Whole cells of Bacillus subtilis AF 1 proved more effective than cell-free and chitinase-based formulations in biological control of citrus fruit rot and groundnut rust

In foliar and postharvest biocontrol systems, the use of active metabolites produced by antagonistic microorganisms is advantageous compared with the use of living microorganisms. Chitinases, a major group of hydrolytic enzymes produced by biocontrol agents, are involved in the lysis of cell walls of pathogenic fungi. In the present study, an attempt was made to test the partially purified beta-1,4-N-acetylglucosaminidase (NAGase) of a biocontrol strain Bacillus subtilis AF 1 for control of rust in groundnut (caused by Puccinia arachidis) and soft rot in lemons (caused by Aspergillus niger). Four proteins of molecular mass 67, 40, 37, and 32 kDa were isolated from the culture filtrates of AF 1 by affinity chromatography, of which the 67-kDa protein has detectable chitinolytic ability. This protein (NAGase) effectively inhibited the in vitro growth of A. niger in microtitre plates. In the presence of NAGase, germination of urediniospores of P. arachidis was reduced by 96% compared with the control. In a detached leaf bioassay, NAGase reduced the rust lesion frequency by >60%. NAGase significantly reduced the incidence of soft rot in harvested lemon fruits. However, fresh cells and (or) alginate formulation of AF 1 were more effective than NAGase in control of both of the test plant - pathogen systems.     

In vitro selection of molecular beacons

While molecular beacons are primarily known as biosensors for the detection of nucleic acids, it has proven possible to adapt other nucleic acid binding species (aptamers) to function in a manner similar to molecular beacons, yielding fluorescent signals only in the presence of a cognate ligand. Unfortunately, engineering aptamer beacons requires a detailed knowledge of aptamer sequence and structure. In order to develop a general method for the direct selection of aptamer beacons we have first developed a selection method for molecular beacons. A pool of random sequence DNA molecules were immobilized via a capture oligonucleotide on an affinity column, and those variants that could be released from the column by a target oligonucleotide were amplified. After nine rounds of selection and amplification the elution characteristics of the population were greatly improved. A fluorescent reporter in the selected beacons was located adjacent to a DABCYL moiety in the capture oligonucleotide; addition of the target oligonucleotide led to release of the capture oligonucleotide and up to a 17-fold increase in fluorescence. Signaling was specific for the target oligonucleotide, and occurred via a novel mechanism, relative to designed molecular beacons. When the target oligonucleotide is bound it can form a stacked helical junction with an intramolecular hairpin in the selected beacon; formation of the intramolecular hairpin in turn leads to release of the capture oligonucleotide. The ability to select molecular beacons may prove useful for identifying available sites on complex targets, such as mRNAs, while the method for selection can be easily generalized to other, non-nucleic acid target classes.     

Influence of ethylene-oxy spacer group on the activity of linezolid: synthesis of potent antibacterials possessing a thiocarbonyl group

The influence of an ethylene-oxy spacer element between the heterocycle and the aromatic ring in linezolid is reported. The introduction of such spacer group generated compounds with inferior antibacterial activity. However, the conversion of the acetamide group present in the linezolid analogues to either thiocarbamate or thioacetamide functionality restored the activity. The synthesis of linezolid analogues possessing the ethylene-oxy spacer group along with SAR studies with different heterocycles and preparation of some thiocarbonyl compounds possessing potent antibacterial property are presented.     

Hemoglobin Einstein: semisynthetic deletion in the B-helix of the alpha-chain

The influence of the deletion of the tetra peptide segment alpha(23-26) of the B-helix of the alpha-chain of hemoglobin-A on its assembly, structure, and functional properties has been investigated. The hemoglobin with the deletion, ss-Hemoglobin-Einstein, is readily assembled from semisynthetic alpha(1-141) des(23-26) globin and human betaA-chain. The deletion of alpha(23-26) modulates the O2 affinity of hemoglobin in a buffer/allosteric effector specific fashion, but has little influence on the Bohr effect. The deletion has no influence on the thermodynamic stability of the alpha1beta1 and the alpha1beta2 interface. The semisynthetic hemoglobin exhibits normal intersubunit interactions at the alpha1beta1 and alpha1beta2 interfaces as reflected by 1H-NMR spectroscopy. Molecular modeling studies of ss-Hemoglobin-Einstein suggest that the segment alpha(28-35) is in a helical conformation, while the segment alpha(19-22) is the nonhelical AB region. The shortened B-helix conserves the interactions of alpha1beta1 interface. The results demonstrate a high degree of plasticity in the hemoglobin structure that accommodates the deletion of alpha(23-26) without perturbing its overall global conformation.     

SPINDLY is a nuclear-localized repressor of gibberellin signal transduction expressed throughout the plant

SPY (SPINDLY) encodes a putative O-linked N-acetyl-glucosamine transferase that is genetically defined as a negatively acting component of the gibberellin (GA) signal transduction pathway. Analysis of Arabidopsis plants containing a SPY::GUS reporter gene reveals that SPY is expressed throughout the life of the plant and in most plant organs examined. In addition to being expressed in all organs where phenotypes due to spy mutations have been reported, SPY::GUS is expressed in the root. Examination of the roots of wild-type, spy, and gai plants revealed phenotypes indicating that SPY and GAI play a role in root development. A second SPY::GUS reporter gene lacking part of the SPY promoter was inactive, suggesting that sequences in the first exon and/or intron are required for detectable expression. Using both subcellular fractionation and visualization of a SPY-green fluorescent protein fusion protein that is able to rescue the spy mutant phenotype, the majority of SPY protein was shown to be present in the nucleus. This result is consistent with the nuclear localization of other components of the GA response pathway and suggests that SPY's role as a negative regulator of GA signaling involves interaction with other nuclear proteins and/or O-N-acetyl-glucosamine modification of these proteins.     

