Ca(2+)-activated myofibrillar ATPase: transient kinetics and the titration of its active sites.

The transient kinetics of rabbit psoas Ca(2+)-activated myofibrillar Mg(2+)-ATPase were studied in a buffer of near physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase and the cleavage and release of products steps were studied by the Pi burst method. The data obtained were interpreted by the simple scheme [formula; see text] represents the myosin heads with or without actin interaction. The constants obtained with myofibrils (where the molecules are highly organized) were compared with those with myosin subfragment 1 (S1) and cross-linked acto-S1 (where the molecules are dispersed in solution). Myofibrils appear to bind ATP as tightly as do S1 and cross-linked acto-S1. This suggests that with them k-2 less than kcat much less than k2, and it is proposed that the ATP chase method can be used to titrate the ATPase sites in myofibrils. The results of titration and single-turnover experiments revealed that myofibrils may contain partially active myosin heads. It is proposed that these heads bind ATP loosely without hydrolysis, as found with S1 [Tesi, C., N. Bachouchi, N., Barman, T., & Travers, F. (1989) Biochimie 71, 363-372]. There were large Pi bursts with the three preparations, showing that with all of them the release of products step (k4) is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of hemolymph polypeptides in Manduca sexta larvae parasitized by Cotesia congregata: A two-dimensional electrophoretic analysis and comparison with major bacteria-induced proteins

Analyses using one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) previously demonstrated that parasitization by the braconid wasp Cotesia congregata significantly alters the normal hemolymph polypeptide profile of host Manduca sexta larvae. In the present study two-dimensional gel analyses corroborated our earlier findings and provided additional evidence that multiple parasitism-specific polypeptides were induced, which varied according to the stage of development of the wasps. Parasitization additionally elicited changes in the total protein concentration detected in the blood. Initially an elevation was observed, with newly parasitized larvae exhibiting a twofold elevation in hemolymph protein concentration by 12–24 h postoviposition. In contrast, terminal-stage hosts with second instar parasites had significantly less protein in the hemolymph, likely due to reduced growth and inhibition of arylphorin synthesis by the fat body during the final stages of parasitism. Comparison of the array of hemolymph polypeptides produced in unparasitized larvae injected with 10⁶cells of the gram-negative bacterium Enterobacter cloacae with those proteins induced by parasitization indicated the two classes are different. Our findings confirm that the hostresponse to parasitism is a specific one, and not mimicked by bacterial challenge. Duringshort-term in vitro culture of wasp larvae dissected from the host hemocoel, several proteins were detected in the medium using SDS - PAGE, with their appearance in vitro suggestive of secretion by the wasps in vivo. Moreover, hemolymph from the parasites had significant amounts of putative host proteins, including an arylphorin - like polypeptide and a protein with a mobility similar to that of insecticyanin. Thus, a dynamic interchange of proteins may occur, with the parasites accumulating host proteins while simultaneously secreting a variety of factors into the host hemocoel.     

Terpenoid compositions, and antioxidant and antimicrobial properties of the rhizome essential oils of different Hedychium species

A phytochemical study of the rhizome essential oils of four different Hedychium species was performed by means of GC and GC/MS analyses. H. ellipticum mainly contained 1,8-cineole, sabinene, and terpin-4-ol, while H. aurantiacum possessed terpin-4-ol, para-cymene, and bornyl acetate as the major entities. Similarly, trans-meta-mentha-2,8-diene and linalool were noticed in H. coronarium. Three different collections (I-III) of H. spicatum showed amazing differences in the relative contents of their essential oils, 1,8-cineole and 10-epi-gamma-eudesmol being identified as markers for samples I and II, terpin-4-ol and sabinene being the major compounds in sample III. The rhizome essential oils of the above species were studied for their antioxidant activities by different methods, including their effect on the chelating properties of Fe(2+), DPPH radical-scavenging activity, and reducing power. Antimicrobial screenings of the oils by the paper-disc method were performed against Staphylococcus aureus, Shigella flexneri, Pasteurella multocida, Escherichia coli, and Salmonella enterica enterica, and the respective minimum-inhibitory-concentration (MIC) values were determined. The rhizome essential oils from all Hedychium species exhibited moderate-to-good Fe(2+) chelating activity. H. spicatum from collection site III showed a completely different DPPH radical-scavenging profile than the samples from the other collection sites.     

