Herpes simplex virus type 1 entry through a cascade of virus-cell interactions requires different roles of gD and gH in penetration.

We examined the entry process of herpes simplex virus type 1 (HSV-1) by using infectious virus and previously characterized noninfectious viruses that can bind to cells but cannot penetrate as a result of inactivation of essential viral glycoprotein D (gD) or H (gH). After contact of infectious virus with the cell plasma membrane, discernible changes of the envelope and tegument could be seen by electron microscopy. Noninfectious virions were arrested at distinct steps in interactions with cells. Viruses inactivated by anti-gD neutralizing antibodies attached to cells but were arrested prior to initiation of a visible fusion bridge between the virus and cell. As judged from its increased sensitivity to elution, virus lacking gD was less stably bound to cells than was virus containing gD. Moreover, soluble gD could substantially reduce virus attachment when added to cells prior to or with the addition of virus. Virus inactivated by anti-gH neutralizing antibodies attached and could form a fusion bridge but did not show expansion of the fusion bridge or extensive rearrangement of the envelope and tegument. We propose a model for infectious entry of HSV-1 by a series of interactions between the virion envelope and the cell plasma membrane that trigger virion disassembly, membrane fusion, and capsid penetration. In this entry process, gD mediates a stable attachment that is likely required for penetration, and gH seems to participate in fusion initiation or expansion.     

Molecular attenuation of vaccinia virus: mutant generation and animal characterization.

These studies demonstrated that the inbred BALB/c mouse strain can be optimized for the assessment of vaccinia virus virulence, growth, and spread from the site of inoculation and immune protection from a lethal vaccinia virus challenge. The studies established that manipulation of the vaccinia virus genome generated mutants exhibiting a wide range of attenuated phenotypes. The nine NYCBH vaccinia virus mutants had intracranial 50% lethal doses that ranged from 2 to greater than 7 log10 units. The decreased neurovirulence was due to decreased replication in brain tissue. Three mutants had a decreased ability to disseminate to the lungs, brains, livers, and spleens of mice after intranasal infection. One mutant had a decreased transmission from mice infected by tail scarification to naive cage mates. Although the mutants, with one exception, grew to wild-type titers in cell culture, they showed a growth potential on the scarified skin of mice that was dramatically different from that of the wild-type virus. Consequently, all of the mutants had significantly compromised immunogenicities at low virus immunization doses compared with that of the wild-type virus. Conversely, at high immunization doses most mutants could induce an immune response similar to that of the wild-type virus. Three Wyeth vaccine strain mutants were also studied. Whereas the thymidine kinase, ribonucleotide reductase, and hemagglutinin mutants had a reduced virulence (50% lethal dose), only the thymidine kinase mutant retained its immunogenicity.     

Molecular and developmental studies of a sperm acrosome antigen recognized by HS-63 monoclonal antibody.

A conserved mouse sperm antigen (MSA-63) recognized by a monoclonal antibody (HS-63) was isolated from mouse testes by single-step immunoaffinity chromatography. Isolated MSA-63 preparation was shown to be a group of proteins ranging from 24-84 kDa and with isoelectric points (pIs) ranging from 4.0-6.0 when analyzed by two-dimensional (2-D) gel electrophoresis. Microsequencing techniques were employed to determine the relationships of various protein spots on 2-D gels. Partial amino acid sequences of some protein spots in isolated MSA-63 preparation were shown to be homologous to mouse actins, while others revealed homology only to the SP-10 protein. Rabbit antisera raised against isolated MSA-63 antigen preparation were used to immunoscreen a mouse testis cDNA library. Isolated cDNA clones carrying a 1.2-kb insert were used to obtain nucleotide sequences containing open-reading frames and to deduce the corresponding amino acid sequence of MSA-63. A high degree of homology was observed between MSA-63 and a known human sperm antigen, SP-10, at DNA/protein levels. Amino acid sequences of tryptic peptides derived from protein spots of 24-47 kDa and pIs of 4.2-4.4 were found to be identical to those deduced from isolated cDNA clones. The gene expression of MSA-63 during spermatogenesis in mice was studied using a specific cDNA probe as well as HS-63. It was observed that MSA-63 was not expressed until the postmeiotic stages of spermatogenesis.     

