Infectivity-deficient vesicular stomatitis virus produced in the presence of interferon has a functional virion core

Interferon induces two antiviral actions against vesicular stomatitis virus by (i) inhibiting viral protein synthesis which leads to a reduction in virion production, and (ii) producing progeny which are deficient in infectivity (VSVIF). At low or physiological concentrations of interferon, while the virion production was decreased by less than 10-fold, the virion infectivity yield was suppressed more than 1000-fold. The VSVIF was found to be deficient (quantitatively) in envelop glycoprotein G and protein M. Tryptic peptide mapping indicated that there was no detectable structural abnormality in the G, M, and N proteins of VSVIF. The virion cores, lacking only the envelop G protein, isolated from VSVIF and control VSV have essentially identical specific infectivity. This indicated that the virion proteins L, N, NS, and M, as well as viral RNA that make up the virion core, must be functionally normal, and the observed deficiency in G protein was likely to be the cause of the functional deficiency of the virion. Low concentrations of DEAE-dextran, which is known to partially overcome the virion's dependence on the G protein for adsorption to the cell during infection, were found to enhance the infectivity of VSVIF more than the control virion. These results together indicated that the loss of infectivity in the VSVIF was due to the deficiency of the surface glycoprotein G.     

Biochemical changes in the gastric mucosa after injury in young and aged rats

Changes in gastric mucosal thymidine kinase (TK) activity (an indicator of proliferative activity) were examined in young (4 month) and aged (24 month) Fischer-344 male rats 6 h after intragastric administration of either 2 M NaCl (1 ml/130 g b.w.) or an equivalent volume of water (control). These changes were related to the expression of c-myc gene, tyrosine kinase (Tyr-K) activity and tyrosine-specific phosphorylation of proteins in the gastric mucosa. Basal gastric mucosal TK activity (data from the controls) in the aged rats was found to be 75% (P less than 0.001) above the young animals. This was accompanied by increased expression of c-myc gene and a 67% (P less than 0.001) enhancement in Tyr-K activity. Intragastric administration of 2 M NaCl resulted in gastric mucosal damage (as evidenced by lesions index) in both age groups. However, in aged rats, the lesions index was found to be about 75% higher than in their younger counterparts. In young rats, mucosal injury resulted in a 95% rise in TK activity, whereas in aged rats it was increased by only 38%, when compared with corresponding controls. This 2-fold rise in TK activity in young rats was also associated with increased expression of the c-myc gene. In young rats, administration of hypertonic saline caused a 90% (P less than 0.001) increment in Tyr-K activity and significantly stimulated tyrosine-specific phosphorylation of five mucosal proteins with an apparent molecular mass of 170, 120, 100, 55 and 43 kDa. On the other hand, administration of hypertonic saline to the aged rats caused only a small 16% (P less than 0.025) increase in Tyr-K activity, and produced no apparent change in either expression of c-myc gene or tyrosine-specific phosphorylation of any of the proteins in the gastric mucosa, when compared with the corresponding controls. We conclude that aging increases the susceptibility of the gastric mucosa to damaging agents and diminishes its regenerative capacity. We also suggest that Tyr-K may play a role in determining these events.     

Expression of vimentin and glial fibrillary acidic protein in human developing spinal cord

Summary Expression of intermediate filament proteins was studied in human developing spinal cord using immunoperoxidase and double-label immunofluorescence methods with monoclonal antibodies to vimentin and glial fibrillary acidic protein (GFAP). Vimentin was found in the processes of radial glial cells in 6-week embryos, while GFAP appeared in vimentin-positive astroglial cells at 8–10 weeks. GFAP and vimentin were present in approximately equal amounts in differentiating astrocytes in 23-week spinal cord. In 30-week fetuses, astrocytes reacted strongly for GFAP, while both the reaction intensity and the number of vimentin-positive cells fluctuated predominantly in the grey matter. No clear-cut transition from vimentin to GFAP was noticed during the development of astrocytes. The majority of ependymal cells in 23-week fetuses contained vimentin but only a few of them reacted for GFAP. The expression of vimentin continued during the whole development of the ependymal layer, in contrast to the reactivity for GFAP which disappeared between the 30th week and term.     

PRODUCTION AND CHARACTERIZATION OF MULTIPLE-LAYERED POPULATIONS OF ANIMAL CELLS

Dense populations containing 129 x 10⁶ Jensen sarcoma, 134 x 10⁶ DON Chinese hamster, 28.9 x 10⁶ WI-38 human diploid, 61.8 x 10⁶ HEp-2 human carcinoma, and 67.4 x 10⁶ WISH human amnion cells were produced from dilute inocula, 0.85 to 5.33 x 10⁶, in 7 to 8 days in a perfusion system using replicate T-60 flasks. Perfusion rates as high as 560 ml medium/day/T-60 were required to maintain pH (to ca ±0.1 unit) and adequate nutrient supplies. The cell densities encountered are described by the term "monolayer equivalents" (M.E.), defined as number of cells per culture divided by number of cells in a monolayer. The M.E.'s for T-60 cultures containing unusually dense populations of 40 x 10⁶ WI-38 and 250 x 10⁶ DON cells (9-day perfusion) were 5 and 17, respectively, and numbers of cells in illustrations of stained cross-sections of membranes from these cultures were in excellent agreement. Threshold M.E.'s exist below which proliferation is the chief cellular activity and above which one or more cell functions may predominate even though proliferation persists. Cellular nutrition and metabolism may change with changes in M.E., as illustrated in different patterns of glutamic acid, proline, and glycine utilization or production in dense vs. dilute WI-38 cell populations. The results indicated that the role of contact inhibition phenomena in arresting cellular proliferation was diminished in perfusion system environments.     

