A complete library of amino acid alterations at N304 in Streptomyces clavuligerus deacetoxycephalosporin C synthase elucidates the basis for enhanced penicillin analogue conversion

N304 of Streptomyces clavuligerus deacetoxycephalosporin C synthase was mutagenized to alter its catalytic ability. Given that N304A, N304K, N304L, and N304R mutant enzymes exhibited significant improvements in penicillin analogue conversions, we advocate that replacement of N304 with residues with aliphatic or basic side chains is preferable for engineering of a hypercatalytic enzyme.     

Macleya cordata and Prunella vulgaris in oral hygiene products - their efficacy in the control of gingivitis

A double-blind, placebo-controlled clinical trial was performed to investigate the effectiveness of a herbal-based dentifrice in the control of gingivitis. Forty volunteers completed the 84-day study. All subjects were balanced for parameters measured - plaque index (PI), community periodontal index of treatment needs (CPITN) and papillary bleeding index (PBI). The dentifrice was effective in reducing symptoms of gingivitis as evaluated by the CPITN and PBI indexes.     

Effect of abietane diterpenes from Plectranthus grandidentatus on T- and B-lymphocyte proliferation

Five known abietane diterpenes of the royleanone and coleon type, namely, fatty acid esters of 7alpha-acyloxy-6beta-hydroxyroyleanone (1), grandidone A (2), 7alpha-acetoxy-6beta-hydroxyroyleanone (3), 6beta,7alpha-dihydroxyroyleanone (4) and coleon U (5), isolated from Plectranthus grandidentatus, were evaluated for their effect on the proliferation of human lymphocytes induced by the mitogen PHA. All except 4, showed a dose-dependent suppressor effect, with 3 yielding the most potent antiproliferative activity, followed by 5. These two compounds, that represent diterpenes of the royleanone and coleon type respectively, were also shown to be potent inhibitors of mouse splenocyte proliferation induced by ConA or LPS mitogens. However, the sensitivity of ConA-stimulated splenocytes to their suppressive effect was higher, suggesting a preferential inhibition of T-lymphocyte proliferation. The antiproliferative activity of 3 seemed to be exerted without affecting the expression of the lymphocyte activation marker CD69. On the contrary, 5 was shown to reduce the expression of CD69 of TCD8(+) and B-cells, suggesting a relationship between its antiproliferative effect and the expression of this early marker of activation on these cell populations. The capacity of 5 to induce apoptosis on ConA-stimulated splenocytes could also be related with its antiproliferative activity.     

Synthesis and characterisation of a new amphotericin B-methoxypoly(ethylene glycol) conjugate

The reaction of methoxypoly(ethylene glycol)-4-nitrophenyl carbonate with amphotericin B has been used to prepare a new conjugate of amphotericin B (mPEG-AmB). A preliminary screening of in vitro antifungal activity has suggested that mPEG-AmB possesses a similar effect and a similar spectrum of activity as the conventional amphotericin B formulated with sodium desoxycholate.     

Purification and characterization of two serine protease inhibitors from the hemolymph of Mythimna unipuncta

Two serine protease inhibitors, trypsin inhibitor and alpha-chymotrypsin inhibitor, were isolated from the hemolymph of Mythimna unipuncta. Mythimna trypsin and alpha-chymotrypsin inhibitors were purified by gel filtration and anion-exchange chromatography. They displayed molecular masses of 52 kDa and 43 kDa, respectively, as determined by electrophoresis under reducing and non-reducing conditions on denaturing polyacrylamide gels. Their isoelectric points were evaluated by isoelectric focusing and two-dimensional electrophoresis. Their N-terminal sequences have been analyzed as APSDTTIAETLTITEEFFPD and FDESFGFQGPSTYEKTPLGEP, respectively. The role of these inhibitors in the regulation of the defense reaction of the insect is discussed.     

Numerical-experimental method for the validation of a controlled stiffness femoral prosthesis

The aim of this paper is to describe a new numerical-experimental method to determine the stiffness of a conceptual proximal femoral prototype. The methodology consists of the comparison of the numerical and experimental displacement distributions of the prosthesis loaded as a cantilever beam to validate a design concept: controlled stiffness prosthesis. The manufactured prototype used to test the applicability of the numerical-experimental procedure integrates a stiff metal core bonded to a composite material made of an epoxy resin reinforced with carbon-glass braided pre-forms. The prosthesis with an embedded controlled stiffness concept was obtained by varying the geometry of the core with the composite layer thickness.     

Tyrosine Hydroxylase Dephosphorylation by Protein Phosphatase 2A in Bovine Adrenal Chromaffin Cells

This study was undertaken to characterise the protein phosphatases in bovine adrenal chromaffin cells acting on tyrosine hydroxylase. Cells were pre-labelled with ³²P_i and permeabilized with digitonin. The extent of dephosphorylation of Ser-8, Ser-19, Ser-31 and Ser-40 on tyrosine hydroxylase was found to be 30%, 38%, 37% and 71% respectively over 5 min. For Ser-19, Ser-31 and Ser-40 the dephosphorylation was entirely due to protein phosphatase 2A, as the dephosphorylation could be completely blocked by microcystin, but not by the protein phosphatase 1 inhibitory peptide. Permeabilization did not change the distribution of protein phosphatase 2A or tyrosine hydroxylase, or the activity of PP2A, from that occurring in intact cells. The dephosphorylation of Ser-8 was not altered by any inhibitor, suggesting the involvement of other protein phosphatases. The method developed here can be used to determine the protein phosphatases acting on substrates in conditions closely approximating those in situ, including the endogenous state of substrate phosphorylation and phosphatase location.     

DNA adduct formation from quaternary benzo[c]phenanthridine alkaloids sanguinarine and chelerythrine as revealed by the 32P-postlabeling technique

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.     

Beta-carbolines as specific inhibitors of cyclin-dependent kinases

Harmine (3), 7-fluoro-1-methyl beta-carboline (35) and 1-(5-methyl-imidazol-4-yl) beta-carboline (41) were potent and specific inhibitors of cyclin-dependent kinases. The degree of aromaticity of the tricyclic ring and the positioning of substituents are important for inhibitory activity. While most beta-carbolines inhibited CDK2 and CDK5 to the same extent, selective inhibition against CDK2 was observed in 1-(2-chlorophenyl)- (12), 1-(2-fluorophenyl)- (15), and 1-(2-chloro-5-nitrophenyl)- (28) beta-carbolines.