenting the web applications are custom implementations of the BLAST program optimized to search specialized data sets. These resources can be accessed through the NCBI home page at www.ncbi.nlm.nih.gov.

Recommendations for the standardization and interpretation of the electrocardiogram: part I: The electrocardiogram and its technology: a scientific statement from the American Heart Association Electrocardiography and Arrhythmias Committee, Council on Clinical Cardiology; the American College of Cardiology Foundation; and the Heart Rhythm Society: endorsed by the International Society for Computerized Electrocardiology.

P Kligfield — 2007-10-20T16:13:46-00:00

Circulation, Vol. 115, No. 10. (13 March 2007), pp. 1306-1324.

This statement examines the relation of the resting ECG to its technology. Its purpose is to foster understanding of how the modern ECG is derived and displayed and to establish standards that will improve the accuracy and usefulness of the ECG in practice. Derivation of representative waveforms and measurements based on global intervals are described. Special emphasis is placed on digital signal acquisition and computer-based signal processing, which provide automated measurements that lead to computer-generated diagnostic statements. Lead placement, recording methods, and waveform presentation are reviewed. Throughout the statement, recommendations for ECG standards are placed in context of the clinical implications of evolving ECG technology.

Investigations of the effects of gender, diurnal variation, and age in human urinary metabolomic profiles.

CM Slupsky — 2007-12-12T15:37:41-00:00

Anal Chem, Vol. 79, No. 18. (15 September 2007), pp. 6995-7004.

Metabolomics may have the capacity to revolutionize disease diagnosis through the identification of scores of metabolites that vary during environmental, pathogenic, or toxicological insult. NMR spectroscopy has become one of the main tools for measuring these changes since an NMR spectrum can accurately identify metabolites and their concentrations. The predominant approach in analyzing NMR data has been through the technique of spectral binning. However, identification of spectral areas in an NMR spectrum is insufficient for diagnostic evaluation, since it is unknown whether areas of interest are strictly caused by metabolic changes or are simply artifacts. In this paper, we explore differences in gender, diurnal variation, and age in a human population. We use the example of gender differences to compare traditional spectral binning techniques (NMR spectral areas) to novel targeted profiling techniques (metabolites and their concentrations). We show that targeted profiling produces robust models, generates accurate metabolite concentration data, and provides data that can be used to help understand metabolic differences in a healthy population. Metabolites relating to mitochondrial energy metabolism were found to differentiate gender and age. Dietary components and some metabolites related to circadian rhythms were found to differentiate time of day urine collection. The mechanisms by which these differences arise will be key to the discovery of new diagnostic tests and new understandings of the mechanism of disease.

p38alpha: A Suppressor of Cell Proliferation and Tumorigenesis.

Lijian Hui — 2007-11-30T14:22:23-00:00

Cell Cycle, Vol. 6, No. 20. (21 July 2007)

The mitogen-activated protein kinase (MAPK) p38alpha is involved in numerous biological processes and is a drug target for inflammation-associated diseases. Genetic analysis in mice demonstrated that fetuses lacking p38alpha are embryonic lethal owing to impaired placental development. The function of p38alpha in mice after birth remained unclear until conditional alleles of p38alpha were used. It was found that p38alpha is essential for lung function in both neonatal and adult mice. Increased proliferation and impaired differentiation are the hallmarks of p38alpha-deficient cells. Moreover, mice deficient in p38alpha are prone to cancer development using carcinogen or oncogene-induced cancer models. p38alpha can suppress cell proliferation by antagonizing the JNK/c-Jun pathway, which is an important regulator of proliferation and apoptosis. These findings suggest that therapeutic inhibition of p38 might lead to unwanted proliferation. Therefore, a combined inhibition of p38 and other pathways, such as the JNK pathway, should be considered for targeting cancer inflammation.

From genome to proteome: developing expression clone resources for the human genome.

G Temple — 2006-05-30T05:28:02-00:00

Hum Mol Genet, Vol. 15 Suppl 1 (15 April 2006)

cDNA clones have long been valuable reagents for studying the structure and function of proteins. With recent access to the entire human genome sequence, it has become possible and highly productive to compare the sequences of mRNAs to their genes, in order to validate the sequences and protein-coding annotations of each (1,2). Thus, well-characterized collections of human cDNAs are now playing an essential role in defining the structure and function of human genes and proteins. In this review, we will summarize the major collections of human cDNA clones, discuss some limitations common to most of these collections and describe several noteworthy proteomics applications, focusing on the detection and analysis of protein-protein interactions (PPI). These human cDNA collections contain principally two types of cDNA clones. The largest collections comprise cDNAs with full-length protein coding sequences (FL-CDS). Some but not all of these cDNA clones may represent the entire mRNA sequence, but many are missing considerable non-coding UTR sequence, usually at the 5' end. A second type of cDNA clone, a 'full-ORF' (F-ORF) expression clone, is one where the annotated protein-coding sequence, excised of 5' UTR and 3' UTR sequence, has been transferred to a vector designed to facilitate transfer to other vectors for protein expression.

