Osteoblasts synthesize and respond to transforming growth factor-type beta (TGF-beta) in vitro

Transforming growth factor-type beta (TGF-beta) has been identified as a constituent of bone matrix (Seyedin, S. M., A. Y. Thompson, H. Bentz, D. M. Rosen, J. M. McPherson, A. Conti, N. R. Siegel, G. R. Gallupi, and K. A. Piez, 1986, J. Biol. Chem. 261:5693-5695). We used both developing bone and bone-forming cells in vitro to demonstrate the cellular origin of this peptide. TGF-beta mRNA was detected by Northern analysis in both developing bone tissue and fetal bovine bone-forming cells using human cDNA probes. TGF-beta was shown to be synthesized and secreted by metabolically labeled bone cell cultures by immunoprecipitation from the medium. Further, TGF-beta activity was demonstrated in conditioned media from these cultures by competitive radioreceptor and growth promotion assays. Fetal bovine bone cells (FBBC) were found to have relatively few TGF-beta receptors (5,800/cell) with an extremely low Kd of 2.2 pM (high binding affinity). In contrast to its inhibitory effects on the growth of many cell types including osteosarcoma cell lines, TGF-beta stimulated the growth of subconfluent cultures of FBBC; it had little effect on the production of collagen by these cells. We conclude that bone-forming cells are a source for the TGF-beta that is found in bone, and that these cells may be modulated by this factor in an autocrine fashion.     

The genetic relationships between the kringle domains of human plasminogen,prothrombin, tissue plasminogen activator,urokinase, and coagulation factor XII

Summary A computer-based statistical evaluation of the optimal alignments of the kringle domains of human plasminogen, human prothrombin, human tissue plasminogen activator, human urokinase, and human coagulation Factor XIIa, as well as the putative kringle of human haptoglobin, has been performed. A variety of different alignments has been examined and scores calculated in terms of the number of standard deviations (SD) of a given match from randomness. With the exception of human haptoglobin, it was found that very high alignment scores (8.9–23.0 SD from randomness) were obtained between each of the kringles, with the kringle 1 and kringle 5 regions of human plasminogen displaying the highest similarity, and the S kringle of human prothrombin and the human Factor XII kringle showing the least similarity. The relationships obtained were employed to construct an evolutionary tree for the kringles. The predicted alignments have also allowed nucleotide mutations in these regions to be evaluated more accurately. For those regions for which nucleotide sequences are known, we have employed the maximal alignments from the protein sequences to assess nucleotide sequence similarities. It was found that a range of approximately 40–55% of the nucleotide bases were placed at identical positions in the kringles, with the highest number found in the alignment of the two kringles of human tissue plasminogen activator and the lowest number in the alignment of the S kringle of prothrombin with the second kringle of tissue plasminogen activator. From both protein and nucleotide alignments, we conclude that haptoglobin is not statistically homologous to any other kringle.Secondary structural comparisons of the kringle regions have been predicted by a combination of the Burgess and Chou-Fasman methods. In general, the kringles display a very high number of -turns, and very low -helical contents. From analysis of the predicted structures in relationship to the functional properties of these domains, it appears as though many of their functional differences can be related to possible conformational alterations resulting from amino acid substitutions in the kringles.     

2,3-Bisphosphoglycerate protects mitochondrial and cytosolic proteins from proteolytic inactivation

2,3-bisphosphoglycerate at physiological concentration similar to that found in many tissues protects effectively ornithine transcarbamoylase (OTC) from proteolytic inactivation by broken lysosomes. 2,3-bisphosphoglycerate protects also many other mitochondrial and cytosolic proteins, such as glutamate dehydrogenase (GDH) an glyceraldehyde-3-phosphate dehydrogenase (GAPDH), from proteolysis by broken lysosomes and other proteases. It is, thus, suggested that 2,3-bisphosphoglycerate may play an important role in the control of the degradative rates of some proteins, which may explain its high concentration in certain cells.     

Analysis by flow cytometry of rat hepatocytes from different acinar zones

Many functional, morphological and biochemical differences among hepatocytes from different acinar zones have been described. Therefore, it will facilitate studies on liver metabolism rapid, non-destructive procedures to isolate hepatocytes from these zones. Flow cytometry is a new powerful tool which, however, has not been used thus far to accomplish the separation of hepatocytes from different acinar zones. We describe here various cytometric parameters which characterize hepatocyte populations, separated by isopycnic centrifugation in Percoll gradients. The intraacinar origin of the different hepatocytes was assessed by enzymatic and morphological measurements.     

Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study

Time-resolved fluorescence anisotropy (TRFA) and steady-state anisotropy measurements and fluorescence intensification microscopic observations were made on RAW264 macrophages labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH). Microscopic analysis revealed that the fluorescent probe DPH was found in association with plasma membranes and small vesicles. Macrophages treated with immune complexes could not be distinguished from untreated cells, indicating that the same membrane compartments were labeled. The probe TMA-DPH was exclusively localized to the plasma membrane. Steady-state anisotropy measurements indicated that in vitro culture conditions did not significantly affect membrane fluidity. TRFA measurements were conducted to determine the physical properties of macrophage membranes during immune recognition and endocytosis. Data were analyzed by iterative deconvolution to yield phi, the rotational correlation time, and r infinity, the limiting anisotropy. These parameters may be interpreted as the "fluidity" and order parameter of the membrane environment, respectively. Typical values for untreated macrophages were phi = 7.8 ns and r infinity = 0.12. Binding and endocytosis of immune complexes prepared in 4-fold antigen excess increase these values to phi = 22.1 ns and r infinity = 0.15. However, receptor-independent phagocytosis of latex beads decreases these values to phi = 2.2 ns and r infinity = 0.10. Addition of catalase before, but not after, immune complex incubation with cells diminishes the effect upon membrane structure, suggesting that H2O2 participates in fluidity changes. Pretreatment of macrophages with the membrane-impermeable sulfhydryl blocker p-(chloromercuri)benzenesulfonic acid also diminished these effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of the Danish (skive) variant of mouse liver alcohol dehydrogenase

The partially inbred Danish (Skive) strain of mice exhibits a form of liver alcohol dehydrogenase (ADH) which differs in electrophoretic mobility from that of all other inbred mouse strains thus far examined, e.g., C57BL/10, DBA/2J, and BALB/c. In order to compare the catalytic and molecular properties of the variant and normal enzyme forms, they were purified to homogeneity by ion-exchange and affinity chromatography. Tryptic peptides of reduced and carboxymethylated subunits of the normal and variant ADH forms were mapped by thin-layer two-dimensional electrophoresis and chromatography and by reversed-phase high-performance liquid chromatography. A unique nonapeptide in the Danish mouse liver ADH which did not appear in enzymes from C57BL/10, DBA/2J, or BALB/c mice was identified by both methods. Amino acid sequencing of this peptide revealed that the Arg residue at position 124, as predicted from the cDNA sequence of ADH in DBA/2J mice, has been replaced by Leu in the Danish variant. The Leu for Arg substitution in the variant form appears to account for its decreased cathodic mobility with electrophoresis in starch gels at pH 7.2. The K _m and V _max of ADH from the Danish strain for three primary alcohols and three aldehydes were similar in value to those of ADH from the C57BL/10, DBA/2J, and BALB/c strains. Based on the X-ray structure of horse liver ADH, position 124 is on the solvent-exposed surface of the catalytic domain. The finding that the kinetic constants are similar for the normal and variant forms is consistent with the observation that this residue is not in the active site and that there is no known role for it in the ADH catalytic mechanism.This work was supported by NIAAA Grant AA-04307.     

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.     

Modifying role of dietary factors on the mutagenicity of aflatoxin B1: in vitro effect of sulphur-containing amino acids

Sulphur-containing amino acids including some derivatives have been tested for their effectiveness in suppressing the mutagenic activity of aflatoxin B1 in Salmonella typhimurium strains provided with a rat liver activation system. Cysteine and N-acetylcysteine have been found to be most effective in the 2 strains tested (TA100 and TA98). Glutathione (oxidised and reduced forms) has shown partial activity, while cystine and methionine are found to be partially effective only in strain TA100. Inhibition of mutagenicity may be due to interaction of these substances with microsomal enzymes resulting in interference with the formation of ultimate mutagenic species.     

Naloxone doesn't determine the response of growth hormone to clonidine in obese patients

The effect of naloxone (opioid receptor blocker) on the impairment of growth hormone (GH) release after clonidine (alfa 2-adrenergic agonist) was investigated in 10 volunteer obese subjects. The patients (4 males and 6 females, 16-22 year old) with fat excess (15 +/- 2 kg) estimated by bioelectrical impedance analysis (BIA) were studied repeatedly. The patients, were perfused by a slow saline infusion. 30 min later they received a bolus dose of clonidine (150 micrograms p.o.), followed 30 min later by a bolus dose of naloxone (10 mg i.v.) or a corresponding volume of isotonic sodium cloride (I.S.) for control. No significant changes occurred in blood GH concentration after clonidine administration and naloxone did not induce GH response at clonidine. These results suggest that in obese subjects the impairment of GH release after clonidine is not mediated via receptors sensitivity to naloxone.