Carbohydrate exposure of human promyelocytic HL60 cells and histiocytic U937 cells during phagocytic differentiation assessed with fluoresceinated lectins

The display of carbohydrate structures was measured in promyelocytic HL60 cells and in histiocytic U937 cells induced to differentiate to phagocytic cellsin vitro during three to seven days of cultivation in the presence of dimethylsulfoxide (DMSO). It was assessed by micro-or spectrofluorometric quantification of the binding of fluorescent lectins. Changes in the cell size and the association and uptake of IgG-or complementopsonized yeast cells (Saccharomyces cerevisiae) were used as signs of phagocyte differentiation.The binding of wheat germ agglutinin (WGA), concanavalin A (Con A),Ricinus communis agglutinin-I (RCA-I) andUlex europaeus agglutinin-I (UEA-I) varied due to the presence of DMSO during cultivation, and without DMSO also on the number of days in culture and the type of cell.Abbreviations DMSO dimethylsulfoxide - PMA phorbol 12-myristate 13-acetate - KRG Krebs-Ringer phosphate buffer with glucose - WGA wheat germ agglutinin - Con A concanavalin A - RCA-I Ricinus communis agglutinin-I - UEA-I Ulex europaeus agglutinin-I     

Seasonal acclimation of bank voles and wood mice: nonshivering thermogenesis and thermogenic properties of brown adipose tissue mitochondria

Summary Seasonal acclimation of nonshivering thermogenesis and brown adipose tissue was studied in wild bank voles (Clethrionomys glareolus), yellow necked field mice and wood mice (Apodemus flavicollis, A. sylvaticus). Both, voles and mice increased their capacity for nonshivering thermogenesis during winter. Thermogenic properties of brown fat (cytochrome c oxidase activity, mitochondrial protein content, GDP-binding of brown fat mitochondria) showed similar changes during seasonal acclimation;Clethrionomys andApodemus spp. both showed lowest thermogenic properties in the summer during August, a rapid increase during fall, and highest levels of thermogenic activity in the winter months. With regard to changes in body weight and brown fat mass these species show different strategies for seasonal acclimation. InClethrionomys a reduction of body mass in the winter was found, both in the wild population as well as in individual animals housed in the laboratory.A. flavicollis showed a reduction of body weight during fall, whereasA. sylvaticus maintained a constant body mass throughout the year. Brown fat mass and cellularity increased in theApodemus spp. during winter, in parallel with the thermogenic properties of brown fat, whereas inClethrionomys brown fat mass and cellularity remained seasonally constant. These species live in the same habitat and were trapped in the same area. It is concluded that seasonal improvements of in vivo and in vitro thermogenesis are very similar in these species, although the physiological basis for this improvement is different inClethrionomys andApodemus.Abbreviations BAT brown adipose tissue - BMR basal metabolic rate (resting metabolic rate at thermoneutrality) - BW body weight - COX cytochrome c oxidase - GDP guanosine diphosphate - MP mitochondrial protein - NA noradrenaline - NST nonshivering thermogenesis - NSTcap NST capacity (NST maximum minus BMR) - T _a ambient temperature     

The cladocera of a ditch at Tienhoven

Summary From 75-08-18 to 75-08-26, the Netherlands Hydrobiological Society investigated during a field research trip the influence of the dimensions of a ditch on its aquatic communities. With regard to theCladocera no direct relationship was found between the dimensions of the ditch and the occurrence of certain species ofCladocera. A decrease in the number of species ofCladocera was assessed in the narrowest part of the ditch.     

Hormonal regulation of sex pheromone biosynthesis in the turnip moth,Agrotis segetum

