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Biomedical subjects

S Usami

Publications and source records attributed to S Usami.

At least 109 records · Page 6Linked to original sources
PubMed1995

Cold stability of pyruvate, orthophosphate dikinase of Flaveria brownii.

The nucleotide sequences of the complementary DNA of pyruvate, Pi dikinase (PPDK) from Flaveria bidentis, a C4 plant which possesses a cold-sensitive form of PPDK, and Flaveria brownii, a 'C4-like' plant which possesses a cold-tolerant form of PPDK, were determined. PPDK was isolated from the leaves of both Flaveria species and purified and the N-terminal amino acid sequences characterised. Together with a maize PPDK cDNA, cDNA inserts which code for the mature form of PPDK of F. bidentis and of F. brownii were expressed in bacteria and the cold sensitivity of the expressed PPDK studied. The cold sensitivity of the PPDK expressed in bacteria mimics the cold sensitivity of PPDK found in vivo in all three plant species. This study indicates that the cold sensitivity of plant PPDK is controlled by the primary structure of the enzyme.

Amino Acid Sequence↗
PubMed1995

Multiple cis-elements mediate shear stress-induced gene expression.

Fluid shear stress activates the expression of immediate early (IE) genes in vascular endothelial cells. The transcriptional regulation can be mediated through the shear stress-sensitive cis-acting elements at the 5' promoter regions of various IE genes such as the monocyte chemotactic protein-1 (MCP-1) gene. We linked wild-type and mutated MCP-1 promoters to the reporter gene luciferase and used such constructs to investigate the role of the phorbol ester TPA responsive element (TRE) in the shear-induced MCP-1 gene expression in vascular endothelial cells. Functional analysis showed that TGACTCC (a divergent TRE) located at nt -54 to -60 is necessary for shear-inducibility in bovine aortic endothelial cells (BAEC). The induction of the wild-type MCP-1 promoter construct by shear stress was attenuated by pretreating the cells with 1 microM dexamethasone or 1 microM retinoic acid 12 h before the shear stress experiments. The induction by shear stress reduced from 13-fold in the untreated cells to 7- and 3-folds in the dexamethasone- and retinoic acid-treated cells, respectively. These results demonstrate that the glucocorticoid receptor and retinoic acid receptor may interfere with the shear stress-activated AP-1/TRE. The reporter activity of HIV(LTR), which is a plasmid construct of the long terminal repeats of the human immunodeficiency virus and contains a kappa B enhancer element, was also activated by shear stress. The results of our investigations indicate that the shear stress-induced IE gene expression can be mediated through multiple cis-elements.

Animals↗
PubMed1995

Plant homologues of components of MAPK (mitogen-activated protein kinase) signal pathways in yeast and animal cells.

As they respond to numerous extracellular and intracellular stimuli, plants develop various morphological features and the capacity for a large variety of physiological processes during their growth. If we are to understand the molecular basis of such developments, we must elucidate the way in which signals generated by such stimuli can be transduced into plant cells and transmitted by cellular components to induce the appropriate terminal events. In yeast and animal systems, signal pathways that are known collectively as MAPK (mitogen-activated protein kinase) cascades have been shown to play a central role in the transmission of various signals. The components of these pathways include the MAPK family, the activator kinases of the MAPK family (the MAPKK family) and the activator kinases of the MAPKK family (the MAPKKK family). The members of each respective family are structurally conserved and signals are transmitted by similar phosphotransfer reactions at corresponding steps that are mediated by a specific member of each family in turn. Both cDNAs and genes that encode putative homologues of these components have recently been isolated from plant sources. Some of them have been shown to be related not only structurally but also functionally to members of the MAPK cascades of other organisms. These findings suggest that plants have signal pathways that are analogues to the MAPK cascades in yeast and animal cells but it remains to be proven that plant homologues do in fact constitute kinase cascades.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Mechanical strain induces monocyte chemotactic protein-1 gene expression in endothelial cells. Effects of mechanical strain on monocyte adhesion to endothelial cells.

