Showing papers by "Osaka University published in 1987"

Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.

Abstract: The iap gene in Escherichia coli is responsible for the isozyme conversion of alkaline phosphatase. We analyzed the 1,664-nucleotide sequence of a chromosomal DNA segment that contained the iap gene and its flanking regions. The predicted iap product contained 345 amino acids with an estimated molecular weight of 37,919. The 24-amino-acid sequence at the amino terminus showed features characteristic of a signal peptide. Two proteins of different sizes were identified by the maxicell method, one corresponding to the Iap protein and the other corresponding to the processed product without the signal peptide. Neither the isozyme-converting activity nor labeled Iap proteins were detected in the osmotic-shock fluid of cells carrying a multicopy iap plasmid. The Iap protein seems to be associated with the membrane.

A hierarchical neural-network model for control and learning of voluntary movement

Abstract: In order to control voluntary movements, the central nervous system (CNS) must solve the following three computational problems at different levels: the determination of a desired trajectory in the visual coordinates, the transformation of its coordinates to the body coordinates and the generation of motor command. Based on physiological knowledge and previous models, we propose a hierarchical neural network model which accounts for the generation of motor command. In our model the association cortex provides the motor cortex with the desired trajectory in the body coordinates, where the motor command is then calculated by means of long-loop sensory feedback. Within the spinocerebellum — magnocellular red nucleus system, an internal neural model of the dynamics of the musculoskeletal system is acquired with practice, because of the heterosynaptic plasticity, while monitoring the motor command and the results of movement. Internal feedback control with this dynamical model updates the motor command by predicting a possible error of movement. Within the cerebrocerebellum — parvocellular red nucleus system, an internal neural model of the inverse-dynamics of the musculo-skeletal system is acquired while monitoring the desired trajectory and the motor command. The inverse-dynamics model substitutes for other brain regions in the complex computation of the motor command. The dynamics and the inverse-dynamics models are realized by a parallel distributed neural network, which comprises many sub-systems computing various nonlinear transformations of input signals and a neuron with heterosynaptic plasticity (that is, changes of synaptic weights are assumed proportional to a product of two kinds of synaptic inputs). Control and learning performance of the model was investigated by computer simulation, in which a robotic manipulator was used as a controlled system, with the following results: (1) Both the dynamics and the inverse-dynamics models were acquired during control of movements. (2) As motor learning proceeded, the inverse-dynamics model gradually took the place of external feedback as the main controller. Concomitantly, overall control performance became much better. (3) Once the neural network model learned to control some movement, it could control quite different and faster movements. (4) The neural netowrk model worked well even when only very limited information about the fundamental dynamical structure of the controlled system was available. Consequently, the model not only accounts for the learning and control capability of the CNS, but also provides a promising parallel-distributed control scheme for a large-scale complex object whose dynamics are only partially known.

Contribution of intra-abdominal fat accumulation to the impairment of glucose and lipid metabolism in human obesity

Abstract: The casual relationship between intraabdominal visceral fat accumulation and metabolic disorders was analyzed in 46 obese subjects (15 males, 31 females) having 34.1 +/- 5.5 of body mass index (BMI). The distribution of fat was determined by our CT scanning technique (Int J Obesity 7:437, 1983). The total cross-cut area, subcutaneous fat area, and intra-abdominal fat area was measured at the umbilical level. The fasting plasma glucose level, area under the plasma glucose concentration curve after oral glucose loading (plasma glucose area), fasting serum triglyceride level, and serum total cholesterol level were all significantly higher or otherwise greater in the group with intraabdominal visceral fat to subcutaneous fat ratio (V/S ratio) of not less than 0.4 than in the group with a lower V/S ratio, when either all or sex-matched obese subjects were examined, though BMI or the duration of obesity was not different between the two groups. The V/S ratio was significantly correlated with the level of plasma glucose area (r = 0.45, P less than .001) under the curve of 75 g oral glucose tolerance test and also with the serum triglyceride (r = 0.65, P less than .001) and total cholesterol levels (r = 0.61, P less than .001). These relationships were also observed when examined in each sex separately and found to be significant after adjustment for BMI and age by multiple regression analyses.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure and expression of human B cell stimulatory factor-2 (BSF-2/IL-6) gene.

