Phosphorylation of residue 131 of HIV-1 matrix is not required for macrophage infection.
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Carrier Proteins↗
Biomedical subjects
M A Martin
Publications and source records attributed to M A Martin.
Efficacy of clean v sterile surgical prep kits.
The goal of preoperative skin preparation is to reduce the risk of postoperative wound infections. This study was designed to determine if a difference exists in the residual microbial flora on the skin of surgical patients who are prepped with clean versus sterile prep kits. The researchers randomly assigned 60 ambulatory surgery patients to two preoperative skin preparation groups (i.e., clean prep kits, sterile prep kits) and obtained cultures of the patients' surgical sites at three different times (i.e., before performing standard povidone-iodine scrub-and-paint skin preps, 10 minutes after the completion of the skin preps, immediately after skin closure). They used repeated mixed factorial analyses of variance to compute the differences in residual microbial counts at the patients' surgical sites. There was no difference in the residual microbial skin flora in the patients prepped with clean or sterile skin prep kits. The study results have significant cost-saving implications for health care facilities and surgical patients.
Adult↗
Infection and pathogenicity of chimeric simian-human immunodeficiency viruses in macaques: determinants of high virus loads and CD4 cell killing.
Chimeric simian-human immunodeficiency viruses (SHIVs) carrying envelope glycoproteins derived from a T cell-macrophage dual-tropic primary isolate (human immunodeficiency virus type 1 [HIV-1] strain DH12) were constructed. When inoculated into macaque monkeys, SHIV(MD14) carrying simian immunodeficiency virus-derived nef established significantly higher virus loads than did SHIV(MD1), which contains the HIV-1 nef gene. Three patterns of CD4 cell depletion were observed in infected monkeys: exponential and irreversible loss to undetectable levels within 10 weeks of infection; marked reduction during acute infection followed by partial recovery and stabilization (lasting from 10 weeks to > 1 year), with a later decline to undetectable levels in some animals; and a transient loss during acute infection. The induced immunodeficiency was accompanied by CD4 cell counts of < 50 cells/microL and was associated with Pneumocystis carinii pneumonia, cytomegalovirus meningoencephalitis, lymphoid depletion, and thymic atrophy.
Amino Acid Sequence↗
Efficient long-term coexpression of a hammerhead ribozyme targeted to the U5 region of HIV-1 LTR by linkage to the multidrug-resistance gene.
Ribozymes as anti-HIV-1 agents hold promise for the treatment of AIDS. They can be delivered into cells either exogenously or through an expression system. For effective protection against HIV-1, sufficient and sustained amounts of the antiviral ribozymes must be delivered into target cells. The coexpression of a dominant selectable marker with ribozymes would serve to enrich for cells containing the molecular antiviral and facilitate prolonged expression of these ribozymes. The multidrug resistance gene (MDR1) is a potential clinically relevant selectable marker and offers many advantages over other known dominant selectable markers, including the use of diverse pharmacologically characterized drug or drug combinations for selection. Harvey sarcoma-based retroviral vectors encoding the MDR1 multidrug transporter with a hammerhead ribozyme targeted to highly conserved sequences within the HIV-1 U5 LTR segment have been constructed in a bicistronic format. The internal ribosome entry site (IRES) from encephalomyocarditis virus was used to initiate translation of the MDR1 mRNA. The ribozyme remained functional despite being tethered to MDR1. Long-term, high-level expression of both the ribozyme and MDR1, as evident by RT-PCR and FACS analysis, was observed in a human T cell line containing the construct selected with vincristine, a cytotoxic substrate for the multidrug transporter.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Live, attenuated simian immunodeficiency virus vaccines elicit potent resistance against a challenge with a human immunodeficiency virus type 1 chimeric virus.
