Roche Diagnostics GmbH Research Articles

The impact of microalbuminuria measured by qualitative or quantitative methods on risk evaluation in hypertensive patients: Results from ICEBERG study

Although microalbuminuria determined by quantitative methods seems to be more accurate, using dipstick is simpler and cheaper, thus making this qualitative method more suitable for screening in primary healthcare settings for hypertensive patients. The primary purpose of Intensive / Initial Cardiovascular Examination regarding Blood pressure levels: Evaluation of Risk Groups (ICEBERG) study protocol is to determine the impact of different laboratory tests on cardiovascular risk stratification of subjects with BP levels ≥130/85 mmHg. This report focuses on the comparison of the impact of quantitative and qualitative microalbuminuria detection on the risk evaluation. ICEBERG is a healthcare organization-based epidemiological study, conducted at 217 centers (20 cardiology -1- and 197 primary healthcare centers -2-). In group 1, microalbuminuria was calculated as urinary albumin/creatinine ratio in random urine samples (quantitative method) and in group 2, urine strips (Micral-test Microalbuminuria®, Roche Diagnostics GmBh) were used (qualitative method). 9268 patients were included in the analysis (765 and 8496 in groups 1 and 2, respectively). Initial risk assessment was performed according to ESC-ESH 2003 guidelines and reassessment was done by using quantitative and qualitative methods. The study patients were 58.4±11.3 years old (female 65.3%). 53.8% and 54.5% of the patients were classified into high (H) or very high (VH) added risk group with baseline evaluation, in groups 1 and 2, respectively. Switching upwards to H/VH risk group was 6.2% and 6.6%, when quantitative and qualitative microalbuminuria tests were taken into account, respectively. Risk group switching upwards were found to be similar in both quantitative and qualitative microalbuminuria groups, thus demonstrating that dipstick microalbuminuria detection has almost the same effectiveness when compared with quantitative.

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Trafficking of the Plasma Membrane γ-Aminobutyric Acid Transporter GAT1: SIZE AND RATES OF AN ACUTELY RECYCLING POOL

Cyclin D1 Inhibits Peroxisome Proliferator-activated Receptor γ-mediated Adipogenesis through Histone Deacetylase Recruitment

The cyclin D1 gene encodes the labile serum-inducible regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. Overexpression of cyclin D1 promotes cellular proliferation and normal physiological levels of cyclin D1 function to inhibit adipocyte differentiation in vivo. We have previously shown that cyclin D1 inhibits peroxisome proliferator-activated receptor (PPAR)gamma-dependent activity through a cyclin-dependent kinase- and retinoblastoma protein-binding-independent mechanism. In this study, we determined the molecular mechanism by which cyclin D1 regulated PPARgamma function. Herein, murine embryonic fibroblast (MEF) differentiation by PPARgamma ligand was associated with a reduction in histone deacetylase (HDAC1) activity. Cyclin D1-/- MEFs showed an increased propensity to undergo differentiation into adipocytes. Genetic deletion of cyclin D1 reduced HDAC1 activity. Reconstitution of cyclin D1 into the cyclin D1-/- MEFs increased HDAC1 activity and blocked PPARgamma-mediated adipogenesis. PPARgamma activity was enhanced in cyclin D1-/- cells. Reintroduction of cyclin D1 inhibited basal and ligand-induced PPARgamma activity and enhanced HDAC repression of PPARgamma activity. Cyclin D1 bound HDAC in vivo and preferentially physically associated with HDAC1, HDAC2, HDAC3, and HDAC5. Chromatin immunoprecipitation assay demonstrated that cyclin D1 enhanced recruitment of HDAC1 and HDAC3 and histone methyltransferase SUV39H1 to the PPAR response element of the lipoprotein lipase promoter and decreased acetylation of total histone H3 and histone H3 lysine 9. Collectively, these studies suggest an important role of cyclin D1 in regulation of PPARgamma-mediated adipocyte differentiation through recruitment of HDACs to regulate PPAR response element local chromatin structure and PPARgamma function.

