Immunocytochemical study of the intracellular localization of M protein of vesicular stomatitis virus

Summary The purpose of this paper is to describe the immunocytochemical localization of M protein of vesicular stomatitis virus (VSV) in infected cells. Vero cells, MDBK cells, Swiss 3T3 cells, and BHK cells were examined at various times after infection. For immunofluorescent staining, the cells were fixed with PLP fixative and then treated with 0.05% Triton X-100 before incubation with antibodies. Three hours after infection, M protein exhibited diffuse immunostaining throughout the cytoplasm and later accumulated along the cell membrane. The localization of M protein differed from the granular localization of the nucleocapsid N protein of VSV in the cytoplasm. For electron microscopy, the cells were fixed first in a mixture of 2% paraformaldehyde and 0.05% glutaraldehyde and then with PLP fixative, this being followed by treatment with 0.05% saponin. They were then immunostained using the immunoperoxidase method. The M protein was found to be distributed throughout the cytoplasm and later under the cell membrane, especially at virus budding sites. We also used postembedding immunostaining and freeze-fracture immunostaining to avoid the translocation of M protein caused by the detergent treatment. These techniques confirmed our previous results. Our findings are consistent with the view that the M protein of VSV is synthesized on free ribosomes and is then associated with the cell membrane where viral assembly may occur.S. Ohno was a visiting fellow from the Fogarty International Center at the National Institutes of Health, USA, from September 1981 to August 1983, while some parts of this work were in progress.     

Isolation and Characterization of an Enterobacter cloacae Strain That Reduces Hexavalent Chromium under Anaerobic Conditions

An Enterobacter cloacae strain (HO1) capable of reducing hexavalent chromium (chromate) was isolated from activated sludge. This bacterium was resistant to chromate under both aerobic and anaerobic conditions. Only the anaerobic culture of the E. cloacae isolate showed chromate reduction. In the anaerobic culture, yellow turned white with chromate and the turbidity increased as the reduction proceeded, suggesting that insoluble chromium hydroxide was formed. E. cloacae is likely to utilize toxic chromate as an electron acceptor anaerobically because (i) the anaerobic growth of E. cloacae HO1 accompanied the decrease of toxic chromate in culture medium, (ii) the chromate-reducing activity was rapidly inhibited by oxygen, and (iii) the reduction occurred more rapidly in glycerol- or acetate-grown cells than in glucose-grown cells. The chromate reduction in E. cloacae HO1 was observed at pH 6.0 to 8.5 (optimum pH, 7.0) and at 10 to 40°C (optimum, 30°C).     

Sea urchin (Anthocidaris crassispina) egg zinc-binding protein. Cellular localization, purification and characterization

Gel-filtration analysis of cytosol fraction obtained from unfertilized sea-urchin (Anthocidaris crassispina) eggs on Sephadex G-75 revealed the presence of two Zn-binding-protein fractions. The major Zn-binding protein fraction had a low molecular weight and a low absorbance at 280 nm, properties similar to those of the metallothionein found in the regenerating rat liver. These fractions were further purified by DEAE-cellulose and Sephadex G-50 chromatography. Homogeneity of the Zn-binding protein was judged by polyacrylamide-disc-gel electrophoresis and gel-permeation chromatography in the presence of 6 M-guanidinium chloride. The molecular weight determined by gel-permeation chromatography was 3900. This value is in good agreement with the minimum molecular weight calculated from the amino acid composition, which was 3655. Zn-binding protein is composed of 36 amino acid residues and the distinctive features include an extremely high content of cysteine, which accounted for one-third of the total amino acid residues, and a complete absence of aromatic amino acids, as well as of methionine, histidine and arginine. Zn-binding protein contained 4.1 g-atoms of zinc per mol and a trace of cadmium, but no copper, iron or calcium. The molar ratio of reactive thiol groups to metal ion was calculated to be 2.73:1. Possible roles of this Zn-binding protein in the homoeostasis of zinc in unfertilized sea-urchin eggs are discussed.     

Changes of lung surfactant and pressure-volume curve in bleomycin-induced pulmonary fibrosis.

We investigated whether alveolar surface force increased and participated in the lung pressure-volume relationship in bleomycin-induced pulmonary fibrosis in hamsters and, if so, whether lung surfactant was hampered in the lungs. On the air-filled pressure-volume curve, decreases of lung volume from control level were significantly higher at 3-8 cmH2O pressure on day 10 than on day 30. Because the change of lung tissue elasticity evaluated from the saline-filled pressure-volume curve was equal for the 2 days, the higher decrease of air volume on day 10 was due primarily to contribution of alveolar surface force. Pressure differences between deflation limbs of air-filled and saline-filled pressure-volume curves, which represented net alveolar surface force, were significantly higher at any lung volume between 50 and 90% total lung capacity on day 10, but almost no significance was observed on day 30. Phospholipid concentration in bronchoalveolar lavage fluid significantly decreased on day 10 but had improved by day 30. Analysis of phospholipid species in purified lung surfactant showed decreased fractions of disaturated phosphatidylcholine and phosphatidylglycerol on day 10. Surface-active properties of the surfactant, measured by a modified Wilhelmy balance, were remarkably hampered on day 10, but most of them had improved by day 30. We consider that the quantitative and functional abnormalities of lung surfactant have a part in the aggravation of lung mechanics in the acute phase of pulmonary fibrosis.     

