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Biomedical subjects

A Kobayashi

Publications and source records attributed to A Kobayashi.

At least 325 records · Page 18Linked to original sources
PubMed1995

Structure of translation initiation region of c-myc mRNA and thermostability of the c-myc mRNA/antisense ODN complex.

We enzymatically synthesized c-myc mRNA fragment containing the translation initiation (TI) region (36mer) and examined the structure by CD, 1H-NMR and RNaseV1 digestion. These study demonstrated that the c-myc mRNA fragment forms a hairpin structure at 37 degrees C. Six 15-mer antisense oligodeoxynucleotides(ODNs), which hybridize to the c-myc mRNA fragment were designed, and synhesized. Thermostabilities of the antisense ODNs-mRNA fragment complexes were examined. Antisense A (complementary to the c-myc mRNA from -9 to +6)-c-myc mRNA fragment complex had the highest melting temperature(Tm), and Antisense D (complementary to the c-myc mRNA from +1 to +15)-c-myc mRNA fragment complex had the most different Tm from a predicted Tm. Furthermore, Antisense D exhibited high inhibitory effect on HL60 cell proliferation, whereas Antisense A did not. The results suggest that the difference in the antisense effect is not simply due to the complex thermostability.

Cell Line↗
PubMed1994

Oxygen free radicals and calcium homeostasis in the heart.

Many experiments have been done to clarify the effects of oxygen free radicals on Ca2+ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analyze the results. It is, however, evident that Ca2+ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Through ATP-independent Ca2+ binding is increased, Ca2+ influx through Ca2+ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca2+ can enter the myocytes is Na(+)-Ca2+ exchanger. Although, the activities of Na(+)-K+ ATPase and Na(+)-H(+) exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na(+) level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na(+)-Ca2+ exchange in Ca2+ influx process from extracellular space. Another question is 'which way does Na(+)-Ca2+ exchange work under oxidative stress? Net influx or efflux of Ca2+?' Membrane permeability for Ca2+ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca(2+)-pump ATPase activity is depressed by oxygen free radicals, Ca2+ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca2+ release from sarcoplasmic reticulum and inhibit Ca2+ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca2+ handling systems could cause the Ca2+ overload due to oxygen free radicals.

Animals↗
PubMed1994

Oxygen free radicals and calcium homeostasis in the heart.

Many experiments have been done to clarify the effects of oxygen free radicals on Ca2+ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analayze the results. It is, however, evident that Ca2+ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Though ATP-independent Ca2+ binding is increased, Ca2+ influx through Ca2+ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca2+ can enter the myocytes is Na(+)-Ca2+ exchanger. Although, the activities of Na(+)-K+ ATPase and Na(+)-Ca2+ exchanger. Although, the activities of Na(+)-H+ exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na+ level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na(+)-Ca2+ exchange in Ca2+ influx process from extracellular space. Another question is 'which way does Na(+)-Ca2+ exchange work under oxidative stress? Net influx or efflux of Ca2+?' Membrane permeability for Ca2+ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca(2+)-pump ATPase activity is depressed by oxygen free radicals, Ca2+ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca2+ release from sarcoplasmic reticulum and inhibit Ca2+ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca2+ handling systems could cause the Ca2+ overload due to oxygen free radicals.

Adenosine Triphosphate↗
PubMed1994

Functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of Pseudomonas origin.

The extradiol ring-cleavage dioxygenases derived from seven different Pseudomonas strains were expressed in Escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. The substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing Pseudomonas putida F1, 1,2-dihydroxynaphthalene dioxygenase from a NAH7 plasmid, and catechol 2,3-dioxygenase from a TOL plasmid pWW0 were compared. Among the dioxygenases, that from Pseudomonas pseudoalcaligenes KF707 showed a very narrow substrate range. Contrary to this, the dioxygenase from pWW0 showed a relaxed substrate range. The seven extradiol dioxygenases from the various Pseudomonas strains are highly diversified in terms of substrate specificity.

