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Biomedical subjects

T Ohtsuki

Publications and source records attributed to T Ohtsuki.

At least 145 records · Page 8Linked to original sources
PubMed1994

A new gerbil model of hindbrain ischemia by extracranial occlusion of the bilateral vertebral arteries.

A new gerbil model of hindbrain ischemia was induced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Carbon black studies, performed at 5 min after occlusion, revealed that the pons-medulla oblongata, and the cerebellum were quite ischemic in all animals. Cardiovascular changes in mean arterial blood pressure (MABP) and heart rate were recorded until 30 min after occlusion, and revealed that the typical cerebral ischemic response (i.e., abrupt increase in MABP, bradycardia, and apnea) was elicited in all animals (n = 10). Thirty minutes after occlusion, animals (n = 4) were decapitated and immersion-fixed. Brain sections were stained with hematoxylin-eosin (HE) and also immunostained for microtubule-associated protein 2 in order to evaluate ischemic neuronal damage from 30 min of ischemia. By HE staining, ischemic lesions were detected bilaterally in the oculomotor, the trigeminal motor, the lateral vestibular, and the cerebellar interpositus nucleus. In addition, immunostaining revealed ischemic lesions in several other hindbrain areas. In conclusion, we could successfully establish a new gerbil model of hindbrain ischemia. Carbon black perfusion and hemodynamic studies revealed that severe and reproducible hindbrain ischemia was produced. By histopathological examination, we could also clearly demonstrate symmetrical ischemic lesions in several hindbrain areas.

Animals↗
PubMed1994

Metabolic and biosynthetic alterations in cultured astrocytes exposed to hypoxia/reoxygenation.

To investigate the astrocyte response to hypoxia/reoxygenation, as a model relevant to the pathogenesis of ischemic injury, cultured rat astrocytes were exposed to hypoxia. On restoration of astrocytes to normoxia, there was a dramatic increase in protein synthesis within 3 h, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of metabolically labeled astrocyte lysates showed multiple induced bands on fluorograms. Levels of cellular ATP declined during the first 3 h of reoxygenation and the concentration of AMP increased to approximately 3.6 nmol/mg of protein within 1 h of reoxygenation. Reoxygenated astrocytes generated oxygen free radicals early after replacement into ambient air, and addition of diphenyliodonium, an NADPH oxidase inhibitor, diminished the generation of free radicals as well as the induction of several bands on fluorogram. Although addition of cycloheximide on reoxygenation resulted in inhibition of both astrocyte protein synthesis and accumulation of cellular AMP, it caused cell death within 6 h, suggesting the importance of protein synthesis in adaptation of hypoxic astrocytes to reoxygenation. Potential physiologic significance of biosynthetic products of astrocytes in hypoxia/reoxygenation was suggested by the recovery of glutamate uptake. These results indicate that the astrocyte response to hypoxia/reoxygenation includes generation of oxygen free radicals and de novo synthesis of products that influence cell viability and function in ischemia.

Adenosine Monophosphate↗
PubMed1994

Proteoglycan form of macrophage colony-stimulating factor binds low density lipoprotein.

We recently isolated a proteoglycan form of macrophage colony-stimulating factor (PG-M-CSF) that carries a chondroitin sulfate glycosaminoglycan chain. Here, we examined the interaction of PG-M-CSF with low density lipoprotein (LDL). When LDL preincubated with PG-M-CSF was fractionated by molecular size sieving chromatography, it was eluted earlier than untreated LDL. When LDL was preincubated with chondroitin sulfate-free 85-kD M-CSF instead of PG-M-CSF, the elution profile of LDL remained unchanged, indicating specific interaction between PG-M-CSF and LDL. The level of PG-M-CSF binding in the wells of a plastic microtitration plate precoated with LDL was significant, this binding being completely abolished by pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate. The addition of exogenous chondroitin sulfate or apolipoprotein B inhibited the binding of PG-M-CSF to LDL in a dose-dependent manner, indicating that the interaction between PG-M-CSF and LDL was mediated by the binding of the chondroitin sulfate chain of PG-M-CSF to LDL apolipoprotein B. PG-M-CSF was also demonstrated in the arterial wall, and there were increased amounts of PG-M-CSF in atherosclerotic lesions. The in vitro interaction between PG-M-CSF and LDL thus appears to have physiological significance.

Aorta↗
PubMed1994

Characteristics of aspartate aminotransferase binding immunoglobulin determined by the isotope method.

