An Analysis of the Amino Acid Clustering Pattern in (α/β)8 Barrel Proteins

The (/)₈ barrel proteins, in spite of having a common fold, do not show any sequence similarity. In order to understand the factors which are responsible for maintaining the common fold, the three-dimensional structures of 36 (/)₈ barrel proteins are analyzed for the presence of identical amino acid clusters or physicochemically similar clusters. The results reveal 14 identical amino acid clusters and a large number of physicochemically similar clusters. Further analysis of the similar clusters points to the conservation of secondary structures, the presence of pairs of residues occupying topologically equivalent secondary structures, and the presence of certain key residues which may play a vital role in directing and stabilizing the (/)₈ barrel fold.     

Analysis of DNA–Protein Contacts in a Complex between Methyltransferase SsoII and a Promoter Region of the SsoII Restriction–Modification Genes

Heterocyclic bases and phosphate groups involved in the DNA–methyltransferase SsoII (M·SsoII) interaction were identified in the regulatory DNA region localized within the promoter region of the SsoII restriction–modification genes by footprinting with the use of formic acid, hydrazine, dimethyl sulfate, and N-ethyl-N-nitrosourea as modifying agents. It has been established that the enzyme interacts with three guanines, one adenine, two thymines, and three phosphate groups of each strand of the DNA duplex. These heterocyclic bases and phosphate groups are disposed symmetrically within the 15-mer inverted repeat of the regulatory DNA region. It has been demonstrated by footprinting with dimethyl sulfate that the C7 atoms of guanines interacting with the enzyme are exposed to the DNA major groove. Two theoretical models were built describing the contacts in a complex between M·SsoII and the regulatory DNA region.     

Deutung und Herkunft des Namens der Lachm?we(Larus ridibundus L.)

Zusammenfassung Der deutsche und ebenso der Linnésche Name der Lachmöwe,Larus ridibundus, stammt von der zuerst mit ihr vereinigten amerikanischen Laughing Gull,Larus atricilla, und ist nach deren Stimme gebildet. Der Name wurde sodann auf die europäische Art übertragen und findet sich nun als Übersetzung in verschiedenen europäischen Sprachen.

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Summary The German name Lachmöwe and the scientific name Larus ridibundus as well of the Black-headed Gull seems to be deriven from the american Laughing Gull(Larus atricilla), which got at first its name fromBrisson after its voice, and was at first united with the Black-headed Gull(Larus ridibundus). This name then is transferred to the european species and is translated in various european languages.

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Herrn Prof. Dr. Ernst Schüz zum 70. Geburtstag gewidmet     

The endogenous development of four new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from Australian geckos

Four new species of Isospora are described from Australian geckoes. Isospora gehyrae n. sp. from Gehyra cf. variegata in South Australia have 18.5-22.5×17.5–20.0 m oöcysts with 10.0-12.5×7.5-9.0 m sporocysts; endogenous stages develop in the host cell cytoplasm. Of the two species found in Heteronotia binoei from northern Queensland, Isospora cytoheteronotis n. sp., with oöcysts of 20.0-26.0×17.5-25.0 m and sporocysts of 10.0-13.5×7.5-11.5 m, undergoes endogenous development in its host cell cytoplasm, whereas I. nucleoheteronotis n. sp., with oöcysts of 17.5-22.5×17.5-21.5 m and sporocysts of 9.0-12.5×6.5-10.0 m, develops in the host cell nucleus. I. oedurae n. sp. from Oedura rhombifer in northern Queensland has oöcysts of 22.5-25.0×22.5-24.0 m and sporocysts of 12.5-14.0×7.5-11.5 m, and undergoes endogenous development in its host cell nuclei.     

Biosynthesis of a blood group H1 antigen by α1,2-fucosyltransferase in PC12 cells

