Leukotriene D4-induced hypotension is reversed by thyrotropin-releasing hormone

Injection of leukotriene D₄ (LTD₄, 20 μg/kg, i.a.) to conscious spontaneous hypertensive (SHR) rats produces a short-lasting pressor and tachycardic response followed by prolonged hypotension and bradycardia. Plasma norepinephrine and epinephrine were elevated at the peak pressor/tachycardic phase as well as at the hypotensive phase. Injection of thyrotropin-releasing hormone (TRH, 2 or 5 mg/kg) at the peak of the LTD₄-induced hypotension resulted in prompt reversal of the hypotension and bradycardia in a dose-related manner. Naloxone (5 mg/kg) had no effect on blood pressure and heart rate LTD₄- treated SHR rats. Pretreatment with TRH (5 mg/kg) did not prevent the depressor effect of LTD₄, but attenuated the bradycardic effect of this leukotriene. In addition, TRH had no effect on LTD₄-induced hypotension in the pithed SHR rat. These results suggest that TRH might exert beneficial effects in hypotensive states mediated by leukotrienes or other mediators of anaphylactic reactions.     

Effect of oxafun on the antibiosis between Bacillus subtilis and Phoma betae

The fungicide oxafun decreased the production of antibiotic substances active against Phoma betae, by Bacillus subtillis. This was confirmed by reduced effectiveness of antibiotic substances against Phoma infection in micropot experiment. Besides, the bacterial metabolites effected cell membrane permeability of mycelium and resulted in some relationships of fungicide and antibiotic substances.     

Characterization of the lipopolysaccharide from the nod mutant of Rhizobium trifolii

Lipopolysaccharides (LPS) from the non-nodulating Rhizobium trifolii 24SM 15 and from the nodulating R. trifolii 24SM 13 were isolated and examined by means of gas-liquid chromatography and mass spectrometry. Analysis of LPS showed these preparations from both strains examined contained Lipid A, 2-keto-3-deoxyoctonate, neutral sugars, amino sugars, and trace amounts of amino acids. In 24SM 13 LPS prevailed glucose and rhamnose whereas LPS from the non-nodulating strain SM 15 contained mainly mannose, galactose and heptose. Quinovosamine and mannosamine were detected only in the nodulating strain. The ratio of glucosamine phosphate to glucosamine was higher in the LPS of the non-nodulating strain SM 15 than in the corresponding material of the nodulating one. An unknown component producing a peak at the position of glyceryl-S-cysteine on amino acid analysis profiles was detected in SM 15 LPS. The differences in LPS composition were associated with the alterations in the sensitivity to phage 3H, and nodulation ability.     

Biochemical characterization of propionyl CoA carboxylase deficiency: Heterogeneity within a single genetic complementation group

Liver tissues and fibroblasts from patients with propionic acidemia assigned to the pcc BC genetic complementation group have previously been shown to contain normal or near-normal quantities of structurally altered propionyl CoA carboxylases (PCC). Biochemical comparisons of PCCs from extracts of three livers and one placenta belonging to the pcc BC complementation group revealed that the K _m values for the enzyme's major substrates, propionyl CoA, bicarbonate, and ATP, and its monovalent activator, potassium, were similar to those of normal PCC. PCC in extracts of one of the livers, however, had an altered isoelectric point (pI = 5.4) compared to that of PCC from normal and other PCC-deficient tissues (pK = 4.6–4.7). Thermostability in the presence of sucrose or ATP differed among several of the mutant PCCs, including the PCC with an altered pI, and from that of normal PCC. To confirm these results and to determine whether valid inferences may be derived from comparisons of mutant and normal PCC in crude extracts, PCC was purified from normal liver and from one of the PCC-deficient livers. The biochemical parameters of the purified carboxylases were similar to those observed in liver extracts. These studies further-more confirmed that, whether purified or in extracts, PCC from the pcc BC group reflects structural mutations. Nevertheless, the abnormal enzyme structure appears to have no corresponding effect on the clinical features of the disorder in various affected individuals. Moreover, there is biochemical heterogeneity within the pcc BC complementation group that probably represents different interallelic gene mutations.This work was supported by NIH Research Grants Am 25675 and AM 26127. B. Wolf is the recipient of NIH Research Career Development Award AM 00677 and is aided by Basil O'Connor Starter Research Grant 5-263 from The National Foundation-March of Dimes. This article is No. 131 from the Department of Human Genetics at the Medical College of Virginia.     