Pheromone production in the female turnip moth, Agrotis segetum, is under the control of a brain factor. This factor was demonstrated to be a proteinaceous substance termed pheromone biosynthesis activating neuropeptide-like substance (PBAN-like substance). The sex pheromone of Swedish A. segetum includes (Z)-5-decenyl acetate, (Z)-7-dodecenyl acetate, and (Z)-9-tetradecenyl acetate as major components. Decapitation of a female decreased pheromone production significantly. Pheromone production was restored by injection of homogenates of either male or female brain-suboesophageal ganglion or the corpora cardiaca alone. Pheromonotropic activity was also found in homogenates of the female thoracic ganglion and abdominal ganglion that were obtained during scotophase. Injection of female brain and thoracic ganglion homogenates made from insects during the scotophase induced two and four times as much Z7-12:OAc, respectively, as injection with similar homogenates from photophase. As little as one-eighth female equivalent (FE) brain homogenate was sufficient to increase the amount of Z7-12:OAc. The effect of brain homogenate on pheromone titer reached its maximum after 30 min. The activity of the PBAN-like substance present in female brain extracts was not correlated to the age of the donor. Injection of hemolymph collected during either photophase or scotophase into decapitated females did not increase the pheromone titer. The target site of the PBAN-like substance was not the pheromone gland, and the ventral nerve cord was not involved in the transportation of the PBAN-like substance, which implies a mode of action different from what has been reported in other moths. Brain homogenates obtained during photophase from females of African A. segetum, Spodoptera littoralis, or Ostrinia nubilalis as well as synthetic Bombyx-PBAN also induced pheromone production in decapitated Swedish female A. segetum. © 1995 Wiley-Liss, Inc.     

Role of differential expression of <Emphasis Type="Italic">sdh1-1</Emphasis> and <Emphasis Type="Italic">sdh1-2</Emphasis> genes in alteration of isoenzyme composition of succinate dehydrogenase in germinating maize seeds

A probable mechanism of alteration of the isoenzyme composition of succinate dehydrogenase (SDH) due to differential expression of genes encoding subunit A was considered. The alteration of SDH activity during maize seed germination was investigated, and its maximal activity on day 4-5 of germination was found. The alteration of the sdh1-1 and sdh1-2 gene expression level during maize seed germination was evaluated using the quantitative polymerase chain reaction method. The presence of four forms of the studied enzymes, providing multiple SDH functions was found in maize inflorescence using electrophoresis in polyacrylamide gel.     

Developmental studies in Drosophila

A study of the puffing patterns of the salivary gland chromosomes of D. pseudoobscura was carried out through several larval, prepupal, and pupal stages of development. A total of 176 puffs were found, 111 of which changed during the stages studied. As described in previous investigations with other Drosophila species there are two major peaks of puffing activity. These two peaks occur during puparium formation and pupation. Additionally, a minor activity-peak occurs during mid-prepupal life. Attempts have been made to establish correlations between the puffing data and those obtained from electrophoretic and ultrastructural studies.Supported in part by grants GM-16736-03 and FR-05426-09 from the U.S. Department of Health, Education, and Welfare. Ann Jacob Stocker was a holder of a University of Texas predoctoral fellowship.     

Identification and purification of a nuclease from Zinnia elegans L.: a potential molecular marker for xylogenesis

A single-strand specific nuclease was identified during a particular stage of a defined cellular differentiation pathway characteristic of xylem development. Using a hormone-inducible system in which cultured mesophyll cells of Zinnia elegans differentiated to xylem cells in synchrony, the enzymatic activity on single-stranded (ss) DNA was highest during the maturation phase of differentiation. Nondifferentiating cells contained little of this activity throughout a similar course of culture. After electrophoresis of extracts from differentiating cells, a 43-kilodalton (kDa) polypeptide was detected by its activity in the gels containing either ssDNA or RNA. Lectins specific for mannose residues on glycoproteins bound to the 43-kDa nuclease and were used to distinguish it from several ribonucleases. The nuclease was purified by a two-step chromatographic procedure: a lectin-affinity column followed by a phosphocellulose column. The purified protein was determined to be a single polypeptide with a relative molecular mass of 43000 by the analysis of its mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme. A sensitive detection system using biotinylated-concanavalin A and avidin was demonstrated to be specific as a probe for the nuclease protein. An N-terminal amino-acid sequence was derived from 5 pmol of the enzyme. The nuclease was most active on ssDNA at pH 5.5 in the presence of Zn²⁺ and dithiothreitol. The purified preparation hydrolyzed RNA and to a lesser extent, native DNA. It digested closed circular duplex DNA by introducing a single endonucleolytic cleavage followed by random hydrolysis. During the induced pathway of synchronous differentiation in Zinnia the 43-kDa nuclease rapidly increased just prior to the onset of visibly differentiated features, and developed to a maximum level during xylem cell maturation. At this time a similar but slightly smaller nuclease appeared and became dominant as differentiation continued, and subsequently both enzymes decayed. After autolysis, a nuclease of about 37 kDa was found together with the 43-kDa enzyme in the culture medium. Complementing these analyses was the examination of the tissue distribution of the 43-kDa enzyme in Zinnia and other dicotyledonous plants, which also indicated an invivo role of the nuclease in autolysis, the terminal stage of vascular differentiation in plants. The Zinnia nuclease is therefore a potential marker for xylogenesis.Abbreviations Con A Canavalia ensiformis (concanavalin) agglutinin - DNase deoxyribonuclease - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - kDa kilodalton - M_r relative molecular mass - RNase ribonuclease - ss single-stranded - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

