Hematopoietic cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Human bone marrow cells that were responsive to the early-acting cytokines Kit ligand and interleukin-3 were forced to a metabolic death. The subfraction remaining represented 1 in 10(5) bone marrow mononuclear cells, were determined to be quiescent by cell cycle analysis, and had a stem cell immunophenotype. The cells were highly enriched for long-term culture-initiating cells, were capable of secondary colony formation, and produced both myeloid and lymphoid progeny. Thus, this technically simple strategy led to the efficient purification of cells with characteristics of hematopoietic stem cells.

Antigens, CD↗

PubMed1995

Expression of the integrin subunit alpha 9 in the murine embryo.

The alpha 9 integrin subunit is expressed in adult skeletal muscle, visceral smooth muscle, hepatocytes, squamous epithelium, and airway epithelium. The in vivo function of this protein is unknown. Thus far, only a single alpha 9-containing integrin has been identified (alpha 9 beta 1) and only a single ligand (tenascin) has been found for this integrin. In order to gain insight into the potential function of alpha 9 integrin(s), we examined the spatiotemporal distribution of the alpha 9 subunit and tenascin during murine embryogenesis. In all tissues where alpha 9 was expressed, its appearance was associated with other evidence of cell differentiation. In developing airway, visceral, and vascular smooth muscles, the onset of alpha 9 expression either coincided with or immediately followed the expression of alpha-SM actin. Expression of alpha 9 in epithelia was restricted to the choroid plexus and the basal cell layer of squamous epithelia where its appearance coincided with the development of stratification. alpha 9 immunostaining was first detected in developing skeletal musculature when skeletal myotubes formed. Tenascin expression was detected in many, but not all tissues found to express alpha 9. For example, the hair germs of maturing hair follicles exhibited high levels of alpha 9 staining, but no tenascin immunoreactivity was detected either within the hair germ themselves or in the adjacent dermis. In some tissues where tenascin expression colocalized with alpha 9, expression patterns were not synchronous. Although alpha 9 expression was associated with the onset of tissue differentiation, its expression was not limited to terminally differentiated cells. In fact, in the skin, alpha 9 expression appeared restricted to cells known to retain the capacity to proliferate, i.e., basal cells and hair germs. Thus, alpha 9 integrin(s) are not likely to contribute to the early steps in organ formation, but probably play a role in the maturation and/or maintenance of a variety of differentiated tissues. The expression of alpha 9 without its only known ligand, tenascin, suggests the existence of additional ligands.

Animals↗

PubMed1995

Role of cyclic plastic deformation in the wear of UHMWPE acetabular cups.

The mechanisms of wear in ultra-high-molecular-weight polyethylene (UHMWPE) acetabular cups were investigated on both laboratory simulator-tested cups and a clinically retrieved component. Two different levels of wear process were identified: one characterized by the formation and detachment of platelet-like flakes from initial machining marks, and the other by the formation of fine ripple and fibrils by repeated passes of microscopic asperities on the femoral head. Both wear processes could be described by a criterion of critical plastic strain. A theoretical model was developed to account for the generation of the microscopic wear particles based on such a critical strain criterion. Its predictions of the dependence of the UHMWPE wear rate on surface roughness and applied load were in excellent agreement with previously published experimental correlations.

Acetabulum↗

PubMed1995

No involvement of Ki-ras or p53 gene mutations in colitis-associated rat colon tumors induced by 1-hydroxyanthraquinone and methylazoxymethanol acetate.

1-Hydroxyanthraquinone (1-HA), which is present in some herbs, and methylazoxymethanol (MAM) acetate, a metabolite of azoxymethane, show synergistic carcinogenicity in rat colon, and 1-HA induces ulcerative changes with simultaneous severe inflammation of the entire colon. In this study, mutations in Ki-ras (exons 1 and 2) and p53 (exons 4-7) were studied by polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) analysis. Of 18 adenomas and 38 adenocarcinomas induced in male F344 rats (52 tumors induced by 1-HA plus MAM acetate, three by 1-HA alone, and one by MAM acetate alone), no mutations in Ki-ras or p53 were detected under two conditions of PCR-SSCP analysis. Because human colon carcinomas from patients with ulcerative colitis have a very low incidence of Ki-ras mutation, this experimental system would be a good animal model of human colon carcinomas with ulcerative colitis and of human colon carcinomas without Ki-ras or p53 mutations.

