Role of humoral immunity in pancreatic islet allo- and xenograft rejection.
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Animals↗
Biomedical subjects
E Chang
Publications and source records attributed to E Chang.
Telomere length and replicative aging in human vascular tissues.
Because repeated injury of the endothelium and subsequent turnover of intimal and medial cells have been implicated in atherosclerosis, we examined telomere length, a marker of somatic cell turnover, in cells from these tissues. Telomere lengths were assessed by Southern analysis of terminal restriction fragments (TRFs) generated by HinfI/Rsa I digestion of human genomic DNA. Mean TRF length decreased as a function of population doublings in human endothelial cell cultures from umbilical veins, iliac arteries, and iliac veins. When endothelial cells were examined for mean TRF length as a function of donor age, there was a significantly greater rate of decrease for cells from iliac arteries than from iliac veins (102 bp/yr vs. 47 bp/yr, respectively, P < 0.05), consistent with higher hemodynamic stress and increased cell turnover in arteries. Moreover, the rate of telomere loss as a function of donor age was greater in the intimal DNA of iliac arteries compared to that of the internal thoracic arteries (147 bp/yr vs. 87 bp/yr, respectively, P < 0.05), a region of the arterial tree subject to less hemodynamic stress. This indicates that the effect is not tissue specific. DNA from the medial tissue of the iliac and internal thoracic arteries showed no significant difference in the rates of decrease, suggesting that chronic stress leading to cellular senescence is more pronounced in the intima than in the media. These observations extend the use of telomere size as a marker for the replicative history of cells and are consistent with a role for focal replicative senescence in cardiovascular diseases.
Adolescent↗
The RNA component of human telomerase.
Eukaryotic chromosomes are capped with repetitive telomere sequences that protect the ends from damage and rearrangements. Telomere repeats are synthesized by telomerase, a ribonucleic acid (RNA)-protein complex. Here, the cloning of the RNA component of human telomerase, termed hTR, is described. The template region of hTR encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (TTAGGG)n. Germline tissues and tumor cell lines expressed more hTR than normal somatic cells and tissues, which have no detectable telomerase activity. Human cell lines that expressed hTR mutated in the template region generated the predicted mutant telomerase activity. HeLa cells transfected with an antisense hTR lost telomeric DNA and began to die after 23 to 26 doublings. Thus, human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.
Animals↗
Shk1, a homolog of the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases, is a component of a Ras/Cdc42 signaling module in the fission yeast Schizosaccharomyces pombe.
We describe a protein kinase, Shk1, from the fission yeast Schizosaccharomyces pombe, which is structurally related to the Saccharomyces cerevisiae Ste20 and mammalian p65PAK protein kinases. We provide genetic evidence for physical and functional interaction between Shk1 and the Cdc42 GTP-binding protein required for normal cell morphology and mating in S. pombe. We further show that expression of the STE20 gene complements the shk1 null mutation and that Shk1 is capable of signaling to the pheromone-responsive mitogen-activated protein kinase cascade in S. cerevisiae. Our results lead us to propose that signaling modules composed of small GTP-binding proteins and protein kinases related to Shk1, Ste20, and p65PAK, are highly conserved in evolution and participate in both cytoskeletal functions and mitogen-activated protein kinase signaling pathways.
Amino Acid Sequence↗
Requirements for transforming growth factor-beta regulation of the pro-alpha 2(I) collagen and plasminogen activator inhibitor-1 promoters.
Experiments were designed to clarify the role of several proteins, junB, retinoblastoma protein (RB), and the transforming growth factor-beta (TGF-beta) receptors that are potential intermediates in TGF-beta activation of the alpha 2(I) collagen promoter. Treatment of NIH-3T3 cells with TGF-beta increased the activity of a transiently transfected murine alpha 2(I) collagen promoter (nucleotides -350 to +54) fused to a luciferase reporter gene 9-fold. Cotransfection of a junB stimulated the basal activity of the alpha 2(I) collagen promoter 93-fold, respectively. Expression of antisense junB RNA attenuated the effect of TGF-beta. Simian virus 40 large T antigen, an inhibitor RB function, did not prevent TGF-beta effects on the alpha 2(I) collagen promoter. A chimeric receptor containing the extracellular domain of the colony-stimulating factor-1 receptor and the intracellular domain of the type I TGF-beta receptor enhanced alpha 2(I) collagen promoter activity 4.8-fold, whereas a similar chimera containing the type II receptor intracellular domain had much weaker effects. Similar results were obtained with a plasminogen activator inhibitor-1 promoter, previously shown to be activated by TGF-beta through AP-1 elements. We conclude that TGF-beta activates the alpha 2(I) collagen and plasminogen activator inhibitor-1 promoters in NIH-3T3 cells through junB and the type I TGF-beta receptor kinase domain.
