Biomedical subjects
Publications and source records attributed to Naoki Yamamoto.
We report a case of severe urinary extravasation after renal contusion and its successful management by endoscopic creation of a neoinfundibulum. When the stenotic infundibulum cannot be traversed with a guide wire, creation of a new infundibulum will offer a secure alternative for accessing the collecting system.
Here we report that sparsomycin, a streptococcal metabolite, enhances the replication of HIV-1 in multiple human T cell lines at a concentration of 400 nM. In addition to wild-type HIV-1, sparsomycin also accelerated the replication of low-fitness, drug-resistant mutants carrying either D30N or L90M within HIV-1 protease, which are frequently found mutations in HIV-1-infected patients on highly active antiretroviral therapy (HAART). Of particular interest was that replication enhancement appeared profound when HIV-1 such as the L90M-carrying mutant displayed relatively slower replication kinetics. The presence of sparsomycin did not immediately select the fast-replicating HIV-1 mutants in culture. In addition, sparsomycin did not alter the 50% inhibitory concentration (IC50) of antiretroviral drugs directed against HIV-1 including nucleoside reverse transcriptase inhibitors (lamivudine and stavudine), non-nucleoside reverse transcriptase inhibitor (nevirapine) and protease inhibitors (nelfinavir, amprenavir and indinavir). The IC50s of both zidovudine and lopinavir against multidrug resistant HIV-1 in the presence of sparsomycin were similar to those in the absence of sparsomycin. The frameshift reporter assay and Western blot analysis revealed that the replication-boosting effect was partly due to the sparsomycin's ability to increase the -1 frameshift efficiency required to produce the Gag-Pol transcript. In conclusion, the use of sparsomycin should be able to facilitate the drug resistance profiling of the clinical isolates and the study on the low-fitness viruses.
Monocytes express on the cell surface several kinds of chemokine receptors that facilitate chemotaxis followed by differentiation in target tissues. In the present study, we found that a large number of monocytes from peripheral blood mononuclear cells (PBMCs) tightly adhered to plastic cell culture plates precoated with a monoclonal antibody (mAb, clone T312) specific for human CCR5 but not an isotype control after overnight incubation. Soluble T312 did not induce such adhesion, indicating that cross-linking of CCR5 is required for the enhanced adhesion of monocytes. The adhesion was blocked by a PI3-K inhibitor and an anti-CD18 blocking mAb. Following the cross-linking of CCR5, monocytes synthesized high levels of M-CSF, RANTES, MIP-1 alpha, and MIP-1 beta associated with a readily detectable down modulation of CD14, CD4, CCR5, and CXCR4 expression. The T312-enriched monocytes differentiated into dendritic cells (DCs) in the presence of interleukin-4 alone. After maturation with beta-interferon, the T312-induced DCs stimulated proliferation of allogeneic naïve CD4(+) T cells accompanied by the synthesis of high levels of gamma-interferon in vitro. Furthermore, the T312-induced DCs were capable of stimulating antigen-specific human T- and B-cell immune responses in our hu-PBL-SCID mouse system. Finally, screening of other anti-chemokine receptor mAbs showed that select clones of mAbs against CXCR4 and CCR3 were also capable of facilitating enrichment of monocytes similar to T312. These results show that cross-linking of chemokine receptors on monocytes by appropriate mAbs leads to activation and differentiation of monocytes and that the method described herein provides an alternate simple strategy for adherence-based isolation of monocytes and generation of functional DCs.
Most patients with acute myocarditis manifest particular clinical signs and symptoms, including marked cardiac failure and/or a high degree of atrioventricular block on admission. However, a 78-year-old man did not have symptoms and was hospitalized as a result of abnormalities observed on an incidentally obtained electrocardiogram (ECG). Several days later, he developed cardiogenic shock and fulminant myocarditis, which required percutaneous cardiopulmonary support; however, the cardiac failure persisted and he died approximately 4 months later. The ECG showed findings similar to those of acute inferior myocardial infarction, and on left ventriculography, diffuse hypokinesis was observed most prominently in the inferoposterior wall. During autopsy, interstitial fibrosis was marked in the inferoposterior wall, with small, round, cell infiltration prominent at the same site. Clustering of these cells is a characteristic feature of chronic myocarditis.
