Genetic relatedness of <Emphasis Type="Italic">Trichoderma</Emphasis> isolates antagonistic against <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">dianthi</Emphasis> inflicting carnation wilt

Twenty-eight isolates of Trichoderma belonging to four different species were screened in vitro for their antagonistic ability against Fusarium oxysporum f.sp. dianthi causing carnation wilt. Three different levels of antagonism observed in dual plate assay were further confirmed by cell-free culture filtrate experiments. Isolates showing class I level of antagonism produced maximum lytic enzymes, chitinases and beta-1,3-glucanases. Genetic variability of 25 selected isolates was assessed by random amplified polymorphic DNA technique and the amplified products were correlated for their level of antagonism. Unweighed pair-group method with arithmetical averages cluster analysis revealed prominent inter-and intraspecific genetic variation among the isolates. Based on their genetic relationship, the isolates were mainly distributed into 3 major groups representing T. atroviride, T. pseudokoningii and T. harzianum, with 20-35% interspecific dissimilarity. However, the polymorphism shown by the isolates did not correlate to their level of antagonism.     

Processing of DNA replication and repair intermediates by the concerted action of RecQ helicases and Rad2 structure-specific nucleases

Processing of DNA replication and repair intermediates is a critical aspect of genome stability maintenance. The coordinated action of RecQ-like helicases with structure-specific nucleases such as Flap Endonuclease 1 plays an important role in the processing of certain DNA structures associated with the replication fork, DNA repair, or telomeres. We will summarize our current understanding of how and in what context these interactions take place, with a particular emphasis on the mechanisms of RecQ helicases in processing of key DNA replication and repair intermediates by their protein interactions with FEN-1 and related structure-specific nucleases.     

Control of foliar diseases of mustard by Bacillus from reclaimed soil

Bacillus subtilis strain UK-9, an isolate from reclaimed soils, was studied for its biological control activity against Alternaria leaf spot disease of mustard. In dual culture, production of antifungal metabolites by the bacteria caused morphological alterations of vegetative cells and spores, disruption and lysis of their cell wall. The antagonist reduced spore germination on leaves and disease incidence of the pathogen in plant trial as well as it also demonstrated plant-growth-promoting ability.     

Neuroprotectin D1 inhibits retinal ganglion cell death following axotomy

Neuroprotectin D1 (NPD1), a docosahexaenoic acid-derived autacoid, is an endogenous neuroprotective and anti-inflammatory mediator that is generated in the retina and brain. The effects of exogenous NPD1 on retinal ganglion cell (RGC) apoptosis and the role of 12/15-lipoxygenase (Alox15) in retina were evaluated after optic nerve transection (ONT). Treatment with NPD1 was associated with significant protection against RGC death. The percentage of RGC survival in NPD1-treated group was 30% at 2 weeks after ONT as compared with 12% of RGC survival in the ONT group without treatment. Endogenous NPD1 was a predominant lipid autocoid in uninjured and axotomized retinas. Alox15 mRNA expression was upregulated in retinas following ONT suggesting that amplification of 12/15-lipoxygenase (12/15-LOX) may represent a neuroprotective response in the rat retina. The density of RGCs was higher in the normal retina of 12/15-LOX-deficient mice as compared with congenic controls. Hence, the resident NPD1 has a potential role in the physiological and pathophysiological responses of the retina.     

A fluorescence detection platform using spatial electroluminescent excitation for measuring botulinum neurotoxin A activity

Current biodetection illumination technologies (laser, LED, tungsten lamp, etc.) are based on spot illumination with additional optics required when spatial excitation is required. Herein we describe a new approach of spatial illumination based on electroluminescence (EL) semiconductor strips available in several wavelengths, greatly simplifying the biosensor design by eliminating the need for additional optics. This work combines EL excitation with charge-coupled device (CCD) based detection (EL-CCD detector) of fluorescence for developing a simple portable detector for botulinum neurotoxin A (BoTN-A) activity analysis. A F?rster Resonance Energy Transfer (FRET) activity assay for BoTN-A was used to both characterize and optimize the EL-CCD detector. The system consists of two modules: (1) the detection module which houses the CCD camera and emission filters, and (2) the excitation and sample module, containing the EL strip, the excitation filter and the 9-well sample chip. The FRET activity assay used in this study utilized a FITC/DABCYL-SNAP-25 peptide substrate in which cleavage of the substrate by BoTN-A, or its light chain derivative (LcA), produced an increase in fluorescence emission. EL-CCD detector measured limits of detection (LODs) were similar to those measured using a standard fluorescent plate reader with valves between 0.625 and 1.25 nM (31-62 ng/ml) for LcA and 0.313 nM (45 ng/ml) for the full toxin, BoTN-A. As far as the authors are aware this is the first demonstration of phosphor-based EL strips being used for the spatial illumination/excitation of a surface, coupled with CCD for point of care detection.     

Characterization of Phosphate-Solubilizing Fluorescent Pseudomonads from the Rhizosphere of Seabuckthorn Growing in the Cold Deserts of Himalayas

Isolation and characterization of fluorescent pseudomonads with high phosphate-solubilizing ability is reported from the alkaline and calcium-rich soils with low P availability in the cold desert region of Lahaul and Spiti in the trans-Himalayas of India. Of 216 phosphate-solubilizing isolates, 12 exhibiting high solubilization of tricalcium phosphate (TCP) in NBRIP liquid culture were identified as Pseudomonas trivialis, P. poae, P. fluorescens, and Pseudomonas spp. on the basis of phenotypic features, whole-cell fatty acids methyl ester (FAME) profiles, and 16S rDNA sequencing. These isolates also showed relatively high solubilization of North Carolina rock phosphate (NCRP) in comparison to the solubilization of Mussoorie rock phosphate (MRP) and Udaipur rock phosphate (URP). The solubilization of phosphate substrates by P. trivialis and P. poae is reported for the first time.     