Monocyte chemotactic protein-1 (MCP-1), a potent monocyte chemoattractant secreted by endothelial cells (ECs), is believed to play a key role in the early events of atherogenesis. Since vascular ECs are constantly subjected to mechanical stresses, we examined how cyclic strain affects the expression of the MCP-1 gene in human ECs grown on a flexible membrane base deformed by sinusoidal negative pressure (peak level, -16 kPa at 60 cycles per minute). Northern blot analysis demonstrated that the MCP-1 mRNA levels in ECs subjected to strain for 1, 5, or 24 hours were double those in control ECs (P < .05). This strain-induced increase was mainly serum independent, and MCP-1 mRNA level returned to its control basal level 3 hours after release of strain. Culture media from strained ECs contained approximately twice the MCP-1 concentration and more than twice the monocyte chemotactic activity of media from control ECs (P < .05). Pretreatment of collected media with anti-MCP-1 antibody suppressed such activity. Monocyte adhesion to ECs subjected to strain for 12 hours was 1.8-fold greater than adhesion to unstrained control ECs (P < .05). A protein kinase C inhibitor, calphostin C, abolished the strain-induced MCP-1 gene expression. In addition, cAMP- or cGMP-dependent protein kinase inhibitors (KT5720 and KT5823, respectively) partially inhibited such expression. Pretreatment with EGTA or the intracellular Ca2+ chelator BAPTA/AM strongly suppressed the strain-induced MCP-1 mRNA. Verapamil, a Ca2+ channel blocker, greatly reduced MCP-1 mRNA levels in both strained and unstrained ECs.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1995

Isolation of bacteria degrading carbazole under microaerobic conditions, i.e. nitrogen gas substituted conditions.

Microorganisms degrading carbazole (CA), a model substrate of heterocyclic nitrogen compounds in crude petroleum oil, were screened under microaerobic conditions, i.e. nitrogen gas substituted conditions. Eight bacteria were isolated and identified. For example, Bacillus sp. KUKK-4 degraded 31% of CA when cultivated for 28 days in a medium initially containing CA at 1000 mg/l with shaking under the microaerobic conditions.

Aerobiosis↗
PubMed1995

Expression of substance P, CGRP, and GABA in the vestibular periphery, with special reference to species differences.

The present study was conducted to elucidate species differences in the distribution of neuroactive substances, including substance P (SP), calcitonin gene-related peptide (CGRP) and gamma-aminobutyric acid (GABA), in the vestibular periphery of various animals (chicken, pigeon, rat, guinea pig and squirrel monkey). SP-like immunoreactivity was found in a subset of the primary vestibular afferents with no marked species differences in the staining pattern. In contrast, the localization of CGRP- and GABA-like immunoreactivities in the efferent nerve fibers varied according to species. This difference in the distribution pattern of neuroactive substances, found in the efferent system, may indicate that each species has a chemically (and probably functionally) distinct efferent system which is related to its specific environment and/or evolution.

Animals↗
PubMed1995

Neuroactive substances in the human vestibular end organs.

In order to evaluate the involvement of neuroactive substances in the human vestibular periphery, the immunocytochemical distribution of substance P (SP), calcitonin gene-related peptide (CGRP), and choline acetyltransferase (ChAT) was examined. SP-like immunoreactivity (LI) was present around and beneath sensory hair cells, probably corresponding to their afferent nerve endings. SP-LI was found predominantly in subpopulations of the primary afferents distributed in the peripheral region of the end organs. ChAT-LI and CGRP-LI were found throughout as small puncta below the hair cell layer, probably corresponding to efferent endings. The present results indicate that these neuroactive substances, previously described in animals, are also distributed in the human vestibular periphery, and almost certainly contribute to human vestibular function.

Calcitonin Gene-Related Peptide↗
PubMed1995

Observation of positional nystagmus with infrared CCD camera.

