Biomedical subjects
M Nakano
Publications and source records attributed to M Nakano.
Protamine dissociation before decondensation of sperm nuclei during in vitro fertilization of pig oocytes.
The correlation between morphological changes and the dynamics of protamine in boar sperm chromatin during in vitro fertilization of pig oocytes matured in vitro was assessed. For this purpose, protamine was purified from boar sperm nuclei and an antiserum against protamine was developed. After affinity purification, the antiserum reacted exclusively with boar protamine during western blotting, showing no crossreactivity with core histones. Immunohistochemical evaluation revealed that only fully developed spermatid nuclei in boar testes stained strongly with the antiserum. When pig oocytes matured in vitro were fertilized in vitro, sperm penetration was observed in 37% of oocytes at 2 h after insemination and the penetration rate increased to 99% by 5 h after insemination, accompanied by an increase in polyspermic penetration. Paraffin wax sections of the inseminated oocytes were examined by immunohistochemical analysis with the antiserum. The proportion of condensed sperm nuclei that reacted with the antiserum was 87% of the sperm nuclei that penetrated by 2 h after insemination, and this decreased to 20 and 13% at 3 and 5 h after insemination, respectively. However, none of the decondensing sperm nuclei or male pronuclei reacted with the antiserum during the entire insemination period. These results indicate that a specific antiserum against boar protamine can be raised and, using this serum, it has been demonstrated that protamine is dissociated from boar sperm nuclei before decondensation during in vitro fertilization.
[Pharmacokinetics of cisplatin and methotrexate in a patient suffering from advanced ureteral tumor accompanied by chronic renal failure, undergoing combined hemodialysis and systemic M-VAC chemotherapy].
The pharmacokinetics of cisplatin and methotrexate were determined in a patient suffering from advanced ureteral tumor accompanied by chronic renal failure undergoing 4 consecutive cycles of M-VAC chemotherapy and hemodialysis. No significant difference was observed in t1/2, AUC or CLtot of total platinum between the patient with the chronic renal failure and patients with normal renal function. The AUC and CLtot of free platinum in the patient with the chronic renal failure were higher and lower, respectively, than in the patients with normal renal function. The free cisplatin rebounded remarkably after the end of dialysis, which may be partly attributed to an increase in the AUC and decrease in CLtot. However, the dialysis index was about 75 and 85% in the 3rd and 4th cycles, respectively. The t1/2 and CLtot of methotrexate in the patient with the chronic renal failure tended to be longer and smaller than those in patients with normal renal function, respectively. Seventy-two hours after administration, the methotrexate level was 0.02 microM, which was not at the high-risk level of high-dose therapy. After four cycles of M-VAC therapy, the rest of the right ureteral tumor was extirpated and the clinical response was CR. In conclusion, it is considered that cisplatin and methotrexate can be given to a patient with chronic renal failure. However, the cisplatin and methotrexate serum levels must be monitored, even after very low doses.
[A case of mesoblastic nephroma in an adult patient].
We treated a rare case of adult mesoblastic nephroma. The patient was a 52-year-old Japanese man with the chief complaint of intermittent gross hematuria and left lumbar pain. Abdominal ultrasonography, computed tomography, excretory urography, retrograde pyelography and angiography revealed a left renal tumor suspected to be a left pelvic tumor. A left nephroureterectomy was performed. The histologic examination showed a mesoblastic nephroma. A total of 38 adult mesoblastic nephroma cases were reviewed.
[Treatment of membranous lupus nephritis].
Explore the source record for details and available documents.
[Evaluation of antibiotic preparations for children from a standpoint of water-solubilities].
The evaluation of seven widely-used antibiotic preparations [five cephem antibiotics; cefaclor (CCL), cefpodoxime proxetil (CPDX-PR), cefdinir (CFDN), cefditoren pivoxil (CDTR-PI), cefcapene pivoxil (CFPN-PI), one macrolide; clarithromycin (CAM) and one penem; faropenem sodium (FRPM)] for children were performed from a standpoint of water-solubilities, both as a preparation and as a component drug. As the results, these preparations showed great differences in the water-solubilities when added 10 ml water to 0.5 g of each preparation. That is, their solubilities differed from about 40% (CFPN-PI) to 100% (FRPM) as a preparation, and from nearly 0% (CAM) to 100% (FRPM, CCL) as a component drug. Additionally, about a half of the insoluble residues were found to be the component drug, in the cases of three preparations (CPDX-PX, CFDN, CDTR-PI) which were solubilized at 80-90%. From these results, it was suggested that the marketed antibiotic preparations for children might be classified into three categories; i.e., [A] preparation for solution and suspension (FRPM, CCL), [B] preparation suitable to suspension (CPDX-PR, CFDN, CDTR-PI), and [C] fine granule preparation for children unsuitable to suspension (CFPN-PI, CAM). Consequently, the names for dosage-forms of these preparations should be standardized.