Biodegradation, Environmental↗
PubMed1994

[Phase analysis of gated blood pool scintigraphy in traumatic myocardial contusion].

It is not easy to make a diagnosis of myocardial contusion following blunt chest trauma, because most patients have many other concurrent injuries with diverse symptoms. The usefulness of phase analysis of gated blood pool scintigraphy (GBPS) for myocardial contusion following blunt chest trauma was evaluated. Thirty-eight patients who had been strongly suspected of having myocardial contusion from clinical symptoms and electrocardiograms underwent phase analysis of GBPS. The results of phase analysis were compared with those of two-dimensional echocardiography (2-D Echo) and CPK-MB fraction measurement in all patients, with those of 201TlCl myocardial scintigraphy in 35 patients and with those of 99mTc-pyrophosphate scintigraphy in 10 patients. In 29 patients (76.3%), the results of phase analysis matched those of 2-D Echo. Two patients (5.3%) who were judged as positive by 2-D Echo and as negative by phase analysis had only rupture of the chordae. Only one of two other patients who were judged as negative by 2-D Echo and as positive by phase analysis was judged as positive by 201TlCl myocardial scintigraphy. The results of both 2-D Echo and phase analysis were not well correlated with those of CPK-MB fraction measurement and 99mTc pyrophosphate scintigraphy. It is concluded that phase analysis of GBPS, as well as 2-D Echo, is useful for diagnosing myocardial contusion, and that phase analysis is most useful for diagnosing myocardial contusion in patients who cannot be examined by 2-D Echo because of the presence of pneumothorax and/or subcutaneous emphysema in the anterior chest wall.

Adolescent↗
PubMed1994

Purification and characterization of ATBP, a novel protein that binds to A/T stretches in three segments of the Sarcophaga lectin gene.

A DNA-binding protein for the Sarcophaga lectin gene, ATBP (A/T-stretches-binding protein) was purified to homogeneity from the nuclear extract of NIH-Sape-4 cells. The molecular mass of ATBP determined under denaturing conditions was 53 kDa, but its native molecular mass estimated by gel-filtration chromatography was 430 kDa, suggesting that it is an octamer of the 53-kDa subunit. This protein bound to at least three DNA fragments from the Sarcophaga lectin gene, two of them are in the 5'-upstream region and the other is in a region containing an intron. These fragments are very AT rich and inlaid with stretches of A or T residues (A/T stretches). ATBP was found to have affinity for poly[d(A-T)]. These results suggest that ATBP binds to A/T stretches in the three DNA fragments from this gene. Furthermore, longer DNA was found to be bound more effectively, suggesting that an octamer of the 53-kDa subunit has multiple binding sites for the DNA fragments and requires a relatively long DNA sequence for binding.

Amino Acid Sequence↗
PubMed1994

Effect of L-carnitine on mitochondrial acyl CoA esters in the ischemic dog heart.

Many studies have shown that L-carnitine has a positive effect on ischemic myocardium, probably by reducing accumulation of long-chain acyl coenzyme A (CoA) esters. Previous studies have involved whole-heart extracts and have not assessed changes of CoA ester levels in mitochondria, the site of translocase inhibition. To more precisely assess L-carnitine effects, we measured long-chain acyl CoA ester levels in cytosol and in mitochondria in the ischemic canine heart. Dogs were divided into four groups: a sham-operated control group; an untreated group; and high- and low-dose L-carnitine-treated groups (30 mg/kg and 100 mg/kg). After 60 min of ischemia, the heart was excised, and the cytosolic and mitochondrial fractions were isolated. CoA esters and the activity of carnitine palmitoylcarnitine transferase (CPT) I and II were measured in both compartments. Approximately 89% of cellular free CoA. 90% of cellular acetyl CoA, 97% of cellular shot-chain acyl CoA, and 92% of cellular long-chain acyl CoA were located in the mitochondrial space under the normal condition. Under the ischemic condition, mitochondrial free CoA was significantly decreased. Conversely, mitochondrial acetyl CoA and long-chain acyl CoA were significantly increased. Treatment with L-carnitine significantly decreased acetyl CoA and long-chain acyl CoA in the ischemic mitochondrial space in a dose-dependent manner. These results support the hypothesis that L-carnitine reduces accumulation of long-chain acyl CoA within the ischemic mitochondrial space and thereby improves mitochondrial function and adenine nucleotide translocation.

