Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis.

Some species of puffer fish have been reported to possess both of tetrodotoxin and saxitoxin, which share one binding site on sodium channels. We purified a novel soluble glycoprotein that binds to these toxins from plasma of the puffer fish, Fugu pardalis, and named puffer fish saxitoxin and tetrodotoxin binding protein (PSTBP). PSTBP possessed a binding capacity of 10.6 +/- 0.97 nmol x mg(-1) protein and a K(d) of 14.6 +/- 0.33 nm for [(3)H]saxitoxin in equilibrium binding assays. [(3)H]Saxitoxin (10 nm) binding to PSTBPs was half-inhibited by the presence of tetrodotoxin and saxitoxin at 12 microm and 8.5 nm, respectively. From the results of gel filtration chromatography (200 kDa) and SDS/PAGE (104 kDa), PSTBP was suggested to consist of noncovalently linked dimers of a single subunit. PSTBP was completely deglycosylated by glycopeptidase F, producing a single band at 42 kDa. Two highly homologous cDNAs to each other coding PSTBP (PSTBP1 and PSTBP2, the predicted amino-acid identity 93%), were obtained from a cDNA library of F. pardalis liver. These proteins consisted to two tandemly repeated homologous domains. The predicted amino-acid sequences of PSTBP1 and 2 were not homologous to that of saxiphilin, a reported saxitoxin binding protein, or sodium channels, but their N-terminus sequences were homologous to that of the reported tetrodotoxin binding protein from plasma of Fugu niphobles, which has not been fully characterized. The partially homologous cDNA sequences to PSTBP1 and 2 were also found in expressed sequence tag clones of nontoxic flounders liver. Presumably, PSTBP is involved in accumulation and/or excretion of toxins in puffer fish.     

The relation between the feeding activity of wintering shovelers (Anas clypeata) and the horizontal distribution of zooplankton in Lake Teganuma,Japan

We investigated seasonal changes in the number and feeding activity of Shovelers wintering in Lake Teganuma in connection with temporal and horizontal fluctuations of zooplankton, and analyzed esophageal contents of a captured Shoveler. Zooplankton community was dominated by rotifers followed by copepods during the study period. Horizontal distribution of both invertebrate taxa differed; rotifers most abundant in the western and eastern parts of the lake in February–March, and cyclopoid copepodids and nauplii more common in the northeast area of the eastern portion of the lake. The number and feeding activity of Shovelers increased from January to March in the northeast area of Lake Teganuma. A Shoveler captured in the area had mostly consumed cyclopoid copepodids despite the fact that rotifers dominated in the zooplankton community at that location. These results suggest that the horizontal distribution and feeding activity of the Shovelers were correlated with the distribution of the cyclopoid copepodids.     

FINE-SCALE GENETIC AND PHENOTYPIC STRUCTURE IN NATURAL POPULATIONS OF BACILLUS SUBTILIS AND BACILLUS LICHENIFORMIS: IMPLICATIONS FOR BACTERIAL EVOLUTION AND SPECIATION

The genetic and phenotypic structure of sympatric populations of wild bacteria traditionally identified as Bacillus subtilis and B. licheniformis was analyzed. Small soil samples were taken from a single, tiny site in the Sonoran Desert of Arizona, USA, to provide a true population analysis, in contrast to many analyses of genetic structure using bacterial strain collections of widely heterogeneous origin. Genetic analyses of isolates used multilocus enzyme electrophoresis, mismatches in restriction fragment length polymorphism, and variants from Southern hybridization with B. subtilis DNA probes. Phenotypic analyses of isolates used the API test system for detection of growth and acid production on specific carbon sources. The two species were distinct both phenotypically and genetically, despite their known potential for genetic exchange in laboratory experiments. Genic and genotypic diversity were high in both species, and only 16% of observed allozyme variants might possibly be common to both species. Hence, there is probably modest genetic exchange, if any, between the species in nature. Clear hierarchies of population-genetic structure were found for both species. Different types of genetic data yield concordant population structures for B. subtilis. For both species, two-locus and multilocus statistical analyses of linkage demonstrated modest to strong disequilibrium at the species level but truly panmictic subunits within each species. The evidence for extensive genetic recombination within these fine-scale subdivisions is unequivocal, indicating that the sexuality of these bacteria can be well expressed in nature. The relation of these results to processes of bacterial evolution and speciation is discussed.     