Antihemolytic effect of Rooibos tea (Aspalathus linearis) on red blood cells of Japanese quails.
The antihemolytic activity of Rooibos and black tea on Japanese quail erythrocytes was studied. Peroxide and hypotonic hemolysis of the red blood cells of quails, either fed with Rooibos tea supplemented food or fed without tea, was performed. Long-term consumption of Rooibos tea did not change the erythrocyte fragility to either peroxide or hypotonia induced hemolysis. However, Rooibos and black teas decreased peroxide induced hemolysis of erythrocytes incubated with each of them, but not hemolysis induced by hypotonic NaCl solution. Stronger inhibition of hemolysis has been obtained when a boiled water extract of Rooibos tea was used for the inhibition. The degree of inhibition was comparable with the effect of ascorbic acid.
Antiviral properties of a mangrove plant, Rhizophora apiculata Blume, against human immunodeficiency virus.
A polysaccharide extracted from the leaf of Rhizophora apiculata (RAP) was assessed in cell culture systems, for its activity against human and simian immunodeficiency viruses. RAP inhibited HIV-1 or HIV-2 or SIV strains in various cell cultures and assay systems. It blocked the expression of HIV-1 antigen in MT-4 cells and abolished the production of HIV-1 p24 antigen in peripheral blood mononuclear cells (PBMC); the 50% effective concentration (EC50) of RAP in HIV-1 infected MT-4 cells and in PBMC was 10.7 and 25.9 microg/ml, respectively. RAP (100 microg/ml) completely blocked the binding of HIV-1 virions to MT-4 cells. RAP also reduced the production of viral mRNA when added before virus adsorption. RAP inhibited syncytium formation in cocultures of MOLT-4 cells and MOLT-4/HIV-1(IIIB) cells. RAP did not prolong activated partial thromboplastin time (APTT) up to 500 microg/ml. These properties may be advantageous should RAP be considered for further development.
Primary structure of hemolytic lectin CEL-III from marine invertebrate Cucumaria echinata and its cDNA: structural similarity to the B-chain from plant lectin, ricin.
CEL-III, a galactose/N-acetylgalactosamine (Gal/GalNAc) specific lectin purified from a marine invertebrate Cucumaria echinata has a strong hemolytic activity especially toward human and rabbit erythrocytes. We determined the primary structure of the CEL-III by examining the amino acid sequences of the protein and the nucleotide sequence of the cDNA. The cDNA encoding CEL-III has 1823 nucleotides and an open reading frame of 1296 nucleotides. CEL-III is composed of 432 amino acid residues with a M(r) of 47¿ omitted¿457 and has six internal tandem repeats, each with of 40-50 amino acids, comprising the N-terminal two-thirds of the molecule. Similar repeats are found in the B-chains of cytotoxic plant lectins, such as ricin and abrin, where six repetitive sequences extend throughout the molecules. A hydropathy plot predicts hydrophobic segments in the C-terminal region of CEL-III. These findings suggest that the N-terminal region of CEL-III plays an important role in binding to carbohydrate receptors on the target cell membranes, an event which triggers an intermolecular hydrophobic interaction of the C-terminal region, the result being oligomerization of CEL-III to lead to pore-formation in erythrocyte membrane.
Identification of genes showing differential expression in antisense K-ras-transduced pancreatic cancer cells with suppressed tumorigenicity.
K-ras point mutation occurs in >80% of pancreatic cancer. We reported previously that the transduction of an antisense K-ras RNA expression vector suppressed the growth of pancreatic cancer cells with K-ras point mutations in vitro and in vivo. The RNA differential display method (DD) was used to compare the mRNA expression profile of the pancreatic cancer cell line AsPC-1 and that of the antisense K-ras-transduced, growth-retarded AsPC-1 cells. cDNA fragments were isolated from 20 bands on the DD gel, and their differential expression between the two cell lines was confirmed. A sequence analysis revealed that all of the 11 clones up-regulated in the antisense-transduced cells were mitochondrial genes. The other nine cDNA clones that were down-regulated in the antisense-transduced AsPC-1 cells included an oncogene PTI-1 (prostate tumor inducing gene-1), matrix metalloproteinase (MMP)-7, the beta3 chain of laminin-5, lysosome-associated membrane protein-2, the H chain of apoferritin, ribosomal protein S6, proteasome subunit XAPC7, and two cDNA fragments with no homology to the GenBank database. In addition to the AsPC-1 cells, reverse transcription-PCR analysis on surgical specimens of pancreatic cancer revealed that the PTI-1 and MMP-7 genes were overexpressed in three and four cases, respectively, of five cases examined. This method offers a unique opportunity to identify a set of genes that may be modulated by K-ras activation, at least in a subset of the pancreatic cancer. The information on such genes may facilitate our understanding of the spectrum of the functional genetic changes in pancreatic cancer.