A woman who had no known underlying diseases showed a persistent elevation (about 300 U/L) of serum aspartate aminotransferase (AST) without other abnormal laboratory findings. Cellolose gel electrophoresis showed that the AST activity in the patient had an atypical band with slower mobility than normal AST. When the sera from the patient and from a patient with acute hepatitis were mixed, the atypical band increased in density and the band of normal size AST disappeared. When the serum was fractionated on Sephadex G-200 gel filtration medium, almost all AST activity was found between the void volume and the gamma-globulin fraction. However, the AST activity in this fraction was not retained on dissociation into small AST by acid treatment. This suggests the loss of enzyme activity in dissociated small AST. The patient's serum was then incubated with iodine 125-labeled porcine AST; when this was fractionated on gel filtration medium, the main radioactivity was eluted in the void volume fraction. The binding activity for 125I-porcine AST was found in the gamma-globulin fraction obtained by gel filtration. The affinity constant of 125I-porcine AST binding to the gamma-globulin fraction was 1.0 x 10(-8) mol/L by Scatchard analysis. The binding gamma-globulin appeared to be (polyclonal) IgG, and the binding site was located in F(ab')2 and Fab fragments. The IgG could be bound with both human and porcine AST but not with chick AST. Thus the IgG appears specific for AST of mammalian species.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1994

Alteration of the proteoglycan form of macrophage colony-stimulating factor produced by a human stromal line stimulated by tumor necrosis factor-alpha.

Immunoblot analysis of macrophage colony-stimulating factor (M-CSF) in KM 102 cell-conditioned medium showed the presence of two M-CSF molecular types, one being 85-kd M-CSF, the other a proteoglycan form (PG-M-CSF) carrying a chondroitin sulfate chain of variable length. When KM 102 cells were stimulated by TNF-alpha, they produced more M-CSF than that produced in unstimulated condition, in which PG-M-CSF had a shorter chondroitin sulfate chain. Although PG-M-CSF has binding affinity for type V collagen, the PG-M-CSF with the shorter chondroitin sulfate chain shows lower affinity. This spreads in type V collagen-containing agarose gel more easily than does PG-M-CSF with a longer chondroitin sulfate chain.

Bone Marrow Cells↗
PubMed1993

Internucleosomal DNA cleavage involved in ischemia-induced neuronal death.

Pyramidal neurons of the hippocampal CA1 are known to be particularly vulnerable to transient ischemia resulting in "delayed neuronal death". Recent studies using aurintricarboxylic acid suggested that ischemia- or excitotoxin-induced neuronal death should share intracellular mechanisms in common with apoptosis. It is, however, unclear about involvement of endonucleases. Here using a transient (5 min) forebrain ischemia model in gerbils, we found that internucleosomal DNA fragmentation developed between 48 and 54 hr recirculations, accompanied with simultaneous or slightly preceding destruction of microtubule-associated protein 2. These results suggest that endonucleases, maybe activated by elevated intracellular Ca2+, play an important role in delayed neuronal death as well as in apoptosis.

Animals↗
PubMed1993

Effect of transient forebrain ischemia on superoxide dismutases in gerbil hippocampus.

Substantial generation of oxygen-derived free radicals has been implicated in pathophysiology of ischemic brain damage. Immunoreactive mitochondrial manganese and cytosolic copper-zinc superoxide dismutases, initial and essential enzymes to scavenge superoxide radical anions, increased in the gerbil hippocampal neurons after transient forebrain ischemia. Neuronal cells responded to oxidative stress in ischemia and induced the protective mechanism to increase superoxide dismutases.

Animals↗
PubMed1993

A possible functional necklace formed by placental antigen X-P2-immunoreactive and intensely acetylcholinesterase-reactive (PAX/IAE) glomerular complexes in the rat olfactory bulb.

The relationship between placental antigen X-P2 (PAX)-immunoreactive glomeruli and intensely acetylcholinesterase-reactive (IAE) patchy regions was evaluated by comparison of neighboring cryostat sections of the rat olfactory bulb. Both groups of distribution show similar necklace patterns. Each IAE region consists of heterologous glomerulus-like structures with variable acetylcholinesterase reactivity: strongly and less-reactive (IAE-S and IAE-L) structures. The PAX-immunoreactive glomeruli were detected as parts of the IAE-L portions. Three heterologous PAX/IAE glomeruli or glomerulus-like structures (IAE-S, IAE-L/PAX and IAE-L/non-PAX structures) locally form a distinct glomerular complex, the 'PAX/IAE glomerular complex'. At the caudal end of the main olfactory bulb, nine to sixteen such complexes occur at intervals and form a circumferential 'necklace'. Since one of them corresponds to the 'modified glomerular complex' involved in rat suckling behavior, the entire 'necklace' may be associated with processing olfactory stimuli eliciting or suppressing the suckling response.

