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A Molla

Publications and source records attributed to A Molla.

At least 37 records · Page 2Linked to original sources
PubMed1998

ABT-378, a highly potent inhibitor of the human immunodeficiency virus protease.

The valine at position 82 (Val 82) in the active site of the human immunodeficiency virus (HIV) protease mutates in response to therapy with the protease inhibitor ritonavir. By using the X-ray crystal structure of the complex of HIV protease and ritonavir, the potent protease inhibitor ABT-378, which has a diminished interaction with Val 82, was designed. ABT-378 potently inhibited wild-type and mutant HIV protease (Ki = 1.3 to 3.6 pM), blocked the replication of laboratory and clinical strains of HIV type 1 (50% effective concentration [EC50], 0.006 to 0.017 microM), and maintained high potency against mutant HIV selected by ritonavir in vivo (EC50, </=0. 06 microM). The metabolism of ABT-378 was strongly inhibited by ritonavir in vitro. Consequently, following concomitant oral administration of ABT-378 and ritonavir, the concentrations of ABT-378 in rat, dog, and monkey plasma exceeded the in vitro antiviral EC50 in the presence of human serum by >50-fold after 8 h. In healthy human volunteers, coadministration of a single 400-mg dose of ABT-378 with 50 mg of ritonavir enhanced the area under the concentration curve of ABT-378 in plasma by 77-fold over that observed after dosing with ABT-378 alone, and mean concentrations of ABT-378 exceeded the EC50 for >24 h. These results demonstrate the potential utility of ABT-378 as a therapeutic intervention against AIDS.

Animals↗
PubMed1998

Brefeldin A inhibits cell-free, de novo synthesis of poliovirus.

Brefeldin A (BFA), an inhibitor of intracellular vesicle-dependent secretory transport, is a potent inhibitor of poliovirus RNA replication in infected cells. We have determined that the unknown mechanism of BFA inhibition of replication is reproduced in the cell-free poliovirus translation, replication, and encapsidation system. Furthermore, we provide evidence suggesting that the cellular mechanism targeted by BFA, the GTP-dependent synthesis of secretory transport vesicles, may be involved in viral RNA replication in the system via a soluble cellular GTP-binding and -hydrolyzing activity. This activity is related to the ARF (ADP-ribosylation factor) family of GTP-binding proteins. ARFs are required for the formation of several classes of secretory vesicles, and some family members are indirectly inactivated by BFA. Peptides that function as competitive inhibitors of ARF activity in cell-free transport systems also inhibit poliovirus RNA replication, and this inhibitory effect can be countered by the addition of exogenous ARF. We suggest that BFA inhibition of replication is diagnostic of a requirement for ARF activity in the cell-free system.

ADP-Ribosylation Factors↗
PubMed1998

In vitro selection and characterization of human immunodeficiency virus type 1 variants with increased resistance to ABT-378, a novel protease inhibitor.

ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 microM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P1' residue Leu to Phe) and p7/p1 (P2 residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 microM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 microM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763-3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662-6670, 1997).

Amino Acid Sequence↗
PubMed1998

Calcium/calmodulin-dependent protein kinase II controls alpha5beta1 integrin-mediated inside-out signaling.

Fibronectin binding on alpha5beta1 integrin is strictly dependent on intracellular calcium. Using an in vitro assay, we previously found that either calcineurin inhibitors or a blocking calcineurin monoclonal antibody added to cell lysates completely abolished the fibronectin/integrin interaction, which suggested that the activity of calcineurin, a calcium/calmodulin-dependent phosphatase, was required to counteract some kinase activity and maintain the high affinity state of alpha5beta1. In this paper, we show that blocking of the calcium/calmodulin kinase II (CaMKII) activity with the specific inhibitor KN-62 or with its pseudosubtrate Autocamtide-2 preserved the high affinity state of the integrin even under experimental conditions that inhibit calcineurin. Conversely, the addition of purified CaMKII to the cell lysate inhibited alpha5beta1 binding to fibronectin in vitro. Consistent with these results, cell adhesion on fibronectin was stimulated by KN-62. Moreover, Scatchard analysis of fibronectin binding on CHO cells revealed that KN-62 decreased the Kd value from 0.3 to 0.05 microM. Finally the expression of exogenous constitutively active CaMKII resulted in a dramatic defect in cell adhesion with no significant modification in alpha5beta1 cell surface expression. In summary our results demonstrate that CaMKII controls the affinity state of the integrin alpha5beta1 in vitro and in living cells.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Autologous dendritic cells derived from CD34+ progenitors and from monocytes are not functionally equivalent antigen-presenting cells in the induction of melan-A/Mart-1(27-35)-specific CTLs from peripheral blood lymphocytes of melanoma patients with low frequency of CTL precursors.

