Tissue producing the serum factor stimulating the release of cathepsin D from lysosomes in vitro

Pancreatectomy as well as thyroparathyroidectomy resulted in the quick disappearance of a serum factor (stimulating cathepsin D release from lysosomes in vitro) from the rat or mouse blood. Extirpation of other organs such as duodenum, stomach, spleen, kidney, submaxillary gland, testis, adrenal gland or hypophysis, showed no effect on the serum factor level. Glucagon (but not insulin or thyroxine) given to the pancreatectomized animals restored the serum factor level in a dose-dependent manner. The serum factor-like activity was detected only in the parathyroids (but not thyroid), and the release of activity from parathyroid-slices was stimulated by glucagon, suggesting that the parathyroid may produce and/or secrete the serum factor under the influence of glucagon.     

Relation of mechanical noise in the rat papillary muscle to the level of its contraction

The existence of mechanical noise (MN) has been demonstrated in isolated papillary muscles of rats at rest. The mean amplitude of the MN was about 1 mg, the mean frequency 1.5 Hz (t 22 degrees C). A good agreement was found between the MN amplitude and the contracture level of the muscle. However, during long contractures, the correlation between the noise and contracture magnitude was disturbed. There was no relationship between the MN amplitude and contracture magnitude during exposures inducing metabolic alterations (hypoxia, NaCN) and upsetting the work of the sarcoplasmic reticulum (caffeine). It is believed that the MN amplitude is in a good agreement with the contracture magnitude and, therefore, with the concentration of intracellular Ca2+, if the sarcoplasmic reticulum and contractile elements of the cells are intact.     

Solution structure of [d(GGTATACC)]2: wrinkled D structure of the TATA moiety

Phase-sensitive two-dimensional nuclear Overhauser effect spectra of [d(GGTATACC)]2 in aqueous deuterium oxide solution at four mixing times were quantified to give all nonoverlapping cross-peak intensities. A structural model for [d(GGTATACC)]2 was built in which the GG- and -CC moieties were in the B-DNA form, while the middle -TATA- moiety was in the wrinkled-D form (BDB model). This model was subjected to energy refinement by molecular mechanics calculations with the program AMBER. Counterions (Na+) were added to neutralize the charges, and water molecules were placed bridging across the minor groove. A complete relaxation matrix analysis was used to calculate two-dimensional nuclear Overhauser effect spectra of [d(GGTATACC)]2 from the above models (before and after energy refinement) and from four other [d(GGTATACC)]2 structural models: regular A, crystalline A, regular B, and energy-minimized B. Among them, the energy-minimized BDB model yielded a set of theoretical spectra that gave the best fit to the experimental spectra. It was also the energetically most stable. Therefore, it is a good representation of the ensemble- and time-averaged structure of the octamer in solution. This model has backbone torsion angles similar to those of B-form DNA in the GG- and -CC moieties and torsion angles similar to those of wrinkled D form DNA in the -TATA- moiety. The base stacking and base pairing are not interrupted at the junctions between the two structural moieties. Its minor groove is narrower than that of B DNA, and the solvent-accessible surface of the minor groove forms a closed hydration tunnel in the middle -TATA- segment.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

Induction of delayed-type hypersensitivity by the T cell line specific to bacterial peptidoglycans

A T cell line specific for the chemically well-defined peptidoglycan of bacterial cell wall, disaccharide tetrapeptide, was established from Lewis rats immunized with the antigen covalently linked to the autologous rat serum albumin. The antigen specificity was examined with various analogues or derivatives of the peptidoglycan. The cell line was reactive to analogues with the COOH-terminal D-amino acid, but least reactive to those with L-amino acid as COOH terminus. Transferring of the T cell line into X-irradiated normal Lewis rats induced delayed-type hypersensitivity in an antigen specific manner.     

