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H Mitsuya

Publications and source records attributed to H Mitsuya.

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

[Two cases of adrenal myelolipoma diagnosed by ultrasonically guided percutaneous biopsy].

We report 2 cases of adrenal myelolipoma which were diagnosed definitively by ultrasonically guided percutaneous biopsy to waive surgery. In case 1, a 39-year-old man presented with abdominal pain. A right adrenal mass was detected by abdominal ultrasonography. In case 2, a 64-year-old man presented with nocturia. A space-occupying lesion above the left kidney was detected by excretory pyelography. Both cases were suspected of adrenal myelolipoma by ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI). The tumor size was 4 x 4 x 3 cm and 5 x 4.5 x 4 cm in cases land 2, respectively. In both cases percutaneous needle biopsy using ultrasonically guided puncture was performed. Histology showed a complex of hematopoietic and adipose tissues, corresponding to adrenal myelolipoma. The course has been uneventful without surgery for 58 months and 24 months, respectively. Percutaneous adrenal biopsy was useful to establish the definitive diagnosis and to select treatment options.

Adrenal Gland Neoplasms↗
PubMed1997

Phosphodiester amidates of unsaturated nucleoside analogues: synthesis and anti-HIV activity.

The effect of introduction of a lipophilic phosphodiester amidate moiety on the HIV activity of inactive unsaturated nucleoside analogues was investigated. Phosphodiester alaninates 5a, 5b, and 6 derived from unsaturated nucleoside analogues 3b, 3c, and 4a were synthesized and investigated as inhibitors of cytopathic effect and replication of HIV-1 in ATH-8 cells. Compound 5a is an inhibitor of HIV-1 whereas analogue 6 is inactive with cytotoxicity appearing above 10 microM and 5b is both inactive and nontoxic. Alkaline or enzymic hydrolysis of 5a gave phosphomonoester alaninate 14, a putative product of intracellular metabolism. Compound 14 as well as adenallene derivative 15c were devoid of anti-HIV activity, and they also failed to inhibit HIV reverse transcriptase. A new regioselective method for preparation of (Z)-4-(benzoyloxy)-1-hydroxy-2-butene, 7, a key intermediate for the synthesis of unsaturated nucleoside analogues of cis configuration such as 3a, 3b, and 3c, is also described.

Animals↗
PubMed1997

Comparative enzymatic study of HIV-1 reverse transcriptase resistant to 2',3'-dideoxynucleotide analogs using the single-nucleotide incorporation assay.

Employing the single-nucleotide incorporation assay using a heteropolymeric RNA template and DNA primers, we defined enzymatic profiles of recombinant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) containing a set of five mutations [A62V, V75I, F77L, F116Y, and Q151M] which confers resistance to multiple 2',3'-dideoxynucleosides (ddNs) on HIV-1. RTs containing other drug-resistance-associated mutations were also examined. The K(m) for dNTPs, the kcat, and the kcat/ K(m) ratios of mutant RTs were all comparable to those of wild-type RT (RTwt). The processive primer extension activity of mutant RTs was also comparable to that of RTwt as examined in the presence of saturating concentrations of dNTPs and heparin. Determination of the Ki values toward 5'-triphosphates (TP) of various ddNs [3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (D4T), 2',3'-dideoxycytidine (ddC), (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), (-)-beta-L-2',3'-dideoxy-5-fluorocytidine (FddC), 2',3'-dideoxyadenosine (ddA), and 2'-beta-fluoro-2',3'-dideoxyadenosine (FddA)] and 9-(2-phosphonylmethoxyethyl)adenine diphosphate (PMEApp) revealed that RTA62V/V75I/F77L/F116Y/Q151M was insensitive to ddATP, AZTTP, D4TTP, FddATP, and ddCTP, but was sensitive to PMEApp, 3TCTP, and FddCTP. RTK65R was less sensitive to ddATP, FddATP, PMEApp, ddCTP, and 3TCTP, while RTM184V was less sensitive only to 3TCTP and ddCTP. The determination of Ki(ddNTP)/K(m)(dNTP) ratios showed that AZTTP, D4TTP, and ddCTP are, as substrates, as efficient for RTwt as their corresponding dNTPs, that ddATP, PMEApp, and 3TCTP are moderately efficient substrates for RTwt, and that FddATP is the least efficient substrate among ddNTPs examined. The observed cross-resistance of HIV-1 RT to various ddNTPs should reflect the alteration of RT's substrate recognition and should provide insights into the molecular mechanism of RT discrimination of ddNTPs from natural substrates.

