Synthesis of selectively radiolabeled hexasaccharides for the determination of enzymatic regioselectivity

Poly-N-acetyllactosamines provide backbone structures for functional modifications such as sialyl Lewis X. To understand how the biosynthesis of poly-N-acetyllactosamines is regulated, two branched oligosaccharides of the structure Gal1,4GlcNAc1, 6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl 1 and 2 were synthesized in which one of the terminal galactose units was selectively radiolabeled. Hexasaccharides 1 and 2 were assembled from the chemically synthesized pentasaccharide precursors GlcNAc1,6(Gal1,4GlcNAc1,2)-Man1,6Man-octyl3 and Gal1,4GlcNAc1,6(GlcNAc1, 2) - Man1,6 Man-octyl 4 respectively, through treatment with UDP-1-[³H]-Gal and 1,4 galactosyltransferase. Compounds 1 and 2 were subsequently incubated with UDP-GlcNAc and the UDP-GlcNAc: Gal1-4Glc(NAc)1,3-N-acetylglucosaminyltransferase (i-GlcNAc transferase) resulting in a partial conversion to a mixture of heptasaccharides which were purified by HPLC. The branch selectivity of the addition of N-acetylglucosamine to compounds 1 and 2 was then characterized by endo--galactosidase digestion of the heptasaccharides, followed by isolation of the resultant pentasaccharides on C18 reverse-phase silica cartridges. Comparison of the amount of radiolabel to a control reaction lacking endo--galactosidase indicated the favored site of GlcNAc addition to be the lower 1,2-branch over the 1,6-branch by a 3:1 ratio.     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

Expression pattern of voltage-dependent calcium channel α1 and β subunits in adrenal gland of N-type Ca2+ channel α1B subunit gene-deficient mice

The Ca²⁺ channel _1B subunit is a pore-forming component capable of generating N-type Ca²⁺ channel activity. Although N-type Ca²⁺ channel plays a role in a variety of neuronal functions, _1B-deficient mice exhibit normal life span without apparent abnormalities of behavior, histology or plasma norepinephrine level, presumably owing to compensation by some other Ca²⁺ channel ₁ or subunit. In this study, we studied the levels of _1A, _1C, _1D, _1E, ₁, ₂, ₃ and ₄ mRNAs in adrenal gland of _1B-deficient mice. The _1A mRNA in homozygous mice was expressed at higher level than in wild or heterozygous mice, but no difference in the expression levels of _1C, _1D, _1E, ₁, ₂, ₃ and ₄ was found among wild, heterozygous and homozygous mice. The protein level of _1A in homozygous mice was also expressed at higher level than in wild or heterozygous mice. To examine whether increased expression is induced by cis-regulatory element within 5-upstream region of _1A gene, we examined lacZ expression in _1B-deficient × _1A6.3-lacZ mice (carrying a 6.3-kb 5-upstream fragment of _1A gene fused to E. coli lacZ reporter gene), which express lacZ in medullar chromaffin cells, but not in cortex. The levels of lacZ expression in homozygous _1B-deficient × _1A6.3-lacZ mice were higher than in wild or heterozygous mice. Therefore, a possible explanation of the normal behavior and plasma norepinephrine level of _1B-deficient mice is that compensation by _1A subunit occurs and that 6.3-kb 5-upstream region of _1A gene contains enhancer cis-element(s) for compensation in adrenal medulla chromaffin cells. (Mol Cell Biochem 271: 91–99, 2005)     

Topography of the subunits of Micrococcus lysodeikticus F1-ATPase

Summary The combined use of proteolytic digestion and lactoperoxidase catalyzed labelling with [¹²⁵I] applied to membrane-bound or soluble pure F₁-ATPase from Micrococcus lysodeikticus has allowed us to establish the topography of its , , and subunits within the protein molecule and with respect to the plane of the membrane.The subunit is most externally located to the membrane bilayer looking towards the cytoplasmic face, a position consistent with its proposed catalytic role. The and subunits lie in an intermediate layer between the subunits and the membrane, in which the subunit occupies a central position within the F₁-ATPase molecule in contact with the subunit. The subunit appears to be tightly bound to the F₀ component of the ATPase complex, probably buried in the membrane bilayer. A molecular arrangement of M. lysodeikticus ATPase is proposed that, taking into account the subunit stoichiometry _{3 3 2 2} (MW 420 000), accommodates the role assigned to each subunit and most, if not all, the known properties of this bacterial energy-transducing protein.     

