Effect of thyrotropin releasing hormone injection on blood growth hormone (GH), TSH and growth hormone releasing hormone (GHRH) concentrations in cancer patients

In order to investigate whether endogenous GHRH and somatostatin were involved in the mechanism of the paradoxical GH rise after TRH injection, changes in serum GH and plasma GHRH were examined before and after TRH injection in 12 cancer patients and changes in serum TSH and GH were similarly studied in 76 cancer patients including 31 GH-responders and 45 GH-nonresponders to TRH. TRH stimulated GH secretions without altering the circulating GHRH concentration in 4 of the 12 cancer patients. There was neither a significant correlation between the increase from the basal to maximum GH and GHRH after TRH injection in the 12 cancer patients nor a reciprocal relationship between the increase in GH and TSH after TRH injection in the 76 cancer patients. These findings suggested that the paradoxical GH rise after TRH injection in cancer patients was exerted by its direct action at the pituitary level, and not mediated through the hypothalamus.     

Growth experiment of Hantaan virus in A549 cells: an attempt to improve the immunofluorescent antibody technique for hemorrhagic fever with renal syndrome

An assay method for the infectivity of Hantaan virus, a causative agent of HFRS (hemorrhagic fever with renal syndrome), was developed by the use of IFA (immunofluorescent antibody technique). With the aid of this method, the growth characteristics of Hantaan virus, 76-118 strain, were followed in A549 cells. At a maximal MOI (multiplicity of infection) of 1.6 VAIU (viral antigen-inducing units) per cell, the conventionally available value, plateau level potencies of the viral antigen and virus infectivity were attained at eight and ten days postinfection, respectively, and most of the infective virus produced accumulated in the culture fluids of infected cells. When infections were defined with MOI values in terms of VAIU per cell, development of the viral antigen was highly consistent and followed a given pattern of kinetics. Based on these findings, a protocol for preparation of the viral antigen in IFA was presented, wherein spot culture and FBS treatment were emphasized as effective procedures to minimize non-specific staining.     

Distinction of G0 cells from senescent cells in cultures of non-cycling human fetal lung fibroblasts by anti-MAP-1 monoclonal antibody staining

On staining with a monoclonal antibody raised against microtubule-associated protein-1 (MAP-1), dot-like structures were seen in the nuclei of interphase cells, but not in those of non-cycling G0-arrested cells. Dots were also not seen in the nuclei of non-cycling senescent human cells (IMR-90). A SV40-DNA-transformed subline of IMR-90 with a limited growth potential showed progressive decrease of cells with nuclei containing dots in the final stage of their lifespan. The dots appeared in G0-arrested IMR-90 cells when these cells were incubated in medium of high osmotic pressure for 3 min. In contrast, no dots appeared in senescent cells or X-ray-irradiated young cells when they were incubated in medium of high osmotic pressure. Thus irreversibly non-cycling cells could be distinguished from G0-phase cells on the level of whole cultures. The results suggest that senescent cells lose their division potential by entering an irreversible cell-cycle stage differing from G0.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of 5 alpha-products as major C19-steroids in estrogen-responsive mouse Leydig cell tumor lines

Homogenates of estrogen-responsive mouse Leydig cell tumors (T 124958-R and T 22137) or 28- and 120-day-old mouse testes were incubated with [3H]progesterone or [14C]4-androstene-3,17-dione in the presence of NADPH, and progesterone metabolism and enzyme activities were estimated. The growth of T 124958-R tumor transplanted in BALB/c mice was markedly stimulated by estrogenization of host mice, but the growth of T 22137 tumor was evidently suppressed by the estrogenization. The major C21-17-OH-steroids and C19-steroids formed from progesterone by both tumors and the testes of immature mice were 5 alpha-steroids, such as 3 alpha,17-dihydroxy-5 alpha-pregnan-20-one, 5 alpha-androstane-3,17-dione, androsterone, 3 beta-hydroxy-5 alpha-androstan-17-one and 5 alpha-androstane-3 alpha,17 beta-diol. In contrast, the major steroids formed by the testes of adult mice were testosterone and 4-androstene-3,17-dione, and no or little 5 alpha-steroids were produced. 5 alpha-Reductase activities in both tumor cells (40-50 nmol/l X 10(8) cells per h) were also found to be approx. 5-6 times higher than that in Leydig cells of adult mouse testes (8 nmol/l X 10(8) Leydig cells per h), though 17-hydroxylase activity was much higher in the Leydig cells of adult testes (730 nmol/l X 10(8) Leydig cells per h) than in both tumor cells (1-7 nmol/l X 10(8) cells per h). Furthermore, the presence of significant amounts of endogenous androsterone and/or 5 alpha-androstane-3 alpha,17 beta-diol was demonstrated in both tumors by radioimmunoassay. The present results demonstrate for the first time that C19-5 alpha-steroids are major C19-steroid products (immature type of testicular androgen production) in Leydig cell tumor lines.     

