Response of cardiac myocytes to a ramp increase of diacylglycerol generated by photolysis of a novel caged diacylglycerol.

To test the responsiveness of living cells to the intracellular messenger diacylglycerol, we developed a prototype caged diacylglycerol compound, 3-O-(alpha-carboxyl-2,4-dinitrobenzyl)-1 ,2-dioctanoyl-rac-glycerol (designated alpha-carboxyl caged diC(8)), that produces dioctanoylglycerol (diC(8)) on photolysis. Alpha-Carboxyl caged diC(8) is biologically inert toward diacylglycerol kinase and protein kinase C in vitro and is readily incorporated into cardiac myocyte membranes, where it has no effect before irradiation. Exposure to near-UV light releases biologically active diC8 in good yield (quantum efficiency = 0.2). Here we examine a cellular response to controlled elevation of diC8 within single cardiac myocytes. Twitch amplitude was monitored in electrically stimulated myocytes, and a ramp increase in the concentration of diC(8) was generated by continuous irradiation of cells loaded with the caged compound. The myocyte response was biphasic with a positive inotropic phase (39% increase in twitch amplitude), followed by a large negative inotropic phase (>80% decrease). The time to peak inotropy for both phases depended on the light intensity, decreasing from 376 +/- 51 S to 44 +/- 5 s (positive phase) and 422 +/- 118 S to 51 +/- 9 S (negative phase) as the light intensity was increased eightfold. Both phases were inhibited by the protein kinase C inhibitor chelethyrine chloride. An increase in extracellular K+ from 5 mM to 20 mM to partially depolarize the cell membrane eliminated the positive inotropic phase, but the negative inotropic response was largely unaltered. The results reveal new features in the response of cardiac muscle to diacylglycerol, including a positive inotropic phase and a complex responsiveness to a simple linear increase in diacylglycerol. The effects of photoreleased diC(8) were similar to the effects of opiate agonists selective for kappa receptors, consistent with a major role for diacylglycerol in these responses.     

Integration ofhup cosmid pHU52 into the chromosomal DNA ofCicer-Rhizobium using Tn5 as an homologous sequence

Cosmid pHU52, which carrieshup genes ofBradyrhizobium japonicum, has been integrated into theCicer-Rhizobium G36-84 genome via Tn5-mediated homologous recombination. Tn5 was inserted into both the cosmid pHU52 and the chromosome ofCicer-Rhizobium to provide a region of DNA homology, without affecting the expression of necessary genes. An incompatible plasmid, pPH1JI, was used to select those few cells that had undergone recombination. The integration of the cosmid was demonstrated by Southern blot analysis. Chromosomal integration of thehup genes maximized stability and minimized the potential for their horizontal transfer to other bacterial species. The integratedhup genes were found to expressex planta as well in nodules. The method described illustrates how a given gene can be stably integrated into the chromosome.     

Activation of ryanodine receptors by imperatoxin A and a peptide segment of the II-III loop of the dihydropyridine receptor

Excitation-contraction coupling in skeletal muscle is believed to be triggered by direct protein-protein interactions between the sarcolemmal dihydropyridine-sensitive Ca2+ channel and the Ca2+ release channel/ryanodine receptor (RyR) of sarcoplasmic reticulum. A 138-amino acid cytoplasmic loop between repeats II and III of the alpha1 subunit of the skeletal dihydropyridine receptor (the II-III loop) interacts with a region of the RyR to elicit Ca2+ release. In addition, small segments (10-20 amino acid residues) of the II-III loop retain the capacity to activate Ca2+ release. Imperatoxin A, a 33-amino acid peptide from the scorpion Pandinus imperator, binds directly to the RyR and displays structural and functional homology with an activating segment of the II-III loop (Glu666-Leu690). Mutations in a structural motif composed of a cluster of basic amino acids followed by Ser or Thr dramatically reduce or completely abolish the capacity of the peptides to activate RyRs. Thus, the Imperatoxin A-RyR interaction mimics critical molecular characteristics of the II-III loop-RyR interaction and may be a useful tool to elucidate the molecular mechanism that couples membrane depolarization to sarcoplasmic reticulum Ca2+ release in vivo.     

Micropropagation of Hemidesmus indicus for cultivation and production of 2-hydroxy 4-methoxy benzaldehyde

Caulogenic responses of various explant types from 12-month-old plants of Hemidesmus indicus were tested. Second and third visible nodes (0.5 cm) from the apex and root segments (0.5 cm) were the most and least regenerative respectively, with the formation of 9.37 and 2.6 shoots in 4 weeks on half strength MS medium supplemented with 2.22 μM BA and 1.07 μM NAA and 4.44 μM BA and 2.69 μM NAA respectively. Caulogenic ability of the nodes decreased with increasing maturity. Shoot buds initiated upon the young nodes on day 10 developed into 7.2 cm long shoots within 4 weeks thereby making a shoot elongation phase unnecessary. Nodal explants of the in vitro raised shoots subcultured in the same medium produced 9.32 shoots of 7.1 cm length in 3–4 weeks, similar to those of the mature plant-derived nodes. Multiplication through subculture of the nodes up to 25 passages of 4 weeks each was achieved without decline. Shoot cultures were rooted in quarter salt strength MS medium containing 9.8 μM IBA and the rooted plants were hardened for establishment in pots at 96% rate. Four months after establishment, the micropropagated plants were stable and showed uniform morphological and growth characteristic. After 12 months of cultivation in the field, on an average micropropagated plant consisted of 4–5 shoots, 5–8 branches per shoot and increased root biomass (13.5 g) compared to the poor growth (single shoot and 2–3 branches) and root production (4.6 g) values obtained with plants raised from conventional rooted stem cuttings. The concentration of the root specific compound, 2-hydroxy 4-methoxy benzaldehyde per plant was 2–3 fold higher in micropropagated plants though on unit dry root biomass (0.12% per g dry wt) basis it remained the same between two sources of plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Differential regulation of endoplasmic reticulum structure through VAP-Nir protein interaction

