The enigma of the clandestine association between chloroquine and HIV-1 infection.

OBJECTIVES: The antimalarial drug chloroquine (CQ) dampens the immune system and is used in the treatment of autoimmune disorders. CQ also shows antiviral activity against nonenveloped and enveloped viruses, including HIV-1. Persistent immune activation in chronic HIV-1infection leads to CD4 T-cell depletion. CQ is envisioned to attenuate immune activation and virus activity in HIV-1-infected patients. The role of CQ in immune activation and virus activity is discussed here. METHODS: To elucidate the effect of CQ on immune activation, a retrospective review of published clinical trials, in vivo experimental studies in animals, and the most relevant in vitro observations in HIV-1-infected cells, together with observations from our own laboratory studies, was carried out and the findings discussed. RESULTS: In a few clinical studies and animal experiments, CQ was ineffective in decreasing immune activation and HIV-1 infection. In vitro, CQ markedly increased HIV-1 infection in astrocytes and other non-CD4 cells. CONCLUSIONS: The use of CQ in HIV-1-infected patients is questionable. The evidence for a dampening of immune activation by CQ is inconclusive.

Pharmacokinetic interaction of some antitubercular drugs with caraway: implications in the enhancement of drug bioavailability.

This study deals with the pharmacokinetic interaction of selected anti-TB drugs with a natural product (CC-1a) derived from caraway (Carum carvi, L.) seed. CC-1a, chemically standardized butanolic fraction, enhanced the plasma levels of rifampicin, pyrazinamide, and isoniazid in Wistar rat, resulting in increased bioavailability indices (C(max) and AUC) of the drugs. Moreover, a 40% reduced dose regimen of these drugs, which additionally contained CC-1a, was equivalent in terms of C(max) and AUC to a normal dose regimen. A permeation-enhancing property of CC-1a across small intestinal absorptive surface was found to be a contributing factor in its bioavailability enhancing profile.

Simultaneous high-performance liquid chromatographic determination of Cedrus deodara active constituents and their pharmacokinetic profile in mice.

A specific and sensitive high-performance liquid chromatographic (HPLC) method with photodiode-array (PDA) ultraviolet detection was developed for the simultaneous determination of three bioactive constituents of Cedrus deodara namely wikstromol, matairesinol and dibenzylbutyrolactol in mouse plasma. In solid-phase extraction (SPE) these constituents were successfully separated using a C18 column by isocratic elution using acetonitrile:water containing hexanesulphonic acid, 32:68 (v/v). The flow rate was set at 1ml/min and detector wavelength at 225nm. Good linearity (r2>0.999) was observed over the studied range of 0.015-5.0microg/ml for wikstromol and 0.030-5.0microg/ml for matairesinol and dibenzylbutyrolactol. The CV values of intra-day precision for wikstromol, matairesinol and dibenzylbutyrolactol were in between 1.8-6.9, 1.7-4.9 and 1.6-4.2% and values of inter-day precision were in between 10.4-12.2, 9.7-11 and 10-11.2%, respectively. The extraction recoveries at low to high concentration were greater than 98, 83 and 87% for each analyte, respectively. The LOQ for wikstromol was 0.015microg/ml and for both matairesinol and dibenzylbutyrolactol it was 0.030microg/ml. The developed method was used to determine the pharmacokinetics of the three analytes in mice after intraperitoneal administration of CD-3.

Herbal modulation of drug bioavailability: enhancement of rifampicin levels in plasma by herbal products and a flavonoid glycoside derived from Cuminum cyminum.

The bioavailability of rifampicin (RIF) in a fixed dose combination (FDC) used for the treatment of tuberculosis remains an area of clinical concern and several pharmaceutical alternatives are being explored to overcome this problem. The present study presents a pharmacological approach in which the bioavailability of a drug may be modulated by utilizing the herb-drug synergism. The pharmacokinetic interaction of some herbal products and a pure molecule isolated from Cuminum cyminum with RIF is shown in this paper. An aqueous extract derived from cumin seeds produced a significant enhancement of RIF levels in rat plasma. This activity was found to be due to a flavonoid glycoside, 3',5-dihydroxyflavone 7-O-beta-D-galacturonide 4'-O-beta-D-glucopyranoside (CC-I). CC-I enhanced the Cmax by 35% and AUC by 53% of RIF. The altered bioavailability profile of RIF could be attributed to a permeation enhancing effect of this glycoside.

Tissue distribution of Diablo/Smac revealed by monoclonal antibodies.