Induction of ovulation and oocyte maturation of amphibian (Rana dybowskii) ovarian follicles by protein kinase C activation in vitro.

We previously reported that protein kinase C (PKC) activation induced meiotic maturation (germinal vesicle breakdown, GVBD) of Rana dybowskii follicular oocytes cultured in vitro without hormone treatment. The experiments reported here were carried out to establish whether ovarian follicles ovulated in response to PKC activation during culture. A phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), was used for PKC activation. TPA addition (10 microM) to cultured ovarian fragments induced ovulation and maturation of the oocytes similar to that seen following addition of frog pituitary homogenate (FPH, 0.05 pituitary/ml) or progesterone (0.5 microgram/ml). Such changes were not observed when ovarian fragments were treated with inactive phorbol ester. The time course of TPA-induced ovulation was similar to that produced by FPH-stimulated ovulation. Both TPA- and FPH-stimulated ovulation and maturation were blocked by treatment with cycloheximide, forskolin (an adenylate cyclase stimulator), and 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7; a PKC inactivator). FPH treatment markedly increased progesterone levels in the medium during ovarian fragment culture whereas TPA treatment failed to elevate progesterone levels. Thus, TPA treatment mimics FPH and progesterone in inducing ovulation and meiotic maturation in cultured amphibian ovarian fragments. The data strongly suggest that PKC plays an important role in regulating ovulation as well as in modulating amphibian oocyte maturation during follicular differentiation.     

Red cell membrane elasticity as determined by flow channel technique.

The elasticity of red cell membrane was determined in a rectangular flow channel under controlled shear flow. The relation between shear stress and cell extension ratio (lambda) has been analyzed with the use of Evans' two-dimensional model. The deformed cell shapes observed experimentally agreed well with the model with lambda up to 1.4. The best correlation was found at lambda = 1.2. The analysis suggests a nonlinear extensional membrane modulus in the low stress range encountered in the flow channel. In terms of an appropriate strain parameter, the elastic modulus is shown to rise toward the level encountered in micropipette aspiration experiments. The implications of the present findings in modeling of cell mechanics and in cell hemolysis are discussed.     

Separation of fructose and glucose mixture by zeolite Y

Na-, K-, Ba-, and Ca-Y were employed for the separation of fructose and glucose in an adsorption column. Effects of temperature, solvent flow rate, amount of mixture injection, and exchangeable cations on the separation were investigated. Efficiency of separation was used as a criterion to characterize the effectiveness of the separation. The transport and kinetic parameters for the column separation were also presented. From simple pulse experiments and moment analysis, the obtained process information of equilibrium and dynamic parameters might be used to design, operate, and control the separation column. (c) 1992 John Wiley & Sons, Inc.     

Incidence of multinucleated and polyploid aortic smooth muscle cells cultured from different age groups of spontaneously hypertensive rats.

Cell size and incidence of multinucleated, polyploid cells in cultured aortic smooth muscle cells from different age groups of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) were compared. Smooth muscle cells from SHR were generally larger than those from WKY, and the percentage of multinucleated smooth muscle cells was always higher in SHR than WKY in the three age groups of rats studied (3-4, 10-12, and 28-30 weeks). In smooth muscle cells from the 3- to 4-week group, there was a positive correlation between cell diameter and the percentage of multinucleated smooth muscle cells. Microdensitometric measurements also showed that the incidence of polyploid smooth muscle cells was always higher in SHR than WKY in the three age groups. There was a positive correlation between DNA density and nuclear area measurements in all the age groups of SHR and WKY. We conclude that cultured aortic smooth muscle cells from different age groups of SHR and WKY contained heterogeneous populations of cells and that, under our culture conditions, the polyploidy of the smooth muscle cells found in vivo was maintained in the SHR and WKY.