Animals↗

PubMed1995

Characterization of six linked open reading frames necessary for pIP501-mediated conjugation.

pIP501 is a broad-host-range gram-positive conjugative plasmid. Previous transposon mutagenesis of a pIP501 derivative, pVA1702, identified two regions, A and B, that were involved in conjugation proficiency. Recently, we reported that the A region contained a functional oriT site similar to those found on plasmids of gram-negative origin (Wang and Macrina, 1995). We report here the nucleotide sequence of the complete A region. Analysis of this DNA sequence (8121 bp) revealed six contiguous open reading frames (orf1-6) immediately downstream from the oriT site. ORF1 was similar to gram-negative plasmid nickase proteins known to play a role in the initiation of conjugal transfer. The remaining ORF proteins (2-6) were unique. Complementation studies suggested that the gene products of ORFs 3-6 were able to function in trans. A single available insertion mutant in orf2 could not be complemented in trans. Thus, although the conjugative apparatus that initiates transfer (oriT and nickase) appears to be conserved between this class of gram-positive plasmid and gram-negative plasmids, most of the A region genes of pIP501 involved in transfer appear unique and their cellular and molecular roles are undefined.

Amino Acid Sequence↗

PubMed1995

Infusion of bile from methylene dianiline-treated rats into the common bile duct injures biliary epithelial cells of recipient rats.

Methylene dianiline (4,4'-diaminodiphenylmethane, DAPM) rapidly causes cholestasis and injury to biliary epithelial cells (BEC) in the liver and common bile duct of rats. Our objective was to determine if the proximate toxicant(s) was present in bile. Bile from DAPM-treated or control rats was infused through the common bile duct of untreated rats via inflow and outflow cannulas for 4 h. Cholestasis, increases in serum constituents, and intrahepatic BEC injury in the livers of DAPM-treated donor rats at 4 h were comparable to previous studies (Kanz et al., Toxicol. Appl. Pharmacol. 117 (1992) 88-97). BEC injury in the common bile duct of rats receiving DAPM bile or control bile was assessed by point counting. Percent necrosis was > 28% in the common bile duct of rats receiving DAPM bile compared to < 5% in rats receiving control bile. These results indicate that bile is a major route of BEC exposure to DAPM proximate toxicant(s) and demonstrate the utility of a new method for investigating mechanisms of biliary toxicants.

Aniline Compounds↗

PubMed1995

Universalism, humanism, and postmodernism.

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Culture↗

PubMed1995

Streptococcal plasmid pIP501 has a functional oriT site.

DNA sequence analysis suggested the presence of a plasmid transfer origin-like site (oriT) in the gram-positive conjugative plasmid pIP501. To test the hypothesis that the putative oriT site in pIP501 played a role in conjugal transfer, we conducted plasmid mobilization studies in Enterococcus faecalis. Two fragments, 49 and 309 bp, which encompassed the oriT region of pIP501, were cloned into pDL277, a nonconjugative plasmid of gram-positive origin. These recombinant plasmids were mobilized by pVA1702, a derivative of pIP501, at a frequency of 10(-4) to 10(-5) transconjugants per donor cell, while pDL277 was mobilized at a frequency of 10(-8) transconjugants per donor cell. These results indicated that the oriT-like site was needed for conjugal mobilization. To demonstrate precise nicking at the oriT site, alkaline gel and DNA-sequencing analyses were performed. Alkaline gel electrophoresis results indicated a single-stranded DNA break in the predicted oriT site. The oriT site was found upstream of six open reading frames (orf1 to orf6), each of which plays a role in conjugal transfer. Taken together, our conjugal mobilization data and the in vivo oriT nicking seen in Escherichia coli argue compellingly for the role of specific, single-stranded cleavage in plasmid mobilization. Thus, plasmid mobilization promoted by pVA1702 (pIP501) works in a fashion similar to that known to occur widely in gram-negative bacteria.