Diablo/Smac is a mammalian pro-apoptotic protein that can antagonize the inhibitor of apoptosis proteins (IAPs). We have produced monoclonal antibodies specific for Diablo and have used these to examine its tissue distribution and subcellular localization in healthy and apoptotic cells. Diablo could be detected in a wide range of mouse tissues including liver, kidney, lung, intestine, pancreas and testes by Western blot analysis. Immunohistochemical analysis found Diablo to be most abundant in the germinal cells of the testes, the parenchymal cells of the liver and the tubule cells of the kidney. In support of previous subcellular localization analysis, Diablo was present within the mitochondria of healthy cells, but released into the cytosol following the induction of apoptosis by UV.

p190-A, a human tumor suppressor gene, maps to the chromosomal region 19q13.3 that is reportedly deleted in some gliomas.

To date, two distinct genes coding for Ras GAP-binding phosphoproteins of 190kDa, p190-A and p190-B, have been cloned from mammalian cells. Rat p190-A of 1513 amino acids shares 50% sequence identity with human p190-B of 1499 amino acids. We have previously demonstrated, using rat p190-A cDNA, that full-length p190-A is a tumor suppressor, reversing v-Ha-Ras-induced malignancy of NIH 3T3 cells through both the N-terminal GTPase (residues 1-251) and the C-terminal Rho GAP (residues 1168-1441) domains. Here we report the cloning of the full-length human p190-A cDNA and its first exon covering more than 80% of this protein, as well as its chromosomal mapping. Human p190-A encodes a protein of 1514 amino acids, and shares overall 97% sequence identity with rat p190-A. Like the p190-B exon, the first exon of p190-A is extremely large (3.7 kb in length), encoding both the GTPase and middle domains (residues 1-1228), but not the remaining GAP domain, suggesting a high conservation of genomic structure between two p190 genes. Using a well characterized monochromosome somatic cell hybrid panel, fluorescent in situ hybridization (FISH) and other complementary approaches, we have mapped the p190-A gene between the markers D19S241E and STD (500 kb region) of human chromosome 19q13.3. Interestingly, this chromosomal region is known to be rearranged in a variety of human solid tumors including pancreatic carcinomas and gliomas. Moreover, at least 40% glioblastoma/astrocytoma cases with breakpoints in this region were previously reported to show loss of the chromosomal region encompassing p190-A, suggesting the possibility that loss or mutations of this gene might be in part responsible for the development of these tumors.

Treatment of ras-induced cancers by the F-actin-bundling drug MKT-077.

A rhodacyanine dye called MKT-077 has shown a highly selective toxicity toward several distinct human malignant cell lines, including bladder carcinoma EJ, and has been subjected to clinical trials for cancer therapy. In the pancreatic carcinoma cell line CRL-1420, but not in normal African green monkey kidney cell line CV-1, it is selectively accumulated in mitochondria. However, both the specific oncogenes responsible for its selective toxicity toward cancer cells, and its target proteins in these cancer cells, still remain to be determined. This study was conducted using normal and ras-transformed NIH 3T3 fibroblasts to determine whether oncogenic ras mutants such as v-Ha-ras are responsible for the selective toxicity of MKT-077 and also to identify its targets, using its derivative called "compound 1" as a specific ligand. We have found that v-Ha-ras is responsible for the selective toxicity of MKT-077 in both in vitro and in vivo. Furthermore, we have identified and affinity purified at least two distinct proteins of 45 kD (p45) and 75 kD (p75), which bind MKT-077 in v-Ha-ras-transformed cells but not in parental normal cells. Microsequencing analysis has revealed that the p45 is a mixture of beta- and gamma-actin, whereas the p75 is HSC70, a constitutive member of the Hsp70 heat shock adenosine triphosphatase family, which inactivates the tumor suppressor p53. MKT-077 binds actin directly, bundles actin filaments by cross-linking, and blocks membrane ruffling. Like a few F-actin-bundling proteins such as HS1, alpha-actinin, and vinculin as well as F-actin cappers such as tensin and chaetoglobosin K (CK), the F-actin-bundling drug MKT-077 suppresses ras transformation by blocking membrane ruffling. These findings suggest that other selective F-actin-bundling/capping compounds are also potentially useful for the chemotherapy of ras-associated cancers.

G proteins, phosphoinositides, and actin-cytoskeleton in the control of cancer growth.