RNA interference-mediated control of hepatitis B virus and emergence of resistant mutant

Background & Aims Present therapy for chronic hepatitis B attains control only in limited proportions. Small interfering RNA (siRNA) offers a new tool with potential therapeutic applications for hepatitis B virus (HBV). Given the importance of sequence identity in the effectiveness of siRNA and the heterogeneity of HBV sequences among different isolates, a short hairpin RNA (shRNA)-expressing plasmid, pSuper/HBVS1, was developed to target a region conserved among major HBV genotypes and assess its effectiveness control of HBV. Methods HBV replication-competent plasmid was cotransfected with pSuper/HBVS1 to HuH-7 cells or to mice. The levels of viral proteins, RNA, and DNA were examined in transfected cells and animals. The effects of pSuper/HBVS1 on clinical isolates with genotypes B and C were also determined. Results pSuper/HBVS1 significantly decreased levels of viral proteins, RNA, and DNA for HBV genotype A in cell culture and in mice. Comparable suppressive effects were observed on clinical isolates of genotypes B and C. A clone with a silent mutation in the target region was identified from a patient with genotype C. This mutant revealed diminished sensitivity to pSuper/HBVS1 and could be selected out in the presence of pSuper/HBVS1 in cell culture. Conclusions These findings indicated that shRNA could suppress HBV expression and replication for genotypes A, B, and C, promising an advance in treatment of HBV. However, the emergence of resistant mutants in HBV quasispecies should be considered.

Positive and Negative Regulation of Nuclear Factor-κB-mediated Transcription by IκB-ζ, an Inducible Nuclear Protein

IkappaB-zeta is an inducible nuclear protein that interacts with nuclear factor-kappaB (NF-kappaB) via its carboxyl-terminal ankyrin-repeats. Previous studies using an NF-kappaB reporter have shown that IkappaB-zeta inhibits the activity of NF-kappaB. In the present study, we dissected the amino-terminal region of IkappaB-zeta, which shows no homology to any other proteins. Indirect immunofluorescence studies demonstrated the presence of a bipartite nuclear localization signal spanning amino acids 163-178. Using GAL4 fusion proteins, we found that internal fragments containing amino acids 329-402 possessed intrinsic transcriptional activation activity. Interestingly, the activity was not detected in GAL4 fusion proteins of the full-length IkappaB-zeta. On the other hand, the GAL4-dependent transcriptional activity was generated by co-expression of the GAL4-NF-kappaB p50 subunit fusion protein and the full-length IkappaB-zeta, neither of which exhibited the activity on their own. A new splicing variant, IkappaB-zeta(D), with a deletion of amino acids 236-429, was found to lack transactivation activity. Forced expression of IkappaB-zeta, but not IkappaB-zeta(D), augmented interleukin-6 production, indicating the functional significance of the transactivation activity. In contrast, tumor necrosis factor-alpha production was inhibited by expression of IkappaB-zeta, highlighting the dual functions of this molecule. These results indicate that IkappaB-zeta harbors latent transcriptional activation activity, and that the activity is expressed upon interaction with the NF-kappaB p50 subunit. In addition to the inhibitory activity on NF-kappaB-mediated transcription, the transcriptional activation activity of IkappaB-zeta should be crucial for the regulation of inflammation.

Value of a standard urinary dipstick test for detecting microalbuminuria in patients with newly diagnosed hypertension.

Microalbuminuria may indicate target organ damage in hypertensive patients. However, testing for microalbuminuria is not yet consistently used in general practice. This may be partly due to a lack of data regarding the diagnostic value of practice-based dipstick testing in newly diagnosed hypertension. Objectives were to assess the diagnostic value of a standard dipstick test for urinary protein excretion. 186 patients who had been newly diagnosed with hypertension were screened for microalbuminuria. A spot urine sample from each of the subjects was evaluated by using a standard dipstick test (Combur 10, Roche Diagnostics GmbH, Mannheim, Germany) in a primary care setting. The albumin/creatinine ratio was used as the "gold standard". Dipstick testing for protein was positive in 31 urine samples (16.7% of the test samples). The albumin/creatinine ratio was elevated in 33 samples (17.7% of the test samples). The sensitivity of detecting microalbuminuria was 26%, specificity 89%, positive predictive value 45%, and the negative predictive was 88%. Repeated dipstick testing 48 hours after the initial testing in 40 randomly selected patients showed a good reproducibility (98%). In a primary care setting a positive standard dipstick test of a random spot urine in patients with newly diagnosed hypertension may indicate the presence of microalbuminuria with high specificity. However, because of its low sensitivity, the standard urinary dipstick test can not be recommended as the sole method of screening for renal target organ damage. In addition standard dipstick testing is important to exclude confounding factors that can falsify the measurement of urinary protein excretion.

