Some properties of β-1,3-glucan hydrolyzing enzymes from the rotifer Brachionus plicatilis

We examined some characteristics of hydrolyticenzymes, especially -1,3-glucanase, to obtain theinformation of cell wall lytic enzymes forrotifers.Crude enzyme (ammonium sulfate fraction) of rotifershydrolyzed starch, -1,3-glucan, glycol chitinand CM-cellulose. Optimum pH for hydrolysis ofstarch and CM-cellulose was 6.5, and that for -1,3glucan and glycol chitin was pH 6.0. Pectic acid,xylan and agarose were not hydrolyzed at pH 3–10.-1,3 glucanase was purified about 73-fold from crudeenzyme by ion-exchange chromatography and gelfiltration. Optimum pH and temperature of the enzymewere 6 and 60 °C, respectively. The molecular weight ofthe enzyme was estimated about 260 kDa by gelfiltration. The enzyme was inhibited byHgCl₂ and MnCl_{_2}.     

Identification of a cDNA/Protein Leading to an Increased P i -uptake in Xenopus laevis Oocytes

In a previous report we documented an increased Na⁺-dependent transport of inorganic phosphate (P _i ) in Xenopus laevis oocytes injected with mRNA isolated from rabbit duodenum (Yagci et al., Pfluegers Arch. 422:211–216, 1992; ref 24). In the present study we have used expression cloning in oocytes to search for the cDNA/mRNA involved in this effect. The identified cDNA (provisionally named PiUS; for P _i -uptake stimulator) lead to a 3-4-fold stimulation of Na⁺-dependent P _i -uptake (10ng cRNA injected, 3–5 days of expression). Na⁺-independent uptake of P _i was also affected but transport of sulphate and l-arginine (in the presence or absence of sodium) remained unchanged. The apparent K _m -values for the induced Na⁺-dependent uptake were 0.26 ± 0.04 mm for P _i and 14.8 ± 3.0 mm for Na⁺. The 1796 bp cDNA codes for a protein of 425 amino acids. Hydropathy analysis suggests a lack of transmembrane segments. In vitro translation resulted in a protein of 60 kDa and provided no evidence of glycosylation. In Northern blots a mRNA of ∼2 kb was recognized in various tissues including different intestinal segments, kidney cortex, kidney medulla, liver and heart. Homology searches showed no similarity to proteins involved in membrane transport and its control. In conclusion, we have cloned from a rabbit small intestinal cDNA library a novel cDNA encoding a protein stimulating P _i -uptake into Xenopus laevis oocytes, but which is not a P _i -transporter itself. Received: 31 July 1996/Revised: 16 October 1996     

Retroviral Oligonucleotide Distributions Correlate with Biased Nucleotide Compositions of Retrovirus Sequences,Suggesting a Duplicative Stepwise Molecular Evolution

A computer-assisted analysis was made of 24 complete nucleotide sequences selected from the vertebrate retroviruses to represent the ten viral groups. The conclusions of this analysis extend and strengthen the previously made hypothesis on the Moloney murine leukemia virus: The evolution of the nucleotide sequence appears to have occurred mainly through at least three overlapping levels of duplication: (1) The distributions of overrepresented (3–6)-mers are consistent with the universal rule of a trend toward TG/CT excess and with the persistence of a certain degree of symmetry between the two strands of DNA. This suggests one or several original tandemly repeated sequences and some inverted duplications. (2) The existence of two general core consensuses at the level of these (3–6)-mers supports the hypothesis of a common evolutionary origin of vertebrate retroviruses. Consensuses more specific to certain sequences are compatible with phylogenetic trees established independently. The consensuses could correspond to intermediary evolutionary stages. (3) Most of the (3–6)-mers with a significantly higher than average frequency appear to be internally repeated (with monomeric or oligomeric internal iterations) and seem to be at least partly the cause of the bias observed by other researchers at the level of retroviral nucleotide composition. They suggest a third evolutionary stage by slippage-like stepwise local duplications. Received: 3 January 1996 / Accepted: 27 March 1996     

Alterations in Phosphatidylcholine Metabolism of Stretch-Injured Cultured Rat Astrocytes

