A single gene directs synthesis of a precursor protein with beta- and alpha-amylase activities in Bacillus polymyxa.

The Bacillus polymyxa amylase gene comprises 3,588 nucleotides. The mature amylase comprises 1,161 amino acids with a molecular weight of 127,314. The gene appeared to be divided into two portions by the direct-repeat sequence located at almost the middle of the gene. The 5' region upstream of the direct-repeat sequence was shown to be responsible for the synthesis of beta-amylase. The 3' region downstream of the direct-repeat sequence contained four sequences homologous with those in other alpha-amylases, such as Taka-amylase A. The 48-kilodalton (kDa) amylase isolated from B. polymyxa was proven to have alpha-amylase activity. The amino acid sequences of the peptides generated from the 48-kDa amylase showed complete agreement with the predicted amino acid sequence of the C-terminal portion. The B. polymyxa amylase gene was therefore concluded to contain in-phase beta- and alpha-amylase-coding sequences in the 5' and 3' regions, respectively. A precursor protein, a 130-kDa amylase, directed by a plasmid, pYN520, carrying the entire amylase gene, had both beta- and alpha-amylase activities. This represents the first report of a single protein precursor in procaryotes that gives rise to two enzymes.     

Amino acid residues stabilizing a Bacillus alpha-amylase against irreversible thermoinactivation

The alpha-amylase of Bacillus licheniformis (BLA) is stable and active at high temperature. More than 80% of its activity is retained after heat treatment at 90 degrees C for 30 min, and the optimum temperature for its activity is 80-85 degrees C. In contrast, the alpha-amylase of Bacillus amyloliquefaciens (BAA), the amino acid sequence of which shows 80% homology with that of BLA, is rapidly inactivated at 90 degrees C. Various chimeric genes were constructed from the structural genes for the two enzymes, and their products were analyzed for stability as to irreversible thermoinactivation. Two regions in the amino acid sequence of BLA comprising Gln178 (region I) and the 255th-270th residues (region II), respectively, were shown to determine the thermostability of BLA. Region I plays a major role in determining the thermostability. By means of site-directed mutagenesis of the BAA gene, deletion of Arg176 and Gly177 in region I and substitutions of alanine for Lys269 and aspartic acid for Asn266 in region II were shown to be responsible for the enhancement of the thermostability. Mutant BAAs containing the above deletion and substitutions showed almost the same thermostability as BLA as to irreversible thermoinactivation. Nevertheless, the mutant BAAs showed a temperature optimum as low as that of BAA (65 degrees C), indicating that they are still susceptible to reversible inactivation at temperatures higher than 65 degrees C.     

Synthesis of biomolecules from N2, CO,and H2O by electric discharge

A model primitive gas containing a mixture of N₂, CO and water vapor over a water pool (300 mL, 37 °C) was subjected to electric discharges. The discharge vessel (7 L in volume) was equipped with a CO₂ absorber (The CO₂ being formed during the discharge), thus simulating possible absorption of CO₂ in the primitive ocean. The vessel also has a cold trap ( –15 °C), which protects the primary products against the further decomposition in the discharge phase by enabling these products to adhere to the trap. Since the partial pressures of CO and N₂ decreased at rates of 1.5–1.7 cmHg day^–1 and 0.1–0.2 cmHg day^–1, respectively, the gases were added at regular intervals. The solution was analyzed at regular intervals for HCN, HCHO and urea, and maximum concentrations of about 50, 2, and 140 mM were observed. The discharge phase was continued for 6 months. In the solution, glycine (5.6% yield based on the carbon), glycylglycine (0.64%), orotic acid (0.004%) and small amounts of the other amino acids were found.     

Prebiotic synthesis of orotic acid parallel to the biosynthetic pathway

By heating an aqueous solution of aspartic acid and urea, carbamylaspartic acid is first formed and then the molecule is cyclized to dihydroorotic acid (DHO) with loss of water. Irradiation of an aqueous solution of DHO with a tungsten lamp yields orotic acid by photo-dehydrogenation of the molecule. This pathway of orotic acid formation is quite similar to that of biosynthesis of the molecule.     