3T3 Cells↗
Changes in phases and crystallinity of plasma-sprayed hydroxyapatite coatings under heat treatment: a quantitative study.
With three kinds of plasma-sprayed hydroxyapatite coatings (HACs) prepared, the objectives of this study were (1) to establish the calibration methods for quantitatively measuring the concentration of impurity phases and the degree of crystallinity of the HACs, and (2) to explore the effects of postheat treatments at various temperatures in vacuo on the changes of phases and crystallinity of the HACs. By the internal standard method used, the concentrations of impurity phases, such as alpha-TCP, beta-TCP, and TP, of the assprayed HACs were significantly higher than those measured by the direct intensity-ratio method, and the CaO phase was lower than the direct intensity-ratio method. When the HACs were heat treated in the temperature interval 630-850 degrees C, the concentrations of impurity phases obviously decreased, and the coating crystallinity apparently increased. After annealing at 850 degrees C, an HAC consisting of at least 95% crystallinity with few impurity phases was obtained. As the annealing temperatures in the interval 850-1000 degrees C were applied, however, the HA phase seriously decomposed, resulting in the appearance of a large number (higher than 20 wt%) of impurity phases in the HACs. This work suggests that the optimum heat treatment conditions in vacuo for maximizing crystallinity and minimizing impurity phases of the HACs do not occur at the same temperature.
Alloys↗
Telomere shortening is associated with cell division in vitro and in vivo.
In humans, the amount of terminal (TTAGGG)n, telomeric DNA decreases during aging of various somatic cell types in vitro and in vivo. While the factors accounting for telomere shortening have not been thoroughly established, the inability of the DNA replication machinery to completely copy chromosomal termini (the "end replication problem") and the absence in somatic cells of telomerase, the enzyme that synthesizes telomeric DNA de novo, is a likely mechanism. One prediction of this hypothesis is that telomere shortening should be dependent on cell division. Thus we analyzed telomere length in actively dividing and quiescent cells in vitro and in vivo. In circular outgrowths of cultured human diploid fibroblasts (HDF), cells at the outer periphery had a significantly lower mean terminal restriction fragment (TRF) length (P = 0.011) and telomeric signal intensity (P = 0.024) than cells at the center. Also, the rate of telomere shortening over time for HDFs held quiescent was not statistically significant (m = -12 bp/day, P = 0.16) while that for serially passaged cells was significant (m = -34 bp/day, P = 0.017). To examine the rate of telomere shortening for quiescent cells in vivo, we measured mean TRF length in brain tissue from adult donors ranging in age from 32-75 years. No significant decrease was observed as a function of donor age (P = 0.087), in contrast to the shortening of telomere length that occurs during in vivo aging of mitotically active cells (P = 0.0001). These observations show that telomere shortening is largely, if not entirely, dependent on cell division and support the end replication problem as a mechanism for this process and the use of telomere length as a biomarker for replicative capacity.
Adult↗
Myelin basic protein does not have a mitogenic effect on adult oligodendrocytes.
Increased numbers of oligodendrocytes and remyelination are frequently observed in multiple sclerosis plaques. It is presumed the increased numbers of oligodendrocytes are due to cell division, but this has not been proven. The mitogens within the lesion which might be responsible for this are unknown. Since oligodendrocyte proliferation occurs in areas in which there is myelin breakdown, we undertook the present study to determine if myelin basic protein (MBP) or its breakdown products could induce oligodendrocyte proliferation. MBP, or MBP digested by the neutral proteinase plasmin, was added in three concentrations to the media of adult bovine oligodendrocytes in culture. Oligodendrocytes were identified by staining for galactocerebroside. Bromodeoxyuridine incorporation was used as a measure of cell division. Oligodendrocytes were found to divide only rarely in regular culture media, in the presence of MBP, plasmin, or MBP digested by plasmin. The results indicate that MBP is not a significant mitogen for the mature oligodendrocyte.