O-acetylation of GD3 prevents its apoptotic effect and promotes survival of lymphoblasts in childhood acute lymphoblastic leukaemia

We have previously demonstrated induction of O-acetylated sialoglycoproteins on lymphoblasts of childhood acute lymphoblastic leukaemia (ALL). These molecules promote survival of lymphoblasts by preventing apoptosis. Although O-acetylated sialoglycoproteins are over expressed, the status of O-acetylation of gangliosides and their role in lymphoblasts survival remains to be explored in ALL patients. Here, we have observed enhanced levels of 9-O-acetylated GD3 (9-O-AcGD3) in the lymphoblasts of patients and leukaemic cell line versus disialoganglioside GD3 in comparison to the normal cells. Localization of GD3 and 9-O-AcGD3 on mitochondria of patient's lymphoblasts has been demonstrated by immuno-electron microscopy. The exogenous administration of GD3-induced apoptosis in lymphoblasts as evident from the nuclear fragmentation and sub G0/G1 apoptotic peak. In contrast, 9-O-AcGD3 failed to induce such apoptosis. We further explored the mitochondria-dependent pathway triggered during GD3-induced apoptosis in lymphoblasts. GD3 caused a time-dependent depolarization of mitochondrial membrane potential, release of cytochrome c and 7.4- and 8-fold increased in caspase 9 and caspase 3 activity respectively. However, under identical conditions, an equimolar concentration of 9-O-AcGD3 failed to induce similar effects. Interestingly, 9-O-AcGD3 protected the lymphoblasts from GD3-induced apoptosis when administered in equimolar concentrations simultaneously. In situ de-O-acetylation of 9-O-AcGD3 with sodium salicylate restores the GD3-responsiveness to apoptotic signals. Although both GD3 and 9-O-acetyl GD3 localize to mitochondria, these two structurally related molecules may play different roles in ALL-disease biology. Taken together, our results suggest that O-acetylation of GD3, like that of O-acetylated sialoglycoproteins, might be a general strategy adopted by leukaemic blasts towards survival in ALL.     

Orally active esters of dihydroartemisinin: Synthesis and antimalarial activity against multidrug-resistant Plasmodium yoelii in mice

A series of artemisinin derived esters 7a-j, incorporating pharmacologically privileged substructure, such as biphenyl, adamantane and fluorene, have been prepared and evaluated for antimalarial activity against multidrug-resistant (MDR) Plasmodium yoelii nigeriensis by oral route. Several of these compounds were found to be more active than the antimalarial drugs beta-arteether 4 and artesunic acid 5. Ester 7i, the most active compound of the series, provided 100% and 80% protection to the infected mice at 24 mg/kg x 4 days and 12 mg/kg x 4 days, respectively. In this model beta-arteether provided 100% and 20% protection at 48 mg/kg x 4 days and 24 mg/kg x 4 days, respectively.     

Orally active 1,2,4-trioxepanes: synthesis and antimalarial activity of a series of 7-arylvinyl-1,2,4-trioxepanes against multidrug-resistant Plasmodium yoelii in Swiss mice

7-Arylvinyl-1,2,4-trioxepanes 7a-d, 8a-d, 9a-d, 10a-d, 11a-c, and 12a-c, prepared by photooxygenation of homoallylic alcohols 5a-d, were evaluated against multi-drug resistant Plasmodium yoelii nigeriensis in Swiss mice by oral and intramuscular routes. Trioxepane 11c, the most active compound of the series, showed more than 98% suppression of parsitaemia at 96 mg/kg by both oral and intramuscular routes. This is the first report on in vivo active 1,2,4-trioxepanes.     