Almost three decades have passed since actin-cytoskeleton (acto-myosin complex) was first discovered in non-muscle cells. A combination of cell biology, biochemistry, and molecular biology has revealed the structure and function of many actin-binding proteins and their physiological role in the regulation of cell motility, shape, growth, and malignant transformation. As molecular oncologists, we would like to review how the function of actin-cytoskeleton is regulated through Ras/Rho family GTPases- or phosphoinosites-mediated signaling pathways, and how malignant transformation is controlled by actin/phosphoinositides-binding proteins or drugs that block Rho/Rac/CDC42 GTPases-mediated signaling pathways.

Treatment of Ras-induced cancers by the F-actin cappers tensin and chaetoglobosin K, in combination with the caspase-1 inhibitor N1445.

UNLABELLED: For transforming normal fibroblasts to malignant cells, oncogenic Ras mutants such as v-Ha-ras require Rho family GTPases (Rho, Rac, and CDC42) that are responsible for controlling actin-cytoskeleton organization. Ras activates Rac through a PI-3 kinase-mediated pathway. Rac causes uncapping of actin filaments (F-actin) at the plus-ends, through phosphatidylinositol 4,5 bisphosphate (PIP2), and eventually induces membrane ruffling. Several distinct F-actin/PIP2-binding proteins, such as gelsolin, which severs and caps the plus-ends of actin filaments, or HS1, which cross-links actin filaments, have been shown to suppress v-Ha-Ras-induced malignant transformation when they are overexpressed. Interestingly, an F-actin cross-linking drug (photosensitizer) called MKT-077 suppresses Ras transformation. Thus, an F-actin capping/severing drug might also have an anticancer potential. PURPOSE: This study was conducted to determine first whether Ras-induced malignant phenotype (anchorage-independent growth) is suppressed by overexpression of the gene encoding a large plus-end F-actin capping protein called tensin and second to test the anti-Ras potential of a unique fungal antibiotic (small compound) called chaetoglobosin K (CK) that also caps the plus-ends of actin filaments. METHODS AND RESULTS: DNA transfection with a retroviral vector carrying the tensin cDNA was used to overexpress tensin in v-Ha-Ras-transformed NIH 3T3 cells. All stable tensin transfectants rarely formed colonies in soft agar, indicating that tensin suppresses the anchorage-independent growth. The anti-Ras action of CK was determined by incubating the Ras-transformants in the presence of CK in soft agar. Two microM CK almost completely inhibited their colony formation, indicating that CK also suppresses the malignant phenotype. However, unlike tensin, CK causes an apoptosis of Ras-transformed NIH 3T3 cells and, less effectively, of normal NIH 3T3 cells, indicating that CK has an F-actin capping-independent side effect(s). CK-induced apoptosis is at least in part caused by CK-induced inhibition of the kinase PKB/AKT. However, a specific ICE/caspase-1 inhibitor called N1445 completely abolished the CK-induced apoptosis by reactivating PKB, but without affecting the CK-induced suppression of Ras transformation. CONCLUSIONS: Like the F-actin cross-linking drug MKT-077, the F-actin capping drug CK may be useful for the treatment of Ras-associated cancers if it is combined with the ICE inhibitor N1445, which abolishes the side effect of CK. Our observations that two distinct F-actin capping molecules (i.e., tensin and CK) suppress Ras-induced malignant phenotype strongly suggest, if not prove, that capping of actin filaments at the plus-ends alone is sufficient to block one of the Ras signaling pathways essential for its oncogenicity. This notion is compatible with the fact that Ras induces the uncapping of actin filaments at the plus-ends through the Rac/PIP2 pathway.

Enteropathogenicity and antimicrobial susceptibility of new Escherichia spp.

To determine the mechanism of enteropathogenicity of the newly described Escherichia species, a total of 50 clinical isolates of Escherichia spp. from diarrhoeal stools were studied. Twelve isolates (24%) were found to be E. vulneris, 6 (12%) E. fergusonii, 2 (4%) E. hermannii, and the rest 30 (60%) were E. coli. Most isolates of the new species were resistant to ampicillin, tetracycline, and co-trimoxazole, but were susceptible to cephalosporins and aminoglycosides. The representative strains of all the new species produced significant fluid accumulation in the rat ileal loops both by live cells and their culture filtrates. E. vulneris, isolated from stools, showed maximum fluid accumulation. Thus, it can be inferred that these species are diarrhoeagenic, but their roles on extra-intestinal infections remain to be determined.

Cytoskeletal tumor suppressors that block oncogenic RAS signaling.