Acute vascular access (VA) obstruction is one of the most common complications for hemodialysis patients and medical professions. We performed early vascular access intervention therapy (VAIVT) to reduce the incidence of acute VA obstruction, VAIVT and fistula reconstruction. During a 2-year period, we performed 125 procedures of VAIVT in 145 cases in 50 patients who underwent arterio-venous fistula angiography. We performed early VAIVT within two weeks since we clinically found VA stenosis. The 125 procedures of VAIVT (95 non-thrombosed cases, 30 thrombosed cases) involved 86 native fistulas (74 non-thrombosed, 12 thrombosed) and 39 graft fistulas (21 non-thrombosed, 18 thrombosed). Prevalence of thrombosed cases was significantly higher in graft fistula than in native fistula (P<0.001). The primary patency rate in 50 patients at 6, 12 and 24 months was 62.1, 46.9 and 41.7%, respectively, while the secondary patency rate was 93.7, 90.7 and 86.6%, respectively. The highest primary patency rate was found in the non-thrombosed group with native fistula and the lowest secondary patency rate was found in the thrombosed group with graft fistula. By comparing the results of this study with the events for the previous 2 years, the total VAIVT number was almost the same. The number of non-thrombosed cases increased significantly from 54 to 95, and those of thrombosed cases decreased from 68 to 30 (P < 0.001). The number of fistula reconstructions significantly decreased from 59 to 22 (P<0.001). Early VAIVT for VA stenosis could decrease acute VA obstruction and fistula reconstruction without increasing the total number of VAIVT.
Structure-activity relationship studies on CXCR4 antagonists, which were previously found by using cyclic pentapeptide libraries, were performed to optimize side-chain functional groups, involving conformationally constrained analogues. In addition, a new lead of cyclic pentapeptides with the introduction of a novel pharmacophore was developed.
D-serine in mammalian brains has been suggested to be an endogenous co-agonist of the NMDA-type glutamate receptor. We have explored the molecules regulating D-serine uptake and release from the rat neocortex cDNA library using a Xenopus oocyte expression system, and isolated a cDNA clone designated as dsm-1 (D-serine modulator-1) encoding a protein that reduces the accumulation of D-serine to the oocyte. dsm-1 is the rat orthologue of the human 3'-phosphoadenosine 5'-phosphosulfate transporter 1 (PAPST1) gene. The hydropathy analysis of the deduced amino acid sequence of the Dsm-1 protein predicts the 10 transmembrane domains with a long hydrophobic stretch in the C-terminal like some amino acid transporters. The dsm-1 mRNA is predominantly expressed in the forebrain areas that are enriched with D-serine and NMDA receptors, and in the liver. The transient expression of dsm-1 in COS-7 cells demonstrates a partially Golgi apparatus-related punctuate distribution throughout the cytoplasm with a concentration near the nucleus. dsm-1-expressing oocytes diminishes the sodium-dependent and -independent accumulation of D-serine and the basal levels of the intrinsic D-serine and increases the rate of release of the pre-loaded D-serine. These findings indicate that dsm-1 may, at least in part, be involved in the D-serine translocation across the vesicular or plasma membranes in the brain, and thereby control the extra- and intracellular contents of D-serine.
D-serine is now considered to be an endogenous co-agonist of the NMDA receptor in mammalian brain. To obtain insight into the molecular mechanisms underlying D-serine metabolism and function, we explored transcripts that are responsive to D-serine in the neocortex of the 8-day-old infant rat by a differential cloning technique, RNA arbitrarily primed PCR. We isolated a novel D-serine inducible transcript, D-serine-responsive transcript-2 (dsr-2), that was exclusively expressed in the brain. Sequence analysis of the corresponding cDNAs to the transcript revealed that the dsr-2 mRNA consists of 7199 nucleotides with an open reading frame encoding 111 amino acids. The dsr-2 gene was located on the reverse strand within an intron of the neurexin-3alpha gene, mapped to rat chromosome 6q24-31. The regional distribution of the basal expression of dsr-2 and its ontogenic changes in the brain closely correlated with those of free D-serine and of NMDA receptor R2B subunit mRNA, but were somewhat different from those of the neurexin-3alpha transcript. These findings suggest that dsr-2 may be involved in D-serine metabolism and/or function, and in the interactions between D-serine, NMDA receptor and neurexin-3alpha, in mammalian brain.