Role of serotonergic input to the ventrolateral medulla in expression of the 10-Hz sympathetic nerve rhythm

We studied the changes in inferior cardiac sympathetic nerve discharge (SND) produced by unilateral microinjections of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists into the ventrolateral medulla (VLM) of urethane-anesthetized, baroreceptor-denervated cats. Microinjection of the 5-HT2 receptor antagonist LY-53857 (10 mM) into either the rostral or caudal VLM significantly reduced (P < or = 0.05) the 10-Hz rhythmic component of basal SND without affecting its lower-frequency, aperiodic component. The selective depression of 10-Hz power was accompanied by a statistically significant decrease in mean arterial pressure (MAP). Microinjection of LY-53857 into the VLM also attenuated the increase in 10-Hz power that followed tetanic stimulation of depressor sites in the caudal medullary raphé nuclei. Microinjection of the 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)2-amino-propane (DOI; 10 microM) into the VLM selectively enhanced 10-Hz SND, and intravenous DOI (1 mg/kg) partially reversed the reduction in 10-Hz SND produced by 5-HT2 receptor blockade in the VLM. Microinjection of the 5-HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OHDPAT; 10 mM), into either the rostral or caudal VLM also selectively attenuated 10-Hz SND and significantly reduced MAP. The reduction in 10-Hz SND produced by 8-OHDPAT was partially reversed by intravenous WAY-100635 (1 mg/kg), which selectively blocks 5-HT1A receptors. These results support the view that serotonergic inputs to the VLM play an important role in expression of the 10-Hz rhythm in SND.     

Histone acetyltransferase-1 regulates integrity of cytosolic histone H3-H4 containing complex

Amounts of soluble histones in cells are tightly regulated to ensure supplying them for the newly synthesized DNA and preventing the toxic effect of excess histones. Prior to incorporation into chromatin, newly synthesized histones H3 and H4 are highly acetylated in pre-deposition complex, wherein H4 is di-acetylated at Lys-5 and Lys-12 residues by histone acetyltransferase-1 (Hat1), but their role in histone metabolism is still unclear. Here, using chicken DT 40 cytosolic extracts, we found that histones H3/H4 and their chaperone Asf1, including RbAp48, a regulatory subunit of Hat1 enzyme, were associated with Hat1. Interestingly, in HAT1-deficient cells, cytosolic histones H3/H4 fractions on sucrose gradient centrifugation, having a sedimentation coefficient of 5–6S in DT40 cells, were shifted to lower molecular mass fractions, with Asf1. Further, sucrose gradient fractionation of semi-purified tagged Asf1-complexes showed the presence of Hat1, RbAp48 and histones H3/H4 at 5–6S fractions in the complexes. These findings suggest the possible involvement of Hat1 in regulating cytosolic H3/H4 pool mediated by Asf1-containing cytosolic H3/H4 pre-deposition complex.     

Interaction of human 3-phosphoglycerate kinase with L-ADP, the mirror image of D-ADP

l-Nucleoside-analogues, mirror images of the natural d-nucleosides, are a new class of antiviral and anticancer agents. In the cell they have to be phosphorylated to pharmacologically active triphosphate forms, the last step seems to involve human 3-phosphoglycerate kinase (hPGK). Here we present a steady state kinetic and biophysical study of the interaction of the model compound l-MgADP with hPGK. l-MgADP is a good substrate with k_cat and K_m values of 685 s⁻¹ and 0.27 mM, respectively. Double inhibition studies suggest that l-MgADP binds to the specific adenosine-binding site and protects the conformation of hPGK molecule against heat denaturation, as detected by microcalorimetry. Structural details of the interaction in the enzyme active site are different for the d- and l-enantiomers (e.g. the effect of Mg²⁺), but these differences do not prevent the occurrence of the catalytic cycle, which is accompanied by the hinge-bending domain closure, as indicated by SAXS measurements.