Animals↗
Prevention of recurrent herpes simplex virus (HSV) infections in HIV-infected persons.
The purpose of this study was to determine the frequency of HSV infections and recurrences among HIV-infected patients and to examine different regimens for suppression of HSV recurrence. A randomized retrospective chart review of HIV-infected patients at a public hospital in Los Angeles County was conducted. We reviewed 224 patients' charts; 26 percent had AIDS based on the 1987 CDC definition. HSV infection was documented as a clinical event in 51 records (23 percent). Patients with an AIDS diagnosis had a greater incidence (53 percent) of HSV infections than did those with a diagnosis of symptomatic or asymptomatic HIV infection (p < 0.001, Fisher's exact test). Recurrences of HSV occurred in 26 (51 percent) of the 51 HSV-infected persons during a period of 1042 patient months. Eighteen patients who had received acyclovir suppression at 600 mg/day had three HSV recurrences in 382 patient months, whereas 14 who received 400 mg/day had eight recurrences in 282 patient months (p = 0.02). HSV infections occur in 23 percent of HIV-infected patients, increasing to 53 percent in AIDS patients. Acyclovir suppression prevents recurrent HSV, and a dosage of 600 mg/day is more effective than 400 mg/day.
Acquired Immunodeficiency Syndrome↗
The AIDS epidemic: 'it ain't over yet'.
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Acquired Immunodeficiency Syndrome↗
Cellular requirements for pancreatic islet xenograft rejection.
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Animals↗
Metabolism of 2',2'-difluoro-2'-deoxycytidine and radiation sensitization of human colon carcinoma cells.
Difluorodeoxycytidine (dFdCyd) is a new antimetabolite with clinical activity in patients with solid tumors but not leukemias. We have studied the metabolism, cytotoxicity, and radiosensitizing properties of dFdCyd in HT-29 human colon carcinoma cells. The results demonstrated that dFdCyd rapidly accumulated as the 5'-triphosphate dFdCTP in HT-29 cells, which was eliminated slowly in the absence of dFdCyd with a half-life of > 12 h. Accumulation of dFdCTP was associated with rapid depletion of cellular dATP pools. Exposure to the concentration that reduces cell survival by 50% of 30 nM dFdCyd decreased dATP levels by > 80% within 4 h. dGTP pools were depleted at higher concentrations of dFdCyd, whereas smaller decreases were effected in dTTP and dCTP pools. These results contrast with previous reports in leukemic cells which demonstrated that dFdCyd exposure depleted the endogenous dCTP pool to a greater extent than the dTTP, dATP, or dGTP pools. Based on these data, we suggest that the profound depletion by dFdCyd of dATP and dGTP pools in HT-29 compared to leukemic cells accounts for the superiority of this agent in solid tumors versus leukemias. Additional studies demonstrated that dFdCyd was a potent radiosensitizer in HT-29 cells. Maximal radiosensitization was observed when cells were irradiated immediately following dFdCyd exposure instead of prior to or in the middle of drug treatment. Radiation sensitization was dose and time dependent, with a noncytotoxic exposure to 10 nM dFdCyd for 24 h or 30 nM dFdCyd for 16 h producing a radiation enhancement ratio of approximately 2. Under these conditions, only the cellular dATP pool was depleted. When cells were exposed to higher concentrations of dFdCyd for 4 h, equivalent radiosensitization with a radiation enhancement ratio of 1.4 was obtained using 0.1, 1.0, or 10 microM dFdCyd. Despite a 15-fold increase in dFdCTP and depletion of dGTP and dCTP pools to < 25% of the control value with 10 microM compared to 0.1 microM dFdCyd, no increase in radiosensitization was observed. These results suggest that dATP depletion is an important factor in the radiosensitizing property of this promising new antitumor compound.