Development and distribution of the early life stages of the longfin pearleye <Emphasis Type="Italic">Benthalbella linguidens</Emphasis> (Aulopiformes: Scopelarchidae) in the western North Pacific

The early life history and development of the scopelarchid Benthalbella linguidens was studied, based on 203 specimens (from 5.3 to 89.7mm in body length: BL) collected from Kuroshio, Oyashio waters and transition waters of the western North Pacific. The early life stages of B. linguidens are distinguished from those of other species of Benthalbella that inhabit the North Pacific by the characters of 62–64 myomeres in the larval stage and 26–28 anal fin rays in juvenile and transforming specimen. Larvae are elongate; notochord flexion begins at ca. 12mm BL and is completed at ca. 15mm BL. The fin ray complements are established at ca. 40mm BL. The single transforming specimen (89.7mm BL) that has peritoneal pigment was collected from transition waters. All larvae were collected from Kuroshio and transition waters from winter to early summer; however, the size of larvae in Kuroshio waters was apparently smaller than that in transition waters, with ranges of 5.3–32.4mm BL (mean 17.1) and 15.3–35.3mm BL (mean 27.5), respectively. Juveniles were distributed in transition and Oyashio waters and were absent in Kuroshio waters, where adults are commonly distributed. These occurrences of larvae and juveniles in the western North Pacific indicate that B. linguidens spawns in Kuroshio waters in winter and uses transition waters as nursery grounds.     

Genetic Determination of Mental Activity Parameters in the Families of Schizophrenia Patients

Psychological parameters of mental activity (30 in total) and their genetic determination were studied in 67 families of schizophrenia patients (67 patients, 107 parent, and 30 sibs). Abnormalities of most of the examined characteristics were found in both the patients and their healthy relatives. Parameters of attention shifting and emotionality revealed the largest genetic component (25–75 and 17–98%, respectively) in all analyzed groups of relatives (probands–affected sibs, probands–healthy sibs, healthy parents–healthy children, affected parents–affected children). Significant impact of genetic factors was also found in parameters stability of attention under conditions of continuous concentration, mediated retention span productivity of voluntary retention by reproduction data, personal anxiety level, reflection of unusual social groups, and self-assessment. The relationships among the characteristics examined in the system of mental activity were established by means of cluster analysis. The results of this study can be used in medical genetic counseling for identifying subjects at high risk for schizophrenia.     

Slow growth phenotype - A possible approach to improved plasmid maintenance in Saccharomyces cerevisiae

Summary Transformation-induced slow growth phenotype (SGP) in yeast is repressed in the presence of 2m plasmids. A full 2m-sequence-based recombinant plasmid (pJB502) was found to be more stable in a 2m-free- [cir] strain of Saccharomyces cerevisiae than in a cir⁺ strain. This could not be attributed to differences in growth rate calculated from kinetic analysis of plasmid loss, but transformed [cir] isolates, which had lost the recombinant plasmid, exhibited varying degrees of SGP in batch culture. One of these isolates was outcompeted in chemostat culture by the recombinant-plasmid-containing strain, suggesting that improved plasmid maintenance can result from SGP in cir hosts.     

Intracellular localization and domain organization of human TRIM41proteins

A human gene previously identified as a partial cDNA homologous to the gene of RET finger protein was characterized. Northern hybridization detected three messages of 3.3, 4.2, and 7.5kb. The coding sequences of the more abundant of the three messages, the 4.2 and the 3.3kb, were determined. The former encodes a 630 amino acid protein (TRIM41) and the latter a 518 amino acid protein (TRIM41). Green fluorescent protein (GFP) fusions of full-length TRIM41 and TRIM41 were both observed as speckles in the cytoplasm and the nucleus. The result was corroborated by Western analysis of cellular fractions. Results with GFP fusions of various segments of the TRIM41 proteins indicated that the nuclear transport of the proteins is mediated by an N-terminal segment common to both isoforms, but independent of a classical nuclear localization signal sequence.     