Acetylcholinesterase↗
PubMed1993

Induced resistance and susceptibility to cerebral ischemia in gerbil hippocampal neurons by prolonged but mild hypoperfusion.

Brief periods of non-lethal cerebral ischemia can induce resistance against subsequent lethal ischemia. In this study, asymptomatic gerbils after unilateral carotid artery ligation were subjected to 5 min of forebrain ischemia. The prolonged but mild hypoperfusion, by carotid occlusion, induced susceptibility at 1 day and tolerance at 30 days to lethal ischemia in the hippocampal neurons. The neuroprotective effect correlated well with induction of heat shock protein 72 in the hippocampal neurons. These results suggested that neuronal cells possess a cellular response to sublethal hypoperfusion and can survive forthcoming ischemic stress.

Animals↗
PubMed1993

A proteoglycan form of macrophage colony-stimulating factor that binds to bone-derived collagens and can be extracted from bone matrix.

We previously found that the human osteoblastic cell line MG-63 produces two molecular types of macrophage colony-stimulating factor (M-CSF). One is an 85-kD M-CSF, and the other is a proteoglycan form of M-CSF (PG-M-CSF) that has a binding affinity to type V collagen. The latter type of M-CSF showed dose-dependent binding to wells coated by pepsin-extracted bone collagens, whereas the 85-kD M-CSF did not. Immunoblot analysis of urea-extracted bone M-CSF revealed the presence of PG-M-CSF. PG-M-CSF contained in bone matrix may have physiological importance in the bone metabolism.

Bone Matrix↗
PubMed1993

Differential vulnerability in the hindbrain neurons and local cerebral blood flow during bilateral vertebral occlusion in gerbils.

Differential vulnerability in the hindbrain neurons was examined immunohistochemically during hindbrain ischemia in the gerbil. Hindbrain ischemia was produced by extracranial occlusion of the bilateral vertebral arteries just before their entry into the transverse foramen of the cervical vertebra. Local cerebral blood flow was measured by quantitative autoradiographic technique after 5 min of ischemia and was reduced to less than 5 ml/100 g per min in the cerebellum, the pons, and the medulla, indicating that severe and reproducible hindbrain ischemia was induced immediately after occlusion. For immunohistochemical investigation, four gerbils each were used for each ischemic period of 5, 10, 15, and 30 min. Immunohistochemical lesions, detected by the reaction for microtubule-associated protein 2, were visible in the lateral vestibular nucleus and the cerebellar interpositus nucleus even after 5 min of ischemia. These results suggested that these areas were more vulnerable than others, although blood flow was markedly reduced in various regions of the hindbrain. In contrast, areas related to respiratory or cardiovascular control were rather resistant to ischemia. The present study suggests that selective vulnerability during hindbrain ischemia depends mainly on different metabolic characteristics inherent to various neurons in the hindbrain.

Animals↗
PubMed1993

Distribution of serum lipoprotein(a) levels--a non-parametric analysis.

The distribution of serum lipoprotein(a) [Lp(a)] concentration among Japanese male adults was evaluated using non-parametric methods. The following results were obtained. 1) Among healthy male adults undergoing a medical checkup, Lp(a) showed a highly skewed distribution towards the low level. The distribution could be regarded as a power normal distribution with the power order of 1/2. The median of Lp(a) level was 14.1 mg/dl (the 25th percentile was 6.2 mg/dl and the 75th percentile was 26.7 mg/dl). 2) The values of serum Lp(a) in subjects with vasospastic angina distributed at a higher level than for subjects with normal coronary arteries as diagnosed by coronary angiography. 3) The observed serum Lp(a) concentration moved to a higher range as the number of branches with significant stenosis on the coronary angiography increased. 4) Serum Lp(a) was one of the risk factors for ischemic heart disease. Its odds ratio when the cut-off value was set at 26.7 mg/dl or 30 mg/dl was 2.52 and 2.94, respectively. Information on the distribution of serum Lp(a) concentration is useful for estimating the coronary atherogenic factor.

Adult↗
PubMed1992

Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocampal neurons.

