Growth-associated modifications of low-molecular-weight thiols and protein sulfhydryls in human bronchial fibroblasts

The thiol redox status of cultured human bronchial fibroblasts has been characterized at various growth conditions using thiol-reactive monobromobimane, with or without the combination of dithiotreitol, a strong reducing agent. This procedure has enabled measurement of the cellular content of reduced glutathione (GSH), total glutathione equivalents, cysteine, total cysteine equivalents, protein sulfhydryls, protein disulfides, and mixed disulfides. Passage of cells with trypsin perturbs the cellular thiol homeostasis and causes a 50% decrease in the GSH content, whereas the total cysteine content is subsequently increased severalfold during cell attachment. During subsequent culture, transient severalfold increased levels of GSH, protein-bound thiols, and protein disulfides are reached, whereas the total cysteine content gradually declines. These changes in the redox balance of both low-molecular-weight thiols and protein-bound thiols correlate with cell proliferation and mostly precede the major growth phase. When the onset of proliferation is inhibited by maintenance of cells in medium containing decreased amounts of serum, the GSH content remains significantly increased. Subsequent stimulation of growth by addition of serum results in decreased GSH levels at the onset of proliferation. In thiol-depleted medium, proliferation is also inhibited, whereas GSH levels are increased to a lesser extent than in complete medium. Exposure to buthionine sulfoximine inhibits growth, prevents GSH synthesis, and results in accumulation of total cysteine, protein-bound cysteine, and protein disulfides. For extracellular cystine, variable rates of cellular uptake correlate with the initial increase in the total cysteine content observed following subculture and with the GSH peak that precedes active proliferation. The results strongly suggest that specific fluctuations in the cellular redox balance of both free low-molecular-weight thiols and protein sulfhydryls are involved in growth regulation of normal human fibroblasts.     

Induction of pregnancy-associated murine protein-1 in dwarf mice by human growth hormone

Pregnancy-associated murine protein-1 (PAMP-1) could not be detected in peripheral blood of female dwarf mice (genotype dw/dw of the DW strain). By contrast the normal size females of the DW strain (genotypes +/+ and +/dw) had PAMP-1 serum levels of 18.9 AU +/- 15.7 AU/ml. Following administration of biosynthetic human growth hormone (hGH) every 2 h for 52 h PAMP-1 was detected in all dwarf females at concentrations of 16.0 AU +/- 3.3 AU/ml. The albumin levels in the circulation of DW females of normal size were significantly higher (P less than 0.05) than those of DW dwarfs, and the hGH administration did not change the serum albumin levels. The present experiment adds weight to the suggestion that the PAMP-1 serum level is regulated by GH.     

Three-dimensional structure in solution of barwin, a protein from barley seed.

The solution structure of a 125-residue basic protein, barwin, has been determined using 1H nuclear magnetic resonance spectroscopy. This protein is closely related to domains in proteins encoded by wound-induced genes in plants. Analysis of the 1H nuclear Overhauser spectrum revealed the assignment of more than 1400 nuclear Overhauser effects. Twenty structures were calculated based on 676 nontrivial distance restraints, 152 torsion angle restraints (92 phi, 56 chi 1, and 4 omega for proline), and stereospecific assignments of 38 chiral centers, using distance geometry, simulated annealing, and restrained energy minimization. None of the distance restraints was violated by more than 0.5 A in any of the 20 structures, and none of the torsion angle restraints was violated by more than 1 degree in any of the structures. The RMS difference between the calculated and target interproton distance restraints is 0.033 A, and the average atomic RMS differences between the 20 structures and their geometric average are 1.23 A for backbone atoms and 1.73 A for all heavy atoms. The dominating structural feature of the protein is a well-defined four-stranded antiparallel beta-sheet, two parallel beta-sheets packed antiparallel to each other and four short alpha-helices. The binding site of barwin to the tetramer N-acetylglucosamine has been qualitatively investigated, and the dissociation constant of the complex has been determined using one-dimensional 1H nuclear magnetic resonance spectroscopy.     