Abstract: The chromosomal DNA segment of human B cell stimulatory factor-2 (BSF-2/IL-6) was isolated and characterized by nucleotide sequence analysis. The human BSF-2/IL-6 gene consists of five exons and four introns and its organization shows a distinctive similarity to granulocyte colony-stimulating factor gene. The two genes have the same number of exons and introns and the size of each exon is strikingly similar. The BSF-2/IL-6 mRNA was found to be constitutively expressed in a human T cell leukemia virus-1 transformed T cell line, TCL-Na1, a bladder cell carcinoma line, T24, and an amnion derived cell line, FL. The BSF-2/IL-6 mRNA was also found to be inducible with interleukin-1 beta in an astrocytoma line, U373 and a glioblastoma line, SK-MG-4. S1 mapping and primer extension analyses showed the presence of multiple initiation sites and the preferential utilization of a different initiation site for each individual tissue tested.

Rheumatoid factor secretion from human Leu-1+ B cells

Abstract: A human B cell subpopulation identifiable by the expression of the cell surface antigen Leu-1 (CD5) is responsible for most of the immunoglobulin M rheumatoid factor secreted in vitro after the cells are stimulated with Staphylococcus aureus. The ability of B cells bearing the Leu-1 marker (Leu-1+) to secrete rheumatoid factor is present early in development and extends to adulthood, since Leu-1+ B cells from cord blood and from peripheral blood lymphocytes of both normal adults and patients with certain autoimmune conditions secrete rheumatoid factor in comparable amounts. The neonatal enrichment of Leu-1+ B cells, the presence of Leu-1+ B cells in increased frequencies in patients with autoimmune disease, and the involvement of Leu-1+ B cells in autoantibody secretion suggest both developmental and functional homologies between this human B cell subpopulation and the murine Ly-1 B cell subpopulation.

Human B-cell differentiation factor defined by an anti-peptide antibody and its possible role in autoantibody production

Abstract: The partial amino acid sequence of the NH2 terminus of a factor named human B-cell differentiation factor or B-cell stimulatory factor 2 (BSF-2) has been determined. Antibodies raised against the synthetic peptide corresponding to residues 1-13 of the NH2-terminal sequence specifically react with BSF-2 generated by a T-cell line and by phytohemagglutinin-stimulated normal T cells. Furthermore, the antipeptide antibodies react with a BSF-2-like factor produced by cardiac myxoma as well as uterine cervical carcinoma cells. The results show that BSF-2 functions in vivo as well and suggest that the constitutive production of BSF-2 may be involved in autoantibody production, since patients with cardiac myxoma and uterine carcinoma showed autoantibody production.

NMDA receptors in the visual cortex of young kittens are more effective than those of adult cats.

Abstract: Acidic amino acids, such as glutamate and aspartate, are thought to be excitatory transmitters in the cerebral neocortex and hippocampus1–8. Receptors for these amino acids can be classified into at least three types on the basis of their agonists. Quisqualate-preferring receptors and kainate-preferring receptors are implicated in the mediation of synaptic transmission in many regions including the hippocampus9,10 and visual cortex11, whereas N-methyl-D-aspartate (NMDA)-preferring receptors are thought to be involved in modulating synaptic efficacy, for example in long-term potentiation, a form of synaptic plasticity in the hippocampus12–14. In the visual cortex of the cat and monkey, it is well established that synaptic plasticity, estimated by susceptibility of binocular responsiveness of cortical neurons to monocular visual deprivation, disappears after the 'critical' period of postnatal development15–17. Here we report that during the critical period in young kittens, a selective NMDA-receptor antagonist blocks visual responses of cortical neurons much more effectively than it does in the adult cat. This suggests that NMDA receptors may be involved in establishing synaptic plasticity in the kitten visual cortex.

Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice

Abstract: The NOD (non-obese diabetic) mouse spontaneously develops insulin-dependent diabetes mellitus (IDDM) characterized by autoimmune insulitis, involving lymphocytic infiltration around and into the islets followed by pancreatic beta (beta) cell destruction, similar to human IDDM. Genetic analysis in breeding studies between NOD and C57BL/6 mice has demonstrated that two recessive genes on independent chromosomes contribute to the development of insulitis. One of the two recessive diabetogenic genes was found to be linked to the major histocompatibility complex (MHC). This is of interest, because the NOD strain has a unique class II MHC: it does not express I-E molecules as no messenger RNA for the alpha-chain of I-E is visible in Northern blot analysis; I-A molecules are not detected with any available monoclonal antibodies or by allo-reactive or autoreactive T-cell clones, although their expression is demonstrated with a conventional antiserum to Ia antigens. To examine whether the unusual expression of class II MHC molecules may be responsible for the development of autoimmune insulitis, we attempted to express I-E molecules in NOD mice selectively, without introducing other genes on chromosome 17 by using I-E-expressing C57BL/6 (B6(E alpha d)) transgenic mice. We report here that the expression of I-E molecules in NOD mice can prevent the development of autoimmune insulitis.

Receptors for B cell stimulatory factor 2. Quantitation, specificity, distribution, and regulation of their expression

Abstract: B cell stimulatory factor 2 receptors (BSF-2-R) were studied using radioiodinated recombinant BSF-2 with a specific activity of 6.16 X 10(13) cpm/g. Kinetic studies showed that binding of 125I-BSF-2 to CESS cells reached maximum level within 150 min at 0 degrees C. There was a single class of receptors with high affinity (Kd 3.4 X 10(-10) M) on CESS, and the number of receptors was 2,700 per cell. Binding of 125I-BSF-2 to CESS was competitively inhibited by unlabeled BSF-2 but not by IL-1, IL-2, IFN-beta, IFN-gamma, and G-CSF, indicating the presence of the receptors specific for BSF-2. EBV-transformed B lymphoblastoid cell lines (CESS, SKW6-CL4, LCL13, and LCL14) expressed BSF-2-R, whereas Burkitt's lines did not. EBV or EBNA2 did not induce the expression of the receptors on Burkitt's cells. The plasma cell lines (ARH-77 and U266) expressed BSF-2-R, fitting the function of BSF-2 as plasma cell growth factor. Several other cell lines, the histiocytic line U937, the promyelocytic line HL60, the astrocytoma line U373 and the glioblastoma line SK-MG-4, in which BSF-2 was inducible with IL-1 or TPA, displayed BSF-2-R with Kd in the range of 1.3-6.4 X 10(-10) M, suggesting the autocrine mechanism in BSF-2 function. The four T cell lines (CEM, HSB, Jurkat, and OM 1) did not express a detectable number of receptors, but normal resting T cells expressed 100-1,000 receptors per cell. BSF-2-R were not present on normal resting B cells but expressed on activated B cells with a Kd of 3.6-5.0 X 10(-10) M, fitting the function of BSF-2, which acts on B cells at the final maturation stage to induce immunoglobulin production.

Sliding movement of single actin filaments on one-headed myosin filaments

Abstract: The myosin molecule consists of two heads, each of which contains an enzymatic active site and an actin-binding site. The fundamental problem of whether the two heads function independently or cooperatively during muscle contraction has been studied by methods using an actomyosin thread, superprecipitation and chemical modification of muscle fibres. No clear conclusion has yet been reached. We have approached this question using an assay system in which sliding movements of fluorescently labelled single actin filaments along myosin filaments can be observed directly. Here, we report direct measurement of the sliding of single actin filaments along one-headed myosin filaments in which the density of heads was varied over a wide range. Our results show that cooperative interaction between the two heads of myosin is not essential for inducing the sliding movement of actin filaments.

Interleukin 4 as an essential factor for in vitro clonal growth of murine connective tissue-type mast cells.