We investigated whether reversible oxidative stress induced by the administration of the superoxide dismutase inhibitor, diethyldithiocarbamate, could induce tolerance to subsequent cerebral ischemia in gerbil hippocampal neurons. Mature male gerbils received intraperitoneal injections of diethyldithiocarbamate (1.0 g/kg), which led to reduced superoxide dismutase activity and increases in thiobarbituric acid-reactive substance in the brain. Cerebral ischemia was produced by occluding the bilateral common carotid arteries for 5 min, either 2 or 4 days after diethyldithiocarbamate injection. One week after ischemia, samples from each brain were stained with hematoxylin-eosin to evaluate ischemic neuronal damage in the hippocampal CA1 sector. Diethyldithiocarbamate treatment 4 days before ischemia had significant protective effects against cerebral ischemia, while diethyldithiocarbamate 2-day pretreatment and vehicle treatment failed to show neuroprotection. Biochemical examinations showed a clear induction of heat shock protein 72 and a significant increase in manganese-containing superoxide dismutase in the hippocampus in animals treated with diethyldithiocarbamate 4 days prior to ischemia. These results suggested that the oxidative stress caused by diethyldithiocarbamate could induced tolerance to ischemia in the gerbil brain, and that the increase in the biosynthesis of manganese-containing superoxide dismutase and heat shock protein 72 could provide a biochemical explanation of the tolerance induced under these conditions.

Animals↗
PubMed1992

A human osteoblastic cell line, MG-63, produces two molecular types of macrophage-colony-stimulating factor.

A human osteoblastic cell line, MG-63, mouse primary osteoblasts, and a mouse osteoblastic cell line, MC3T3-E1, were shown to produce macrophage-colony-stimulating factor (M-CSF) by bone-marrow-cell colony assay, using a specific neutralizing antibody for M-CSF. Immunoblot analysis of M-CSF, produced by MG-63 cells, revealed the presence of a higher-molecular-weight species of M-CSF, in addition to the 85-kDa M-CSF. The higher-molecular-weight species had a high affinity to the DEAE-Sephacel column and was sensitive to chondroitinase ABC and AC. These physico-chemical profiles were wholly compatible with those of the proteoglycan form of M-CSF (PG-M-CSF), which was recently identified by our group in the conditioned medium of Chinese hamster ovary cells transfected with the 4.0-kb cDNA of the M-CSF gene. Conditioned medium of MG-63 cells was fractionated by DEAE-Sephacel column chromatography, and the M-CSF of each fraction was measured by both enzyme-linked immunosorbent assay and bone-marrow-cell colony assay. The fractions eluted by 0.3-0.6 M NaCl, which were shown to contain only PG-M-CSF on immunoblot analysis, also have macrophage-colony-stimulating activity.

Animals↗
PubMed1992

Biological activity of a proteoglycan form of macrophage colony-stimulating factor and its binding to type V collagen.

Two different types of macrophage colony-stimulating factors (M-CSF) were found, one with an apparent molecular mass of 85 kDa and the other greater than 200 kDa. The high molecular mass M-CSF was identified as a proteoglycan carrying chondroitin sulfate glycosaminoglycan and was designated as the proteoglycan form of M-CSF (PG-M-CSF). In this study, we compared the biological activity of the 85-kDa M-CSF and PG-M-CSF and examined the binding properties of these two M-CSF to certain extracellular matrix proteins, i.e. types I-V collagen and fibronectin, using a modified enzyme-linked immunosorbent assay. PG-M-CSF was capable of supporting the formation of murine macrophage colonies, and pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate, did not alter its colony-stimulating activity. The specific activity of PG-M-CSF was similar to that of the 85-kDa M-CSF. The 85-kDa M-CSF had no apparent affinity for the extracellular matrix proteins examined, whereas PG-M-CSF had an appreciable binding capacity to type V collagen, but did not bind to types I, II, III, and IV collagen or to fibronectin. Pretreatment of PG-M-CSF with chondroitinase AC completely abolished the binding of the species to type V collagen. Addition of exogenous chondroitin sulfate inhibited the binding of PG-M-CSF to type V collagen in a dose-dependent manner. These data indicated that the interaction between PG-M-CSF and type V collagen was mediated by the chondroitin sulfate chain of PG-M-CSF. PG-M-CSF bound to type V collagen could stimulate the proliferation of bone marrow macrophages, indicating that the matrix protein-bound PG-M-CSF retained its biological activity. This interaction between PG-M-CSF and type V collagen implies that the role of PG-M-CSF may be distinct from that of 85-kDa M-CSF.

Animals↗