Identification of a second endogenous Porphyromonas gingivalis insertion element.

In this study a second endogenous Porphyromonas gingivalis insertion element (IS element) that is capable of transposition within P. gingivalis was identified. Nucleotide sequence analysis of the Tn4351 insertion site in a P. gingivalis Tn4351-generated transconjugant showed that a complete copy of the previously unidentified IS element, designated PGIS2, had inserted into IS4351R in Tn4351. PGIS2 is 1,207 bp in length with 19-bp imperfect terminal inverted repeats, and insertion resulted in a duplicated 10-bp target sequence. Results of Southern hybridization of chromosomal DNA isolated from several P. gingivalis strains with a PGIS2-specific probe demonstrated that the number of copies of PGIS2 per genome varies among different P. gingivalis strains. Computer analysis of the putative polypeptide encoded by PGIS2 revealed strong homologies to the products encoded by IS1358 from Vibrio cholerae, ISAS1 from Aeromonas salmonicida, and H-rpt in Escherichia coli K-12.     

cAMP-stimulated termination of vitellogenesis in Hyalophora cecropia: formation of a diffusion barrier and the loss of patency

Activation of cAMP-dependent protein kinase (PKA) by cell-permeable analogs of cAMP causes early and mid-vitellogenic follicles of Hyalophora cecropia to terminate vitellogenin uptake [[Wang and Telfer, 1996], Insect Biochem. Mol. Biol. 26, 85-94 (1996)]. The response is shown here to entail the formation of an epithelial diffusion barrier. Follicle cells that have been loosely organized to provide intercellular pathways for the movement of vitellogenin to the oocyte surface transform into a tight epithelium within 1-2h of exposure to PKA activators. The follicle cells can now prevent the escape of Lucifer yellow CH that has been iontophoresed into the space surrounding the oocyte, and the entry of labeled vitellogenin from the medium. As they form this functional equivalent of a tight junction, the follicle cells further reduce the intercellular spaces by enlarging and pressing against each other, and by slowing the secretion of the sulfated glycosaminoglycan matrix that separates them during vitellogenesis. The activation of PKA in early and mid-vitellogenic follicles thus appears to trigger prematurely a set of changes that do not normally occur until the follicle has grown to a length of about 2.0mm.     

δ-Glutamate Receptors Are Differentially Distributed at Parallel and Climbing Fiber Synapses on Purkinje Cells

Abstract: Neurons containing multiple excitatory inputs may sort and target glutamate receptor subtypes to subsets of synapses. A good model for testing this hypothesis is the Purkinje cell, which expresses significant levels of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate, kainate, N -methyl- d -aspartate, δ-, and metabotropic glutamate receptors. Purkinje cells receive two excitatory inputs, the parallel and climbing fibers; the combined effect of stimulation of these two inputs is to produce long-term depression of parallel fiber/Purkinje cell neurotransmission. Distribution of glutamate receptors in these two synapse populations in rat cerebella was studied using preembedding immunocytochemistry with antibodies to GluR1, GluR2/3, GluR5-7, NR1, δ1/2, and mGluR1α. Moderate/dense postsynaptic staining was most frequent in postsynaptic densities and spines of both parallel and climbing fiber synapses with mGluR1α antibody, was intermediate in frequency with GluR2/3 and GluR5-7 antibodies, and was least frequent with GluR1 and NR1 antibodies. The most striking finding was the absence of significant postsynaptic staining with δ1/2 antibody in climbing fiber synapses in adult animals, even though postsynaptic staining was prevalent in parallel fiber synapses with this antibody. In contrast to adults, moderate/dense postsynaptic immunolabeling of climbing fiber synapses with δ1/2 antibody was common in rats at 10 days postnatal. This study provides direct morphological evidence that δ-glutamate receptors are differentially targeted to synapse populations. Our results support previous suggestions that δ2 is involved in development of parallel and climbing fiber synapses and in long-term depression of parallel fiber/Purkinje synaptic responses in adults.     

Lack of evidence for hepatitis G virus replication in the livers of patients coinfected with hepatitis C and G viruses.

The pathogenic implications of hepatitis G virus (HGV) infection are still unclear. We searched for the presence of HGV RNA and HCV RNA sequences in liver and serum samples from 10 patients with chronic liver disease, 9 of whom were coinfected with HCV. All livers were negative for the presence of the HGV RNA minus strand and only six were positive for the presence of the positive strand, albeit at low levels. In striking contrast, the HCV RNA positive strand was detectable in the liver samples from all nine HCV-positive patients in titers ranging from 10(2) to 10(8) genomic eq/microg of RNA, and the negative HCV RNA strand was present in all but two of these patients. However, the positive-strand RNA titers in serum for the two viruses had similar ranges. These findings imply that the liver is not the primary replication site for HGV, at least in the population of HCV/HGV-coinfected patients. Absence of replication in liver tissue may explain the reported lack of influence of HGV coinfection on the course of chronic hepatitis C.     

Adeno-associated virus type 2-mediated transfer of ecotropic retrovirus receptor cDNA allows ecotropic retroviral transduction of established and primary human cells.