Metabolism of i.v. administered 45 kDa epidermal growth factor in the rat

The 45 kDa epidermal growth factor (EGF-(45 kDa)) has been purified from rat urine. We have investigated the distribution and the processing of i.v. injected 125I-labeled EGF-(45 kDa) in the rat. 2.5 min after the i.v. injection only 12% of the label remained in the blood. Most of the label was found in the liver (54%), in the kidneys (7%) and in the skin (4%). The submandibular glands, stomach, small intestine, colon, spleen and lungs contained 1% or less of the radioactivity. Some of the 125I-EGF-(45 kDa) was processed to 125I-EGF-(6 kDa) immunoreactivity in the liver and in the kidneys. The kidneys excreted 125I-EGF-(45 kDa) in the urine, but we were not able to demonstrate 125I-EGF-(6 kDa) in urine. In conclusion, this study shows that homologous EGF-(45 kDa) is cleared from the circulation of rats within a few minutes, mainly by the liver and the kidneys. In vivo both the liver and the kidneys are able to process some of the EGF-(45 kDa) to EGF-(6 kDa) immunoreactivity.     

A serum-free system for culturing human placental trophoblasts

Summary We have compared hormone production by early gestation and term human placental trophoblasts cultured in Ham's F10 medium containing 10% fetal bovine serum with that by cells cultured in serum-free HB102 medium. Mean daily production of progesterone on Days 3 to 7 was approximately 25% less by both early gestation and term cells cultured in HB102 as compared to Ham's F10, but production was maintained at a stable level for at least 7 d longer than the cells in Ham's. Estradiol production from 10⁻⁶ M dehydroepiandrosterone by both early gestation and term cells was comparable in both media. Human placental lactogen production on Days 3 to 7 was 40% less by cells cultured in HB102. Human chorionic gonadotropin (hCG) output by early gestation cells was also 50% less in HB102 but term cells in HB102 produced twice as much hCG as those in Ham's F10. 3B-Hydroxysteroid dehydrogenase (3BHSD) activity in early gestation and term cells and 11B-hydroxysteroid dehydrogenase (11BHSD) activity of early gestation cultures was comparable in the two media. 11BHSD activity was decreased in the term cultures, and this decrease was more marked in Ham's than in HB102. Sulfatase and aromatase activities in the early gestation cultures were comparable in both media; sulfatase activity was comparable and aromatase activity only 20% less in the term cells cultured in HB102. These results indicate that serum-free HB102 supports differential function of human trophoblast cells and is useful for studies of placental activity for as long as 14 d in culture.     

Spermatogenesis and sperm ultrastructure in the polychaete genusOphryotrocha (Dorvilleidae)

The details of spermatogenesis and spermiogenesis are described forOphryotrocha puerilis. The ultrastructure of mature sperm is shown forO. puerilis, O. hartmanni, O. gracilis, O. diadema, O. labronica, andO. notoglandulata. Clusters of sixteen cells each are proliferated by two stem cells in each setigerous segment ofO. puerilis representing the very early stages of both oogenesis and spermatogenesis. In each spermatocyte-I cluster, the cells are interconnected by cytoplasmic bridges. Early, clusters are enveloped by peritoneal sheath cells. These transient gonad walls break down prior to meiosis. The meiotic processes may start in the clusters with the cells still interconnected, or during breakdown of the original cluster, giving rise to smaller subclusters of both spermatocytes I and spermatocytes II with various numbers of cells. Finally, spermatid tetrads are present. As spermiogenesis progresses, the tetrads disintegrate. Golgi vesicles in both spermatocytes and spermatids contain electron-dense material, presumably preacrosomal. The acrosome is formed by such vesicles. In the six species studied here, the acrosomes appear to be of a similar overall structure but are of different shape. Centrioles are usually located beneath the acrosome. The distal centriole forms the basal body of a flagellum-like cytoplasmic process. The microtubules of these flagellar equivalents do not show a normal ciliar arrangement. The flagellar equivalent appears to be non-motile. InO. hartmanni and inO. notoglandulata, a flagellar equivalent is missing. Microtubules originating from the proximal end of the distal centriole stretch to the nuclear envelope. This feature appears to be especially conspicuous inO. puerilis and inO. labronica. InO. labronica and inO. notoglandulata, bundles of microtubules paralleling the cell perimeter appear to stabilise the sperm. Various numbers of mitochondria are either randomly distributed around the nucleus or accumulate on one side, often directly under the acrosome. Parts of the present paper were presented at the 2^nd International Polychaete Conference, Copenhagen 1986 and at the 3^rd International Polychaete Conference, Long Beach, Ca. 1989.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.