Animals↗
PubMed1994

Change in growth kinetics of hybridoma cells entrapped in collagen gel affected by alkaline supply.

The growth yields for glucose and glutamine of murine hybridoma cells entrapped in collagen gel particles were examined during the growth phase. The immobilized hybridoma cells were cultivated in a fluidized bed fermenter where the medium was circulating to supply oxygen separately. Procedures to supply an alkaline solution for adjusting the pH level strongly affected the growth yields. A direct supply of the alkaline solution to the cultivation system reduced both the growth yields for glucose and glutamine, probably due to a local increase in pH level. On the other hand, when fresh medium in which the pH was adjusted to around 8.5 was added to the cultivation system, the growth yields were unchanged even at the same pH level as when direct alkaline supply was used. These results suggest that an indirect alkaline supply could be recommended to adjust the pH level when using medium-circulating-fermenters.

Animals↗
PubMed1994

Analysis of MHC class II antigens in Japanese IDDM by a novel HLA-typing method, hybridization protection assay.

We examined HLA Class II antigens in 116 Japanese IDDM patients [84 typical IDDM (T-IDD); 32 slowly progressive IDDM (S-IDD)] by the hybridization protection assay (HPA) which is a novel HLA typing method based on hybridization of acridinium-ester-labeled DNA probes to amplified DNA. We detected HLA-DRB1, -DQA1 and -DQB1 genes by this method which is capable of analyzing over 50 samples within 4 h with high sensitivity. Positive associations were found in DRB1*0405, DRB1*0802, DRB1*0901, DQA1*0301, DQB1*0303 and DQB1*0401, negative correlations in DRB1*0403, DR2, DR12, DRB1*0801 or 03, DQA1*0101 or 02, DQA1*0501, DQB1*0301 and DQB1*0602 alleles. The absence of aspartic acid (Asp) at position 57 of the DRB1 chain and the presence of arginine (Arg) at position 52 of the DQA1 chain correlated positively with both types of IDDM. There were no significant differences in HLA between T-IDD and S-IDD. These results suggest that the absence of Asp at position 57 of the DRB1 chain and the presence of Arg at position 52 of the DQA1 chain are significant Japanese IDDM patients and that DRB1*0802, in which the amino acid at position 57 is aspartic acid, may play a role in the pathogenesis of IDDM. Also, T-IDD and S-IDD have common bases in the HLA gene.

Adolescent↗
PubMed1994

The expression of osteoclast markers on foreign body giant cells.

The expression of some candidate osteoclast markers, tartrate-resistant acid phosphatase (TRAP), macrophage associated antigens (M phi Ag), and vitronectin receptor (VNR) on foreign body giant cells (FBGCs) was investigated in peri-implant tissues of loosened total joint arthroplasties. Osteoclasts showed distinct staining characteristics. They were strongly TRAP-positive at tartrate concentrations of 50-200 mM and expressed VNR and a restricted range of M phi Ag. In contrast, FBGCs were shown to be significantly heterogeneous. Significant numbers of FBGCs were TRAP-positive at a 100 mM tartrate concentration and some were more intense than osteoclasts. A population of FBGCs did not express M phi Ag such as CD11b, but expressed VNR. It was demonstrated that these candidate osteoclast markers were also positive on FBGCs. These results have highlighted the difficulty in distinguishing these two cell lineages and suggested that there might be some uncertainty in defining osteoclast-like cells in culture studies.

Acid Phosphatase↗
PubMed1994

An S-alkylating reagent with positive charges as an efficient solubilizer of denatured disulfide-containing proteins.

