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Biomedical subjects

M Nakai

Publications and source records attributed to M Nakai.

At least 145 records · Page 8Linked to original sources
PubMed1996

Mutational analysis of two zinc finger motifs in HIV type 1 nucleocapsid proteins: effects on proteolytic processing of Gag precursors and particle formation.

To clarify the physiological function of two zinc finger motifs in the nucleocapsid (NC) domain of the Gag protein of human immunodeficiency virus type 1 (HIV-1), we changed cysteine to serine in either of the two motifs or both by site-directed mutagenesis. Viral infectivity was lost by any of the mutations, but their effects appeared differently in the respective mutants. Northern blot analysis showed that the first finger mutant was far less efficient (approximately 10% of the wild type) in genomic RNA encapsidation and that the dual mutant of both fingers completely failed to encapsidate the RNA. In contrast, the second finger mutant retained its ability for RNA encapsidation with an efficiency similar to that of the wild type. Immunoblot analysis of the lysates of CD4-positive M8166 cells transfected with the mutant proviral DNAs showed that the processing of Gag precursors was delayed in two mutant viruses having alterations in the first finger sequence, whereas the processing of the second finger mutant appeared to be normal. On the other hand, immunoblot analysis of the virus particles showed that the second finger mutant particles contained some proteins that were thought to be degradation products of p24CA. Electron microscopic observation showed that all particles of these mutant viruses were morphologically alike except that they had a slightly larger diameter than that of the wild type. These results indicate that these finger motifs of HIV-1 NC protein do not function equivalently. Namely, the first finger is primarily responsible for RNA encapsidation and the second is required for stabilization of virus particles.

Amino Acid Sequence↗
PubMed1996

Comparison of the sensitivities of two non-isotopic reverse transcriptase (RT) assays for human immunodeficiency virus type 1 RT.

The sensitivities of two reverse transcriptase (RT) assays, an enzyme-linked oligonucleotide sorbent assay (ELOSA)-RT assay and a non-radioisotopic (non-RI) RT assay were compared. For measuring recombinant HIV-1 RT, the ELOSA-RT assay was 8 times less sensitive in dilution endpoint and 16 times less sensitive in measurement of RT from pelleted HIV-1 than the non-RI RT assay. Higher level of interference by an RNA-DNA hybrid observed in the former assay may indicate that the reduction in sensitivity was due to the presence of viral RNA in the sample of pelleted virus. The ELOSA-RT assay was interfered with to a great extent than the non-RI RT assay by fetal bovine serum and thus may be unsuitable for measuring RT from HIV-1 in a culture supernatant.

Enzyme-Linked Immunosorbent Assay↗
PubMed1996

Binding of mitochondrial presequences to yeast cytosolic heat shock protein 70 depends on the amphiphilicity of the presequence.

The interactions between a yeast cytosolic hsp70, Ssa1p, and various synthetic peptides, including mitochondrial presequences, have been studied. The interactions were monitored both indirectly, by measuring the effects of the presequences on the ATPase activity and oligomeric state of the enzyme, and directly, by measuring the increased steady-state fluorescence polarization of fluorescent derivatives of the presequences as they bind to Ssa1p. The presequences are all able to convert Ssa1p from an oligomeric to a monomeric form in a concentration-dependent manner. The presequences are also able to stimulate the ATPase activity of the enzyme at similar concentrations. Quantification of the binding by fluorescence polarization showed that the affinity for Ssa1p is directly related to the physical properties of the presequences. The most amphiphilic presequences, as measured by retention times on reversed-phase high pressure liquid chromatography or surface activity in lipid monolayers, had the highest affinity for Ssa1p. The least amphiphilic presequences, which had previously been shown to be ineffective as mitochondrial targeting sequences, had relatively low affinity for Ssa1p. The results show that Ssa1p interacts with a broad range of amino acid sequences and that the strength of these interactions is related to the physical properties of the sequence. That the physical properties recognized by Ssa1p are identical to those necessary for the targeting function of mitochondrial presequences suggests that Ssa1p may interact with mitochondrial precursor proteins in the cytosol. The interactions may serve a variety of purposes: the maintenance of precursors in translocation-competent forms, the prevention of improper association of precursors with non-mitochondrial membranes, and the delivery of precursors to the mitochondrial surface.

Adenosine Triphosphatases↗
PubMed1996

Complete inhibition of human immunodeficiency virus Gag myristoylation is necessary for inhibition of particle budding.

