Effect of chemical modification on the binding of gossypol by gossypin (11S protein) and congossypin (7S protein) of cottonseed

Binding of gossypol by gossypin and congossypin and their succinylated and sulfhydryl group-blocked derivatives has been measured. The binding by gossypin and congossypin is characterized by weak interaction. Succinylation of gossypin decreases the binding affinity whereas that of congossypin increases it. Blocking of sulfhydryl groups of both the proteins does not significantly affect gossypol binding, Succinylation dissociates gossypin and causes conformational changes whereas it does not dissociate congossypin but causes conformational changes. Sulfhydryl group blocking does not dissociate gossypin or congossypin, nor does it cause any conformational changes.     

Light-adaptation in the photophobic response by Stentor coeruleus

Effects of preillumination on photophobic response (light-adaptation) and recovery of the photophobic sensitivity in the dark (dark-adaptation) in Stentor coeruleus were examined. When the cells were preilluminated with white light of 7.80 W/m² for 2 min, the fluence-rate response curve of photophobic response was shifted toward higher light intensities by half an order of magnitude compared to the one without preillumination. Preillumination with a higher light intensity resulted in a further shift of the fluencerate response curve. An action spectrum for light-adaptation showed a primary peak at 610 nm and secondary peaks at 540 and 480 nm which are almost identical to the peaks observed in the photophobic action spectrum.The light-adapted cells showed a recovery of their photophobic sensing ability following dark treatment. Dark-adaptation resulted in total recovery of photophobic sensing ability in 8 minutes for the most cases examined.     

Chromosomal location, cloning and nucleotide sequence of the Bacillus subtilis cdd gene encoding cytidine/deoxycytidine deaminase

Summary The Bacillus subtilis cdd gene encoding cytidine/2-deoxycytidine deaminase has been located by transduction at approximately 225 degrees on the chromosome, and the gene order rpC-lys-cdd-aroD was established. The gene was isolated from a library of B. subtilis DNA cloned in D69 by complementation of an Escherichia coli cdd mutation. Minicell experiments revealed a molecular mass of 14000 dalton for the cytidine deaminase subunit encoded by the cloned DNA fragment. The molecular weight of the native enzyme was determined to be 58000, suggesting that it consists of four identical subunits. The nucleotide sequence of 1170 bp, including the cdd gene, was determined. An open reading frame encoding a polypeptide with a calculated molecular mass of 14800 dalton was deduced to be the coding region for cdd. The deduced amino acid composition of the 136-amino acid-long subunit shows that it contains six cysteine residues. A computer search in the GenBank DNA sequence library revealed that the 476 bp HindIII fragment containing the putative promoter region and the first ten codons of cdd is identical to the P43 promoter-containing fragment previously isolated by Wang and Doi (1984). They showed that the fragment contained overlapping promoters transcribed by B. subtilis 43 and 37 RNA polymerase holoenzymes during growth and stationary phase.Abbreviations SDS sodium dodecyl sulphate - Ap ampicillin resistance - Tet^r tetracycline resistance - Km^r kanamycin resistance     

Field response of chickpea to inoculation withGlomus versiforme

The response ofCicer arietinum to inoculation withGlomus versiforme under field conditions was investigated in a phosphorus deficient sandy loam soil. Inoculation with the mycorrhizal fungusGlomus versiforme increased the rate of VAM development in chickpea. The weight of nodules and the number of nodules per plant were higher in inoculated than in uninoculated plants. The phosphorus content of the shoots and its total uptake, were increased by either the application of single super-phosphate, or by inoculation withG. versiforme. Inoculation increased shoot dry weights and grain yields by 12% and 25% respectively, as compared with the 33% and 60% increases respectively produced by P-treated plants.     

The association of H-2Ld with human beta-2 microglobulin induces localized conformational changes in the alpha-1 and- 2 superdomain

We have analyzed changes in the antigenicity of major histocompatibility complex class I molecules resulting from the association of human beta-2 micro-globulin (B2m) with the mouse class I heavy chain. In particular, the H-2L^d molecule exhibited enhanced crossreactivity for the 34-1-2 monoclonal antibody. In order to assess the nature of this structural alteration induced by human B2m, we utilized H-2 class I hybrid molecules in the mapping of the 34-1-2 determinant to the helical region of the alpha-1 domain. H-2L^d class I hybrid molecules were then used to establish the importance of the alpha-2 and- 3 domains in the observed increase of 34-1-2 cross-reactivity following exchange with human B2m. The H-2L^d hybrids suggest that alterations in interdomain contact are responsible for enhanced 34-1-2 cross-reactivity on the H-2L^d molecule. It is likely that this alteration arises through changes in class I conformation at regions of the molecule distant from points of contact between B2m and the class I molecule. This suggests that perturbations induced by association of human B2m with H-2L^d can affect the conformation of the alpha-1 and- 2 superdomain. That class I antigenic determinants are altered by the association of human B2m with mouse class I further suggests that the class I molecule is structurally flexible and may reflect the ability of the class I molecule to bind and present a vast array of disparate peptides to the T-cell receptor.     