A novel S-alkylating reagent, N-(3-bromopropyl)-N,N,N',N',N'-pentamethyl-1,3-propanedi(ammonium bromide) (TAP2-Br) which carries two positive charges in the molecule, was prepared to increase the solubility or to decrease the hydrophobicity of cysteine-containing denatured proteins (or peptides). S-Alkylation with TAP2-Br introduces two positive charges per cysteine residue, which will effectively shift the net charge of a protein in the positive direction. Disulfide-containing proteins, such as hen egg-white lysozyme, RNase A, BSA, and soybean trypsin inhibitor (Kunitz type), were reduced and S-alkylated with TAP2-Br to evaluate the potential of this reagent compared with other S-alkylating reagents such as monoiodoacetic acid, bromosuccinic acid and (3-bromopropyl)trimethylammonium bromide. The solubilities of these denatured proteins in the pH range of 2-10 indicated that S-alkylation with TAP2-Br effectively solubilized not only basic proteins (lysozyme and RNase) but also an acidic protein containing a fairly large number of cysteine residues (BSA). Moreover, the retentions of cysteine-containing tryptic peptides derived from lysozyme on reversed-phase HPLC were greatly reduced by S-alkylation with TAP2-Br. These results indicate that TAP2-Br is very useful to increase the solubility of some cysteine-containing denatured proteins and to decrease the hydrophobicity of peptides containing cysteine residue(s).

Alkylating Agents↗
PubMed1994

Expulsion of Hymenolepis nana from mice with congenital deficiencies of IgE production or of mast cell development.

The roles of IgE and mast cells on expulsion of adult Hymenolepis nana from the intestine were examined in mice. IgE-dependency was determined by comparing congenitally IgE-deficient SJA/9 and IgE-producing SJL/J mice infected with 50 H. nana eggs. Anti-H. nana IgE antibody was detected at three weeks post infection (p.i.) in SJL but not in SJA mice. The number of adult worms in the intestines of SJA and of SJL mice were similar at two weeks, but significantly more were found in SJA mice at three weeks p.i. Treatment of mice with anti-epsilon antibody also resulted in an increased worm burden at three weeks, suggesting participation of IgE in expulsion of H. nana. Intestinal mastocytosis was induced by infection regardless of the IgE status of the mice. Mast cell-dependency was tested in mast cell-deficient W/Wv and in normal littermate +/+ mice infected with 100 H. nana eggs. Anti-H. nana antibody was detected in both groups of mice at three weeks p.i. Worm expulsion seemed to be mast cell dependent because expulsion was less complete in W/Wv mice at three weeks p.i. Peripheral blood eosinophilia was comparable at three weeks p.i. in both IgE and mast cell sufficient and deficient mice. These results suggest that IgE and mast cells participate in the expulsion of H. nana adults from intestine in mice.

Animals↗
PubMed1994

Simultaneous measurement of intracellular Na+ and Ca2+ during K(+)-free perfusion in isolated myocytes.

To study the relationship between intracellular Na+ concentration ([Na+]i) and intracellular Ca2+ concentration ([Ca2+]i), guinea pig ventricular myocytes were loaded with both the Na(+)-sensitive probe, sodium-binding benzofuran isophthalate (SBFI), and the Ca(2+)-sensitive probe, fluo 3. [Na+]i was measured from the ratio image at 510 nm when excited at 340/380 nm. [Ca2+]i, expressed as the percent change of corrected fluo 3 fluorescence, was measured at 540 nm when excited at 500 nm. The fluorescent spectra of these probes were sufficiently different to allow for simultaneous measurement. After 30 min perfusion of K(+)-free solution, [Na+]i of rod-shaped cells increased from 6.4 +/- 0.5 to 20.6 +/- 2.6 mM, and [Ca2+]i increased to 256 +/- 36% of the control. [Ca2+]i was higher in spontaneously contracting cells and shortened cells than in rod-shaped cells at similar levels of [Na+]i. When Ca(2+)-free solution or Ni2+ (5 mM) was applied, [Ca2+]i was lower than in cells perfused with K(+)-free solution alone. It was suggested that extracellular Ca2+ and the Na(+)-Ca2+ exchange were involved in the increase in [Ca2+]i. In conclusion, we have developed a new method for the simultaneous measurement of [Na+]i and [Ca2+]i in isolated myocytes, which should be useful to study the relation between [Na+]i and [Ca2+]i.