Adult T-cell leukemia (ATL), an aggressive malignancy of CD4+ T cells associated with human T-cell leukemia virus type I (HTLV-I) infection, carries a very poor prognosis because of the resistance of leukemic cells to any conventional regimen, including chemotherapy. We examined the effect of ritonavir, an HIV protease inhibitor, on HTLV-I-infected T-cell lines and primary ATL cells and found that it induced apoptosis and inhibited transcriptional activation of NF-kappaB in these cells. Furthermore, ritonavir inhibited expression of Bcl-xL, survivin, c-Myc, and cyclin D2, the targets of NF-kappaB. In nonobese diabetic/severe combined immunodeficient (NOD/SCID)/gammacnull (NOG) mice, ritonavir very efficiently prevented tumor growth and leukemic infiltration in various organs of NOG mice at the same dose used for treatment of patients with AIDS. Our data indicate that ritonavir has potent anti-NF-kappaB and antitumor effects and might be clinically applicable for treatment of ATL. These results would provide a new concept and novel platform for new drug development of leukemia and solid cancer as well.
Assembly and deposition of amyloid beta-protein (Abeta) in the brain is a fundamental process of Alzheimer's disease (AD). We previously hypothesized that GM1 ganglioside-bound Abeta (GAbeta) is an endogenous seed for Abeta assembly in brain. Recently, we have succeeded in generation of a monoclonal antibody specific to GAbeta. Notably, this antibody, 4396C, per se substantially inhibits Abeta assembly in vitro. Here we report that the peripheral administration of Fab fragments of 4396C into transgenic mice expressing a mutant amyloid precursor protein gene, following the conjugation of the protein transduction domain of the Tat protein, markedly suppressed Abeta deposition in the brain. This result further supports our previous hypothesis and also provides a new insight into develop AD therapy through targeting seed Abeta in the brain, which selectively inhibits the initial step of the pathological process of AD.
Laboratory-grown miniature tomato (Lycopersicon esculentum) cultivar Micro-Tom has attracted attention as a host for functional genomics research. In this study, we generated 35,824 expressed sequence tags (ESTs) from leaves and fruits of Micro-Tom. The ESTs comprised 10,287 unigenes (5007 contigs and 5280 singletons), including 1858 novel tomato unigenes. Of the 18 unigenes that shared strong homology with tobacco chloroplast genome sequences, one unigene was likely derived from polyadenylated transcripts of the atpH gene. Interestingly, ESTs for vacuolar invertase, pectate lyase and alcohol acyl transferase were underrepresented in the Micro-Tom data set. From all of the ESTs, we mined 2039 candidate single nucleotide polymorphisms (SNPs) and 121 candidate insertions and deletions (indels) based on homology with four tomato inbred lines, E6203, R11-13, Rio Grande PtoR and R11-12, and a wild relative, L. pennellii TA56, for which sequence data was publicly available with more than 5000 entries. Direct genome sequencing of several SNP or indel sites in Micro-Tom and L. esculentum E6203 suggested that more than 69% of the candidate sites were truly polymorphic, making them useful for the preparation of DNA markers.
We have recently shown constitutive IkappaB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.
Several new phorbol derivatives having ethereal substituents at the 12-position were synthesized and subjected to biological evaluation to find new candidates of an anti-HIV agent. Among them, 12-O-(methoxymethyl)phorbol 13-decanoate showed potent inhibitory activity against infection of HIV-1 in MT-4 cells (EC50: 1.3 ng/mL) and relatively low cytotoxicity (CC50: 8.3 microg/mL). This compound was also found to have sufficient stability in mouse plasma compared with the corresponding 12-acetate derivative, which was an equipotent HIV-1 inhibitor, but with an activity that decreased considerably after plasma treatment.
Overexpression of CD30 and constitutive nuclear factor-kappaB (NF-kappaB) activation are hallmarks of the malignant Hodgkin Reed-Sternberg (H-RS) cells. Previous investigations have demonstrated that both proliferation and survival of H-RS cells require constitutive NF-kappaB activity, which is comprised of the p50 and RelA subunits. We report here enhanced expression of NF-kappaB2/p52 and RelB-containing NF-kappaB DNA-binding activity in Epstein-Barr virus-negative H-RS cells. Kinetic studies revealed that a proteasome inhibitor MG132 induced p100 accumulation with reduced p52 expression in H-RS cells, suggesting proteasome-dependent processing of p100. In addition, treatment with a protein synthesis inhibitor cycloheximide rapidly downregulated inhibitor of NF-kappaB (IkappaB) kinase activity in H-RS cells. We also demonstrate that overexpression of CD30 in rat fibroblasts at levels comparable to those in H-RS cells results in constitutive IkappaB kinase activation, proteasome-dependent p100 processing, and NF-kappaB-dependent cell transformation. Our results thus indicate that CD30 triggers the noncanonical NF-kappaB activation pathway, and suggest that deregulated CD30 signaling contributes to the neoplastic features of H-RS cells.
