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K Fukuhara

Publications and source records attributed to K Fukuhara.

At least 91 records · Page 5Linked to original sources
PubMed1994

Characteristics of a de novo designed protein.

A series of 204 amino acid proteins intended to form TIM (triose phosphate isomerase) barrel structures were designed de novo. Each protein was synthesized by expression of the synthetic gene as a fusion protein with a portion of human growth hormone in an Escherichia coli host. After BrCN treatment, the protein was purified to homogeneity. The refolded proteins are globular and exist as monomers. One of the designed proteins is stable toward guanidine hydrochloride (GuHCl) denaturation, with a midpoint of 2.6 M determined from CD and tryptophan fluorescence measurements. The GuHCl denaturation is well described by a 2-state model. The NMR spectra, the thermal denaturation curves, and the 1-anilino-8-naphthalene sulfonic acid binding imply that the stability of the protein arises mainly from hydrophobic interactions, which are probably of a nonspecific nature. The protein has a similar shape to that of rabbit triosephosphate isomerase, as determined by electron microscopy.

Amino Acid Sequence↗
PubMed1994

Stress elicits trans-synaptic activation of adrenal neuropeptide Y gene expression.

Stress triggers responses important to maintain internal homeostasis, and yet when prolonged can cause medical consequences that are most likely mediated by changes in gene expression. In this study we examine the alterations in gene expression of neuropeptide Y (NPY), a potent vasoconstrictor, with stress. Stressors, such as immobilization and cold, were found to increase adrenal NPY gene expression in rats. Adrenal prepro-NPY mRNA levels were elevated by a relatively short period of stress. A single immobilization was sufficient for an increase in prepro-NPY mRNA, that remained elevated for as long as one day later. This rise in adrenal NPY mRNA was abolished by the transcriptional inhibitor actinomycin D. Repeated (2 and 7) daily immobilizations led to a further rise and sustained elevations of prepro-NPY mRNA levels. This increase persisted 2-3 days after the cessation of repeated stress. The stress-elicited increase in NPY gene expression is transsynaptic requiring splanchnic innervation and mediated by nicotinic receptors. Hypophysectomy did not prevent the stress elicited rise in adrenal prepro-NPY mRNA levels. These results suggest that long lasting changes in NPY gene expression might be an important component in the homeostatic mechanisms triggered by chronic stress.

Adrenal Medulla↗
PubMed1994

Increase in rat adrenal phenylethanolamine N-methyltransferase mRNA level caused by immobilization stress depends on intact pituitary-adrenocortical axis.

The effects of a single and of repeated immobilization stress on the expression of the final enzyme involved in epinephrine biosynthesis, phenylethanolamine N-methyltransferase (PNMT), are described. A single immobilization (whether lasting 5 or 120 min) caused a severalfold increase of the adrenal PNMT mRNA level as measured 2 h after the beginning of the procedure. This elevation was of a transient nature, peaked 3-6 h after the 2-h immobilization, and returned to control values by 12 h after the stress. When the animals were immobilized for 2 h/day for seven consecutive days, an increase in content of PNMT mRNA of a similar magnitude was observed, which persisted for at least 2 days after the seventh immobilization. The immobilization-induced increase was completely abolished in hypophysectomized animals, whereas adrenal denervation failed to prevent it. These data suggest that the immobilization-induced increase in adrenal PNMT mRNA level depends primarily on pituitary-adrenocortical regulation.

Adrenal Cortex↗
PubMed1994

Detection of nitro-azabenzo[a]pyrene derivatives in the semivolatile phase originating from airborne particulate matter, diesel and gasoline vehicles.

Mutagens in the semivolatile phase of airborne particulate matter, diesel and gasoline engine emissions were investigated using chemical and biological assays. The previously unknown mutagens, 1-(3-)nitro-6-azabenzo[a]pyrenes (1-N-6-ABP and 3-N-6-ABP) and 1-(3-)nitro-6-azabenzo[a]pyrene-N-oxides (1-N-6-ABPO and 3-N-6-ABPO), were detected in the semivolatile phase, which was adsorbed onto XAD-4 resin combined with a Teflon-coated fibre filter. Dichloromethane extracts of materials adsorbed onto XAD-4 resin were divided into acidic, neutral and basic by liquid-liquid separation. These chemicals in the basic fraction were the major mutagens in the semivolatile phase when they were bioassayed using Salmonella typhimurium TA98 in the absence of S9 mix; the mutagenicity of the basic fraction contributed at the rate of 42.9-68.8% of that of crude extracts for airborne, diesel and gasoline emission materials. As the mutagens were found to be difficult to analyse by gas chromatography (because of their probable absorption onto the packing) they were purified by HPLC and analysed by mass spectrometry. The concentrations of 1-N-6-ABP, 3-N-6-ABP, 1-N-6-ABPO and 3-N-6-ABPO detected were 1.1, 1.2, 0.8 and 0.3 ng/g, respectively, of materials in the airborne sample, and 4.9, 7.7, 2.2 and 3.8 ng/g, respectively, of materials in the diesel emission. Only 1-N-6-ABP (3.4 ng/g) and 3-N-6-ABP (4.9 ng/g) were detected in the semivolatile phase of the gasoline engine emission.(ABSTRACT TRUNCATED AT 250 WORDS)