Strategic Approach to Fit-for-Purpose Biomarkers in Drug Development.

John A Wagner — 2007-10-30T03:48:56-00:00

Annu Rev Pharmacol Toxicol (15 October 2007)

blacksquare, square, filled Abstract The strategic, fit-for-purpose use of the combination of robust target engagement and well-qualified disease-related biomarkers enhances understanding of the mechanism of action, ties together preclinical and clinical data, enables the assessment of target engagement, facilitates early proof of concept and dose focusing, and increases the efficiency of early clinical development with improved quality of decision making. Significant progress in biomarker discovery, validation, and qualification has increased drug-development decision making and regulatory applications. Target engagement biomarkers are present early in a pathophysiologic cascade and inform on physical or biological interactions with the molecular target of the drug. Disease-related biomarkers are present late in the pathophysiologic cascade and are linked to clinical benefit; thus, they assess a drug's effect on a particular disease. Together, these concepts lay the groundwork for high-quality drug-development decision making and a framework for the acceptance and qualification of biomarkers for regulatory use. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 48 is January 6, 2008. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.

Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR.

Ingeborg Hein — 2007-10-30T02:11:29-00:00

J Microbiol Methods (17 September 2007)

Unspecific background DNA in quantitative universal real-time PCR utilizing a hydrolysis probe was completely suppressed by the addition of EMA or PMA to the PCR mix via cross-linking of the dyes to DNA during 650 W visible light exposure. The proposed procedure had no effect on the sensitivity of the real-time PCR reaction.

TLR4/MD-2 Monoclonal Antibody Therapy Affords Protection in Experimental Models of Septic Shock

Bruno Daubeuf — 2007-10-29T04:31:20-00:00

J Immunol, Vol. 179, No. 9. (1 November 2007), pp. 6107-6114.

Overactivation of the immune system upon acute bacterial infection leads to septic shock. Specific bacterial products potently stimulate immune cells via toll-like receptors (TLRs). Gram-negative bacteria induce a predominantly TLR4-driven signal through LPS release. To neutralize LPS signaling in experimental models of sepsis, we generated mAbs toward the TLR4/myeloid differentiation protein-2 (MD-2) complex. The binding properties of an array of selected rat mAbs differed in respect to their specificity for TLR4/MD-2 complex. The specificity of one such mAb, 5E3, to murine TLR4 was confirmed by its recognition of an epitope within the second quarter of the ectodomain. 5E3 inhibited LPS-dependent cell activation in vitro and prevented proinflammatory cytokine production in vivo following LPS challenge in a dose-dependent manner. Furthermore, 5E3 protected mice from lethal shock-like syndrome when applied using both preventative and therapeutic protocols. Most notably, in the colon ascendens stent peritonitis model of polymicrobial abdominal sepsis, administration of a single dose of 5E3 (50 microg) protected mice against mortality. These results demonstrate that neutralizing TLR4/MD-2 is highly efficacious in protecting against bacterial infection-induced toxemia and offers TLR4/MD-2 mAb treatment as a potential therapy for numerous clinical indications.

The EGF receptor is required for efficient liver regeneration

Anuradha Natarajan — 2007-10-25T14:59:47-00:00

Proceedings of the National Academy of Sciences, Vol. 104, No. 43. (23 October 2007), pp. 17081-17086.

Mice lacking the EGF receptor (EGFR) die between midgestation and postnatal day 20 with various defects in neural and epithelial organs. Here, we generated mice carrying a floxed EGFR allele to inactivate the EGFR in fetal and adult liver. Perinatal deletion of EGFR in hepatocytes resulted in decreased body weight, whereas deletion in the adult liver did not affect body mass. Although liver function was not affected, after partial hepatectomy mice lacking EGFR in the liver showed increased mortality accompanied by increased levels of serum transaminases indicating liver damage. Liver regeneration was delayed in the mutants because of reduced hepatocyte proliferation. Analysis of cell cycle progression in EGFR-deficient livers indicated a defective G1S phase entry with delayed transcriptional activation and reduced protein expression of cyclin D1 followed by reduced cdk2 and cdk1 expression. Impaired liver regeneration was accompanied by compensatory up-regulation of TNFalpha in the serum and prolonged activation of c-Jun. Moreover, p38alpha and NF-kappaB activation was reduced in regenerating mutant livers, indicating an impaired stress response after hepatectomy. Our studies demonstrate that EGFR is a critical regulator of hepatocyte proliferation in the initial phases of liver regeneration. 10.1073/pnas.0704126104

c-Jun/AP-1 controls liver regeneration by repressing p53/p21 and p38 MAPK activity

Ewa Stepniak — 2007-10-21T12:20:28-00:00

Genes Dev., Vol. 20, No. 16. (15 August 2006), pp. 2306-2314.

