Biomedical subjects
Publications and source records attributed to H Ochiai.
Two genomic DNA clones encoding cyclophilin (CyP) from Arabidopsis thaliana were isolated. The deduced protein products of these genes appeared to be cytosol-localized isoforms given the absence of a specific presequence for targeting to cellular compartments. Thus, multiple CyPs may exist and function in the cytosol of Arabidopsis thaliana.
Adherent cells derived from human palatine tonsils were isolated and cultivated. Exponentially growing adherent cells (TAC) were observed by phase-contrast microscopy and transmission electron microscopy. Immunocytochemical studies were also performed. TAC were composed of relatively monotonous cells with polygonal or spindle shapes and high proliferative activity. In addition to the development of rough endoplasmic reticulum and lysosomes, the TAC possessed a moderate amount of pinocytotic vesicles and a few microfilaments. All of the TAC strongly expressed fibroblastic markers and partial monocyte/macrophage markers, such as beta-subunit of prolyl 4-hydroxylase (DAKO-fibroblast), lysozyme, anti-alpha-1-antichymotrypsin (alpha ACT), and CD68 (KP-1, EBM/11). It was noted that, as the TAC were cultured for a longer period, they gradually increased the reactivity with the monoclonal antibody PG-M1. Furthermore, the TAC expressed myocytic phenotype, such as alpha-smooth muscle actin (alpha SMA) with various intensity. Moreover, as to extracellular matrix, TAC stained for collagen type I, collagen type III, laminin, and fibronectin. Collagen type IV was weakly positive. The results presented here showed that the TAC expressed three different phenotypes of fibroblasts, histiocytes and smooth muscle cells at the same time. The monoclonal antibody raised against the TAC reacted strongly with the subendothelial pericytes and/or smooth muscle cells in the extrafollicular area in human tonsils. The present results also suggested that the origin of the TAC was probably subendothelial pericytes and/or smooth muscle cells of the microvasculatures in the tonsil.
T-5575 and T-5578, novel 2-carboxypenams in which a carboxyl group has been introduced into the C-2 beta position of the nucleus, were evaluated for their in vitro antibacterial properties. The spectrum of activity of T-5575 was similar to that of aztreonam. However, it showed stronger activities than those of aztreonam against most gram-negative bacteria. T-5575 also showed potent activities against isolates of Enterobacter cloacae, Citrobacter freundii, and Pseudomonas aeruginosa resistant to ceftazidime, with MICs at which 90% of the isolates were inhibited of 0.39, 0.39, and 3.13 microgram/ml, respectively. T-5578 showed moderate levels of activity against gram-negative bacteria, compared with those of T-5575. Its activity against P. aeruginosa, however, was superior to those of T-5575 and the reference drugs tested. The most characteristic feature of T-5578 was its potent activities against ceftazidime-, imipenem-, and gentamicin-resistant P. aeruginosa isolates, with MICs at which 90% of the isolates were inhibited at 0.39, 3.13, and 3.13 microgram/ml, respectively. These two compounds were unfortunately poorly active against gram-positive bacteria, such as Staphylococcus aureus and streptococci. Both compounds were found to be stable for hydrolysis by various kinds of beta-lactamases and to have low affinities for these enzymes, with Ki values of > 100 microM. These novel penams bound most tightly to penicillin-binding protein 3 of Escherichia coli and P. aeruginosa. These results indicate that T-5575 and T-5578 can be regarded as promising 2-carboxypenams specially targeted against gram-negative pathogens.
We evaluated the characteristics of platelet alpha 2-adrenoceptors in 12 patients with effort angina pectoris, 11 patients with variant angina pectoris and 11 normal control subjects. alpha 2-Adrenoceptors were quantified using a radioligand binding assay with radiolabelled rauwolscine, an alpha 2-selective antagonist. In addition, plasma norepinephrine concentrations were measured by high performance liquid chromatography. The mean value of the maximal number of binding sites (Bmax) in patients with effort angina (205.1 +/- 11.3 fmol/mg protein) was significantly lower than that in control subjects (293.0 +/- 10.2 fmol/mg protein). Bmax did not differ between patients with variant angina (322.9 +/- 45.4 fmol/mg protein) and control subjects. There was no significant difference in the dissociation constant (Kd) among the 3 groups. The plasma norepinephrine concentration tended to be higher in patients with effort angina or variant angina than in normal controls, but this difference was not statistically significant. In addition, studies in another group of young volunteers (n = 20) revealed a negative correlation (r = -0.50, p < 0.05) between the Bmax of 3H-rauwolscine binding to platelets and the percent change in the plasma norepinephrine concentration when subjects moved from the supine to the standing position. This suggests a functional correlation between platelet alpha 2-adrenoceptors and those located at presynaptic sites. If platelet alpha 2-adrenoceptors correlate with presynaptic alpha 2-adrenoceptors, the current findings of decreased alpha 2-adrenoceptor density in platelets from patients with effort angina could represent attenuated negative feedback of norepinephrine by presynaptic alpha 2-adrenoceptors.
