Extracellular matrix components and proteolytic enzymes in uterine cervical carcinoma

The important components of mucopolysaccharides and collagen have been analyzed in tissues of control and carcinoma of uterine cervix. Among these components hyaluronic acid and chondroitin sulphate levels were found to be increased, whereas decreased level of collagen was observed in uterine cervical carcinoma. Serum cathepsin B, D and acid and alkaline phosphatases have also been analyzed in controls and carcinoma patients before and after treatments. The activities of these enzymes have been found to increase prominently in advanced stages. Among these enzymes cathepsin B and alkaline phosphatase have exhibited remarkable increase in activity in uterine cervical carcinoma. Different modes of treatment exerted reversion of the elevated activities of these enzymes. However, combined therapy type II (radiation combined with cisplatin and cyclophosphomide) seems to be more effective in reverting the activities of these enzymes to normal levels.     

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H⁺,K⁺-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K⁺-stimulated ATPase, activity of at least 4–5 fold compared to basal ATPase activity. The H⁺,K⁺-ATPase showed hydroxylamine-sensitive phosphorylation and K⁺-dependent dephosphorylation of the phospho-enzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K⁺-dependent H⁺-transport — properties characteristic of gastric proton pump One notable difference with H⁺,K⁺-ATPase of other species has been the observation of valinomycin-independent H⁺ transport in such membrane vesicles. It is proposed that such H⁺,K⁺-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H⁺,K⁺-ATPase-function in relation to HCl secretion.     

In vivo effects of vanadate on hepatic glycogen metabolizing and lipogenic enzymes in insulin-dependent and insulin-resistant diabetic animals

The insulin-mimetic action of vanadate is well established but the exact mechanism by which it exerts this effect is still not clearly understood. The role of insulin in the regulation of hepatic glycogen metabolizing and lipogenic enzymes is well known. In our study, we have, therefore, examined the effects of vanadate on these hepatic enzymes using four different models of diabetic and insulin-resistant animals. Vanadate normalized the blood glucose levels in all animal models. In streptozotocin-induced diabetic rats, the amount of liver glycogen and the activities of the active-form of glycogen synthase, both active and inactive-forms of phosphorylase, and lipogenic enzymes like glucose 6-phosphate dehydrogenase and malic enzyme were decreased and vanadate treatment normalized all of these to near normal levels. The other three animal models (db/db mouse, sucrose-fed rats and fa/fa obese Zucker rats) were characterized by hyperinsulinemia, hypertriglyceridemia, increases in activities of lipogenic enzymes, and marginal changes in glycogen metabolizing enzymes. Vanadate treatment brought all of these values towards normal levels. It should be noted that vanadate shows differential effects in the modulation of lipogenic enzymes activities in type I and type II diabetic animals. It increases the activities of lipogenic enzymes in streptozotocin-induced diabetic animals and prevents the elevation of activities of these enzymes in hyperinsulinemic animals. The insulin-stimulated phosphorylation of insulin receptor subunit and its tyrosine kinase activity was increased in streptozotocin-induced diabetic rats after treatment with vanadate. Our results support the view that insulin receptor is one of the sites involved in the insulin-mimetic actions of vanadate.     

Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke's encephalopathy

Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme -ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency.     

Covalent control of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase: insights into autoregulation of a bifunctional enzyme.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.     