Recurrent cerebral infarctions and del(10)(p14p15.1) de novo in HDR (hypoparathyroidism, sensorineural deafness, renal dysplasia) syndrome.
We report on a Japanese boy with HDR syndrome (hypoparathyroidism, sensorineural deafness, renal dysplasia) and recurrent cerebral infarctions in the basal ganglia. The patient experienced cerebral infarctions four times between age 7 months and age 20 months. Chromosome analysis of the patient demonstrated a 46,XY, del(10)(p14p15.1) de novo. This suggests that the putative gene responsible for HDR syndrome is located at 10p14-p15.1.
Characteristics of chemiluminescence observed in the horseradish peroxidase-hydrogen peroxide-tyrosine system.
Electrolysis or horseradish peroxidase (HRP)-catalyzed oxidation of tyrosine and bityrosine in aqueous solution at pH 7.4 resulted in light emission in the visible region. Electrolysis of tyrosine emitted light which peaked at 490 nm and was almost completely quenched by superoxide dismutase (SOD), while emission by bityrosine peaked at 530 nm. In the HRP-H(2)O(2)-tyrosine system the oxidation-reduction of tyrosine emitted light with two prominent peaks, 490 and 530 nm, and was not quenched by SOD. The phenoxyl neutral radical of the tyrosine in HRP-H(2)O(2)-tyrosine system was detected by electron spin resonance (ESR) spectrometry using tert-nitrosobutane as a spin trap; the spin adduct was found to adhere to the HRP molecule during the enzymatic reaction. Further, bityrosine was detected in the HRP-H(2)O(2)-tyrosine reaction system. Changes in absorption spectra of HRP and chemiluminescence intensities during HRP-catalyzed oxidation of tyrosine suggest that for photon emission compound III is a candidate superoxide donor to the phenoxyl cation radical of tyrosine on the enzyme molecule. The luminescence observed in this study might be originated from at least two exciplexes involved with the tyrosine cation radical (Tyr(*+)) and the bityrosine cation radical (BT(*+))
Singlet oxygen ((1)Delta(g)O(2)) as the principal oxidant in myeloperoxidase-mediated bacterial killing in neutrophil phagosome.
Intraphagosomal viability of wild type E. coli and lycopene (a powerful (1)O(2) quencher)-producing transformant E. coli was investigated using human polymorphonuclear leukocytes as the cells for phagocytosis of opsonized viable bacteria. While the viability of both wild type and the transformant E. coli decreased very rapidly in the phagosome, but the viability of the lycopene-transformant in phagosomes was about 1.7 times higher than that of wild type E. coli after 5 min of incubation. The results were very similar to the results obtained when E. coli strains were exposed to (1)O(2) generated in myeloperoxidase-H(2)O(2)-Br(-) system (a pure (1)O(2) generating system) at pH 4.5. The reason for HOCl, which may be generated in the myeloperoxidase-H(2)O(2)-Cl(-) system under physiological conditions but does not become involved in bactericidal action, could be explained by the near neutral pH in phagosomes at which bacterial killing by chlorination is extensively attenuated. This is the first report which proved (1)O(2)-mediated bacterial killing in neutrophil-bacterial phagosomal system.
Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase. A possible additional family in the aldo-keto reductase superfamily.
Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP(+), was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 +/- 1,600. The enzyme showed a single absorption peak at 280 nm (E(1%) = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBank(TM) accession number D89205). The gene corresponding to this protein was expressed in Escherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with human Shaker-related voltage-gated K(+) channel beta2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.
A Na+-H+ exchange inhibitor (SM-20550) protects from microvascular deterioration and myocardial injury after reperfusion.
