Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Clustering, haplotype diversity and locations of MIC-3: a unique root-specific defense-related gene family in Upland cotton (Gossypium hirsutum L.)

MIC-3 is a recently identified gene family shown to exhibit increased root-specific expression following nematode infection of cotton plants that are resistant to root-knot nematode. Here, we cloned and sequenced MIC-3 genes from selected diploid and tetraploid cotton species to reveal sequence differences at the molecular level and identify chromosomal locations of MIC-3 genes in Gossypium species. Detailed sequence analysis and phylogenetic clustering of MIC-3 genes indicated the presence of multiple MIC-3 gene members in Gossypium species. Haplotypes of a MIC-3 gene family member were discovered by comparative analysis among consensus sequences across genotypes within an individual clade in the phylogram to overcome the problem of duplicated loci in the tetraploid cotton. Deficiency tests of the SNPs delimited six A_t-genome members of the MIC-3 family clustered to chromosome arm 4sh, and one D_t-genome member to chromosome 19. Clustering was confirmed by long-PCR amplification of the intergenic regions using A_t-genome-specific MIC-3 primer pairs. The clustered distribution may have been favored by selection for responsiveness to evolving disease and/or pest pressures, because large variants of the MIC-3 gene family may have been recovered from small physical areas by recombination. This could give a buffer against selection pressure from a broad range of pest and pathogens in the future. To our knowledge, these are the first results on the evolution of clustering and genome-specific haplotype members of a unique cotton gene family associated with resistant response against a major pathogen.     

Electrophoretic elution of nucleic acids from acrylamide and agarose gels

A simple method for electrophoretic elution of nucleic acids from gel slices is described. The procedure utilizes a standard tube gel system and can be completed in as little as one hour. Nucleic acids are recovered in a small volume with almost 100% efficiency. The procedure is applicable equally to acrylamide and agarose gels, and small as well as large RNA and DNA molecules. The eluted nucleic acids are essentially undegraded and are suitable for a variety of structural and biological analyses.     

Some characteristics of a raw starch digestion inhibitory factor fromAspergillus niger

Summary The effect of an inhibitory factor (IF) fromAspergillus niger 19 on raw starch digestion by pure glucoamylase I of blackAspergillus, pure glucoamylae ofRhizopus niveus, bacterial -amylase, fungal -amylase and various combination was investigated. The IF caused higher inhibition of raw starch hydrolysis by the combined action of glucoamylase and fungal -amylase than of hydrolysis by the individual enzymes. A protein moiety of IF might play an active part in this inhibition phenomenon. The IF was bound to starch granules, preventing hydrolysis by the enzymes, and caused decreased raw starch hydrolysis yields.     

Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum.

The presence of a phospholipase A2 (PLA2) activity in rabbit neutrophil membrane preparation that is able to release [1-14C]oleic acid from labelled Escherichia coli has been demonstrated. The activity is critically dependent on the free calcium concentration and marginally stimulated by GTP gamma S. More than 80% of maximal activity is reached at 10 microM-Ca2+. The chemotactic factor, fMet-Leu-Phe, does not stimulate the PLA2 activity in this membrane preparation. Pretreatment of the membrane preparation, under various experimental conditions, or intact cells, before isolation of the membrane with phorbol 12-myristate 13-acetate (PMA), does not affect PLA2 activity. Addition of the catalytic unit of cyclic AMP-dependent kinase to membrane preparation has no effect on PLA2 activity. Pretreatment of the intact neutrophil with dibutyryl-cAMP before isolation of the membrane produces a small but consistent increase in PLA2 activity. The activity of PLA2 in membrane isolated from cells treated with the protein kinase inhibitor 1-(5-isoquinolinesulphonyl)-2-methyl piperazine dihydrochloride (H-7) is significantly decreased. Furthermore, although the addition of PMA to intact rabbit neutrophils has no effect on the release of [3H]arachidonic acid from prelabelled cells, it potentiates significantly the release produced by the calcium ionophore A23187. This potentiation is not due to an inhibition of the acyltransferase activity. H-7 inhibits the basal release of arachidonic acid but does not inhibit the potentiation by PMA. These results suggest several points. (1) fMet-Leu-Phe does not stimulate PLA2 directly, and its ability to release arachidonic acid in intact neutrophils is mediated through its action on phospholipase C. (2) The potentiating effect of PMA on A23187-induced arachidonic acid release is most likely due to PMA affecting either the environment of PLA2 and/or altering the organization of membrane phospholipids in such a way as to increase their susceptibility to hydrolysis. (3) The intracellular level of cyclic AMP probably does not directly affect the activity of PLA2.     

Demonstration of two protein kinases in extracts of Legionella micdadei

Protein kinases I (PK I) and II (PK II) were purified 253- and 13.5-fold, respectively, from an extract of sonically disrupted cells of Legionella micdadei by ion-exchange chromatography on QAE-Sephadex, by histone affinity chromatography, and by HPLC-gel filtration chromatography. Both enzymes catalysed the phosphorylation of calf thymus histones, with a Km of 2.7 mg ml-1 for PK I and 2.9 mg ml-1 for PK II. Histone H2b was the best protein kinase substrate for both PK I and PK II. The pH optima were 6.8 and 7.0 for PK I and PK II respectively. The Km for ATP was 0.29 mM for PK I and 0.33 mM for PK II. PK II activity was stimulated by either cAMP or cGMP, whereas PK I was inhibited by both cyclic nucleotides. The activity of PK I was unaffected by addition of calmodulin, diacylglycerol and mixtures of Ca2+ and acidic phospholipids, but these additions increased PK II activity threefold. The activity of PK II was stimulated by spermine and spermidine, but PK I was inhibited by these compounds. PK I and PK II were both strongly inhibited by heparin.     

Origin of the Koreans: a population genetic study.

A population genetic study was undertaken to investigate the origin of Koreans. Thirteen polymorphic and 7 monomorphic blood genetic markers (serum proteins and red cell enzymes) were studied in a group of 437 Koreans. Genetic distance analyses by both cluster and principal components models were performed between Koreans and eight other populations (Koreans in China, Japanese, Han Chinese, Mongolians, Zhuangs, Malays, Javanese, and Soviet Asians) on the basis of 47 alleles controlled by 15 polymorphic loci. A more detailed analysis using 65 alleles at 19 polymorphic loci was performed on six populations. Both analyses demonstrated genetic evidence of the origin of Koreans from the central Asian Mongolians. Further, the Koreans are more closely related to the Japanese and quite distant from the Chinese. The above evidence of the origin of Koreans fits well with the ethnohistoric account of the origin of Koreans and the Korean language. The minority Koreans in China also maintained their genetic identity.