Myristoylation of human immunodeficiency virus (HIV) Gag protein is essential for virus particle budding. Two reactions are involved; activation of free myristate to myristoyl-CoA and transfer of the myristoyl residue to the Gag N-terminal glycine. We have investigated the effects of triacsin C, an inhibitor of long chain acyl-CoA synthetase, on release of HIV Gag virus-like particle (VLP) produced using the recombinant baculovirus system. First, inhibition of acyl-CoA formation by triacsin C was confirmed using the membrane fractions of insect Sf9 cells as an enzyme source. Second, when HIV Gag protein was expressed in the presence of triacsin C (0-48 microM), Gag myristoylation was inhibited in a dose-dependent manner. Budding of Gag VLP, however, did not follow similar inhibition kinetics but appeared unaffected up to 24 microM, yet was completely abolished at 48 microM when the myristoylation of Gag protein was also completely inhibited. The "all-or-none" inhibition of Gag VLP budding suggests that although inhibition of acyl-CoA synthetase blocks the production of myristoylated Gag protein, only complete inhibition of Gag myristoylation prevents VLP budding. Thus, relatively few myristoylated Gag molecules are sufficient for plasma membrane targeting and VLP budding.

Acyl Coenzyme A↗
PubMed1996

Cerebral blood flow decreases following microinjection of sodium nitroprusside into the nucleus tractus solitarii of anesthetized rats.

The present study was undertaken to examine the effects of microinjection of sodium nitroprusside (SNP), which releases nitric oxide (NO) spontaneously, into the nucleus tractus solitarii (NTS) on cerebral circulation. Cerebral blood flow (CBF) was measured in urethane-anesthetized (1.5 g middle dotkg-1, i.p.), paralysed and artificially ventilated rats using labeled microspheres or laser Doppler flowmetry. The CBF was significantly decreased by microinjection of SNP (5 nmol, n=10, microsphere technique; 0.5 nmol, n=6, laser Doppler flowmetry) into the unilateral NTS. Microinjection of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of the formation of NO, prevented cerebral vasoconstrictor responses induced by microinjection of L-glutamate into the NTS (n=10). Microinjection of NG-monomethyl-D-arginine (D-NMMA) had no effect on the cerebral vasoconstrictor responses induced by L-glutamate (n=11). Unilateral microinjections of L-NMMA into the NTS (n=9), of SNP into the area adjacent to the NTS (n=9), of vehicle solution into the NTS (n=10), and of light-inactivated SNP into the NTS (n=6) had no effect on cerebral circulation. Cerebral autoregulation was well maintained in our protocols (n=9). These results indicate that microinjection of SNP, an NO donor, into the NTS decreases CBF.

Animals↗
PubMed1996

Spinal cord lactate concentration during chemical stimulation of the nucleus tractus solitarii in anesthetized rats.

This study was conducted to determine the mechanism of spinal cord blood flow (SCBF) decrease following the nucleus tractus solitarii (NTS) activation. In urethane-anesthetized, paralyzed and artificially ventilated rats, neurons in the NTS were chemically stimulated by microinjection of L-glutamate (1.7 nmol; 50 nl) and the lactate concentration, one of indicators of local neuronal metabolism, in the spinal cord was monitored in real time using an enzyme electrode. Before the chemical stimulation study, the responses of the enzyme electrode and its specificity were tested in vitro and in vivo. The electrode responded to step changes in lactate concentration and a calibration plot and regression line were obtained in vitro. The lactate concentration was significantly (P < 0.01) increased during induced apnea in vivo (n = 8). The lactate concentration in the spinal cord was not significantly changed by chemical stimulation of the NTS when arterial blood pressure (ABP) remained above the lower limit of spinal cord autoregulation (n = 21). When chemical stimulation of the NTS decreased ABP to below the lower limit of autoregulation (n = 18), the lactate concentration in the spinal cord was significantly (P < 0.01) increased. This may only be due to hypotensive effects because the lactate concentration was also significantly (P < 0.01) increased when the ABP was passively decreased below the lower limit of autoregulation by controlled hemorrhage in intact (n = 11) and sinoaortic denervated rats (n = 10). Intravenous lactate injection produced no significant increase in the current from the enzyme electrode in the spinal cord (n = 4). Using the electrode with inactivated enzyme solution, the current from the electrode did not change with the increase in lactate in the spinal cord. These findings indicate that the enzyme electrode can detect rapid changes of lactate, a product of anaerobic metabolism. These results also indicate that the spinal cord vasoconstrictor response elicited by chemical stimulation of the NTS, which was performed above the lower limit of spinal cord autoregulation in our previous study, may be due to neurogenic regulatory mechanism, but not to the secondary effects of changes in metabolism.