Anti-HIV Agents↗
PubMed1997

HIV-1 protease does not play a critical role in the early stages of HIV-1 infection.

We asked whether human immunodeficiency virus type 1 (HIV-1) protease plays a major role in the early stages of infection (i.e. from viral entry to reverse transcription) by using various protease inhibitors (saquinavir, ritonavir, and KNI-272). When assessed in the two-day multinuclear activation of a galactosidase indicator (MAGI) assay, involving a single cycle of HIV-1 replication, all protease inhibitors failed to block infection of HeLa-CD4-LTR-beta-gal cells by HIV-1, while reverse transcriptase (RT) inhibitors (AZT and ddI) completely blocked the infection. Moreover, when HIV-1 proviral DNA synthesis was examined by polymerase chain reaction in HeLa-CD4-LTR-beta-gal cells exposed to HIV-1 and cultured in the presence of protease inhibitors, a significant amount of proviral DNA was detected, while no proviral DNA synthesis was detected when the cells were cultured in the presence of RT inhibitors. Protease inhibitors also failed to block chloramphenicol acetyltransferase (CAT) expression in HLCD4-CAT cells exposed to HIV-1, while RT inhibitors completely suppressed CAT expression. These results strongly suggest, contrary to a previous report by Nagy et al. (1994), that HIV-1 protease does not play a major role in the early stages of infection.

Chloramphenicol O-Acetyltransferase↗
PubMed1997

HIV-1 acquires resistance to two classes of antiviral drugs through homologous recombination.

Genetic recombination contributes to the genomic heterogeneity of human immunodeficiency virus type 1 (HIV-1). In the present study, we demonstrate that HIV-1 readily develops resistance to two classes of anti-HIV-1 drugs through in vitro genetic recombination involving large segments of the viral genome. Co-transfection of COS-7 cells with an HIV-1 plasmid (pSUM13) carrying five mutations in the reverse transcriptase (RT)-encoding region (A62V, V75I, F77L, F116Y, Q151M), conferring resistance to multiple dideoxynucleoside analogs (ddNs), and another HIV-1 plasmid (pSUM431) carrying five mutations in the protease-encoding region (V321, L33F, K451, 184V, L89M), conferring resistance to protease inhibitors such as KNI-272, readily produced HIV-1 carrying both sets of mutations when propagated in MT-2 cells in the presence of azidothymidine (AZT) and KNI-272. The resultant HIV-1 variant was highly resistant to both ddNs and KNI-272. Co-infection of MT-2 cells with HIV-1SUM13 carrying the RT mutations and HIV-1SUM431 carrying the mutations in the protease also generated HIV-1 with both sets of mutations when cultured with AZT and KNI-272. We also report here that the problematic artifactual recombination occurring during genetic analyses of heterogeneous nucleic acid sequences using polymerase chain reaction can be successfully obviated.

Animals↗
PubMed1997

Effect of combination therapy with zidovudine and didanosine on neuropsychological functioning in patients with symptomatic HIV disease: a comparison of simultaneous and alternating regimens.

