Molecular and Biochemical Diversity Among Isolates of Radopholus spp. from Different Areas of the World

Eleven isolates of Radopholus similis from various banana-growing areas around the world and one isolate of R. bridgei from turmeric in Indonesia were compared using DNA and isoenzyme analysis. The polymerase chain reaction (PCR) was used to amplify a fragment of ribosomal DNA (rDNA), comprising the two internal transcribed spacers (ITS) and the 5.8S gene. Restriction fragment length polymorphisms (RFLPs) in this rDNA fragment were used to compare the 10 isolates. The analysis of this rDNA region revealed little variation among the isolates tested. However, data also were obtained by random amplified polymorphic DNA (RAPD) analysis of total DNA, and a hierarchical cluster analysis of these data arranged the R. similis isolates into two clusters. The first cluster consisted of isolates from Nigeria, Cameroon, Queensland, and Costa Rica; the second was comprised of isolates from Guinea, Guadeloupe, the Ivory Coast, Uganda, and Sri Lanka. The isolate of R. bridgei from turmeric in Indonesia appeared to be more divergent. This grouping was consistent with that obtained when phosphate glucose isomerase (PGI) isoenzyme patterns were used to compare the R. similis isolates. The results from both RAPD analysis and PGI isoenzyme studies indicate that two gene pools might exist within the R. similis isolates studied. No correlation could be detected between the genomic diversity as determined by RAPD analysis and either geographic distribution of the isolates or differences in their pathogenicity. The results support the hypothesis that R. similis isolates have been spread with banana-planting material.     

Bacterial adhesion measurements on soft contact lenses using a Modified Vortex Device and a Modified Robbins Device

S. marcescens 8100 andP. aeruginosa 15442 were used to study bacterial adhesion to hydrogel contact lenses which had not been worn. Bacterial removal from unworn lens materials was assessed with a calibrated vortex device modified with a digital rpm readout and fitted with a test tube attachment (MVD). The MVD, which relies on a whirlpool-like force to remove the bacteria, showed that bacteria adhered to the same degree to etafilcon A, vifilcon A and polymacon lenses under standardized conditions. Tracking the isoenzyme patterns of these bacterial species over time showed instability ofS. marcescens upon repeated passage. This instability was not evident withP. aeruginosa. Bacterial adhesion ofP. aeruginosa 15442, to human worn and unworn etafilcon A materials was determined with a Modified Robbins Device. The MRD was closed off at both ends stopping medium and bacterial movement after 1 h of fluid flow over the lens surface. The results show that immediately following this 1-h period more bacteria adhere to unworn contact lenses than to worn lenses. However, bacterial counts were equivalent on worn and unworn lenses following 5 h of static incubation.     

白蚁巢上生长的子囊菌——黑柄炭角菌的深层培养及其生理研究

黑柄炭角菌Xylaria nigripes(K1.)Sacc.是著名的滋补药用真菌,尤其是菌核部位生长在白蚁废弃的蚁巢中,生态条件特殊,资源稀少,研究和开发该菌具有重要意义。为了提高黑柄炭角菌菌丝体和多糖的产量,以便于组织工业化生产,本文探索了该菌的营养条件,测定其生长过程中菌丝体和多糖形成的动态变化,并研究了酯酶同工酶谱和多糖含量变化的相关性。结果表明:在深层培养时,碳源以葡萄糖、淀粉最佳;花生、黄豆、蛋白胨是适宜菌丝生长的良好氮源;Mg~(2+),Zn~(2+)等是其生长的必需元素;有机酸中仅柠檬酸对其菌丝生长有促进作用,在蚁酸影响下,pH值为4.0时,菌丝生长受抑制。在上述实验基础上,进行了产多糖和菌丝体的适宜培养基筛选试验,在适宜条件下,能培养出密集、白色、均匀的菌球和丰富的多糖,经4天培养的培养液中粗多糖量可达15.35g/L,菌丝干重为26.89g/L,符合了工业化生产要求。该菌的菌丝体酯酶同工酶谱较稳定,均为二条酶带,但它们的酶活性有所不同,与菌丝体生长的天数有关。并与多糖含量变化具有一定关系。     