Peptide presentation by autologous dendritic cells (DCs) is a new tool to activate tumor antigen-specific T cells in melanoma patients. However, it is not known whether autologous DCs, differentiated by two of the most efficient protocols (from CD34+ progenitors or from monocytes), are equally effective as professional antigen-presenting cells (APCs) when the patients have a low frequency of peptide-specific precursors. To this end, a limiting dilution assay was applied to evaluate the frequency of antigen-specific CTL precursors (CTLps) in peripheral blood of HLA-A*0201+ melanoma patients. Then, from two melanoma patients showing low frequency of CTLps to melanoma antigen-A/melanoma antigen recognized by T cell (Melan-A/Mart-1)(27-35) peptide, autologous DCs were differentiated from granulocyte colony-stimulating factor-mobilized CD34+ progenitors or from monocytes. CD34+- and monocyte-derived DCs were characterized by a similar proportion of CD1a+ cells expressing HLA class II antigens and CD54, CD80, and CD86 molecules. Both types of DC presented Melan-A/Mart-1(27-35) and tyrosinase(369-377) peptides to melanoma-specific CTL clones and were equally effective as peptide-pulsed APCs in the activation of influenza A matrix(58-66)-specific CTLs from high-frequency precursors (1294/10(6) and 1789/10(6) lymphocytes in the two patients). However, efficient activation of Melan-A/Mart-1(27-35)-specific CTLs from low-frequency precursors (158/10(6) and 77/10(6) lymphocytes) of the two patients was markedly dependent on the use of peptide-loaded CD34+-derived DCs. These results suggest that CD34+- and monocyte-derived DCs are not functionally equivalent APCs for the activation of low-frequency peptide-specific CTLps.

Antigen-Presenting Cells↗
PubMed1997

Impact of a single megadose of vitamin A at delivery on breastmilk of mothers and morbidity of their infants.

OBJECTIVES: To evaluate the effect of vitamin A supplementation 24 h after delivery on breastmilk retinol concentration. METHODS: Fifty low income women were randomly assigned to a single oral dose of 209 micromol of Vitamin A or none at delivery. Maternal serum and breastmilk retinol levels and infant morbidity and anthropometry were serially assessed. RESULTS: Mean (95% CI) serum retinol levels increased in the supplemented mothers at 2.77 (2.3, 3.2) compared to 1.15 (0.9, 1.4) micromol/l in controls (P < 0.05) and remained at a significantly higher level of 1.59 (1.4, 1.8) micromol/l compared to 1.33 (1.8, 1.5) micromol/l in the control group (P < 0.001) up to a period of three months. Breastmilk retinol concentration was also greater at 24 h after supplementation, mean (CI) 11.34 (9.0, 13.7) micromol/l, compared to 2.95 (2.3, 3.6) micromol/l in the control group (P < 0.0001), and remained higher for the next six months at 1.06 (0.9, , 1.3) micromol/l compared to 0.73 (0.6, 0.8) micromol/l in the control group (P < 0.02). Infants of the supplemented mothers had reduced mean duration of respiratory tract infection of 3.1 (2.7, 3.5) days compared to 3.7 (3.3, 4.2) days (P < 0.03) and mean incidence of febrile illness 0.1 (0.1, 0.1) compared to control infants 0.3 (0.3, 0.3) days, (P < 0.002). CONCLUSION: Vitamin A supplementation of malnourished mothers maintains higher breastmilk retinol concentration for at least six months and reduces the duration of respiratory tract infection and febrile illness in their breastfed infants.