龙眼体胚发生相关未知蛋白DlUP-3基因的克隆与表达分析

在龙眼体胚发生早期的蛋白质组学研究中,发现1个体胚发生相关未知蛋白DlUP-3,通过简并引物结合RACE技术进行其基因全长序列克隆。结果显示:(1)克隆到的龙眼体胚发生相关未知蛋白基因DlUP-3的全长cDNA序列为1 681bp,开放阅读框由1 017个核苷酸组成,编码338个氨基酸(GenBank登录号为GQ167202)。(2)生物信息学分析发现,该基因推导蛋白分子量为36 854.2Da,pI为9.05;该蛋白为Ras蛋白质家族成员,具有ATP/GTP-binding site motif A(P-loop)结合位点和1个典型的Ras_like_GTPase superfamily组件,无典型信号肽结构,但有跨膜螺旋的亲水性蛋白;不规则卷曲是其最大量的结构元件,散布于整个蛋白质中。(3)实时荧光定量PCR分析显示,该基因在龙眼体胚发生过程中均有表达,其中以胚性愈伤组织阶段表达量最低,而球形胚阶段最高。研究表明,DlUP-3基因在龙眼体胚发生过程尤其是球形胚阶段有重要的作用,为进一步研究该基因在龙眼体胚发生过程中的功能奠定了基础。     

Zur Induktion der Nitrat- und Nitritreduktase in vollsynchronen Chlorella-Kulturen

Summary Nitrate- and nitrite-reductase (E.C. 1.6.6.1. and E.C. 1.6.6.4.) activities were determined during the light-dark changes in two completely synchronized Chlorella strains. A sharp increase of both enzyme activities during the first light hour was found in Chl. vulgaris forma tertia, a smaller one in Chl. pyrenoidosa. The rise in enzyme activity could only be inhibited by actidione but not by antibiotics which inhibit plastidic protein synthesis. It can therefore be concludet that light causes a de novo synthesis of both enzymes on cytoplasmic ribosomes. It is assumed that light effects a release of substances to the cytoplasm, probably formed during CO₂-fixation, which can effect a cytoplasmic protein synthesis.

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Abkürzungen DCMU 3-(3,4 dichlorphenyl)-1,1-Dimethylharnstoff - DTE 2,3-dihydroxy-1,4-dithiolbutan - FMN Flavinmononucleotid - GAP Glycerinaldehyd-3-phosphat - LDW Licht-Dunkel-Wechsel - NAR Nitratreduktase - NIR Nitritreduktase - PGS 3-Phosphoglycerinsäure - RudPCo Ribulose-1,5-diphosphat Carboxylase, 7 - 10 7 bzw 10 Dunkelstunden     

Sex-specific pleiotropic changes in emotional behavior and alcohol consumption in human α-synuclein A53T transgenic mice during early adulthood

Point mutations in the α-synuclein coding gene may lead to the development of Parkinson's disease (PD). PD is often accompanied by other psychiatric conditions, such as anxiety, depression, and drug use disorders, which typically emerge in adulthood. Some of these point mutations, such as SNCA and A30T, have been linked to behavioral effects that are not commonly associated with PD, especially regarding alcohol consumption patterns. In this study, we investigated whether the familial PD point mutation A53T is associated with changes in alcohol consumption behavior and emotional states at ages not yet characterized by α-synuclein accumulation. The affective and alcohol-drinking phenotypes remained unaltered in female PDGF-hA53T-synuclein-transgenic (A53T) mice during both early and late adulthood. Brain region-specific activation of ceramide-producing enzymes, acid sphingomyelinase (ASM), and neutral sphingomyelinase (NSM), known for their neuroprotective properties, was observed during early adulthood but not in late adulthood. In males, the A53T mutation was linked to a reduction in alcohol consumption in both early and late adulthood. However, male A53T mice displayed increased anxiety- and depression-like behaviors during both early and late adulthood. Enhanced ASM activity in the dorsal mesencephalon and ventral hippocampus may potentially contribute to these adverse behavioral effects of the mutation in males during late adulthood. In summary, the A53T gene mutation was associated with diverse changes in emotional states and alcohol consumption behavior long before the onset of PD, and these effects varied by sex. These alterations in behavior may be linked to changes in brain ceramide metabolism.

