Defective T-cell differentiation in acquired immune deficiency syndrome (AIDS).

A decline in T-cell lymphocyte number is the central characteristic of acquired immune deficiency syndrome (AIDS). The reason for the loss of these cells is not well understood. We investigated the hypothesis that defects in T-cell differentiation contributed to T-cell loss using an in vitro colony assay that measures T-cell precursor (CFU-T) frequency. The results indicate a substantial generalized decrease in CFU-T in people with AIDS (P less than 0.01), most of whom have Kaposi's sarcoma, and an occasionally severe decrease in CFU-T in people with ARC. Some of the cells from low colony formers suppressed colony formation by control cells. In addition, plasma from people with AIDS was less supportive of colony growth than control plasma. Decreased Ia expression on adherent mononuclear cells did not correlate with colony formation. A defect in T-cell repopulation can help explain the loss of T cells associated with AIDS.

Processing of procollagen types III and I in cultured bovine smooth muscle cells.

The processing of type III and type I procollagen molecules in cultured bovine aortic smooth muscle cells was investigated. The molecular identities of the processing intermediates of type III and type I procollagen were characterized by analysis of the radioactive collagenous components using mammalian collagenase and pepsin digestions and cyanogen bromide peptide mapping. The results indicate that the processed intermediates for procollagen type III and type I are their respective pC components. Although the processing pathways for both collagen types are the same, data from pulse-chase experiments suggest that the rates at which the processing occurs are different. Type I procollagen is processed more rapidly to its intermediate than is type III procollagen. The type I pC intermediate is almost completely processed to alpha-chains and a significant portion of these fully processed molecules remains in a soluble form even after 11 h. In the same time period, the type III pC intermediate is slowly converted to alpha-chains. Since beta-aminopropionitrile was not employed in these studies, significant accumulation of collagen chains into the insoluble extracellular matrix was observed during the chase period.

Cell-free translation of calf type III collagen. Effect of magnesium on ribosome movement during elongation.

Calf aortic smooth muscle cell cultures produce both type III and type I collagen. Polyadenylated mRNA species purified from these cells direct the synthesis of prepro-alpha 1(III), prepro-alpha 1(I), and prepro-alpha 2(I) in a rabbit reticulocyte cell-free system. These polypeptides were identified by specific immunoprecipitation, cyanogen bromide peptide mapping, and bacterial collagenase digestion. Lower molecular weight collagenase susceptible polypeptides were also produced in translation reactions incubated under conditions optimized for incorporation of radiolabeled amino acids. Their presence did not appear to result from ribonuclease or protease involvement or from premature termination. Increasing the Mg2+ concentration in the translation system significantly reduced the production of these lower molecular weight species. Pulse-chase experiments indicate that the time required for completion of full length preprocollagen at the high Mg2+ concentration is greatly decreased compared to the low concentration. Additional experiments suggest that the incomplete collagen polypeptides result from pausing of ribosome movement during elongation. The relative synthesis of type III and type I chains was examined as a function of mRNA concentration in the cell-free system. At levels of RNA above saturation, the relative production of type III decreased with respect to type I. These data suggest that the ability of the alpha 1(III) mRNA to initiate translation is less efficient than the mRNAs of alpha 1(I) and alpha 2(I).

Cell density and estradiol modulation of procollagen type III in cultured calf smooth muscle cells.

