Morphological aspects of the hypothalamic-hypophyseal system

Summary The ultrastructure of the tanycyte ependyma in male 160–180 g Wistar albino rats was studied under normal conditions and in experiments involving long-term suppression of ACTH secretion and its long-term stimulation. The former was accomplished by daily (for 8 days) intraperitoneal administration of dexamethasone phosphate at low (5 g/100 g) and high (100 g/100 g) concentrations. The effectiveness of suppression of the hypothalamic-hypophyseal-adrenal system in the experimental animals was judged by their reaction to two-minute ether stress (determination of plasma corticosterone) and by the results of measurement of the adrenal weights. Stimulation of ACTH secretion was achieved by bilateral adrenalectomy; the animals were examined on days 8, 10, 14, and 22 following the operation. The results obtained were in agreement with the previously established fact that there is a negative correlation between tanycyte activity and hypophyseal adrenocorticotrophic function (Akmayev and Fidelina, 1974). They also testified to the predominant involvement of the median eminence tanycyte ependyma (beta-tanycytes according to the authors' nomenclature) in these relationships.It is supposed that these correlations are regulated by a feedback mechanism and attest to the involvement of beta-tanycytes in the inhibiting control of hypophyseal adrenocorticotrophic function. The mechanism of this control may be explained alternatively: either the tanycytes transport ACTH-suppressing substances (catecholamines, corticosteroids, ACTH) from the CSF to the hypophyseal portal system or they themselves secrete substances possessing ACTH-suppressive activity. The authors distinguish several types of vesicles in the beta-tanycytes, the number of which changed with experimentally induced shifts in hypophyseal adrenocorticotrophic function. These vesicles are discussed in connection with the transport and secretory activity of the tanycytes and are considered to be a possible substrate of the hypothalamic inhibiting effect on ACTH secretion.     

Chemical modification of lysine residues in bacterial formate dehydrogenase

Specific modification of 4.4 lysine residues per molecule of formate dehydrogenase, from the methylotrophic bacterium Achromobacter parvulus I by pyridoxal, results in complete inactivation of the enzyme. The concentration effect of the modifying agent and substrates on the inactivation of formate dehydrogenase has been studied. Coenzymes do not protect the enzyme from inactivation. Complete maintenance of enzyme activity was achieved in the presence of saturating concentrations of the formate and upon formation of the ternary complex, enzyme-NAD-azide. Formate specifically protects two lysine residues per dimer molecule of the enzyme from modification. The presence of one essential lysine residue in the substrate-binding region of the enzyme active site is assumed.     

Constructing a test system for the identification of plague microbe based on genetic probes]

DNA probes for detection of the plague agent Yersinia pestis were made on a basis of its three typical extrachromosomal replicons. The recombinant plasmid pBS2 including pBR327 vector and SalGI-BspRI fragment of the plasmid pFra was constructed. The above fragment is connected with synthesis of Y. pestis capsular antigen and it is a 400 bp species-specific DNA probe called F1 which is suitable for identification of Y. pestis species that bears the 60 mdal plasmid. The DNA probes called P1 was made on a basis of the plasmid pPst; it is the 460 BglII-BamHI fragment of the fibrinolysin-coagulase gene suitable for species-specific detection of Y. pestis species that bears the 60 mdal plasmid. The P1 fragment was cloned into the pAT153 vector and the constructed recombinant plasmid was called pEK7. The recombinant plasmid pCL1, including the pBR325 vector and the 6th BamHI fragment of Y. pestis EV plasmid pCad was constructed. The above fragment includes the replication origin of the pCad and it is hybridized to the pCad-bearing strains of Y. pestis and Y. tuberculosis only. Thus, it may be a basis for a bi-species-specific DNA probe making. These three recombinant plasmids are considered as a test-system for detection of both typical and atypical strains of Y. pestis.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Studies on the mechanism of the changes in serum and liver gamma-glutamyl transpeptidase activity. I. Experimental extrahepatic cholestasis in rabbits.

Serum, liver and renal gamma-glutamyl transpeptidase (GGT) activities were studied in four groups of rabbits: controls, rabbits with obstructive extrahepatic cholestasis, rabbits with obstructive anuria, and animals with combined obstructive extrahepatic cholestasis and obstructive anuria. Serum GGT was essentially increased in rabbits with obstructive extrahepatic cholestasis, showing peak values in the combined cholestasis + obstructive anuria group, and practically normal values in animals with anuria. Liver GGT was increased in both cholestasis groups, but the increase was less prominent than the increase in serum GGT and there was no correlation between them. In both anuric groups renal GGT was reduced, probably as a result of inhibited enzyme synthesis secondary to the altered conditions for adequate renal function. The results obtained are suggestive of a probable renal involvement in the formation of the serum GGT activity level.