Increased cotransformation of distant markers and altered patterns of DNA-cell interactions following the exposure of transforming DNA to two carcinogenic and mutagenic alkylating agents,diethyl and dimethyl sulfate

Transforming DNA exposed to either diethyl sulfate (diES) or dimethyl sulfate (diMS) is inactivated. The rate of inactivation depends on the marker tested and on the chemical used: diMS is more active than diES. Cotransformation of linked markers is similarly depressed. In contrast, there is a transient increase in the cotransformation of distant, unlinked markers. These observations indicate that some of the intermolecular complexes of transforming DNA created in the test tube by the treatment with diES and diMS are biologically active. Radioactively labeled DNA treated with diES or diMS changes its patterns of interaction with cellular surfaces that are characteristic of untreated DNA. A possibility is considered that such alterations in DNA-protein interactions as well as the ability of these alkylating agents to transpose fragments of chromosomal material may play an important role in the processes of mutagenesis and, especially, carcinogenesis.     

Action of cholera toxin on dispersed acini from rat pancreas. Post-receptor modulation involving cyclic AMP and calcium

In dispersed acini from rat pancreas, cholera toxin caused a significant increase in cellular cyclic AMP but little or no change in amylase secretion. The presence of a secretagogue that causes mobilization of cellular calcium (e.g., cholecystokinin, carbamylcholine, bombesin or ionophore A23187) caused a substantial increase in the effect of cholera toxin on enzyme secretion. Cholera toxin did not alter calcium transport or the changes in calcium transport caused by other secretagogues, and secretagogues that mobilize cellular calcium did not alter cellular cyclic AMP or the increase in cyclic AMP caused by cholera toxin. These results indicate that in dispersed acini from rat pancreas there is post-receptor modulation of the action of cholera toxin by secretagogues that mobilize cellular calcium and that this modulation is a major determinant of the effect of the toxin on enzyme secretion.     

Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.     

F-actin in guinea pig spermatozoa: its role in calmodulin translocation during acrosome reaction.

The presence of actin has been determined in mammalian spermatozoa. However, its function in these cells is still almost unknown. Only in boar spermatozoa has evidence for F-actin and a possible function for it been presented. In this work, actin distribution and F-actin were determined in uncapacitated, capacitated, and acrosomal-reacted guinea pig spermatozoa, by means of monoclonal and polyclonal antibodies, using an indirect immunoperoxidase technique, and by the use of rhodamine-phalloidin. With the last probe we found filamentous actin in these cells. By both techniques, actin was detected in the acrosome and in the entire tail. In some cells with acrosomal reaction, actin was also detected in the equatorial and in the postacrosomal regions. SDS-PAGE and Western blots immunostained with monoclonal and polyclonal anti-actin antibodies confirmed the presence of actin in extracts of guinea pig spermatozoa. Actin was also detected in preparations of Percoll-purified spermatozoa. We have communicated that guinea pig spermatozoa show a change on calmodulin location during the acrosome reaction. They present it first in the equatorial region and later in the postacrosomal region. To determine if F-actin participates in this calmodulin translocation, we studied the effect of cytochalasin D. It was found that the number of cells with calmodulin in the equatorial region increased in the presence of cytochalasin D while the number of cells with calmodulin in the postacrosomal region decreased. We also found that after cytochalasin D treatment acrosome loss was increased and sperm motility was slightly inhibited. Our results suggest that actin participate in calmodulin translocation to the postacrosomal region during acrosome reaction, in maintaining the acrosome structure, and perhaps also in sperm motility.     

Mutational analysis of the transin (rat stromelysin) autoinhibitor region demonstrates a role for residues surrounding the "cysteine switch"

The family of mammalian extracellular matrix metalloproteases (MMPs) are secreted by cells in an inactive (latent) proenzyme form. A highly conserved amino acid sequence, PRCGVPDV, is found near the COOH-terminal end of the pro-domain of these MMPs and believed to act as an "autoinhibitor." Recent studies (Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., and Wart, H. E. V. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 364-368) indicate the Cys of this sequence ligands to the active-site zinc keeping the proenzyme in an inactive state, and mutational analysis (Sanchez-Lopez, R., Nicholson, R., Gesnel, M. C., Matrisian, L. M., and Breathnach, R. (1988) J. Biol. Chem. 263, 11892-11899) suggests that the conserved residues surrounding this Cys are required for latency. We have constructed 16 new site-directed mutations of the PRCGVPDV autoinhibitor region of the MMP transin (rat stromelysin) and tested whether these mutant enzymes are produced in a latent or activated form. We find that the conserved Arg as well as the Cys are essential for maintaining latency. The Cys cannot be replaced by other zinc-liganding amino acids, and the Arg cannot be replaced by Lys. Residues immediately surrounding the Cys are sensitive to even conservative amino acid substitutions. We show that a synthetic peptide PRCGVPDV is capable of acting as a weak inhibitor of transin and that replacement of the Cys with a Ser abolishes inhibition by the peptide. A review of the current knowledge of MMP substrate specificity in combination with these new results suggests that the PRCGVPDV sequence does not inhibit activity by mimicking the known substrates of the protease.