OBJECTIVE: To evaluate the effects of treatment with alternating and simultaneous regimens of zidovudine and didanosine on neuropsychological function in patients with symptomatic HIV-1 disease, focusing on patients with possible HIV-1-associated central nervous system (CNS) compromise at entry. DESIGN: Randomized non-blinded clinical trial. SETTING: Government medical research center. PATIENTS: Thirty-eight patients with symptomatic HIV-1 disease, of whom 21 had evidence of CNS compromise at entry. RESULTS: After 12 weeks of therapy, overall significant improvements in memory (P < 0.01) and focused attention (P < 0.001) were seen on both regimens. These gains, however, were largely limited to those patients with HIV-1-associated CNS compromise at entry (P < 0.05). Improvements were also noted in receptive vocabulary, reading, perceptual discrimination and reasoning, divided attention, motor strength, and in mood and affect. Improvements in those latter functions were generally of limited magnitude and were of comparable size for both compromised and non-compromised patients. There was no overall difference between the two drug regimens in the effects on CNS parameters. CONCLUSIONS: Therapy-related improvements were noted particularly for patients with HIV-1-associated CNS compromise. Neuropsychological functions that have been implicated in AIDS dementia--memory and attention--showed the greatest gains. In contrast to the previously described superiority of the simultaneous regimen with regard to immunologic and virologic parameters, there was no difference between the regimens with regard to CNS measures. This supports the contention that the CNS constitutes a relative independent compartment in terms of HIV disease and treatment.

AIDS Dementia Complex↗
PubMed1997

Removal of human immunodeficiency virus type 1 (HIV-1) protease inhibitors from preparations of immature HIV-1 virions does not result in an increase in infectivity or the appearance of mature morphology.

The processing of gag and gag-pol polyproteins by human immunodeficiency virus type 1 (HIV-1) protease is a crucial step in the formation of infectious HIV-1 virions. In this study, we examine whether particles produced in the presence of inhibitors of HIV-1 protease can subsequently undergo gag polyprotein cleavage with restoration of infectivity following removal of the inhibitors. Viral particles produced during 7 days of culture in the presence of the protease inhibitors KNI-272 (10 microM) and saquinavir (5 microM) contained predominantly p55gag polyprotein but little or no p24gag cleavage product. Following resuspension of the particles in medium free of the inhibitor, some gag polyprotein processing was detected in particles produced from the KNI-272-treated cells, but not from the saquinavir-treated cells within the first 3 h. However, the majority of the protein remained as p55gag throughout a 48-h experimental period. The infectivity (50% tissue culture infective dose per milliliter) of the viral particles from KNI-272-treated cells was 10(6)-fold lower than that of control particles and did not significantly increase over the 48 h after the inhibitor was removed, despite the apparent return of protease function in a subset of these virions. This failure to restore infectivity was due neither to a reduction in the number of particles produced by protease inhibitor-treated cells nor to a failure of HIV RNA to be packaged in the virions. These particles also failed to express the mature phenotype by electron microscopy. Thus, while some processing of the gag polyprotein can occur in isolated HIV virions, this does not appear to be sufficient to restore infectivity in the majority of particles. This finding suggests that there may be constraints on postbudding polyprotein processing in the production of viable particles. These results should have positive implications regarding the use of protease inhibitors as anti-HIV drugs.

Blotting, Western↗
PubMed1997

In vitro induction of human immunodeficiency virus type 1 variants resistant to 2'-beta-Fluoro-2',3'-dideoxyadenosine.

2'-beta-Fluoro-2',3'-dideoxyadenosine (F-ddA) is an acid-stable purine dideoxynucleoside analog active against a wide spectrum of human immunodeficiency virus type 1 (HIV-1) and HIV-2 strains in vitro. F-ddA is presently undergoing a phase I clinical trial at the National Cancer Institute. We induced HIV-1 variants resistant to F-ddA by exposing wild-type HIV-1 (HIV-1LAI) to increasing concentrations of F-ddA in vitro. After 18 passages, the virus was fourfold less sensitive to F-ddA than HIV-1LAI. Sequence analyses of the passage 18 virus revealed changes in three amino acids in the reverse transcriptase (RT)-encoding region of the pol gene: P to S at codon 119 (P119S; present in 3 of 13 and 28 of 28 molecular clones before and after F-ddA exposure, respectively), V179D (0 of 13 and 9 of 28, respectively), and L214F (9 of 13 and 28 of 28, respectively). Drug sensitivity assays using recombinant infectious clones confirmed that P119S was directly responsible for the reduced sensitivity of HIV-1 to F-ddA. Various infectious clones with single or multiple amino acid substitutions conferring viral resistance against nucleoside RT inhibitors, including HIV-1 variants with multi-dideoxynucleoside resistance, were generally sensitive to F-ddA. The moderate level of resistance of HIV-1 to F-ddA, together with the lack of conferment of significant cross-resistance by the F-ddA-associated amino acid substitutions, warrants further investigation of F-ddA as a potential antiviral agent for use in treatment of HIV-1 infection.

