Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture.

Rabbit articular chondrocytes in suspension culture synthesize Type II collagen [3alpha1(II)] in the absence of extracellular Ca2+ and Type I collagen [2alpha1(I) - alpha2] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca2+ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl2. If added directly to the suspension culture medium containing no CaCl2, calcitonin stimulates an active efflux of Ca2+ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Simultaneous analysis of substrates,products, and inhibitors of xanthine oxidase by high-pressure liquid chromatography and gas chromatography

Procedures are presented for the simultaneous analysis of hypoxanthine, xanthine, allopurinol, oxipurinol, and uric acid in standard mixtures and physiological fluids using gas chromatography (gc) or high-pressure liquid chromatography (hplc). Excellent correlation was obtained between the two methods for hypoxanthine, xanthine, oxipurinol, and uric acid. There are advantages and disadvantages to both methods. hplc requires no prior derivatization, uses isocratic elution with a buffer containing no organic solvent, and has 50- to 100-fold greater sensitivity than gc. Simpler methods of prepurification, readily adapted to clinical laboratories, can be used for hplc analysis. Although substances that are found in some urine samples from cancer patients interfere with hplc, separations by gc are not affected by these substances.     

Structure of bovine beta-lactoglobulin at 6A resolution.

Bovine β-lactoglobulin is a dimer with a molecular weight of 2 × 18,400. In solution it undergoes a pH-dependent transition at pH 7.0 between two alternative structures, named N and R. The structures of four different crystal forms have been determined by multiple isomorphous replacement with heavy-atoms. Two of them, lattices K and X, were crystallised at pH 6.5, corresponding to the N state in solution; and the other two, lattices Y and Z, were crystallised at pH 7.5, corresponding to the R state in solution. The figures of merit of the phase angles determined for these lattices were 0.76, 0.77, 0.80 and 0.80, respectively. The four structures that emerged are similar and show certain features suggestive of α-helices and pleated sheets, but the resolution is insufficient to trace the entire course of the polypeptide chain. No clear distinction can yet be made between the structures above or below pH 7.0, nor between the native molecule and the molecule from which the C-terminal leucine and histidine residues have been cleaved. Analyses at higher resolution are in progress.     

Pathways of d-fructose catabolism in species of Pseudomonas

Cell-free extracts of d-fructose grown cells of Pseudomonas putida, P. fluorescens, P. aeruginosa, P. stutzeri, P. mendocina, P. acidovorans and P. maltophila catalyzed a P-enolpyruvate-dependent phosphorylation of d-fructose and contained 1-P-fructokinase activity suggesting that in these species fructuse-1-P and fructose-1,6-P₂ were intermediates of d-fructose catabolism. Neither the 1-P-fructokinase nor the activity catalyzing a P-enolpyruvate-dependent phosphorylation of d-fructose was present in significant amounts in succinate-grown cells indicating that both activities were inducible. Cell-free extracts also contained activities of fructose-1,6-P₂ aldolase, fructose-1,6-P₂ phosphatase, and P-hexose isomerase which could convert fructose-1,6-P₂ to intermediates of either the Embden-Meyerhof pathway or Entner-Doudoroff pathway. Radiolabeling experiments with 1-¹⁴C-d-fructose suggested that in P. putida, P. aeruginosa, P. stutzeri, and P. acidovorans most of the alanine was made via the Entner-Doudoroff pathway with a minor portion being made via the Embden-meyerhof pathway. An edd ^- mutant of P. putida which lacked a functional Entner-Doudoroff pathway but was able to grow on d-fructose appeared to make alanine solely via the Embden-Meyerhof pathway.Non-Standard Abbreviations cpm counts per min - edd ^- mutant lacking Entner-Doudoroff dehydrase (6-PGA dehydrase) - EDP Entner-Doudoroff pathway - EMP Embden-Meyerhof pathway - FDP fructose-1,6-P₂ - FDPase FDP phosphatase - F-1-P fructose-1-P - F-6-P fructose-6-P - FPTs PEP: d-fructose phosphotransferase system - G-6-P glucose-6-P - KDPG 2-keto-3-deoxy-6-P-gluconate - PEP P-enolpyruvate - 1-PFK 1-P-fructokinase - 6-PFK 6-P-fructokinase - 6-PGA 6-P-gluconate     

