Neoflavonoids and the cinnamylphenol kuhlmannistyrene from Machaerium kuhlmannii and M. Nictitans

The trunkwood of Machaerium kuhlmannii contains methyl palmitate, 3-O-acetyloleanolic acid and sitosterol; the benzene derivatives 2,3-dimethoxyphenol, 2,6-dimethoxyphenol, 2-hydroxy-3-methoxyphenol, 2,3-dimethoxybenzaldehyde and methyl 3-(2-hydroxy-4-methoxyphenyl)-propionate; the isoflavonoids formononetin and (6aS,11aS)-medicarpin; the neoflavonoids (R)-3,4-dimethoxydalbergione, (R)-3,4-dimethoxydalbergiquinol, kuhlmanniquinol [(R)-3-(4-hydroxyphenyl)-3-(5-hydroxy-2,3,4-trimethoxyphenyl)-propene], dalbergin, kuhlmannin (6-hydroxy-7,8-dimethoxy-4-phenylcoumarin) and kuhlmannene (6-hydroxy-7,8-dimethoxy-4-phenylchrom-3-ene), as well as the cinnamylphenol kuhlmannistyrene [Z-1-(5-hydroxy-2,3,4-trimethoxybenzyl)-2-(2-hydroxyphenyl)-ethylene]. Five of these compounds, in addition to (R)-4′-hydroxy-3,4-dimethoxydalbergione, were also isolated from a trunkwood extract of M. nictitans. Structural assignments were confirmed by chemical interconversion and by the synthesis of (±)-kuhlmanniquinol.     

Cytosolic calcium dependent neutral proteinase of human erythrocytes: the role of calcium ions on the molecular and catalytic properties of the enzyme

The soluble neutral proteinase of human erythrocytes dissociates into constituent subunits of 80k and 30k in the presence of mM concentrations of Ca²⁺. Similarly the soluble natural inhibitor of this proteinase, of approximate molecular weight 240k, is dissociated into 60k subunits by mM concentrations of Ca²⁺. Removal of Ca²⁺ restores the native oligomeric structure of the proteinase and of the natural inhibitor. The formation of the native active enzyme or of the inactive enzyme-inhibitor complex depends on reversible association-dissociation processes mediated by Ca²⁺ concentration.     

Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily

The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.     

Synthetic guinea pig insulin B-chain C-terminal decapeptide: a novel immunogen for generating immunocytochemical-grade antisera

Antisera to guinea pig insulin are not commonly available, largely because of the short supply and limited immunogenicity of the intact hormone. To overcome these problems we have employed a novel reagent, synthetic guinea pig insulin B-chain C-terminal decapeptide, as a hapten for raising antibodies that react with intact guinea pig insulin. The decapeptide, coupled to bovine serum albumin, was successfully used as an immunogen in rabbits. The resulting anti-serum was employed for immunocytochemical staining of guinea pig insulin in pancreatic sections. The specificity of the staining was verified by both pre-absorption and pre-immune serum controls. The utility of this new antiserum for investigations of guinea pig insulin physiology is discussed.     

Single-crystal resonance Raman spectroscopy of site-directed mutants of cytochrome c peroxidase

Resonance Raman spectra are reported for single crystals of cytochrome c peroxidase (CCP) mutants, taken by using a microscope equipped with a variable-temperature stage. The spectra are similar to those observed for the mutant proteins in solution, but there are detectable differences having to do with the coordination and spin state of the heme. The Asn-235 mutant contains a mixture of six-coordinate high- and low-spin states with a detectably higher fraction of the former than in solution. Upon cooling even to 223 K, the heme is converted mostly to the low-spin form. The Phe-191 mutant likewise shows a high/low-spin six-coordinate mixture, together with a preponderant population of five-coordinate heme. Upon cooling, the high-spin six-coordinate population converts immediately to the low-spin form, while the five-coordinate population does so more slowly. This behavior is intermediate between that of native CCP and the Asn-235 mutant, consistent with an ancillary role for the normal Trp-191-Asp-235 H-bond in the proximal anchoring of the heme Fe. The Phe-51 mutant shows a dominant high-spin five-coordinate heme population in the single crystal, whereas in solution the six-coordinate form is dominant. This difference is mimicked by adding 2-methyl-2,4-pentanediol (MPD) to the solution and is attributed to the dehydrating effect of MPD, which is present during crystallization. Upon lowering the temperature, the five-coordinate heme converts partially to a six-coordinate high-spin form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the hydrogen peroxide-enzyme reaction for two cytochrome c peroxidase mutants

The bimolecular reaction between Escherichia coli-produced cytochrome-c peroxidase (CcP(MI)) and hydrogen peroxide is identical to that of native yeast cytochrome-c peroxidase (CcP) and hydrogen peroxide in the neutral pH region. Both enzymes have pH-independent bimolecular rate constants of 46 microM-1.s-1 for the reaction with hydrogen peroxide. A second mutant enzyme, E. coli-produced cytochrome-c peroxidase mutant with phenylalanine at position 191 (CcP(MI, F191)), has a pH-independent bimolecular rate constant for the hydrogen peroxide reaction of 65 microM-1.s-1, 40% larger than for CcP or CcP(MI). The initial peroxide-oxidation product of CcP(MI, F191) is an oxyferryl porphyrin pi-cation radical intermediate in contrast to the oxyferryl amino-acid radical intermediate formed upon oxidation of CcP or CcP(MI) with hydrogen peroxide. The reactions of all three enzymes with hydrogen peroxide are pH-dependent in KNO3-containing buffers. The reactions are influenced by an ionizable group, which has an apparent pKa of 5.4 in all three enzymes. The enzymes react with hydrogen peroxide when the ionizable group is unprotonated. Both CcP(MI) and CcP(MI, F191) have slightly smaller pH stability regions compared to CcP as assessed by the hydrogen peroxide titer and spectral analysis. The alteration in structural stability must be attributed to differences in the primary sequence between CcP and CcP(MI) which occur at positions -2, -1, 53 and 152.     

Preliminary study on the operating variables of SCP production from grape must

Summary The fermentation of grape must by Candida utilis ISS 28 was studied at different substrate concentrations, pH values, and nutrient supplementation in a shaken-flask fermenter, by using a composite design experiment.The experimental biomass yields were fitted to the only statistically significant factors with a mean standard error less than 8%, by using multiple regression analysis.Optimal conditions for maximum cell yield were established by plotting a series of loci at constant biomass yield and then verified experimentally, thus confirming the remarkable accuracy of the model