Kinetic investigation of the hydrolysis of aminoacyl p-nitroanilides by dipeptidyl peptidase IV from human and pig kidney

Dipeptidyl peptidase IV (dipeptidyl-peptide hydrolase, EC 3.4.14.5), an enzyme that participates in the catabolism of bradykinin and Substance P as well as the post-translational processing of various other peptides, has been purified from human and pig kidney. The assay reaction involved the cleavage of p-nitroaniline (pNA) from various dipeptidyl p-nitroanilides. The specific activities of the human and pig enzyme (with Gly-Pro-pNA at pH 7.6) were 49.2 and 45.8, respectively. The dependence of initial reaction velocity on substrate concentration was determined for a variety of dipeptidyl p-nitroanilides over the concentration range 0.05 to 2.0 mM. Most of the substrates tested produced significant non-hyperbolic behavior for the function v vs. S at concentrations above 0.5 mM. As to differences between the two enzymes, the pig enzyme exhibited featureless (i.e., hyperbolic) behavior with Glu-Pro-pNA concentrations as high as 2.0 mM, whereas the human enzyme produced significant non-hyperbolic behavior for the function v vs. S, beginning at S = 0.4 mM. Thus, the human and pig dipeptidyl peptidases IV are kinetically distinct enzyme forms.     

Chromosome 16 microdeletion in a patient with juvenile neuronal ceroid lipofuscinosis (Batten disease).

The gene that is involved in juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease--CLN3--has been localized to 16p12, and the mutation shows a strong association with alleles of microsatellite markers D16S298, D16S299, and D16S288. Recently, haplotype analysis of a Batten patient from a consanguineous relationship indicated homozygosity for a D16S298 null allele. PCR analysis with different primers on DNA from the patient and his family suggests the presence of a cytogenetically undetectable deletion, which was confirmed by Southern blot analysis. The microdeletion is embedded in a region containing chromosome 16-specific repeated sequences. However, putative candidates for CLN3, members of the highly homologous sulfotransferase gene family, which are also present in this region in several copies, were not deleted in the patient. If the microdeletion in this patient is responsible for Batten disease, then we conclude that the sulfotransferase genes are probably not involved in JNCL. By use of markers and probes flanking D16S298, the maximum size of the microdeletion was determined to be approximately 29 kb. The microdeletion may affect the CLN3 gene, which is expected to be in close proximity to D16S298.     

The energetics of middle-distance running

In order to assess the relative contribution of aerobic processes to running velocity (v), 27 male athletes were selected on the basis of their middle-distance performances over 800, 1500, 3000 or 5000 m, during the 1987 track season. To be selected for study, the average running velocity (v) corresponding to their performances had to be superior to 90% of the best French v of the season. Maximum O2 consumption (VO2max) and energy cost of running (C) had been measured within the 2 months preceding the track season, which, together with oxygen consumption at rest (VO2rest) allowed us to calculate the maximal v that could be sustained under aerobic conditions: vamax = (VO2max - VO2rest) x C-1. The treadmill running v corresponding to a blood lactate of 4 mmol.l-1 (vla4), was also calculated. In the whole group, C was significantly related to height (r = -0.43; P less than 0.03). Neither C nor VO2max (with, in this case, the exception of the 3000 m athletes) were correlated to v. On the other hand, vamax was significantly correlated to v over distances longer than 800 m. These v were also correlated to vla4. However vla4 occurred at 87.5% SD 3.3% of vamax, this relationship was interpreted as being an expression of the correlation between vamax and v. Calculation of vamax provided a useful means of analysing the performances. At the level of achievement studied, v sustained over 3000 m corresponded to vamax.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of usual interstitial pneumonia pattern using RNA-Seq and machine learning: challenges and solutions

Background

We developed a classifier using RNA sequencing data that identifies the usual interstitial pneumonia (UIP) pattern for the diagnosis of idiopathic pulmonary fibrosis. We addressed significant challenges, including limited sample size, biological and technical sample heterogeneity, and reagent and assay batch effects.

Results

We identified inter- and intra-patient heterogeneity, particularly within the non-UIP group. The models classified UIP on transbronchial biopsy samples with a receiver-operating characteristic area under the curve of ~?0.9 in cross-validation. Using in silico mixed samples in training, we prospectively defined a decision boundary to optimize specificity at ≥85%. The penalized logistic regression model showed greater reproducibility across technical replicates and was chosen as the final model. The final model showed sensitivity of 70% and specificity of 88% in the test set.

Conclusions

We demonstrated that the suggested methodologies appropriately addressed challenges of the sample size, disease heterogeneity and technical batch effects and developed a highly accurate and robust classifier leveraging RNA sequencing for the classification of UIP.

     

Tau Kinetics in Neurons and the Human Central Nervous System

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Lipid levels,insulin resistance and cardiovascular risk over 96 weeks of antiretroviral therapy: a randomised controlled trial comparing low-dose stavudine and tenofovir

Background

HIV infection and antiretroviral treatment are associated with changes in lipid levels, insulin resistance and risk of cardiovascular disease (CVD). We investigated these changes in the first 96 weeks of treatment with low-dose stavudine or tenofovir regimens.

Methods

This is a secondary analysis of a double blind, randomised controlled trial performed in South-Africa, Uganda and India comparing low-dose stavudine (20 mg twice daily) with tenofovir in combination with efavirenz and lamivudine in antiretroviral-naïve adults (n?=?1067) (Clinicaltrials.gov, NCT02670772). Over 96 weeks, data were collected on fasting lipids, glucose and insulin. Insulin resistance was assessed with the HOMA-IR index and 10-year CVD risk with the Framingham risk score (FRS). A generalized linear mixed model was used to estimate trends over time.

Results

Participants were on average 35.3 years old, 57.6% female and 91.8% Black African. All lipid levels increased following treatment initiation, with the sharpest increase in the first 24 weeks of treatment. The increase in all lipid subcomponents over 96 weeks was higher among those in the stavudine than the tenofovir group. Insulin resistance increased steadily with no difference detected between study groups. FRS rose from 1.90% (1.84–1.98%) at baseline to 2.06 (1.98–2.15%) at week 96 for the total group, with no difference between treatment arms (p?=?0.144). Lipid changes were more marked in Indian than African participants.

Conclusion

Lipid levels increased in both groups, with low-dose stavudine resulting in a worse lipid profile compared to tenofovir. Insulin resistance increased, with no difference between regimens. CVD risk increased over time and tended to increase more in the group on stavudine. The low CVD risk across both arms argues against routine lipid and glucose monitoring in the absence of other CVD risk factors. In high risk patients, monitoring may only be appropriate at least a year after treatment initiation.