Value of a standard urinary dipstick test for detecting microalbuminuria in patients with newly diagnosed hypertension.

Microalbuminuria may indicate target organ damage in hypertensive patients. However, testing for microalbuminuria is not yet consistently used in general practice. This may be partly due to a lack of data regarding the diagnostic value of practice-based dipstick testing in newly diagnosed hypertension. Objectives were to assess the diagnostic value of a standard dipstick test for urinary protein excretion. 186 patients who had been newly diagnosed with hypertension were screened for microalbuminuria. A spot urine sample from each of the subjects was evaluated by using a standard dipstick test (Combur 10, Roche Diagnostics GmbH, Mannheim, Germany) in a primary care setting. The albumin/creatinine ratio was used as the "gold standard". Dipstick testing for protein was positive in 31 urine samples (16.7% of the test samples). The albumin/creatinine ratio was elevated in 33 samples (17.7% of the test samples). The sensitivity of detecting microalbuminuria was 26%, specificity 89%, positive predictive value 45%, and the negative predictive was 88%. Repeated dipstick testing 48 hours after the initial testing in 40 randomly selected patients showed a good reproducibility (98%). In a primary care setting a positive standard dipstick test of a random spot urine in patients with newly diagnosed hypertension may indicate the presence of microalbuminuria with high specificity. However, because of its low sensitivity, the standard urinary dipstick test can not be recommended as the sole method of screening for renal target organ damage. In addition standard dipstick testing is important to exclude confounding factors that can falsify the measurement of urinary protein excretion.

N-acetyltransferase 2 polymorphisms in patients with Behcet's disease

It is possible that dietary, environmental factors and/or genetic polymorphisms in xenobiotic-metabolizing enzymes may contribute to the development of Behcet's disease. As N-acetyltransferase (NAT) 2 is an important xenobiotic-metabolizing enzyme and theoretically the nonacetylated xenobiotics may induce an autoimmune mechanism, the aim of the present study was to investigate whether the genetic polymorphism of NAT2 plays a role in susceptibility to Behcet's disease. Forty Behcet's disease patients and 82 control subjects were enrolled in the study. NAT2*5A, NAT2*6A, NAT27*A/B and NAT2*14A polymorphisms were detected by using real time PCR with LightCycler (Roche Diagnostics GmbH, Mannheim, Germany). The NAT2*5A and NAT2*6A mutant genotypes carried an increased risk of developing Behcet's disease [odds ratio (OR) = 66.29, 95% confidence interval (CI) = 8.21-535.33; and OR = 24; 95% CI = 2.04-304.98, respectively]. The NAT2*7A/B and NAT2*14A gene polymorphisms were not an increased risk for developing Behcet's disease. As a result of this study we conclude the NAT2 slow acetylator status may be a determinant in susceptibility to Behcet's disease. This finding may have implications for the theories of the pathogenesis of the disease as well as for therapeutic aspects.

E4F1, a Novel Estrogen-Responsive Gene in Possible Atheroprotection, Revealed by Microarray Analysis

Estrogen has been postulated to be involved in inhibition of vascular smooth muscle cell (VSMC) proliferation mainly via estrogen receptor (ER), but the detailed mechanism has remained primarily unknown. Therefore, in this study, microarray analysis was used in two types of cultured human VSMCs: one positive for ER alpha, and the other for ER beta, which were treated by estrogens to detect the estrogen-responsive genes. We also used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to evaluate mRNA levels of selective target gene (TG) in these cells. We further studied whether the TG product was involved in inhibition of proliferation using small interfering RNA (siRNA) of the TG transfection. We subsequently used quantitative RT-PCR and in situ hybridization analysis to evaluate the expression of these gene products in human aorta. E4F1, a possible inducer of cell growth arrest, was markedly increased only in ER alpha-positive VSMCs by estrogens in both microarray and RT-PCR analyses. Blocking of E4F1 using siRNA suppressed estrogenic inhibition of ER alpha-positive VSMC proliferation. E4F1 mRNA was abundant in premenopausal female aorta with mild atherosclerotic changes. E4F1 is therefore considered one of the estrogen-responsive genes involving ER alpha-mediated inhibition of VSMC proliferation and may play an important role in estrogen-related atheroprotection of human aorta.