Antimetabolites, Antineoplastic↗
Nerve growth factor derived from terminals selectively increases the ratio of p75 to trkA NGF receptors on mature sympathetic neurons.
Exposure of neuronal terminals to target-derived NGF has been hypothesized to regulate cell body responses at a distance. To test this hypothesis and, more specifically, to determine whether NGF distally regulates the synthesis of its two characterized receptors, we developed the following paradigm. Sympathetic neurons of the superior cervical ganglion (SCG) that project either to the eye or to the ear were labeled, in adult animals, with the retrograde tracers Fast Blue and Fluoro-Gold, respectively. NGF was then injected daily into the anterior chamber of one eye, exposing the terminals of the ipsilateral eye neurons to increased NGF. To control for systemic and/or localized injury effects, cytochrome C or PBS were injected into the contralateral eye of the same animals. In situ hybridization and image analysis were then used to determine neuronal levels of p75 NGF receptor, trkA, and T alpha 1 alpha-tubulin mRNAs, with the latter providing a correlative measure of neuronal sprouting. Morphological measurements revealed that exogenous, terminally-derived NGF increased the mean cross-sectional area of eye neurons by 37%. Grain counts for p75 NGF receptor mRNA increased from 2- to 6-fold in the NGF-treated neurons, and grain densities, which accounted for neuronal hypertrophy, also increased significantly. In contrast, grain counts for trkA mRNA were not significantly increased by this treatment, while T alpha 1 alpha-tubulin mRNA levels increased only 1.5- to 2-fold. No increase in grain density was detected for either of these mRNAs. The NGF-induced increased in p75 NGF receptor mRNA levels was accompanied by terminal sprouting and by an increase in the density of p75 NGF receptors on terminal neurites, as indicated by IgG-192 immunostaining of the NGF-treated iris. These data therefore suggest that, in addition to promoting local sprouting, increased target-derived NGF increases the levels of p75 NGF receptor relative to trkA on terminal neurites, by differentially regulating receptor synthesis. Such a direct regulatory feedback loop may well play an integral role in precisely modulating neuronal responses as a function of the amount of available trophic support and/or target tissue.
Animals↗
Intraneural ulnar nerve pressure changes related to operative techniques for cubital tunnel decompression.
To evaluate the effect of critical anatomic structures on the ulnar nerve after cubital tunnel decompression, we determined the intraneural ulnar nerve pressure in 50 fresh cadavers after the following surgical procedures: simple decompression, medial epicondylectomy, subcutaneous transposition, and submuscular transposition by the Learmonth and by the musculofascial lengthening technique. Intraneural pressure was measured in 0 degrees, 30 degrees, 60 degrees, and 90 degrees elbow flexion at locations that were proximal, within, and distal to the cubital tunnel. Statistical analysis compared the mean change in intraneural pressure between the "postoperative" and the baseline "preoperative" pressure measurements for the different surgical strategies. While both the simple decompression and the medial epicondylectomy had significantly lower intraneural pressures than the Learmonth or the subcutaneous transposition, each of these four techniques resulted in elevated intraneural pressures. The musculofascial lengthening technique for submuscular transposition was the only surgical strategy that reduced intraneural ulnar pressure at each site of measurement and for all degrees of elbow flexion, this reduction of pressure being significant in comparison with the other surgical techniques.
Cadaver↗
The influence of spinal cord injury on coping styles and self-perceptions two years after the injury.
This study is a two year follow-up of previous longitudinal research which investigated the effects of spinal cord injury (SCI) on perceptions of control, self-esteem and coping styles over the first year of SCI. Persons with SCI and a demographically matched able-bodied control group completed standardised questionnaires on four occasions over two years. The instruments included the Locus of Control of Behaviour Scale (LCB), Rosenberg's Self-Esteem Scale, and an adapted Mental Adjustment to Cancer Scale (MAC), which measures coping styles, including fighting spirit, helplessness/hopelessness and fatalism. Results obtained in the first year were replicated in the two year data, except for the LCB Scale. After one year, the SCI group were found to perceive their life to be externally controlled, to be lower in self-esteem, and have more helpless/hopeless and fatalistic attitudes than the controls. There were no differences in self esteem and coping styles after two years for the SCI group. However, locus of control fluctuated over the two years, though there was a trend for the SCI group to be more externally focussed. There were no significant interactions between group and time. Implications for the adjustment of SCI persons are discussed.
