Fluorine nuclear magnetic resonance spectra of rabbit carbonmonoxyhemoglobin

Carbonmonoxyhemoglobin prepared from protein isolated from rabbits maintained on a diet supplemented with 4-fluorophenylalanine (Phe (4F)) has been studied by fluorine NMR spectroscopy. Substitution of Phe(4F) appears to take place randomly at the sixteen nonequivalent phenylalanine positions of the globins; examination of hybrid hemoglobins in which only one type of globin chain contained the fluorinated amino acid, as well as changes in the spectrum upon exposure to oxygen, aided in the assignment of fluorine resonances from the alpha- or beta-globin chains. The effects of modification of Cys-beta-93 with a spin label and variation of pH and sample temperature on the spectrum were also examined. These data in association with theoretical estimates of aromatic ring current and van der Waals effects on chemical shifts were used to support tentative assignments of several signals observed to specific amino acid residues. Evidence suggesting the presence of two conformational forms of the protein, possibly due to disorder of the heme groups, is described.     

Regulation of thyroid hormone metabolism in rat liver fractions

The nature of the conversion of thyroxine (T₄) to triiodothyronine (T₃) and reverse triiodothyronine (rT₃) was investigated in rat liver homogenate and microsomes. A 6-fold rise of T₃ and 2.5-fold rise of rT₃ levels determined by specific radioimmunoassays was observed over 6 h after the addition of T₄. An enzymic process is suggested that converts T₄ to T₃ and rT₃. For T₃ the optimal pH is 6 and for rT₃, 9.5. The converting activity for both T₃ and rT₃ is temperature dependent and can be suppressed by heat, H₂O₂, merthiolate and by 5-propyl-2-thiouracil. rT₃ and to a lesser degree iodide, were able to inhibit the production of T₃ in a dose related fashion. Therefore the pH dependendy, rT₃ and iodide may regulate the availability of T₃ or rT₃ depending on the metabolic requirements of thyroid hormones.     

The differential protection by WR2721 of skin versus growing cartilage following irradiation in weanling rats

The potential for radioprotection of growing cartilage by the thiophosphate WR2721 was evaluated in weanling rats using single fractions of irradiation. Protection of acute skin toxicity was monitored simultaneously. Single doses of 600, 1200, 1800, or 2400 cGy were administered to the left tibia of CrL:CD(SD)BR female rats in groups of 12. Identically treated groups were injected with 310 mg/kg WR2721 (2/3 the determined LD50/30) in a concentration of 26 mg/ml intraperitoneally 15 min prior to irradiation. Rats untreated or given WR2721 without radiation served as control groups. Radiographs of the irradiated and unirradiated tibiae for each animal were obtained weekly to the date of sacrifice at 80 days following the initial treatment. Skin toxicity was assessed weekly starting on the second week using Moulder's scale (J.E. Moulder, J.J. Fischer, and A. Casey, Radiology 115, 465-470 (1975]. No significant difference in bone growth as measured by tibial lengths for the WR2721-treated or untreated animals was observed. Skin toxicity including moist desquamation occurred in irradiated limbs and was substantially less in rats treated with WR2721. As opposed to previous work with cysteamine, WR2721 as administered had no significant radioprotective effect on tibial growth in weanling rats but substantially reduced the accompanying skin toxicity.     

Structure of the sugar-phosphate moiety of lipid A from lipooligosaccharide of Neisseria meningitidis group B,strain BC5S No. 125

On the basis of chemical and NMR data the partial structure of lipid A from lipooligosaccharide (LOS) of Neisseria meningitidis group B, strain BC₅S No 125 was established. Lipid A consisted of disaccharide 2-deoxy-6-O-[2-deoxy-2-(3-hydroxytetradecanoylamino)--gluco-pyranosyl]-2-(3-hydroxytetradecanoylamino)--glucopyranose carrying the -(2-aminoethyl)pyrophosphate residue at 0–4 and the pyrophosphate or phosphate residue at 0–1. On hydrolysis of the acidic form of LOS with 1% acetic acid the substituent at 0–1 was practically completely removed whereas that at 0–4 was stable. The analogous hydrolysis of the Mg-salt of LOS was accompanied by splitting off the pyrophosphate linkage in the substituent at 0–4. Hydrolysis of LOS at pH 4.5 in the presence of SDS led mainly to a lipid A preparation retaining both pyrophosphate residues.Abbreviations KDO 2-keto-3-deoxyoctulosonic acid - LA-I, LA-II preparations of lipid A - LOS lipooligosaccharide - LOS-H⁺ the acidic form of LOS - OS oligosaccharide - TLC thin-layer chromatography - GLC-MS gas-liquid chromatography/mass spectrometry     

Metalloproteinases mediate extracellular matrix degradation by cells from mouse blastocyst outgrowths.