Anesthesia↗
PubMed1996

Vasodilatation and enhanced oxidative metabolism of the cerebral cortex provoked by the periaqueductal gray matter in anaesthetized rats.

The aim of the present study was to evaluate the possible contribution of the cerebral cortical oxidative metabolism to the cortical vasodilator action of the periaqueductal gray. In 70 rats with cervical cordotomy, we found that unilateral stimulation of the caudal third of the lateral longitudinal column of the periaqueductal gray with N-methyl-D-aspartate bilaterally provoked the greatest increase in cortical blood flow (laser-Doppler flowmetry and/or microsphere flowmetry). The response was widespread over the entire neocortical regions, elicited in a dose-dependent manner, with little change in arterial blood pressure. The flow was increased effectively by a submaximal dose of the amino acid (1 mM, 100 nl), attaining a peak increase by 99 +/- 41% of the baseline level (mean +/- S.D., n = 30), and was associated with an enhancement of the cortical metabolic rate for oxygen by 51 +/- 26%. We then compared the flow increase with that induced by cold exposure (by 52 +/- 29%, n = 27), the latter response being tightly coupled to an enhanced metabolic rate for oxygen (by 41 +/- 23%). It was thus found that the increase in cortical blood flow provoked by the subdivision was dependent on the cerebrovasodilator mechanism that may be coupled to the cortical oxidative metabolism to the extent of one-half, and on certain other mechanisms for the remaining half. In view of the fact that this particular region serves to generate vigorous defence reactions that involve flight behaviour, the region should also help to meet the urgent demand for an increased cortical blood flow, so as to prepare for the possible generation of cortical hyperactivity in coping vigorously with a threatening emergency.

Anesthesia↗
PubMed1996

Retrospective study on the usefulness of radius and lumbar bone density in the separation of hemodialysis patients with fractures from those without fractures.

We measured bone mineral density (BMD) at the lumbar spine (LS-BMD), 1/3 radius (1/3R-BMD), and ultradistal radius (UDR-BMD) in 59 men (4 with spine fractures and 4 with nonspine fractures) and 65 women (10 with spine fractures and 9 with nonspine fractures), all receiving maintenance hemodialysis (HD). The BMD at each site expressed absolutely in g/cm2 was significantly lower in women than in men (p = 0.0001). In men, the absolute and age-matched values of both 1/3R- and UDR-BMD were inversely and significantly correlated with the duration of HD, and with serum alkaline phosphatase and parathyroid hormone levels (p < 0.05), whereas such relationships were obscure in women. On the other hand, the absolute values of BMD at each site in women but not in men were inversely and significantly correlated with patient age (p < 0.001). In both sexes, R-BMD was significantly lower in both the spine and nonspine fracture groups than in the nonfracture group (p < 0.05 and p < 0.01, respectively), whereas the only significant difference in LS-BMD was that it was lower in women with spine fractures than in women without fractures, when expressed as its absolute value (p < 0.05). By receiver operating characteristic analyses, both the absolute and age-matched values of R-BMD were better than LS-BMD as a determinant of non-spine fracture histories, and were similar to absolute LS-BMD as a determinant of spine fracture histories. We conclude that R-BMD is more valuable than LS-BMD for discriminating HD patients with all types of fractures from those without fractures.

Adult↗
PubMed1996

Ultrastructure and morphogenesis of human immunodeficiency virus.

The ultrastructure and morphogenesis of human immunodeficiency virus (HIV) were elucidated by observation with several techniques including immunoelectron microscopy and cryo-microscopy. The virus particle consists of an envelope, a core and matrix. The virus particles were observed extracellularly as having one of three profiles: (1) a centric or an eccentric electron-dense core, (2) rod-shaped electron-dense core, and (3) doughnut-shaped. HIV-1 particles in the hydrated state were observed by high resolution electron cryo-microscopy to be globular, and the lipid membrane was clearly resolved as a bilayer. Many projections around the circumference were seen to be knob-like. The shapes and sizes of the projections, especially head parts, were found to vary in each projection. By isolation with Nonidet P40 and glutaraldehyde, HIV-1 cores were confirmed to consist of p24 protein by immunogold labeling. When the virus enters the cell, two entry modes were found: membrane fusion and endocytosis. No structures resembling virus particles could be seen in the cytoplasm after viral entry. In HIV-1-infected cells, positive reactions by immuno-labeling suggest that HIV-1 Gag may be produced in membrane-bound structures and transported to the cell surface by cytoskeletons. Then a crescent electron-dense layer was first formed underneath the cell membrane. Finally, the virus particle was released from the cell surface. Several cell clones producing defective particles were isolated from MT-4/HIV-1 cells. Among them, doughnut-shaped or teardrop-shaped particles were seen to be produced in the extracellular space. In the doughnut-shaped particles, Gag p17 and p24 proteins faced each other against the inner electron dense ring, suggesting that the inner ring consists of a precursor Gag protein.