Adaptation, Psychological↗
Anti-idiotypic antibodies to delineate epitope specificity of anti-amiloride antibodies.
Amiloride and related compounds have found widespread use as cation transport inhibitors. We have previously raised a series of polyclonal anti-amiloride antibodies using different amiloride-protein conjugates as immunogens, where amiloride was coupled to protein either through its guanidino moiety or through its 5-aminopyrazinyl moiety. The anti-amiloride antibodies recognized distinct sites on amiloride, and the site of attachment of amiloride to carrier protein was a critical factor in determining which part of the amiloride molecule was recognized by the anti-amiloride antibody. The specificity of binding of amiloride analogues to these polyclonal anti-amiloride antibodies mimicked the specificity of binding of amiloride analogues to selected isoforms of the epithelial Na+ channel or the Na+/H+ exchanger, suggesting that antigen binding site of these antibodies might be similar in structure to amiloride binding sites on selected Na+ transport proteins. We previously generated monoclonal anti-idiotypic antibodies RA2.4 and RA6.3 by an auto-anti-idiotypic approach, using amiloride coupled to albumin through the guanidinium moiety (amiloride-A1). We have now raised a series of monoclonal anti-idiotypic antibodies, T6, T26, T40, and T181, using amiloride coupled to keyhole limpet hemocyanin through the 5-aminopyrazinyl moiety (amiloride-A5) as an immunogen with the same auto-anti-idiotypic approach. These monoclonal anti-idiotypic antibodies recognized both polyclonal anti-amiloride-A1 and anti-amiloride-A5 antibodies, suggesting that idiotype-anti-idiotype interaction was not epitope restricted.(ABSTRACT TRUNCATED AT 250 WORDS)
Amiloride↗
Regional localization of 56 new human chromosome 18-specific yeast artificial chromosomes.
Fifty-six chromosome 18-specific yeast artificial chromosomes (YAC) were isolated from a human monochromosomal somatic cell hybrid DNA library. Fluorescence in situ hybridization revealed that the clones were evenly distributed throughout the chromosome. The clones, with an average insert size of 250 kb, cover about 18% of the euchromatic part of chromosome 18. Of 90 STS markers tested, 17 were represented in this YAC collection. Together with our previously reported set of unique chromosome 18-specific YACs, we now have available 111 regionally mapped, essentially nonchimeric, clones that provide more than 30% coverage and form a framework for the complete physical map.
Animals↗
A radiation hybrid map of human chromosome 18.
A human x Chinese hamster (CH) somatic cell hybrid subclone deficient in HPRT and containing only human chromosome 18 was irradiated with 7000 rad and fused to a thymidine kinase deficient CH cell line. Radiation-rescued hybrid cell lines, selected in HAT medium, were analyzed for human DNA with human interspersed-repeat sequence primers. Size and number of human chromosome fragments retained in a subset of hybrids were determined by FISH. A panel of 98 radiation hybrids (RH) was selected and analyzed for 90 chromosome 18-specific STSs by PCR, and for the D18Z1 centromeric marker by Southern blotting. STSs were developed from previously mapped RFLP loci and from published sequences. In addition, 32 novel STSs were generated from an 18-specific lambda library and from 18-specific YACs previously localized to chromosome bands by FISH. Marker retention frequency varied from 8-65% with an average of 24%. In selected RH the STS typing data were correlated with the chromosome 18 regions retained using 'reverse FISH' of IRS-PCR products from the RH to normal metaphase chromosomes. The order and intermarker distances of loci were determined using two-point and multipoint maximum likelihood methods. The resulting RH map covers most of chromosome 18 with four groups of tightly linked markers and three regions of loosely linked markers, one around the centromere and two on the long arm. More than a third of the markers are polymorphic and allow integration with the linkage map. This RH map provides a framework for establishing a clone contig of the entire chromosome 18.
Animals↗