Adult↗
PubMed1997

Expression and function of receptors for extracellular matrix molecules in the differentiation of human megakaryocytes in vitro.

The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34+ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA-2), fibronectin (VLA-4 and VLA-5) and laminin (VLA-6) during the maturation of Mks. We have shown that a significant percentage of CFU-MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM-CD34+ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA-2, VLA-5 and VLA-6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA-4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA-4 may be involved in the adhesion of CFU-MK and immature Mks on fibronectin, then replaced by VLA-5 in the final stages of maturation. The expression of VLA-6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.

Antigens, CD34↗
PubMed1997

Multiple-dose pharmacokinetics of ritonavir in human immunodeficiency virus-infected subjects.

The multiple-dose pharmacokinetics of ritonavir were investigated in four groups of human immunodeficiency virus-positive male subjects (with 16 subjects per group) under nonfasting conditions; a 3:1 ritonavir:placebo ratio was used. Ritonavir was given at 200 (group I), 300 (group II), 400 (group III), or 500 (group IV) mg every 12 h for 2 weeks. The multiple-dose pharmacokinetics of ritonavir were moderately dose dependent, with the clearance for group IV (6.8 +/- 2.7 liters/h) being an average of 32% lower than that for group I (10.0 +/- 3.2 liters/h). First-pass metabolism should be minimal for ritonavir. The functional half-life, estimated from peak and trough concentrations, were similar among the dosage groups, averaging 3.1 and 5.7 h after the morning and evening doses, respectively. The area under the concentration-time curve at 24 h (AUC24) and apparent terminal-phase elimination rate constant remained relatively time invariant, but predose concentrations decreased 30 to 70% over time. Concentration-dependent autoinduction is the most likely mechanism for the time-dependent pharmacokinetics. The Km and initial maximum rate of metabolism (Vmax) values estimated from population pharmacokinetic modeling (nonlinear mixed-effects models) were 3.43 microg/ml and 46.9 mg/h, respectively. The group IV Vmax increased to 68 mg/h after 2 weeks. The maximum concentration of ritonavir in serum (Cmax) and AUC after the evening doses were an average of 30 to 40% lower than the values after the morning doses, while the concentration at 12 h was an average of 32% lower than the predose concentration, probably due to protracted absorption. Less than 2% of the dose was eliminated unchanged in the urine. Triglyceride levels increased from the levels at the baseline, and the levels were correlated with baseline triglyceride levels and AUC, Cmax, or predose concentrations.

Adult↗
PubMed1996

Novel azacyclic ureas that are potent inhibitors of HIV-1 protease.

A series of novel, azacyclic ureas which are highly potent inhibitors of the HIV-1 protease (IC50 = 4.1 to < 0.5 nM) were synthesized. Aqueous solubilities of this series of compounds were improved by incorporating polar functional groups at the P1' P2 and P2' positions. These compounds also possess good anti-viral activity by inhibition of the cytopathic effect of HIV-13B in MT-4 cells in vitro.

Amino Acid Sequence↗
PubMed1996

A novel, picomolar inhibitor of human immunodeficiency virus type 1 protease.

The design, synthesis, and molecular modeling studies of a novel series of azacyclic ureas, which are inhibitors of human immunodeficiency virus type 1 (HIV-1) protease that incorporate different ligands for the S1', S2, and S2' substrate-binding sites of HIV-1 protease are described. The synthesis of this series is highly flexible in the sense that the P1', P2, and P2' residues of the inhibitors can be changed independently. Molecular modeling studies on the phenyl ring of the P2 and P2' ligand suggested incorporation of hydrogen-bonding donor/acceptor groups at the 3' and 4-positions of the phenyl ring should increase binding potency. This led to the discovery of compound 7f (A-98881), which possesses high potency in the HIV-1 protease inhibition assay and the in vitro MT-4 cell culture assay (Ki = approximately 5 pM and EC50 = 0.002 microM). This compares well with the symmetrical cyclic urea 1 pioneered at DuPont Merck.

Binding Sites↗
PubMed1996

Cotranscription of two RNA coding for the cell adhesion regulator and its variant in Reh leukemia cells.