Base Sequence↗

PubMed1995

Delayed activation of altered fusion glycoprotein in a chronic measles virus variant that causes subacute sclerosing panencephalitis.

We compared the intracellular processing of the fusion (F) glycoproteins of an acute measles virus (MV) Nagahata strain and its relative Biken strain that caused subacute sclerosing panencephalitis (SSPE), Nagahata strain synthesizes a precursor F0 which acquires three asparagine (N)-linked oligosaccharide chains sequentially in 1 h. One oligosaccharide chain on the partially glycosylated F0 is less accessible to endo-beta-N-acetylglucosaminidase H (endo-H) but becomes accessible as the protein becomes fully glycosylated, suggesting a protein conformational change. Biken strain SSPE virus synthesizes a similarly glycosylated F0. However, one oligosaccharide chain on the Biken F0 remains less accessible to endo-H even after the protein is fully glycosylated. The Nagahata F0 is cleaved into the F1 and F2 subunits with a half life of 1 h. The Biken F0 is cleaved with a half life of 4 h. We cloned the F genes of Nagahata and Biken strains and showed by transfection that the defect causing delayed cleavage of F0 resides in the Biken F gene. Sequence analysis predicts a mutation in the cleavage recognition sequence, a truncated carboxyl-terminus, and multiple mutations in F1 of the Biken F protein. Expression of chimeric F genes showed the mutated cleavage recognition sequence and the carboxyl-terminal truncation do not delay cleavage of F0. Instead, delayed F0 cleavage is due to multiple mutations in the extracellular domain of F1, and four amino acid substitutions near the transmembrane region impair endo-H access to the oligosaccharide chain. These results provide detailed information on the normal maturation process of the F protein of MV and additional clues to the mechanisms of MV persistence in the CNS.

Animals↗

PubMed1995

Delayed activation of altered fusion glycoprotein in a chronic measles virus variant that causes subacute sclerosing panencephalitis.

We compared the intracellular processing of the fusion (F) glycoproteins of an acute measles virus (MV) Nagahata strain and its relative Biken strain that caused subacute sclerosing panencephalitis (SSPE). Nagahata strain synthesizes a precursor F0 which acquires three asparagine (N)-linked oligosaccharide chains sequentially in 1 h. One oligosaccharide chain on the partially glycosylated F0 is less accessible to endo-beta-N-acetylglucosaminidase H (endo-H) but becomes accessible as the protein becomes fully glycosylated, suggesting a protein conformational change. Biken strain SSPE virus synthesizes a similarly glycosylated F0. However, one oligosaccharide chain on the Biken F0 remains less accessible to endo-H even after the protein is fully glycosylated. The Nagahata F0 is cleaved into the F1 and F2 subunits with a half life of 1 h. The Biken F0 is cleaved with a half life of 4 h. We cloned the F genes of Nagahata and Biken strains and showed by transfection that the defect causing delayed cleavage of F0 resides in the Biken F gene. Sequence analysis predicts a mutation in the cleavage recognition sequence, a truncated carboxyl-terminus, and multiple mutations in F1 of the Biken F protein. Expression of chimeric F genes showed the mutated cleavage recognition sequence and the carboxyl-terminal truncation do not delay cleavage of F0. Instead, delayed F0 cleavage is due to multiple mutations in the extracellular domain of F1, and four amino acid substitutions near the transmembrane region impair endo-H access to the oligosaccharide chain. These results provide detailed information on the normal maturation process of the F protein of MV and additional clues to the mechanisms of MV persistence in the CNS.

