Mutational analysis of simian immunodeficiency virus from African green monkeys and human immunodeficiency virus type 2

We constructed ten mutants of simian immunodeficiency virus isolated from African green monkey (SIVAGM), and nine mutants of human immunodeficiency virus type 2 (HIV-2) in vitro. Their infectivity, cytopathogenicity, transactivation potential, virus RNA, and protein synthesis were examined by transfection and infection experiments. Mutations in three structural (gag, pol, env) and two regulator (tat, rev) genes abolished the infectivity of both viruses, but vpx, vpr (HIV-2), and nef were dispensable and mutant viruses were indistinguishable phenotypically from wild type virus. A vif mutant of HIV-2 showed poor infectivity in cell-free condition, whereas SIVAGM mutants grew equally well with wild type virus. In transient transfection assays, rev mutants derived from both viruses produced mainly small mRNA species and no detectable virus proteins and particles. Transactivation potential of tat mutants originated from both viruses was about three- to ten-fold less than that of respective wild type DNAs, generating small amounts of virus.     

Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.     

The existence of activin A/erythroid differentiation factor and its inhibitor in human serum: comparison of normal and chronic renal failure sera.

Activin A/EDF, initially found as a differentiation inducer of murine Friend erythroleukemia, also has a stimulatory effect on erythropoiesis in vitro and in vivo. Here we proved activin A/EDF activity in human serum. The activin A/EDF level in 18 normal human serum samples was measured by a specific bioassay and was found to be 8.3 +/- 4.6 ng/ml, indicating that there exists sufficient activity to affect erythropoiesis in normal serum. In contrast, activin A/EDF activity was reduced in the chronic renal failure patients and 23/26 serum samples examined showed levels below 1.2 ng/ml. Further analysis using HPLC revealed that chronic renal failure serum actually contained as much activin A/EDF as normal serum, and that the difference between normal and patient serum existed in the content of a specific inhibitor of activin A/EDF. This observation suggests the possibility that the inhibitor is participating in the regulation of activin A/EDF activity in vivo in chronic renal failure patients and also the possibility of activin A/EDF could be utilized in the therapy of the anemia of such patients.     

Gene expression of metalloproteinase and its inhibitor in mesangial cells exposed to high glucose.

To clarify the roles of metalloproteinases and their inhibitor (TIMP) in diabetic glomerulopathy, we studied the effect of a high glucose concentration on the gene expression of metalloproteinase transin and TIMP as well as collagen type IV and laminin in cultured rat mesangial cells (MCs). In the high glucose group, collagen type IV, laminin, and TIMP mRNA levels were all elevated in a concentration-dependent manner, whereas transin expression was suppressed. Osmotic control of high glucose with mannitol selectively stimulated TIMP expression. We hypothesize that high glucose decreases matrix-degrading activity as well as increases matrix productivity in MCs.     

Comparison of muscle force,muscle endurance,and electromyogram activity during an expedition at high altitude

Handgrip force (HF), maximal pinch force (MF), muscle endurance (ME), and the median power frequency (MdPF) of the activity shown in the electromyogram (EMG) were studied at various altitudes in eight normal healthy subjects. MF and ME were measured between the index finger and thumb, and all measurements were obtained at altitudes ranging from 610 to 4860 m during an expedition in the Qinghai Plateau in China. With the change in altitude HF, ME, and MF showed no significant change. Compared to the MdPF at 2260 m on ascent, the MdPF at other altitudes showed a significant decrease (P<0.01). Thus, we conclude that muscle performance (HF, MF, and ME) was not affected by the environment at high altitude. However, MdPF was affected and the mean MdPF at 610 m after the expedition did not recover to initial values of MdPF. We suggest these results may have been affected by fatigue and chronic exposure to the hypobaric hypoxic environment, since the members of the expedition party expressed feelings of sluggishness and fatigue after the expedition.     

Intradermal administration of lipopolysaccharide in treatment of human cancer

 Lipopolysaccharide (LPS) has been recognized as a potent antitumor agent in animal tumor models; however, its use in human cancer therapy has been limited to only one trial, in which LPS from Salmonella was given intravenously. It was not very successful because of poor tumor response and was also toxic. We originally developed LPS prepared from Pantoea agglomerans (LPSp), and this was a well-purified, small-molecular-mass (5 kDa) agent. We chose intradermal rather than intravenous administration in the hope that the former would release LPS slowly into the bloodstream, and thus be less toxic while preserving antitumor activity. In our animal tumor models, intradermal administration was indeed less toxic and more beneficial for tumor regression than intravenous administration. We made a pilot study with intradermal administration of LPSp on the treatment of ten advanced cancer patients. Five of them had evaluable tumor, which had failed earlier to respond to conventional chemotherapy. Cyclophosphamide was also administered in this trial, in anticipation of its synergistic effect with LPSp. In this study LPSp was injected intradermally into each patient twice a week, starting with an initial dose of 0.4 ng/kg, and raising it to 600 or 1800 ng/kg. A 400-mg/m² dose of cyclophosphamide was given intravenously every 2 weeks. After completion of the dose escalation, the treatment was continued for at least 4 months, and it was found that 1800 ng/kg LPSp was well tolerated. A significant level of cytokines was observed in the sera for at least 8 h. These results indicate higher tolerable doses and remarkably more continuous induction of the cytokines than were reported in a previous study by others using intravenous administration. Three of the five evaluable tumors showed a significant response to our combined therapy. Intradermally administered, LPS was less toxic and elicited a tumor response in combination with cyclophosphamide; it can thus can be applied to cancer treatment even in humans. Received: 3 August 1995 / Accepted: 2 April 1996     

Complete1H NMR assignments of synthetic glycopeptides from the carbohydrate-protein linkage region of serglycins

We present complete¹H NMR assignments for two synthetic glycopeptides representative of the carbohydrateprotein linkage region of serglycin proteoglycans. The peptides are: Ser(Galp-Xylp)-Gly-Ser-Gly-Ser(Galp-Xylp)-Gly and, Ser(Galp-Xylp)-Gly-Ser(Galp-Xylp)-Gly-Ser(Galp-Xylp)-Gly. A number of 2D NMR spectra together with a 3D NOESY-TOCSY spectrum were acquired at 600 MHz to complete the assignments of the glycopeptides dissolved in water with 40% trifluoroethanol. Preliminary analysis of the NMR data suggests folded structures for the glycopeptides.A preliminary account of this work was presented at an International Symposium held at the University of Alabama at Birmingham in November, 1994 on the occasion of the 65th birthday of Professor Lennart Rodén.     

Expression of NADH-Dependent Glutamate Synthase in Response to the Supply of Nitrogen in Rice Cells in Suspension Culture

The effects of inorganic and organic nitrogen on the levelsof mRNA for NADH-dependent glutamate synthase (GOGAT) and theprotein were examined in rice cells in suspension culture. Asupply of NH⁺₄, NO^-₃, glutamine, or asparagine induced the accumulationof the protein and mRNA, but levels of mRNA for ferredoxin-GOGATwere hardly affected. ¹Present address: P.C. Center Wakuya-cho, Toda-gun, Miyagi,Japan.     

Postnatal reorganization of actin filaments and differentiation of intercellular boundaries in the rat aortic endothelial cells

Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is though to be correlated with the change of fluid shear stress loaded in the aortic endothelium.