We have reported previously that 17 beta-estradiol reduces the amount of procollagen type III produced in cultured bovine aortic smooth muscle cells. The effect of cell density and estradiol on the production of collagen in second passage cultures has been further evaluated. In agreement with our previous findings, at day 14, significant differences in [14C]hydroxyproline production were detected between hormone-treated and control cultures. To further relate this observation to cell culture density and estradiol treatment, collagen production was evaluated after a 1-h pulse period. Quantities of assembled procollagen molecules and the individual collagenous polypeptides were determined by DEAE-cellulose chromatography or by immunoprecipitation. As cell numbers increased with time in culture, an augmented production of both procollagen types I and III was observed. The increased production of total type III collagen and procollagen molecules lagged behind type I production. Thus, it was found that the ratio of procollagen type I to type III decreased with days in culture from 2.7 to 1.0, and the ratio of the total of each type decreased from 7.5 to 3.4. In the presence of 1.0 x 10(-8) M estradiol, the level of type III collagen was reduced by approximately 50% at all culture times. The production of procollagen type I was not affected and remained equivalent in both hormone-treated and control cultures. The different ratios obtained by the two methods indicates that procollagen type I is processed more rapidly than procollagen type III in the 1-hour pulse period.

Effects of 17 beta-estradiol on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells.

The effect of 17 beta-estradiol (E2) on the biosynthesis of collagen in cultured bovine aortic smooth muscle cells was explored. Cells treated with various concentrations of the hormone for 14 days following subcultivation were subjected to growth studies. The cultures were also evaluated for [14C]hydroxyproline formation, the presence of collagenase-susceptible protein, prolyl hydroxylase activity, and procollagen types. There were no effect of E2 on the growth of these cells. At 10(-8) M E2, the hydroxylation of proline when compared to control cultures was reduced by 25-30%; however, little difference in extractable prolyl hydroxylase activity or total [14C]proline incorporation into protein was observed. The effect on collagen synthesis appears to be dose dependent over concentrations of E2 ranging from 10(-6) to 10(-12) M when measured by collagenase susceptibility. Procollagen typing on diethylaminoethylcellulose displayed reduced amounts of procollagen type I and type III fractions as well as other collagenous components. More importantly, however, the ratio of these two procollagen types were also altered. Similar results were obtained from the medium or cell layer. It is concluded that aortic smooth muscle cells cultured in the presence of 17 beta-estradiol display a decreased production of collagen in addition to altering the ratio of type I to type III procollagen fractions produced.

Use of collagen-hydroxyethylmethacrylate hydrogels for cell growth.

Collagen-hydroxyethylmethacrylate hydrogels were prepared by polymerizing monomeric hydroxyethylmethacrylate in the presence of various concentrations of soluble native collagen. The resulting transparent hydrogels were evaluated as substrata for growth of IMR-90 human embryonic lung fibroblasts. Without collagen no significant growth occurred, whereas a dose-response curve expressing maximal cell growth against collagen concentration could be constructed quite readily by the use of appropriate hydrogels. The method allows for quantification of the collagen contribution to cell growth and, in a more general sense, provides the foundation for a relatively easy procedure to probe mechanisms of cell adhesion and cell differentiation.

The interaction of collagen with alpha1-acid glycoprotein.

The influence of alpha1-acid glycoprotein on the formation of fibrous long spacing fibers of collagen has been investigated. It was observed that addition of the glycoprotein to dialyzed collagen solutions caused a significant decrease in the intensity of the circular dichroic spectrum of collagen. This phenomenon, which displays an optimum with respect to glycoprotein, is consistent with previous observations of fibrous long spacing fiber formation. Changes in viscosity of collagen initially dissolved in acetic acid were monitored during dialysis. It was found that a significant increase in viscosity must occur during dialysis of collagen before fibrous long spacing formation could take place. This increase in viscosity can be related directly to removal of acetic acid from the collagen solution. Removal of all sialyl residues from the alpha1-acid glycoprotein with neuraminidase prevents fibrous long spacing formation while removal of up to 35% of the sialyl residues has no effect on the interaction of glycoprotein with collagen. Amino acid composition and radioactivity studies suggest that 45-55% of the insoluble fibrous long spacing fibers is glycoprotein. In contrast to native collagen fibers, reduced fibrous long spacing fibers do not contain histidinohydroxymerodesmosine or hydroxylysinonorleucine. Instead, they contain significant quantities of allysine aldol and epsilon-hydroxynorleucine.