Enantioselective reductive amination of alpha-keto acids by papain-based semisynthetic enzyme.

Alkylation of a cysteine residue in papain with a pyridoxamine (PX) cofactor was carried out. The resulting semisynthetic enzyme (papain-PX) has no detectable protease activity but has the ability to catalyze enantioselective reductive amination of alpha-keto acids. The conjugate was characterized by ion-exchange chromatography, and the optimal reaction conditions were found. We report that papain-PX reductively aminates the alkyl side chain of functionalized alpha-keto acids to give the respective alpha-amino acids with high enantioselectivities, greater than 70%. Based on these studies, we propose a new model for the catalytic activity of the semisynthetic enzyme with Interchem software. The results of the study demonstrate the effectiveness of the modified enzyme and its potential for engineering new catalytic specificity.

Structural features and anti-HIV-1 activity of novel polysaccharides from red algae Grateloupia longifolia and Grateloupia filicina.

Since sulphated polysaccharides have antiviral activity in vitro, we examined the structure and antiretroviral activity of native sulphated galactans extracted from the red algae, Grateloupia filicina (GFP) and Grateloupia longifolia (GLP). The sulphate contents of GFP and GLPE (the 1,4-alpha-d-glucan-glucanohydrolase digest of GLP) were 25.7 and 18.5%, respectively. The sulphate ester groups were located at carbon 2 for GFP and at carbon 2 and 6 for GLPE. Antiretroviral activity was investigated with a primary isolate (PI) of HIV-1 and human peripheral blood mononuclear cells (PBMCs) rather than T-cell line adapted (TCLA) HIV-1 and T-cell lines because it is more representative of the in vivo situation. Both compounds and their derivatives had potent anti-HIV-1 activity when added at the time of infection, and 2h post-infection (EC50s 0.010-0.003microM, EC(90s) 0.87-0.33microM) and low cytotoxicity. Their potential medical application as virucidal vaginal formulations is discussed.

Effect of phenothiazines on Hymenolepis diminuta with special reference to the brush border Ca2+-dependent ATPase.

Incubation of Hymenolepis diminuta with the calmodulin antagonist trifluoperazine causes lesions in the brush border of the cestode. Exposure to a phenothiazine of lower lipophilicity, trifluoperazine sulphoxide, had little effect. Characterisation of isolated brush border revealed two forms of Ca2+-ATPase which exhibited maximum activity at pH 5.5 and 7.5. Both forms were Ca2+-dependent but only the latter was influenced by calmodulin and trifluoperazine. It is suggested that the Ca2+-ATPase present in the tapeworm brush border may be the site of trifluoperazine toxicity.

Evidence for a Ca2+-dependent activator protein in the rat tapeworm Hymenolepis diminuta.

A low molecular weight, acidic, heat stable protein has been characterised from the rat tapeworm Hymenolepis diminuta. This protein was found to activate cyclic 3', 5'-nucleotide phosphodiesterase in a Ca2+-dependent manner. The activation process was inhibited by the phenothiazine drug trifluoperazine. The biochemical properties of this protein clearly resemble those of ovine brain calmodulin. Our investigation thus concludes that there is a calmodulin-like activator protein in this cestode.

Sulphated glycoproteins in synaptosomes.

Synaptosomes from sheep brain were incubated with Na2(35)SO4. After lysis at least twelve 35S-containing proteins were identified in the synaptosomal soluble fraction with molecular weights ranging between 13,000 and 150,000. Extraction of the particulate fraction with SDS/urea revealed a further eight [35S]proteins with 27,000-140,000 molecular weights. A glycopeptide rich in fucose, sialic acid, N-acetylglucosamine and galactose was isolated from papain treated synaptosomal material. Over half of the hexosamine and neutral sugar residues were found to contain 35S. These findings suggest that sulphation of glycoproteins can occur in isolated nerve terminals.

Bovine corneal stroma contains a structural glycoprotein located in the gap region of the collagen fibrils.

Treatment of bovine corneal stroma using SDS-containing extracting solutions removes a 135,000 MW glycoprotein from the main collagen framework of the tissue. Low-angle synchrotron X-ray diffraction patterns obtained from corneas extracted in this way indicate that the glycoprotein has been removed from the gap regions of the collagen fibrils and is thus an important structural component of the corneal stroma. The glycoprotein (GP 135) shares a number of properties with one of the subunits of type VI collagen, but tests have so far failed to establish their identity.

Laccase-mediated system pretreatment to enhance the effect of hydrogen peroxide bleaching of cotton fabric.

This study evaluates the bleaching efficiency of the hydrogen peroxide bleaching process combined with laccase-mediated system pretreatment (LMS-HPBP) in the treatment of scoured cotton fabric. By changing the factors of laccase-mediated system pretreatment and the hydrogen peroxide bleaching process and examining the subsequent whiteness value and retained tensile strength of the samples, we find three LMS-HPBP processes that are more environment friendly than the conventional hydrogen peroxide bleaching process (CHPBP): (i) bleaching with lower dosage of hydrogen peroxide; (ii) bleaching at reduced temperature; (iii) bleaching for shortened duration. Whiteness, retained tensile strength and K/S values of cotton fabric samples treated by i-iii processes were similar to or higher than those by CHPBP. X-ray diffraction (XRD) analysis also demonstrated that the three processes rendered fabric of both lower crystallinity and bigger crystallite size than those by CHPBP. In addition, the "green" short-flow process was developed to treat cotton fabric and the results obtained shows this method is feasible as a new energy-saving process.

Classification and differentiation of the genus Peganum indigenous to China based on chloroplast trnL-F and psbA-trnH sequences and seed coat morphology.