Different impacts of two species of aphid-attending ants with different aggressiveness on the number of emerging adults of the aphid’s primary parasitoid and hyperparasitoids

The impacts of two species of ants (Lasius niger and Pristomyrmex pungens) attending the cotton aphid (Aphis gossypii) on the number of emerging adults of the aphids primary parasitoid (Lysiphlebus japonicus) and hyperparasitoids were assessed experimentally at a no-pesticide mandarin grove. L.japonicus females were observed foraging frequently in aphid colonies attended by either ant species, with more females in P.pungens-attended than in L.niger-attended colonies, but rarely in aphid colonies where ants were artificially excluded. Females were less often attacked by P.pungens workers than by L.niger workers, so that they stayed longer and oviposited into more aphids in P.pungens-attended than in L.niger-attended colonies. Attendance by each ant species reduced predator numbers in aphid colonies, compared to colonies where ants were absent, although P.pungens was slightly less effective in repelling predators. Therefore, both ant species incidentally protected parasitized aphids from predators. Consequently, L.japonicus-mummies were abundant in colonies attended by either ant species, with more mummies in P.pungens-attended colonies, and were scarce in ant-excluded colonies. The hyperparasitism and predation on L.japonicus larvae within mummies occurred more frequently in P.pungens-attended than in L.niger-attended colonies, but mummy predation rate was only 20% in the former. As a result, the number of emerging L.japonicus adults did not differ significantly between aphid colonies attended by the two ant species, but significantly more hyperparasitoids emerged in colonies attended by the relatively less aggressive P.pungens than in colonies attended by L.niger.     

Polyphosphoinositide mono- an diphosphoesterases of three subfractions of rat brain myelin

Phosphomonoesterase and diesterase that cleave phosphatidylinositol-4-phosphate (diphosphoinositide, DPI) and phosphatidylinositol-4,5-bisphosphate (triphosphoinositide, TPI) were detected in three subfractions of purified rat brain myelin, and some properties of the enzymes were studied. Monoesterase activity was stimulated by KCl, maximally at a concentration of 25 mM, and inhibited at KCl concentrations above 50 mM. Addition of boiled pH 5 supernatant of rat brain homogenate doubled the enzymic activity; EDTA was inhibitory. The specific activities were nearly equal in the low density, medium density, and heavy density myelin fractions but about 30% lower than in whole brain homogenate. The monophosphatase could be solubilized by extraction with 0.2% Triton X-100. The phosphodiesterase activity was inhibited by EDTA and EGTA and not stimulated by KCl or pH 5 supernatant. Specific activities were nearly equal in whole brain and myelin but were by about 60 percent elevated in the heavy density over the low density myelin fraction. These results show that the hydrolases operative in the fast turnover of the inositide phosphate groups are distributed over the entire myelin structure.     

Oligoadenylates formation on an oligouridylate template in the presence of a lead catalyst

Summary Oligouridylates with more than eight chain units can serve as a template for the template-directed condensation of ImpA catalyzed by Pb²⁺ ion. The templates and the Pb²⁺ ion catalyst facilitate the formation of longer oligoadenylates with five or more units. The ratio of 3–5 linked oligomers to the 2–5 isomers increases with increasing chain length of the oligouridylate template. Short oligouridylates up to a hexamer tend to decrease the yield of oligoadenylates, and do not affect the selectivity of internucleotide linkage.Abbreviations EDTA ethylenediaminetetracetic acid - Tris tris(hydroxymethyl)aminomethane - A adenosine - ImpA adenosine 5-phosphorimidazolide - pA adenosine 5-phosphate - Ap adenosine 2(3)-phosphate - poly A polyadenylic acid - AppA P₁,P₂-diadenosine 5-diphosphate - pAp adenosine 2(3),5-diphosphate - ApA adenylyl adenosine - (pA)_n (n = 2,3,) oligomers of pA - ImpApA 5-phosphorimidazolide of ApA - U uridine - pU uridine 5-phosphate - Up uridine 2(3)-phosphate - poly U polyuridylic acid - pUp uridine 2(3),5-diphosphate - (pU)_n (n = 2,3,) oligomers of pU - (pU)_n – (pA)_m cooligomers composed of (pU)_n and (pA)_m units - AppUpUpUpUp pyrophosphate derived from pA and (pU)₄ - AppUp P₁-(adenosine 5)-P₂-(uridine 2(3)-phosphate 5) -pyrophosphate - BAP bacterial alkaline phosphatase - VPD venom phosphodiesterase - N.P₁ nuclease P₁ - RNase A pancreatic ribonuclease - A^* radioactive adenosine     

Genetic control and racial variation of β-glucosidase isozymes in maize (Zea mays L.)

