Reaction rate measurements of proteases and glycosidases with chromogenic methods

Simultaneous azo-coupling and indigogenic methods were evaluated for the quantitative histochemical assay of the plasma membrane proteases gamma-glutamyl transpeptidase (EC 2.3.2.2) and dipeptidyl peptidase IV (EC 3.4.14.5) and the glycosidases maltase-glucoamylase and glucoamylase (EC 3.2.1.20) in decidual cells, jejunal enterocytes and renal proximal tubulocytes. Using kinetic (continuous) microdensitometry, a linear increase in the final reaction product was found from 3 up to 10 min, depending on the substrate concentration and the plasma membrane glycosidase or protease under investigation. Combined continuous and end point (static) microdensitometry revealed a linear relationship between the section thickness (enzyme concentration) and final reaction product up to 12 microns for the proteases and up to 16 microns for the glycosidases. Apparent Km and Vmax values were calculated with a computerized version of the direct linear plot and compared with the results obtained with the linear transformations according to Lineweaver-Burk, Eadie-Hofstee and Hanes. Apparent Km and Vmax values for the proteases were calculated separately for each animal and were 1.82 mM and 1.02 mM and 2.43 arbitrary units (a.u.) and 1.67 a.u. (gamma-glutamyl transpeptidase, decidua) and 0.42 mM and 0.38 mM and 0.29 and 0.26 a.u. (dipeptidyl peptidase IV, decidua). For the alpha-D-glucosidases, the corresponding values were 0.23 mM and 0.15 a.u. (kidney) and 0.55 mM and 0.20 a.u. (jejunum). The results show the suitability of the indigogenic methods for quantitative histochemical measurements of plasma membrane alpha-D-glucosidases, whereas the simultaneous azo-coupling procedures seemed to be less suitable for the quantification of surface membrane proteases, due to, for example, interactions of diazonium salts with amino acid or peptide substrates, reaction products and peptide activators.     

Proliferation of AXC/SSh rat prostate cancer cells in vitro is androgen modulated

We used heterogeneous parental cultures of AXC/SSh rat prostate cancer cells to isolate clonally derived prostate cancer cell lines. Light and electron microscopic analyses established that parental and clonally isolated cells possess features characteristic of secretory epithelium. Biochemical analyses showed that these cells contained androgen receptors and acid phosphatase and 5 alpha-reductase activity; phenotypic markers characteristic of differentiated prostate epithelium. Content of these prostate epithelial cell markers was variable and cell line specific. We used selected cell lines to examine androgen modulation of AXC/SSh rat prostate cancer cell proliferation in vitro. We found that proliferation of C-family or D-family cells, those respectively maintained on medium without additions or medium containing 10(-7) M 5 alpha-dihydrotestosterone, was not affected by changes in medium testosterone concentration through the range 10(-6)-10(-9) M. In contrast, testosterone modified proliferation of T-family cells, those maintained on medium containing 10(-7) M testosterone, and effects were antagonized by the anti-androgen RU 23908. Preliminary studies established that AXC/SSh rat prostate cancer cells elaborate polypeptide components which stimulate in vitro cell proliferation. Both the ability to elaborate these components and their effects on in vitro cell proliferation appeared to be cell line specific.     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

Allium cepa L. Cultivars from Four Continents Compared by Flow Cytometry show Nuclear DNA Constancy

In 1965 Van't Hof estimated the nuclear DNA amount of an unidentifiedAllium cepa L. cultivar as 2C = 33.55 pg (Experimental CellResearch39: 8–58). This value has been adopted by commonusage as the main calibration standard for angiosperm DNA C-valueestimations. However, different cultivars have been used whileassuming species DNA C-value constancy. Surprisingly this assumptionhas never been tested. A. cepa is an outbreeder with telomericheterochromatic segments, so intraspecific variation in C-value,possibly correlated with environmental factors as seen in Zeamays L., might be expected. We used laser flow cytometry tocompare nuclear DNA amounts in roots of six A. cepa cultivarsused as calibration standards or from different environments.Tissues from one cultivar, or similar volumes of tissue fromtwo cultivars, were run and the variance between nuclei in 2Cpeaks compared. Only one shoulderless 2C peak was seen for allpairs of co-chopped cultivars. Thus, no large differences inC-value between cultivars from different environments were found.Moreover, comparing cultivars run singly or as pairs showedno evidence for increased variation in 2C peaks in the latter,and hence of critical differences in DNA amounts between ‘AilsaCraig’ and another cultivar. Such variation was insufficientto make their use as alternative calibration standards, or thepractice of imputing Van't Hof's original C-value estimate tothem, unacceptable for most practical purposes. Given the mechanismsknown which can generate genome size variation, the degree ofconstancy in DNA C-value found seems remarkable. Copyright 2000Annals of Botany Company Allium cepa, onion cultivars, calibration standards, DNA C-value constancy, flow cytometry     

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1 complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1 PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10-day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02 microgram/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71-86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1-100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.     

Genetic variation maintained in multilocus models of additive quantitative traits under stabilizing selection.

Stabilizing selection for an intermediate optimum is generally considered to deplete genetic variation in quantitative traits. However, conflicting results from various types of models have been obtained. While classical analyses assuming a large number of independent additive loci with individually small effects indicated that no genetic variation is preserved under stabilizing selection, several analyses of two-locus models showed the contrary. We perform a complete analysis of a generalization of Wright's two-locus quadratic-optimum model and investigate numerically the ability of quadratic stabilizing selection to maintain genetic variation in additive quantitative traits controlled by up to five loci. A statistical approach is employed by choosing randomly 4000 parameter sets (allelic effects, recombination rates, and strength of selection) for a given number of loci. For each parameter set we iterate the recursion equations that describe the dynamics of gamete frequencies starting from 20 randomly chosen initial conditions until an equilibrium is reached, record the quantities of interest, and calculate their corresponding mean values. As the number of loci increases from two to five, the fraction of the genome expected to be polymorphic declines surprisingly rapidly, and the loci that are polymorphic increasingly are those with small effects on the trait. As a result, the genetic variance expected to be maintained under stabilizing selection decreases very rapidly with increased number of loci. The equilibrium structure expected under stabilizing selection on an additive trait differs markedly from that expected under selection with no constraints on genotypic fitness values. The expected genetic variance, the expected polymorphic fraction of the genome, as well as other quantities of interest, are only weakly dependent on the selection intensity and the level of recombination.     

1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).