Human genomic DNA analysis reveals the existence of polymorphisms at the cell molecular adhesion regulator (CMAR) locus. In order to choose between the two possible open frames deduced from the variant sequence, we have sequence both the human 5' non-coding region and the mouse CMAR variant DNA. We found that both mRNA species coexist in human cells.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1996

Ordered accumulation of mutations in HIV protease confers resistance to ritonavir.

Analysis of the HIV protease gene from the plasma of HIV-infected patients revealed substitutions at nine different codons selected in response to monotherapy with the protease inhibitor ritonavir. Mutants at valine-82, although insufficient to confer resistance, appeared first in most patients. Significant phenotypic resistance required multiple mutations in HIV protease, which emerged subsequently in an ordered, stepwise fashion. The appearance of resistance mutations was delayed in patients with higher plasma levels of ritonavir. Early mutants retained susceptibility to structurally diverse protease inhibitors, suggesting that dual protease inhibitor therapy might increase the duration of viral suppression.

Codon↗
PubMed1995

Beta 1 integrins mediate adherent phenotype of human erythroblastic cell lines after phorbol 12-myristate 13-acetate induction.

We investigated the effects of phorbol ester (phorbol 12-myristate 13-acetate; PMA) treatment on the adhesive behaviour of three erythroleukaemia cell lines: HEL, LAMA-84 and AP217. In the three cell lines PMA induced an increase in expression of a megakaryocytic marker: alpha IIb beta 3 integrin, but did not promote activation of this receptor. Indeed, an antibody specific for the activated form of alpha IIb beta 3 failed to react with the three cell lines. PMA induction led to different adhesive phenotypes depending on the cell line; in fact LAMA-84 and HEL cells became adherent while AP217 cells remained non-adherent. By studying cell surface receptors we found that the major difference between the adherent and the non-adherent cells was the expression of beta 1 integrins. After PMA induction, beta 1 integrin expression was totally abolished in AP217 cells and the amount of beta 1 mRNA was reduced preventing new synthesis of the subunit. In HEL and LAMA-84 cells, PMA treatment did not alter the overall level of beta 1 integrin but induced a new pattern of alpha-subunit expression: up-regulation of alpha 2 and alpha v subunits and down-regulation of alpha 4 and alpha 5 subunits. Function-perturbing antibodies against beta 1, alpha 4, alpha 5, alpha v and alpha 2 reduced adhesion of HEL cells to fibronectin or collagen, whereas antibodies against beta 3 or alpha v beta 3 did not. Our results favour the involvement of beta 1 integrins in PMA-induced adhesion of erythroleukaemia cells.

Base Sequence↗
PubMed1995

Rice oral rehydration solution hastens recovery from dysentery.

Using a three-cell study design over two years, we assessed the efficacy of rice ORS (R-ORS) for home management of dysentery in rural Bangladesh involving 1,911 children of less than five years of age who were matched for socioeconomic status, morbidity, nutrition and other factors. Dysentery was defined as passing blood and/or mucus in the stool, and the total numbers of episodes studies were: 1472, 1335, and 1557 in the R-ORS, glucose ORS (G-ORS), and comparison areas respectively. Packaged R-ORS and G-ORS were supplied free of cost. The comparison area continued the usual treatment of dysentery as practised in the community. During the two-year period, weekly observations were made in all areas on the incidence, management, and outcome of all episodes of dysentery. We demonstrated that the use of R-ORS was associated with reduction in duration and a lower incidence of prolonged diarrhoea compared to treatment with G-ORS or drugs. The mean durations of dysenteric diarrhoea were 7.6 +/- 4.9, 10.1 +/- 6.3, and 14.0 +/- 8.0 days in the R-ORS, G-ORS, and comparison areas respectively. Similarly, the cumulative recovery rates on day 7 of the illness were 61%, 42% and 21% in R-ORS, G-ORS, and comparison areas respectively. Based on these observations, we conclude that R-ORS may be an effective treatment of dysentery and deserves further investigation.

Bangladesh↗
PubMed1994

Stimulation of poliovirus proteinase 3Cpro-related proteolysis by the genome-linked protein VPg and its precursor 3AB.