     

Identification,cloning, and characterization of β-glucosidase from <Emphasis Type="Italic">Ustilago esculenta</Emphasis>

Hydrolytic enzymes responsible for laminarin degradation were found to be secreted during growth of Ustilago esculenta on laminarin. An enzyme involved in laminarin degradation was purified by assaying release of glucose from laminaribiose. Ion-exchange chromatography of the culture filtrate followed by size-exclusion chromatography yielded a 110-kDa protein associated with laminaribiose hydrolysis. LC/MS/MS analysis of the 110-kDa protein identified three peptide sequences that shared significant similarity with a putative glucoside hydrolase family (GH) 3 β-glucosidase in Ustilago maydis. Based on the DNA sequence of the U. maydis GH3 β-glucosidase, a gene encoding a putative GH3 β-glucosidase in U. esculenta (Uebgl3A) was cloned by PCR. Based on the deduced amino acid sequence, the protein encoded by Uebgl3A has a molecular mass of 91 kDa and shares 90% identity with U. maydis GH3 β-glucosidase. Recombinant UeBgl3A expressed in Aspergillus oryzae released glucose from β-1,3-, β-1,4-, and β-1,6-linked oligosaccharides, and from 1,3-1,4-β-glucan and laminarin polysaccharides, indicating that UeBgl3A is a β-glucosidase. Kinetic analysis showed that UeBgl3A preferentially hydrolyzed laminaritriose and laminaritetraose. These results suggest that UeBgl3A is a key enzyme that produces glucose from laminarioligosaccharides during growth of U. esculenta on laminarin.     

Effects of respiratory activity on starvation survival of marine vibrios

The marine bacterium Vibrio fluvialis NCTC11328 responded to nutrient depletion by a reduction in cell volume, and this was prevented by conditions that eliminated respiration as a source of energy. Addition of the protonophore, CCCP, removal of oxygen and introduction of mutations leading to defects of the respiratory chain prevented size reduction during periods of nutrient limitation. Further, survival of the wild-type strain during starvation was reduced under anaerobic conditions and survival of respiratory mutants under aerobic conditions was reduced compared with that of the parent strain. Removal by mutation of the respiratory Na⁺ pump from Vibrio alginolyticus did not inhibit size reduction or lead to reduced viability in starved cultures.Abbreviations ANa medium A containing 0.4 M sodium chloride - ANaS ANa containing 50 mM sodium succinate - CCCP carbonyl cyanide-m-chlorophenylhydrazone - CFU colony forming units - FMNH flavine mononucleotide - MMS modified Morita's salts solution - MMSGC MMS containing 20 mM glucose and 0.1% (w/v) casamino acids - MMST MMS buffered to pH 8.5 with 50 mM tricine - NM nutrient Morita's broth - Ox oxidase - DH dehydrogenase - NTG N-methyl-N'-nitro-N-nitrosoguanidine     

26. Valdeyernos,Toledo Mountains (central Spain)

The seasonal variation of Aspergillus in the atmosphere of Mexico City was determined, using an Andersen air sampler, at different times of the day, over a period of one year. Aspergillus represented a low percentage of the total mould count, 5%, 4%, and 2% in the morning, noon, and night respectively. It was possible only in the morning to observe a seasonal trend for Aspergillus, with high colony numbers during the dry season. However, the highest CFUm^-3 were detected in April for the three sampling times of the day.

Different percentages of negative growth plates were recorded during the sampling time, but a large percentage was recorded at noon.