Aniline Compounds↗
PubMed1994

Regulation of [Na+]i and [Ca2+]i in guinea pig myocytes: dual loading of fluorescent indicators SBFI and fluo 3.

To investigate the mechanisms regulating intracellular Na+ and Ca2+ concentrations ([Na+]i and [Ca2+]i, respectively), we monitored both ion concentrations simultaneously using fluorescent probes, Na(+)-binding benzofuran isophthalate (SBFI) and fluo 3, in unstimulated guinea pig ventricular myocytes. After the addition of 500 microM strophanthidin, [Na+]i increased gradually from 6.6 +/- 0.6 to 20.1 +/- 1.6 mM (mean +/- SE) at 50 min. [Ca2+]i, expressed as the percentage change of corrected fluo 3 fluorescence, was kept at the low level during the first 20 min and then began to increase to 447 +/- 81% of the control at 50 min. The addition of 1 microM hexamethylene amiloride prevented the increases in both [Na+]i and [Ca2+]i. The perfusion of Ca(2+)-free solution or 5 mM Ni(2+)-containing solution suppressed the increase in [Ca2+]i. In cells that exhibited spontaneous contractile activities, [Ca2+]i increased further than that in quiescent cells, whereas [Na+]i levels were similar. In the presence of 1 microM ryanodine, both the spontaneous contractile activities and the further increase in [Ca2+]i were eliminated. These findings indicated that 1) the pathway of Na+ entry was mainly through Na(+)-H+ exchange and that the elevated [Na+]i induced Ca2+ entry mediated by the reverse mode of Na(+)-Ca2+ exchange, and 2) the entered Ca2+ triggered the ryanodine-sensitive Ca2+ release from the sarcoplasmic reticulum, causing the dissociation in the relationship between [Na+]i and [Ca2+]i.

Amiloride↗
PubMed1994

Receptor-mediated regulation of differentiation and proliferation of hepatocytes by synthetic polymer model of asialoglycoprotein.

Morphology and responses of hepatocytes are investigated using an artificial asialoglycoprotein model polymer--lactose-carrying polystyrene (PVLA) as a culture substratum, especially in focusing on the effect of the surface density of the PVLA substratum. The surface density of PVLA on polystyrene dishes was determined using fluorescein-labeled PVLA as a probe under a fluorescence laser microscope. PVLA-coated surfaces were observed by scanning electron microscope and atomic force microscopies under air and water, which showed that PVLA molecules were adsorbed patchily on low density surfaces and uniformly concentrated all over the dish on high density surfaces. It is suggested from the requirement of the Ca2+ ion, inhibition of galactosyl substances, and localization of receptors that the adhesion of hepatocytes to both low and high PVLA-density surfaces is mediated by galactose-specific interactions between PVLA and asialoglycoprotein receptors. At low PVLA densities (0.07 micrograms cm-2), the hepatocytes were flat and expressed high levels of 3H-thymidine uptake and low levels of bile acid secretion. Contrastingly, at high PVLA densities (1.08 micrograms cm-2), they were round and expressed a low level of 3H-thymidine uptake and a high level of bile acid secretion. The shapes, proliferation, and differentiation of hepatocytes could be regulated by varying the densities of PVLA adsorbed to polystyrene dishes. We assume that there are two recognition mechanisms operating between PVLA and hepatocytes: (1) adhesion through highly concentrated or clustered galactose-specific interaction; and (2) responses in shape, proliferation, and differentiation by PVLA-coating densities.

Animals↗