Purification and properties of cathepsin D from the mammary glands of lactating rabbits

Cathepsin D was purified from the lactating rabbit mammary gland by a rapid procedure, which included fractionation with (NH4)2SO4, acid precipitation, double affinity chromatography on pepstatin-Sepharose 4B and gel filtration on Sephadex G-100, resulting in approximately 360-fold purification of the enzyme over the homogenate and approximately 16% recovery. After isoelectric focusing, the enzyme dissociated into four (pI 5.8, 6.3, 6.5 and 7.2) multiple forms, but appeared homogeneous on polyacrylamide gel electrophoresis. Cathepsin D has a Mr of 45 kDa as determined by Sephadex G-100 column chromatography. On sodium dodecylsulfate/polyacrylamide gel electrophoresis the enzyme gave a single protein band, corresponding to Mr of 45 kDa. The amino acid composition of the enzyme is similar to that of cathepsins D from other tissues. A single N-terminal amino acid was glycine. Cathepsin D contains 6.4% carbohydrates consisting of mannose, galactose, fucose and glucosamine at a ratio of 3:9:2:2. Cathepsin D is inhibited by pepstatin with Ki of 2.5 X 10(-9) M and irreversibly by N-diazoacetyl-N'-2.4-dinitrophenyl-ethylene diamine. The enzyme hydrolyzes bovine hemoglobin with the maximal activity at pH 3.0 with Km = 10(-5) M and HLeu-Ser-Phe(NO2)-Nle-Ala-Leu-OMe with Km = 4 X 10(-5) M and Rcat = 0.95 s-1. The major cleavage sites were Leu15-Tyr16, Phe24-Phe25 and Phe25-Tyr26 during hydrolysis of the oxidized insulin B-chain by cathepsin D.     

Study of the mechanism of initiation of enzymatic NADPH-dependent lipid peroxidation in membranes of the endoplasmic reticulum

The localization and mechanism of generation of active oxygen species in the enzymatic NADPH-dependent lipid peroxidation system in liver microsomes were studied. Using the spin-trapping method, the key role of active oxygen species in the initiation of NADPH-dependent enzymatic lipid peroxidation was confirmed. It was shown that active oxygen species are generated via consecutive one-electron reduction of the oxygen molecule by NADPH-cytochrome P-450 reductase.     

Progesterone metabolism in T47Dco human breast cancer cells--II. Intracellular metabolic path of progesterone and synthetic progestins

We show here that progesterone added to the medium of proliferating T47Dco human breast cancer cells is metabolized with a half life of 2-4h. The final metabolic product, 5 alpha-pregnan-3 beta,6 alpha-diol-20-one, (P-metabolite) is released into the medium. This structure suggested that the intracellular metabolism of progesterone involves the enzymes 5 alpha-reductase, 3 beta-hydroxysteroid dehydrogenase, and 6 alpha-hydroxylase. To investigate this pathway, the cells were incubated with a variety of potential substrates. In addition to progesterone, only precursors with the 5 alpha-configuration served as substrates for the enzymes leading to P-metabolite formation. Some precursors with a 5 beta-configuration were also metabolized by T47Dco cells. This metabolism reflected activity by either 3 beta-hydroxysteroid dehydrogenase and/or 6 alpha-hydroxylase but, in contrast to progesterone metabolism, the rates were different and the products were often mixtures. In T47Dco and MCF-7 human breast tumor cells, the reduction at C-3 followed by 6 alpha-hydroxylation, appear to be the major, and possibly only, route of progesterone metabolism. In contrast, preliminary data suggest that in normal human breast epithelial cells, this is not an exclusive route. Androgens are partially subject to the same metabolic enzymes, but synthetic progestins are not metabolized by T47Dco during an 18 h incubation.     

Immunological status of monkeys during acclimatization and its correction with levamisole

The work presents the results of investigations, carried out in different monkey species, on the physiological norms as regards the bactericidal factors of neutrophilic polymorphonuclear leukocytes in the blood, nonenzymatic lysosomal cationic proteins and myeloperoxidase, as well as on changes in these characteristics in monkeys at different periods of their acclimatization at the Institute of Experimental Pathology and Therapy, Sukhumi. The possibility of correcting the characteristics under study by means of the immunostimulating agent levamisole is shown.