Prevalence of the C282Y, H63D, and S65C Mutations of theHFEGene in 1,146 Newborns from a Region of Northern Spain

In Spain, 85% of patients with genetic hemochromatosis (GH) are homozygous for the C282Y mutation of the HFE gene. H63D and S65C mutations of HFE may also play some role in the disease. The aim of this study was to establish the prevalence of C282Y, H63D, and S65C mutations of the HFE gene in newborns in Catalonia, Spain. One thousand one hundred forty-six newborn screening cards were selected randomly. DNA from these cards was extracted and HFE mutations were analyzed with the LightCycler equipment (Roche Diagnostics Gmbh, Mannheim, Germany). Sufficient DNA sample was obtained to screen for the three mutations in 1,043 cases (91%). The allelic frequencies of C282Y, H63D, and S65C mutations were 0.03 (IC 95% 0.022-0.037), 0.2 (IC 95% 0.19-0.22), and 0.01 (95% confidence interval [CI] 0.006-0.015), respectively. The frequency of C282Y homozygous newborns was 0.001 (95% CI 0.0005-0.0014). The frequencies of newborns doubly heterozygous for C282Y/H63D and C282Y/S65C were 0.01 (95% CI 0.005-0.02) and 0.002 (95% CI 0.0002-0.01), respectively. The allelic frequency of C282Y mutation is similar to that observed in Southern France, in the Czech Republic and in some areas of Italy. The allelic frequency of H63D mutation in Catalonia is the highest reported to date. Nevertheless, S65C is infrequent. These data should be kept in mind when designing hemochromatosis genotypic screening programs in Catalonia.

Citrus tristeza closterovirus

<i>Citrus tristeza closterovirus</i>

First Report of the Occurrence of Tomato chlorosis virus and Tomato infectious chlorosis virus in Taiwan.

Pronounced yellowing symptoms on the lower leaves of tomato plants, similar to those caused by nitrogen deficiency, were observed in the spring of 1998 in The Asian Vegetable Research and Development Center and in farmers' fields in southern Taiwan. However, the brittleness of the discolored leaves, occasional upward leaf rolling, and abundance of whiteflies on these plants suggested the involvement of Tomato chlorosis virus (ToCV) and Tomato infectious chlorosis virus (TICV) that belong to the group of whitefly-transmitted, phloem-limited criniviruses (family Closteroviridae). Leaves of symptomatic and healthy plants were collected, and total nucleic acids were extracted from 0.2 g of leaf tissue (1). The total nucleic acids were precipitated by ethanol and dissolved in 160 μl of sterile water. Eight microliters of total nucleic acids were observed on positively charged nylon membranes (Roche Diagnostic GmbH, Roch Applied Science, Germany). Two digoxigenin-labeled riboprobes, transcribed from pTIC8-44 (complementary to the 3'-end region of TICV RNA 1) and pToC 78 (corresponding to the coat protein region of ToCV RNA 2), were used in hybridization tests to detect TICV and ToCV, respectively (2). Six of seventeen symptomatic tomato plant samples were positive with the ToCV probe, whereas none of the 13 samples reacted with the TICV probe. Similar symptoms as described above for tomato were observed on zinnia plants in the same locations. Five of eight zinnia samples gave a positive reaction with the ToCV probe. One of the ToCV positive samples also gave a positive reaction with the TICV probe. Electron microscopic examination from leaf-dip preparations of ToCV-positive leaf tissues, stained in 1% uranyl acetate, showed the presence of flexuous filamentous particles approximately 800 to 850 nm long. To our knowledge, this is the first evidence of the presence of ToCV and TICV in zinnia and ToCV in tomato in Taiwan.