-Glucosidase (-D-glucoside glucohydrolase, E.C. 3.2.1.21, -Glu) isozyme variants were studied in a large number of inbred lines, crosses, and races of maize (Zea mays L.). The pattern of Mendelian inheritance demonstrated for -GLU variants indicated that they are under nuclear gene control. Twenty-two allelic forms at a single locus were identified in the materials studied by starch gel electrophoresis. Genetic data indicate that -GLU in maize is functionally a dimer. Variation of -GLU isozymes in 51 racial collections of maize from Mexico showed little correlation with morphological or geographical data. In 39 collections from Central America, variation patterns appeared to have some association with altitude.This work was supported in part by NIH Research Grant GM 11546.Paper No. 5040 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Bioenergetic aspects of aerobic growth of Klebsiella aerogenes NCTC 418 in carbon-limited and carbon-sufficient chemostat culture

Carbon-limited chemostat cultures of Klebsiella aerogenes NCTC 418 consumed more oxygen per unit of cell synthesis when growing on mannitol or glycerol than when growing on glucose; and since the maintenance requirements were similar, this suggested that the extra reducing equivalents present in these compounds were oxidized wastefully. By comparison with carbon-limited cultures, carbon-sufficient cultures that were ammonia-, sulphate- or phosphate-limited generally consumed considerably more oxygen per unit of cell synthesis, particularly at low growth rates. Thus, according to the theory of Pirt, these carbon-sufficient cultures had a greatly increased maintenance energy requirement but nevertheless used the remaining energy with a much increased efficiency compared with carbon-limited cultures. This, we suggest, is a false conclusion which stems from the basic assumption that the maintenance requirement does not change with growth rate. Thus we propose an alternative theory which allows for this possibility, and present evidence to show that it may be applicable to both carbon-limited and carbon-sufficient chemostat cultures. Finally we offer an explanation of the high maintenance rate of oxygen consumption found with carbon-sufficient cultures, and consider this phenomenon in relation to the loose coupling between respiration and growth extant in most microbial cultures.     

Concerted evolution of mammalian cardiac myosin heavy chain genes

We have recently determined the complete nucleotide sequences of the cardiac - and -myosin heavy chain (MyHC) genes from both human and Syrian hamster. These genomic sequence data were used to study the molecular evolution of the cardiac MyHC genes.Between the - and -MyHC genes, multiple gene conversion events were detected by (1) maximum parsimony tree analyses, (2) synonymous substitution analyses, and (3) detection of pairwise identity of intron sequences. Approximately half of the 40 cardiac MyHC exons have undergone concerted evolution through the process of gene conversion with the other half undergoing divergent evolution. Gene conversion occurred more often in exons encoding the a-helical myosin rod domain than in the globular head domain, and an apparent directional bias was also observed, with transfer of genetic material occurring more often from to .     

Pleiotropic transport mutants of Escherichia coli lack porin, a major outer membrane protein.

Summary Four pleiotropic transport mutants of Escherichia coli B/r with decreased affinity for the uptake of most nutrients were found to lack a major outer membrane protein of 36,500 daltons (porin) previously shown to produce transmembrane diffusion channels in in vitro reconstitution experiments. Consequent decrease in outer membrane permeability was confirmed by measuring the transmembrane diffusion rate of 6-aminopenicillanic acid. Quantitative considerations on the porin-dependent permeability of the outer membrane show that (a) there may be very large differences in the actual rates of penetration, even among the permeable substances and (b) the numbers of porin molecules present in wild type cells is several orders of magnitude higher than that necessary for the uptake of rapidly diffusing substrates such as glocose from ordinary culture media. The absence of porin and the pleiotropic transport defect were always contransduced, and the mutation was mapped at 73.7 min between aroB and malT by P1 transduction. When revertants able to grow on low concentrations of lactose were selected, in addition to true revertants suppressor strains with increased amounts of non-porin membrane proteins were isolated.This paper corresponds to paper XVI of the series dealing with the bacterial outer membrane from the laboratory of H.N. The preceding paper in the series is Nikaido, Bavoil, and Hirota, J. Bacteriol., in press     