The AP-1 transcription factor c-Jun is a key regulator of hepatocyte proliferation. Mice lacking c-Jun in the liver (c-jun Deltali*) display impaired liver regeneration after partial hepatectomy (PH). This phenotype correlates with increased protein levels of the cdk-inhibitor p21 in the liver. We performed PH experiments in several double-knockout mouse models to genetically identify the signaling events regulated by c-Jun. Inactivation of p53 in c-jun Deltali* mice abrogated both hepatocyte cell cycle block and increased p21 protein expression. Consistently, liver regeneration was rescued in c-jun Deltali* p21 -/- double-mutant mice. This indicated that c-Jun controls hepatocyte proliferation by a p53/p21-dependent mechanism. Analyses of p21 mRNA and protein expression in livers of c-jun Deltali* mice after PH revealed that the accumulation of p21 protein is due to a post-transcriptional/post-translational mechanism. We have investigated several candidate pathways implicated in the regulation of p21 expression, and observed increased activity of the stress kinase p38 in regenerating livers of c-jun Deltali* mice. Importantly, conditional deletion of p38alpha in livers of c-jun Deltali* mice fully restored hepatocyte proliferation and attenuated increased p21 protein levels after PH. These data demonstrate that c-Jun/AP-1 regulates liver regeneration through a novel molecular pathway that involves p53, p21, and the stress kinase p38alpha. 10.1101/gad.390506

The origins of oncomice: a history of the first transgenic mice genetically engineered to develop cancer.

D Hanahan — 2007-10-15T04:02:52-00:00

Genes Dev, Vol. 21, No. 18. (15 September 2007), pp. 2258-2270.

This perspective describes the concurrent development in the 1980s of the first transgenic mice genetically engineered to express dominant oncogenes, involving independent researchers who were largely unaware of each other's strategies and progress. We relate the experimental designs, the pitfalls and challenges encountered, and the eventual success in developing distinctive mouse models of cancer, wherein tumors arose heritably in various organs. These early oncomice have produced a wealth of new knowledge, become topics of intellectual property, and spawned a vibrant field of cancer research that is revealing mechanisms of tumorigenesis and suggesting new therapeutic strategies for treating the human disease.

Software for optimization of SNP and PCR-RFLP genotyping to discriminate many genomes with the fewest assays.

SN Gardner — 2007-09-24T03:51:02-00:00

BMC Genomics, Vol. 6, No. 1. (2005)

BACKGROUND: Microbial forensics is important in tracking the source of a pathogen, whether the disease is a naturally occurring outbreak or part of a criminal investigation. RESULTS: A method and SPR Opt (SNP and PCR-RFLP Optimization) software to perform a comprehensive, whole-genome analysis to forensically discriminate multiple sequences is presented. Tools for the optimization of forensic typing using Single Nucleotide Polymorphism (SNP) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) analyses across multiple isolate sequences of a species are described. The PCR-RFLP analysis includes prediction and selection of optimal primers and restriction enzymes to enable maximum isolate discrimination based on sequence information. SPR Opt calculates all SNP or PCR-RFLP variations present in the sequences, groups them into haplotypes according to their co-segregation across those sequences, and performs combinatoric analyses to determine which sets of haplotypes provide maximal discrimination among all the input sequences. Those set combinations requiring that membership in the fewest haplotypes be queried (i.e. the fewest assays be performed) are found. These analyses highlight variable regions based on existing sequence data. These markers may be heterogeneous among unsequenced isolates as well, and thus may be useful for characterizing the relationships among unsequenced as well as sequenced isolates. The predictions are multi-locus. Analyses of mumps and SARS viruses are summarized. Phylogenetic trees created based on SNPs, PCR-RFLPs, and full genomes are compared for SARS virus, illustrating that purported phylogenies based only on SNP or PCR-RFLP variations do not match those based on multiple sequence alignment of the full genomes. CONCLUSION: This is the first software to optimize the selection of forensic markers to maximize information gained from the fewest assays, accepting whole or partial genome sequence data as input. As more sequence data becomes available for multiple strains and isolates of a species, automated, computational approaches such as those described here will be essential to make sense of large amounts of information, and to guide and optimize efforts in the laboratory. The software and source code for SPR Opt is publicly available and free for non-profit use at http://www.llnl.gov/IPandC/technology/software/softwaretitles/spropt.php.

Distinct functions of junD in cardiac hypertrophy and heart failure.

