Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.     

A polymerase chain reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region.

Under appropriate conditions, specific double-stranded DNA product was generated after amplification of genomic DNA sequences in a polymerase chain-like reaction that contained only a single primer. This type of amplification reaction was performed with a variety of primers and substrate DNAs. In addition to nonspecific heterogeneous products, 5 of 11 primers reproducibly directed synthesis of double-stranded DNA that corresponded to the region of the template that contained the authentic primer annealing site. Three of these amplified products were cloned and their ends were sequenced. All three contained a copy of the primer at both 5' ends, and the position of one of the primers represented the authentic primer binding site. In each case, the location of the second copy of the primer indicated that it had initially hybridized to a partially homologous sequence in the template DNA. This single primer reaction makes it possible to amplify and clone a DNA region of unknown sequence that is adjacent to a known DNA sequence. One of the single primer reaction products described here included sequence to the 5' side of the coding region of a serotonin receptor gene that contained a functional promoter.     

Plasmodium falciparum: gene structure and hydropathy profile of the major merozoite surface antigen (gp195) of the Uganda-Palo Alto isolate

The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.     

Amino acids of the Torpedo marmorata acetylcholine receptor alpha subunit labeled by a photoaffinity ligand for the acetylcholine binding site

The acetylcholine-binding sites on the native, membrane-bound acetylcholine receptor from Torpedo marmorata were covalently labeled with the photoaffinity reagent [3H]-p-(dimethylamino)-benzenediazonium fluoroborate (DDF) in the presence of phencyclidine by employing an energy-transfer photolysis procedure. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or presence of carbamoylcholine were cleaved with CNBr and the radiolabeled fragments purified by high-performance liquid chromatography. Amino acid and/or sequence analysis demonstrated that the alpha-chain residues Trp-149, Tyr-190, Cys-192, and Cys-193 and an unidentified residue(s) in the segment alpha 31-105 were all labeled by the photoaffinity reagent in an agonist-protectable manner. The labeled amino acids are located within three distinct regions of the large amino-terminal hydrophilic domain of the alpha-subunit primary structure and plausibly lie in proximity to one another at the level of the acetylcholine-binding sites in the native receptor. These findings are in accord with models proposed for the transmembrane topology of the alpha-chain that assign the amino-terminal segment alpha 1-210 to the synaptic cleft. Furthermore, the results suggest that the four identified [3H]DDF-labeled residues, which are conserved in muscle and neuronal alpha-chains but not in the other subunits, may be directly involved in agonist binding.     

Purification of duck growth hormone and cloning of the complementary DNA

Duck growth hormone (GH) was isolated and purified from duck pituitaries by salt precipitation and HPLC on reverse-phase C18 columns. The duck GH was homogeneous as shown by SDS-polyacrylamide gel electrophoresis with a molecular weight of 22,000. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The positive clones were selected and sequenced. The full-length duck GH cDNA contains 820 nucleotide pairs with an open reading frame coding for the precursor form duck GH of 216 amino-acid residues. The partial amino-acid sequence from the protein completely agrees with that derived from the cDNA, with Phe as the first residue in mature duck GH preceded by a 27-residue hydrophobic signal peptide. The duck GH is almost completely homologous to the chicken GH, with only three conservative substitutions (Ser for Thr, His for Tyr and Lys for Arg) and one deletion (Ala) in the duck GH sequence. Comparison of amino-acid sequence of duck GH with that of various species reveals 56%, 73% and 40% homologies with GHs of human, rat and salmon, respectively.     

Cloning and sequencing of bullfrog growth hormone complementary DNA

Total mRNA was isolated from the pituitary glands of bullfrog (Rana catesbeiana), purified by affinity chromatography with oligo(dT)-cellulose columns. The cDNA was synthesized and cloned in Escherichia coli using EcoRI linkers and pBR322 as vector. The cDNA library was screened by hybridization with 32P-labeled duck growth hormone (GH) cDNA. A positive clone was selected and sequenced. The full-length bullfrog GH cDNA contains 950 nucleotide pairs with an open reading frame coding for the precursor GH of 215 amino-acid residues. The partial amino-acid sequence from the protein confirms that derived from the cDNA, with Phe as the first residue in the mature bullfrog GH preceded by a 25-residue hydrophobic signal peptide. The bullfrog GH shares sequence homology with those of other vertebrate species in the following order: duck (61% protein sequence homology; 67% cDNA homology), rat (56%; 61%), human (47%; 57%) and salmon (42%; 50%).     

The effect of sulfhydryl oxidation on the morphology of immature hamster epididymal spermatozoa induced to acquire motility in vitro

Immotile spermatozoa from the caput epididymidis become progressively motile when incubated in medium containing theophylline, seminal plasma, and albumin. We previously reported that under these incubation conditions the spermatozoa induced to acquire motility exhibited a marked flagellar angularity, with the sperm head or midpiece bent 90-180 degrees towards the tail. In addition, we demonstrated that sperm flagellar bending did not occur when the sulfhydryl oxidant diamide was added to the motility induction medium. In the present study, we examined further the effect of sulfhydryl oxidation on the morphology and sulfhydryl content of immature caput spermatozoa induced to acquire motility in vitro. We found that flagellar bending was prevented and sperm flagellar straightness was maintained in a dose-dependent manner by diamide. Moreover, flow cytometric analysis of caput sperm sulfhydryls using the sulfhydryl reagent monobromobimane (mBBr) revealed that 1) diamide oxidizes caput sperm sulfhydryls, and 2) less than 15% of the total reactive sperm sulfhydryls were oxidized at diamide concentrations capable of preventing sperm angulation. Sodium tetrathionate (NaTT), another sulfhydryl oxidant, and hamster cauda epididymal fluid (CEF) containing sulfhydryl oxidase enzyme activity also maintained flagellar straightness in induced caput spermatozoa and oxidized sperm sulfhydryls. The flagellar straightness in caput spermatozoa treated with sulfhydryl oxidants, however, was temporary; with extended incubation, diamide- or CEF-treated spermatozoa exhibited flagellar bending. Additional studies showed that the flagellar straightness observed in sulfhydryl-oxidized spermatozoa was sustained when nitrofurantoin, an inhibitor of glutathione reductase, was included in the induction medium. Flow cytometric analysis of nitrofurantoin-treated spermatozoa showed that nitrofurantoin maintained the sperm disulfides formed by diamide and prevented the reduction of sperm disulfides back to sulfhydryls. Taken together, these studies demonstrate the significance of sulfhydryl oxidation in maintaining the morphology of immature caput epididymal spermatozoa induced to acquire motility in vitro and suggest that sulfhydryl oxidation may be important in the development of motility during sperm epididymal maturation in vivo.     

Syntheses and properties of circular dichroism active phospholipids

A group of circular dichroism (CD) active phospholipids has been synthesised, in which one or both acyl chains has been replaced with a cinnamoyl or azobenzene chromophore-containing acid. Studies on the structure, CD activity and thermodynamic property of liposome membranes composed of CD active phospholipids were carried out. CD active liposomes were found to be stable, normal liposomes of approximately 550 A diameter based on the electron micrograph and dynamic light scattering, and to have thermodynamic property similar to the conventional phospholipid membranes without serious perturbation by aromatic bulk groups based on DSC. Liposomes composed of phospholipid having two trans-azobenzene chromophores showed an extremely large CD enhancement even well above Tc. This CD enhancement was drastically changed by the presence of cis-azobenzene chromophore and cis-cis isomer content after irradiation was higher than the theoretical value, suggesting the importance of interchromophore interaction in the liposome membranes.