CD4-Positive T-Lymphocytes↗
PubMed1996

Identification of a repeated epitope recognized by human serum antibodies in a surface protein antigen of Streptococcus mutans.

This study determined the antigen determinants of a 190-kDa protein antigen of Streptococcus mutans that is involved in the initial attachment to the tooth surface. In 5 subjects, the reactivities of serum antibodies to 7 overlapping surface protein antigen fragments covering the entire antigen molecule and 19 sequential overlapping synthetic 19-mer peptides covering the entire A-region of the surface protein antigen were examined with enzyme-linked immunosorbent assay (ELISA). The study showed that the A-region of the antigen is strongly immunogenic in humans and contains several widely distributed epitopes. In addition, an amino acid sequence of the one of dominant epitopes in a certain subject was identified as LTAENTAI with ELISA inhibition assays using the relevant truncated peptides. This epitope was located both at the positions from L-346 to I-364 and L-430 to E-437 of the antigen molecule, and serum antibodies against the epitope were found in 3 of the 5 subjects.

Adult↗
PubMed1996

[Follow-up of HIV carriers by a laboratory method. I: Virus isolation is useful for prognostication of the onset of symptoms].

We followed 54 HIV-1 carriers (44 asymptomatic carriers and 10 AIDS patients) by virus isolation and immunological examination and evaluated their usefulness for prognostication of the onset of symptoms. From 37 carriers (27 asymptomatic carriers and 10 symptomatic), 132 HIV-1 strains were isolated; the virus isolation rate was 60% in the asymptomatic carriers (AC) but 100% in the symptomatic. In the AC, the isolation rate was 54.5% in the group showing stable in the CD4+ level but 95.5% in the group showing a decrease in the CD4+ level. With progression of the disease, the culture time required for virus isolation was shortened, and the percentage of isolates showing infectivity to the T-cell line (MT-4 cells) increased. These findings suggest that the virus in the body is changed with progression of the disease to that showing rapid replication, T-cell tropic, and high pathogenicity. Indeed, progression of the disease was observed in all carriers in whom a highly pathogenic virus was detected; some developed the disease within 1 year, some showed temporary recovery in the CD4+ level after AZT administration followed by progression to ARC, and others showed a rapid decrease in the CD4+ level. In contrast, in carriers with only slightly pathogenic virus, the CD4+ level was maintained for a long period. These results suggest that the detection of a highly pathogenic virus is one of the most reliable marker for the prognostication of the onset of the disease. The detection of HIV-1 antigen in the plasma and a decrease in the gag antibody were also associated with the progression of the disease. However, the reliability of these markers seems to be lower than that of virus isolation.

Acquired Immunodeficiency Syndrome↗
PubMed1996

[Prognosis and evaluation of drug therapy by V3 and RT gene analysis of HIV-1].

We have reported on the investigation of 54 HIV-1 patients concerning the isolation and clinical marker in the preceding paper. We have attempted the analysis of the V3 and RT genes. HIV-1 from a patient who had rapidly taken a turn for the worse had basic amino acid at position 11 (Arg) and lost an acidic amino acid at position 25 (Gln) of V3. This sequence pattern was a distinguished feature of a virus with a rapid-high, syncytium inducing (SI) and T-cell-line tropic phenotype. In contrast, patients with no or mild clinical symptoms had sequences characterized as slow-low, non-synsytium inducing (NSI) and macrophage tropic. We then investigated the appearance of resistant to AZT and ddI. It was shown that the virulent virus obtained drug resistant variants faster than the wild type by analysis of the RT gene. We consider that these data concerning virus isolation and gene analysis are useful for prognosis and strategy for clinical therapy.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Diabetic renal failure and serum accumulation of the creatinine oxidative metabolites creatol and methylguanidine.