Abstract: We investigated the biological activity of IL-4 to murine connective tissue-type mast cells (CTMC). When purified peritoneal mast cells, typical CTMC, were incubated with pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) in methylcellulose, about one-fifth of mast cells showed clonal growth. Recombinant IL-4 alone did not stimulate the clonal growth, and purified IL-3 alone induced development of a small number of tiny clusters. In contrast, addition of IL-4 to IL-3 increased the number of clusters by a factor of 10. The number and size of clusters induced by the combination of IL-3 and IL-4 were comparable to those of mast cell clusters induced by PWM-SCM. The present results indicate that IL-4 is an essential factor for in vitro clonal growth of CTMC.

A functional role of cholinergic innervation to neurons in the cat visual cortex.

Abstract: 1. Effects of microionophoretic application of acetylcholine (ACh) and its antagonists on neuronal responses to visual stimuli and to electrical stimulation of the lateral geniculate nucleus were studied in the cat striate cortex. 2. Responses elicited visually and electrically were facilitated by ACh in 74% of the cells tested, whereas the responses were suppressed in 16%. These ACh effects were blocked by a muscarinic antagonist, atropine, but not by a nicotinic antagonist, hexamethonium, indicating that the ACh effects are mediated through muscarinic receptors. A single application of atropine suppressed visual responses of cells facilitated by ACh, whereas it enhanced those of cells inhibited by ACh, suggesting that endogenous ACh may tonically modulate visual responsivity of cortical neurons. 3. In most cells with the facilitatory ACh effect, responses with single spikes to the electrical stimulation became more consistent, often with double spikes, during the ACh application. The suppressive effects of ACh were noted most often in cells with a longer response latency to electrical stimulation of lateral geniculate nucleus. 4. In most of the facilitated cells the spontaneous activity remained null or very low during ACh application, in spite of marked enhancement of visual responses, suggesting that ACh may improve the signal-to-noise ratio (S/N) of cortical neuron activity. To confirm this suggestion, we calculated a S/S + N index by counting the total number of spikes in the responses (S) and that in peristimulus time histogram (S + N) and found that it was improved during the ACh application in about a half of the cells, whereas it became worse in about one-fifth. 5. In most of the facilitated cells, ACh enhanced visual responses not only to optimal but also to nonoptimal stimuli, resulting in no improvement or even worsening of the orientation selectivity. This was also the case in the selectivity of direction of stimulus movement. 6. The laminar location of the facilitated cells was biased toward layers V and VI of the cortex, although they also made up the majority in layers II + III and about half the tested cells in layers IVab and IVc. 7. In the light of recent understanding of cortical circuitry, these results suggest that the cholinergic innervation to cortical neurons may play a role in improvement of the S/N ratio of information processing in the striate cortex and in facilitation of sending processed informations to other visual centers.

Stochastic random network model in Ge and Si chalcogenide glasses.