Quantification of Parvovirus B19 DNA Using COBAS AmpliPrep Automated Sample Preparation and LightCycler Real-Time PCR

The COBAS AmpliPrep instrument (Roche Diagnostics GmbH, D-68305 Mannheim, Germany) automates the entire sample preparation process of nucleic acid isolation from serum or plasma for polymerase chain reaction analysis. We report the analytical performance of the LightCycler Parvovirus B19 Quantification Kit (Roche Diagnostics) using nucleic acids isolated with the COBAS AmpliPrep instrument. Nucleic acids were extracted using the Total Nucleic Acid Isolation Kit (Roche Diagnostics) and amplified with the LightCycler Parvovirus B19 Quantification Kit. The kit combination processes 72 samples per 8-hour shift. The lower detection limit is 234 IU/ml at a 95% hit-rate, linear range approximately 10(4)-10(10) IU/ml, and overall precision 16 to 40%. Relative sensitivity and specificity in routine samples from pregnant women are 100% and 93%, respectively. Identification of a persistent parvovirus B19-infected individual by the polymerase chain reaction among 51 anti-parvovirus B19 IgM-negative samples underlines the importance of additional nucleic acid testing in pregnancy and its superiority to serology in identifying the risk of parvovirus B19 transmission via blood or blood products. Combination of the Total Nucleic Acid Isolation Kit on the COBAS AmpliPrep instrument with the LightCycler Parvovirus B19 Quantification Kit provides a reliable and time-saving tool for sensitive and accurate detection of parvovirus B19 DNA.

Hepsin is Highly Over Expressed in and a New Candidate for a Prognostic Indicator in Prostate Cancer

Other cDNA microarray studies have shown that hepsin is one of the highly over expressed genes in prostate cancer tissue compared with nonmalignant and benign prostatic hyperplasia tissue. We quantitatively analyzed hepsin gene expression with real-time polymerase chain reaction and calculated its relationships with clinicopathological parameters in a large cohort of samples. Matched prostate tissue samples from the cancerous and noncancerous parts of the same prostates were obtained from 90 patients with prostate cancer who underwent radical prostatectomy. Quantitative reverse transcriptase-polymerase chain reaction was performed using LightCycler Fast Start DNA Master SYBR Green I on a LightCycler (Roche Diagnostics GmbH, Mannheim, Germany) system. The ratio of hepsin-to-beta-actin (a housekeeping gene) was used to normalize data. Hepsin over expression in cancerous compared with noncancerous tissue was found in 81 of the 90 patient samples (90%, p <0.001). In 48 patients (53%) hepsin over expression was more than 10-fold in cancerous tissue. The ratio of cancerous-to-noncancerous hepsin expression was significantly higher in the 39 patients with grade 3 tumors compared with the 51 with grade 2 tumors (median 15.5 vs 9.6, p = 0.031). For the prognosis a cutoff at the 75th percentile provided a significant difference between patients at lower risk (pT2, G2 and Gleason score less than 7) and higher risk (pT3/4, G3 and Gleason score 7 or greater) for relapse. This report of the quantitative analysis of hepsin expression, which is the first to our knowledge, shows strong and significant over expression in prostate cancer tissue. Hepsin expression may be a new prognostic marker that could be used for assessing prostate cancer aggressiveness.

Report of a trial with lyophilised QC sera in the CMCH/ACBI EQAS programmes in India

2000 vials of lyophilized QC of two different levels (low and high) were donated by Roche Diagnostics GmbH, through the IFCC and received by CMCH in June 2001. A total of 240 la boratories were enrolled for this 6 month pilot study. In addition to the 12 analytes in the liquid QC programme, six additional analytes, LDH, triglyceride, urate, total bilirubin, phosphate and amylase were included. It was also possible to measure sodium and potassium by ion selective electrode (ISE) methods in the QC for the first time.The performance of the laboratories for the existing 12 analytes using liquid stabilized QC was compared to the performance using lyophilized QC. Using a statistical comparison of the methodwise mean variance index score (MVIS) values, five assays viz glucose, albumin, cholesterol, and SGOT and SGPT performance was the same in liquid QC and lyophilized QC. Three assays viz urea, calcium and creatinine were significantly better, and 4 assays total protein, sodium, potassium and ALP were significantly worse. However the overall VIS (OMVIS) for the laboratories was the same and the ranking pattern of this 6 month OMVIS was also unaltered.The lyophilized QC scheme highlighted a negative bias between flame and ISE methods for sodium and potassium, and a definite standardization problem in reporting LDH and amylase results, but triglyceride, urate and total bilirubin assays were performing well.It was concluded that the introduction of lyophilized QCs will not cause any deterioration of performance to participating laboratories. Stability of the material seems to be good and the laboratories are generally using a good reconstitution technique.