We have examined the expression of GDP-fucose: glycosphingolipid fucosyltransferase activity in PC12 cells and PC12 sublines in relation to the neuronal differentiation induced by nerve growth factor (NGF) or dexamethasone. Transfer of fucose to paragloboside (nLc₄Cer) yielded a product which was determined to be a blood group H₁ antigen (Fuc1-2Gal1-4GlcNAc1-3Gal1-4Glc-Cer) by gas chromatography/mass spectrometry analysis and enzymatic hydrolysis, suggesting that PC12 cells have an 1,2-fucosyltransferase. Lactosylceramide was also fucosylated at a reduced rate. When the differentiation of PC12 cells and PC12 subline cells, PC12D and MR31, was induced by exposure to either NGF or dexamethasone, the fucosyltransferase activity for nLc₄Cer was found to decrease in both cell lines, suggesting the association with cell differentiation. This is the first report of the presence of an 1,2-fucosyltransferase in cultured neuronal cell lines which catalyses thein vitro biosynthesis from nLc₄Cer of a type-2 chain glycosphingolipid having the blood group H₁ determinant. The disaccharides, -lactose andN-acetyllactosamine, were also fucosylated by PC12 cell enzyme, although the specificity for the carbohydrate structure was different from that for glycosphingolipids.Abbreviations Glc d-glucose - Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Fuc l-fucose - Cer ceramide - nLc₄Cer neolactotetraosylceramide (paragloboside) - GDP guanosine diphosphate - CDP cytidine diphosphate - CTP cytidine triphosphate - NGF nerve growth factor - DX dexamethasone - GC/MS gas chromatography/mass spectrometry     

The Catalytic Transition State in ATP Synthase

The catalytic transition state of ATP synthase has been characterized and modeled by combined use of (1) Mg-ADP–fluoroaluminate, Mg-ADP–fluoroscandium, and corresponding Mg-IDP–fluorometals as transition-state analogs; (2) fluorescence signals of -Trp331 and -Trp148 as optical probes to assess formation of the transition state; (3) mutations of critical catalytic residues to determine side-chain ligands required to stabilize the transition state. Rate acceleration by positive catalytic site cooperativity is explained as due to mobility of -Arg376, acting as an arginine finger residue, which interacts with nucleotide specifically at the transition state step of catalysis, not with Mg-ATP- or Mg-ADP-bound ground states. We speculate that formation and collapse of the transition state may engender catalytic site / subunit-interface conformational movement, which is linked to -subunit rotation.     

DNA reduplication during meiotic prophase in the oocytes of Carausius morosus Br. (Insecta,Cheleutoptera)

Cytological investigations combined with cytophotometric DNA determinations on Feulgen stained squash preparations of ovarioles from third and fourth instar nymphs of Carausius morosus revealed that during meiotic prophase a largely despiralized stage follows pachytene and that in this stage an extra reduplication of the nuclear DNA takes place (pachyreduplication phase). Meiotic prophase then proceeds with tetrapachytene, a pachytene-like stage with twice the amount of DNA as compared to oocyte nuclei in earlier meiotic stages, and with more than half the somatic number of elements, being probably autobivalents.Supported by Deutsche Forschungsgemeinschaft.     

β2-Adrenoceptor activation by zinterol causes protein phosphorylation,contractile effects and relaxant effects through a cAMP pathway in human atrium

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both ₁-adrenoceptors (₁AR) and ₂-adrenoceptors (₂AR) mediate positive inotropic effects but that only ₁AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both ₁ AR and ₂ AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose -AR comprise around 2/3 of ₁AR and 1/3 of ₂AR, whether or not ₂AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate ₂AR, we used the ₂AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t_1:2 of relaxation) effects with EC₅₀ values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the ₂AR-selective antagonist ICI 118551 (50 nM) which reduced both EC₅₀ values to 1 M. Zinterol stimulated adenylyl cyclase activity with an EC₅₀ of 30 nM and intrinsic activity of 0.75 with respect to (–)-isoprenaline (600 M); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (–)-[¹²⁵I] cyanopindolol with higher affinity for ₂AR than for - 1 AR; the binding to ₂AR but not to - BAR was reduced by GTPyS (10 M). In the presence of CGP 20712A (300 nM) (–)-isoprenaline (400 M); (to activate both ₁AR and ₂AR maximally) and zinterol (10 M); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (–)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (–)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both ₁AR and ₂AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.     

α- andβ-adrenergic control of thermogenin mRNA expression in brown adipose tissue

By the use of an earlier characterised cDNA clone, CIN-1, corresponding to a sequence of the mRNA coding for the brown-fat specific uncoupling protein, thermogenin, the amount of thermogenin mRNA found in the brown adipose tissue of mice was quantitatively investigated under different physiological and pharmacological conditions.It was found that a 4 hr cold stress led to a 7-fold increase in the amount of thermogenin mRNA; injection of norepinephrine had a significant but smaller effect. Most notably, isoprenaline (-agonist) and phenylephrine (-agonist) had in themselves no effect, but when injected together were able to increase the mRNA level synergistically. In 4 hr cold-stressed mice, norepinephrine, isoprenaline and cholera toxin could all further potentiate the effect of the cold stress itself on the mRNA level. Insulin and the glucocorticoid dexamethasone both had weak stimulatory effects on the mRNA level.It is concluded that an increase in intracellular cAMP levels is a necessary and perhaps sufficient stimulus for the increase in thermogenin gene expression. However, at least underin vivo conditions, this increase requires stimulation of both - and-adrenergic pathways.     

Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity in in vitro pear cultivar shoots

This study was made to clarify the effects of cytokinin type and their concentrations on shoot proliferation and hyperhydricity in in vitro Pyrus pyrifolia N. (`Hosui' and `Kosui') shoots. The shoots were subcultured in a woody plant medium supplemented with 0.5 M 3-indolyl-butyric acid, 3% (w/v) sorbitol, 0.8% (w/v) agar, and with cytokinins kinetin, 6-benzylaminopurine (BA), N-(2-chloro-4-pyridyl)-N9-phenylurea (CPPU), 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) added at concentrations 0.44, 4.40, 11.0 and 44.0 M. The highest number of shoots was induced by adding BA at concentration 11.0 M, then by 4.4 M BA, in both cultivars. TDZ and CPPU caused greater hyperhydricity in cultured explants than BA and kinetin. `Kosui' was more susceptible to hyperhydricity compared with `Hosui'.     

A meta-analysis of frontalis EMG levels with biofeedback and alternative procedures

The lack of comparative reviews of the efficacy of EMG frontalis biofeedback versus alternative procedures for reduction of muscle tension prompted the present meta-analytic treatment of literature previously concluded to be equivocal. Twenty studies comparing EMG frontalis biofeedback with other tension-reduction procedures produced a total of 68 separate effect sizes suitable for meta-analysis. Differences between clinical and normal samples were nonsignificant, and data analyses revealed that EMG frontalis biofeedback was significantly superior to control (p<.05) but that alternative forms of muscle relaxation, while effective, did not reach statistical significance.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

Identification of hybrids betweenQuercus petraea andQ. robur (Fagaceae): Results obtained with RAPD markers confirm allozyme studies based on theGot-2 locus

RAPDs were employed as genetic markers to detect interspecific hybridization between the closely related oak speciesQuercus robur andQ. petraea. Fourteen primers were used in order to check the genetic status (pure or hybrid) of individuals classified morphologically. Among the 147 PCR fragments obtained 11 appear to be species-specific. In the phenotypically intermediate individuals different combinations of these species-specific bands were obtained. The patterns in these putative hybrids were not additive, which may be either the result of repeated backcrossing and introgression between the two species or of heterozygosity within the parental species. The results of the RAPD study are consistent with morphological analyses and allozyme data obtained for theGot-2 locus. Thus the RAPD markers used in this study may provide a powerful genetic tool for the identification of hybrids and the discrimination between the two pure species.     

Enzymic synthesis of the trisaccharide core region of the carbohydrate chain ofN-glycoprotein

Transmannosylation from mannotriose (Man1-4Man1-4Man) to the 4-position at the nonreducing end N-acetylglucosaminyl residue ofN,N-diacetylchitobiose was regioselectively induced through the use of -d-mannanase fromAspergillus niger. The enzyme formed the trisaccharide Man1-4GlcNAc1-4GlcNAc (3.7% of the enzyme-catalysed net decrease ofN,N-diacetylchitobiose) from mannotriose as a donor andN,N-diacetylchitobiose as an acceptor. Mannobiose (Man1-4Man) was also shown to be useful as a donor substrate for the desired trisaccharide synthesis.Abbreviations Man d-mannose - (M _n) (n=1–5) -linkedn-mer of mannose - GlcNAc₂ 2-acetamido-2-deoxy--d-glucopyranosyl-(1–4)-2-acetamido-2-deoxy-d-glucose     

Identification of a cDNA encoding a plant Lewis-type alpha1,4-fucosyltransferase

Recently, it has been found that plants, including tomato (Lycopersicon esculentum), express the Lewis-a epitope, Gal1,3(Fuc1,4)GlcNAc, on some N-glycans. By searching the EST database, it was possible to identify a tomato cDNA encoding a protein, designated FucTC, of 413 amino acids with homology to plant and mammalian 1,3/4-fucosyltransferases. The cDNA was expressed in Pichia pastoris and the recombinant enzyme was found to transfer fucose from GDP-Fuc (K_m 16 M) to lacto-N-tetraose (Gal1,3GlcNAc1,3Gal1,4Glc; K_m 80 M) as well as to 1,3- and 1,4-galactosylated N-glycans. It is concluded that FucTC is responsible for the biosynthesis of Lewis-a on N-glycans in tomato.     