荧光探针在大鼠胃粘膜表面疏水性研究中的应用

采用１－萘胺－８－磺酸（ＡＮＳ）为疏水探针,对大鼠胃粘膜表面疏水性作了研究,结果表明：以ＡＮＳ（２５μｍｏｌ／Ｌ）与胃粘膜表面刮取物（胃粘液凝胶层）混合后的萤光强度（正常为１．２３±０．１９ＲＦＵ／胃）可代表该粘液层的疏水性;以不同浓度ＡＮＳ与胃粘液混合后的萤光强度呈饱和趋势,可用Ｓｃａｔｃｈａｒｄ作图法求得粘液中ＡＮＳ的最大萤光强度（２．４６７±０．６３８ＲＦＵ／胃）和相对亲和系数（０．０３２±０．０１６）,它们可分别代表胃粘液中疏水基团的总量和单个基团的疏水性,从而可阐明胃粘膜被盐酸损伤后凝胶层粘液的ＡＮＳ萤光减弱,系其疏水基团总量减少,而非单个基团的疏水性改变所致。     

Characterization of neurotensin binding to rat gastric smooth muscle receptor sites

The binding of monoiodo 125I-Trp11-neurotensin to purified rat gastric fundus smooth muscle plasma membranes was characterized. Specific binding of ligand in subcellular fractions from rat fundus smooth muscle showed a distribution that paralleled that of several plasma membrane marker enzymes. 125I-Trp11-neurotensin binding to smooth muscle plasma membranes at 25 degrees C was maximal at 30 min, reversible and saturable. Scatchard analysis of equilibrium data indicated the existence of two classes of binding sites with dissociation constants (Kd) of 56 pmol and 1.92 nM, and corresponding binding capacities (Bmax) of 6.6 fmol/mg and 11.4 fmol/mg of membrane protein. Analogues and fragments of neurotensin competed for 125I-Trp11-neurotensin binding with a rank order of potency similar to that previously reported for their contracting effect in rat fundus strips. Na+ decreased in a concentration dependent manner the binding of labelled ligand to the high affinity site. At 100 mM, Na+ induced a 6-fold increase in the IC50 of neurotensin for inhibition of 125I-Trp11-neurotensin binding. At this concentration of Na+, the IC50 for neurotensin was 1 nM, a value close to the Kd of the low affinity site.     

Intrinsic factor secretion from isolated human gastric mucosal cells

Human gastric mucosal cells were isolated from the resected fundic mucosa of peptic ulcer patients. The intracellular content and secretion of intrinsic factor were estimated by binding to cyano[⁵⁷Co]cobalamin. The content was maximal in the enriched parietal cell fraction which also displayed the highest H⁺ production as measured by amino[¹⁴C]pyrine uptake. Secretagogues evoked full response after 15 min of incubation: pentagastrin (181% of basal secretion), carbachol (208%), histamine (250%) and dibutyryl cyclic adenosine monophosphate (304%). The phosphodiesterase inhibitor isobutylmethylxanthine was slightly more effective even than dibutyryl cAMP. The response to histamine was abolished by ranitidine, indicating activation of adenylate cyclase via histamine H₂ receptors, but remained unaffected by atropine, which in turn blocked the carbachol effect, whereas ranitidine was ineffective. The mean formation rate was 8.4 fmol intrinsic factor/10⁶ cells per h under basal conditions and 14.3 fmol in response to histamine.     

Changes in cytoplasmic and lysosomal enzyme activities in cultured rat heart cells: The relationship to cell differentiation and cell population in culture

Summary Postnatal rat heart cells in culture enriched with respect to muscle cells were obtained by either high density seeding or by the replating technique. [³H]Thymidine incorporation to DNA and the enzymatic pattern of cytoplasmic and lysosomal enzymes have been studied as a function of the culture’s age, of seeding density, and replating. It was shown that (a) replating maintains predominance of myocyte population for at least 2 wk in culture; (b) heavy seeding density allows homogeneous myocyte population for the 1st wk in culture; and (c) the enzyme profile of the culture may serve as an indicator for the type of cell population in culture and its state of differentiation. This study was done as partial fulfilment of the M.Sc. thesis in Biochemistry (SY). Supported by grants from The Chief Scientist, Ministry of Health, State of Israel; The Ministry of Education and Sciences, State of Niedersachssen (FRG); and The Foundation for Heart Research from Mr. and Mrs. D. Vidal-Madjar, Paris, France.