Abstract: The primary objective of this study was to determine the influence of stretch-induced cell injury on the metabolism of cellular phosphatidylcholine (PC). Neonatal rat astrocytes were grown to confluency in Silastic-bottomed tissue culture wells in medium that was usually supplemented with 10 µM unlabeled arachidonate. Cell injury was produced by stretching (5–10 mm) the Silastic membrane with a 50-ms pulse of compressed air. Stretch-induced cell injury increased the incorporation of [³H]choline into PC in an incubation time- and stretch magnitude-dependent manner. PC biosynthesis was increased three- to fourfold between 1.5 and 4.5 h after injury and returned to control levels by 24 h postinjury. Stretch-induced cell injury also increased the activity of several enzymes involved in the hydrolysis [phospholipase A₂ (EC 3.1.1.4) and C (PLC; EC 3.1.4.3)] and biosynthesis [phosphocholine cytidylyltransferase (PCT; EC 2.7.7.15)] of PC. Stretch-induced increases in PC biosynthesis and PCT activity correlated well (r = 0.983) and were significantly reduced by pretrating (1 h) the cells with an iron chelator (deferoxamine) or scavengers of reactive oxygen species such as superoxide dismutase and catalase. The stretch-dependent increase in PC biosynthesis was also reduced by antioxidants (vitamin E, vitamin E succinate, vitamin E phosphate, melatonin, and n-acetylcysteine). Arachidonate-enriched cells were more susceptible to stretch-induced injury because lactate dehydrogenase release and PC biosynthesis were significantly less in non-arachidonate-enriched cells. In summary, the data suggest that stretch-induced cell injury is (a) a result of an increase in the cellular level of hydroxyl radicals produced by an iron-catalyzed Haber-Weiss reaction, (b) due in part to the interaction of oxyradicals with the polyunsaturated fatty acids of cellular phospholipids such as PC, and (c) reversible as long as the cell's membrane repair functions (PC hydrolysis and biosynthesis) are sufficient to repair injured membranes. These results suggest that stretch-induced cell injury in vitro may mimic in part experimental traumatic brain injury in vivo because alterations in cellular PC biosynthesis and PLC activity are similar in both models. Therefore, this in vitro model of stretch-induced injury may supplement or be a reasonable alternative to some in vivo models of brain injury for determining the mechanisms by which traumatic cell injury results in cell dysfunction.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Isolation of the transposable element hupfer from the entomopathogenic fungus Beauveria bassiana by insertion mutagenesis of the nitrate reductase structural gene

A transposable element has been isolated from the entomopathogenic fungus Beauveria bassiana by trapping it in the nitrate reductase structural gene, which has been cloned from this species. The element had inserted in the first exon of the nia gene and appeared to have duplicated the sequence TA at the site of insertion. It was 3336 bp long with 30-bp imperfect, inverted, terminal repeats. The element, called hupfer, contained an open reading frame encoding a 321-amino acid protein similar to the IS630- or mariner-Tc1-like transposases, and a residual sequence of about 2 kb which was not significantly similar to any published sequence. There are fewer than five copies of this transposable element present per genome in the fungus. Received: 12 February 1997 / Accepted: 2 May 1997     

Point mutations in the Drosophila sodium channel gene para associated with resistance to DDT and pyrethroid insecticides

The gene para in Drosophila melanogaster encodes an α subunit of voltage-activated sodium channels, the presumed site of action of DDT and pyrethroid insecticides. We used an existing collection of Drosophila para mutants to examine the molecular basis of target-site resistance to pyrethroids and DDT. Six out of thirteen mutants tested were associated with a largely dominant, 10- to 30-fold increase in DDT resistance. The amino acid lesions associated with these alleles defined four sites in the sodium channel polypeptide where a mutational change can cause resistance: within the intracellular loop between S4 and S5 in homology domains I and III, within the pore region of homology domain III, and within S6 in homology domain III. Some of these sites are analogous with those defined by knockdown resistance (kdr) and super-kdr resistance-associated mutations in houseflies and other insects, but are located in different homologous units of the channel polypeptide. We find a striking synergism in resistance levels with particular heterozygous combinations of para alleles that appears to mimic the super-kdr double mutant housefly phenotype. Our results indicate that the alleles analyzed from natural populations represent only a subset of mutations that can confer resistance. The implications for the binding site of pyrethroids and mechanisms of target-site insensitivity are discussed. Received: 9 May 1997 / Accepted: 21 July 1997     

布氏田鼠肥满度分析和小型兽类肥满度指标K与KWL（重长指标）的比较

通过对两个肥满度指标的理论和生物学意义分析,以及对布氏田鼠肥满度的研究和实际应用的讨论,认为描述动物的肥满度时,重长指标ＫＷＬ优于指标Ｋ。两指标的最大差别是成体的ＫＷＬ值大于幼体,而成体的Ｋ值小于幼体。布氏田鼠肥满度没有性别差异;有异著的年龄差异,成体鼠的肥满度高于幼鼠;有显著的季节变化,鼠种群春季肥满度最高,夏季降低,秋季回升;有显著的年际变化,高数量年的肥满度高于低数量年。     

Comparative study of seed albumins in the Old-WorldLupinus species (Fabaceae) by reversed-phase HPLC

Seed albumins and 2S proteins isolated from the albumin fraction of 36 accessions representing 10 Old-WorldLupinus species (5 smooth- and 5 rough-seeded) were studied using reversed-phase high-performance liquid chromatography. In addition, the globulin fraction was analyzed to determine its 2S protein content. The performed separations showed the suitability of RP-HPLC technique in the analysis of variation of the seed albumin composition in lupins. In the group of rough-seeded lupins, 3 types of RP-HPLC elution profiles of albumins were distinguished: (1)L. atlanticus, (2)L. cosentinii andL. digitatus, (3)L. palaestinus andL. pilosus. All the species of this group were found to have proteins not observed in smooth-seeded species. Smooth-seeded species exhibited more abundant protein spectra, each species distinguishing by its specific RP-HPLC elution profile. It was found that 2S proteins classified as 2S albumins were responsible for the observed variation. Depending onLupinus species, the 2S albumin class consists of two to six proteins.