Diabetic patients (n = 23) with chronic renal failure (CRF) accumulate the creatinine (Cr) oxidative metabolites creatol (CTL) and methylguanidine (MG; a uremic toxin) in their sera. Analysis of serum CTL, a key intermediate in mammalian Cr catabolism into MG, is shown to offer some useful diagnostic information on CRF, especially in the determination of an initial stage of pathological renal failure. The sera of all diabetic (n = 23) and nondiabetic (n = 20) patients with CRF (s-Cr > 1.25 mg/dl) contained s-CTL (> 2 micrograms/dl), whereas those from normal subjects (n = 18) and diabetic patients (n = 18) without CRF contained no detectable s-CTL. A similar accumulation of s-MG was observed, but only when s-Cr was higher than 2.0 mg/dl. Although each s-CTL (Y: microgram/dl, Y': mol/l) and s-MG level (Z: microgram/dl, Z': mol/l) is highly correlated with s-Cr (X: mg/dl, X': mol/l) in a normal equation, Y or Z = AX + B, an alternative correlation in a second-order equation, Y or Z = alpha X2 + beta X, could also fit well. Since the quadratic equation can be convertible to Y/X or Z/X = alpha X + beta [Y'/X' or Z'/X' = alpha' X+ beta'] and active oxygen species, especially hydroxyl radicals, convert Cr into CTL, Y/X, Y'/X', Z/X and Z'/X' values which might be a kind of indices for oxygen stress (oxidative stress) increased in proportion to the increased severity of CRF in such patients. Although its meaning and interpretation are still debatable, diabetic CRF patients had a significantly higher alpha' value (2.2) than that (0.89) of nondiabetic CRF patients. All serum values for Cr, CTL and MG were measured with HPLC.

Creatinine↗
PubMed1996

Experimental hepatitis induced by Campylobacter jejuni infection in Japanese quail (Coturnix coturnix japonica).

To establish an experimental model for vibrionic hepatitis caused by Campylobacter jejuni, Japanese quails (Coturnix coturnix japonica) were inoculated with C. jejuni strains isolated from chicken hepatitis (BL107) and human diarrhea (HP5113). Necrotic liver lesions were formed by intra-pancreaticoduodenal vein injection by which the bacteria reached the liver directly via the portal vein, but not by intra-gastric infection. These liver lesions were observed from day 1 to 7 after the infection. The pathological changes were weak and no clinical signs were observed throughout the experimental period. By immunohistochemistry, the bacterial antigens were detected in the hepatocytes, and intercellular spaces between the hepatocytes, and in the macrophages during the early stage of the infection. When focal hepatocyte necrosis was formed, the antigen was detected more frequently in the intact hepatocytes at the periphery of the lesions than within necrotic foci. The bacteria were not detected from the liver, spleen or blood according to raising the serum agglutination titer. In contrast, the bacteria immediately invaded the bile in 5 min post-infection and were retained in the gallbladder for long periods. The present study showed that necrotizing hepatitis was formed by intra-pancreaticoduodenal vein infection of the quail with C. jejuni.

Animals↗
PubMed1996

[Gore-tex covering of ultraflex stent and its usefulness for malignant esophageal stenosis: preliminary clinical results].

Malignant esophageal stenoses develop esophagorespiratory fistulae or perforations so frequently that esophageal stents must be required. We devised a covered stent with a thin Gore-Tex sheet and a nitinol stent system, Ultraflex, without increasing the size of the introducer assembly. Nine patients with malignant esophageal stenoses, including seven patients with perforation or fistulation, were carefully treated with the covered stent. All stent deployments were successfully carried out under fluoroscopic guidance. The average time required for full self-expansion was two weeks. The average grade of dysphagia improved from 3.7 to 1.2. Clinical symptoms due to esophagorespiratory fistulae were improved in three of four patients. No clinical or technical complications, such as migration, were observed, but a fistula developed at the bare site of the stent. This covering method for the flexible stent was simple and safe, and was considered to be useful in the treatment of malignant esophageal stenosis.

Adenocarcinoma↗
PubMed1996

[Experience with surgery for synchronous double cancer of rectum and lung detected after long-term follow-up for pneumoconiosis].

A 60-year-old man has been followed up for pneumoconiosis for 7 years. He was admitted to our hospital because of melena. He was undergone a colonofiberscopy and was diagnosed as rectal cancer. We performed anterior resection of the rectum (Stage I). About 5 months after the first operation, abnormal shadows of the both lungs were growing in size. Bronchoscopic examination revealed adenocarcinoma, we performed left upper lobectomy (Stage I) and this case was judged a synchronous double cancer.

Adenocarcinoma↗