Calcium-independent induction of cytocidal activity of polymorphonuclear leukocytes by phorbol myristate acetate-like tumor promoters

Tumor promoters were tested for the ability to induce cytocidal activity of polymorphonuclear leukocytes (PMNs), and the extracellular calcium-dependency of their PMN cytotoxicities were examined in comparison with that by some immunomodulators. Immunomodulators such as linear beta-1, 3-D-glucan (TAK) induced potent cytocidal activity of PMNs. The induction was dependent on extracellular Ca2+. Tumor promoters such as phorbol 12-myristate 13-acetate (PMA) and its derivatives, teleocidin which is structurally unrelated to PMA, and croton oil as an example of mixture also induced potent PMN cytotoxicities. In the latter cases, however, the induction was not dependent on extracellular Ca2+. The ability of these tumor promoters to induce PMN cytotoxicity correlated well with their skin-tumor promoting activities. These results indicate that inductions by PMA-like tumor promoters are distinguishable from those by TAK-like immunomodulators in not being Ca2+-dependent. The application of Ca2+-independent PMN cytotoxicity to detect PMA-like tumor promoters is discussed.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

OH-induced free radicals in purine nucleosides and their homopolymers: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by X-irradiation of N2O-saturated aqueous solutions of purine nucleosides (2'-deoxyadenosine, adenosine, 2'-deoxyguanosine, 3'-deoxyadenosine, guanosine and inosine) and the corresponding homopolymers (poly A and poly I) have been investigated by the technique of spin-trapping and e.s.r. spectroscopy. 2-Methyl-2-nitrosopropane was used as a spin-trap. For 2'-deoxyadenosine and 2'-deoxyguanosine, the resulting spin-adducts were separated by Bio-Gel P-2 column chromatography and analysed by e.s.r. spectroscopy. For homopolymers, e.s.r. spectra were recorded at 50 degrees C after enzymatic digestion to obtain signals with narrower line width. The e.s.r. signal consisting of only a primary triplet without further splittings, which is consistent with assignment to the trapping of an H-abstraction radical at the C4' position of the sugar moiety, was observed in all cases. For 2'-deoxyguanosine an e.s.r. signal consisting of a secondary triplet was observed. Examinations using other spin-trapping reagents such as PBN, 4-PyOBN and DMPO provided no positive evidence supporting the proposal that this was due to an alpha-nitrogen. The e.s.r. signal consisting of a secondary doublet which further splits into a doublet was observed for 2'-deoxyadenosine, adenosine, 3'-deoxyguanosine, 2'-deoxyguanosine, and inosine, and tentatively associated with a radical centered in the sugar moiety.     

Conservation of DNA sequences for plasmid-mediated citrate utilization within the enterobacteria.

Southern blot DNA-DNA hybridization experiments with a cloned Cit+ DNA fragment as a probe showed that the plasmid-mediated Cit+ determinants from four Cit plasmids (R726, pOH3001, pOH3035, and pOH30221) were all homologous. Sequences homologous to the plasmid-borne Cit+ gene were also found in total bacterial DNA isolated from Salmonella paratyphi B, Salmonella enteritidis, Salmonella typhimurium LT-2, Citrobacter freundii, ATCC 8090, Citrobacter amalonaticus ATCC 25405, Klebsiella pneumoniae I and IID 977, and Enterobacter aerogenes ATCC 13048. The DNA digest from C. amalonaticus ATCC 25405 contained a 1.4-kilobase BamHI-HincII DNA fragment that was strongly homologous with and identical in size to the plasmid Cit+ probe.     

Solid-state ethanol fermentation by means of inert gas circulation

A new method for solid-state ethanol fermentation (the SSEF system) was experimented on for the ethanol production from solid starchy materials, where a packedbed-type fermentor was used. Both cultivation of Aspergillus saitoi and enrichment of a saccharifying enzyme were effective for hydrolysis of the starch. Ethanol production was set in by a form of parallel fermentation using a respiration-deficient mutant of Saccharomyces cerevisiae. Produced ethanol was simultaneously stripped by circulating inert gas and separated in a condenser. Average ethanol concentration in the condensate was over 200 g/L, and over 90% of produced ethanol was recovered from the packed bed during 15 or 16 days of stripping. The fermentation efficiency was about 80%, which was evaluated much higher than those of conventional solid-state fermentations. The residue had lesser volume and a higher solids content compared with the distillery wastewaters of conventional liquid-state fermentations. This means an advantage for the treatment and the effective conversion of the residue into fetilizers or animal feeds.