1H NMR studies on ferricytochromec 3 fromDesulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I

Summary The¹H NMR signals of the heme methyl, propionate and related chemical groups of cytochromec ₃ fromDesulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry,29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val¹⁸. All the results contradicted the heme assignments forD.v. MF cytochromec ₃ made on the basis of electron spin resonance (Gayda et al. (1987)FEBS Lett.,217 57–61). Based on these assignments, the interaction of cytochromec ₃ withD.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochromec ₃ was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochromec ₃ molecules per monomer of ferredoxin I and 10⁸ M^–2 (at 53 mM ionic strength and 25°C), respectively.     

Measurement of the exchange rates of rapidly exchanging amide protons: Application to the study of calmodulin and its complex with a myosin light chain kinase fragment

Summary A technique is described for measuring the approximate exchange rates of the more labile amide protons in a protein. The technique relies on a comparison of the intensities in¹H–¹⁵N correlation spectra recorded with and without presaturation of the water resonance. To distinguish resonance attenuation caused by hydrogen exchange from attenuation caused by cross relation, the experiment is repeated at several different pH values and the difference in attenuation of any particular amide resonance upon presaturation is used for calculating its exchange rate. The technique is demonstrated for calmodulin and for calmodulin complexed with its binding domain of skeletal muscle myosin light chain kinase. Upon complexation, increased amide exchange rates are observed for residues Lys⁷⁵ through Thr⁷⁹ located in the central helix of calmodulin, and for the C-terminal residues Ser¹⁴⁷ and Lys¹⁴⁸. In contrast, a decrease in amide exchange rate is observed at the C-terminal end of the F helix, from residues Thr¹¹⁰ through Glu¹¹⁴.Istituto Guido Donegani, Novara, Italy     

3D structure of bovine pancreatic ribonuclease A in aqueous solution: An approach to tertiary structure determination from a small basis of1H NMR NOE correlations

Summary A method is proposed to generate initial structures in cases where the distance geometry method may fail, such as when the set of¹H NMR NOE-based distance constraints is small in relation to the size of the protein. The method introduces an initial correlation between the and backbone angles (based on empirical observations) which is relaxed in later stages of the calculation. The obtained initial structures are refined by well-established methods of energy minimization and restrained molecular dynamics. The method is applied to determine the solution structure of Ribonuclease A (124 residues) from a NOE basis consisting of 467 NOE cross-correlations (97 intra-residue, 206 sequential, 23 medium-range and 141 long-range) obtained at 360 MHz. The global shape and backbone overall fold of the eight final refined structures are close to those shown by the crystal structure. A meaningful difference in the positioning of the catalytically important His¹¹⁹ side chain in the solution and crystal structures has been detected.     

Some factors influencing the proportion of periplasmic hepatitisB virus pre-S2 antigen in the recombinant yeastHansenula polymorpha

Summary A central composite design (CCD) was used to evaluate, for the purpose of future process optimization, the influence of pH, yeast extract and ammonium chloride concentrations on the proportion of periplasmic hepatitisB pre-S2 antigen in the recombinant yeastHansenula polymorpha. Each factor was tested at five levels, and a second order polynomial model for the proportion of periplasmic antigen was fitted to the treatment combinations. pH showed the greatest effect: the proportion of periplasmic antigen was greatly increased at the higher pH levels. At the higher pH levels used, the proportion of periplasmic antigen was enhanced by a high concentration of ammonium chloride. Additional experiments have confirmed both the validity of the selected model and the optimal conditions found. A significant correlation was found between the proportion of periplasmic antigen and the total yield of antigen. These results indicated that is should be possible to modulate the distribution of the pre-S2 antigen between the periplasm and the cytoplasm of the yeast.     

Purification and properties of extracellularβ-fructofuranosidases fromAureobasidium sp. ATCC 20524

Summary Two extracellular -fructofuranosidases (E-1 andE-2) fromAureobasidium sp. ATCC 20524, producing 1-kestose (1^F--fructofuranosyl-sucrose) from sucrose, were purified to homogeneity. Molecular weights of the enzymes were estimated to be about 304000 (E-1) and 315000 (E-2) Da by gel filtration. The enzymes contained 33% (w/w) (E-1) and 27% (w/w) (E-2) carbohydrate. TheK _m values for sucrose ofE-1 andE-2 andE-2 were 0.34 and 0.28 M, respectively. were 0.34 and 0.28 M, respectively. The enzymatic profiles of these enzymes were almost identical to intracellular enzymesP-1 andP-2 except for the differences in carbohydrate content andK _m values ofE-2 andP-2.     

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila Serogroup 1

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.     

~(125)Ⅰ标记单抗LC-I与肺腺癌细胞体内外结合特性的研究

本文用~(125)Ⅰ标记LC-1进行了一些体内外实验。实验结果表明:LC-1单抗的结合常数为4.8×10~8M~(-1),LC-1针对的SPC-A_1细胞表面抗原的位点数为7.2×10~4/细胞;LC-1与LAC-122两单抗针对的抗原决定簇没有交叉;用蛋白酶和过碘酸钠处理SPC-A_1细胞,前者对LC-1的结合抑制39%,后者抑制66%;LC- 1不但有较强的体外结合靶细胞的能力,从LC-1在荷瘤裸鼠中的组织器官分布来看,LC-1与肿瘤有较高的体内亲和性,并且是特异性的结合。     

An antigen present in the Drosophila central nervous system only during embryonic and metamorphic stages

We report here about an antigen that is expressed in the central nervous system (CNS) of Drosophila only during the embryonic and metamorphic stages. In Drosophila, axonogenesis and synaptogenesis occur twice during the development: first in the embryonic and second in the metamorphic stages. We generated monoclonal antibodies (MAbs) in order to obtain molecular probes for analyzing axonogenesis or synaptogenesis in the CNS on the assumption that good candidates for molecules responsible for such phenomena must be present in the neuropil during those stages exclusively. As a result, we found MAb 66B2 whose intense immunoreactivity in the neuropil of the CNS was observed exclusively in the embryo and pupa, and not in the larva and adult. Immunoblot analyses showed that MAb 66B2 binds specifically to a protein with an apparent molecular weight of 350 K and neutral pl in the prepupal CNS. A significant amount of the antigen was isolated in forms that were soluble without detergent. Results of immunohistochemistry with MAb 66B2 in a primary culture of embryos showed that some live cells in the ganglion-like cluster were stained, and that neuronal cell bodies and neurites emanating from there were negative. These results strongly suggest that the 66B2 antigen observed in the CNS is an extracellular matrix component secreted from nonneuronal cells. These developmental changes in the 66B2 immuno-reactivity in the CNS presumably reflect dynamic changes of an extracellular matrix in the CNS that are accompanied by axonogenesis or synaptogenesis. © 1992 John Wiley & Sons, Inc.     

Interferon-γ activates the cleavage of double-stranded RNA by bovine seminal ribonuclease

Bovine seminal ribonuclease (BS-RNase), a dimeric homologue of RNase A, cleaves both single- and double-stranded RNA and inhibits the growth of tumor cells. Its catalytic activity against double-stranded RNA, either homopolymeric ([³H]polyA/polyU) or mixed sequence, is enhanced by bovine or human recombinant interferon-γ (IFN-γ). Activation is seen with as little as 4–10 interferon units per assay. Enhancing the degradation of double-stranded RNA, an intermediate in the growth cycle of many viruses, could contribute to IFN-γ's ability to control cell growth and induce an antiviral state.