Several distinct peptides or drugs that block the Rho family GTPases-mediated pathways were found to suppress RAS-induced malignant phenotype. They include (1) C3 enzyme that selectively inactivates Rho, (2) ACK42, a peptide that blocks the interaction of CDC42 with its effectors such as ACKs, (3) PAK18, a peptide that blocks the activation of PAK and membrane ruffling, and (4) actin-binding drugs, chaetoglobosin K (CK) and MKT-077, that block membrane ruffling by capping and bundling actin filaments, respectively.

The GTPase and Rho GAP domains of p190, a tumor suppressor protein that binds the M(r) 120,000 Ras GAP, independently function as anti-Ras tumor suppressors.

p190 is a Tyr-phosphorylatable G protein of M(r) 190,000 that binds NH2-terminal SH2 domains of GAP1, a Ras GAP of M(r) 120,000. p190 contains at least two functional domains: a GTPase domain at the NH2 terminus and a GAP domain at the COOH terminus that can attenuate signal-transducing activity of three distinct G proteins (Rac, Rho, and CDC42). Here, we demonstrate that overexpression of either an antisense p190 RNA or a dominant negative mutant (Asn36) of p190 GTPase domain (residues 1-251) but not the wild-type p190 GTPase domain is able to transform normal NIH/3T3 fibroblasts. Furthermore, overexpression of either the wild-type p190 GTPase domain or the COOH-terminal GAP domain can suppress v-Ha-Ras-induced malignant transformation. These results indicate that p190 contains at least two distinct anti-Ras tumor suppressor domains, the GTPase and GAP domains, and suggest that one of the mechanisms underlying the suppression of Ras-transformation by p190 is the attenuation by p190 GAP domain of Rac/Rho/CDC42 signalings, which are essential for Ras-transformation. In fact, the p190 GAP domain alone suppresses the expression of the c-Fos gene, which is mediated by Rac/Rho/CDC42 and is required for oncogenicity of Ras.

Production of the new cholera toxin by environmental isolates of Vibrio cholerae non-O1.

One of five strains of Vibrio cholerae non-O1 isolated from environmental sources caused fluid accumulation in an initial rabbit ileal loop (RIL) test. The four strains that caused little or no accumulation of fluid gave a positive response after one-to-three consecutive passages through RILs. The amount of fluid produced increased after each passage. Filtrates of cultures of all five environmental isolates caused fluid accumulation similar to that produced by live cells. The enterotoxin showed a precipitin band with new cholera antitoxin and was neutralised completely by new cholera antitoxin diluted 1 in 32, indicating its close immunobiological relationship to the new cholera toxin. The present study indicates that V. cholerae non-O1 strains produce an enterotoxin that is similar to the new cholera toxin.

Development of an improved synthetic medium for a better production of the new cholera toxin and its immunological relationship with the toxin produced by Vibrio cholerae O139 strains.

An improved synthetic medium (M4) comprising syncase medium supplemented with sodium chloride (1%) and sucrose (0.5%) pH adjusted to 7.4 was developed for a better production of the new cholera toxin (NCT). The culture filtrates prepared in the M4 medium caused significantly (P > 0.05) more fluid accumulation than that in syncase medium. Crude toxin, prepared in the M4 medium with V. cholerae O1 strains (X-392 and 2740-80) caused a reaction similar to that of the same amount of NCT (32 micrograms) prepared in the syncase medium. The neutralization of the optimal loop reacting dose of the NCT prepared in the M4 medium by anti-NCT raised against syncase prepared toxin indicates the release of the same kind of toxin in both media. These observations indicate that the modified M4 medium may be used for NCT preparation and further characterization. All the strains of Vibro cholerae O139 used in this study produced a toxin antigenically similar to NCT.

The minimal fragments of c-Raf-1 and NF1 that can suppress v-Ha-Ras-induced malignant phenotype.

v-Ha-Ras, an oncogenic Ras mutant, causes malignant transformation of mammalian cells by recruiting c-Raf-1, a cytosolic Ser/Thr kinase, to the plasma membranes/cytoskeleton. The kinase activity of c-Raf-1 resides in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf257, residues 1-257) that binds the Ras-GTP complex and can compete Ras GTPase-activating proteins such as NF1 for binding to Ras. However, it still remains to be clarified whether overexpression of Raf257 or its minimal Ras-binding fragment alone is sufficient to suppress Ras-induced malignancy. In this paper we demonstrate for the first time that the 81-amino acid fragment (Raf81, residues 51-131), the minimal Ras-binding fragment of Raf, indeed can suppress v-Ha-Ras-induced malignant phenotype. A further deletion of the first 6 amino acids causes 65% reduction in the Ras binding of Raf81. The resultant 75 amino acid fragment (Raf75, residues 57-131) consists of a single alpha-helix, five anti-paralleled beta-sheets and five loops. We have found that a further deletion of either the first beta-sheet/loop or the last two beta-sheets/loops completely abolishes Ras binding. In addition we have found that the removal of the C-terminal 35 amino acids from a Ras-binding 91-amino acid fragment of NF1 (NF91, residues 1441-1531) does not abolish its ability to suppress the Ras-induced malignancy.

