Genetic analysis and molecular mapping of seedling survival drought tolerance gene in lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis> Medikus)

Lentil populations were developed from crosses between ‘JL-3’ (sensitive to drought stress) and ‘PDL-1’ and ‘FLIP-96-51’ (tolerant to drought stress), to study the inheritance of drought tolerance and to identify the markers associated with it. The parental types, F₁, F₂, F₃, and backcross (BC) generations were screened for drought tolerance using seedling survivability and drought scores. The F₁ hybrids responded similar to the drought-tolerant parent, indicating dominance of seedling drought tolerance over sensitivity. Segregation for seedling survival drought tolerance versus sensitivity in F₂ generation was in complete agreement with monogenic 3:1 ratio. The F₃ families and backcross data additionally confirmed monogenic tolerance based on seedling survival under drought. Out of 51 SSR markers screened, thirteen markers were polymorphic between the parental types. Seven markers among them were found to be associated with seedling survival drought tolerance through bulk segregant analysis. Association of these markers with seedling survival drought tolerance was further confirmed through their screening on 10 drought-tolerant and drought-sensitive genotypes. These seven markers were screened in F₂ mapping population (JL-3 × PDL-1) of 101 individuals to map their position in relation to the gene for seedling survival drought tolerance. Linkage analysis mapped the seven markers within a map distance of 133.2 cM. A single major gene Sdt was identified with a LOD value of 19.9 and phenotypic variation (R ²) of 69.7 %. The Sdt locus was obtained in the marker interval of PLC_105–PBA_LC_1480 spanning 24.9 cM with the closest marker PLC_105 at a distance of 9.0 cM on the obtained linkage group. This is the first report on genetic control and linkage of SSR markers for drought tolerance in lentil. These linked markers can be used in molecular breeding programmes for introgression of seedling survival drought tolerance gene in high-yielding cultivars.     

Measurement of pulmonary blood flow by fractal analysis of flow heterogeneity in isolated canine lungs

Barman, Scott A., Laryssa L. McCloud, John D. Catravas, andIna C. Ehrhart. Measurement of pulmonary blood flow by fractalanalysis of flow heterogeneity in isolated canine lungs. J. Appl. Physiol. 81(5):2039-2045, 1996.Regional heterogeneity of lung blood flow can bemeasured by analyzing the relative dispersion (RD) of mass(weight)-flow data. Numerous studies have shown that pulmonary bloodflow is fractal in nature, a phenomenon that can be characterized bythe fractal dimension and the RD for the smallest realizable volumeelement (piece size). Although information exists for theapplicability of fractal analysis to pulmonary blood flow in wholeanimal models, little is known in isolated organs. Therefore, thepresent study was done to determine the effect of blood flow rate onthe distribution of pulmonary blood flow in the isolated blood-perfusedcanine lung lobe by using fractal analysis. Four different radiolabeledmicrospheres (¹⁴¹Ce,⁹⁵Nb,⁸⁵Sr, and⁵¹Cr), each 15 µm in diameter,were injected into the pulmonary lobar artery of isolated canine lunglobes (n = 5) perfused at fourdifferent flow rates ( flow1 = 0.42 ± 0.02 l/min;flow2 = 1.12 ± 0.07 l/min;flow 3 = 2.25 ± 0.17 l/min; flow 4 = 2.59 ± 0.17 l/min), and the pulmonary blood flow distribution was measured. Theresults of the present study indicate that under isogravimetric bloodflow conditions, all regions of horizontally perfused isolated lunglobes received blood flow that was preferentially distributed to themost distal caudal regions of the lobe. Regional pulmonary blood flowin the isolated perfused canine lobe was heterogeneous and fractal innature, as measured by the RD. As flow rates increased, fractal dimension values (averaging 1.22 ± 0.08) remained constant, whereas RD decreased, reflecting more homogeneous blood flowdistribution. At any given blood flow rate, high-flow areas of the lobereceived a proportionally larger amount of regional flow, suggestingthat the degree of pulmonary vascular recruitment may also be spatially related.

     

Cell Lysis of Bacillus subtilis Caused by Intracellular Accumulation of Glucose-1-Phosphate

Mutants deficient in both glucose-6-phosphate dehydrogenase and phosphoglucose isomerase lysed 4 to 5 h after growth in nutrient medium containing glucose, or after prolonged incubation if the medium contained galactose. The lysis could be prevented by the addition of any other rapidly metabolizable carbon source such as fructose, glucosamine, or glycerol. The glucose-induced lysis was also abolished by introduction of a third mutation lacking phospho-glucose mutase activity but not by a third mutation lacking uridine diphosphate-glucose pyrophosphorylase or teichoic acid glucosyl transferase activity. Galactose-induced lysis was prevented only if the additional mutation abolished the uridine diphosphate-glucose pyrophosphorylase activity. The results showed that lysis was caused by the intracellular accumulation of glucose-1-phosphate, which in turn inhibited at least one of the two enzymes that convert glucosamine-6-phosphate to N-acetyl glucosamine-6-phosphate.     

