Analysis of non-TIR NBS-LRR resistance gene analogs in <Emphasis Type="Italic">Musa acuminata</Emphasis> Colla: Isolation,RFLP marker development,and physical mapping

Background  

Many commercial banana varieties lack sources of resistance to pests and diseases, as a consequence of sterility and narrow genetic background. Fertile wild relatives, by contrast, possess greater variability and represent potential sources of disease resistance genes (R-genes). The largest known family of plant R-genes encode proteins with nucleotide-binding site (NBS) and C-terminal leucine-rich repeat (LRR) domains. Conserved motifs in such genes in diverse plant species offer a means for isolation of candidate genes in banana which may be involved in plant defence.     

A noninvasive cell-based assay for monitoring proteolytic activity within a specific subcellular compartment

A noninvasive cell-based assay has been developed to monitor the proteolytic activity of cathepsin L within a specific subcellular compartment, the lysosome. The green fluorescent protein (GFP) of Aequorea victoria was selected as a substrate. Targeting to lysosomes was achieved by fusing GFP to preprocathepsin L, which also ensures colocalization of the enzyme and the substrate. Stably transfected HeLa-rtTA (reverse tetracycline-controlled transactivator) cells were induced with doxycycline and cultured in the presence of various concentrations of cysteine protease inhibitors for 48 h. In the absence of inhibitor, proteolytic degradation of GFP leads to loss of fluorescence, which is due almost exclusively to the action of recombinant cathepsin L. However, a dose-dependent increase of GFP fluorescence is observed for cells treated with the potent cathepsin L inhibitor benzyloxycarbonyl-LeuLeuTyr-CHN(2). Fluorescence is also observed when GFP is fused to an inactive preprocathepsin L (C25A mutant). Targeting of GFP to an acidic cellular compartment can destabilize the protein and render it susceptible to proteolytic degradation. The approach should be generally applicable for proteases localized in acidic environments. Such an assay can be of great value in validating the participation of a specific enzyme in a given process or in testing the ability of putative inhibitors to reach their intracellular target.     

Hsp72-mediated suppression of c-Jun N-terminal kinase is implicated in development of tolerance to caspase-independent cell death

Pretreatment with mild heat shock is known to protect cells from severe stress (acquired thermotolerance). Here we addressed the mechanism of this phenomenon by using primary human fibroblasts. Severe heat shock (45 degrees C, 75 min) of the fibroblasts caused cell death displaying morphological characteristics of apoptosis; however, it was caspase independent. This cell death process was accompanied by strong activation of Akt, extracellular signal-regulated kinase 1 (ERK1) and ERK2, p38, and c-Jun N-terminal (JNK) kinases. Suppression of Akt or ERK1 and -2 kinases increased cell thermosensitivity. In contrast, suppression of stress kinase JNK rendered cells thermoresistant. Development of thermotolerance was not associated with Akt or ERK1 and -2 regulation, and inhibition of these kinases did not reduce acquired thermotolerance. On the other hand, acquired tolerance to severe heat shock was associated with downregulation of JNK. Using an antisense-RNA approach, we found that accumulation of the heat shock protein Hsp72 is necessary for JNK downregulation and is critical for thermotolerance. The capability of naive cells to withstand moderate heat treatment also appears to be dependent on the accumulation of Hsp72 induced by this stress. Indeed, exposure to 45 degrees C for 45 min caused only transient JNK activation and was nonlethal, while prevention of Hsp72 accumulation prolonged JNK activation and led to massive cell death. We also found that JNK activation by UV irradiation, interleukin-1, or tumor necrosis factor was suppressed in thermotolerant cells and that Hsp72 accumulation was responsible for this effect. Hsp72-mediated suppression of JNK is therefore critical for acquired thermotolerance and may play a role in tolerance to other stresses.     