An anti-Ras function of neurofibromatosis type 2 gene product (NF2/Merlin).

Previously, we have cloned a candidate for the 595-amino acid neurofibromatosis type 2 tumor suppressor called NF2 or Merlin, with striking sequence similarity in its N-terminal half to an F-actin-binding protein family called TERM, which includes talin, ezrin, radixin, and moesin (Trofatter, J. A., MacCollin, M. M., Rutter, J. L., Murrell, J. R., Duyao, M. P., Parry, D. M., Eldridge, R., Kley, N., Menon, A. G., Pulaski, K., Haase, V. H., Ambrose, C. M., Munro, D., Bove, C., Haines, J. L., Martuza, R. L., MacDonald, M. E., Seizinger, B. R., Short, M. P., Buckler, A. J., and Gusella, J. F. (1993) Cell 72, 791-800). In an attempt to determine whether NF2 serves as a tumor suppressor and if so whether its N-terminal half is involved in its anti-oncogenicity, both full-length NF2 and its N-terminal half (NF2-N, residues 9-359) have been expressed in v-Ha-Ras-transformed NIH/3T3 cells. Like neurofibromatosis type 1 (NF1) fragments (Nur-E-Kamal, M. S. A., Varga, M., and Maruta, H. (1993) J. Biol. Chem. 268, 22331-22337), full-length NF2 can reverse the Ras-induced malignant phenotype, i.e. anchorage-independent growth in a soft agar, and restore contact inhibition of cell growth, indicating that NF2 is indeed a tumor suppressor. Furthermore, NF2-N also suppresses the Ras-induced malignant phenotype, although it appears to be less effective than the full-length NF2. These observations indicate that the anti-Ras function of NF2 resides in part in its N-terminal half. Thus, NF2 appears to be a new member of the tumor suppressor family of actin-cytoskeleton-associated proteins, which includes vinculin, alpha-actinin, tropomyosin-1, gelsolin, and tensin.

Influence of animal passage on haemolysin and enterotoxin production in Vibrio cholerae O1 biotype El Tor strains.

Of 43 strains of Vibrio cholerae O1 biotype El Tor isolated over a span of almost three decades (1964-1990) from stools of children and adults with diarrhoea (25 isolates) and from sewage (three) and water from the river Ganges (15) examined for production of haemolysin and its correlation with enterotoxin production, 17 isolates showed haemolysis. The majority of isolates (26), including 68% of diarrhoeal and 50% of environmental origin, were non-haemolytic. The titre of haemolysin produced was 4-16 HU/ml, irrespective of the source of isolation. Haemolytic strains caused significantly more fluid accumulation than the non-haemolytic strains in the rabbit ileal loop (RIL) test. Twenty nine (67.4) V. cholerae biotype El Tor isolates--all the haemolytic and most (61.5%) of the non-haemolytic isolates tested--caused fluid accumulation. The remaining non-haemolytic strains that caused little or no accumulation of fluid did so after one to four consecutive passage(s) through RIL without change in haemolytic character; these strains required more consecutive passage through rabbit gut to show haemolysis. All these strains reverted to their original non-haemolytic character on repeated subculture or on storage in the laboratory but continued to show enterotoxic activity. The present study indicated that El Tor haemolysin is not responsible for fluid accumulation in rabbit gut.

Hematological lesions in rat following heavy alcohol ingestion.

Hematological fluctuations following large alcohol oral administration (2 mL per animal per day) in rats were monitored at intervals ranging from 10 to 22 weeks and the findings were compared to those in control animals that were fed sucrose isocalorically. Following alcohol ingestion, there was a significant decrease in the total blood cellularity at all treatment intervals. A fall in the erythrocyte count per millimeter3 was accompanied by decreased hematocrit values and hemoglobin levels in the alcohol-treated animals. On the other hand, the MCV and MCH values were significantly elevated after alcohol ingestion. Despite overall leukopenia observed after alcohol ingestion, there was a significant increase in the absolute neutrophil count. The absolute lymphocyte count, however, fell significantly in the alcohol-treated animals. Eosinophils and basophils were not significantly altered by alcohol toxicity.