Purified recombinant poliovirus polypeptide 3AB interacts with 3CDpro and 3Dpol as shown by coimmunoprecipitation with anti-3Dpol antibodies. A consequence of this interaction is an accelerated autoprocessing of 3CDpro to produce 3Cpro and 3Dpol. The activation of 3Dpol polymerase activity by cleavage of 3CDpro, a polypeptide that has no polymerase activity, can be shown by template- and primer-dependent poly(U) synthesis. Anti-VPg antibodies (VPg = 3B) added to HeLa translation extracts programmed with poliovirion RNA inhibit cleavage of 3CDpro whereas addition of purified 3AB or VPg to these translation reactions increases 3CDpro processing. 3AB stimulates also 3Cpro-related proteolysis of 2BC, a poliovirus-specific, nonstructural processing intermediate. In contrast, 3CDpro-specific cleavage of the structural precursor P1 is inhibited by the addition of 3AB as shown by a decrease in the production of VP0 and VP3. These data shed new light on a phenomenon in the regulation of expression of poliovirus genetic information: whereas the proteinase 3CDpro is needed for processing of the capsid precursor, the cleavage product of this relatively stable precursor is required for RNA replication.

3C Viral Proteases↗
PubMed1994

Studies of a putative amphipathic helix in the N-terminus of poliovirus protein 2C.

Poliovirus protein 2C contains near its N-terminus a putative amphipathic helix which is well conserved among picornaviruses. Three mutants were constructed within this region by site-directed mutagenesis. In the first mutant (pT7XL2-2C-N1) two glutamic acids were replaced with valines at the boundary of the charged and uncharged faces of the helix. The second mutant (pT7XL2-2C-N2) contains an isoleucine to lysine change in the hydrophobic half; in the third mutant (pT7XL2-2C-N3) two lysines were replaced with threonines in the hydrophilic half of the helix. Upon transfection of HeLa cells with RNA transcripts made from these plasmids only pT7XL2-2C-N1 yielded viable virus (W1-2C-N1) which had a small-plaque phenotype. A large-plaque revertant of this virus, W1-2C-N1R, was found to contain the original glutamic acid at one of the mutated sites (E19). There is no detectable minus-stranded RNA synthesis following transfection of HeLa cells with transcript RNAs of the other two plasmids, pT7XL2-2C-N2 and -N3. In vitro translation of these two mutant RNA transcripts in HeLa extracts revealed processing abnormalities in the P2/P3 region of the polyprotein. This leads to a nearly complete absence of 2C and 3AB, which might be the primary cause of defective viral RNA synthesis. The putative amphipathic helix was found to overlap a consensus binding site for double-stranded RNA.

Amino Acid Sequence↗
PubMed1994

Is rice based oral rehydration therapy effective in young infants?

Rice based oral rehydration therapy (ORT) solutions have been shown to be superior to glucose oral rehydration salts (World Health Organisation (WHO) ORS) in reducing stool volume and duration of diarrhoea in children and adults. Rice based ORT has been used only sparingly in young infants, however, because of theoretical concerns about digestibility. A randomised controlled trial of rice based ORT (50 g rice and electrolytes identical to WHO ORS) and WHO ORS was carried out in 52 male infants less than 6 months old with moderately severe acute diarrhoea to evaluate efficacy and digestibility. Nineteen (70%) of 27 children who received rice based ORT and 18 (72%) of 25 children who received WHO ORS were treated successfully. The mean (SD) diarrhoeal stool output for the first 24 hours of treatment was significantly lower in the infants receiving the rice based ORT than in those receiving WHO ORS (101.0 (60.5) v 137.1 (74.6) g/kg). The stool output was also significantly less in the rice based ORT group in the second 24 hours. Infants in the rice based ORT group drank significantly less rehydration solution than infants in the WHO ORS group (mean (SD) 165.4 (77.4) v 217.9 (86.1) during the first 24 hours of treatment. There was no difference in the duration of diarrhoea between the groups. The volume of breast and formula feeding was similar in the two groups. No difference was seen in the frequency of finding reducing substances or acid pH in the stools of either group of children. The results suggest that rice based ORT is as effective as WHO ORS in infants with moderately severe diarrhoea and that rice based ORT is as well tolerated as WHO ORS in infants.

Citrates↗