Activation of quiescent ovaries by administration of PMSG after LH-RH analogue treatment in heifers

The effect of pregnant mare serum gonadotrophin (PMSG) treatment on activation of quiescent ovaries was examined in heifers. Groups of thirteen, twenty and twelve heifers which showed ovulation within 2 d and corpus luteum (CL) development after injection with a luteinizing hormone releasing hormone analogue (LH-RH-A) were supplementally injected with 500 IU of PMSG (Group I); 500 IU of PMSG and 500 mug of Prostaglandin F(2alpha) analogue (PGF(2alpha)-A; Group II); and 500 mug of PGF(2alpha)-A (Group III) on Day 6 after the injection of 200 mug of LH-RH-A (Day 0), respectively. Estrus appeared in 33.3 to 45.0% of the heifers of the respective groups after the treatment. Ovulation occurred at a significantly (P<0.01) higher rate in Groups I (100%) and II (90.0%) than in Group III (41.7%). The ovarian cyclic activity was initiated in all the heifers that ovulated. Plasma progesterone levels decreased significantly (P<0.05) to about 1 ng/ml on Day 8 and Day 7 in Group I and Groups II and III, respectively. Plasma estradiol-17beta (E(z)) levels increased significantly (P<0.05), reaching a peak on Days 7 to 7.5 in Groups I and II but not in Group III. It is concluded that PMSG treatment stimulates maturation and E(z) secretion of a follicle, thus promoting ovulation and the onset of ovarian cyclic activity.     

Features of two hepatitis B virus (HBV) DNA integrations suggest mechanisms of HBV integration.

Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.     

Alteration of rat liver proteoglycans during regeneration.

Sepharose CL-6B column chromatography of crude extracts from the slices of regenerating rat livers after partial hepatectomy and sham-operated controls labeled with [35S]sulfuric acid revealed an enhancement of [35S]sulfate incorporation into proteoglycan fractions during regeneration. The 35S-labeled proteoglycans contained heparan sulfate (more than 80% of the total) and chondroitin/dermatan sulfate. The 35S-incorporation into both glycosaminoglycans increased to maxima 3-5 days after partial hepatectomy and decreased thereafter toward the respective control levels. When [35S]sulfuric acid was replaced by [3H]glucosamine, similar results were obtained. These results suggest that the maximal stimulation of proteoglycan synthesis in regenerating rat liver follows the maximal mitosis of hepatic cells 1-2 days after partial hepatectomy. The 35S-labeled proteoglycans from regenerating liver 3 days after partial hepatectomy and control were analyzed further. They were similar in chromatographic behavior on a gel filtration or an anion-exchange column and in glycosaminoglycan composition. Their glycosaminoglycans were indistinguishable in electrophoretic mobility. However, these proteoglycans were slightly but significantly different in their affinity to octyl-Sepharose and in the molecular-weight distribution of their glycosaminoglycans.     

A method for removal of toxic chromium using dialysis-sac cultures of a chromate-reducing strain of Enterobacter cloacae

Summary A method for removal of toxic hexavalent chromium (chlomate: CrO _inf4 ^sup2– ) was developed by use of dialysis-sac cultures of a chromate-reducing strain of Enterobacter cloacae (HO1). E. cloacae strain HO1 cells were put in dialysis (semipermeable membrane) sacs, and the sacs were submerged in water containing toxic CrO _inf4 ^sup2– . The dialysis sacs allowed CrO _inf4 ^sup2– to diffuse into the culture, and CrO _inf4 ^sup2– was reduced anaerobically in the dialysis sacs by the E. cloacae cells. Because reduced chromium readily formed insoluble chromium hydroxides in the dialysis sacs, the greater part of reduced chromium was unable to diffuse out through the semipermeable membrane. Thus the dialysis culture of E. cloacae strain HO1 could successfully remove toxic chromium from the surrounding water. If the initial concentration of CrO _inf4 ^sup2– was less than 4mM (208 ppm as chromium), about 90% of the total chromium could be removed from water by the described method. Offprint requests to: H. Ohtake     

Cloning and characterization of a Pseudomonas aeruginosa gene involved in the negative regulation of phosphate taxis.

Pseudomonas aeruginosa PAO1 exhibited a positive chemotactic response to P(i). The chemotactic response was induced by P(i) limitation. An alkaline phosphatase (AP) constitutive mutant showed a chemotactic response to P(i), regardless of whether the cells were starved for P(i). Sequence analysis and complementation studies showed that the P. aeruginosa phoU gene was involved both in the regulation of AP expression and in the induction of P(i) taxis. However, unlike AP expression, P(i) taxis was not regulated by the phoB gene product.