Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.     

A rapid colorimetric assay for heparinase activity.

A rapid, sensitive, assay for enzymes that degrade heparin is described. The procedure is based on the interference of heparin with color development during the interaction of protein with the dye Coomassie brilliant blue. The loss of this property when the glycosaminoglycan is degraded by heparinase can be used to quantify activity of the enzyme in pure form, or in complex biological samples such as tissue homogenates or serum. The assay is also suitable for studying dependence of heparinase activity under conditions such as varying pH and temperature.     

Halophytes – A resource for the future

Wetlands Ecology and Management -     

Significance of mirex-caused hypoglycemia and hyperlipidemia in rats

Treatment of rats with mirex (40 ppm in diet) caused hypoglycemia, liver enlargement, and inhibition of adrenal corticosteroid-synthesizing enzyme activity. At toxic dosages (20,000 ppm mirex in diet, which has a lethal toxicity-50 [LT-50] of ten days) poisoned female rats showed severe hypoglycemia, fatty liver, adrenal hyperplasia, hypophagia, lipid mobilization, and body weight (bw) loss. A 50 μg/kg intraperitoneal (IP) dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in male rats caused similar effects two days posttreatment. Hypoglycemia could be overcome by prednisone (which also inhibited adrenocorticoid-synthesizing enzyme activities) but not by streptozotocin treatment, indicating that hypoglycemia may be related to glucocorticoid deficiency resulting from inhibition of their synthesis and not by direct effects on pancreatic β-cells. Glucocorticoid deficiency could also cause increased release of adrenocorticoid hormone (ACTH), which may enhance fat mobilization caused by hypophagia.     

Mechanistic studies on aromatase and related C---C bond cleaving P-450 enzymes

Some P-450 systems, notably aromatase and 14-demethylase catalyse not only the hydroxylate reaction but also the oxidation of an alcohol into a carbonyl compound as well as a C---C bond cleavage process. All these reactions occur at the same active site. A somewhat analogous situation is noted with 17-hydroxylase-17,20-lyase that participates in hydroxylation as well as C---C bond cleavage process. The C---C bond cleavage reactions catalysed by the above enzymes conform to the general equation:

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It is argued that all three types of reaction catalyzed by these enzymes may be viewed as variations on a common theme. In P-450 dependent hydroxylation the initially formed Fe^III---O---O^. species is converted into Fe^III---O---OH and the heterolysis of the oxygen—oxygen bond of the latter then gives the oxo-derivative for which a number of canonical structures are possible; for example Fe^V = O ↔ (+^.)Fe^IV = O ↔ Fe^IV---O^.. One of these, Fe^IV---O^. behaves like an alkoxyl radical and participates in hydrogen abstraction from C---H bond to produce Fe^IV---OH and carbon radical. The latter is then quenched by the delivery of hydroxyl radical from Fe^IV---OH. The latter species may thus be regarded as a carrier of hydroxyl radical. We have proposed that the C---C bond cleavage reaction occurs through the participation of the Fe^III---O---OH species that is trapped by the electrophilic property of the carbonyl compound giving a peroxide adduct that fragments to produce an acyl—carbon cleavage. Scientific developments leading up to this conclusion are considered. In the first author's views,

“The study of mechanisms is not a scientific but a cultural activity. Mechanisms do not aim at an absolute truth but are intended to be a “running” commentary on the status of knowledge in a field. As the structural knowledge in a field advances Mechanisms evolve to take note of the new findings. Just as a constructive “running” commentary provides the stimulus for higher standards of performance, so Mechanisms call for better and firmer structural information from their practitioners”.     

Effects of mot gene expression on the structure of the flagellar motor

Direct freezing procedures have enabled us to visualize distinctive intramembrane particle ring structures in the cytoplasmic membranes of peritrichously flagellated bacteria by means of freeze-fracture electron microscopy. These structures were identified as flagellar motor components because their distribution matched that of flagella, and because they were absent in non-flagellated mutants of Escherichia coli. Particle rings were present in both the Gram-positive Streptococcus and the Gram-negative E. coli. In E. coli, a non-functional mocha operon produced flagellated but immotile cells lacking the particle rings. Simultaneous introduction of the motA and motB genes, led to recovery of both motility and the ring structures but neither gene alone was sufficient. The concomitant loss of the rings and motility is consistent with the ring particles having a central role in the flagellar motor.     

The use of the probit model to predict pregnancy status of buffalo based on physio-chemical properties of estrual mucus

Improper timing of artificial insemination with respect to ovulation is one of the major factors hampering the conception rate in buffalo. The present study was an attempt to relate physio-chemical changes in estrual mucus to subsequent pregnancy status in order to find their optimal values for determining the time for artificial insemination (AI). Serum estradiol, total protein and dry matter contents of estrual mucus were evaluated to predict the subsequent pregnancy in 36 buffalo during October 1988 to February 1989. Serum estradiol was determined by radioimmunoassay (RIA); spinnbarkeit, dry matter and total protein were determined by standard methods. Multivariate probit analyses were carried out to relate these variables to subsequent pregnancy status. Elasticity and protein concentration were significantly related to prediction probability of pregnancy status, and they predicted the pregnancy status 86% of the times correctly (P < 0.05). The probability of pregnant animals being correctly classified was 0.76, whereas the corresponding value for non-pregnant animals was 0.95. The present study demonstrated the possibility of using such a statistical model on mucus characteristics for determining proper AI time for better conception rates in Nili-Ravi water buffalo.