Assessment of Amyloid β Protein in Cerebrospinal Fluid as an Aid in the Diagnosis of Alzheimer's Disease

Abstract: The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39–42-amino-acid protein, amyloid β protein (Aβ). This study examined whether the measurement of Aβ levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and Aβ concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean Aβ value for the AD group (15.9 ± 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 ± 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. Aβ values did not correlate with age ( r = −0.05, p = 0.59), severity of cognitive impairment ( r = 0.22, p = 0.21), or duration of AD symptoms ( r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of Aβ in CSF. Aβ immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of Aβ reactivity. In conclusion, the total CSF Aβ level is not a useful marker for current diagnosis of AD.     

Production of (R)-1-phenylethanol through enantioselective oxidation of (S)-isomer from their racemic mixture by Pachysolen tannophilus IFO 1007

Summary Stereoselective oxidation of (S)-isomer of rac-1-phenylethanol (1-PA) by the yeast Pachysolen tannophilus IFO 1007 immobilized into calcium-alginate gels was investigated to produce (R)-isomer. Continuous production of (R)-isomer was accomplished for more than 80 h with an enantiomeric excess of > 90% using a bioreactor of a fluidized-bed type.     

Direct high-level secretion into the culture medium of tuna growth hormone in biologically active form byBacillus brevis

The characteristic features of theBacillus brevis system developed by us are very high productivity of heterologous proteins and very low extracellular proteinase activity. However, the production level of eucaryotic proteins with this system was generally one or two orders of magnitude lower than that of bacterial proteins. Therefore, we have explored methods for increasing the production efficiency as to animal proteins. Signal peptide modification was found to be very effective for high-level secretion of tuna growth hormone (tGH). Modification of the signal peptide with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a ten-fold increase in tGH production. Further elevation of the tGH yield to 240 mg/l was achieved by using a low proteinase mutant and a stable plasmid, and by culturingB. brevis under optimal conditions with the addition of some chemicals. Thus, biologically active tGH can be efficiently produced directly in the medium with thisB. brevis system.     

Interaction of α2-macroglobulin with alkaline proteinase from an alkalophilicBacillus sp. grown in an extraordinarily alkaline environment

The interaction of ₂-macroglobulin (₂M) with an alkaline serine proteinase (ALPase I) from alkalophilicBacillus sp. grown in an extraordinarily alkaline environment was investigated. Stoichiometry of the reaction showed that ALPase I bound to ₂M in a molar ratio of about 21. The ₂M-ALPase I complex showed about 80% of the proteinase activity shown by ALPase I in the hydrolysis of succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanyl-4-methyl-coumaryl-7-amide (Suc-Ala-Ala-Pro-Phe-MCA) and casein. The conformational changes in the ₂M molecule caused by the complex formation at pH 7.5 were determined from electron micrographs and difference spectra. The antigenic activity of the ₂M-ALPase I complex with the anti-ALPase I antiserum was found to be completely abolished. Immunoelectrophoresis of the complex incubated at pH 7.5 after 48 h showed no appreciable change, and the complex was recognized as exhibiting enhanced stability at pH 7.5.     

Controlled release of adeno-associated virus from alginate hydrogel microbeads with enhanced sensitivity to ultrasound

Adeno-associated virus (AAV)-based gene therapy holds promise as a fundamental treatment for genetic disorders. For clinical applications, it is necessary to control AAV release timing to avoid an immune response to AAV. Here we propose an ultrasound (US)-triggered on-demand AAV release system using alginate hydrogel microbeads (AHMs) with a release enhancer. By using a centrifuge-based microdroplet shooting device, the AHMs encapsulating AAV with tungsten microparticles (W-MPs) are fabricated. Since W-MPs work as release enhancers, the AHMs have high sensitivity to the US with localized variation in acoustic impedance for improving the release of AAV. Furthermore, AHMs were coated with poly-l -lysine (PLL) to adjust the release of AAV. By applying US to the AAV encapsulating AHMs with W-MPs, the AAV was released on demand, and gene transfection to cells by AAV was confirmed without loss of AAV activity. This proposed US-triggered AAV release system expands methodological possibilities in gene therapy.