Novel anti-inflammatory mechanisms of N-Acetyl-Ser-Asp-Lys-Pro in hypertension-induced target organ damage

High blood pressure (HBP) is an important risk factor for cardiac, renal, and vascular dysfunction. Excess inflammation is the major pathogenic mechanism for HBP-induced target organ damage (TOD). N-acetyl-Ser-Asp-Lys-Pro (Ac-SDKP), a tetrapeptide specifically degraded by angiotensin converting enzyme (ACE), reduces inflammation, fibrosis, and TOD induced by HBP. Our hypothesis is that Ac-SDKP exerts its anti-inflammatory effects by inhibiting: 1) differentiation of bone marrow stem cells (BMSC) to macrophages, 2) activation and migration of macrophages, and 3) release of the proinflammatory cytokine TNF-alpha by activated macrophages. BMSC were freshly isolated and cultured in macrophage growth medium. Differentiation of murine BMSC to macrophages was analyzed by flow cytometry using F4/80 as a marker of macrophage maturation. Macrophage migration was measured in a modified Boyden chamber. TNF-alpha release by activated macrophages in culture was measured by ELISA. Myocardial macrophage activation in mice with ANG II-induced hypertension was studied by Western blotting of Mac-2 (galectin-3) protein. Interstitial collagen deposition was measured by picrosirius red staining. We found that Ac-SDKP (10 nM) reduced differentiation of cultured BMSC to mature macrophages by 24.5% [F4/80 positivity: 14.09 +/- 1.06 mean fluorescent intensity for vehicle and 10.63 +/- 0.35 for Ac-SDKP; P < 0.05]. Ac-SDKP also decreased galectin-3 and macrophage colony-stimulating factor-dependent macrophage migration. In addition, Ac-SDKP decreased secretion of TNF-alpha by macrophages stimulated with bacterial LPS. In mice with ANG II-induced hypertension, Ac-SDKP reduced expression of galectin-3, a protein produced by infiltrating macrophages in the myocardium, and interstitial collagen deposition. In conclusion, this study demonstrates that part of the anti-inflammatory effect of Ac-SDKP is due to its direct effect on BMSC and macrophage, inhibiting their differentiation, activation, and cytokine release. These effects explain some of the anti-inflammatory and antifibrotic properties of Ac-SDKP in hypertension.     

The total cavopulmonary connection resistance: a significant impact on single ventricle hemodynamics at rest and exercise

Little is known about the impact of the total cavopulmonary connection (TCPC) on resting and exercise hemodynamics in a single ventricle (SV) circulation. The aim of this study was to elucidate this mechanism using a lumped parameter model of the SV circulation. Pulmonary vascular resistance (1.96+/-0.80 WU) and systemic vascular resistances (18.4+/-7.2 WU) were obtained from catheterization data on 40 patients with a TCPC. TCPC resistances (0.39+/-0.26 WU) were established using computational fluid dynamic simulations conducted on anatomically accurate three-dimensional models reconstructed from MRI (n=16). These parameters were used in a lumped parameter model of the SV circulation to investigate the impact of TCPC resistance on SV hemodynamics under resting and exercise conditions. A biventricular model was used for comparison. For a biventricular circulation, the cardiac output (CO) dependence on TCPC resistance was negligible (sensitivity=-0.064 l.min(-1).WU(-1)) but not for the SV circulation (sensitivity=-0.88 l.min(-1).WU(-1)). The capacity to increase CO with heart rate was also severely reduced for the SV. At a simulated heart rate of 150 beats/min, the SV patient with the highest resistance (1.08 WU) had a significantly lower increase in CO (20.5%) compared with the SV patient with the lowest resistance (50%) and normal circulation (119%). This was due to the increased afterload (+35%) and decreased preload (-12%) associated with the SV circulation. In conclusion, TCPC resistance has a significant impact on resting hemodynamics and the exercise capacity of patients with a SV physiology.     

Purification, immobilization and characterization of tannase from Penicillium variable

Tannase from Penicillium variable IARI 2031 was purified by a two-step purification strategy comprising of ultra-filtration using 100 kDa molecular weight cutoff and gel-filtration using Sephadex G-200. A purification fold of 135 with 91% yield of tannase was obtained. The enzyme has temperature and pH optima of 50 degrees C and 5 degrees C, respectively. However, the functional temperature range is from 25 to 80 degrees C and functional pH range is from 3.0 to 8.0. This tannase could successfully be immobilized on Amberlite IR where it retains about 85% of the initial catalytic activity even after ninth cycle of its use. Based on the Michaelis-Menten constant (Km) of tannase, tannic acid is the best substrate with Km of 32 mM and Vmax of 1.11 micromol ml(-1)min(-1). Tannase is inhibited by phenyl methyl sulphonyl fluoride (PMSF) and N-ethylmaleimide retaining only 28.1% and 19% residual activity indicating that this enzyme belongs to the class of serine hydrolases. Tannase in both crude and crude lyophilized forms is stable for one year retaining more than 60% residual activity.