Abstract: A stochastic random network model is proposed for the structure of ${\mathrm{Ge}}_{\mathrm{x}}$${\mathrm{S}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$, ${\mathrm{Ge}}_{\mathrm{x}}$${\mathrm{Se}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$, ${\mathrm{Si}}_{\mathrm{x}}$${\mathrm{S}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$, and ${\mathrm{Si}}_{\mathrm{x}}$${\mathrm{Se}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$ (x\ensuremath{\le}0.33) glasses. This model is constructed to explain the existence of two types of microcrystalline states induced by photoirradiation above or below the threshold intensity. This model characterizes the glass structure by one parameter P which is related to the existing probability of the edge-sharing bonds between the tetrahedral ${\mathrm{MX}}_{4}$ molecules relative to the corner-sharing bonds. P depends only on the species of atoms forming the glass and not on x. In order to prove the validity of the present model, Raman scattering experiments were made and the x dependence of the intensity ratio of the ${A}_{1}^{c}$ companion peak to the ${A}_{1}$ peak, I(${A}_{1}^{c}$)/I(${A}_{1}$), was obtained. From the viewpoint of phonon localization, the ${A}_{1}$ mode is assigned to the breathing mode of ${\mathrm{MX}}_{4}$ molecules and the ${A}_{1}^{c}$ mode to the vibration of chalcogen atoms on the edge-sharing double bonds. The x dependence of the intensity ratio I(${A}_{1}^{c}$)/I(${A}_{1}$) calculated by the present model is in good agreement with the experimentally obtained ratio. The P obtained increases in order from ${\mathrm{Ge}}_{\mathrm{x}}$${\mathrm{S}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$, ${\mathrm{Ge}}_{\mathrm{x}}$${\mathrm{Se}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$, ${\mathrm{Si}}_{\mathrm{x}}$${\mathrm{Se}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$ to ${\mathrm{Si}}_{\mathrm{x}}$${\mathrm{S}}_{1\mathrm{\ensuremath{-}}\mathrm{x}}$ with the same order of tendency of getting edge-sharing bonds in the crystals. The value of P is independent of the method of making the amorphous but it can be changed by photoirradiation. P decreases with irradiation below the threshold intensity, but it increases with irradiation above the threshold. The local energy in the glass is lower in the corner-sharing bonds, but the total energy is lowest in the same structure as the crystal. The threshold irradiation intensity for Se glass is less than one-hundredth of that for ${\mathrm{GeSe}}_{2}$ glass.

Stable expression and replication of hepatitis B virus genome in an integrated state in a human hepatoma cell line transfected with the cloned viral DNA.

Abstract: A human hepatocellular carcinoma cell line (Huh6-c15) was transfected with a recombinant DNA molecule that consists of tandemly arranged hepatitis B virus (HBV) genome and a neomycin-resistant gene. One clone resistant to G-418 produces and releases surface antigen and e antigen into medium at a high level and accumulates core particles intracellularly. This clone has a chromosomally integrated set of the original recombinant DNA and produces a 3.5-kilobase transcript corresponding to the pregenome RNA as well as HBV DNAs in an extrachromosomal form. Most of these DNAs were in single-stranded or partially double-stranded form and were packaged in the intracellular core particles. In the medium, particles were detected that contained HBV DNA and were morphologically indistinguishable from Dane particles. These results demonstrate that the HBV genome in an integrated state acted as a template for viral gene expression and replication. The cells were maintained for more than 6 months without losing the ability to produce the extrachromosomal HBV DNA and Dane-like particles. Thus, the cells can be used as a model system for analyses of gene expression and DNA replication of HBV in human hepatocytes.

A short amino-terminal segment of microsomal cytochrome P-450 functions both as an insertion signal and as a stop−transfer sequence

Abstract: Co-translational insertion of liver microsomal cytochrome P-450 into the endoplasmic reticulum membrane is mediated by the signal recognition particle (SRP) and the presence in the cytochrome molecule of a signal sequence that can be recognized by SRP has been postulated. To locate this signal sequence, six hybrid cDNAs were constructed in which various segments of a cDNA for a rabbit liver cytochrome P-450 are fused with a cDNA or its fragment encoding yeast porin (an outer mitochondrial membrane protein) or with a cDNA for pre-interleukin 2 (a secretory protein) from which the 5'-terminal portion encoding most of its signal sequence had been removed. These hybrid cDNAs were inserted into an SP-6 transcription vector and transcribed in vitro. The mRNAs thus synthesized were translated in a cell-free system in the presence of rough microsomes. It was thus found that only those chimeric proteins containing (at their amino-terminal end) the amino-terminal cytochrome P-450 segments consisting of greater than or equal to 29 amino acid residues were co-translationally inserted into the membrane in an SRP-dependent fashion. These proteins were, however, neither processed nor translocated across the membrane. These findings, coupled with the observation that the major portion of these proteins, when inserted into the membrane, was degraded by trypsin, led to the conclusion that a short amino-terminal segment (less than 29 residues) of the cytochrome P-450 functions not only as an insertion signal but also as a stop-transfer sequence. This segment is, therefore, similar to the internal signal of type II plasma membrane proteins, but differs from the latter in the topogenic function.