Air Pollutants↗
PubMed1994

Crystallization and preliminary crystallographic data of the alpha-amylase inhibitors, Haim I and Paim I.

Two proteins that act as alpha-amylase inhibitors, Haim I and Paim I, were crystallized and preliminary X-ray diffraction studies on them were carried out. We also sequenced Haim I prepared from Streptomyces griseosporeus YM-25 and confirmed that it is composed of 78 amino acid residues. Crystals of Haim I were grown from ammonium sulfate solution mixed with ethanol by the vapor diffusion technique. The crystals grew as hexagonal bipyramids and diffracted X-rays beyond 2.0 A resolution. They belong to the space group P6(1)22 (or P6(5)22) with unit cell dimensions of a = b = 36.7 A, c = 192.4 A, and contain one molecule per asymmetric unit. Paim I, a protein of 39 amino acid residues produced by Streptomyces corchorusii, was crystallized under similar conditions to Haim I. The crystals diffracted X-rays beyond 2.5 A. They belong to the space group P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 65.4 A, c = 96.1 A, and contain three molecules per asymmetric unit.

Amino Acid Sequence↗
PubMed1994

p53 gene mutations in human skin cancers and precancerous lesions: comparison with immunohistochemical analysis.

Mutations of exons 3 through 9 of the p53 gene in skin lesions were screened in 23 cases of squamous cell carcinoma (SCC), 25 cases of basal cell carcinoma (BCC), two cases of Bowen's disease, 10 cases of solar keratosis, and five cases of keratoacanthoma by polymerase chain reaction--single strand conformation polymorphism analysis. Mutations of the p53 gene were detected in seven of 23 SCCs (30%), three of 25 BCCs (12%), and none in all cases of Bowen's disease, solar keratosis, or keratoacanthoma. Of 23 cases of SCC, mutations were detected in four of 15 SCCs (27%) that originated in the sunlight-exposed skin region, in two of three SCCs (67%) that originated in the scar tissue, and in one of three SCCs (33%) that originated in radiation dermatitis. Mutations of C-->T transition predominated in SCC and BCC that originated in the sunlight-exposed skin region. Mutations of C-->A or CC-->AT observed in tumors that originated in the predisposed conditions, presumably unrelated to UV light, are different from those found in UV light-related SCC or BCC. Twelve cases of SCC were comparatively analyzed with the immunohistochemical staining with anti-p53 antibody. Two of four cases with positive staining had missense mutations, and three of eight cases with negative staining had nonsense mutations. Based on these findings, immunohistochemical results do not necessarily mean the presence or absence of p53 gene mutations in skin tumors, and sequence analysis is essential for determining whether the gene is mutated.

Aged↗
PubMed1994

Effects of immobilization on the capsule of the canine glenohumeral joint. A structural functional study.

The effects of immobilization on the function of the glenohumeral joint and its capsule were investigated in eight beagle dogs. One foreleg of each animal was immobilized in a spica cast, and the dogs were euthanized after 4, 8, 12, and 16 weeks of immobilization in groups of two. The contralateral limb and the forelegs of two normal beagles were used as controls. At the time of euthanasia, the range of motion, intraarticular filling volume during passive movements of the joint, and intraarticular filling volume to rupture the capsule were measured. The synovium, including the capsule and the subscapular bursa, were examined histologically. In the immobilized limb, there was progressive restriction in the range of motion with increases in intraarticular pressure, and the filling volume that was required to cause a rupture of the capsule diminished. Morphologically, the capsule and the subscapular bursa showed focal adhesions. Thus, it appeared that immobilization of these canine forelimbs produced changes in the glenohumeral joint that resembled those of frozen shoulder in humans.

Animals↗
PubMed1994

The pathogenesis of club foot. A histomorphometric and immunohistochemical study of fetuses.