The genus Peganum (Zygophyllaceae) consists of six species and one subspecies; three of which are distributed in China, P. harmala Linn, P. nigellastrum Bunge and P. multisectum (Maxim.) Bobr. A probable new or intermediate species, Peganum sp., has been suggested in the wild in northwest China. Traditional classification in genus Peganum has focused on hairs on the plant surface. In this study, seed coat characteristics of Peganum species were investigated using light and scanning electron microscopy, demonstrating clear differences in morphology between species. In addition, DNA sequence data from two sequences (chloroplast: trnL-F, psbA-trnH) were used to differentiate Peganum sp. and study polygenetic relationships. Diversity in DNA sequences among Peganum species was found, with inter-specific sequence divergence ranging from 0.6% to 5.6% in psbA-trnH, and 0.0% to 1.8% in trnL-F. The variations within species were low: from 0.0% to 0.4% in psbA-trnH and 0.0% to 0.4% in trnL-F. Therefore, Peganum species can now be easily identified as separate entities based on variations in DNA sequences. Phylogenetic trees were constructed from the combined data set for the two gene fragments, and the results indicate that Peganum sp. is monophyletic and sister to P. harmala and P. nigellastrum, while P. multisectum is also monophyletic. DNA data further confirmed that P. multisectum is an independent species and that a new species, Peganum sp., exists within the genus Peganum growing wild in China.

The distribution of calmodulin/calmodulin binding proteins in the rat tapeworm, Hymenolepis diminuta.

Live tapeworms have been fixed to retain antigenicity of their proteins, and subsequently prepared for electron microscopy. Thin sections of tapeworms were prepared from resin blocks. Sections were immunocytochemically labelled using a colloidal gold probe and viewed using transmission electron microscopy. Calmodulin was detected associated with cellular structures to which calmodulin has previously been linked in other higher eukaryotes. Calmodulin would appear to have a similar role of importance in tapeworms, as it does in higher eukaryotes although tapeworms are prevalently a syncitium.

Astragaloside IV and polysaccharide production by hairy roots of Astragalus membranaceus in bioreactors.

Hairy roots of Astragalus membranaceus were grown in bioreactors up to 30 l for 20 d. Cultures from a 30 l airlift bioreactor gave 11.5 g l dry wt with 1.4 mg g(-1) astragaloside IV, similar to cultures from 250 ml and 1 l flasks, but greater than yields from a 10 l bioreactor (dry wt 9.4 g l(-1), astragaloside IV 0.9 mg g(-1)). Polysaccharide yields were similar amongst the different bioreactors (range 25-32 mg g(-1)). The active constituent content of the cells approached that of plant extracts, indicating that large scale hairy root cultures of A. membranaceus has the potential to provide an alternative to plant crops without compromising yield or pharmacological potential.

Subunit interactions and catalytic activity of triose phosphate isomerase.

Rabbit and chicken triose phosphate isomerase were labelled with iodo-(1-14C)-acetamide. The efficiencies of renaturation after denaturation in guanidinium chloride were similar to those of the unlabelled enzymes. The enzymes were immobilised to Sepharose 4B and after guanidinium treatment only immobilised monomers remained on the gel. Hybridisation studies demonstrated that the dimeric form could be reformed on the denatured gels with recovery of the original specific activity. Under similar conditions formation of rabbit-chicken dimers were observed. However, competition studies demonstrated a preferential formation of the homogeneous dimers.

Ca2+ inhibition of brush border alkaline phosphatase activity in Hymenolepis diminuta.

The brush border membrane of Hymenolepis diminuta contains several Ca2+-dependent enzymes. Following our isolation of a Ca2+-dependent modulator protein we examined the kinetic properties of the brush border marker alkaline phosphatase from fractionated and crude tegument. We show that this enzyme is inhibited by Ca2+ concentrations approaching those in the calcareous corpuscles of H. diminuta.

Biochemical and ultrastructural investigation of the effect of Stelazine (trifluoperazine) on Hymenolepis diminuta (Cestoda).

The effects of the phenothiazine, Stelazine, on Hymenolepis diminuta were investigated. The cestode was incubated for 10 min at 37 degrees C with 1 mM trifluoperazine, in the presence and absence of Ca2+. Assay of brush border enzymes showed that drug treatment lowered the activities of alkaline phosphatase, Ca2+-ATP'ase, 5'-nucleotidase and type 1 phosphodiesterase. This occurred in parallel with a significant reduction in tegumental protein. Under these conditions gross changes in ultrastructural appearance and cellular organization were observed. There was a lack of ordered microtriches and the distal cytoplasm was absent. Glycogen granules were scattered throughout the cytoplasm within the subtegumental layer. The connective tissue also appeared to be in some disarray. The effects of Stelazine appeared to be dependent on time and were significantly increased when Ca2+ was included in the incubation medium. Incubation with the less hydrophobic phenothiazine trifluoperazine sulphoxide had minimal effect on the integrity of the cestode. The results reported here support the premise that certain phenothiazines may be considered as potential cestocidal agents.

Identification of ADPR-transferase activity in the rat tapeworm, Hymenolepis diminuta.

1. The nuclear fraction of the rat tapeworm Hymenolepis diminuta (Cestoda) contains the enzyme adenosine diphosphoribosyl transferase (ADPR-transferase). 2. The enzyme catalyzes the postsynthetic modification of some nuclear proteins by the covalent attachment of the (ADP-ribose) moiety of NAD to such proteins. 3. The reaction is dependent on DNA which contains strand-breaks, and chain lengths equivalent to (ADP-ribose) is estimated. 4. The formation of polynucleotide products was competitively inhibited by 3-acetamidobezamide, with a Km of 125 microM. 5. The catalytic properties of ADPR-transferase in Hymenolepis diminuta are similar to those in T. brucei.