Host range temperature-sensitive mutants of herpes simplex virus type 2.

Two small-plaque mutants of herpes simplex virus type 2 (HSV-2) (strain 333), whose growth at 39 C was blocked in certain cell types (cell-dependent temperature sensitivity), were compared compared with parental virus in a number of biological assays. One mutant (no. 69) was found to produce a large number of morphologically normal, but noninfectious, particles; under nonpermissive conditions, these mutant particles were able to interfere with the replication of wild-type HSV-2. The other mutant (no. 74), which is known to belong to a different complementation group, appeared to direct little virus DNA synthesis, even at the permissive temperature. Progeny production and virus DNA synthesis in cells infected by mutant 74 were delayed in comparison with wild-type virus-infected cells. Both mutants were found to be more sensitive to UV irradiation than the parental virus; this was especially marked in the case of mutant 74. Moreover, this mutant was found to have a high transforming efficiency at much lower doses of irradiation than those needed to abolish the cytopathic effect of wildtype HSV-2.     

Herpes simplex virus DNA in transformed cells: sequence complexity in five hamster cell lines and one derived hamster tumor.

Analyses of the hybridization kinetics of labeled herpes simplex virus 2 (HSV-2) DNA with DNA from five hamster cell lines transformed by UV light-irradiated HSV-2 revealed the following. (i) Viral DNA sequences were detected in all five cell lines tested. (ii) None of the cell lines contained the full complement of HSV-2 DNA. (iii) The amount of viral DNA present in the cells varied in different transformed cell lines and ranged from 8 to 32% of the HSV-2 DNA genome in 1 to 3 copies/cell. (iv) Two parallel passages of the same cell line (333-2-29) differed in the amount of viral DNA they contained. We also compared the viral DNA sequences present in (i) one transformed cell line (333-8-9) propagated serially in culture for 80 passages, (ii) a tumor produced by inoculation of a newborn hamster with the 333-8-9 cells, and (iii) a cell line derived from a hamster tumor as above and propagated in culture for 32 passages. The results show that viral DNA present in the hamster tumor and in the cells derived from the tumor had a lower sequence complexity than that present in the original serially passaged 333-8-9 cell line.     

The development by cytomegalovirus-infected cells of binding affinity for normal human immunoglobulin.

After infection with human cytomegalovirus (CMV), cells develop an affinity for normal human immunoglobulin G (IgG). This was demonstrated using 125iodine-labeled purified IgG. It was further demonstrated that the immunoglobulin molecule binds to CMV-infected cells via its Fc portion, and competition for binding to infected cells occurred between purified preparations of human IgG and the Fc fragment of human IgG. Whole sera from individuals with or without a high titer of anti-CMV antibody were labeled with 125iodine and it was demonstrated that serum from individuals with no anti-CMV antibody had an affinity for CMV-infected cells which probably reflected binding of IgG via its Fc fragment. The possible significance of these results in immunologic studies of human CMV is considered.     

Temperature-sensitive host range mutants of herpes simplex virus type 2.

Herpesviruses are capable of several types of infection of a host cell. To investigate the early events which ultimately determine the nature of the virus-host cell interaction, a system was established utilizing temperature-sensitive mutants of herpes simplex virus type 2. Four mutants have been isolated which fail to induce cytopathic effects and do not replicate at 39 C in hamster embryo fibroblast cells. At least one mutant is virus DNA negative. Since intracellular complementation is detectable between pairs of mutants, a virus function is known to be temperature sensitive. However, all four mutants induce cytopathic effects and replicate to parental virus levels in rabbit kidney cells at 39 C. This suggests that a host cell function, lacking or nonfunctional in HEF cells but present in rabbit kidney cells at 39 C, is required for the replication of these mutants in hamster embryo fibroblasts cells at 39 C. Therefore, we conclude that these mutants are both temperature sensitive and exhibit host range properties.     

Quantitative assay for transformation of 3T3 cells by herpes simplex virus type 2.