Three rhesus macaques, previously immunized with SIVdelta3 or SIVdelta2, each an attenuated derivative of SIVmac239, and two naive monkeys were challenged with 30,000 50% tissue culture infective doses of SHIV, an SIV/human immunodeficiency virus type 1 (HIV-1) chimeric virus bearing the dual-tropic envelope of HIV-1DH12. By several criteria, including virus isolation, serological assays, and PCR (both DNA and reverse transcriptase), SHIV levels were reduced to barely detectable levels in the circulating blood of vaccinated animals. The resistant SIV-vaccinated macaques had no preexisting neutralizing antibodies directed against SHIV, nor did they produce neutralizing antibodies at any time over a 14-month observation period following SHIV challenge. Interestingly, SIV sequences, derived from the vaccine, could be amplified from numerous tissue samples collected at the conclusion of the experiment, 60 weeks postchallenge, but SHIV-specific sequences (viz., HIV-1 env) could not. These results demonstrate that live attenuated SIV vaccines provide strong long-term protection even against challenge strains with highly divergent envelope sequences.
Animals↗
Incorporation of Pr160(gag-pol) into virus particles requires the presence of both the major homology region and adjacent C-terminal capsid sequences within the Gag-Pol polyprotein.
The determinants critical for the incorporation of Pr160(gag-pol) into human immunodeficiency virus type 1 (HIV-1) particles were examined by cotransfecting cells with (i) a plasmid expressing wild-type Gag protein and (ii) a series of chimeric Gag-Pol expression plasmids in which individual murine leukemia virus (MLV) Gag regions and subdomains precisely replaced their HIV-1 counterparts. The presence of the MLV MA and NC Gag regions in the chimeric Gag-Pol precursor had no detectable effect on the incorporation of Gag-Pol into progeny virions. In contrast, the entire HIV-1 CA region was required to achieve wild-type levels of Gag-Pol assembly into particles; both the CA major homology region and the adjacent C-terminal CA sequences play dominant roles in this process yet, when assayed in the context of a chimeric Gag-Pol polyprotein, restored the defect affecting Gag-Pol incorporation to approximately half of the wild-type level.
Capsid↗
The role of gamma-tubulin in mitotic spindle formation and cell cycle progression in Aspergillus nidulans.
gamma-Tubulin has been hypothesized to be essential for the nucleation of the assembly of mitotic spindle microtubules, but some recent results suggest that this may not be the case. To clarify the role of gamma-tubulin in microtubule assembly and cell-cycle progression, we have developed a novel variation of the gene disruption/heterokaryon rescue technique of Aspergillus nidulans. We have used temperature-sensitive cell-cycle mutations to synchronize germlings carrying a gamma-tubulin disruption and observe the phenotypes caused by the disruption in the first cell cycle after germination. Our results indicate that gamma-tubulin is absolutely required for the assembly of mitotic spindle microtubules, a finding that supports the hypothesis that gamma-tubulin is involved in spindle microtubule nucleation. In the absence of functional gamma-tubulin, nuclei are blocked with condensed chromosomes for about the length of one cell cycle before chromatin decondenses without nuclear division. Our results indicate that gamma-tubulin is not essential for progression from G1 to G2, for entry into mitosis nor for spindle pole body replication. It is also not required for reactivity of spindle pole bodies with the MPM-2 antibody which recognizes a phosphoepitope important to mitotic spindle formation. Finally, it does not appear to be absolutely required for cytoplasmic microtubule assembly but may play a role in the formation of normal cytoplasmic microtubule arrays.
Aspergillus nidulans↗
Comparison of the sympathetic nervous system activity between spontaneously hypertensive and Wistar-Kyoto rats to respond to blood pressure reduction.