A highly potent CXCR4 antagonist, compound 2, was previously found by using two orthogonal cyclic pentapeptide libraries involving conformation-based and sequence-based libraries based on the pharmacophore of a 14-mer peptidic antagonist, 1. Herein, cyclic tetrapeptides derived from replacements of the dipeptide unit (Nal-Gly) with a gamma-amino acid and pseudopeptides cyclized by disulfide and olefin bridges were synthesized to find novel scaffold structures different from that of cyclic pentapeptides. These compounds contain a reduced number of peptide bonds compared to compound 2. Furthermore, several analogues with chemical modification of the side chain of Arg(4) in 2 were also prepared. From these, several new leads possessing high to moderate CXCR4-antagonistic activity were characterized.
Vpr, one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), affects aspects of both viral and cellular proliferation, being involved in long terminal repeat (LTR) activation, arrest of the cell cycle at the G2 phase, and apoptosis. We have discovered a novel role for Vpr as a regulator of the splicing of pre-mRNA both in vivo and in vitro. We found, by RT-PCR and RNase protection analysis, that Vpr caused the accumulation of incompletely spliced forms of alpha-globin 2 and beta-globin pre-mRNAs in cells that had been transiently transfected with a Vpr expression vector. We postulated that this novel effect of Vpr might occur via a pathway that is distinct from arrest of the cell cycle at G2. By analyzing splicing reactions in vitro, we showed that Vpr inhibited the splicing of beta-globin pre-mRNA in vitro. The splicing of intron 1 of alpha-globin 2 pre-mRNA was modestly inhibited by Vpr but the splicing of intron 2 was unaffected. Interestingly, an experimental infection system which utilizes high-titered HIV-1/vesticular stomatitis virus G protein showed that Vpr expressed from an HIV-1 provirus was sufficient to accumulate endogenous alpha-globin 2 pre-mRNA. Thus, it is likely that Vpr contributes to selective inhibition of the splicing of cellular pre-mRNA.
The mechanisms underlying regional amyloid beta-protein (Abeta) deposition in brain remain unclear. Here we show that assembly of hereditary variant Dutch- and Italian-type Abetas, and Flemish-type Abeta was accelerated by GM3 ganglioside, and GD3 ganglioside, respectively. Notably, cerebrovascular smooth muscle cells, which compose the cerebral vessel wall at which the Dutch- and Italian-type Abetas deposit, exclusively express GM3 whereas GD3 is upregulated in the co-culture of endothelial cells and astrocytes, which forms the cerebrovascular basement membrane, the site of Flemish-type Abeta deposition. Our results suggest that regional Abeta deposition is induced by the local gangliosides in the brain.
A water-soluble extract of fermented Polygonum tinctorium Aiton (Polygonaceae) called Sukumo, exhibited a potent inhibitory activity against HIV type 1 in vitro. The extract potently suppressed acute HIV-1 (IIIB) infection in MT-4 cells with EC50 values of 0.5 microg/ml but exhibited low cytotoxicity to MT-4 cells even at a high concentration (CC50 > 1000 microg/ml). It also inhibited giant cell formation in co-cultures of HIV-infected cells and uninfected Molt-4 cells. Sukumo extract was found to interact with both the viral envelope glycoprotein and cellular receptors, thus blocking virus-cell binding and virus-induced syncytium formation. There was a good correlation between the extract's anti-HIV-1 activity and its inhibitory effects on HIV-1 binding. It also suppressed replication of herpes simplex virus type 1 in Vero cells with an EC 50 of 11.56 microg/ml. On the other hand, there was no appreciable activity against influenza A virus, poliovirus or SARS corona virus when tested at concentrations ranging from 3.2-400 microg/ml as shown by microscopic image analysis for cytopathic effect (CPE). Physico-chemical studies revealed that the anti-HIV activity in the extract was essentially maintained after boiling at 100 degrees C in 1N HCl or 1N NaOH, and after treatment with 100 mM NaIO4. The inhibitory activity of the extract was also not reduced after pronase digestion. The active factor in the extract is likely to be a novel compound(s) having a polyanionic substructure and a molecular weight of 10,000-50,000.