We studied 16 club feet and 27 normal feet from spontaneously aborted human fetuses in the second trimester of gestation and measured the length of the spring ligament, and the declination angle and size of the talus. We also studied the cellular characteristics of the spring ligament and the immunohistochemical features of the medial ankle ligaments using monoclonal antibodies against type-III collagen, desmin, vimentin, and smooth muscle actin. Histomorphometric results indicated that the talar deformity was not the primary lesion. Histological and immunohistochemical findings showed that the cells and collagen fibres of the medial ankle ligaments of club feet appeared to be the site of the earliest changes, in that they had lost their spatial orientation and had contracted. In severe club feet before the third trimester of gestation, myofibroblast-like cells seemed to create a disorder of the ligaments resembling fibromatosis. This led to contraction and resulted in typical club-foot deformity.

Ankle↗
PubMed1993

Purification and characterization of kumamolysin, a novel thermostable pepstatin-insensitive carboxyl proteinase from Bacillus novosp. MN-32.

We have found a novel type of thermostable, pepstatin-insensitive carboxyl proteinase in the culture filtrate of Bacillus novosp. MN-32. The carboxyl proteinase, which was named kumamolysin, was purified about 8,300-fold by column chromatography including DEAE-Sepharose CL-6B, Sephadex G-100, and TSKgel DEAE-5PW. The purified kumamolysin gave a single band corresponding to 41 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of kumamolysin was estimated to be 40 kDa by gel filtration. The isoelectric point of kumamolysin was estimated to be pH 3.5 by isoelectric focusing. Kumamolysin has maximum proteolytic activity at 70 degrees C and at pH 3.0. Kumamolysin specifically hydrolyzed the Leu15-Tyr16 peptide bond in oxidized insulin B-chain (Km = 9.0 x 10(-5) M, Kcat = 71 s-1; at pH 3.0, 30 degrees C), and additional cleavage at Phe25-Tyr26 was detected at a considerably lower rate. Kumamolysin is insensitive to the known carboxyl proteinase inhibitors pepstatin, diazoacetyl-DL-norleucine methyl ester, and 1,2-epoxy-3-(p-nitrophenoxy)propane. Kumamolysin has no similarity to the thermostable acid protease thermopsin from Sulfolobus acidocaldarius (Lin, X.-L., and Tang, J. (1990) J. Biol. Chem. 265, 1490-1495). Thus, the substrate specificity, the inhibitor sensitivity, the molecular mass, and the thermostability all suggest that kumamolysin is a novel type of carboxyl proteinase.

Amino Acid Sequence↗
PubMed1993

Recurrence of hepatocellular carcinoma in the liver remnant after hepatic resection.

We analyzed the results of 19 patients who had intrahepatic recurrence of hepatocellular carcinoma (HCC) among 47 patients who were discharged from the hospital after having a hepatic resection in order to evaluate the factors affecting recurrence and survival. Recurrence-free survival rates were 80%, 44%, and 28% at 1, 3, and 5 years, respectively. Of the 19 patients with recurrence, 14 had multiple lesions, and 16 of the recurrences were detected within 3 years of surgery. None of the following factors correlated with recurrence: hepatic function; tumor size; presence of tumor capsule; capsular invasion; vascular invasion and intrahepatic metastasis; extent of hepatic resection; and resection with tumor-free margins. Patients having multiple recurrent HCCs, however, had larger-sized tumors at the time of resection than those with a solitary recurrence. The survival rates after recurrence were significantly better in patients with a solitary recurrence, and these patients were treated with transcatheter arterial embolization (TAE) therapy. Early detection as well as TAE for recurrent HCCs is necessary to improve long-term survival.

Carcinoma, Hepatocellular↗
PubMed1993

Light microscopic autoradiographical analysis of [125I]epidermal growth factor binding in basal cell epithelioma and squamous cell carcinoma of the skin.