Two types of calcium antagonists, diltiazem and nicardipine, were separately infused in 23-28 week-old spontaneously hypertensive (SHR) and age-matched normotensive Wistar-Kyoto (WKY) rats (under sodium thiobutabarbital anesthesia and ventilation, n = 4) through the left femoral vein, resulting in the reduction of blood pressure. In each rat, mean arterial blood pressure, heart rate and the concentration of plasma catecholamines (CAs), norepinephrine (NE) and epinephrine (E), were concomitantly determined and the correlations between these three values were studied for each calcium antagonist. Plasma concentration of CAs were measured in blood samples collected during the infusion from the right femoral artery of each rat by the automatic sensitive and selective detection system. The reduction of blood pressure induced by the calcium antagonists brought about an increase in plasma CAs levels. The blood pressure correlated well with the logarithm of plasma NE and E concentration and the relations were expressed as Y = -alpha log(X)+m (Y, blood pressure; X, concentration of plasma NE or E; alpha, slope; and m, intercept). The alpha s of SHR rats were greater than those of WKY rats for the calcium antagonists employed, meaning that the increment of plasma CAs responding to a decrease in blood pressure was smaller in SHR than in WKY rats. It was concluded that the contribution of the sympathetic nervous system to maintaining blood pressure reduced by diltiazem and nicardipine is less in SHR than in WKY rats.
Animals↗
Myopathy and spontaneous Pasteurella pneumotropica-induced abscess formation in an HIV-1 transgenic mouse model.
In an effort to augment human immunodeficiency virus type 1 (HIV-1) gene expression in transgenic mice, an infectious proviral DNA clone was modified by deleting the two NF kappa B binding sites and some adjacent upstream LTR sequences and replacing them with the core enhancer of Moloney murine leukemia virus (MLV). Two independent lines of MLV/HIV transgenic mice were established that expressed HIV-1-specific RNA in lymphoid tissue, striated skeletal muscle, and the eye lens. Heterozygous animals from each transgenic line spontaneously developed an inflammatory disease of the eye associated with the production of copious amounts of purulent lacrimal secretions beginning at 2 weeks of age. Periorbital abscess formation became grossly apparent by 2 months of age and Pasteurella pneumotropica was cultured from the harderian glands and conjunctival surfaces of many of the MLV/HIV animals but not their nontransgenic, cohabiting littermates. This gram-negative commensal bacterium has been previously associated with a similar disease phenotype in immunocompromised (e.g., nude mice) rodent colonies. MLV/HIV mice developed normally until 15 weeks of age, when weight loss and wasting occurred, culminating in premature death (as earlier as 6 months of age). The cachexia was associated with an initially focal and subsequently progressive myopathy, coinciding with age-related increases of HIV gene expression in muscle.
Abscess↗
Infection of human immunodeficiency virus 1 transgenic mice with Toxoplasma gondii stimulates proviral transcription in macrophages in vivo.
Human immunodeficiency virus (HIV) 1 transgenic mice expressing low or undetectable levels of viral mRNA in lymphoid tissue were infected with the intracellular protozoan Toxoplasma gondii. Exposure to this parasite resulted in an increase in HIV-1 transcript in lymph nodes, spleens, and lungs during the acute phase of infection and in the central nervous system during the chronic stage of disease. In vivo and ex vivo experiments identified macrophages as a major source of the induced HIV-1 transcripts. In contrast, T. gondii infection failed to stimulate HIV-1 transcription in tissues of two HIV-1 transgenic mouse strains harboring a HIV-1 proviral DNA in which the nuclear factor (NF) kappa B binding motifs from the viral long terminal repeats had been replaced with a duplicated Moloney murine leukemia virus core enhancer. A role for NF-kappaB in the activation of the HIV-1 by T. gondii was also suggested by the simultaneous induction of NF-kappaB binding activity and tumor necrosis factor alpha synthesis in transgenic mouse macrophages stimulated by exposure to parasite extracts. These results demonstrate the potential of an opportunistic pathogen to induce HIV-1 transcription in vivo and suggest a mechanism for the in vivo dissemination of HIV-1 by macrophages.
AIDS-Related Opportunistic Infections↗
Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virions.
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Cell Line↗
Sporadic MERRF/MELAS overlap syndrome associated with the 3243 tRNA(Leu(UUR)) mutation of mitochondrial DNA.