Animals↗
PubMed1996

Phosphodiester amidates of allenic nucleoside analogues: anti-HIV activity and possible mechanism of action.

Lipophilic phosphodiester amidates 2a, 2b, 4a, 4b, and 6 derived from anti-HIV agent adenallene 1a, 3a, inactive hypoxallene 1b, 3b, and 9-(4-hydroxy-2-butyn-1-yl)adenine (5) were synthesized and studied as inhibitors of HIV-1 in ATH8 cell system. All phosphodiester amidates were more biologically active than their parent nonphosphorylated compounds. Analogues 2a and 4a derived from (+/-)-adenallene 1a and (R)-enantiomer 3a are effective anti-HIV agents with EC50 approximately 0.88 and 0.21 microM, respectively. Both analogues are 16 and 28 times more effective than parent compounds 1a and 3a, respectively. Some anti-HIV activity of hypoxallene derivatives 2b and 4b was noted in the range of 0.1-10 microM but the dose-response relationship was poor. Phosphodiester amidate analogue 6 also exhibited anti-HIV activity in the range of 0.1-100 microM, but this effect was accompanied by cytotoxicity. Hydrolytic studies performed at pH 9.8 and with pig liver esterase at pH 7.4 have shown that analogue 2a gives adenallene 4'-phosphoralaninate (10a) as the major product. These results can be interpreted in terms of initial hydrolysis of phosphodiester amidates 2a, 2b, 4a, 4b, and 6 catalyzed by intracellular esterase(s) to give stable phosphomonoester amidate intermediates with a free carboxyl group. The results obtained with hypoxallene phosphoramidates 2b and 4b indicate that the aminosuccinate-fumarate enzyme system responsible for activation of AIDS drug ddIno (didanosine, Videx) can also, albeit less efficiently, activate hypoxallene 4'-phosphate (9b) and the respective (R)-enantiomer released inside the HIV-infected cells.

Adenosine Monophosphate↗
PubMed1996

Lipophilic, acid-stable, adenosine deaminase-activated anti-HIV prodrugs for central nervous system delivery. 3. 6-Amino prodrugs of 2'-beta-fluoro-2',3'-dideoxyinosine.

A series of 6-substituted amino analogs of 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) purines (F-ddN) has been synthesized and characterized with the objective of finding compounds which might be superior to existing drugs for the treatment of HIV in the central nervous system. These compounds are intended to be more lipophilic than the currently approved anti-HIV drugs for better blood-brain barrier penetration. Subsequent adenosine deaminase (ADA)-catalyzed hydrolysis of these prodrugs in the brain is expected to produce the anti-HIV agent, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine (F-ddI). The new compounds, synthesized from the corresponding 6-chloro analog, include F-ddN which contain methylamino, ethylamino, dimethylamino, hydroxylamino, methoxyamino, benzyloxyamino, hydrazino, and nitro substituents in the 6-position. The 6-nitro analog was isolated as an unexpected product during the preparation of the 6-chloro derivative. Among the analogs with anti-HIV activity, the ethylamino and dimethylamino compounds are ca. 100 times more lipophilic than ddI or F-ddI. As expected, 2'-fluoro substitution protects the compounds from acid-catalyzed glycosylic cleavage. Only the hydroxylamino and nitro analogs underwent any nonenzymatic hydrolysis at pH 1.0 or 7.4. This reaction, however, results in hydrolysis of the group in the 6-position rather than glycosylic bond cleavage. ADA catalyzes the hydrolysis of the 6-substituents at rates which vary from slightly slower (NO2, 1.7x) to much slower (NHEt, 5000x) than F-ddA. The 6-dimethylamino analog is the only compound which possesses anti-HIV activity (ED50 18 microM) without ADA hydrolysis. With the exception of the two inactive alkoxyamino compounds, the other prodrugs exhibited cellular protection in the HIV-1/PHA-PBM system with IC50 potencies of 7-40 microM.