Search for microbial insect growth regulators

Abstract: Allosamidin, a novel insect chitinase inhibitor, was isolated from the mycelium of Streptomyces sp. It showed strong inhibitory activity against the chitinases of the silkworm, Bombyx mori, in vitro, and also insecticidal activity by preventing its ecdysis in vivo.

The mode of hepatitis B virus DNA integration in chromosomes of human hepatocellular carcinoma.

Abstract: Nineteen DNA samples that carry integrated hepatitis B virus (HBV) DNA were isolated from seven independent human hepatomas by molecular cloning, and their structures were determined. The results, combined with reported data, were analyzed so that one can obtain insights into the mechanisms of integration of this virus DNA and possible rearrangements that occur subsequently. The distribution of DNA junctions along the virus genome suggests that there are recombination-proficient regions. Thus, about half of the integrants were the Coh type, viz., one of their virus-cell DNA junctions fell within the so-called cohesive end region that lies between two 11-bp direct repeats (DR1 and DR2) in the virus genome where transcription and replication of the genome are initiated. All the integrated virus genomes were defective at least in one site around the cohesive end region, particularly within the X gene. The recombination-proficient regions are used not only for formation of virus-cell but also of virus-virus junctions. Neither virus nor cell DNA show unique sequences at the junctions, and targets for integration lie on many different chromosomes.

Specific intermediate-valence state of insulating 4f compounds detected by L3 x-ray absorption.

Abstract: The intermediate valence of formally tetravalent compounds has been detected by ${L}_{3}$ x-ray-absorption near-edge structure (XANES) in ${\mathrm{CeO}}_{2}$ and in ${\mathrm{PrO}}_{2}$ but not in ${\mathrm{UO}}_{2}$, which have the same ${\mathrm{CaF}}_{2}$ structure and large f and ligand mixing. The intermediate valence has been found both in ${\mathrm{CeO}}_{2}$ and in Ce(${\mathrm{SO}}_{4}$${)}_{2}$\ensuremath{\cdot}${4\mathrm{H}}_{2}$O, which have similar local structure but different crystal structure. We show that ${L}_{3}$ XANES final states are a direct probe of configuration interaction between 4${f}^{n}$ and 4${f}^{n+1}$L configurations in the ground state and that the weight of the 4${f}^{n+1}$L in the ground state can be deduced. The many-body final states arise from the characteristic properties of these materials: (i) the presence of localized 4f level above the oxygen 2p band separated by a gap \ensuremath{\delta}\ensuremath{\varepsilon} with relevant correlation energy ${U}_{\mathrm{ff}}$ (${U}_{\mathrm{ff}}$\ensuremath{\ge}\ensuremath{\delta}\ensuremath{\varepsilon}) and (ii) mixing of 4f localized states with ligand valence orbitals such that the hybridization energy V is of the same order of magnitude as the energy separation \ensuremath{\Delta}E between the many-body configuration 4${f}^{n}$ and 4${f}^{n+1}$L (V\ensuremath{\ge}\ensuremath{\Delta}E). These insulating materials, which cannot be classified as standard mixed-valence systems, are called here interatomic intermediate-valence systems.

Plastic deformation of the intermetallic compound Al3Ti

Abstract: Polycrystalline specimens of the intermetallic compound Al3Ti with the D022-type structure have been deformed under compression at 25–860°C. At temperatures below 620°C, specimens fracture with very small fracture strain since extremely localized deformation occurs and cracks are initiated in intensely deformed regions almost at yielding. At temperatures above 620°C, good compression ductility is observed. Yield stress starts decreasing rapidly with increasing temperature at around 330°C and becomes less temperature dependent above 630°C. The major mode of deformation of Al3Ti is twinning of the type (111)[112] which does not disturb the D022 symmetry of the lattice. However, at high temperatures, the four (111)[112]-type twinning systems are augmented by slip of the types [110], [100] and [010]. This would supply an explanation for the good compression ductility observed at temperatures above 620°C. Suggestions are made for further experimental work on alloying additions which may improve the du...