The interaction of herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) with Swiss/3T3 cells was investigated. Virus-induced cytopathic effects developed in the absence of production of infectious virus. HSV-2 inactivated with UV light (2, 4, 6, and 8 min) also induced cell death in the absence of virus replication. Cell death was not detectable after infection by HSV-2 that had been inactivated by UV irradiation for 10, 12, and 14 min. 3T3 cells infected with UV-inactivated virus (10 and 12 min) continued to replicate past the contact-inhibited monolayer normally associated with these cells. Infection of 3T3 cells with UV-irradiated USV-2 also induced the development of transformed foci. Transformed cells with an epithelioid of fibroblastoid morphology were identified and isolated. All HSV-2-transformed cell lines contained HSV-2-specific antigens detectable by immunofluorescence techniques. The maximum frequency of HSV-2-induced transformation was 3 times 105 PFU per transformed focus, and the observed transformation could be inhibited by pretreatment of the virus with specific antiserum. No type C particles were detected within five cell culture passages after transformation by HSV-2. Type C virus particles were detected after 10 cell culture passages of the HSV-2-transformed cell lines.     

Database resources of the National Center for Biotechnology Information

In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI's Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap'99, Human-Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri-tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih. gov.     

Implication of apoE isoforms in cholesterol metabolism by primary rat hippocampal neurons and astrocytes

Apolipoprotein E (apoE) has been genetically linked to late-onset Alzheimer's disease. From the three common alleles (epsilon2, epsilon3 and epsilon4), epsilon4 has been suggested to promote amyloid beta (Ass) plaque fibrillation, one hallmark of Alzheimer's disease. It has been demonstrated that altered lipid content of hippocampal plasma membrane coincides with the disease. In this study, we show for the first time that the apoE dependent cholesterol metabolism in hippocampal neurons is higher than that of hippocampal astrocytes. Further, apoE-bound cholesterol is highly incorporated in membranous compartments in hippocampal neurons, whereas hippocampal astrocytes show higher intracellular distribution. This is an effect that coincides with cell-type dependent difference of low density lipoprotein receptor (LDLR) family member expression. Hippocampal neurons express high levels of the LDLR related protein (LRP), whereas hippocampal astrocytes are highly positive for LDLR. We could also demonstrate an apoE isoform (apoE2, apoE3 and apoE4) dependent cholesterol uptake in both cells types. In hippocampal neurons, we could find a decreased apoE4-bound cholesterol uptake. In contrast, hippocampal astrocytes show decreased internalization of apoE2-bound cholesterol. In addition, lipidated apoE4 is little associated with neurites in hippocampal neurons in comparison to the other two isoforms. In contrary, hippocampal astrocytes show faint apoE2 immunocytostaining intensity. Data presented indicate that the role of apoE4 in cholesterol homeostasis and apolipoprotein cell association is more pronounced in hippocampal neurons, showing significant alterations compared to the other two isoforms, suggesting that hippocampal neurons are affected by apoE4 associated altered cholesterol metabolism compared to hippocampal astrocytes.     

Parameter-dependent transitions and the optimal control of dynamical diseases

Many theoretical studies in biological and physical sciences consider the dynamical behavior of ann-dimensional ordinary differential equation that contains a large number of independent parameters. A frequently asked question is, are there permissible parameter sets that result in periodic or chaotic behavior? The large number of distinct parameters often limits the feasibility of trial and error calculations. The large dimension and nonlinearity of the system make application of analytic methods at best difficult and at worst effectively impossible. It is shown here that a computational search for parameter-dependent transitions of attractor topology can be effected by constrained optimization of quantitative measures of dynamical behavior (Hurwitz polynomials, Floquet coefficients, Lyapunov exponents and correlation dimension). As an example, we examine a three-dimensional nonlinear ordinary differential equation containing seven parameters that was constructed by Goldbeter and Segel to model periodic synthesis of cyclic AMP inDictyostelium. A search for bifurcations to periodic solutions is made by minimizing Hurwitz coefficients subject to parameter constraints. By comparing four optimization algorithms, the defects and advantages of the procedure are identified. It is also argued that it may be possible to use this characterization of dynamics to construct optimal responses to dynamical diseases (those disorders that result from parameter-dependent bifurcations in physiological control systems).