Animals↗

PubMed1995

Interleukin-10 enhances human immunodeficiency virus type 1 expression in a chronically infected promonocytic cell line (U1) by a tumor necrosis factor alpha-independent mechanism.

TNF-alpha enhances HIV-1 replication in acutely and chronically infected cells and likely contributes to the wasting associated with the acquired immunodeficiency syndrome. Agents that inhibit TNF-alpha activity should theoretically delay the progression of disease, and several are currently in clinical trials. We hypothesized that IL-10, a cytokine that suppresses the gene expression and synthesis of TNF-alpha in monocytic cells, might inhibit HIV-1 replication. As expected, IL-10 suppressed PMA-induced TNF-alpha production in U1 cells; however, when U1 cells were cultured in the presence of PMA and increasing doses of IL-10, a dose-dependent increase in HIV-1 expression was observed. IL-10 also enhanced IL-1 beta-, TNF-alpha-, and GM-CSF-induced HIV-1 expression in U1 cells, and this occurred, at least in part, at the level of transcription. We next stimulated cells under conditions of TNF-alpha blockade. When PMA-induced TNF-alpha activity and HIV-1 replication were blocked by the presence of soluble TNF receptors, IL-10 independently enhanced HIV-1 replication. In contrast, other agents that are capable of blocking TNF-alpha synthesis or TNF-alpha activity either had no effect (IL-13 and IL-4) or inhibited HIV-1 expression (soluble TNF receptors and pentoxifylline) in U1 cells. These data suggest that IL-10, while inhibiting TNF-alpha synthesis, has an independent mechanism of action that enhances HIV-1 replication. Therefore, IL-10 may have undesirable effects in HIV-1-infected patients.

Acquired Immunodeficiency Syndrome↗

PubMed1995

Chemoprevention of the naturally occurring carcinogen 1-hydroxyanthraquinone-induced carcinogenesis by the nonsteroidal anti-inflammatory drug indomethacin in rats.

The chemopreventive effect of the nonsteroidal anti-inflammatory drug indomethacin (IMC) on 1-hydroxyanthraquinone (1-HA)-induced carcinogenesis was investigated in a total of 69 male ACI/N rats. Animals in Group 1 were fed the diet containing 1.5% 1-HA for 48 weeks. The rats in Group 2 were given the 1-HA diet together with 16 ppm IMC in the drinking water for 48 weeks. Group 3 was given IMC alone throughout the study. Group 4 was served as an untreated control. At the end of the study, the incidences of large bowel and forestomach tumors in Group 2 were significantly smaller than those in Group 1 (large bowel tumors: 0/14 rats, 0% in Group 2 versus 12/27 rats, 44% in Group 1, p < 0.002; forestomach tumors: 2/14 rats, 14% in Group 2 versus 14/27 rats, 52% in Group 1, p < 0.01). Also, the incidences of inflammatory changes including ulcerative colitis and melanosis coli in colonic mucosa of rats of given 1-HA together with IMC were significantly reduced compared with those in rats given 1-HA alone (ulcerative colitis: 2/14 rats, 14% in Group 2 versus 20/27 rats, 74% in Group 1, p < 0.004). Concurrent administration of IMC with 1-HA caused a significant decrease in the bromodeoxyuridine labeling index of the colonic mucosa. These results indicate that the nonsteroidal anti-inflammatory drug IMC inhibits 1-HA-induced carcinogenesis and that this effect in decreasing the cell proliferation.

Animals↗

PubMed1995

[Current progress in the study on neurotrophic factors of motoneurons].

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Animals↗

PubMed1995

[Preparation method for compound Hirudo oral liquid].

The extraction efficiency of alcohol, water and alcohol followed by water for Hirudo and the principal constituents of compound Hirudo oral liquid were compared with the extraction rate of nitric substances and pharmacological activity as the evaluation marker. Based on results of the above experiments, a new preparation method for the oral liquid was established.

Animals↗