The Interactions of Superoxide Ion(O-2) with Metallo-Porphyrins [(C18TPP)M, M = FE,MN,CO,ZN]; Models for Biological Systems and Superoxide Dismutases

In dimethylformamide superoxide ion forms a l:l adduct with tctrakis (2.6-dichlorophenyl) porphinatoiron, (Cl₈ TPP)FeOO^-, as well as with its manganese analogue, (Cl₈ TPP)MnOO^-. On the basis of their electrochemical, spectroscopic, and magnetic properties these adducts have a metal-oxygen covalent bond (PorM-OO^-), oxygen-centered redox chemistry. and reactivities that are similar to the hydroperoxide ion (HOO^-). Addition of ^-OH to a solution of PorFe and O₂ results in the formation of PorFe(OH)(OO^-), which can be electrochemically oxidized to PorFeOH plus O₂ (-0.2 V vs SCE). Addition of protons to the PorM-OO^- adducts promotes their rapid decomposition to PorM, HOOH. and O₂. This chemistry provides insight to the reactions of biological superoxide and superoxide dismutases.     

Induction of erythropoietin responsiveness in murine hematopoietic cells by the gag-myb-ets-containing ME26 virus.

ME26 virus, which was generated by inserting the coding region of the acute avian leukemia-inducing virus E26 into a murine retrovirus vector, encodes a 135-kDa gag-myb-ets fusion protein. Amphotropic murine leukemia virus pseudotypes of ME26 virus induce a high incidence of erythroleukemia 2 to 4 months after injection into newborn NFS/N mice. Spleen cells from the majority of these mice proliferate to high levels in the presence of the erythroid hormone erythropoietin (Epo) and can easily be established as permanent Epo-dependent cell lines. The cell lines contain multiple copies of ME26 viral DNA and express viral message and protein. An Epo receptor mRNA of normal size can be detected in these cells, and binding studies reveal a single class of lower-affinity Epo receptor with an affinity for Epo that is in the range of that previously reported for erythroid cells. The ME26 virus-induced Epo-dependent cell lines, however, appear more immature than previously described erythroid cell lines and more closely resemble early hematopoietic precursor cells, suggesting that the virus may be activating the Epo receptor in hematopoietic cells that do not normally express it. Consistent with this idea, we are able to infect an interleukin-3-dependent myeloid cell line, FDC-P2, with ME26 virus and convert it to Epo dependence. The ME26 virus-infected FDC-P2 cells, even before growth on Epo, showed a large increase in the amount of Epo receptor mRNA. However, no ME26 viral integrations can be detected adjacent to the Epo receptor gene, indicating that the virus is not activating the Epo receptor gene by promoter/enhancer insertion. Our results are more consistent with the hypothesis that the gag-myb-ets-encoded viral fusion protein, which is known to bind DNA, is directly or indirectly activating the expression of the Epo receptor gene in these cells.     

Receptors for prostaglandins F2 alpha and E2 in ovine corpora lutea during maternal recognition of pregnancy.

Plasma membrane receptors for prostaglandins (PG) F2 alpha and E2 were quantified in ovine corpora lutea obtained from nonpregnant and pregnant ewes on Days 10, 13, and 15 post-estrus, and from additional ewes on Days 25 and 40 of pregnancy. Regardless of reproductive status or day post-estrus, concentrations of luteal receptors for PGF2 alpha were 7- to 10-fold greater than those for PGE2. In pregnant ewes the concentration of receptors for PGF2 alpha was highest on Day 10 (35.4 +/- 2.8 fmol/mg) and lowest on Day 25 (22.3 +/- 2.5 fmol/mg). A difference in the concentration of luteal receptors for PGF2 alpha between pregnant and nonpregnant ewes was apparent only on Day 15 post-estrus, at which time the concentration of receptors for PGF2 alpha was higher in pregnant ewes than in nonpregnant ewes (27.1 +/- 2.7 vs. 17.7 +/- 2.7 fmol/mg). Concentrations of receptors for PGE2 in pregnant ewes were similar (p > 0.05; 2.8 +/- 0.3 to 3.7 +/- 0.2 fmol/mg) between Days 13 and 40 but were higher (p < 0.05) than in corpora lutea obtained from nonpregnant ewes on Days 10 (5.0 +/- 0.4 vs. 4.1 +/- 0.2 fmol/mg) and 15 (3.7 +/- 0.2 vs. 2.0 +/- 0.4 fmol/mg) post-estrus. Although concentrations of receptors for both PGF2 alpha and PGE2 were lowest in corpora lutea obtained from nonpregnant ewes on Day 15, this was not due to luteal regression since the weights and concentrations of progesterone in corpora lutea on Day 15 were not lower than those for corpora lutea obtained on Days 10 and 13.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of autoclave-induced carbohydrate hydrolysis on the growth ofBeta vulgaris cells in suspension