Uptake of transmitter amino acids by glial plasmalemmal vesicles from different regions of rat central nervous system

From rat hippocampal homogenate, we recently isolated a novel subcellular fraction richly containing glial plasmalemmal vesicles (GPV), which takes up glutamate remarkably as a synaptosomal fraction [Y. Nakamura et al. (1993) Glia, 9, 48–56]. In the present study, we prepared GPV from different regions of rat CNS, namely olfactory bulb (Ob), cerebral cortex (Cx), caudatoputamen (Cp), hippocampus (Hp), cerebellum (Ce) and spinal cord (Sc), and analyzed their activities of Na⁺-dependent uptake of following neurotransmitters and a related compound; glutamate, -aminobutyrate (GABA), glycine, dopamine and choline. The uptake activities of these amino acids were not significantly different between GPV and synaptosomes in each region. Regionally, however, the activities were varied considerably. The activities of glutamate uptake revealed in the following rank order: Cx, Hp, Cp>Ce, Ob>Sc. GABA uptake activities were: Ce>Ob, Cx, Hp>Cp, while glycine uptake activities were: Sc, Ce>Ob, Cp, Cx, Hp. On the other hand, the uptake activities of dopamine and choline were quite different between GPV and synaptosomes. Synaptosomal fraction from Cp took up dopamine in a high activity; however, GPV from the same tissue hardly showed the uptake activity. Choline was taken up by synaptosomes prepared from Hp but not by GPV.     

Ubiquitin and ubiquitin-protein conjugates in PC12h cells: Changes during neuronal differentiation

Ubiquitin and ubiquitin-protein conjugates in PC12h cells were detected with in vitro [¹²⁵I]ubiquitination, and quantified by immunoblotting. These levels were altered by nerve growth factor (NGF), which promotes neuronal differentiation. (i) Levels of high molecular weight (HMW) ubiquitin-protein conjugates ranging from 40 to 1,000kDa were increased by 2 days of NGF treatment, and remained high up to 10 days of NGF treatment. (ii) Ubiquitin and a 23-kDa conjugate tended to be decreased from days 2 to 10 of NGF treatment. 10-Day culture with 10 nM staurosporine, an protein kinase inhibitor, that blocks NGF-induced neurite outgrowth suppressed the NGF-induced increases in levels of HMW conjugates. Cyclic AMP and forskolin, both of which promote neurite outgrowth, mimicked the NGF-induced changes in ubiquitin and HMW conjugates, but phorbol ester and epidermal growth factor had little effect. These findings suggest that changes in ubiquitin-protein conjugates are closely coupled with neuronal differentiation.     

Anti-Human IgE Monoclonal Antibodies Recognizing Epitopes Related to the Binding Sites of High and Low Affinity IgE Receptors

Anti-human IgE monoclonal antibodies (mAbs) were produced and eight clones recognizing epitopes on native IgE were selected. Epitopes were mapped by a competitive inhibition enzyme-linked immunosorbent assay, Western blotting and a multi-pin peptide technology. Four sites (one each in the Cε1, Cε2, Cε2/Cε3 junction and Cε3) were recognized by the mAbs. The relationship between the four epitopes and the binding sites of high and low affinity IgE receptors (FcεRI and FcεRII, respectively) was studied using a monovalent Fab fragment of each mAb as a binding inhibitor. The IgE-FcεRII binding was clearly inhibited by the mAb recognizing the Cε2/Cε3 junction, suggesting that FcεRII binds to a rather limited area around the Cε2/Cε3 junction. The IgE-FcεRI binding, on the other hand, was scarcely inhibited by any single mAb. However, the binding was inhibited when the epitope in Cε2 was blocked simultaneously with that at the Cε2/Cε3 junction or with that in Cε3, indicating that these three distinct epitopes are related to the FcεRI binding sites. When these three epitopes were shown in the stereograph of human IgE, the FcεRI binding area was spread largely on the groove side between Cε2 and Cε3 domains. These results suggest that FcεRI acquires the high affinity through multiple bindings.     