Antibodies Against Synthetic Peptides Predicted from the Nucleotide Sequence of D2 Receptor Recognize Native Dopamine Receptor Protein in Rat Striatum

Two peptides corresponding to amino acid sequences predicted from the nucleotide sequence of the dopamine D2 receptor were synthesized. Peptide I (CGSEG-KADRPHYC) and peptide II (NNTDQNECIIY), corresponding to 24-34 and 176-185 from the NH2 terminus, respectively, were conjugated to keyhold limpet hemocyanin and injected into rabbits. Peptide I showed a greater immunogenic response than did peptide II. Both peptide antibodies exhibited high titer for the homologous antigens, but showed little or no cross-reactivity with heterogeneous peptides. Peptide I antibodies reacted with striatal membrane proteins of apparent molecular masses of 120, 90, 85, and 30 kDa on a western blot. Furthermore, the 90-kDa band was identified as denatured D2 receptor by its high affinity for the D2 selective photoaffinity probe 125I-N'-azidospiperone (125I-NAPS). Photoaffinity labeling of the 90-kDa protein by 125I-NAPS was reduced by 40% in the presence of the peptide I antibody. In addition, evidence is also presented to show the low level of 90-kDa protein in cerebellum which contains little or no D2 ligand binding sites. The antibody to peptide I inhibited the binding of [3H]YM-09151-2, a dopamine D2 receptor selective antagonist, to striatal membranes in a concentration-dependent manner; a 50% inhibition was obtained at a 1:500 dilution of the antisera with 20 pM ligand concentration. The data on the equilibrium inhibition kinetics of [3H]YM-09151-2 binding to striatal membranes were examined in the presence of antibody and showed a 25-30% decrease in Bmax (203.5 +/- 11.0 and 164.6 +/- 3.3 fmol/mg of protein in presence of preimmune and immune sera, respectively) with no change in KD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cryoenzymic studies on actomyosin ATPase. Evidence that the degree of saturation of actin with myosin subfragment 1 affects the kinetics of the binding of ATP

The initial steps of actomyosin subfragment 1 (acto-S1) ATPase (dissociation and binding of ATP) were studied at -15 degrees C with 40% ethylene glycol as antifreeze. The dissociation kinetics were followed by light scattering in a stopped-flow apparatus, and the binding of ATP was followed by the ATP chase method in a rapid-flow quench apparatus. The data from the chase experiments were fitted to E + ATP in equilibrium (K1) E.ATP----(k2) E*ATP, where E is acto-S1 or S1. The kinetics of the binding of ATP to acto-S1 were sensitive to the degree of saturation of the actin with S1. There was a sharp transition with actin nearly saturated with S1: when the S1 to actin ratio was low, the kinetics were fast (K1 greater than 300 microM, k2 greater than 40 s-1); when it was high, they were slow (K1 = 14 microM, k2 = 2 s-1). With S1 alone K1 = 12 microM and k2 = 0.07 S-1. With acto heavy meromyosin (acto-HMM) the binding kinetics were the same as with saturated acto-S1, regardless of the HMM to actin ratio. The dissociation kinetics were independent of the S1 to actin ratio. Saturation kinetics were obtained with Kd = 460 microM and kd = 75 S-1. The data for the saturated acto-S1 could be fitted to a reaction scheme, but for lack of structural information the abrupt dependence of the ATP binding kinetics upon the S1 to actin ratio is difficult to explain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Specificity and affinity of neuraminic acid exhibited by canine rotavirus strain K9 carbohydrate‐binding domain (VP8*)

The outer capsid spike protein VP4 of rotaviruses is a major determinant of infectivity and serotype specificity. Proteolytic cleavage of VP4 into 2 domains, VP8* and VP5*, enhances rotaviral infectivity. Interactions between the VP4 carbohydrate‐binding domain (VP8*) and cell surface glycoconjugates facilitate initial virus‐cell attachment and subsequent cell entry. Our saturation transfer difference nuclear magnetic resonance (STD NMR) and isothermal titration calorimetry (ITC) studies demonstrated that VP8*_64‐224 of canine rotavirus strain K9 interacts with N‐acetylneuraminic and N‐glycolylneuraminic acid derivatives, exhibiting comparable binding epitopes to VP8* from other neuraminidase‐sensitive animal rotaviruses from pigs (CRW‐8), cattle (bovine Nebraska calf diarrhoea virus, NCDV), and Rhesus monkeys (Simian rhesus rotavirus, RRV). Importantly, evidence was obtained for a preference by K9 rotavirus for the N‐glycolyl‐ over the N‐acetylneuraminic acid derivative. This indicates that a VP4 serotype 5A rotavirus (such as K9) can exhibit a neuraminic acid receptor preference that differs from that of a serotype 5B rotavirus (such as RRV) and the receptor preference of rotaviruses can vary within a particular VP4 genotype.     

