Nuclear magnetic resonance and molecular modeling studies on O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-se rine, a carbohydrate-protein linkage region fragment from connective tissue proteoglycans

The solution conformation of O-beta-D-galactopyranosyl-(1----4)-O-beta-D-xylopyranosyl-(1----0)-L-ser ine (GXS), a carbohydrate-protein linkage region fragment from connective tissue proteoglycans, was investigated by two-dimensional NMR spectroscopy and molecular modeling calculations. Specifically, the 1H and 13C resonances were assigned by 2D-COSY and by 1H-13C heteronuclear correlation spectroscopy methods. 2D-NOESY was used to generate distance constraints between the galactose and xylose and between the xylose and serine residues. The 1H vicinal coupling constants for the sugars and the serine were also determined. A general molecular modeling methodology suitable for complex carbohydrates was developed. This methodology employed molecular dynamics and energy minimization procedures together with the application of inter-residue spatial constraints across the linkages derived from 2D-NOESY. The first step in this methodology is the generation of a wide variety of starting conformations that span the (phi, psi) space for each linkage. In the present study, nine such conformations were constructed for each linkage using the torsion angles phi and psi corresponding to the gauche+, gauche-, and trans configurations across each of the two bonds constituting the linkage. These conformations were subjected to a combined molecular dynamics/energy minimization refinement using the NOESY derived constraints as pseudoenergy functions. Families of conformations for the whole molecule were then constructed from the structures derived for each linkage. Characterization of GXS using this methodology identified a single family of conformations that are consistent with the solution phase NMR data on this molecule.     

Monoclonal antibodies to a nitroxide lipid hapten

The isolation and characterization of a hybridoma cell line producing a monoclonal IgG₁ antibody against a spin-label nitroxide group is described. The antibody recognizes a synthetic hapten containing linked dinitrophenyl and 2,2,6,6-tetramethylpiperidinyl 1-oxy groups, having an affinity of 3.6±1.0·10⁶ M^?1 for the soluble hapten at 25°C. The antibody binds to phospholipid vesicles containing 2 mol% of spin label-derivitized lipid (lipid hapten) with an affinity of 1.5±0.2·10⁸ M^?1. This monoclonal IgG₁ mediates the binding of hapten-bearing lipid vesicles to mouse macrophage RAW264 cells bearing Fc receptors. The cellular responses to this binding are similar to those observed previously using polyclonal rabbit anti-hapten IgG. As with the heterogeneous antibodies, the monoclonal IgG₁ is more efficient in mediating cellular uptake when the vesicles are in the ‘fluid’ physical state (dimyristoylphosphatidylcholine at 37°C) compared to ‘solid’ (dipalmitoylphosphatidylcholine at 37°C). Despite the enhanced binding of ‘fluid’ phospholipid vesicles to cells, only the ‘solid’ vesicles triggered a significant respiratory burst in RAW264 macrophages.     

Pentacyclic steroids,part XVI,studies on the total syntheses of racemic 1,6-dithiabenz[3,4]estra-3, 5(10),8,14-tetraen-17-one and its D-homo analogue

The total syntheses of racemic 1,6-dithiabenz[3,4]-estra-3,5(10), 8,14-tetraen-17-one [VII]and 1,6-dithiabenz-[3,4]-D-homoestra-3,5(10),8,14-tetraen-17a-one [IX]starting fron isothiochroman-4-one [I]are described.     

Evolution of chromosomal abnormalities in sequential cytogenetic studies of ataxia telangiectasia

Summary Sequential cytogenetic studies of four patients with ataxia telangiectasia showed the progressive development of lymphocyte clones, each marked with a rearranged chromosome 14. Initial studies had shown random chromosomal breaks and rearrangements. Later studies in all patients showed nonrandom rearrangement of chromosome 14 with a breakpoint at 14q12 and with the distal segment translocated to either chromosome 14 or 7. The proportion of circulating lymphocytes carrying the marker tended to increase with time, accounting for the majority of the lymphocytes eventually in one case. The marked lymphocyte clones evolved further, as a result of loss of the small centric portions of the rearranged chromosome 14 (14pter14q12).Perhaps the abnormal clones in ataxia telangiectasia escape immunologic surveillance and flourish in an immunologically impaired environment. Subsequent to the loss of the centric portion of the rearranged chromosome 14, the cells may acquire additional capabilities that enhance malignant transformation.     