The cellular receptors that mediate binding and internalization of retroviruses have recently been identified. The concentration and accessibility of these receptors are critical determinants in accomplishing successful gene transfer with retrovirus-based vectors. Murine retroviruses containing ecotropic glycoproteins do not infect human cells since human cells do not express the receptor that binds the ecotropic glycoproteins. To enable human cells to become permissive for ecotropic retrovirus-mediated gene transfer, we have developed a recombinant adeno-associated virus type 2 (AAV) vector containing ecotropic retroviral receptor (ecoR) cDNA under the control of the Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter (vRSVp-ecoR). Established human cell lines, such as HeLa and KB, known to be nonpermissive for murine ecotropic retroviruses, became permissive for infection by a retroviral vector containing a bacterial gene for resistance to neomycin (RV-Neo(r)), with a transduction efficiency of up to 47%, following transduction with vRSVp-ecoR, as determined by the development of colonies that were resistant to the drug G418, a neomycin analog. No G418-resistant colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. Southern and Northern blot analyses revealed stable integration and long-term expression, respectively, of the transduced murine ecoR gene in clonal isolates of HeLa and KB cells. Similarly, ecotropic retrovirus-mediated Neo(r) transduction of primary human CD34+ hematopoietic progenitor cells from normal bone marrow was also documented, but only following infection with vRSVp-ecoR. The retroviral transduction efficiency was approximately 7% without prestimulation and approximately 14% with prestimulation of CD34+ cells with cytokines, as determined by hematopoietic clonogenic assays. No G418-resistant progenitor cell colonies were present in cultures infected with either vRSVp-ecoR or RV-Neo(r) alone. These results suggest that sequential transduction of primary human cells with two different viral vectors may overcome limitations encountered with a single vector. Thus, the combined use of AAV- and retrovirus-based vectors may have important clinical implications for ex vivo and in vivo human gene therapy.     

Two distinct CCR5 domains can mediate coreceptor usage by human immunodeficiency virus type 1.

The chemokine receptor CCR5 is the major fusion coreceptor for macrophage-tropic strains of human immunodeficiency virus type 1 (HIV-1). To define the structures of CCR5 that can support envelope (Env)-mediated membrane fusion, we analyzed the activity of homologs, chimeras, and mutants of human CCR5 in a sensitive gene reporter cell-cell fusion assay. Simian, but not murine, homologs of CCR5 were fully active as HIV-1 fusion coreceptors. Chimeras between CCR5 and divergent chemokine receptors demonstrated the existence of two distinct regions of CCR5 that could be utilized for Env-mediated fusion, the amino-terminal domain and the extracellular loops. Dual-tropic Env proteins were particularly sensitive to alterations in the CCR5 amino-terminal domain, suggesting that this domain may play a pivotal role in the evolution of coreceptor usage in vivo. We identified individual residues in both functional regions, Asp-11, Lys-197, and Asp-276, that contribute to coreceptor function. Deletion of a highly conserved cytoplasmic motif rendered CCR5 incapable of signaling but did not abrogate its ability to function as a coreceptor, implying the independence of fusion and G-protein-mediated chemokine receptor signaling. Finally, we developed a novel monoclonal antibody to CCR5 to assist in future studies of CCR5 expression.     

Anthropometrics Do Not Influence Dual X-ray Absorptiometry (DXA) Measurement of Fat in Normal to Obese Adults: A Comparison with In Vivo Neutron Activation Analysis (IVNA)

Dual-energy X-ray absorptiometry (DXA) is now a commonly used method for the determination of bone mineral status and body composition in humans. The purposes of this study were to compare fat mass by in vivo neutron activation analysis (FM_IVNA) with that by DXA (FM_DXA) in an anthropometrically heterogeneous sample of healthy adult men (n=33) and women (n=36) (19=≤BMI≤39), and to determine whether differences in fat mass estimates between the two methods (ΔFM) were attributable to subject anthropometry as defined by several circumference (waist, iliac crest, thigh) and skinfold thickness (umbilical, suprailiac, abdominal) measurements. No significant differences between FM_DXA and FM_IVNA were observed in men (p=0.46) or women (p=0.09). The two methods were very highly correlated in both sexes (women r²=0.97, p<0.001, men r²=0.91, p<0.001), although the regression line for men was significantly different from the line of identity (p=0.043). These results suggest modest trends toward underestimation of FM_DXA in men when FM_IVNA<18 kg, and overestimation in men when FM_IVNA>18 kg. ΔFM (IVNA-DXA) was not significantly related to any combination of skinfold thicknesses and circumferences in either gender. Age explained 27% of the variance in ΔFM for the men (p=0.008). Furthermore, ΔFM was not significantly related to inter-method disparity in total-body bone mineral measurements in men or women (p<0.05). The present study demonstrates strong correlation in fat measurements between IVNA and DXA in men and women ranging from normal to markedly obese. Correction for subject anthropometry does not significantly improve this relationship.     

Experimental study and mechanism analysis on bioeffects by nanosecond electromagnetic pulses

The athermal bioeffects caused by nanosecond electromagnetic pulses with body cells was studied by using a broad band transverse EM-wave cell (BTEM CELL). The experimental system and preliminary mechanism analysis were presented.