Collagen synthesis by cells synchronously replicating DNA.

Replication and the performance of a differentiated function have been considered antagonistic processes. When cells in culture are partially tially synchronized with 5-fluoro-2'-deoxyuridine (FUdR), the synthesis of the specialized protein (collagen) is not reduced during chromosomal replication (S period). Collagen synthesis varies with general protein synthesis through the S period.

The clementine mission to the moon: scientific overview.

In the course of 71 days in lunar orbit, from 19 February to 3 May 1994, the Clementine spacecraft acquired just under two million digital images of the moon at visible and infrared wavelengths. These data are enabling the global mapping of the rock types of the lunar crust and the first detailed investigation of the geology of the lunar polar regions and the lunar far side. In addition, laser-ranging measurements provided the first view of the global topographic figure of the moon. The topography of many ancient impact basins has been measured, and a global map of the thickness of the lunar crust has been derived from the topography and gravity.

Purification and characterization of a collagenous protein secreted by a murine teratocarcinoma-derived cell line.

A collagenous protein could be precipitated by (NH4)2SO4 from the culture medium of a murine teratocarcinoma-derived cell line (Ko, C.Y., Johnson, L.D. and Priest, R.E. (1979) Biochim. Biophys. Acta 581, 252-259). Further purification of this protein was achieved by combining DEAE-cellulose chromatography with either CM-cellulose or molecular sieve chromatography. The collagenous polypeptides had subunit molecular weights of 160 000, if determined by molecular sieve chromatography, or 190 000, if determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and they are not linked by disulphide bridges. Amino acid composition of this collagen is similar to that of a murine type IV collagen isolated from EHS sarcoma (Timpl et al. (1978) Eur. J. Biochem. 84, 43-52). The most prominent peptides resulting from cleavage of the protein by CNBr had estimated molecular weights of 25 000, 23 000, 11 700 and 9400. Pepsin treatment of this collagen under non-denaturing conditions produced three major fragments having molecular weights of 70 000, 45 000 and 43 000. We conclude that the collagen secreted by the murine teratocarcinoma-derived cell culture is a type IV basement membrane collagen. Therefore, this culture system should provide a continuous source of type IV collagen, which may be used to study the interaction of this collagen with other basement membrane components.

Isolation and characterization of hydroxyproline-containing proteins secreted by a murine carcinoma cell culture.

A collagenous protein was isolated from a murine carcinoma cell culture, which has been shown to synthesize basement membrane. The molecular weight of this protein was estimated to be 155 000. It eluted from carboxymethyl-cellulose in the region near the alpha 1 and beta 11 components of calf skin collagen. 63--69% of the peptide-bound prolines were hydroxylated, and the 4-/3-hydroxyproline ratios ranged from 12 : 1 to 14 : 1. About 95% of the hydroxylysines in the peptide were glycosylated, and almost all of them were in the glucosylgalactosyl dissacharide form. Judging from the posttranslational characteristics, this collagenous protein is probably of basement membrane type.

The phytohemagglutinin response of human peripheral blood lymphocytes as a function of donor age: a re-examination using BrdU-Hoechst flow cytometry.

Mononuclear cells were isolated from peripheral blood by a standard Ficoll-Hypaque technique from 127 healthy donors, ranging in age from newborns to 86 years of age. As a measure of their in vitro growth response, the fraction of non-cycling cells was determined at 48 and 72 h after phytohemagglutinin (PHA) exposure by means of BrdU-Hoechst flow cytometry. This technique provides an optimal assay system for the non-cycling cell fraction, since all cycling cells will have incorporated BrdU thereby quenching the fluorescence of the Hoechst 33258 fluorochrome. Lymphocytes from prepubertal donors showed significantly decreased non-cycling cell fractions, as did lymphocytes from an additional group of 14 adults with hypogonadism due to the 45, XO condition (Turner-Syndrome). Much to our surprise, we found no definitive correlation between donor age and the non-cycling fraction of cells from the adult lymphocyte donors. Nor did we find any age-related increase in the variance of the non-cycling cell fraction. These observations suggest that the previously reported age-related decline in the PHA response of human PBL may reflect an increasing delay, rather than an overall diminution, of the PHA response as a function of donor age.

HLA typing of cultured amniotic fluid cells.

HLA typing was performed on 18 cultures of human amniotic fluid cells using cytotoxicity and absorption technics. Confirmation of antigen assignments was obtained in nine of ten instances, where HLA typing also was performed on cord blood. Three major problems were encountered in performing these studies: (1) complement cytotoxicity, (2) false-positive reactions, and (3) false-negative reactions. False-positive and false-negative reactions occurred more frequently with sera defining HLA-B locus specificities than with sera defining HLA-A locus specificities. Absorption studies were helpful in making antigen assignments when false reactions occurred. Preliminary studies suggest that the frequency of false-positive reactions can be decreased by absorbing HLA typing sera with antigen-negative amniotic fluid cultured cells, buffy coat, or platelets. Accurate antigen assignment is difficult when parental HLA types are unavailable.