Soil-binding pilosity and camouflage in ants of the tribes Basicerotini and Stegomyrmecini (Hymenoptera,Formicidae)

Summary Workers of the tropical ant tribes Basicerotini and Stegomyrmecini possess two dominant forms of setae on the dorsal surfaces of the body and outer surfaces of the legs: longer brush hairs with splintered distal ends, and shorter holding hairs that vary among species from plumose to blade-shaped or filiform. The two usually but not invariably occur together to create a double layer. The brush hairs evidently scrape or otherwise capture fine particles of soil, while the holding hairs help to keep them in place next to the surface exoskeleton. As the worker ages, the soil accumulates as a thin, mud-like layer, greatly enhancing the overall camouflage of the body. The material appears to be primarily or entirely exogenous; no special secretory cells were found (in Basiceros manni, studied for this purpose) that might contribute cryptically colored chemicals or adhesive substances to hold the soil in place.     

The significance of pattern in vegetation

Résumé A l'encontre de Scurfield (1956), il est inexact d'attribuer le pattern (écart à une distribution au hasard des espèces et des individus) dans les groupements végétaux, exclusivement ou principalement à la concurrence. Il peut être dû à la modification du régime physique ou à la morphologie, ou au comportement reproductif des espèces en question. L'hypothèse de Scurfield d'une tendance à une régularité croissante, inhérente à la végétation en évolution, n'est pas confirmée, Au contraire, il semble qu'il y ait une tendance à une diminution de l'intensité de pattern, et un accroissement de l'échelle de pattern au cours de la succession. L'étude du pattern peut constituer un instrument écologique utile.     

Field performance of lines derived from haploid and diploid tissues of Hordeum vulgare

Summary Plant tissue culture technology is of increasing interest to plant breeders. As part of a continuing investigation into breeding methods with spring barley two studies were conducted to assess the field performance of the progenies of material regenerated in tissue culture. The first study involved two spring barley cultivars, Golden Promise and Mazurka and compared lines produced from immature embryo (IE) derived callus with those from embryos developed by the Hordeum bulbosum (Hb) technique of chromosome elimination. In general the mean values for the seven characters scored were lower for the IE than the Hb material. In the second study F₁ hybrid material (Golden PromisexMazurka) was used and doubled haploid lines produced by the H. bulbosum and microspore culture (M) techniques were compared with single seed descent (SSD) material. Analysis of these F samples indicated that the mean values for the M lines were significantly lower than those of the Hb and SSD lines. Furthermore, data from the M lines showed significant evidence of variation created during the culture phase. The implications of these findings for barley breeding are discussed.     

Immunocytochemical localization of a novel pituitary polypeptide “7B2” in the gastro-intestinal tract of the rat

Summary Immunoreactivity to the polypeptide designated 7B2 recently isolated from human and porcine pituitary glands, appears to be consistently confined to neuroendocrine and endocrine cells in various tissues. In rat gut, immunoreactive 7B2 was found in endocrine-paracrine cells. Highly labeled cells were found in the antrum of the stomach and, cells with lower concentrations, in the fundus, duodenum, jejunum and ileum. Except for a few cells which were simultaneously positive for 5-hydroxytryptamine, and a few others showing Grimelius's reaction, 7B2 cells do not exhibit argentaffin and/or argyrophil character. The 7B2 polypeptide seems to be distributed amongst several different types of endocrine cells in the gut.     

Detection of DC electric dipoles in background fields by the nurse shark

Summary Two nurse sharks (Ginglymostoma cirratum) were trained to respond to the galvanic dipole fields of steel spheres with and without a background electric field. Detection ranges were increased by a factor of 1.6 with background-field-on over background-field-off for dc fields in the range of 0.01 V/cm to 0.04 V/cm independent of background field strength. No increase in detection range was found for a background field strength of 0.005 V/cm. Similar results were obtained for an ac background field of 0.005 V/cm zero-to-peak at frequencies from 0.2 Hz to 1.6 Hz. These results indicate that threshold sensitivity was increased by a factor of 4 with the background field on. Further observations made on one shark, electric dipoles, and a dc background field of 0.02 V/ cm, are consistent with the factor of four increase in threshold sensitivity with the background field on found using the steel balls. The reason for the observed increase in sensitivity is unknown.