Understanding factors that limit the productivity of suspension-based perfusion cultures operated at high medium renewal rates

One of the key parameters in perfusion culture is the rate of medium replacement (D). Intensifying D results in enhanced provision of nutrients, which can lead to an increase in the viable cell density (X(v)). The daily MAb production of hybridoma cells can thus be increased proportionally without modifying the bioreactor scale, provided that both viable cell yield per perfusion rate (Y(Xv/D)) and specific MAb productivity (q(MAb)) remain constant at higher D. To identify factors prone to limit productivity in perfusion, a detailed kinetic analysis was carried out on a series of cultures operated within a D range of 0.48/4.34 vvd (volumes of medium/reactor volume/day) in two different suspension-based systems. In the Celligen/vortex-flow filter system, significant reductions in Y(Xv/D) and q(MAb) resulting from the use of gas sparging were observed at D > 1.57 vvd (X(v) > 15 x 10(6) cells/mL). Through glucose supplementation, we have shown that the decrease in Y(Xv/D) encountered in presence of sparging was not resulting from increased cellular destruction or reduced cell growth, but rather from glucose limitation. Thus, increases in hydrodynamic shear stress imparted to the culture via intensification of gas sparging resulted in a gradual increase in specific glucose consumption (q(glc)) and lactate production rates (q(lac)), while no variations were observed in glutamine-consumption rates. As a result, while glutamine was the sole limiting-nutrient under non-sparging conditions, both glutamine and glucose became limiting under sparging conditions. Although a reduction in q(MAb) was observed at high-sparging rates, inhibition of MAb synthesis did not result from direct impact of bubbles, but was rather associated with elevated lactate levels (25-30 mM), resulting from shear stress-induced increases in q(lac), q(glc), and Y(lac/glc). Deleterious effects of sparging on Y(Xv/D) and q(MAb) encountered in the Celligen/vortex-flow filter system were eliminated in the sparging-free low-shear environment of the Chemap-HRI/ultrasonic filter system, allowing for the maintenance of up to 37 x 10(6) viable cells/mL. A strategy aimed at reducing requirements for sparging in large-scale perfusion cultures by way of a reduction in the oxygen demand using cellular engineering is discussed.     

Developmental changes in endothelium-derived vasorelaxant factors in cerebral circulation

Endothelium-derived prostanoids are predominant vasorelaxant factors in the cerebral circulation of newborn pigs in vivo, whereas in older pigs nitric oxide (NO)-mediated responses also contribute to the regulation of cerebral vascular tone. We compared the expression and activities of NO synthase and cyclooxygenase in the cerebral microcirculation of newborn and adult pigs. In adult animals, expression and activity of endothelial NO synthase in cerebral microvessels and in cultured cerebral endothelial cells is two- to threefold higher than in newborn pigs; acetylcholine and bradykinin cause a greater increase in NO production in adult pigs. Expression and activity of cyclooxygenase in cerebral microvascular endothelial cells is similar in newborn and adult pigs; acetylcholine and bradykinin stimulated dilator prostanoid production to the same degree in both age groups. Endothelial prostanoid synthesis in cerebral microvessels and cultured endothelial cells was inhibited 30-70% by NS-398, reflecting a large contribution of COX-2 in both newborn and adult animals. These data indicate that in the cerebral circulation of pigs, NO synthase is age-dependently upregulated, whereas endothelial cyclooxygenase is not altered during postnatal development.     

SH3BP4 Regulates Intestinal Stem Cells and Tumorigenesis by Modulating β-Catenin Nuclear Localization

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Candidate SNP of CACNA2D1 Gene Associated with Clinical Mastitis and Production Traits in Sahiwal (Bos taurus indicus) and Karan Fries (Bos taurus taurus × Bos taurus indicus)

The present study was conducted to identify polymorphisms in CACNA2D1 gene and their association with clinical mastitis and production traits. Exon 18 and its flanking regions were screened for the presence of SNPs. Statistical analysis was performed to identify association of period of birth, breed, and genotype with mastitis incidence on randomly selected 103 Sahiwal and 102 Karan Fries cattle. PCR-RFLP analysis revealed that g.38819398G?>?A mutation in exon 18 (269?bp amplicon) of CACNA2D1 gene resolved into AA, AG, and GG genotypes in Sahiwal and Karan Fries cattle. Wald chi-square analysis revealed that the period of birth, breed, and genotype were significantly associated with mastitis incidence. GG genotyped cattle were found to be less susceptible to mastitis. Least square analysis revealed that GG and AG genotype animals of G38819398A SNP of CACNA2D1 gene in Sahiwal as well as in Karan Fries cattle were associated with higher average milk yields during 1st, 2nd, and 3rd lactations (P?<?0.01). These observations and their differential association with the incidence of mastitis and production traits can be utilized as an aid to selection for simultaneous improvement of both antagonistic traits; however, validation of results on large number of animals is warranted.