Is tissue culture a viable system with which to examine environmental and hormonal regulation of cold acclimation in woody plants?

In vitro-grown saskatoon berry (Amelanchier alnifolia Nutt.) plantlets were exposed to various hormonal treatments, dormancy-inducing and cold acclimation conditions to determine if this in vitro system would be viable for dormancy/hardiness studies in woody plants. Low temperature induced significant hardiness levels in plantlets to ?27°C after 6 weeks at 4°C but did not approach liquid nitrogen levels of fully hardened, field-grown buds. Control plantlets were consistently killed at ?5°C throughout this period. Significant hardiness was attained under both short and long day/low temperature conditions; however, hardiness was reduced under continuous light or dark treatments. A pre-exposure to the typical short photoperiod regime of woody plants did not significantly increase the rate of acclimation in these plantlets. The presence/absence of phytohormones in the media have a pronounced influence on the ability of plantlets to cold acclimate. Hormone-free media increased hardiness to ?10.5°C after 2 weeks in treatment. Addition of abscisic acid (ABA) increased cold hardiness levels (?12°C) while addition of benzylaminopurine (BAP) to this hormone-free media decreased hardiness to ?5.3°C. A combination of BAP and ABA treatments produced LT₅₀ values intermediate between individual applications of either hormone. Conversely, α-naphthaleneacetic acid (NAA) could not counteract the ABA-induced hardiness. ABA treatments alone were not able to harden plantlets to the extent attained under low temperature acclimation conditions. Further, ABA could not maintain the hardiness levels of cold-acclimating treatments and plantlets de-acclimated to ?9°C in BAP + ABA media. Subculturing in itself significantly elevated cold hardiness in plantlets to ?9°C on BAP + NAA media within 3 days after subculture and thereafter plantlets dehardened to ?5°C. While tissue culture has value in specific cases, caution should be taken when using tissue-cultured plantlets as a system to evaluate environmental regulation of cold acclimation in woody plants, in part, due to the influence of phytohormones in the media.     

Characterization of an abundant short interspersed nuclear element (SINE) present in Canis familiaris

A short interspersed nuclear element (Can SINE) of ∼130–150 bp was cloned and characterized from Canis familiaris. We demonstrate that this element is interspersed, present approximately every 5–8.3 kbp, and many are sufficiently close to allow IRS (interspersed repetitive DNA sequences) PCR. Sequence analysis of >20 Can SINEs from the dog has identified a conserved region that was used to design oligonucleotides for IRS PCR. Since Can SINEs are not present in human or rodent genomes, IRS PCR using oligonucleotides directed to the conserved region of Can SINEs can be used to simplify analysis of canid DNA in somatic cell hybrids, as well as in large insert cloning vectors. We demonstrate that the canid IRS products are polymorphic and could be developed as genetic markers for filter-based genotyping in this organism. Received: 23 July 1997 / Accepted: 9 September 1997     

Chromosome-damaging action of isoniazid and thiacetazone on human lymphocyte cultures in vivo

Summary Antitubercular drugs in general are given in various combinations, one being isoniazid and thiacetazone. In the present study, was evaluated the in vivo chromosome-damaging effects of a combination of these two drugs in 72 h lymphocyte cultures.Chromosome aberrations were significantly increased in the patients treated with INH and thiacetazone as compared with two types of controls: (1) tuberculosis patients before starting the drug treatment and (2) individuals from the general population. The most frequently observed aberrations were chromatid breaks and gaps.It has been shown that individually, isoniazid may not be clastogenic on human chromosomes in therapeutic doses. The effects of thiacetazone on human chromosomes are not known. Consequently, the enhancement in chromosomal aberrations in the drug-exposed patients may be due to a synergistic effect of isoniazid and thiacetazone or to the clastogenic effects of thiacetazone alone.     

KvLQT1 modulates the distribution and biophysical properties of HERG. A novel alpha-subunit interaction between delayed rectifier currents

Cardiac repolarization is under joint control of the slow (IKs) and rapid (IKr) delayed rectifier currents. Experimental and clinical evidence indicates important functional interactions between these components. We hypothesized that there might be more direct interactions between the KvLQT1 and HERG alpha-subunits of IKs and IKr and tested this notion with a combination of biophysical and biochemical techniques. Co-expression of KvLQT1 with HERG in a mammalian expression system significantly accelerated HERG current deactivation at physiologically relevant potentials by increasing the contribution of the fast component (e.g. upon repolarization from +20 mV to -50 mV: from 20 +/- 3 to 32 +/- 5%, p < 0.05), making HERG current more like native IKr. In addition, HERG current density was approximately doubled (e.g. tail current after a step to +10 mV: 18 +/- 3 versus 39 +/- 7 pA/picofarad, p < 0.01) by co-expression with KvLQT1. KvLQT1 co-expression also increased the membrane immunolocalization of HERG by approximately 2-fold (p < 0.05). HERG and KvLQT1 co-immunolocalized in canine ventricular myocytes and co-immunoprecipitated in cultured Chinese hamster ovary cells as well as in native cardiac tissue, indicating physical interactions between HERG and KvLQT1 proteins in vitro and in vivo. Protein interaction assays also demonstrated binding of KvLQT1 (but not another K+ channel alpha-subunit, Kv3.4) to a C-terminal HERG glutathione S-transferase fusion protein. Co-expression with HERG did not affect the membrane localization or ionic current properties of KvLQT1. This study shows that the alpha-subunit of IKs can interact with and modify the localization and current-carrying properties of the alpha-subunit of IKr, providing potentially novel insights into the molecular function of the delayed rectifier current system.