R Ricci — 2007-09-21T08:54:39-00:00

Genes Dev, Vol. 19, No. 2. (15 January 2005), pp. 208-213.

Cardiac hypertrophic stimuli induce both adaptive and maladaptive growth response pathways in heart. Here we show that mice lacking junD develop less adaptive hypertrophy in heart after mechanical pressure overload, while cardiomyocyte-specific expression of junD in mice results in spontaneous ventricular dilation and decreased contractility. In contrast, fra-1 conditional knock-out mice have a normal hypertrophic response, whereas hearts from fra-1 transgenic mice decompensate prematurely. Moreover, fra-1 transgenic mice simultaneously lacking junD reveal a spontaneous dilated cardiomyopathy associated with increased cardiomyocyte apoptosis and a primary mitochondrial defect. These data suggest that junD promotes both adaptive-protective and maladaptive hypertrophy in heart, depending on its expression levels.

Novel insights into the regulation of systemic phosphate homeostasis and renal phosphate excretion.

CA Wagner — 2007-09-18T16:06:27-00:00

J Nephrol, Vol. 20, No. 2. (r 2007), pp. 130-134.

Stimulation of soluble guanylyl cyclase inhibits mesangial cell proliferation and matrix accumulation in experimental glomerulonephritis.

B Hohenstein — 2007-09-05T09:19:24-00:00

Am J Physiol Renal Physiol, Vol. 288, No. 4. (April 2005)

To date, no specific treatment is established in mesangial proliferative glomerulonephritis in humans. Specific stimulation of soluble guanylyl cyclase (sGC), an enzyme catalyzing the synthesis of cGMP from GTP, can be achieved by the novel pyrazolopyridine derivative BAY 41-2272. The effect of sGC stimulation via BAY 41-2272 on mesangial proliferation was assessed in vivo using a mesangial proliferative glomerulonephritis model in rats (anti-Thy1 model). Renal biopsies, as well as glomerular isolates, urine samples, and blood samples were compared in BAY 41-2272- and placebo-treated groups during anti-Thy1 nephritis. The sGC beta(1)-subunit is upregulated during anti-Thy1 nephritis and mainly confined to mesangial areas by immunohistochemistry. Specific therapeutic sGC stimulation during anti-Thy1 nephritis in vivo was achieved via BAY 41-2272 treatment as demonstrated by increased glomerular cGMP levels causing inhibition of mesangial proliferation, glomerular matrix accumulation, and proteinuria compared with placebo-treated animals. sGC is tightly regulated in glomeruli during experimental glomerulonephritis. Considering its beneficial antiproliferative, antifibrotic, and antiproteinuric effect in experimental glomerulonephritis, the therapeutic stimulation of sGC could become a promising future goal in mesangial proliferative glomerulonephritis in humans.

Angiotensin II-induced ERK1/ERK2 activation and protein synthesis are redox-dependent in glomerular mesangial cells.

Y Gorin — 2007-09-05T08:33:53-00:00

Biochem J, Vol. 381, No. Pt 1. (1 July 2004), pp. 231-239.

Angiotensin II (Ang II) stimulates hypertrophy of glomerular mesangial cells. The signalling mechanism by which Ang II exerts this effect is not precisely known. Downstream potential targets of Ang II are the extracellular-signal-regulated kinases 1 and 2 (ERK1/ERK2). We demonstrate that Ang II activates ERK1/ERK2 via the AT1 receptor. Arachidonic acid (AA) mimics the action of Ang II on ERK1/ERK2 and phospholipase A2 inhibitors blocked Ang II-induced ERK1/ERK2 activation. The antioxidant N-acetylcysteine as well as the NAD(P)H oxidase inhibitors diphenylene iodonium and phenylarsine oxide abolished both Ang II- and AA-induced ERK1/ERK2 activation. Moreover, dominant-negative Rac1 (N17Rac1) blocks activation of ERK1/ERK2 in response to Ang II and AA, whereas constitutively active Rac1 resulted in an increase in ERK1/ERK2 activity. Antisense oligonucleotides for Nox4 NAD(P)H oxidase significantly reduce activation of ERK1/ERK2 by Ang II and AA. We also show that protein synthesis in response to Ang II and AA is inhibited by N17Rac1 or MEK (mitogen-activated protein kinase/ERK kinase) inhibitor. These results demonstrate that Ang II stimulates ERK1/ERK2 by AA and Nox4-derived reactive oxygen species, suggesting that these molecules act as downstream signal transducers of Ang II in the signalling pathway linking the Ang II receptor AT1 to ERK1/ERK2 activation. This pathway involving AA, Rac1, Nox4, reactive oxygen species and ERK1/ERK2 may play an important role in Ang II-induced mesangial cell hypertrophy.