Reversible alteration of morphology in an invertebrate erythrocyte: properties of the natural inducer and the cellular response

The normal shape of the erythrocytes of the bivalves known as blood clams is maintained by a marginal band (MB) of microtubules. When hemolymph (or "blood") is withdrawn from the animal, its erythrocytes change, within minutes, from the normal smooth-surfaced, flattened ellipsoids (N-cells) to spheroids with folded surfaces (X-cells). This alteration can be prevented by rapidly diluting the hemolymph with physiological medium, yielding N-cells for use in studying the transformation to X-cells. Bioassays showed that shape transformation was induced by a hemolymph activity (Hx) and was a function, in part, of cell responsiveness to this activity. Eventually the shape of the cells spontaneously returned to normal, at a rate dependent upon the concentration of the cells and of Hx; recovery was correlated with loss of Hx. The X-cells contained an intact but highly deformed MB, but this was not the effector of the transformation. Erythrocytes made to lack MBs still changed shape, although they did not recover as completely as did the MB-containing controls. When clams were cooled before hemolymph was withdrawn, the concentration of Hx was reduced. Hx was retained after dialysis of hemolymph, and initial filtration and chromatography indicated that its Mr was greater than 500,000. Shape transformation was blocked by EGTA, by serine protease inhibitors, and by sodium azide; the last indicates ATP-dependence. Although the mechanism responsible for shape transformation remains to be determined, the data suggest that the change is triggered by a coagulation-related activity in response to the removal of hemolymph from the animal.     

Development of special hyphal branches of Phyllactinia corylea on host and non-host surfaces

Hyphae of Phyllactinia corylea produce two kinds of special branches on the host surface: adhesion bodies which serve as fungal attachment and stomatopodia which enter the leaf through stomata. Conidial germination on host and non-host surfaces was examined with a scanning electron microscope to explain the stimuli responsible for development of the special branches, and the involvement of host recognition in the process. Conidia germinated within 4 h on host and non-host surfaces, but on non-host surfaces the emergence of the germ tube was not always directed towards the substratum. Adhesion bodies were formed from the tips of germ tubes at the first contact point on host and non-host surfaces. Development of stomatopodia was more specific and they were formed precisely over stomata on the host surface. Stomatopodia-like structures were occasionally formed over finely ridged leaf veins on the host surface and over some fine scratches on synthetic surfaces. The experiments showed that while conidial germination and development of adhesion bodies are in response to contact stimuli, the development of stomatopodia is a response to precise topographical signals, and the directional emergence and attached growth of germ tubes involve host recognition.This revised version was published online in October 2005 with corrections to the Cover Date.     

Development of acetylcholinesterase silica sol-gel immobilized biosensor--an application towards oxydemeton methyl detection

An amperometric silica sol-gel film immobilized biosensor doped with acetylcholinesterase was fabricated in the laboratory finding application in organophosphate detection based on enzyme inhibition. The substrate used was acetylthiocholine chloride and thiocholine released from the enzymatic hydrolysis was electrochemically oxidized giving larger anodic current at 0.5-0.6 V (versus Ag/AgCl reference). The dependencies of the current response on pH, enzyme loading and thermal stability of the developed biosensor were evaluated. The analytical performance of enzyme electrode towards substrate and inhibitor was investigated. Oxydemeton methyl was taken as a model compound for the inhibition studies. Linear calibration for oxydemeton methyl was obtained in the range of 2-200 ppb under the optimized conditions following an incubation time of 20 min. Treatment of the inhibited enzyme with pyridine-2-aldehyde restored 92% of its original activity. The sensor stored at -20 degrees C had a good storage and operational stability retaining 85% of its original activity for 60 successive measurements.     

Perinatal transmission of SHIV-SF162P3 in Macaca nemestrina

We developed a SHIV/macaque model of transmission from infected dams to their infants. Ten pregnant dams were infected intravenously with 100 MID(50) of macaque-titered SHIV-SF162P3 during the second trimester. Nine infants were born; the seven surviving beyond day of birth suckled for 6 months. Four of nine infants were infected (transmission rate = 44.4%), with one infection in utero, and three intrapartum and/or immediately post-birth via suckling. Varying levels of binding and neutralizing antibodies were transplacentally transferred to infants. Passive antibodies were detected in plasma on the day of birth and persisted for 5 weeks. Infants infected at or after birth controlled acute and post-acute viremia. Exposure to maternal SHIV-SF162P3 during birth and suckling in the presence of autologous maternal neutralizing antibodies may have affected transmission or pathogenesis in the infants. This transmission model can allow investigation of key parameters involved in perinatal transmission of HIV.     