Characterization and differential expression of human vascular smooth muscle myosin light chain 2 isoform in nonmuscle cells

The 20-kDa regulatory myosin light chain (MLC), also known as MLC-2, plays an important role in the regulation of both smooth muscle and nonmuscle cell contractile activity. Phosphorylation of MLC-2 by the enzyme MLC kinase increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. We have isolated and characterized an MLC-2 cDNA corresponding to the human vascular smooth muscle MLC-2 isoform from a cDNA library derived from umbilical artery RNA. The translation of the in vitro synthesized mRNA, corresponding to the cDNA insert, in a rabbit reticulocyte lysate results in the synthesis of a 20,000-dalton protein that is immunoreactive with antibodies raised against purified chicken gizzard MLC-2. The derived amino acid sequence of the putative human smooth muscle MLC-2 shows only three amino acid differences when compared to chicken gizzard MLC-2. However, comparison with the human cardiac isoform reveals only 48% homology. Blot hybridizations and S1 nuclease analysis indicate that the human smooth muscle MLC-2 isoform is expressed restrictively in smooth muscle tissues such as colon and uterus and in some, but not all, nonmuscle cell lines. Previously reported MLC-2 cDNA from rat aortic smooth muscle cells in culture is ubiquitously expressed in all muscle and nonmuscle cells, and it was suggested that both smooth muscle and nonmuscle MLC-2 proteins are identical and are probably encoded by the same gene. In contrast, the human smooth muscle MLC-2 cDNA that we have characterized from an intact smooth muscle tissue is not expressed in skeletal and cardiac muscles and also in a number of nonmuscle cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Primary photoprocesses of phytochrome. Picosecond fluorescence kinetics of oat and pea phytochromes

The primary photoprocesses of etiolated oat and pea phytochromes (Pr forms) are diffusion-modulated by the microscopic viscosity within the chromophore pocket. The chromophore pocket is preferentially accessible to glycerol but not to Ficoll. Glycerol preferentially retarded the rate (rate constant ca. 1-2 X 10(10) s-1) of the initial reaction from the Qy excited state of phytochrome, whereas it increased the long fluorescence lifetime (nanosecond) component that can be attributed to either an emitting intermediate or to modified/conformationally heterogeneous phytochrome populations. The picosecond time-resolved fluorescence spectra of different phytochrome preparations (i.e., full-length vs 6/10-kDa NH2-terminus truncated forms of phytochromes from monocot and dicot plants) revealed no significant differences. The spectra in the picosecond time scale showed no spectral shifts, but at longer time scales of up to approximately 1.90 ns, significant blue spectral shifts were observed. The shifts were more in the truncated than in the full-length pea phytochrome. Comparison of the fluorescence decay data and the picosecond time-resolved fluorescence spectra suggests differences in conformational flexibility/heterogeneity among the preparations of the monocot vs dicot phytochromes and the full-length native vs the amino terminus truncated phytochromes.     

Clonorchis sinensis: purification and characterization of a cysteine proteinase from adult worms

1. Adult Clonorchis sinensis, the Chinese liver fluke, is known to migrate to the bile ducts of its mammalian host and cause significant pathology. 2. An acidic, thiol-dependent proteinase with a native mol. wt of approximately 18,500 was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. By SDS-polyacrylamide gel electrophoresis, the mol. wt of the enzyme was estimated to be 15,000. 3. The enzyme was similar to cathepsin B-like cysteine proteinases based on pH optimum, substrate specificity, and inhibitor sensitivity. 4. Antisera from human clonorchiasis and C. sinensis-infected rabbits reacted in immunoblots with the partially purified proteinase. The C. sinensis proteinase may be useful for serodiagnosis of clonorchiasis.     