Na+-H+ exchange inhibitors may reduce myocardial damage after reperfusion. However, their effects on microvascular deterioration are not known. We examined the potency of a novel Na+-H+ exchange inhibitor, SM-20550 [ N-(Aminoiminomethyl)-1,4-dimethyl-1H-indole-2-carboxamide methanesulfonate], and its effects on microvascular damage after reperfusion. In an in vitro study, the Na+-H+ exchange inhibiting activity of SM-20550 was about 10 times greater than that of ethylisopropyl amiloride. In in vivo experiments, we occluded the left circumflex coronary artery in 29 dogs for 2 h and then reperfused for 5 h. SM-20550 was administered either before ischemia (n = 11) or before reperfusion (n = 7). Another 11 dogs served as controls. We found that SM-20550 not only improved coronary vasodilator responses to acetylcholine and adenosine after reperfusion, but also reduced infarct size (P < 0.01). Intramyocardial bleeding, which should reflect microvascular damage, was not found in dogs with SM-20550 treatment. Infarct size was correlated inversely with collateral blood flow in control (both, P < 0.01) but not in SM-20550-treated animals. Furthermore, SM-20550 significantly suppressed ventricular fibrillation during both ischemia and reperfusion. These results suggest that protective effects of Na+-H+ exchange inhibitors on reperfused myocardium are due at least in part to microvascular protection.
Lipopolysaccharide tolerance in murine peritoneal macrophages induces downregulation of the lipopolysaccharide signal transduction pathway through mitogen-activated protein kinase and nuclear factor-kappaB cascades, but not lipopolysaccharide-incorporation steps.
Endotoxin/lipopolysaccharide (LPS) tolerance, a hyporesponsive state to endotoxin or LPS stimulation, was induced in murine peritoneal macrophages by previous exposure of macrophages to LPS. Expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 mRNA in response to LPS stimulation was suppressed in LPS-tolerant macrophages. Tyrosine phosphorylations in response to LPS of 40-45-kDa proteins in non-tolerant macrophages were also suppressed in LPS-tolerant macrophages. These proteins corresponded to two members of the mitogen-activated protein kinase (MAPK) family, ERK and p38. In addition to these proteins, another MAPK family protein, JNK, was also suppressed in LPS-tolerant macrophages. Activation of Raf-1, located in the upstream portion of ERK cascades, was also suppressed by LPS-tolerance induction. These suppressions in LPS-tolerant macrophages were exhibited against stimulation by an LPS agonist like taxol, but not towards stimulation by an unrelated activator like phorbol ester (PMA). Activation of the transcription factor NF-kappaB, which is supposed to be one of the components of another important pathway for transduction of LPS-stimulated cytokine producing signals, was strongly suppressed and degradation of IkappaB, an inhibitor of NF-kappaB, was also severely diminished in LPS-tolerant macrophages. Although a monosaccharide lipid A analog, GLA-58, was able to stimulate macrophages to activate ERK proteins without cytokine production, pretreatment of macrophages with this compound suppressed both LPS-stimulated activation of ERK and cytokine production. Furthermore, downregulation of LPS-uptake in LPS-tolerant macrophages was not observed. Based on all these findings, LPS tolerance might be caused by the previous activation of some components on LPS-signaling pathways. This may then induce a refractory state in key LPS-signal transducer molecules located downstream of the cell membrane LPS receptor and upstream of the branching point in intracellular cascades for activation of MAPK and NF-kappaB, probably in some initial steps of intracellular signaling.
Glutathione S-transferase (GST) M1, T1, P1, N-acetyltransferase (NAT) 1 and 2 genetic polymorphisms and susceptibility to colorectal cancer.
A case-control study was carried out to examine the relation between genetic polymorphisms of five genes, cigarette smoking and colorectal cancer risk. We collected blood samples from 106 colorectal cancer patients and 100 healthy persons, then analyzed them to identify genotypes for glutathione S-transferase (GST) M1, T1, P1, N-acetyltransferase (NAT) 1 and 2 by the PCR method. We also collected smoking history data from all participants by questionnaire. From statistical evaluation on various combinations of genotypes, we observed that the cancer risk of those who have both GSTM1 present genotype and GSTP1 Adenine/Adenine homozygous genotype was significantly less than those who have other combinations of genotypes for two genes. For other combinations of genes, there was no significant association between genotype and cancer risk. There was also no significant association between amount of cigarette smoking and the cancer risk. These findings suggest that it is valuable to study cancer risk when examining genotypes of more than two genes at the same time. For further study, we need to collect more samples to increase statistical reliability, and besides cigarette smoking, include the nutrition data as an environmental factor.