Using infrared CCD camera and electronystagmography we analyzed positional nystagmus in 33 patients with peripheral positional vertigo. Patients were classified into three types; (a) lateral type: nystagmus occurs in lateral position, (b) sagittal type: nystagmus occurs in head-hanging position (so-called benign paroxysmal positional vertigo, BPPV), and (c) mixed type. Nystagmus in the lateral type was horizontal and that in the sagittal type was rotatory. In all types, the vertigo and nystagmus began with a few seconds' latency after head tilt and briefly increased to a maximum, and then gradually decreased. The duration of horizontal nystagmus in the lateral and mixed type was significantly longer than that of rotatory nystagmus in the sagittal type mixed type. The mean value of the former was 90.6 s and that of the latter 18.1 s. We suggest that the velocity storage mechanism of the horizontal vestibulo-ocular reflex (VOR) is considerably different from that in the vertical VOR.

Adolescent↗
PubMed1994

Fluid shear stress induces a biphasic response of human monocyte chemotactic protein 1 gene expression in vascular endothelium.

The focal distribution of atherosclerotic lesions in the arterial tree is related to the local shear stress generated by blood flow, but the molecular basis of the atherogenic response of endothelial cells in these lesion-prone areas is still unclear. We report that shear stress mediates a biphasic response of monocyte chemotactic protein 1 (MCP-1) gene expression in vascular endothelial cells (EC). Northern blot analysis indicated that the level of MCP-1 mRNA in human umbilical vein EC (HUVEC) subjected to a shear stress of 16 dynes/cm2 (1 dyne = 10 microN) for 1.5 hr increased by 2- to 3-fold when compared with static cells. The MCP-1 gene expression decreased to the basal level at 4 hr and then declined further to become completely quiescent at 5 hr after the onset of shear. Once the gene expression was fully suppressed, it remained quiescent even after static incubation for 1.5 hr and would not respond to reshearing after this static incubation. However, if the postshearing incubation extended from 1.5 to 24 hr, the MCP-1 mRNA returned to the basal level and was then able to increase after the reapplication of shear stress. Nuclear run-on experiments showed that the shear-induced increased MCP-1 mRNA in HUVEC was regulated at the transcriptional level. By using cycloheximide, it was shown that de novo protein synthesis was not necessary for the induction of MCP-1 by shear stress. The biphasic response of MCP-1 gene expression was found in experiments in which the applied shear stress was 6, 16, or 32 dynes/cm2, and it was observed not only in HUVEC but also in HeLa cells, glioma cell lines, and skin fibroblasts. This in vitro study demonstrates that the response of MCP-1 gene to shear stress represents an immediate early gene activation and suggests that this gene is probably suppressed in EC that have been exposed to a constant shear stress.

Biomechanical Phenomena↗
PubMed1993

A simplified method for culture of endothelial cells and analysis of adhesion of blood cells under conditions of flow.

We have developed a simplified technique for culturing human umbilical vein endothelial cells under shear flow conditions, using prefabricated glass microcapillary tubes ("microslides") with a well-defined rectangular cross section and good optical quality. These microslides have been incorporated into a controlled flow system for quantitative video-microscopic analysis of the adhesion of blood cells to endothelial cells. Microslides were pretreated with 3-aminopropyltriethoxy-silane, or gelatin, and then loaded with a suspension of endothelial cells. After the cells had settled and attached to the substrate, the microslides were inserted into a flow-based culture system. Medium was drawn through them at intervals or continuously until confluency was reached (approximately 24 hr). Cells were cultured at wall shear stresses over a range 0.06 to 2.2 Pa. For adhesion assays, the endothelialized microslides were attached to microscope slides, and suspensions of blood cells were drawn through at desired wall shear stresses (0.02-0.5 Pa). Adhesion of malarial-infected red blood cells and of neutrophilic granulocytes was quantitated by direct microscopic observation. The adhesive behavior of both cell types closely resembled that previously described by ourselves and others using flow chambers incorporating endothelial-coated glass coverslips. The use of microslides represents a significant simplification of methodology for endothelial cell growth and adhesion studies under flow conditions.

Blood Cells↗
PubMed1993

Design and construction of a linear shear stress flow chamber.

A new parallel plate flow chamber that has a linear variation of shear stress, starting from a predetermined maximum value at the entrance and falling to zero at the exit, has been designed and tested. This is in contrast to the usual rectangular channel plan which produces a constant shear stress over the entire length. The new design is based on the theory of Hele-Shaw flow between parallel plates. To verify the efficacy of the flow channel, the effect of fluid shear stress on platelet adhesion to a fibrinogen-coated glass surface was tested. The percentage of attached platelets after 5 min of shear stress is shown to be a function of shear stress. With this new flow chamber, cell-cell interactions can be studied efficiently over a wide range of shear stress using a single run at constant discharge.