Fozivudine: BM 211290, fozivudine tidoxil, FZT, HDP 990002, W 09726867.

Fozivudine [BM 211290, fozivudine tidoxil, FZT, HDP 990002, W 09726867] is a thioether lipid conjugate of zidovudine (AZT) in clinical development with Heidelberg Pharma Holdings for the potential treatment of HIV infection. Heidelberg Pharma's proprietary Linker-Cleavag-Enzyme Technology (LCE_Technology) is used to conjugate specific carriers to parent compounds, significantly improving their pharmacological, toxicological and/or clinical profile. LCE_Technology was originated at Boehringer Mannheim, which was acquired by, and integrated into, Roche. In 1999, when Heidelberg Pharma was founded, following a management buy-out from Roche Diagnostics GmbH, the new company acquired full ownership of the intellectual property and documentation related to various proprietary compounds, the LCE_Technology and its applications. According to Heidelberg Pharma's pipeline, published in August 2003, the product candidate, HDP 99.0002 (the company-specific code for fozivudine) will be in phase III development until early 2006, at which time the company hopes to file an NDA. A phase II study of fozivudine in 72 antiretroviral-naive patients with HIV infection was completed in the US and France by Roche.

Reliability of IFCC Method for Lactate Dehydrogenase Measurement in Lithium-Heparin Plasma Samples

In a recent Technical Brief on the IFCC-recommended method for lactate dehydrogenase (LD) measurement, Bakker et al. (1) questioned the reliability of this method for heparin samples. According to their findings, the IFCC method produced an excessive frequency of significantly discordant duplicate measurements (17.8%; 27 of 152 samples) compared with the formerly applied French Society (SFBC)-recommended method (1.4%; 2 of 140 samples). Neither increased centrifugation time and speed or the use of plasma separators to reduce the plasma contamination with blood cells, nor LD measurements without concurrent measurements of other analytes to eliminate possible interferences lowered the reported high rate of duplicate errors. The problem was also not attributable to the clinical chemistry analyzer because measurements on both Hitachi 911 and 717 analyzers (Roche Diagnostics GmbH) showed results similar to those obtained with the previously used Modular analyzer (Roche Diagnostics GmbH). Only when serum samples or secondary tubes for centrifuged plasma samples were used was there a significantly lower frequency of duplicate errors: 2.6% (3 of 114) for serum and 1.1% (1 of 94) for secondary plasma tubes. The authors pointed out that the differences in buffer composition of the IFCC (pH 9.40 ± 0.05) (2) and SFBC (pH 7.4–7.8) methods influenced the integrity of platelets and erythrocytes. In this case, the presence of cells might play a role in the high frequency of duplicate errors. We have been measuring LD activity on three Roche Hitachi 917 analyzers according to the recommendations of the IFCC (2) in our laboratory since February 2003. The within-run imprecisions (CV) for a heparin-plasma sample (n = 20) were 0.8% (197.4 U/L) and 0.7% (321.3 U/L), and the between-run CV were 1.4% for the Roche Precipath U (178.3 U/L; n = 28) and 1.1% for the Roche Precinorm U calibrators (241.0 U/L; n = 28). …

Matrix Metalloproteinases 2 and 9 Mediate Epidermal Growth Factor Receptor Transactivation by Gonadotropin-releasing Hormone

Rapid engagement of the extracellular signal-regulated kinase (ERK) cascade via the Gq/11-coupled GnRH receptor (GnRHR) is mediated by transactivation of the epidermal growth factor receptor (EGFR). Here we show that the cross-talk between GnRHR and EGFR in gonadotropic cells is accomplished via gelatinases A and B (matrix metalloproteinases (MMPs) 2 and 9), identifying gelatinases as the first distinct members of the MMP family mediating EGFR transactivation by G protein-coupled receptors. Using a specific MMP2 and MMP9 inhibitor, Ro28-2653, GnRH-dependent EGFR transactivation was abrogated. Proving the specificity of the effect, transient transfection of alphaT3-1 cells with ribozymes directed against MMP2 or MMP9 specifically blocked EGFR tyrosine phosphorylation in response to GnRH stimulation. GnRH challenge of alphaT3-1 cells furthered the release of active MMP2 and MMP9 and increased their gelatinolytic activities within 5 min. Rapid release of activated MMP2 or MMP9 was inhibited by ribozyme-targeted down-regulation of MT1-MMP or MMP2, respectively. We found that GnRH-induced Src, Ras, and ERK activation were also gelatinase-dependent. Thus, gelatinase-induced EGFR transactivation was required to engage the extracellular-signal regulated kinase cascade. Activation of c-Jun N-terminal kinase and p38 MAPK by GnRH was unaffected by EGFR or gelatinase inhibition that, however, suppressed GnRH induction of c-Jun and c-Fos. Our findings suggest a novel role for gelatinases in the endocrine regulation of pituitary gonadotropes.