Media of de Greef & Jacobs (1979) were autoclaved either with all the nutrient components in a single vessel (medium 1) or with the following components in separate vessels: FeNa–EDTA+CaCl₂ (medium 2), FeNa–EDTA+NaH₂PO₄ (medium 3) or sucrose (medium 4). Medium 5 was prepared by autoclaving FeNa–EDTA+NaH₂PO₄ and sucrose in two separate vessels. It was found that the dry mass yield of cell suspensions ofBeta vulgaris was lowest in medium 1, followed by media 2 and 3. There was no significant difference among media 3, 4, and 5.The plot of dry mass yield of the cell suspensions against the rates of cyanide-initiated oxygen consumption which indicate the extent of carbohydrate hydrolysis of the media during autoclaving, indicated the presence of a threshold rate of about 17–20 nmol ml^–1 min^–1. Dry mass yield of the suspensions decreased rapidly when the rate exceeded this value.For media with glucose as the source of carbohydrate, the rate of cyanide-initiated oxygen consumption exceeded the threshold value by a factor of 1.5 to 2, depending on the volume of the media autoclaved.Abbreviations FeNa-EDTA ferric monosodium ethylenediamine-tetraacetic acid     

Calcium-free calmodulin is a substrate of proteases from human immunodeficiency viruses 1 and 2

Calcium-free calmodulin-(CaM) is rapidly hydrolyzed by proteases from both human immunodeficiency viruses (HIV) 1 and 2. Kinetic analysis reveals a sequential order of cleavage by both proteases which initiates in regions of the molecule known from X-ray crystallographic analysis of Ca2+/CaM to be associated with calcium binding. Although HIV-1 and HIV-2 proteases hydrolyze two bonds in common, the initial site of cleavage required for subsequent events differs in each case. The first bond hydrolyzed by the HIV-1 protease is the Asn-Tyr linkage in the sequence, -N-I-D-G-D-G-Q-V-N-Y-E-E-, found in the fourth calcium binding loop. In contrast, it is an Ala-Ala bond in the third calcium loop, -D-K-D-G-N-G-Y-I-S-A-A-E-, that is first hydrolyzed by the HIV-2 enzyme, followed in short order by cleavage of the same Asn-Tyr linkage described above. Thereafter, both enzymes proceed to hydrolyze additional peptide bonds, some in common, some not. Considerable evidence exists that inhibitors are bound to the protease in an extended conformation and yet all of the cleavages we observed occur within, or at the beginning of helices in Ca2+/CaM, regions that also appear to be insufficiently exposed for protease binding. Molecular modeling studies indicate that CaM in solution must adopt a conformation in which the first cleavage site observed for each enzyme is unshielded and extended, and that subsequent cleavages involve further unwinding of helices.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the mouse thymidine kinase gene to the distal portion of chromosome 11

We report the cytogenetic mapping of the thymidine kinase (tk-1) gene in the mouse using two complementary and independent analyses: (1) investigation of chromosome aberrations associated with tk-1 gene inactivation in the L5178Y TK+/- -3.7.2C cell line, and (2) fluorescence in situ molecular hybridization of cloned tk-1 cDNA probes to mitotic chromosomes of this cell line. The consensus location from both analyses is 11E1-E2. Consideration of the mouse tk-1 gene localization, along with evidence that the homologous human TK1 gene is located distally on the large arm of chromosome 17, appears to extend the region of homology between MMU11 and HSA17 to the distal end of both chromosomes.