Levels of epidermal growth factor (EGF) receptor expression were investigated in five basal cell epitheliomas (BCEs) and 10 primary lesions from squamous cell carcinoma (SCC) of the skin, using light microscopic autoradiography with [125I]EGF. All of the BCEs were clinically the pigmented type and histologically the solid type. All of them showed an EGF binding level similar to that of the basal and suprabasal layers of the normal epidermis. The SCCs included one case of Bowen's disease (SCC in situ), five of the well differentiated type, three of the moderately differentiated type and one of the poorly differentiated type. Eight of the 10 SCCs showed an EGF binding level similar to that of the normal epidermal basal and suprabasal cell layers. One of the two remaining SCCs, a moderately differentiated type, showed highly increased EGF binding. The other one, a poorly differentiated type, showed very little EGF binding in a large region consisting of poorly differentiated cells, although a small area composed of more differentiated, nest-forming cells had an EGF binding level similar to that of the basal and suprabasal cells. Metastasis was found in three of these 10 SCCs after surgery. Two of the three SCCs with metastasis showed increased or decreased EGF binding levels in primary lesions as described above; in contrast, primary lesions of the seven SCCs without metastasis had EGF binding levels similar to those of the normal epidermal basal and suprabasal cells. Abnormally increased or decreased EGF binding level in SCC of the skin may be indicative of a poor prognosis, although it is necessary to examine more SCCs to confirm this assumption.

Aged↗
PubMed1993

Effects of single or repeated immobilization on release of norepinephrine and its metabolites in the central nucleus of the amygdala in conscious rats.

The release of norepinephrine (NE) and its metabolites in the central nucleus of the amygdala was measured using in vivo microdialysis during immobilization (IMMO) stress in conscious rats. Animals underwent 2-hour periods of IMMO either once or daily for 7 days. Extracellular fluid concentrations of NE, dihydroxyphenylglycol (DHPG), methoxyhydroxyphenylglycol (MHPG), and the dopamine metabolite dihydroxyphenylacetic acid (DOPAC) were measured before, during, and after IMMO. Microdialysate levels of NE and DHPG attained 2- to 3-fold increments during the 1 h of IMMO and declined thereafter, whereas MHPG and DOPAC levels attained maximal levels of about twice basal concentrations during the 2- or 3-h after initiation of IMMO. After the sixth IMMO basal levels of NE, DHPG, MHPG, and DOPAC were decreased, and NE, DHPG, and DOPAC responses during the seventh IMMO failed to attain levels found during the first IMMO, although the absolute changes during IMMO were similar between animals subjected to IMMO once or seven times. The results indicate that acute IMMO increases synthesis, release, and metabolism of NE in the central nucleus of the amygdala and that repetition of IMMO decreases basal catecholamine synthesis and noradrenergic turnover in this brain region, without inhibiting acute noradrenergic responses.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Effect of single and repeated immobilization stress on the heat shock protein 70/90 system of the rat: glucocorticoid-independent, reversible reduction of Hsp90 in the liver and spleen.

Heat shock proteins (hsps) and glucocorticoids are produced in response to many common stressors. In addition, hsps interact physically with the intracellular glucocorticoid receptor (GR) and facilitate its activation by the hormone. To study the effect of stress on the hsp70/90 system and the potential association of the system with the hypothalamic-pituitary-adrenal (HPA) axis, we subjected 3 month-old male Harlan-Sprague Dawley rats to single or repeated (once daily for 6 consecutive days) immobilizations, and measured hsp70/90 steady-state levels in a panel of tissues, as well as circulating ACTH and corticosterone concentrations before, during and after immobilization. Single or repeated immobilizations had, as expected, a profound stimulatory effect on the HPA axis but did not influence the steady-state levels of hsp70/90 in any of the gross brain regions (pituitary, hypothalamus, hippocampus or brain cortex) or most peripheral tissues (thymus, adrenal glands, testes) examined. The hsp90 levels, however, were markedly and reversibly decreased in the liver and spleen both by single and repeated immobilizations. The potential inhibitory effect of glucocorticoids on liver and spleen hsp90 was investigated in bilaterally adrenalectomized rats treated with placebo or oral pharmacologic doses of corticosterone for 1 week. Neither adrenalectomy nor corticosterone treatment had an effect on the hsp70/90 system, suggesting that factors other than glucocorticoids mediate the effect of stress on hsp90 concentrations in the liver and spleen. We conclude that acute and chronic stress are associated with a reversible reduction of hsp90 in the liver and spleen, and this appears independent of glucocorticoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Adrenalectomy augments in vivo release of norepinephrine in the paraventricular nucleus during immobilization stress.