Anaerobic metabolism of Nitrosomonas europaea

Nitrosomonas europaea is capable of maintaining an anaerobic metabolism, using pyruvate as an electron donor and nitrite as an electron acceptor; utilization of nitrite depends upon supply of both pyruvate and ammonia. The role of ammonia in this reaction was not determined. N europaea also assimilates CO₂ anaerobically into cell material in the presence of nitrite (0.5–1.0 mM), pyruvate and ammonia. This reaction was partially inhibited by nitrite which apparently competed with CO₂ for reducing power. Anaerobic nitrite respiration is sensitive to ionophores, FCCP being the most effective.Non-standard-abbreviations TCA trichloroacetic acid - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazon     

Identification of beta subunit of the rhesus monkey chorionic gonadotropin (rmCG)

Chorionic gonadotropin (CG) is a placental derived hormone that plays a crucial role in successful implantation and establishment of early pregnancy in the primates. The rhesus monkey was chosen as a model to understand the feasibility of developing human DNA immuno-contraceptive. The coding region of rhesus monkey CG -subunit (rmCG) was isolated by the TDRT-PCR method. The nucleotide sequence including the leader peptide was 499 nucleotide long and encoded 166 amino acids. In comparing with the previous known primates CG -subunits, the rmCG was the highest degree of homology with baboon CG -subunit at the deduced amino acid sequence (94%), 79.5% homology with human CG -subunit and 70.4% homology with marmoset monkey CG -subunit. The eukaryotic expression vector pCMV4-rmCG inserted full-coding cDNA sequence of rmCG was constructed, and the expression of rmCG -subunit in HeLa cells transient expressing system in vitro and BALB/c mice in vivo was determined. The results demonstrated that the recombinant PCMV4-rmCG eukaryotic expression vector could express rmCG -subunit in vitro and in vivo.     

A BamHI family of highly repeated DNA sequences of Nicotiana tabacum

Summary HRS60.1, a monomer unit (184 bp) of a highly repeated nuclear DNA sequence of Nicotiana tabacum, has been cloned and sequenced. Following BamHI digestion of tobacco DNA, Southern hybridization with HRS60.1 revealed a ladder of hybridization bands corresponding to multiples of the basic monomer unit. If the tobacco DNA was digested with restriction endonucleases which have no target site in HRS60.1, the larger part of DNA homologous to HRS60.1 remained as uncleaved relic DNA. These results suggest a tandem arrangement of this DNA repeat unit. Four other clones of tobacco nuclear DNA cross-hybridized with HRS60.1, thus forming a HRS60-family. Sequencing their inserts has shown their strong mutual homology. HRS60-family comprised about 2% of the nuclear genome of N. tabacum. Computer comparisons with other tandem plant-repeated DNA sequences could not detect any other homologous sequence.     

GADD45γ, down-regulated in 65% Hepatocellular Carcinoma (HCC) from 23 Chinese patients,inhibits cell growth and induces cell cycle G2/M arrest for Hepatoma Hep-G2 cell lines

Growth-arrest and DNA-damage inducible (GADD) genes and Myeloid differentiation primary response (MyD) genes represent a family of genes that play a key role in negative control of cell growth. In the present study, following clone and location of human GADD45 (MyDL) gene, we have found that its mRNA expression level was down-regulated in 15/23 cases of clinic hepatocellular carcinoma (HCC) by comparing the northern hybridization results between the tumor tissues and adjacent normal tissues. Transient transfection of GADD45 cDNA with intact open reading frame sequence into the human hepatoma cells Hep-G2 resulted in dramatic growth suppression in colony formation assays. Furthermore, flow cytometry analysis indicated that GADD45 caused cell cycle arrest at G2/M transition when transfected into Hep-G2 cells. Therefore, the possible role of GADD45 in cell growth control was further confirmed in this paper.     

Negative complementation of recA protein by recA1 polypeptide: in vivo recombination requires a multimeric form of recA protein

Summary Recombination in vivo was studied in recA ^- heterozygous lacZ merodiploids by performing -galactosidase assays after infection with precA ⁺. Recombination as measured by -galactosidase production was a linear function of pecA ⁺ multiplicity of infection (MOI) when the strain contained a deletion of the chromosomal recA gene. However, when the strain carried a recA1 missense allele, a higher precA ⁺ MOI was required to obtain levels of recombination comparable to the (recA) strain, and the slope of the dose-response curve increased to approximately two. It is proposed that negative complementation occurs in mixed tetramers of wild-type and missense recA polypeptides, and that in vivo recombination is a property of a multimeric form of recA protein.