Modifications and substitutions of the RNA extraction module in the ViroSeq HIV-1 genotyping system version 2: effects on sensitivity and complexity of the assay.

Genotypic testing for HIV-1 resistance to anti-retroviral drugs has become accepted widely as a routine method to guide anti-retroviral therapy. However, implementation into routine high-throughput laboratory diagnosis is difficult due to the complexity of the assay. A commercially available assay is the ViroSeq HIV-1 Genotyping System (Applied Biosystems, Weiterstadt, Germany). We modified and substituted the RNA extraction module to optimize the proportion of samples amplified successfully as follows: 1 ml plasma was concentrated by ultracentrifugation and extracted according to the manufacturer's instructions (Kit), by substituting the lysis buffer (Roche, Roche Diagnostics GmbH, Mannheim, Germany), and by using the QIAamp Viral RNA Kit (Qiagen GmbH, Hilden, Germany) with elution volumes of 60 (Q60) or 50 micro l (Q50). Overall Q50 showed a higher success rate (97%) than the other extraction modules used (range 88-91%). In samples with a viral load range of 1,000-4,999 copies/ml, Q50 was superior (95 vs. 65% to 83%), while in samples with a viral load range of 5,000-9,999 copies/ml or those with 10,000 or more copies/ml, the success rate of the extraction procedures showed no significant differences. In 18 samples, which were negative using the Kit or Roche extraction, Q60 resulted in 7/18 positive results; in addition the Q50 was successful in amplifying 7/10 of the Q60 negative samples. When investigating samples with a measurable viral load of less than 1,000 copies/ml or lower, Q50 had the highest success rate with 80% compared to the other procedures (33-63%). A statistically significant new cut-off could be defined for Q50 at a value of 250 copies/ml. The results showed clearly that the ViroSeq System is suitable for analyzing the HIV-1 genotype over a wide range of viral loads but could be improved significantly when substituting the RNA extraction module with Q50 without using a nested PCR protocol. This is of great importance as it avoids further time- and cost-intensive steps.

Establishing canine clinical chemistry reference values for the Hitachi � 912 using the International Federation of Clinical Chemistry (IFCC) recommendations

The aim of this study was to establish population-based canine clinical chemistry reference values for the Hitachi 912 (Roche Diagnostics GmbH, Germany) with regard to age, sex, breed, housing and intended use. Reference biochemistry values for 22 variables are presented from 308 clinical healthy dogs, 145 females and 163 males, approximately 1 month to 13 years of age and of various breeds. For each variable the data were examined for homogeneity and, when suspected, outliers were excluded using the range test. Non-parametric analysis was used to calculate the conventional central 95% interval. Then the two-sided non-parametric 0.9 confidence interval of each percentile was determined. Finally, the effects of subgrouping were examined using the Kruskal–Wallis test and p <0.05 was considered significant. Significant age influences were found for 20 of the 22 variables, 14 showing clinical relevance. Significant breed effects could be found for 10 of the 22 parameters, including clinically relevant lower total protein concentrations for retrievers, lower lipase activity for sled dogs, lower total bilirubin concentrations for terriers and higher total bilirubin for Molossians. Differences between male and female were present for six of the 22 variables but had no clinical relevance. Housing and intended use influenced some of the values, but these differences were of no clinical significance. We successfully established canine clinical chemistry reference values for the Hitachi 912. The IFCC Recommendations on Reference Values offered a good framework for establishing standardised reference values, and make it possible for several laboratories to share the same values. Our results clearly indicate that subgrouping according to age and breed is necessary to obtain accurate reference values.