We studied a patient with a mitochondrial encephalomyopathy characterized by the presence of all the cardinal features of both myoclonic epilepsy and ragged-red fibers (MERRF) and mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) syndromes. Muscle biopsy showed ragged-red fibers (RRF). Some RRF were cytochrome c oxidase (COX)-negative while some others stained positive for COX. Muscle biochemistry revealed defects of complexes I and IV of the respiratory chain. Both muscle and blood mitochondrial DNA from the patient showed the presence of the mutation at nucleotide position 3243 in the tRNA(Leu(UUR)) gene and the absence of point mutations related to MERRF syndrome. The proportions of mutant mtDNA were 70% in muscle and 30% in blood. The mutation was absent in blood from all maternal relatives, in hair follicles from the mother, and in muscle from one sister of the proband. Therefore, there was no evidence of maternal inheritance.
Adolescent↗
Abnormal carnitine distribution in the muscles of patients with idiopathic inflammatory myopathy.
OBJECTIVE: To analyze the levels of free carnitine and carnitine esters in the muscles of patients with inflammatory myopathies. METHODS: Six men and 7 women with inflammatory myopathy and 25 age-matched healthy controls were studied. Free carnitine and carnitine esters in muscle homogenates were measured by a radiochemical procedure. Muscle histochemical staining and measurement of respiratory chain enzyme activity were also performed. RESULTS: Eleven patients had muscle carnitine insufficiency. Five of them had subsarcolemmal oxidative accumulations, 5 had lipid droplets, and 4 had defects of the respiratory chain enzyme complexes. CONCLUSION: Abnormal distribution of muscle carnitine is present in patients with inflammatory myopathies and could impair muscle function. Coexistent mitochondrial dysfunction may contribute to carnitine insufficiency.
Adolescent↗
Differentiating inclusion complexes from host molecules by tapping-mode atomic force microscopy.
Tapping-mode atomic force microscopy imaging under different cantilever vibration amplitudes has been used to differentiate the host beta-cyclodextrin nanotubes from retinal/beta-cyclodextrin inclusion complex nanotubes. It was observed that both compounds were deformed differently by the applied probe force because of their different local rigidity. This change in the elasticity properties can be explained as a consequence of the inclusion process. This method shows that tapping-mode atomic force microscopy is an useful tool to map soft sample elasticity properties and to distinguish inclusion complexes from their host molecules on the basis of their different mechanical response.
Biophysical Phenomena↗
Use of micellar media for the fluorimetric determination of ellipticine in aqueous solutions.
Ellipticine is a pyridocarbazole alkaloid with interesting antitumour activity. Use of neutral ellipticine is hampered by its very low water solubility and therefore this compound has been administered as a salt; however, nitrogen quaternization modifies the antitumour properties of ellipticine. Potential alternatives to quaternization include the use of cyclodextrins, and also the use of micellar media. The latter possibility is explored in this work as an analytical tool. The results obtained with model anionic (SDS), cationic (CTAB) and neutral (Brij-35) surfactants are described. Fluorimetric analysis shows that ellipticine solubilizes completely in the presence of all these compounds, as a result of its aromatic, planar structure. The use of micellar media considerably increases the slopes of the calibration curves with improved correlation coefficients (e.g. 0.8904 in water and 0.9982 with SDS). Micellar media also modify proton transfer processes, as a consequence of the apolar environment of the micellar phase. Deprotonation of ellipticine is hampered in SDS because of the relationship between this process and the surface charge of the micelles. Finally, fluorescence quenching in micellar media has been studied, it being found that surfactants provide protection towards this phenomenon.
Antineoplastic Agents↗
Influence of the presence of trace amounts of metals on the polymorphism of tolbutamide.