Adenosine Deaminase↗
PubMed1996

In vitro selection and molecular characterization of human immunodeficiency virus type 1 with reduced sensitivity to 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA).

9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA) is an acyclic nucleotide with potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). The present study was undertaken to determine whether HIV-1 resistance to PMEA could be generated by in vitro selection and if so, to determine which mutations in reverse transcriptase (RT) were responsible. HIV-1LAI was serially passaged for 10 months in the presence of increasing concentrations of PMEA up to a maximum of 40 microM. After 40 passages, the 50% inhibitory concentration (IC50) of PMEA had increased almost 7-fold from 4.45 to 30.5 microM. Some cross-resistance to 2',3'-dideoxycytidine (ddC, zalcitabine), 2',3-dideoxyinosine (ddI, didanosine), and 3'-thiacytidine (3TC, lamivudine) was also observed, but no cross-reactive resistance to 3'-azido-3'-thymidine (AZT, zidovudine). Sequencing of the RT encoding region of each of eight pol clones from resistant isolates revealed a Lys-65-->Arg (K65R) substitution. HIV with the K65R mutation inserted by site-directed mutagenesis also had decreased sensitivity to PMEA in H9 cells and a similar cross-resistance profile. Thus, HIV can develop decreased sensitivity to PMEA after long-term in vitro exposure and this change is associated with a K65R substitution. Additional studies will be needed to determine whether a similar mutation in HIV RT develops in patients receiving PMEA or its orally bioavailable prodrug adefovir dipivoxil (bis-POM PMEA).

Adenine↗
PubMed1996

Human immunodeficiency virus type 1 infection of H9 cells induces increased glucose transporter expression.

A clone obtained from a differential display screen for cellular genes with altered expression during human immunodeficiency virus (HIV) infection matched the sequence for the human GLUT3 facilitative glucose transporter, a high-velocity-high-affinity facilitative transporter commonly expressed in neurons of the central nervous system. Northern (RNA) analysis showed that GLUT3 expression increased during infection. Flow cytometry showed that GLUT3 protein expression increased specifically in the HIV-infected cells; this increase correlated with increased 2-deoxyglucose transport in the HIV-infected culture. HIV infection therefore leads to increased expression of a glucose transporter normally expressed at high levels in other cell types and a corresponding increase in glucose transport activity. If HIV infection places increased metabolic demands on the host cell, changes in the expression of a cellular gene that plays an important role in cellular metabolism might provide a more favorable environment for viral replication.

Biological Transport↗
PubMed1996

2',3'-Didehydro-3'-deoxythymidine: regulation of its metabolic activation by modulators of thymidine-5'-triphosphate biosynthesis.

The anti-human immunodeficiency virus (anti-HIV) agent 2',3'-didehydro-3'-deoxythymidine (D4T), like other 2',3'-dideoxynucleosides, requires conversion to its 5'-triphosphate to exert its pharmacological effect. Although D4T-triphosphate is unusually potent as an inhibitor of HIV-1 reverse transcriptase, the phosphorylation of the drug at low dose levels is inefficient because of its low affinity as an alternate substrate for the initial phosphorylation enzyme thymidine kinase. Because thymidine kinase is under feedback regulatory control by the physiological deoxynucleoside-5'-triphosphate dTTP, we examined the effect on D4T phosphorylation and thus, potentially, on its antiviral activity, of a variety of agents that lower intracellular dTTP pools. We found that agents that inhibit the de novo pyrimidine biosynthetic pathway have the ability to increase D4T phosphorylation, the most effective being two inhibitors of thymidylate formation, methotrexate and 5-fluoro-2'-deoxyuridine, compounds that block the enzymes dihydrofolate reductase and thymidylate synthetase, respectively. Because HIV itself lacks the capacity to synthesize dTTP and the other deoxynucleoside triphosphates essential for viral replication, combinations of D4T with modulatory agents that deplete host-cell dTTP, unlike conventional anti-HIV drug monotherapy directed solely at viral enzymes, have the ability to inhibit replication of mutant HIV strains as well as of wild-type virus.

Amides↗