The endoplasmic reticulum (ER) exhibits a characteristic tubular structure that is dynamically rearranged in response to specific physiological demands. However, the mechanisms by which the ER maintains its characteristic structure are largely unknown. Here we show that the integral ER-membrane protein VAP-B causes a striking rearrangement of the ER through interaction with the Nir2 and Nir3 proteins. We provide evidence that Nir (Nir1, Nir2, and Nir3)-VAP-B interactions are mediated through the conserved FFAT (two phenylalanines (FF) in acidic tract) motif present in Nir proteins. However, each interaction affects the structural integrity of the ER differently. Whereas the Nir2-VAP-B interaction induces the formation of stacked ER membrane arrays, the Nir3-VAP-B interaction leads to a gross remodeling of the ER and the bundling of thick microtubules along the altered ER membranes. In contrast, the Nir1-VAP-B interaction has no apparent effect on ER structure. We also show that the Nir2-VAP-B interaction attenuates protein export from the ER. These results demonstrate new mechanisms for the regulation of ER structure, all of which are mediated through interaction with an identical integral ER-membrane protein.     

Isolation and molecular characterization of Toxoplasma gondii from chickens from Sri Lanka

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 100 free-range chickens (Gallus domesticus) from Sri Lanka was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). Antibodies were found in 39 chickens with titers of 1:5 in 8, 1:10 in 8, 1:20 in 4, 1:40 in 5, 1:80 in 5, 1:160 in 5, 1:320 in 2, 1:640 or more in 2. Hearts and brains of 36 chickens with MAT titers of 1:5 or more were bioassayed in mice. Tissues of 3 chickens with doubtful titers of 1:5 were pooled and fed to a cat; the cat shed T. gondii oocysts in its feces. Tissues from 61 chickens with titers of less than 1:5 were pooled and fed to 2 T. gondii-free cats; the cats did not shed oocysts. Toxoplasma gondii was isolated from 11 of 36 seropositive chickens by bioassay in mice. All 12 T. gondii isolates were avirulent for mice. Genotyping of 12 isolates using the SAG2 locus indicated that 6 were type III, and 6 were type II. This is the first report of genetic characterization of T. gondii from any host in Sri Lanka.     

A comparison of several serologic tests to detect antibodies to Toxoplasma gondii in naturally exposed bottlenose dolphins (Tursiops truncatus)

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In a previous study, 138 of 141 (97.8%) bottlenose dolphins (Tursiops truncatus) from the coasts of Florida and California had antibodies to T. gondii by the modified agglutination test (MAT). Although the MAT has been found to be highly sensitive and specific for T. gondii antibodies from several species of terrestrial animals, it has not yet been validated for T. gondii infections in marine mammals. Furthermore, T. gondii has yet not been isolated from dolphins. In the present study, sera from 146 (60 from the 2004 samples and 86 from the 2003 samples) T. truncatus from the coastal areas of South Carolina and Florida were tested for antibodies to T. gondii. Sera from 2004 were tested by the MAT, the indirect fluorescent antibody test (IFAT), the Sabin-Feldman dye test (DT), an indirect hemagglutination test (IHAT), an enzyme-linked immunosorbent assay (ELISA), and Western blot. All 60 dolphins were seropositive, with MAT titers of 1:20 in 3, 1:40 in 19, 1:80 in 29, 1:160 in 2, 1:1,280 in 3, 1:2,560 in 2, and 1:5,120 or higher in 2, and these results were confirmed in another laboratory. The DT titers of these dolphins were <1:10 in 53, 1:800 in 3, 1:1,600 in 2, and 1:3,200 in 2. The IHAT titers were <1:64 in 52, 1:128 in 1, 1:512 in 2, and 1:2,048 in 5. The IFAT titers were <1:20 in 3, 1:20 in 11, 1:40 in 36, 1:80 in 2, 1:160 in 1, and 1:320 or higher in 7. All 7 DT-positive dolphins had high MAT titers, but 2 were negative by the IHAT. Western blot results closely followed MAT results; ELISA results matched MAT results, which were 1:40 or higher. In sera from the 2003 samples, MAT antibodies were found in 86 of 86 dolphins with titers of 1:25 in 29, 1:50 in 23, 1:100 in 27, 1:200 in 3, 1:1,600 in 1, and 1:3,200 in 3; these sera were not tested by other means. Overall, MAT antibodies were found in all 146 dolphin sera tested. Because marine mammals are considered sentinel animals indicative of contamination of the coastal and marine waters by T. gondii oocysts, serologically positive infections need to be validated by the detection of T. gondii organisms in the tissues of seropositive animals.