Endothelium, Vascular↗
PubMed1993

Active motion of polymorphonuclear leukocytes in response to chemoattractant in a micropipette.

A novel experimental method of producing and observing the active motion of polymorphonuclear leukocytes (PMNs) using a micropipette technique has been recently developed (Usami et al., 1992). The present paper develops a quantitative theory for the chemoattractant gradients and cell locomotion observed in these experiments. In previous experimental methods (e.g., the Boyden chamber, the Zygmond chamber and the Dunn chamber) for study chemotaxis of leukocytes, fibroblasts, and PMNs, the exact nature of the concentration gradient of the chemoattractant is unknown. The cells may themselves modify the local gradient of the chemoattractant. In experiments using the micropipette, an internal source of chemoattractant provides well-defined boundary and initial conditions which allow the computation of the chemoattractant concentration gradient during the active locomotion of the PMNs. Since the cell completely fills the pipette lumen, convection is limited to the motion of the cells themselves. In coordinates moving with cell, it is assumed that diffusion is the only mechanism of mass transport of the chemoattractant (fMLP). Computations of the fMLP concentration during locomotion of the cell were carried out for a range of rates of fMLP binding by the receptors expressed on the front face of the cell membrane. The results show that the front face of the cell is subjected to increasing fMLP concentration during the cell motion. The sequence of events involve receptor binding of fMLP, signal transduction, polymerization of the cell cytoskeleton at the membrane of the front face, spatially dependent adhesion to the pipette wall, and localized contraction of the cytoskeleton. This sequence of events leads to the steady locomotion of the leukocytes in the micropipette. The computation of the distribution of the fMLP concentration during cell locomotion with constant velocity in micropipette experiments shows that the cell is exposed to increasing concentration of fMLP. This suggests that chemotaxis maybe induced by temporal gradient of an attractant.

Cell Movement↗
PubMed1993

Sustained increase in adrenergic activity in gerbil striatum following transient ischemia.

We investigated the changes in striatal monoaminergic functions, focusing on the release and metabolism, in a cerebral ischemic model induced by a 5-min bilateral occlusion of the carotid arteries (BOCA) and reperfusion in anesthetized gerbils. In the microdialysis study, the striatal extracellular level of dopamine (DA) markedly increased (144-fold) immediately after BOCA. Although norepinephrine (NE) and 5-hydroxytryptamine (5-HT) could not be detected in the dialysates throughout the baseline period, they increased to detectable levels after BOCA. On the contrary, the tissue contents of NE and 5-HT decreased or tended to decrease up to 4 hr following reperfusion. Striatal DA contents did not show any changes in the early period after ischemia-reperfusion and slightly increased at 4 hr or later. Tissue contents of 3-methoxytyramine (3-MT), a metabolite of DA by catechol-O-methyltransferase (COMT), increased 0 and 5 min after reperfusion. Normethanephrine (NMN), which is a metabolite of NE by COMT, also increased not only 5 min after but also up to 4 hr after ischemia-reperfusion, indicating a sustained increase in NE release. These results suggested that the neuronal activity of NE, which is supposed to exert a protective effect on ischemic damage, was enhanced for a longer period than that of DA after transient ischemia.

Animals↗
PubMed1993

Immunocytochemical localization of substance P and neurofilament proteins in the guinea pig vestibular ganglion.

To examine the cytochemical features of primary vestibular afferent neurons, we examined the immunocytochemical distribution of substance P(SP) and neurofilament proteins (NFP) in the guinea pig vestibular endorgans and ganglion. 84.9% of vestibular ganglion cells showed SP-like immunoreactivity. SP immunoreactive cells were small in diameter (25.4 microns), and randomly distributed in the ganglia. NFP immunoreactive cells (34.2%) were relatively large in diameter (31.1 microns). By double-staining immunocytochemistry it was possible to distinguish three different cell types: SP-positive/NFP-negative, SP-negative/NFP-positive, and SP/NFP-positive vestibular ganglion cells. Vestibular ganglion cells can thus be classified on the basis of size and immunocytochemical characteristics. These chemically distinct subpopulations of vestibular ganglion cells may be an indication of functional differences within vestibular afferent neurons.

