Discrete limbal epithelial stem cell populations mediate corneal homeostasis and wound healing

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Assay for measuring relative potency of leptospiral bacterins containing serovar pomona

An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of leptospiral antigen in bacterins containing Leptospira interrogans serovar pomona type kennewicki. A monoclonal antibody (MAb), 2D7, which is directed against a surface antigen on whole cells of L. interrogans serovar pomona type kennewicki, was used in the assay. The capture of antigen in bacterins by a polyclonal antiserum was followed by the addition of the 2D7 ascites fluid, an anti-mouse conjugate and substrate. Biologicals evaluated with this system included preparations containing type kennewicki antigen (homologous) and those not containing type kennewicki antigen (heterologous). Heterologous bacterins gave optical density (OD) values comparable to those of blank wells. Homologous bacterins yielded OD values equal to or greater than those of the National Veterinary Services Laboratories (NVSL) reference pomona bacterin. The relative potencies (RP) of 84 licensed commercial Leptospira pomona bacterin serials were evaluated against the NVSL reference pomona bacterin using the NVSL Relative Potency computer program. Random samples of 1, 2, 3 and 5 ml dose products were selected for evaluation with this system. All products tested passed the hamster potency assay required for leptospiral bacterins. This ELISA system enables detection of antigen in bacterins containing L. interrogans serovar pomona type kennewicki and demonstrates the potential for in vitro testing of leptospiral bacterins.     

Proteolytic sensitivity of the α subunit in luciferases ofPhotobacterium species

Bacterial luciferases isolated from strains of three species ofPhotobacterium (P. leiognathi, P. phosphoreum andP. fischeri) have been found to be considerably more sensitive to trypsin inactivation than luciferase fromBeneckea harveyi. P. leiognathi luciferase has a pseudo-first-order rate constant of inactivation of 0.14 min⁻¹ when exposed to 1 μg/ml (42 nM) trypsin. As judged by sodium dodecyl sulfate electrophoresis of the products of proteolysis, only the α subunit of the αβ heterodimeric luciferases ofPhotobacterium species was attacked by trypsin. After treatment of these enzymes with trypsin, the β subunit of eachPhotobacterium species retained its ability to reform active luciferase when renatured with native complementary α subunit.     

Vibrio fischeri Outer Membrane Protein OmpU Plays a Role in Normal Symbiotic Colonization

The nascent light-emitting organ of newly hatched juveniles of the Hawaiian sepiolid squid Euprymna scolopes is specifically colonized by cells of Vibrio fischeri that are obtained from the ambient seawater. The mechanisms that promote this specific, cooperative colonization are likely to require a number of bacterial and host-derived factors and activities, only some of which have been described to date. A characteristic of many host-pathogen associations is the presence of bacterial mechanisms that allow attachment to specific tissues. These mechanisms have been well characterized and often involve bacterial fimbriae or outer membrane proteins (OMPs) that act as adhesins, the expression of which has been linked to virulence regulators such as ToxR in Vibrio cholerae. Analogous or even homologous mechanisms are probably operative in the initiation and persistence of cooperative bacterial associations, although considerably less is known about them. We report the presence in V. fischeri of ompU, a gene encoding a 32.5-kDa protein homolog of two other OMPs, OmpU of V. cholerae (50.8% amino acid sequence identity) and OmpL of Photobacterium profundum (45.5% identity). A null mutation introduced into the V. fischeri ompU resulted in the loss of an OMP with an estimated molecular mass of about 34 kDa; genetic complementation of the mutant strain with a DNA fragment containing only the ompU gene restored the production of this protein. The expression of the V. fischeri OmpU was not significantly affected by either (i) iron or phosphate limitation or (ii) a mutation that renders V. fischeri defective in the synthesis of a homolog of the OMP-regulatory protein ToxR. The ompU mutant grew normally in complex nutrient media but was more susceptible to growth inhibition in the presence of either anionic detergents or the antimicrobial peptide protamine sulfate. Interestingly, colonization experiments showed that the ompU null mutant initiated a symbiotic association with juvenile light organ tissue with only about 60% of the effectiveness of the parent strain. When colonization did occur, it proceeded more slowly and resulted in an approximately fourfold-smaller bacterial population. Surprisingly, there was no evidence that in a mixed infection with its parent, the ompU-defective strain had a competitive disadvantage, suggesting that the presence of the parent strain provided a shared compensatory activity. Thus, the OmpU protein appears to play a role in the normal process by which V. fischeri initiates its colonization of the nascent light organ of juvenile squids.     

Establishment of a Cre-rat resource for creating conditional and physiological relevant models of human diseases

Transgenic Research - The goal of this study is to establish a Cre/loxP rat resource for conditional and physiologically predictive rat models of human diseases. The laboratory rat (R. norvegicus)...     

Viral modulation of the host response via crmA/SPI-2 expression.

Viruses have evolved numerous strategies to modulate the host response to infection. Poxviruses cause acute infections and need to replicate quickly to promote efficient transmission. Consequently, it is not surprising to learn that poxviruses encode a large number of proteins designed to target various arms of the host inflammatory response. One of the earliest described and most well-studied viral modulatory proteins is crmA/SPI-2. While the biochemical targets and possible modes of action have been well characterized in vitro, the role that crmA/SPI-2 plays during natural infection is less clear. It may have effects in modulating host responses involving apoptosis and inflammation. It is important to further understand the precise mode of action of viral proteins, such as crmA/SPI-2, because this may lead to better therapeutic strategies to combat a range of inflammatory and autoimmune diseases.     

Intercellular connections between germ cells in the developing human ovary

Summary An electron microscopic examination of human fetal ovaries reveals the presence of intercellular bridges between developing germ cells. The bridges are characterized by a band of electron-dense material beneath the lateral limiting membrane, and cell organelles are seen within the confines of these connections. Their general morphology is similar to that described in ovaries of other species. The possible functional significance of these connections is discussed.This work was supported by grants from the Edward G. Schlieder Educational Foundation, New Orleans, Louisiana State University and HD-03288 from the National Institute of Child Health and Human Development.The authors would like to thank Miss Cathy Chase for her technical assistance.