Origin of cells in human amniotic fluid cultures: ultrastructural features.

Two classes of cells can be subcultured from human amniotic fluid (F and AF). Ultrastructural features of these cultrued cells were determined using scanning and transmission electron microscopy with particular attention to extracellular material. Results obtained from these two cell types are compared to one another and to cultured human dermal fïbroblasts. The F-type cultures have an abundance of extracellular type i collagen fïbers, identical with cultured human dermal fïbroblasts. No type I collagen fïbers are seen in cultures of AF cells. Instead, fïne fïlamentous material is admixed with amorphous material adjacent to cell membranes where epithelial basement membrane glycoprotein has been localized. By scanning electron microscopy parallel arrays of cells are present at confluency of F cultures, typical of cultured dermal fïbroblasts. AF cultures show a much looser growth pattern at confluency. On the basis of accumulated evidence, we believe that F-type cells arise from fïbrous connective tissue and are fïbroblasts, whereas AF cells, the predominant type in early cultures used for prenatal genetic diagnosis, arise from fetal membranes and retain features of trophoblast.

Endurance training in the rat. I. Myocardial mechanics and biochemistry.

The effects of physical training and detraining on cardiac structure and myocardial mechanics were studied in young and adult male rats trained by graded treadmill running for 12 wk and compared with sedentary controls. Detraining was produced by training for 12 wk followed by 6 sedentary wk. A training effect was demonstrated by increased succinate dehydrogenase activity in skeletal muscle (trained 10.0 +/- 1.2 mumol . g-1 . min-1; sedentary 6.4 +/- 0.8 mumol . g-1 . min-1; P less than 0.05). Although heart weight-to-body weight ratios were increased in trained rats of both ages, left ventricular fiber diameters and myocardial RNA, DNA, and collagen content were unchanged by training. Active and passive mechanics (myocardial contractility and stiffness) were studied in left ventricular papillary muscles and did not differ significantly between groups, with the exception of depressed contractility observed in young trained rats [(e.g., papillary peak developed isometric tension at Lmax (length at peak tension)] was 2.64 +/- 0.24 g/mm2 in trained vs. 3.59 +/- 0.22 g/mm2 in sedentary (P less than 0.01). This difference was abolished by detraining. Papillary muscle contractile responses to calcium, norepinephrine, and hypoxia were not altered by training or detraining. In conclusion, moderate endurance training did not result in significant cardiac hypertrophy, altered myocardial stiffness, or consistent changes in myocardial contractility.

Characterization of two types of crystalloids in pleomorphic adenomas of minor salivary glands. A light-microscopic, electron-microscopic, and histochemical study.

Crystalloids have been previously described in salivary gland tumors. In order to ascertain the incidence of these structures, the authors reviewed a series of 294 minor salivary gland tumors. One hundred thirty pleomorphic adenomas were identified, and 6 of these contained crystalloids. No crystalloids were found in other benign or malignant salivary gland tumors. These six file cases and a recent seventh case containing crystalloids were studied by light and electron microscopy and with histochemistry. Two types of crystalloids were found. One case contained previously described tyrosine-rich crystalloids, and the other six contained crystalloids composed of radially arranged collagen fibers. Both types of crystalloids are further characterized and discussed.

The characterization of hCG regulation in cultured human amniotic fluid cells.

The media from primary cultures and subcultures of second trimester human amniotic fluid (AF) cells were assayed by radioimmunoassay to quantitate production of human chorionic gonadotropin (hCG). Primary AF cultures produce more hCG per cell than do the corresponding subcultures. Sodium butyrate (2mM) stimulates AF subcultures to produce 5-13 times more hCG per cell or per mg of cellular protein than do untreated subcultures. This stimulatory effect of sodium butyrate is dose dependent between 0 and 5 mM. Addition of sodium butyrate 24 hours after subculture, while stimulating production of hCG during the subsequent 3 days, also results in fewer cells and less protein per culture. This effect on cell growth is also dose-dependent. Previous investigators have proposed that the stimulation of hCG by sodium butyrate in other types of cell cultures is due to an effect of that agent on culture growth. Therefore, in these studies AF cells are allowed to grow to confluency before sodium butyrate was added. Production of hCG was stimulated by sodium butyrate about four-fold during the next 5 days although no significant changes were observed either in number of cells or amount of cellular protein per culture. These results suggest that stimulation of hCG by sodium butyrate is not dependent on its effect on growth of the cultures.