Genetic analysis of disease resistance to all strains of BaYMV in a Chinese barley landrace, Mokusekko 3

 A Chinese landrace of barley, Mokusekko 3, is unique in being completely resistant against all strains of barley yellow mosaic virus (BaYMV). The present investigation revealed that the resistance of Mokusekko 3 is governed by two recessive genes. As one of the resistance genes was known to be tightly linked with alleles at the Est complex locus, consisting of the Est1, Est2 and Est4 loci for esterase isozymes, each of the resistance genes could be separated by means of marker-assisted selection using an isozyme allelic combination as a marker. One of the resistance genes, ym1, is linked to K (hooded lemma) and gl3 (glossy leaf 3) with recombination values of 25.3% and 9.7% respectively, and these three genes are located in the order K-gl3-ym1 on chromosome 4. Another newly designated resistance gene, ym5, is linked to alleles at the Est complex locus and cu2 (curly growth 2), with recombination values of 1.9% and 19.5% respectively, in the order cu2-Est-ym5 from proximal to distal on the long arm of chromosome 3. The complete resistance of Mokusekko 3 is caused by combining two resistance genes, ym1 and ym5. However, almost all the “resistant” cultivars derived from crosses with Mokusekko 3 are susceptible to the recently detected strain BaYMV-III in Japan, since they contain only one resistance gene, ym5. Marker-assisted selection to combine resistance genes into a cultivar is discussed for the breeding of stabilizing resistance to BaYMV. Received: 23 September 1996 / Accepted: 8 November 1996     

Arthrobacter ureafaciens sialidase isoenzymes, L, M1 and M2, cleave fucosyl GM1

Among bacterial, fungal and viral sialidases, the sialidase from Arthrobacter ureafaciens has the unique property of cleaving sialic acids linked to the internal galactose of gangliotetraose. In this study, we examined the ability to cleave the internal sialic acids of GM1 and fucosyl GM1 of sialidases from several bacterial and fungal origins, including Clostridium perfringens and Vibrio cholerae. We found that A. ureafaciens sialidase could liberate the sialic acid of GM1 at the highest rate, and was the only enzyme which could cleave fucosyl GM1 among the sialidases examined.The affinity-purified sialidase derived from the culture medium of A. ureafaciens was comprised of four isoenzymes with different molecular weights and isoelectric points, the isoenzymes that cleaved fucosyl GM1 being L (88 kDa, pI 5.0), M1 (66 kDa, pI 6.2) and M2 (66 kDa, pI 5.5), but not S (52 kDa, pI 6.2) which showed the highest specific activity toward colominic acid among the four isoenzymes. Abbreviations: SA, sialic acid; PBS, phosphate-buffered saline; PVP, polyvinylpyrrolidone; FABMS, fast atom bombardment mass spectrometry; Galint, internal galactose of Gg4Cer; Galext, external galactose of Gg4Cer     

Clustered genes within the genome of Arabidopsis thaliana encoding pectin methylesterase-like enzymes

We focused our attention on the isolation and regulation of genes encoding for pectin methylesterase (PME) isoenzymes in Arabidopsis thaliana (At). The present data reports the identification of two PME-like genes, named AtPME1 and AtPME2, that are closely linked in the At genome. These genes possess different structural organisations. While all higher plant PME characterised so far possess an intron at a similar location relative to their coding sequence, AtPME2 shows an additional intron whose presence within other higher plant PME-like genes has not been previously reported. Sequence comparison of the N-terminal region suggests that the secretory pathways of the putative AtPME1 and AtPME2 isoforms are different, and that this region may contribute to specify a biological function to each isoform. Moreover, phylogenetic analysis reflects the possible existence of functional groups of PME isoforms in higher plant species that seem to have been conserved during evolution.     

