The potency of tissue-type plasminogen activator (TPA) determined with chromogen and clot-lysis assays

An assessment was made of two methods for determining the potency of tissue-type plasminogen activator (TPA). A chromogenic microtitre plate assay was established which contained TPA, plasminogen, a synthetic plasmin substrate (H-D-valyl-L-leucyl-L-lysyl-p-nitroaniline dihydrochloride, S2251) and any one of the following stimulators: native fibrinogen, enzymatic and chemical digests of fibrinogen, poly-D-lysine (PDL) and chemical derivatives of the latter. The chromogen assay was compared with an automated clot-lysis (turbidimetric) assay for sensitivity, reproducibility and validity for potency determination. Reference preparations of TPA were titrated in both assays: in the chromogen assay the dose-response curves were non-parallel, whereas parallelism was observed in the clot-lysis assay. Thus, the chromogen assay was restricted in its applicability and disqualified from any routine regulatory use. The potency of individual lots of recombinant (r)TPA could only be estimated in International Units (IU) of TPA activity with the automated clot-lysis assay and the potency values obtained (IU/vial) were in remarkably close agreement with the manufacturers' values.     

Effets sur le spermatozoïde humain du Diuron (3-(3,4-dichlorophényl)-1,1-diméthyl-urée) et de l’un de ses produits de transformation, la 3,4-dichloroaniline (3,4-DCA) (Etude préliminaire)

Diuron belongs to the family of halogenophenylureas, one of the main groups of herbicides used for more than 40 years. These herbicides absorb sunlight and can be photochemically transformed in the environment (herbicides are transformed on the soil surface exposed to sunlight) or biotransformed by microorganisms present in soil or in water. The metabolites (chlorohydroxyphenylurea, chlorophenylaniline, respectively) are more toxic than the parent compound, as demonstrated by a bioluminescence inhibition assay performed with a marine bacterium (Vibrio fischeri toxicity test). The lipophilicity of these pesticides makes the cell membrane a target for their action, especially the spermatozoa cell membrane. The aim of this study is to use human spermatozoa to evaluate the effect of this urea pesticide and its biotransformed product on the spermatozoa membrane. We investigated the structural and functional effects of these environmental pollutants on spermatozoa. Three million spermatozoa purified on a 95/47.5% Percoll gradient were suspended in 250 μl of modified Earle’s medium (without phenol red) supplemented with 7.5% of human decomplemented serum. Pesticides (Diuron or 3,4-dichloroaniline (3,4-DCA)) were added at a final concentration of 0.1; 1 and 5 mM. Samples were incubated at room temperature for 24 hours. We show that both Diuron and 3,4-DCA decrease motility and vitality of spermatozoa incubated with the highest concentration of pesticides. Our preliminary results show that the effects are more rapid and more intense with the biotransformed product (3,4-DCA) than with Diuron. Addition of herbicide to human spermatozoa increases membrane fluidity, assessed by measuring the fluorescence polarisation anisotropy with a fluorescent probe: 1,6-diphenyl-1,3,5-hexatriene (DPH). Changes in membrane fluidity may be a primary toxic effect of these herbicides. These results suggest that human spermatozoa may constitute a valuable indicator of the toxic effects of pesticides.     

Role of the adrenal cortex and sodium in the pathogenesis of human hypertension.

After 30 years of continuous research into the mechanisms of human hypertension, we summarize the results obtained by the members of the multidisciplinary research group on hypertension of the Clinical Research Institute of Montreal on the disturbances of minerlocorticoid activity in a rigorously selected group of patients with early, mild essential hypertension. We attempt to integrate these findings with those of many other groups working on other aspects of hypertensive cardiovascular diseases. On the assumption that the increased peripheral resistance responsible for hypertension results from an imbalance or a disturbance of the equilibrium between the sympathetic nervous system and norepinephrine on one hand, and the vascular tone, sensitivity and responsiveness of the arterial smooth muscle to norepinephrine and to angiotensin II on the other hand, three models that fit the experimental and clinical facts as known at present are described.     

Effect of lipid-protein interaction on the color of bacteriorhodopsin

Detergent solubilization and subsequent delipidation of bacteriorhodopsin (bR) results in the formation of a new species absorbing maximally at 480 nm (bR480). Upon lowering the pH, its absorption shifts to 540 nm (bR540). The pK of this equilibrium is 2.6, with the higher pH favoring bR480 (Baribeau, J. and Boucher, F. (1987) Biochim. Biophysica Acta, 890, 275-278). Resonance Raman spectroscopy shows that bR480, like the native bR, contains a protonated Schiff base (PSB) linkage between the chromophore and the protein. However, the Schiff base vibrational frequency in bR480, and its shift upon deuteration, are quite different from these in the native bR, suggesting changes in the Schiff base environment upon delipidation. Infrared absorption and circular-dichroism (CD) spectral studies do not show any net change in the protein secondary structure upon formation of bR480. It is shown that deprotonation of the Schiff base is not the only mechanism of producing hypsochromic shift in the absorption maximum of bR-derived pigments, subtle changes in the protein tertiary structure, affecting the Schiff base environment of the chromophore, may play an equally significant role in the color regulation of bR-derived pigments.     

On the classification of regular systems of inbreeding

Regular systems of inbreeding are defined as those with discrete, nonoverlapping generations and with the same number of individuals and mating pattern in every generation. Given the number of individuals in a generation, there are many possible regular mating systems. A notion of when two such mating systems are equivalent is introduced, and several necessary conditions are given for such an equivalence. The use of these conditions is illustrated for N = 2, 3, 4 and 5 individuals, and a complete enumeration has been found for these cases: the numbers of inequivalent mating systems are 1, 5, 57 and 858, respectively. The maximal eigenvalue of the matrix q that specifies the recursion relations satisfied by the probabilities of identity have also been found for these cases. For N = 3 and 4 (and 2 trivially), circular mating gives the slowest rate of approach to genetic uniformity of those systems that do evolve to uniformity, but for N = 5 there are two other mating systems that have a slower rate of convergence, and for N = 6 partial results show that there are many such examples.Deceased     

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes

The alpha-like globin gene cluster in rabbits contains embryonic zeta- globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1- zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.      

The limited photochemical activity of solid aggregated forms of bovine rhodopsin

The visual pigment rhodopsin has been purified and depleted of detergent. Under these conditions, the pigment strongly aggregates. When dried, a significant fraction of these aggregates appear insensitive to light. We have characterized them by means of absorption and photoacoustic spectroscopies and we find that their photochemical behavior is best explained by a limited activity that does not reach photointermediates beyond the lumirhodopsin step in the bleaching sequence of rhodopsin. We interpret this result as an indication of a significant conformational change of the protein during the transition from lumi- to meta-rhodopsin.