The 4993-bp cryptic plasmid, pMA2, from a cyanobacterium, Microcystis aeruginosa f. aeruginosa Kützing, originally derived from Kasumigaura lake, was completely sequenced and analyzed. Plasmid pMA2 had a unique sequence motif CTTGATT, which was proposed to be a nicking site on the smaller plasmid pMA1 (2287-bp) by the presence of single-stranded DNA susceptible to S1 nuclease. We had detected the occurrence of the single-stranded pMA1 in the living M. aeruginosa cells. This was also the case of pMA2. Thus, we suggest that both plasmids, pMA1 and pMA2, replicate through the rolling circle mechanism. By computer analysis of the pMA2 sequence, two open reading frames were found: one had a predicted molecular size of 30,440 Da and the other on a complementary one had a predicted molecular size of 10,852 Da. No rep protein was detected. Replication mechanisms of the plasmids are discussed.
This investigation was performed to determine whether ethanol affects induction on Chang liver cells of the c-met protooncogene product (c-met), a cell-surface receptor for hepatocyte growth factor (HGF). These cells were cultured for 4 weeks with 5, 50, or 100 mmol/L ethanol, or 10, 100, or 200 mumol/L acetaldehyde. Ethanol was found to inhibit [3H]thymidine incorporation by cells in a dose-dependent manner. In addition, acetaldehyde at 100 mumol/L also inhibited [3H]thymidine uptake. Treatment with recombinant HGF led to enhanced [3H]thymidine incorporation by cells treated with ethanol or acetaldehyde as well as by those left untreated, with no significant differences in the rates of increase among these three cell groups. The amount of c-met messenger RNA, however, was unaffected by treatment with ethanol or acetaldehyde. Expression of c-met as measured by cell ELISA was also unchanged by both treatments. These results suggest that ethanol has no effect on c-met induction on Chang liver cells and that HGF may not be involved in the ethanol induced inhibition of DNA synthesis.
The 64-kDa membrane-bound glycoprotein of the cellular slime mold Polysphondylium pallidum (referred to as gp64), seems to be implicated in cell-cell adhesion. Previously we have isolated a full-length gp64 cDNA, determined its nucleotide sequence, and found that all cysteine residues in the protein are involved in the formation of disulfide bonds. The disulfide arrangement of the 36 cysteines in gp64 was established by analysis of proteolytically cleaved protein and sequence analysis of cystine-containing fragments. Since gp64 has 36 Cys residues, 18 disulfide bonds must exist and the positions of 15 of them were determined. The 15 disulfide bonds in gp64 constitute five characteristic, so-called Sushi domains. In a Sushi domain, the first Cys in a sequence is connected to the third one and the second Cys to the fourth one. This is the first report describing the presence of Sushi domains in a cellular slime mold protein. From these data, gp64 appears to be distinct from all other previously described cell-adhesion proteins.
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The cellular slime mold Polysphondylium pallidum expresses a cell surface glycoprotein (referred to as gp64), which seems to be implicated in cell-cell adhesion. We identified a near full-length gp64 cDNA (1,104 base) upon screening a P. pallidum lambda gt11 library with a monoclonal antibody. The open reading frame encodes a protein of 320 amino acids with a molecular mass of 32,752 Da; the protein includes hydrophobic segments at both a NH2- and a COOH-terminal ends. By an Edman degradation analysis of S-pyridylethylated gp64 and its COOH-terminal peptide, it was found that the NH2- and COOH-terminal segments are both removed from the precursor protein of gp64. The COOH-terminal segment was isolated from a lysyl endopeptidase digest of gp64 by an affinity method. The COOH-terminal segment was identified at positions 266-279 in the primary sequence deduced from the cDNA sequence. The mature gp64 consisted of 279 amino acid residues and extremely rich in Cys residues (36 Cys/279 amino acids = 12.9%). Although there was already maximal accumulation of gp64 mRNA in vegetative cells, the protein reached a maximal level during aggregation stage, decreased, and then leveled off through the developmental cycle.