Modulation of phosphoinositide metabolism in rat brain slices by excitatory amino acids,arachidonic acid,and GABA

In rat brain slices the synthesis of [³H]phosphoinositides and the production of [³H]inositol monophosphate (IP₁) induced by norepinephrine (NE) were inhibited by glutamate. Calcium concentrations were varied to test if these inhibitory effects of glutamate were mediated by a calcium-dependent process. Although reducing calcium or addition of the calcium antagonist verpamil reduced the inhibitory effects of glutamate, these results were equivocal because reduced calcium directly decreased agonist-induced [³H]phosphoinositide synthesis. The inhibitory effects of glutamate were mimicked by quisqualate in a dose-dependent manner, but none of a variety of excitatory amino acid receptor antagonists modified the inhibition caused by quisqualate. It is suggested that glutamate activates a quisqualate-sensitive receptor (for which an antagonist is not available) and causes inhibition of phosphoinositide hydrolysis mediated in part by a direct or indirect inhibitory effect of calcium on phosphoinositide synthesis. Modulatory effects of arachidonic acid were examined because glutamate and calcium can activate phospholipase A₂. Arachidonic acid caused a rapid and dose-dependent inhibition of [³H]phosphoinositide synthesis and of NE-stimulated [³H]IP₁ production. A similar inhibition of the response to carbachol also occurred. The inhibition caused by arachidonic acid was unchanged by addition of inhibitors of cyclooxygenase or lipoxygenase. Activation of phospholipase A₂ with melittin caused inhibitory effects similar to those of arachidonic acid. Inhibitors of phospholipase A₂ were found to impair phosphoinositide metabolism, likely due to their lack of specificity for phospholipase A₂. Further studies were carried out in slices that were prelabelled with [³H]inositol in an attempt to separate modulatory effects on [³H]phosphoinositide synthesis and agonist-stimulated [³H]IP₁ production. Several excitatory amino acid agonists inhibited NE-stimulated [³H]IP₁ production. This inhibitory inter-action could be due to impaired synthesis of [³H]phosphoinositides because, even though the slices were prelabeled, addition of unlabelled inositol reduced NE-stimulated [³H]IP₁ production, indicating that continuous regeneration of [³H]phosphoinositides is required. In contrast to the inhibitory effects of the excitatory amino acids, gamma-aminobutyric acid (GABA) enhanced the response to NE in cortical and hippocampal slices. GABA also enhanced the response to carbachol in hippocampal and striatal slices and to ibotenic acid in hippocampal slices. Baclofen potentiated the response to NE similarly to the effect of GABA and baclofen partially blocked the inhibitory effect of arachidonic acid but did not alter that of quisqualate.Abbreviations AMPA -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid AP₄ dl-2-amino-4-phosphonobutyric acid - BPB bromphenacyl bromide - BSA bovine serum albumin - CNQX 6-cyano-7-nitroquinoxaline-2,3-dione - DFMO -difluoromethylornithine - DIDS diisothiocyanotostilbene-2,2-disulfonic acid - EGTA ethyleneglycol-bis-N - N, N N-tetraacetic acid - GABA -aminobutyric acid - GDEE glutamate diethyl ether - -GG -glutamylglycine - IP₁ inositol monophosphate - IP₂ inositol bisphosphate - IP₃ inositol trisphosphate - NDGA nordihydroguaiaretic acid - NE norepinephrine - NMDA N-methyl-d-aspartate     

Inhibitory insulin-like growth factor-binding protein: cloning, complete sequence, and physiological regulation.

In this study we report the preparation of a human osteosarcoma cell cDNA library and describe the isolation and sequence determination of a clone encoding the complete sequence of a novel human insulin-like growth factor (IGF)-binding protein (hIGFBP-4). Previous work indicated that hIGFBP-4 is the predominant IGFBP expressed by human osteoblast-like cells, and that IGFBP-4 binds and inhibits the mitogenic activities of IGF-I and IGF-II. Sequence determination revealed that hIGFBP-4 is a unique gene product with significant amino- and carboxy-terminal sequence similarity to three other known IGFBPs. Identical alignment of 18 cysteines in IGFBP-4 and the three other IGFBPs is a key structural feature of this protein family. In vitro studies of human osteoblast-like cells suggest that PTH regulates the expression of hIGFBP-4 and that the PTH effect is mediated through a cAMP mechanism. hIGFBP-4 mRNA was also expressed in skin fibroblasts, and thus, this inhibitory IGFBP could be an important physiological regulator of IGF actions in bone cells and other cell types as well.