Cell culture and animal models of viral hepatitis. Part I: hepatitis B

Despite the existence of a preventative vaccine, HBV represents a substantial threat to public health, suggesting the need for research to develop new treatments to combat the disease. The authors review the available in vitro and in vivo models, including recently developed transgenic and chimeric mouse models.     

Somatic embryogenesis in Centella asiatica L. an important medicinal and neutraceutical plant of India

Leaf segments excised from Centella asiatica, a medicinal and neutraceutical plant, produced abundant somaticembryoswhen cultured onMS mediumwith 9.29 Mkinetin in combination with 2.26 M2,4-D. Granular, white,shiny clusters of callus developed after 1 week of culture, and then formed heart and cotyledonary stage embryoson the same medium after 4 weeks. Somatic embryos matured and germinated in the presence of MS mediumcontaining 2.32 M kinetin with (2.89M) GA3. Plantlets were successfully transferred to pots containing amixture of soil and vermiculite (1:1).     

Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.     

Yeast and mammalian alpha-glucosidase inhibitory constituents from Himalayan rhubarb Rheum emodi Wall.ex Meisson

The methanolic extract of rhizome of Himalayan rhubarb Rheum emodi displayed mild yeast as well as mammalian intestinal alpha-glucosidase inhibitory activity. However, further fractionation of active extract led to the isolation of several potent molecules in excellent yields, displaying varying degrees of inhibition on two test models of alpha-glucosidase. Rhapontigenin, desoxyrhapontigenin, chrysophanol-8-O-beta-d-glucopyranoside, torachrysone-8-O-beta-d-glucopyranoside displayed potent yeast alpha-glucosidase inhibition. However chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside displayed potent to moderate mammalian alpha-glucosidase inhibitory activity. Other compounds displayed mild activity on both the tests. Except desoxyrhapontigenin and rhapontigenin that increased Vmax, other compounds including crude extract decreased the Vmax significantly (p<0.02) in yeast alpha-glucosidase test. Further kinetic analysis on mammalian alpha-glucosidase inhibition showed that chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin and torachrysone-8-O-beta-d-glucopyranoside may be classified as mixed-noncompetitive inhibitors. However, desoxyrhapontigenin and rhapontigenin may be classified as modulators of enzyme activity. Presence and position of glycoside moiety in compounds appear important for better inhibition of mammalian alpha-glucosidase. This is the first report assigning particularly, mammalian intestinal alpha-glucosidase inhibitory activity to these compounds. Chrysophanol-8-O-beta-d-glucopyranoside, desoxyrhaponticin, desoxyrhapontigenin and rhapontigenin have been isolated in substantial yields from R. emodi for the first time. Therefore, these compounds may have value in the treatment and prevention of hyperglycemia associated diabetes mellitus.     

Analysis of substance P in rat brain by means of immunoaffinity capture and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry

Interest in the analysis of low abundance neuropeptides particularly using matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) is increasing because these neuropeptides are essential to the mechanism of transportation and the metabolism. This article describes an immunoprecipitation procedure that is suitable for MALDI-MS analysis of substance P (SP), a neuropeptide, in rat brain tissues. Substance P was precipitated from brain tissue extracts by immunoprecipitation with antibodies directed against SP, and are analyzed by MALDI-TOF-MS. Mass spectrometric analysis showed a singly charged [M+H]+ ion peak that corresponded to the SP molecular mass and was observed with a detection error of 1.6%. The average mass errors between the observed and theoretical molecular mass were within the 0.11 Da range. Capillary zone electrophoresis analysis was subsequently performed, and the effects of the different separation parameters were examined. Beginning with milligram quantities of brain tissue, picomole quantities of SP could be detected using this method.     

The synthesis of [([beta-D-ribofuranosyloxy)methyl]nucleosides

The coupling reaction of acetoxymethoxy ribofuranoside 4 with nucleic acid bases 5a-f to synthesize novel (ribofuranosyloxy)methyl uracil, thymine, cytosine, adenine, guanine derivatives 6a-g respectively in preference to the expected formation of natural nucleosides 2',3',5'-tri-O-benzoyl uridine, methyluridine, cytidine, adenosine and guanosine 7a-g is described. Detailed study of these reactions catalysed by Lewis acids TMSOTf and SnCl4 is described. TMSOTf exhibited selectivity for the formation of ribofuranosyloxy methyl derivatives 6a-g rather than 7a-g. Reason for formation of 6a-g is explained by HSAB principle.