Proton NMR study of iron(II)-bleomycin: Assignment of resonances by saturation transfer experiments

The assignment of the paramagnetically shifted resonances of the Fe(II)-bleomycin complex in D₂O has been accomplished using the transfer of saturation method. A number of additional resonances arising from labile N$\text{H}$ protons which are shifted by the metal ion are observed in the ¹H spectrum of the complex in H₂O. The temperature dependence of the chemical shifts is consistent with the formation of an isolated 1:1 complex, but does not obey either the Curie Law or the Curie-Weiss Law. The magnitude of the shifts suggests that the valeric acid hydroxyl (or carbonyl) group, the α-amino group, the imidazole N^π, the carbamoyl oxygen, the pyrimidine N₁ and/or the secondary amino group may be coordinated to the iron(II).     

Purification and properties of milk-clotting enzyme from Bacillus subtilis K-26

A milk-clotting enzyme from Bacillus subtilis K-26 was purified by gel filtration and ion-exchange chromatography resulting in a 24-fold increase in specific activity with an 80% yield. Polyacrylamide gel electrophoresis and ultracentrifugel analysis revealed that the purified enzyme was homogeneous and had a molecular weight of 27,000 and a K_m of 2.77mg/ml for κ-casein. The enzyme was most stable at pH 7.5 and showed increasing clotting activity with decrease in milk pH up to 5.0. The maximum milk-clotting activity was obtained at 60°C, but the enzyme was inactivated by heating for 30 min at 60°C. The enzyme was irreversibly inhibited by EDTA and unaffected by DFP. Heavy-metal ions (Hg²⁺, Pb²⁺) inactivated the enzyme.     

Differential phosphorylation by GTP and ATP in isolated rod outer segments of the retina.

Isolated bovine rod outer segment protein is phosphorylated with GTP-gamma-32P and ATP-gamma 32P and to a much lesser extent by CTP-gamma-32P and UTP-gamma-32P. Phosphorylation with both GTP (GTP-kinase activity) and ATP (ATP-kinase activity) is markedly stimulated by light; phosphorylation with GTP is lower in dark-adapted and higher in light-adapted rod outer segments than is phosphorylation with ATP. Km values of 20 and 200 muM and Vmax values of 2.1 and 5.9 nmol/(mg min(-1)) were calculated using ATP and GTP, respectively, in light-adapted outer segments. When outer segments are incubated with GTP-gamma-32P under the usual conditions employed in these experiments, no formation of ATP-gamma-32P was detected by the techniques of high-pressure liquid chromatography and thin-layer chromatography. In intact, light-bleached outer segments, GTP appears to specifically phosphorylate rhodopsin. Histone and phosvitin are not phosphorylated to any appreciable extent by GTP. Histone appears to block rhodopsin phosphorylation by GTP while histone and, to some extent, phosvitin, both act as substrates for ATP-kinase activity. Cyclic AMP and other adenine derivates have a marked inhibitory effect on GTP-kinase activity. Phosphate also inhibits GTP-kinase activity but stimulates ATP-kinase activity. Such differences in phosphorylation with GTP and ATP indicate that these activities are either due to separate enzyme systems or, if only one enzyme is involved, the activities are under separate physiological control in the photoreceptor unit.     

Studies on the production of bacterial rennet in a pilot plant fermentor

An attempt was made to find out the optimum aeration and agitation rates on the production of bacterial rennet from Bacillus sublilis K-26 using 5% wheat bran medium in a 13 liter fermentor. The enzyme activity and the growth rate were shown to increase with an increase in the rate of agitation. The fermentation experiments carried out at an agitation rate of 400 rpm showed an approximate threefold increase in enzyme activity with a considerable decrease in the fermentation time over those agitated at 200 and 300 rpm. The beneficial effect of a higher oxygen rate was observed for enzyme production occurring at a lower agitation rate. The inoculum activity and the varying amounts of antifoam agent which were added showed no apparent effect either on the total incubation time or on the final enzyme activity. It has been suggested that an agitation rate of 400 rpm with an aeration level of 3000 cc/min are the optimum values for the efficient production of bacterial rennet from B. subtilis K-26 using 5% wheat bran medium in a 13 liter fermentor.     

NMR study of in vivo RIF-1 tumors: Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the ³¹P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the ³¹P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The ¹H and ¹³C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.     

A Combination Supplement of Fructo- and Xylo-Oligosaccharides Significantly Abrogates Oxidative Impairments and Neurotoxicity in Maternal/Fetal Milieu Following Gestational Exposure to Acrylamide in Rat

Neurochemical Research - Prebiotic oligosaccharides are demonstrated to confer a wide spectrum of physiological benefits during pregnancy. In view of this, focused attempts are being directed...