Phosphate effects in the fermentation of α-amylase by Bacillus amyloliquefaciens

Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding.     

Effect of water activity on enzymatic synthesis of cephalexin

Summary In enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D--phenylglycine methyl ester (PGM) using an acylase fromXanthomonas citri, it was found that the synthetic activity and conversion yield were enhanced markedly by depressing the water activity (a _w ) of reaction system. This enhancement was probably resulted from the change of thermodynamic equilibrium and maximized at a range ofa _w from 0.96 to 0.97.     

Heat generation in laser irradiated tissue

Many medical applications involving lasers rely upon the generation of heat within the tissue for the desired therapeutic effect. Determination of the absorbed light energy in tissue is difficult in many cases. Although UV wavelengths of the excimer laser and 10.6 microns wavelength of the CO2 laser are absorbed within the first 20 microns of soft tissue, visible and near infrared wavelengths are scattered as well as absorbed. Typically, multiple scattering is a significant factor in the distribution of light in tissue and the resulting heat source term. An improved model is presented for estimating heat generation due to the absorption of a collimated (axisymmetric) laser beam and scattered light at each point r and z in tissue. Heat generated within tissue is a function of the laser power, the shape and size of the incident beam and the optical properties of the tissue at the irradiation wavelength. Key to the calculation of heat source strength is accurate estimation of the light distribution. Methods for experimentally determining the optical parameters of tissue are discussed in the context of the improved model.     

Molecular dynamics of the alpha-helical epitope of a novel synthetic lipopeptide foot-and-mouth disease virus vaccine

A novel synthetic foot-and-mouth disease virus (FMDV) peptide vaccine consisting of a synthetic B-cell and macrophage activator covalently linked to an amphiphilic alpha-helical T-cell epitope was developed. The low molecular weight vaccine of 3400 daltons is composed of virus VP1 antigenic determinant and the immunologically active lipotripeptide tripalmitoyl-S-glyceryl-cysteinyl-seryl-serine (P3CSS) as built-in adjuvant. The vaccine, tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl-FMDV-VP1 (VP1 = serotype O1K 135-154) induces protection against homologous challenge and serotype-specific virus neutralizing antibodies in guinea pigs after single administration without further adjuvants or carriers. A P3CSS conjugate with the FMDV-VP1 segment 135-154 of strain O Wuppertal produced only poor cross-protection against challenge with O1K virus. The antigenic determinant VP1(135-154) is an amphiphilic alpha-helix, as shown by CD. Molecular dynamics simulations (MDS) carried out using the highly homologous alpha-helical alcohol dehydrogenase (ADH) segment H3 as starting conformation for VP1(138-149) suggest that the FMDV segment 138-149 may adopt alpha-helical conformation during binding to its T-cell receptor, and that the development of the system during MDS may be considered as the dissociation step of the complex.     

New mutations fts-36, lts-33, and ftsW clustered in the mra region of the Escherichia coli chromosome induce thermosensitive cell growth and division.

Three new mutants of Escherichia coli showing thermosensitive cell growth and division were isolated, and the mutations were mapped to the mra region at 2 min on the E. coli chromosome map distal to leuA. Two mutations were mapped closely upstream of ftsI (also called pbpB), in a region of 600 bases; the fts-36 mutant showed thermosensitive growth and formed filamentous cells at 42 degrees C, whereas the lts-33 mutant lysed at 42 degrees C without forming filamentous cells. The mutation in the third new thermosensitive, filament-forming mutant, named ftsW, was mapped between murF and murG. By isolation of these three mutants, about 90% of the 17-kilobase region from fts-36-lts-33 to envA could be filled with genes for cell division and growth, and the genes could be aligned.     

Identification of an unusual deletion within homologous repeats of human reticulocyte beta-spectrin and probable peptide polymorphism

We screened two different human reticulocyte cDNA libraries with beta-spectrin(beta Sp)-specific polyclonal antibodies and with our original radiolabeled human BSP cDNA probe (encoding beta Sp). Of the 20 independent clones, the largest had about a 2.5-kb insert corresponding to the deduced amino acid (aa) sequence of the beta-7 to beta-14 repetitive segments. Among these segments, segment 12 was 7 aa shorter than the other repetitive segments. We showed that this truncation was not a result of (i) cloning artifact, (ii) alternate splicing, or (iii) common genomic polymorphism by additional examination of 14 individual human chromosomes. Recently, another laboratory described the BSP nucleotide (nt) sequence overlapping partially with our sequence. These overlapping sequences were homologous with the exception of two nt differences at the positions 1342 and 1514. The discrepancy at nt 1342 changes the His to Arg. This newly derived probe has been used to find an additional example of BSP restriction fragment length polymorphism.     

Identification of an amino acid substitution in the benA, beta-tubulin gene of Aspergillus nidulans that confers thiabendazole resistance and benomyl supersensitivity

We are using molecular genetic techniques to identify sites of interaction of beta-tubulin with benzimidizole anti-microtubule agents. We have developed a marker-rescue technique for cloning mutant alleles of the benA, beta-tubulin gene of Aspergillus nidulans and have used the technique to clone two mutant benA alleles, benA16 and benA19. These are the only A. nidulans alleles known to confer resistance to the benzimidazole antimicrotubule agent thiabendazole and supersensitivity to other benzimidazole antimicrotubule agents including benomyl and its active breakdown product, carbendazim. benA16 has been shown, moreover, to reduce thiabendazole binding to beta-tubulin. We have sequenced the two mutant alleles and have found that they carry different nucleotide changes that cause the same single amino acid substitution, valine for alanine at amino acid 165. Since thiabendazole and carbendazim differ at only one side chain, the R2 group, we conclude that the region around amino acid 165 is involved in the binding of the R2 group of benzimidazole antimicrotubule agents to beta-tubulin.     

Ethylene production by sunflower cell suspensions : effects of plant growth retardants

From a variety of undifferentiated plant cell suspensions, 2,4-dichlorophenoxyacetic acid-dependent cells of sunflower (Helianthus annuus L. Spanners Allzweck) produced large quantities of ethylene. The maximum rate was about 1 nanomole × gram fresh weight⁻¹ × hour⁻¹ during the exponential growth phase. The action of various compounds known to interfere with ethylene formation in plant tissue was studied in sunflower cell suspensions. The influence on ethylene, 1-aminocyclopropanecarboxylic acid (ACC), and N-malonyl-ACC (MACC) levels suggested that the final steps in ethylene synthesis resemble those of other plant systems. This makes sunflower cells suitable for analyzing the effects of biologically active compounds on cellular ethylene biosynthesis. In particular, plant growth retardants of the norbornenodiazetine and triazole type inhibited ethylene production of sunflower cells. On the other hand, the ACC level was considerably elevated while that of MACC did not change significantly. It is assumed that the conversion of ACC to ethylene catalyzed by the ethylene-forming enzyme was influenced.