Anti-bacterial antibodies in Epstein-Barr virus (EBV)-transformed oligoclonal B-cell lines established from normal persons and autoimmune disease patients

We have established 950 and 430 oligoclonal B-lymphoblastoid cell lines (LCL) from two normal persons and eight autoimmune disease patients, respectively by using Epstein-Barr virus (EBV)-induced transformation. To re-evaluate the EBV technique for production of human monoclonal antibodies (mAb) related to infectious disease, we screened these oligoclonal LCLs for antibodies against 31 bacterial strains systematically. A total of 74 cultures out of 1380 were reactive to a total of 18 strains out of 31. Among these, eight cultures showed 10^-3 antibody (Ab) titers to Pseudomonas aeruginosa serotypes C, E, F and I, Staphylococcus aureus, Serratia marcescens and Bacillus cereus. Ten cultures showed 10^-2 Ab titers to Ps. aeruginosa serotypes D, E, F and I, Ps. maltophilia, Staph, epidermidis, Klebsiella ozaenae, Ser. marcescens and B. subtilis. The results reveal the further possibilities for the EBV technique to produce various infectious disease-related human mAbs.     

Staining method of poly(3-hydroxyalkanoic acids) producing bacteria by nile blue

Summary Using an ethanol solution of nile blue, we have developed an efficient method to detect the colonies of poly(3-hydroxyalkanoic acids) (PHA) producing bacteria on the agar plate. When the bacterial colonies with PHA granules were stained with nile blue, the stained colonies fluoresced bright orange on the irradiation of UV light. In the fluoresce emission spectra, fluorescence intensity increased with an increase in the PHA content of bacterial cells.Alcaligenes eutrophus andA.latus colonies with poly(3-hydroxybutyric acid) (PHB) homopolymer exhibited an emission maximum at 580nm on the excitation at 490nm. On the other hand,Pseudomonas oleovorans andP.putida with medium-chain-length (mcl-) PHA copolymers of C6, C8 and C10 units exhibited an emission maximum at 570nm.     

Global bifurcation structure of a Bonhoeffer-van der Pol oscillator driven by periodic pulse trains

The Bonhoeffer-van der Pol (BVP) oscillator is a valuable dynamical system model of pacemaker neurons. Isochrons, phase transition curves (PTC), and two dimensional bifurcation diagrams served to analyze the neuron's response to periodic pulse stimuli. Responses are described and explained in terms of the nonlinear dynamical system theory. An important issue in the generation of spikes by pacemaker neurons is the existence of both slow and fast dynamics in the state point's trajectory in the phase plane. It is this feature in particular that makes the BVP oscillator a faithful model of living pacemaker neurons. Comparison of the model's responses with those of a living pacemaker was based also on return maps of interspike intervals. Analyzed in detail were the complex discharges called stammering which involve interspike intervals that arise unpredictably and exhibit histograms with several modes separated by the equal intervals.Supported by Trent H. Wells Jr. Inc.     

Cloning, nucleotide sequence and expression of a hemolysin gene of Clostridium septicum

Abstract A genomic library of Clostridium septicum NCTC547 strain was made in Escherichia coli by means of λgt10. The DNA insert of a hemolysin-positive (Hly⁺) λ-clone was transferred into pUC19. The resulting plasmid, pCS21, confers a Hly⁺ phenotype on E. coli . Crude lysates of E. coli (pCS21) possessed a strong lytic activity on human erythrocytes and also a lethal effect on mice, characteristic of an α toxin. Nucleotide sequence analysis revealed that the insert DNA (5.2 kb) in pCS21 included at least one open reading frame of 1380 bp. The coding frame for hemolysin was predicted to be 1329 bp in size and to encode a protein of 49.8 kDa. It coincided with the molecular mass (48 kDa) of the α toxin secreted by C. septicum . Taken together, the data indicated that plasmid pCS21 indeed encoded an α toxin gene of C. septicum .     

Energy minimization method using automata network for sequence and side-chain conformation prediction from given backbone geometry

Globular proteins have high packing densities as a result of residue side chains in the core achieving a tight, complementary packing. The internal packing is considered the main determinant of native protein structure. From that point of view, we present here a method of energy minimization using an automata network to predict a set of amino acid sequences and their side-chain conformations from a desired backbone geometry for de novo design of proteins. Using discrete side-chain conformations, that is, rotamers, the sequence generation problem from a given backbone geometry becomes one of combinatorial problems. We focused on the residues composing the interior core region and predicted a set of amino acid Sequences and their side-chain conformations only from a given backbone geometry. The kinds of residues were restricted to six hydrophobic amino acids (Ala, Ile, Met, Leu, Phe, and Val) because the core regions are almost always composed of hydrophobic residues. The obtained sequences were well packed as was the native sequence. The method can be used for automated sequence generation in the de novo design of proteins. © 1994 Wiley-Liss, Inc.     

Stable isotopic structure of aquatic ecosystems

Isotopic, biogeochemical and ecological structure can provide a new dimension for understanding material flows, and the simultaneous function and structure of an ecosystem. Distributions ofδ ¹³C andδ ¹⁵N for biogenic substances in the Nanakita river estuary involving Gamo lagoon in Japan were investigated to construct isotope biogeochemical and ecological structure for assessing fate and transfer of organic matter, and food web structure. The isotopic framework of the ecosystem was successfully described in aδ ¹⁵N–δ ¹³C map. In this estuary the variations of isotope ratios of biogenic substances were clearly explained by the mixing of land-derived organic matter, and marine-derived organic matter. A trophic-level effect of¹⁵N enrichment was clearly observed. Organisms were classified into three groups depending upon the contribution of land-derived organic matter in a food chain. Almost all biota except mollusca in the lagoon depend on organic matter of marine origin. The contributions of both land and marine organic matter were comparable for mollusca in the lagoon.     

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.     

Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

W-heterochromatin of chicken; its unusual DNA components,late replication,and chromatin structure

About 65% of DNA in the chicken W chromosome has been shown to consist ofXhoI andEcoRI family repetitive sequences. These sequences showed remarkable delay in the electrophoretic mobility at low temperature on a polyacrylamide gel. Three dimensional structures of the 0.7-kbXhoI and the 1.2-kbEcoRI family repeating units were estimated to be irregular solenoids using a computer program based on wedge angles of all the 16 dinucleotide steps. Fluorescencein situ hybridization demonstrated that these two family sequences were localized in a major heterochromatic body in an interphase nucleus. Incorporation of bromodeoxyuridine into the W chromosome in the synchronous culture of MSB-1 cells occurred about 1 h later than the peak of S phase. The chromatin structure formed alongXhoI andEcoRI family sequences was suggested to be different from the total chromatin or chromatin containing the β-actin gene sequence in that the linker DNA lengths of the former were significantly longer. Fractionation of theHaeIII-digested MSB-1 nuclei yielded a chromatin fraction in whichXhoI family sequences were partially enriched. Several DNA-binding proteins showing higher affinity for theXhoI family sequence were present in this fraction.