Human cytomegalovirus maturational proteinase: expression in Escherichia coli, purification, and enzymatic characterization by using peptide substrate mimics of natural cleavage sites.

The proteolytic processing of the human cytomegalovirus (HCMV) assembly protein, resulting in truncation of its C terminus, is an essential step in virion maturation. The proteinase responsible for this cleavage is the amino-terminal half of the protein encoded by the UL80a open reading fame. We have obtained high expression levels of this 256-amino-acid HCMV proteinase, assemblin, in Escherichia coli. In addition to the 28-kDa proteinase, a 15-kDa protein comprising the first 143 amino acids and a 13-kDa protein comprising the last 113 amino acids of the 28-kDa HCMV proteinase were present. Both the 28-kDa proteinase and the 15-kDa protein were purified by a two-step chromatographic procedure utilizing anion exchange in urea and dithiothreitol and size exclusion in NaSCN and dithiothreitol. Activation of the purified 28-kDa proteinase required denaturation in urea as well as complete reduction of all five cysteine residues in the molecule. Removal of the urea by dialysis with retention of the reducing agent yielded an active proteinase. Addition of glycerol to 50% enhanced the activity. The HCMV proteinase cleaved the peptides RGVVNASSRLAK and SYVKASVSPE, which are mimics of the maturational (M)- and release (R)-site sequences, respectively, in the UL80a-encoded protein. The cleavage site in the peptides was at the same Ala-Ser scissile bond as observed in the UL80a protein. The Km value for the cleavage of RGVVNASSRLAK (M-site mimic) by the proteinase was similar to that for SYVKASVSPE (R-site mimic), but the turnover (kcat) of the M-site peptide mimic substrate by the proteinase was six to eight times faster. The peptide homologs of the herpes simplex virus type 1 M- and R-site sequences in the UL26-encoded protein were also cleaved by the HCMV proteinase, although at rates slower than those for the HCMV substrates. The HCMV proteinase was inhibited by Zn2+ and by alkylating agents, but only at very high inhibitor concentrations. The purified 15-kDa protein, subjected to the same activation conditions as the 28-kDa proteinase, had no enzymatic activity against the HCMV M- and R-site peptide substrates.     

Depressant effects of prostacyclin in human atrial fibers and cardiomyocytes

The aim of this study was to characterize the electropharmacological effects of prostacyclin (PGI₂) in human atrial fibers and cardiomyocytes. Atrial tissues obtained from the hearts of 28 patients undergoing corrective cardiac surgery were used. Transmembrane action potentials were recorded using a conventional microelectrode technique, and twitch force by a transducer. Effects of PGI₂ (1 nM–10 µM) on action potential characteristics and contraction of atrial fibers were evaluated in normal [K]_o (4 mM) and high [K]_o (27 mM) in the absence and presence of cardiotonic agents. In addition, atrial and ventricular myocytes were isolated enzymatically from atrial tissues and hearts of 4 patients undergoing cardiac transplant. The effects of PGI₂ on Na- and Ca-dependent inward currents (I_Na and I_Ca) of cardiomyocytes were tested. In 9 human atrial fibers showing fast-response action potentials (mean dV/dt_max = 101 ± 15 Vs^–1) in 4 mM [K]_o, PGI₂ did not influence dV/dt_max of phase 0 depolarization even at 1 µM. However, at a concentration as low as 10 nM, PGI₂ depressed spontaneous rhythms or slow-response action potentials in high-K-depolarized fibers. PGI₂ also depressed delayed afterdepolarizations and aftercontractions induced by cardiotonic agents. In isolated cardiomyocytes, PGI₂ reduced I_Ca but not I_Na. The present findings show that, in human atrial fibers and cardiomyocytes, PGI₂ induces greater depressant effects on the slow-response action potential, I_Ca and triggered activity than on the fast-response action potential. It is suggested that PGI₂ may act through a selective reduction of transmembrane Ca influx.     

Immune-complex-trapping cells in the spleen of the oriental fire-bellied toad,Bombina orientalis

Summary The spleen of the oriental fire-bellied toad, Bombina orientalis, consists of well-developed white pulp, separated from the lymphocytic marginal zone by the connective tissue boundary layer. Injection of peroxidase-conjugated rabbit anti-peroxidase revealed that these immune complexes were localized on the surface of acid-phosphatase-positive and non-specific-esterasepositive cells in the white pulp. The majority of immunecomplex-trapping cells were present around the blood vessels. Cell processes of some of these cells penetrated into the wall of blood vessels. The significance of the present findings is discussed with respect to the evolution of immune-complex-trapping cells in the spleen.