Animals↗
PubMed1993

Immunocytochemical localization of intermediate filaments in the guinea pig vestibular periphery with special reference to their alteration after ototoxic drug administration.

The present study examined the immunocytochemical localization of various intermediate filaments (IFs), 68 kDa, 160 kDa and 200 kDa neurofilament protein (NFP), cytokeratin (CK) 1, 8, 10 and 19, vimentin, and glial fibrillary acidic protein (GFAP) in the vestibular end-organs and ganglia of normal and streptomycin-treated guinea pigs. In normal animals, 68 kDa, 160 kDa and 200 kDa NFP were found in afferent nerve fibers and nerve terminals (probably nerve chalices). Fine nerve fibers (probably efferent and/or sympathetic nerve fibers) were also immunoreactive to NFP. In the vestibular ganglia, 68 kDa and 160 kDa NFP were predominantly distributed in larger cells, whereas 200 kDa NFP was also found in some small ganglion cells. Cytokeratin 8 and 19 were located in supporting cells, transitional cells, dark cells of vestibular end-organs, and the epithelial cell lining of the membranous labyrinth. Vimentin was observed in the hair cells distributed in the central region of the end organs, supporting cells, most connective tissue cells, and Schwann cells of the vestibular ganglion. Although GFAP-like immunoreactivity was evident in glial cells of the proximal vestibular nerve, no immunoreactivity was detected in the distal portion of the vestibular nerve, vestibular ganglion, or vestibular end-organs. These highly distinct staining patterns of IFs indicated that they may play different roles in the different cell types, and that they may serve as a specific marker for each cell type. In streptomycin-treated guinea pigs, immunoreactivities for NFP and vimentin (found in the hair cells) decreased after treatment, whereas immunoreactivities for the other IFs were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

[Lipid peroxide and free radical scavengers in congenital heart disease with pulmonary hypertension].

There is a strong possibility that lipid peroxide (LPO) exerts a great influence on the persistence of pulmonary vascular obstruction (PVO) after radical operation of congenital heart disease with pulmonary hypertension (PH). We investigated the relationship between LOP and PVO, and discussed the effects of scavengers. Fourteen cases of infantile open heart surgery were investigated. LPO, superoxide dismutase SOD and reduced glutathione (GSH) were measured in blood and lung tissue. In the cases of PH group, the levels of lung tissue and plasma LPO showed significantly higher than those of PS group (p < 0.05) before and after radical operation. The levels of Pp/Ps and pulmonary vascular resistance (PVR) of PH cases showed still higher than those of PS group (p < 0.05) even after radical operation. In addition, the levels of plasma and lung tissue SOD, lung tissue GSH of PH cases were lower than those of PG group. These suggest that LPO plays an important role as the cause of PVO before operation and which remains unchanged even after operation. It is to be expected that increase of the free radical scavengers will be effective to suppress the generation of LPO and at last to reduce the level of PVR after operation.

Free Radical Scavengers↗
PubMed1993

Mechanical models of pseudopod formation.

The active locomotion of polymorphonuclear leukocytes into a glass pipette has been recently reported using formyl-methionyl-leucyl-phenylalanine (fMLP) as a chemoattractant. The frontal portion of the leukocyte appears clear and free of granules as observed in pseudopod formation. Three possible mechanisms for pseudopod formation are considered: (1) pressure-flow generated by actin-myosin contraction at the rear of the cell or at the base of the pseudopod; (2) osmotic pressure generated at the cell membrane, interior to the cell; and (3) actin polymerization of the cell membrane at the leading edge of the pseudopod. Experimental data on the movement of F-actin toward the rear of the cell, away from the front, favors polymerization of G-actin to F-actin at the leading edge. The active role of osmotic pressure and contraction at the base of the pseudopod are possible but not yet proven.

Actins↗