The maintenance and developmental remodeling of extracellular matrix is crucial to such processes as uterine implantation and the cell migratory events of morphogenesis. When mouse blastocysts are placed in culture they adhere to extracellular matrix, and trophoblast giant cells migrate out onto the matrix and degrade it. The secretion of functional proteinases by developing mouse embryos increases dramatically at the time of implantation. By zymography we identified the major secreted gelatin-degrading proteinase, also known as type IV collagenase, as one migrating at 92 x 10(3) Mr. Several casein-degrading proteinases were also secreted. The tissue inhibitor of metalloproteinases (TIMP) inhibited all of the embryo-derived proteinases detected by gelatin gel zymography, indicating that they are metalloproteinases, whereas TIMP did not inhibit all of the caseinases. Urokinase was also secreted. Addition of TIMP at 5-500 nM effectively inhibited the degradation of matrix by the trophoblast outgrowths. Blocking antibodies directed against 92 x 10(3) Mr gelatinase abolished matrix degradation by the trophoblast cells. These observations suggest that several metalloproteinases are regulated in early development and that 92 x 10(3) Mr gelatinase, in particular, has a rate-limiting function in degradation of the maternal extracellular matrix by trophoblast cells.     

Somatic recombination rather than uniparental disomy suggested as another mechanism by which genetic imprinting may play a role in the etiology of Prader-Willi syndrome

Summary Six Prader-Willi syndrome (PWS) patients with normal karyotypes and their parents were analyzed to determine the nature of the molecular aberrations present in the proximal region of 15q and to determine the parental origin of the aberrant chromosome 15. In addition, the likehood that uniparental disomy plays a significant role in the etiology of PWS patients with normal karyotypes was studied. Restriction fragment length polymorphisms (RFLPs) recognized by seven probes [pML34 (D15S9), pTD3-21, pCGS0.9, pCGS1.1 (D15S10), IR4.3 (D15S11), IR10.1 (DS15S12), p189-1 (D15S13), IR39 (D15S18), and CMW-1 (D15S24)] mapping to the Prader-Willi chromosome region (PWCR) and an additional two probes [pMS1-14 (D15S1); the cDNA of neuromedin B] mapping elsewhere on chromosome 15 were analyzed in the six PWS patients and their parents. Copy number of each locus within the PWCR was determined by densitometry. Molecular rearrangements of the proximal region of 15q were observed in all of the six probands and the origin of the aberrant chromosome 15 when determined was consistently paternal in origin. While data obtained from our six patients does not support the mechanism of disomy, results obtained from three of the six patients show more complex rearrangements hypothesized to have resulted from somatic recombination. These rearrangements have resulted in acquired homozygosity and the lack of a paternal allele at various loci within the PWCR. The presence of only a maternal contribution at certain loci as the result of somatic recombination may be another mechanism by which genetic imprinting plays a role in the presentation of the PWS phenotype.     

Effect of subunit III removal on control of cytochrome c oxidase activity by pH

Studies were undertaken to assess the postulated involvement of subunit III in the proton-linked functions of cytochrome c oxidase. The effect of pH on the steady-state kinetic [corrected] parameters of subunit III containing and subunit III depleted cytochrome oxidase was determined by using beef heart and rat liver enzymes reconstituted into phospholipid vesicles. The TNmax and Km values for the III-containing enzyme increase with decreasing pH in a manner quantitatively similar to that reported by Thornstrom et al. [(1984) Chem. Scr. 24, 230-235], giving three apparent pKa values of less than 5.0, 6.2, and 7.8. The maximal activities of the subunit III depleted enzymes (beef heart and rat liver) show a similar dependence on pH, but the Km values are consistently higher than those of the III-containing enzyme, an effect that is accentuated at low pH. The pH dependence of TNmax/Km for both forms of the enzyme (+/- subunit III) indicates that protonation of a group with an apparent pKa of 5.7 lowers the affinity for substrate (cytochrome c) independently of a continued increase in maximal velocity. N,N'-Dicyclohexylcarbodiimide (DCCD) decreases the pH responsiveness of the electron-transfer activity to the same extent in both III-containing and III-depleted enzymes, indicating that this effect is mediated by a peptide other than subunit III. Control of intramolecular electron transfer by a transmembrane pH gradient (or alkaline intravesicular pH) is shown to occur in cytochrome oxidase vesicles with cytochrome c as the electron donor, in agreement with results of Moroney et al. [(1984) Biochemistry 23, 4991-4997] using hexaammineruthenium(II) as the reductant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure and function of Y chromosomal DNA

The sequence organization of four different families of Y chromosomal repetitive DNA is characterized at three levels of spatial extension along the Y chromosome of Drosophila hydei. At the lowest level of resolution, DNA blot analysis of Y chromosomal fragments of different lengths and in situ hybridization experiments on metaphase chromosomes demonstrate the clustering of each particular sequence family within one defined region of the chromosome. At a higher level of resolution, family specific repeats can be detected within these clusters by crosshybridization within 10–20 kb long continuous stretches of cloned DNA in EMBL3 phages. At the highest level of resolution, detailed sequence analysis of representative subclones about 1 kb in length reveals a satellite-like head to tail arrangement of family specific degenerated subrepeats as the building scheme common to all four families. Our results provide the first comparative sequence analysis of three novel families of repetitive DNA on the long arm of the F chromosome of D. hydei. Additional data are presented which support the existence of two related subfamilies of repetitive DNA on the short arm of the Y chromosome.