Evidence of putative sensory receptors from snout and tongue in an upstream amphihaline migratory fish hilsa <Emphasis Type="Italic">Tenualosa ilisha</Emphasis>

Epidermal sensory structures of adults and juveniles of amphihaline migratory fish hilsa Tenualosa ilisha were studied from two habitats, i.e., freshwater (FW) and marine water (MW). Every year, adults and sexually mature hilsa migrate upstream from marine habitat to riverine freshwater habitat for breeding. This report provides evidences of chemoreception on their upstream migration through several characteristic features on their body, especially on the head and oral cavity. Scanning electron microscopy (SEM) reveals that freshwater adult hilsa (FH) has abundant solitary chemosensory cells (SCCs) on the snout epidermis (around the openings of the epidermal pit) and upper lip, whereas marine water adult hilsa (MH) moderately possesses such sensory structures. The juveniles returning to marine water completely lack SCCs. Immunohistochemical studies revealed the expression of PLC β2 on the snout of FH and tongue of both FH and MH. Further analysis (immunofluorescence, immunoblot and densitometry) of the epidermis confirms the presence of chemosensory structures through strong expression and localization of G-proteins (Gαq and Gα s/olf) from the snout as well as tongue in freshwater hilsa. The SEM also confirms the presence of two types of taste buds in FH, viz. type I (TB I) and type III (TB III). Whereas TB I and TB III are observed on the upper palatine and lips, most of the TB III are located on the tongue region of freshwater and marine hilsa. The juvenile hilsa are devoid of such structures. The presence of dense and rich SCCs and taste sensory cells in adults could be a characteristic feature for strong sensory reception to recognize odour and food-related environmental cues from habitats where they often migrate.     

Optical and chemical identification of kinetic steps in trypsin- and chymotrypsin-catalysed reactions

Previous interpretations of the mechanism of trypsin- and chymotrypsin-catalysed reactions in terms of two intermediates, the Michaelis complex and an acyl-enzyme, were based on steady-state studies and on the observation of individual steps under sub-optimum conditions. In the present paper new methods for the rapid analysis of chemical events and for the spectrophotometric detection of individual steps are applied to these two enzymes. These methods can be used to study reactions with specific amino acid ester substrates. It can be shown that there are at least three distinct steps between the Michaelis complex and the release of ethanol; the latter is likely to correspond to acyl-enzyme formation. The relative rates of these three steps are measured by rapid-flow techniques from observations of the displacement of chromophoric inhibitors and reactions with specific substrates containing chromophores, as well as from ethanol analyses during a single turnover of the enzyme reactions. It is concluded that the reactions of trypsin and chymotrypsin with their specific substrates involve the formation of a specially reactive conformation of the enzyme–substrate complex and that the rate constants involved in this rearrangement are at least as important for the overall reaction as those of the subsequent formation and decomposition of the acyl-enzyme.     

Lipid raft disruption by cholesterol depletion enhances influenza A virus budding from MDCK cells

Lipid rafts play critical roles in many aspects of the influenza A virus life cycle. Cholesterol is a critical structural component of lipid rafts, and depletion of cholesterol leads to disorganization of lipid raft microdomains. In this study, we have investigated the effect of cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) treatment on influenza virus budding. When virus-infected Madin-Darby canine kidney cells were treated with MbetaCD at the late phase of infection for a short duration, budding of virus particles, as determined by protein analysis and electron microscopy, increased with increasing concentrations and lengths of treatment. However, infectious virus yield varied, depending on the concentration and duration of MbetaCD treatment. Low concentrations of MbetaCD increased infectious virus yield throughout the treatment period, but higher concentrations caused an initial increase of infectious virus titer followed by a decrease with a longer duration. Relative infectivity of the released virus particles, on the other hand, decreased with increasing concentrations and durations of MbetaCD treatment. Loss of infectivity of virus particles is due to multiple effects of MbetaCD-mediated cholesterol depletion causing disruption of lipid rafts, changes in structural integrity of the viral membrane, leakage of viral proteins, a nick or hole on the viral envelope, and disruption of the virus structure. Exogenous cholesterol increased lipid raft integrity, inhibited particle release, and partially restored the infectivity of the released virus particles. These data show that disruption of lipid rafts by cholesterol depletion caused an enhancement of virus particle release from infected cells and a decrease in the infectivity of virus particles.