Multiple regresional analyses indicate that significant correlations exist between Aspergillus CFUm^-3 and vapor pressure and temperature inversion in the morning, and with temperature and daily range of temperature at night. Fourteen species of Aspergillus were identified, but A. niger, A. fumigatus, and A. flavus were the most abundant.     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

Control of Brennandania lambi (Acari: Pygmephoroidea) by freezing: evaluation of its efficacy and effects on mushroom growth and yield

Laboratory and field studies were conducted to evaluate freezing as a control measure against Brennandania lambi (Krczal) which infests cultivated mushrooms (Agaricus bisporus) in Shanghai primarily through contaminated spawn. Laboratory experiments showed that exposure of contaminated spawn at -10° C for 24 h killed all stages of B. lambi. These mites probably died due to the freezing of body tissues during sustained exposure at -10° C for 24 h. because the supercooling points for the egg, active larva, quiescent larva, non-gravid adult and gravid adult of B. lambi were respectively -11.34, -10.96, -11.43, -7.65 and -11.20° C, whereas the freezing points were respectively -6.68, -6.84. -6.56, -3.96 and -6.75 °C. Semi-field experiments showed that compost inoculated with B. lambi-infested spawn that had been exposed at -10 °C for 24 h had no mite infestation during spawn running and thus mycelium growth was normal. Laboratory and field experiments during 1989 to spring 1990 showed that freezing spawn had no effect on mushroom yield. Further field experiments in two farms during fall 1990 showed no effect of freezing spawn on yield in one farm and increased yield in the other farm. A field experiment during 1991 to 1992 also indicated that freezing spawn -10 °C for 24 h to 34 h had no effect on mushroom yield.     

Absence of ribosomes in Capsicum chromoplasts

Ribosome development was followed by electron microscopy and gel electrophoresis of ribosomal (r)RNAs in the plastids of fully expanded fruits of Capsicum annuum L. during ripening. Chloroplasts from young Capsicum leaves were used as a structural and electrophoretic standard. Four stages were distinguished on the basis of colour changes during fruit ripening. Chloroplasts of the green fruit had a lower content of 16S and 23S rRNAs than leaf chloroplasts. They contained only a few ribosomes, some more discrete ribosomal particles, and the contrast of ribosomal structures was faint. From the outset of ripening, most of the ribosomal structures in the plastid stroma disappeared. A continuous decrease in plastid rRNAs occurred during ripening. Fully differentiated chromoplasts of the red fruit did not contain rRNAs or ribosomes. Throughout plastid development, DNA nucleoids were evident and there was only a small decrease in the DNA peak on electrophoretograms. The loss of ribosomes during the chloroplast-to-chromoplast conversion in Capsicum fruit is discussed in relation to the variations in pigments and enzymic systems in both plastid types.Abbreviations Developmental stages of leaves and fruits: A four-week-old green leaf - B green fruit - C brownish fruit - D orange fruit - E red fruit - ptRNA, DNA plastid RNA - DNA; rRNA ribosomal RNA     

Characterization of endo-1,3–1,4-β-glucanases in GH family 12 from Magnaporthe oryzae

We have cloned three putative endoglucanase cDNAs, designated MoCel12A, MoCel12B, and MoCel12C, from Magnaporthe oryzae. The deduced peptide sequences of both MoCel12A and MoCel12B contain secretion signal peptides and a catalytic core domain that classify them into GH subfamily 12-1. In contrast, the deduced peptide sequence of MoCel12C consists of a signal peptide, a catalytic core domain, and a fungal-type carbohydrate binding module belonging to GH subfamily 12-2. Although most GH family 12 endoglucanases hydrolyze β-1,4-glucans such as carboxymethylcellulose or phosphoric acid-swollen cellulose, MoCel12A that was prepared by overexpression in M. oryzae and Brevibacillus choshinensis hydrolyzed specifically 1,3–1,4-β-glucans, such as barley β-glucan and lichenan. The specific activity of MoCel12A overexpressed in M. oryzae was about 20 times higher than that prepared from B. choshinensis. Furthermore, MoCel12B prepared by overexpression in B. choshinensis also revealed preferential hydrolysis of endo-1,3–1,4-β-glucans with limited hydrolysis on carboxymethylcellulose. In comparison with MoCel12A, the activity of MoCel12B was more stable under alkaline conditions. Levels of mRNA encoding MoCel12A were constitutively high during infection and spore formation. The overexpression and disruption of the MoCel12A gene did not affect germination, appressorium formation, or invasion rate; however, M. oryzae overexpressing MoCel12A produced larger numbers of spores than the wild type or a mutant in which the MoCel12A gene was disrupted. These results suggest that MoCel12A functions in part to hydrolyze 1,3–1,4-β-glucan during infection and spore formation.