Adrenalectomy (ADX) activates CRH neurons in the paraventricular nucleus (PVN) of the hypothalamus and increases hypothalamic norepinephrine (NE) turnover in vitro. Immobilization (IMMO) markedly increases the release of NE into extracellular fluid in the PVN. The present study assessed whether ADX affects the release of NE in the PVN in vivo in conscious rats at baseline and during IMMO and whether cortisol (CORT) treatment attenuates the effects of ADX. Concentrations of NE, the NE metabolites dihydroxyphenylglycol and methoxyhydroxyphenylglycol, and the dopamine metabolite dihydroxyphenylacetic acid were measured in microdialysate samples beginning 24 h after implantation of a microdialysis probe in the PVN. Seven to 10 days after ADX or ADX plus CORT (20 mg/kg.day via osmotic minipump), animals underwent IMMO for 2 h. Adrenalectomized rats had slightly higher baseline microdialysate NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, and dihydroxyphenylacetic acid levels and much larger IMMO-induced responses of these compounds than did sham-operated rats. CORT treatment abolished the ADX-induced augmentation of these responses. The results indicate that ADX, by removing endogenous glucocorticoids, augments IMMO-induced release and turnover of NE and amplifies the responses of catecholamine synthesis. Glucocorticoids, therefore, appear to exert feedback inhibition on stress-induced increments in the release of NE and catecholamine biosynthesis in the PVN.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Endogenous glucocorticoids restrain catecholamine synthesis and release at rest and during immobilization stress in rats.

Cardiovascular and metabolic adjustments during stress involve participation of the sympatho-adrenal and hypothalamic-pituitary-adrenocortical systems, which interact at several levels. The present study investigated the effects of removal of endogenous glucocorticoids on indices of norepinephrine release and metabolism and of catecholamine biosynthesis in sympathetic nerves, at baseline and in response to immobilization stress (IMMO) in rats. Plasma levels of the catecholamine precursor dihydroxyphenylalanine, norepinephrine (NE), the NE metabolites dihydroxyphenylglycol and methoxyhydroxyphenylglycol, dopamine, and the dopamine metabolites dihydroxyphenylacetic acid and homovanillic acid were measured in adrenalectomized, adrenal-medullectomized, or sham-operated conscious rats, with or without glucocorticoid treatment (25 mg/kg.24 h cortisol for 7 days by osmotic minipumps), at rest and after 5, 20, 60, and 120 min of IMMO. Adrenalectomy eliminated plasma levels of epinephrine and corticosterone and augmented IMMO-induced increments in levels of NE, dihydroxyphenylglycol, methoxyhydroxyphenylglycol, dihydroxyphenylalanine, dihydroxyphenylacetic acid, and homovanillic acid. Adrenal-medullectomy reduced plasma epinephrine, but not corticosterone, to undetectable levels at baseline and did not augment stress-induced responses of any of the measured compounds. Cortisol administration (plasma levels approximately 600 pmol/ml) reversed the augmentation of catecholaminergic responses in adrenalectomized rats. The results indicate that adrenalectomy stimulates several aspects of sympatho-neural function, including stress-induced increments in NE release, reuptake, metabolism, and turnover and in catecholamine biosynthesis. This augmentation was absent in adreno-medullectomized rats, indicating that the loss of adrenomedullary hormones after adrenalectomy does not appear to contribute to the augmentation. Since cortisol treatment reversed or prevented this augmentation, loss of feedback inhibition by endogenous glucocorticoids appears to be the basis of the enhanced responses. Thus, the results suggest that endogenous glucocorticoids restrain responses of catecholamine turnover, synthesis, release, reuptake, and metabolism in sympathetic nerves in this stress model.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1993

Effects of highly purified ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E) on rabbit platelets.

The effects of ethyl all-cis-5,8,11,14,17-icosapentaenoate (EPA-E), highly purified ethyl ester of icosapentaenoic acid (EPA), on rabbit platelets were studied. In in vitro, highly purified EPA (62.5-3000 microM) suppressed the platelet aggregation induced by collagen, arachidonic acid (AA) and adenosine diphosphate (ADP). In ex vivo, a single administration of EPA-E (300 and 1000 mg/kg, p.o.) and repeated administrations (30 and 300 mg/kg/d, p.o.) for 2 weeks showed no effects on collagen-, AA- and ADP-induced platelet aggregation. Repeated administrations (30 and 300 mg/kg/d, p.o.) for 4 weeks suppressed the collagen-induced platelet aggregation, but not the AA- and ADP-induced platelet aggregation. Repeated administrations for 4 weeks also suppressed thromboxane B2 (TXB2) formation induced by collagen, but a single administration and repeated administrations for 2 weeks failed to inhibit TXB2 formation. The EPA level in the platelet phospholipids increased slightly with a single administration, and increased markedly with repeated administrations for 2 and 4 weeks. The AA level in the phospholipids showed practically no changes with a single and repeated administrations. These results suggested that highly purified EPA-E could reduce platelet aggregability by the change of the EPA level in the platelet phospholipids and should allow for a reasonable period of administration.

Animals↗