The polymorphism phenomenon causes an important problem in the preparation of many pharmaceutical dosage forms, including oral suspensions, tablets, creams and suppositories. In this work the influence of the presence of certain metallic elements (Ca(II), Fe(III), Mn(II), Zn(II), Cd(II), Ni(II) and Co(II)) in trace amounts on the polymorphism of tolbutamide is described. For these studies, known amounts (5-500 ppm) of different cations were added to ethanol-water solutions of the pharmacologically active polymorph A of tolbutamide prior to its crystallization. These cations were chosen as being among those that commonly appear in drug solutions as a consequence of elution from glass containers. IR studies show that the presence of cations prevents the appearance of the polymorph B of the tolbutamide. Thus, the peaks at 2980, 2940, 2890, 905, 851, 816 and 727 cm-1 (polymorph A) appeared but not the ones at 1045 and 847 cm-1 corresponding to polymorph B. Scanning electron microscopy studies reveal that, in the presence of Ca2+, a third kind of crystal different from polymorphs A and B appears. This form of olbutamide cannot be identified with "polymorph III" of Al-Saieq and Rileys [S.S. Al-Saieq and G.S. Riley, Pharm. Acta. Helv., 56 (1981) 125-129] due to their different IR data. Dissolution rate tests and X-ray diffractometry were also employed in this study. All these results showed that divalent cations partially avoid the transformation of polymorph A into polymorph B.
Cadmium↗
Domains of the human immunodeficiency virus type 1 matrix and gp41 cytoplasmic tail required for envelope incorporation into virions.
We recently demonstrated that a single amino acid substitution in matrix residue 12 (12LE) or 30 (30LE) blocks the incorporation of human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins into virions and that this block can be reversed by pseudotyping with heterologous retroviral envelope glycoproteins with short cytoplasmic tails or by truncating the cytoplasmic tail of HIV-1 transmembrane glycoprotein gp41 by 104 or 144 amino acids. In this study, we mapped the domain of the gp41 cytoplasmic tail responsible for the block to incorporation into virions by introducing a series of eight truncation mutations that eliminated 23 to 93 amino acids from the C terminus of gp41. We found that incorporation into virions of a HIV-1 envelope glycoprotein with a deletion of 23, 30, 51, or 56 residues from the C terminus of gp41 is specifically blocked by the 12LE matrix mutation, whereas truncations of greater than 93 amino acids reverse this defect. To elucidate the role of matrix residue 12 in this process, we introduced a number of additional single amino acid substitutions at matrix positions 12 and 13. Charged substitutions at residue 12 blocked envelope incorporation and virus infectivity, whereas more subtle amino acid substitutions resulted in a spectrum of envelope incorporation defects. To characterize further the role of matrix in envelope incorporation into virions, we obtained and analyzed second-site revertants to two different matrix residue 12 mutations. A Val-->Ile substition at matrix amino acid 34 compensated for the effects of both amino acid 12 mutations, suggesting that matrix residues 12 and 34 interact during the incorporation of HIV-1 envelope glycoproteins into nascent virions.
Amino Acid Sequence↗
Infection of chimpanzee peripheral blood mononuclear cells by human immunodeficiency virus type 1 requires cooperative interaction between multiple variable regions of gp120.
We have recently reported the isolation and molecular cloning of a human immunodeficiency virus type 1 isolate (HIV-1 DH125) that exhibits rapid replication kinetics and marked cytopathicity in both human and chimpanzee peripheral blood mononuclear cells (PBMC). To identify the viral determinants responsible for infectivity of chimpanzee PBMC, chimeric viruses containing the following components were constructed: (i) the entire envelope gene; (ii) gp120 sequences; (iii) gp41 sequences; and (iv) individual or various combinations of the gp120 variable regions of HIV-1 DH125 inserted into the backbone of another HIV-1 isolate (HIV-1 AD8), which is unable to infect chimpanzee PBMC. Analyses of virus replication kinetics in human and chimpanzee PBMC revealed that gp120 contains determinants which confer infectivity for chimpanzee PBMC and that the capacity to establish such an infection requires the cooperative interaction between multiple variable regions of the HIV-1 DH125 gp120.
Animals↗