Alanine dehydrogenase (AlaDH) was purified to homogeneity from cell-free extracts of a non-N2-fixing filamentous cyanobacterium, Phormidium lapideum. The molecular mass of the native enzyme was 240 kDa, and SDS-PAGE revealed a minimum molecular mass of 41 kDa, suggesting a six-subunit structure. The NH2 terminal amino acid residues of the purified AlaDH revealed marked similarity with that of other AlaDHs. The enzyme was highly specific for L-alanine and NAD+, but showed relatively low amino-acceptor specificity. The pH optimum was 8.4 for reductive amination of pyruvate and 9.2 for oxidative deamination of L-alanine. The Km values were 5.0 mM for L-alanine and 0.04 mM for NAD+, 0.33 mM for pyruvate, 60.6 mM for NH4+ (pH 8.7), and 0.02 mM for NADH. Various L-amino acids including alanine, serine, threonine, and aromatic amino acids, inhibited the aminating reaction. The enzyme was inactivated upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The copresence of NADH and pyruvate largely protected the enzyme against the inactivation by PLP.
BACKGROUND: It has been shown that the development of coxsackievirus B3 (CB3) myocarditis is regulated by T cells and not by B cells. Interleukin-2 (IL-2) is a T-cell-derived cytokine that stimulates the growth of T cells. This study was carried out to determine the effects of IL-2 on CB3-infected BALB/c mice. METHODS AND RESULTS: In two separate experiments, recombinant human IL-2 (5 x 10(4) U) was administered subcutaneously to 30 mice early (days 0 to 7) and 30 mice late (days 7 to 14) after infection with CB3. Each experiment had a control group of infected animals that did not receive IL-2. On days 7 and 10, splenic natural killer (NK) cell activity determined by 51Cr release assay and the distribution of myocardial lymphocyte subsets were compared in the treated and untreated groups. In the early treatment experiment, survival at 7 days was higher in treated compared with control animals, myocardial virus titers were lower, inflammatory cell infiltration was less (as was the severity of necrosis at the time the mice were killed), and NK cell activity was higher. However, in the late treatment experiment, survival at 14 days was lower in treated compared with control animals, and there was more infiltration, more severe necrosis, and more T-cell infiltration, but the NK cell activity did not differ significantly. In a third experiment similar to the late experiment described above but involving infected athymic nude mice, we confirmed the lack of effect of late in vivo administration of IL-2 on outcome. CONCLUSIONS: IL-2 has the capacity to limit CB3 myocarditis by enhancing NK cell activity in the acute viremic stage, resulting in a reduction of cardiac pathology. However, in the subacute aviremic stage, in contrast, IL-2 exacerbates the course and severity of the disease by increasing the number of T cells infiltrating the myocardium. That is, IL-2 has differential effects on acute CB3 myocarditis. IL-2 is beneficial if treatment is given early but later in murine CB3 myocarditis.
Single-stranded plasmid DNA of pPF1 from Phormidium foveolarum that was specifically degraded by S1 nuclease was detected by Southern hybridization. This is also the case of the homologous plasmid pPB1 from Plectonema boryanum. These observations suggest that such small cryptic plasmids as pPF1 and pPB1, both from Gram-negative and filamentous cyanobacteria, replicate by a rolling circle mechanism in their living cells.
gamma-Aminobutyric acid (GABA)-like immunoreactive neuronal profiles and their synaptic relations in the area postrema of the rat were studied by ultrastructural immunocytochemistry using an antibody directed against GABA. GABA-like immunoreactive (GABA-LI) perikarya and dendrites usually received synaptic inputs from nonimmunoreactive axon terminals. These synaptic contact zones were mostly asymmetrical. Conversely, GABA-LI axon terminals were found to make synapses with nonimmunoreactive perikarya and dendrites, usually of the symmetrical type. Furthermore, GABA-LI axon terminals in synaptic contact with GABA-LI perikarya and dendrites were sometimes found. Although some GABA-LI axon terminals were in close vicinity of other nonimmunoreactive axon terminals, no clear synaptic contacts could be found. On the other hand, GABA-LI profiles could be found close to the capillaries, although they were always separated by a thin layer of glial processes. The above results suggest that the GABAergic neuron receives information from both the circulating blood and other neurons and then performs its depressor function through other neurons.
The membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell-cell adhesion protein identified by adhesion-blocking antibody fragments. Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell-cell adhesion protein. This study reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Although the anchor resistance to phosphatidylinositol-specific phospholipase C can be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol anchors, this was not the case. After a mild-base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected but, after strong acid hydrolysis, we detected both amide-linked fatty acids and a long-chain base. The long-chain base was further analysed by gas-chromatography/mass spectrometry and was found to be phytosphingosine. Both fatty acids and myo-inositol were also analysed by gas-chromatography/mass spectrometry. These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo-inositol.
In the present paper, the distribution of a neuron-specific phosphoneuroprotein 14 (PNP 14) in cell and tissue was investigated in detail by the immunoblot method using affinity-purified antibody against this protein. The immunoblot of the supernatant fractions of various tissue homogenates of rat clearly demonstrated that PNP 14 was enormously rich in the brain. The content in rat brain was as much as 0.1% of the homogenate. The immunocytochemical study showed that the protein was localized at nerve endings in the cerebellum. Existence of the protein was also confirmed in cultured neuronal cells from postnatal rat midbrain, but not in glial cells. Examination of subcellular localization of PNP 14 indicates that the protein was present in synaptic plasma membranes and synaptic supernatant fractions, but not in synaptic vesicles. During the development of rat brain, PNP 14 came into existence after birth and it's amount linearly increased to a maximum at 21-28 days after birth. The content of the protein then remained at the same level for more than 10 months. We concluded that this protein is neuron specific and supposed that it may be involved in neuronal formation and function.
The effect of prenatal X-irradiation on the ontogenesis of corticospinal tract (CST) neurons was examined in rats using retrograde labeling with Fast blue and intracellular Lucifer yellow staining. In prenatally irradiated rats, the cortical laminar architecture of the CST neurons was confused and many cells demonstrated migratory disturbances. Migratory-disordered CST neurons at deeper cortical levels resembled pyramidal cells, but their apical dendrites were oriented in various directions and the development of their dendrites was poor. Migratory-disordered CST neurons near the ependymal layer demonstrated round somata and many thin dendrites with spokewise radiation, suggesting a maturation disturbance. These results suggested that prenatal X-irradiation impeded the migration and maturation of CST neurons. These findings may form the basis for analyzing the mechanisms of radiation-induced mental retardation and behavioral changes.
A 64-kDa membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell/cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth-phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysylendopeptidase-digested peptides of gp64 were determined by protein analysis; all six asparagine-linked glycosylation sites (Asn-Xaa-Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins.
We characterized four monoclonal antibodies that recognize monomeric and/or tetrameric forms of Dictyostelium lectin (discoidin I) to study intracellular localization of this lectin in early development. Three different PAGEs (native-, urea-, and SDS-PAGE) following immunoblot showed that three of the four mAbs exhibit preference for the tetrameric form while mAb DC2 reacts only with unreduced monomer. By immunofluorescence studies of Dictyostelium NC-4, we found that the anti-tetramer antibodies mainly stain multilamellar bodies, which are food vacuoles to be externalized from cells. In contrast, mAb DC2 stains the cytosol weakly but not the multilamellar bodies. The same pattern of distribution was confirmed by immunoelectron microscopy. These results clearly indicate that, upon packaging discoidin I into multilamellar bodies, the tetrameric form is selectively packaged from the cytosolic pool. In addition, to clarify the relation between the multilamellar bodies and the tetramer, we examined the localization of tetramer using both the cells fed by Escherichia coli and the A3 cells axenically grown. When the cells were fed by E. coli, the cells made multilamellar bodies that contained no lectin; instead the tetramer was found in the cytoplasm and the cells still exclude the lectin around the cells. In contrast, A3 cells grown axenically without bacteria do not make multilamellar bodies but the tetramer also was found in the cytoplasm evenly (also, see W.F. Loomis, Dictyostelium Discoideum, pp. 160-161, Academic Press, New York, 1975). The data suggest that, under these conditions, the tetramer can be excluded from cells via some organelle such as normal secretory vacuole but not multilamellar bodies. Moreover this represents the first example of differential localization of tetramer and monomer of discoidin I in the cellular slime mold cells.