A meiotic DNA polymerase from Coprinus cinereus: further purification and characterization

A meiotic DNA polymerase that is present at a high level of activity in meiotic cells of a basidiomycete, Coprinus cinereus, was purified to near homogeneity using synthetic RNA homopolymer [poly(C)] cellulose column chromatography. This report presents the first extensive purification and characterization of any eukaryotic DNA polymerase having a role in meiosis. This enzyme is a single polypeptide with a molecular mass of 65,000. Activity in this enzyme requires magnesium ions and occurs at an optimal pH of 7.5. It is strongly inhibited by dideoxythymidine triphosphate but is relatively insensitive to aphidicolin and N-ethylmaleimide and can use poly(C)/oligo(dG)_12–18 as a template-primer. Polymerase activity can be found only in cells at meiotic prophase, even though the enzyme has been identified in somatic cells in an inactive state using immunoblot analysis. Its distinctive distribution makes possible a genetic and biochemical analysis of functional role of a meiotic DNA polymerase in meiotic recombination, repair and synthesis.Abbreviations ddTTP 2,3-dideoxythymidine 5-triphosphate - NEM N-ethylmaleimide - PMSF phenylmethylsulfnylfluoride - BSA bovine serum albumin     

Production of a fungal protein,Taka-amylase A,by protein-producingBacillus brevis HPD31

Summary An expression-secretion vector, pMK300, was constructed to express theAspergillus oryzae Taka-amylase A (Taa) cDNA. The promoter and signal peptide regions of the HWP (a major cell wall protein ofBacillus brevis HPD31) gene on pMK300 were efficiently utilized inB. brevis HPD31 and a large amount of Taa (22 mg/l) was secreted into the medium. The HWP signal peptide utilized for secretion of Taa was correctly processed during the protein transport across the membrane. The enzymatic properties of Taa produced byB. brevis HPD31 were the same as those of theAspergillus oryzae Taa in several respects; specific activity thermal and pH stabilities, and temperature and pH optima. These results, in combination with previous results, indicate thatB. brevis HPD31 could be used to produce extracellularly foreign proteins of diverse orgins as functional proteins.     

Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution

Summary All the backbone ¹H and ¹⁵N magnetic resonances (except for Pro residues) of the GDP-bound form of a truncated human c-Ha-ras proto-oncogene product (171 amino acid residues, the Ras protein) were assigned by ¹⁵N-edited two-dimensional NMR experiments on selectively ¹⁵N-labeled Ras proteins in combination with three-dimensional NMR experiments on the uniformly ¹⁵N-labeled protein. The sequence-specific assignments were made on the basis of the nuclear Overhauser effect (NOE) connectivities of amide protons with preceding amide and/or Cprotons. In addition to sequential NOEs, vicinal spin coupling constants for amide protons and C protons and deuterium exchange rates of amide protons were used to characterize the secondary structure of the GDP-bound Ras protein; six strands and five helices were identified and the topology of these elements was determined. The secondary structure of the Ras protein in solution was mainly consistent with that in crystal as determined by X-ray analyses. The deuterium exchange rates of amide protons were examined to elucidate the dynamic properties of the secondary structure elements of the Ras protein in solution. In solution, the -sheet structure in the Ras protein is rigid, while the second helix (A66-R73) is much more flexible, and the first and fifth helices (S17-124 and V152-L171) are more rigid than other helices. Secondary structure elements at or near the ends of the effector-region loop were found to be much more flexible in solution than in the crystalline state.     

A novel Cys-214-Ser mutation in the peripherin/RDS gene in a Japanese family with autosomal dominant retinitis pigmentosa

We have screened for possible disease-causing mutations in the peripherin/retinal degeneration slow (RDS) gene in 13 Japanese families with autosomal dominant retinitis pigmentosa (ADRP). Using polymerase chain reaction-single strand conformation polymorphism analysis, a novel mutation at codon 214 was found in which the highly conserved cysteine was replaced with a serine in one family. The mutation at codon 214 was found in all three affected siblings of this family, but none of the 40 normal control individuals had this mutation. These results strongly suggest, that the mutation is pathogenic for RP in this family. The clinical phenotype for this family is a late-onset form of ADRP.