Detection of lactate dehydrogenase B4 in human hemolysate of patients deficient in lactate dehydrogenase B subunit activity using enzyme immunoassay

We developed a sensitive enzyme immunoassay system specific for human lactate dehydrogenase (LDH)- B₄ with antiacetylated LDH-B₄ Fab-horse-radish peroxidase conjugate. The enzyme immunoassay system was not interfered with by up to 0.3 mg/tube of hemoglobin. Thus, we measured LDH-B₄ concentrations in the hemolysate of seven heterozygous individuals deficient in LDH-B subunit activity and eight normal individuals. We could not find a significant difference between the LDH-B₄ concentrations in heterozygous and those in normal individuals. These results demonstrate that heterozygous individuals deficient in LDH-B subunit activity produce enzymatically inactive B subunits.This work was supported in part by grants in aid for Scientific Research from the Ministry of Education, Japan (59570998), and from the Clinical Pathology Research Foundation of Japan.     

Peptides isolated from human liver with specific inhibitory effects on reassociation/reactivation of in vitro dissociated lactic dehydrogenase (LDH-M4 and −H4) isozymes

Two different peptides have been purified from human liver, similar to those previously reported (Schoenenberger, G.A., and Wacker, W.E.C. (1966) Biochemistry 5, 1375–1379) to be present in human urine, which may serve as metabolic regulators of lactate dehydrogenase (EC 1.1 1.27) isoenzymes (LDH-M₄ = muscle type; LDH-H₄ = heart type). By trichloroacetic acid precipitation, ultrafiltration, Sephadex G-25 and Bio-Gel P-2 columns, affinity chromatography on immobilized LDH-isozymes and HPLC two peptides which differed with respect to molecular weight, retention on the affinity columns and amino acid composition were isolated. No effect was observed when native, tetrameric lactate dehydrogenase was incubated with these peptides. However, when lactate dehydrogenase was dissociated to monomers at low pH and allowed to reassociate by adjusting the pH to 7.5 complete inhibition of the reactivation occurred when the inhibitors were incubated together with respective reassociating monomeric isozymes. The two peptides showed no cross-specificity, i.e. each peptide exhibited inhibitory activity only on one of the two isozymes LDH-M₄ or LDH-H₄. From the amino acid analyses, gel-filtration and PAGE + SDS, molecular weight of 1800 for the M₄ and ≈2700 for the H₄ inhibitor were calculated. An apparent K_i of ≈3 × 10^?5 mM for the H₄ and ≈7 × 10^?5 mM for the H₄ inhibitor was estimated. The interaction of the inhibitors with the enzyme system showed strong cooperativity with Hill coefficients of 2.9 (LDH-M₄-specific) and 2.4 (LDH-H₄-specific). Mathematical modelling of the reassociation and reactivation of lactate dehydrogenase and its specific inhibition by the peptides led to the conclusion that the peptides reacts with monomers, dimers or a transition state during the tetramerisation process. k₁ for the dimerisation step of M₄ = 2.0 × 10⁵ M^?1 · s^?1 and of H₄ = 8.2 × 10⁴ M^?1 · s^?1; k₂ for the tetramerisation step of M₄ = 2.8 × 10⁵ M^?1 · s^?1 and of H₄ = 1.2 × 10⁵ · M^?1 · s^?1, were calculated, the second step still being the faster one.     

Characterization of anther differentiation in cytoplasmic male sterile maize using a specific isozyme system (esterase)

Summary During anther development, characterized in maize plants with N cytoplasm, certain esterase isozymes in non-microspore cells decrease in amount with anther age and new isozymes appear in the developing microspores. In anthers from male sterile plants with cms T or cms C cytoplasm, neither of these changes in esterase patterns occurred. In anthers from plants with cms S cytoplasm, the decrease in the esterases of non-microsporogenous cells was observed but not the appearance of microspore esterases. In lines carrying cms S cytoplasm and nuclear restorer genes, esterase changes during anther development were as in normal fertile anthers. These results are discussed with respect to the phenomenon of cytoplasmic male sterility in the different maize genotypes.