Limiting dilution analysis of the frequency of antigen-reactive lymphocytes isolated from the central nervous system of Lewis rats with experimental allergic encephalomyelitis

Lymphocytes were isolated from the spinal cord and draining lymph nodes of Lewis rats with acute experimental allergic encephalomyelitis (EAE) 12 days after immunization with myelin basic protein (MBP) and tetanus toxoid (TT). An average of 8.0 +/- 2.0 X 10(6) cells was obtained from the spinal cord. Of these 71.1 +/- 8.6% expressed the helper-T-cell marker W3/25 and 14.8 +/- 6.2% expressed the killer/suppressor-T-cell marker OX8. By limiting dilution analysis of cells exhibiting an antigen-specific proliferative response, the average frequencies of cells reactive to MBP and TT were 3.36 +/- 2.4 and 7.60 +/- 4.1 per 10(4), respectively. In the draining lymph nodes, the frequencies of cells reactive to MBP and TT were 2.24 +/- 1.7 and 2.69 +/- 2.5 per 10(4). At a relatively early stage of clinical EAE, MBP-reactive T cells comprise only a small minority of the cells which can be isolated from the spinal cord; lymphocytes reactive to a protein antigen irrelevant to EAE pathogenesis are present in comparable numbers. This finding suggests that most of these cells accumulate as a result of mechanisms not specific for MBP-reactive lymphocytes.     

Effects of purified measles virus components on proliferating human lymphocytes.

Preparations of purified measles virus and both core and membrane-rich fractions suppressed mitogen-induced proliferation of human lymphocytes without reducing viability or the number of T lymphocytes in culture. The suppressive activity of the measles virus was heat labile. The virus preparations by themselves did not induce lymphocyte proliferation.     

G3 domains of aggrecan and PG-M/versican form intermolecular disulfide bonds that stabilize cell-matrix interaction

The extracellular matrix plays a critical role in maintaining tissue integrity. Among the matrix molecules, the large aggregating chondroitin sulfate proteoglycans are the major structural molecules and are the primary contributors to the stability for some tissues such as cartilage. The notable exceptions are nanomelic cartilage and arthritic cartilage: the former contains a point mutation leading to a stop codon before translating to the C-terminal G3 domain; the latter contains a large proportion of aggrecan from which the G3 domain has been cleaved. These phenomena suggest that the G3 domain may be important in cartilage stability. Here, we demonstrated for the first time that the G3 domains of aggrecan and another proteoglycan, PG-M/versican, formed intermolecular disulfide bonds, and all subdomains were involved. Further studies indicated that each of the 10 cysteine residues of the aggrecan G3 domain could potentially form intermolecular disulfide bonds in vitro. The disulfide bonds were disrupted in the presence of reducing reagent beta-mercaptoethanol and dithiothreitol. As a result, normal chondrocyte-matrix interaction was disrupted, and the structure of the extracellular matrix was altered. Furthermore, disruption of disulfide bonds also reduced the role of PG-M/versican G3 domain in mediating cell adhesion. Our study provides strong evidence of the importance of proteoglycan interactions through intermolecular disulfide bonds in cartilage firmness and cell-matrix stability.     

Conformational coupling,bridge helix dynamics and active site dehydration in catalysis by RNA polymerase

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP–NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant β R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge α-helix, the extended fork loop, the active site, and the trigger loop–trigger helix is apparent and adversely affected in β R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site “latch” assembly that includes a key trigger helix residue Tt β′ H1242 and highly conserved active site residues β E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.     

Rift Valley Fever Virus Incorporates the 78 kDa Glycoprotein into Virions Matured in Mosquito C6/36 Cells

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.     

Insights from diploblasts; the evolution of mesoderm and muscle

The origin of both mesoderm and muscle are central questions in metazoan evolution. The majority of metazoan phyla are triploblasts, possessing three discrete germ layers. Attention has therefore been focused on two outgroups to triploblasts, Cnidaria and Ctenophora. Modern texts describe these taxa as diploblasts, lacking a mesodermal germ layer. However, some members of Medusozoa, one of two subphyla within Cnidaria, possess tissue independent of either the ectoderm or endoderm referred to as the entocodon. Furthermore, members of both Cnidaria and Ctenophora have been described as possessing striated muscle, a mesodermal derivative. While it is widely accepted that the ancestor of Eumetazoa was diploblastic, homology of the entocodon and mesoderm as well as striated muscle within Eumetazoa has been suggested. This implies a potential triploblastic ancestor of Eumetazoa possessing striated muscle. In the following review, I examine the evidence for homology of both muscle and mesoderm. Current data support a diploblastic ancestor of cnidarians, ctenophores, and triploblasts lacking striated muscle.     

Versican protects cells from oxidative stress-induced apoptosis.

Oxidant injury plays a critical role in the degenerative changes that are characterized by a decline in parenchymal cell numbers and viability, and occur with aging and in the etiology of many diseases. The extracellular proteoglycan versican is widely distributed in the extracellular matrix surrounding the cells. This study examines whether versican plays a role in protecting cells from free radical-induced apoptosis. Stable expression of versican or its C-terminal domain significantly decreased H(2)O(2)-induced cellular apoptosis. Cells in adherent monolayer were more resistant to H(2)O(2)-induced apoptosis than cells cultured in suspension. While vigorous trypsinization caused integrin cleavage and rendered the cells more susceptible to H(2)O(2)-induced damages, expression of versican or its C-terminal domain enhanced cell attachment and expression of beta1 integrin and fibronectin. Enhanced cell-matrix interaction by addition of manganese (MnCl(2)) to cultures also significantly diminished H(2)O(2)-induced apoptosis. The results suggest that versican plays an important role in reducing oxidant injury through an enhancement of cell-matrix interaction.     

Improved Understanding of the Bacterial Vaginal Microbiota of Women before and after Probiotic Instillation

The vaginal bacterial microbiota of 19 premenopausal women was examined by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of the V2-V3 region of the 16S rRNA gene. Ten of the women were studied further to investigate the effect and persistence of vaginally inserted capsules containing viable lactobacilli. PCR-DGGE indicated that most subjects had a microbiota represented by one to three dominant DNA fragments. Analysis of these fragments revealed that 79% of the women possessed sequences with high levels of similarity to Lactobacillus species sequences. Sequences homologous to Lactobacillus iners sequences were the most common and were detected in 42% of the women tested. Alteration of the vaginal microbiota could be detected by PCR-DGGE in several women after the instillation of lactobacilli. Additionally, randomly amplified polymorphic DNA analysis of lactobacilli isolated from selective media demonstrated that the exogenous strains could be detected for up to 21 days in some subjects. This study demonstrates that non-culture-based techniques, such as PCR-DGGE, are useful adjuncts for studies of the vaginal microbiota.