The action of cyclic somatostatin on hypoglycemia due to prolonged fasting in normal animals]

The authors have examined the action of cyclic Somatostatin on blood glucose levels in normal rats and in rats starved for 36 and 50 hours. The infusion of 0,235 gamma/min. of Somatostatin for thirty minutes in the normals induced a slight increase in blood glucose levels that was statistically non significative. Under the same condition, the cyclic Somatostatin increased, in a statistically significant way, the levels of plasma glucose in both starved groups of rats.     

A fluorescent probe study of salmine AI

A fluorescent probe, 1-p-toluidinylnapthalene-8-sulfonate (1,8-TNS), was used to study the nonpolar sites on salmine AI. Fluorescence enhancement resulting from binding between the probe and the protein occurs at a wavelength of maximum emission of 497-500 nm, indicating the existence of moderately nonpolar binding sites on salmine AI.Fluorescence enhancement decreases as the ionic strength of the solvent is increased from 0.002 M to 0.050 M. Fluorescence increases with increasing acidity although this effect is not correlated to the pKa of 1,8-TNS. Positive cooperative binding takes place between 1,8-TNS and salmine AI. Equilibrium dialysis indicates that binding occurs only under conditions resulting in significant fluorescent enhancement. The binding was also studied using thin film dialysis, which is much faster than equilibrium dialysis and avoids the observed changes in probe-protein interaction that occur over long time periods with the latter system.     

Lower azure B methylene blue ratios in Giemsa type blood and malaria stains

Starting from ancient reports that rare samples of methylene blue were apparently sufficiently contaminated with azures to give red plasmodial and red purple nuclear chromatin in Chenzinsky type methylene blue eosin stains, it was decided to determine how little azure B would suffice for such staining in methylene blue eosin stains. The traditional 1902 Giemsa had an azure : methylene blue : eosin ratio of about 6 : 3 : 6.3 : 10; Lillie's 1943 formula had a 5 : 7 : 10 ratio. In the current series of tests 5 : 7 : 10 (I), 4 : 8 : 10 (II), 3 : 9 : 10 (III), 2 : 10 : 10 (IV), 1 : 11 : 10 (V), and 0 : 12 : 10 (VI) were used. Malaria and blood stains were better than the standard 5 : 7 : 10 (I) in III, IV and II in that order. Normal and leukemic human blood, mouse blood with Plasmodium berghei, and monkey blood with the CDC strain of Pl. falciparum were used as test materials. The staining mixtures were made from highly purified samples of azure B and methylene blue. Staining mixtures contained 12 ml 0.1% thiazin dye, 10 ml 0.1% eosin, 2 ml each of glycerol, methanol and 0.1 M phosphate buffer pH 6.5, 3 ml acetone as accelerator, and distilled water to make 40 ml; staining times of 10--30 min were used.     

Immunocytochemical visualization of coated pits and vesicles in human fibroblasts: relation to low density lipoprotein receptor distribution.

The coated pit-coated vesicle system has a key role in the uptake of plasma low density lipoprotein (LDL) and other receptor-bound proteins in human fibroblasts. To study the distribution of coated pits and coated vesicles in fibroblasts by immunochemical techniques at both the light and electron microscopic levels, we immunized rabbits with coat protein extracted from bovine brain-coated vesicles. The resulting anti-coat protein antibody was directed predominantly against clathrin, the 180,ooo dalton protein that constitutes the major component of coat protein. By indirect immunoperoxidase electron microscopy, the anti-coat protein antibody was observed to bind specifically to coated pits on the surface of human fibroblasts and to coated vesicles within the cell. Indirect immunofluorescence and immunoperoxidase staining techniques at the light microscopic level revealed that the coat protein was distributed in fibroblasts in two distinctive patterns: as discrete foci on or near the cell surface that were linearly aligned in association with phase-dense cellular fibers (first pattern), and as intracellular foci that were randomly arranged around the cell nucleus (second pattern). The distribution of coat protein in fibroblasts was compared with the distribution of ferritin-labeled LDL, which was studied with the use of similar electron microscopic and immunofluorescence techniques. As previously reported, electron microscopic studies revealed that the LDL-ferritin binding sites at 4 degrees C were clustered in coated pits. By immunofluorescence microscopy, the LDL-ferritin that was bound to receptors within coated pits was shown to be arranged linearly over the cell surface in a pattern that was similar to the linear arrangement of coat protein (first pattern). Considered together, the current data indicate that coated pits in human fibroblasts contain a protein analogous to clathrin, and that those coated pits which contain receptors for LDL are located over intracellular fibers most likely corresponding to stress fibers. These observationa may have relevance to the mechanisms by which the coated pit-coated vesicle system efficiently delivers recptor-bound ligands to lysosomes.     

Studies on DNA damage: Discordant responses of rate of DNA disentanglement (viscosimetrically evaluated) and alkaline elution rate,obtained for several compounds

Alkaline elution is a well-known method for detecting DNA damage. Recently we have developed a viscosimetric method that is even more sensitive than alkaline elution. Here we report that the two methods, although apparently both revealing alkaline DNA fragmentation, can give dramatically different results for a significant series of compounds. We suspect that alkaline elution might reveal not only DNA fragmentation but also the extent of disentanglement of chromatin structure, whereas this DNA disentanglement rate, when evaluated viscosimetrically, is more strictly correlated with the initiation of DNA unwinding.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

Human T cell responses to gp63, a surface antigen of Leishmania

gp63, an abundant and conserved leishmania cell surface protein, has been implicated in the ability of these parasitic protozoa to infect macrophages in vitro and has shown potential as a protective immunogen in mice. However, little is known regarding human immune responses to this glycoprotein Ag. In this study, human T lymphocyte responses to Leishmania amazonensis native gp63 and to recombinant gp63 (rgp63) produced in Escherichia coli were evaluated in individuals with active or cured cutaneous, mucosal or visceral leishmaniasis. Both native and rgp63 elicited strong proliferative responses in all patients tested. In addition, IFN-gamma was produced in response to stimulation with both forms of the protein. T cell lines generated from PBMC by stimulation with native or rgp63 were phenotypically similar, and proliferated and produced IFN-gamma in response to stimulation with both forms of the molecule. These results suggest that gp63 is a strong T cell immunogen and that the recombinant and native forms can elicit the same type of T cell response from infected patients. In order to compare the immunogenic properties of these two forms of gp63, PBMC from naive (uninfected) donors were sensitized in vitro with native or rgp63. T cell lines generated against rgp63 proliferated in response to rgp63, but failed to proliferate in response to native gp63 or to promastigote lysate. Thus, rgp63 was effective in eliciting T cell responses from patients with active or cured leishmania infection, but did not effectively induce T cell responses under the conditions used.     

Buffering of intracellular calcium in response to increased extracellular levels in mortal, immortal, and transformed human breast epithelial cells.

Extracellular levels of calcium at 1.05 mM or higher induce terminal differentiation and senescence in the mortal (MCF-10M) line of human breast epithelial cells, but does not retard the growth or induce differentiation in the immortal (MCF-10A) and oncogene transformed (MCF-10AneoT) lines. Intracellular levels of calcium and inositol triphosphate were determined in MCF-10M, MCF-10A, and MCF-10AneoT, under conditions of low and high extracellular calcium. We hereby report that increases in extracellular calcium is translated into significant increases in intracellular levels of calcium and inositol triphosphate in MCF-10M, but not in MCF-10A and MCF-10AneoT. This difference in the apparent calcium buffering capacity between the mortal and the immortalized human breast epithelial cells could account for the latter's unperturbed growth potential in high extracellular calcium environment.