Coronary capillary network remodeling and hypofibrinolysis in aged obese diabetic rats: Implications for increased myocardial vulnerability to ischemia

Despite the known abnormalities of cardiac function in patients with overt non-insulin dependent diabetes mellitus (NIDDM) the temporal changes of coronary capillary network remodeling leading to potential microcirculatory dysfunction have not been elucidated. To this end, left ventricular subendocardial capillary network of Otsuka Long-Evans Tokushima Fatty (OLETF) rats, characterized by hypertension, obesity, hyperglycemia, hyperinsulinemia and mild NIDDM, and control Long-Evans Tokushima (LETO) rats were investigated. Total capillary density in OLETF was significantly higher than that in LETO at 20 weeks, suggesting compensatory improvement of O₂ transport at early stages of NIDDM. The increase in capillary density in OLETF was lost at 40 and 60 weeks due to the decreases of intermediate capillary portions and venular capillary portions. Although capillary domain area (area innervated by single capillary) in OLETF was lower than that in LETO at 20 weeks, the values were similar between OLETF and LETO at 40 and 60 weeks, suggesting that adaptive improvement in the capacity for O₂ transport with a high perfusion was lost in late stages of NIDDM. Activity of plasma plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of proteo(fibrino)lysis, in OLETF was higher than that in LETO at 40 and 60 weeks, suggesting that increase of PAI-1 may downregulate compensatory adaptive capillary network remodeling by inhibiting proteolysis and angiogenesis in the cardiac interstitium. Loss of adaptive myocardial microcirculation may therefore contribute to increased vulnerability in ischemic injury and to cardiac dysfunction in NIDDM.     

Glycosaminoglycan structures required for strong binding to midkine,a heparin-binding growth factor

Midkine (MK), a heparin-binding growth factor, binds strongly to oversulfated structures in chondroitin sulfates (CSs) and heparan sulfate. To elucidate the carbohydrate structure actually involved in the strong binding, dissected brains from 13-day mouse embryos were incubated with [14C]-glucosamine. The labeled glycosaminoglycans were fractionated by MK-agarose affinity chromatography to a weakly binding fraction, which was eluted by 0.5 M NaCl, and a strongly binding fraction, which was eluted by higher NaCl concentrations. Among the unsaturated disaccharides released from the strongly binding fraction by chondroitinase ABC, DeltaDi-diSE with 4,6-disulfated N-acetylgalactosamine accounted for 32.3%, whereas its content was lower in the weakly binding fraction. Artificial CS-E structure was formed using N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase purified from squid or recombinant human enzyme. Analysis of the products and their interaction with MK revealed that E units without 3-O-sulfation of glucuronic acid are sufficient for strong binding, provided that they are present as a dense cluster. Among the sulfated disaccharides released by heparitinase digestion, the trisulfated one, DeltaDiHS-triS, was the most abundant in the strongly binding fraction and was lower in the weakly binding fraction. Together with results of previous studies, we concluded that the multivalent trisulfated heparin-like unit is another structure involved in strong binding to MK.     

Effects of endocrine disruptors on the formation of prostaglandin and arachidonoyl-CoA formed from arachidonic acid in rabbit kidney medulla microsomes

Under physiological conditions, small amounts of free arachidonic acid (AA) are released from membrane phospholipids, and cyclooxygenase (COX) and acyl-CoA synthetase (ACS) competitively act on this fatty acid to form prostaglandins (PGs) and arachidonoyl-CoA (AA-CoA). To explore the possible actions of endocrine disruptors on the metabolic fate of free AA into these two pathways, we investigated the effects of nonylphenol (NP), bisphenol A (BPA), di-n-butyl phthalate (DBP), benzyl-n-butyl phthalate (BBP) and di-2-ethylhexyl phthalate (DEHP) on the formation of PG and AA-CoA from 5 microM AA (close to the physiological concentration of the substrate) in rabbit kidney medulla microsomes. The kidney medulla microsomes were incubated with 5 microM [(14)C]-AA in 0.1 M Tris/HCl buffer (pH 8.0) containing cofactors of COX (reduced glutathione and hydroquinone) and cofactors of ACS (ATP, MgCl(2) and CoA). After incubation, PG (as total PGs) and AA-CoA were separated by selective extraction using petroleum ether and ethyl acetate. NP (1-200 microM) strongly enhanced the AA-CoA formation with a coincident decrease in the PG formation. BPA, DBP, BBP and DEHP failed to show any effect on the PG and AA-CoA formation up to 200 microM. Experiments utilizing 60 microM AA as the substrate concentration indicated that, under a low concentration of AA, NP decreases PG formation by inhibiting the COX activity, and reduces the AA flow into the COX pathway through inhibition on the COX activity, increasing availability of the substrate for the ACS and leading to enhanced AA-CoA formation. These results firstly show that NP has the potential to disturb the balance of PG and AA-CoA formations under normal physiological conditions.     

Regulation of the expression of two female-predominant CYP3A mRNAs (CYP3A41 and CYP3A44) in mouse liver by sex and growth hormones

A second female-predominant murine CYP3A, CYP3A44, was isolated from liver and its mRNA expression was compared with that of the previously described CYP3A41. The expression of CYP3A44 was relatively constant after birth in females, whereas it gradually declined in males after 5 weeks of age. The expression of CYP3A41 increased with age in females after 3 weeks of age, whereas it gradually declined in males after 5 weeks of age. Hypophysectomy and growth hormone replacement indicated that expression of both CYP3A mRNAs in females was dependent on the feminine plasma growth hormone profile. Estradiol induced the expression of both mRNAs and the effect was dependent on the presence of the pituitary gland. These observations suggest that endocrine control of expression might be similar, but not identical, for two female-predominant CYP3A mRNAs.     

Interleukin-1beta expression in human gastric carcinoma with Epstein-Barr virus infection

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1beta (IL-1beta) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1beta mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1beta increased the cell growth of a GC cell line, TMK1. Thus, IL-1beta may act as an autocrine growth factor in EBVaGC.     

Cloning and chromosomal mapping of human glucuronyltransferase involved in biosynthesis of the HNK-1 carbohydrate epitope

The HNK-1 carbohydrate is expressed on various cell adhesion molecules in the nervous system and is suggested to play a role in cell-cell and cell-substrate interactions. Here we describe the isolation of a cDNA encoding human glucuronyltransferase (GlcAT-P), which is a key enzyme in the biosynthesis of the HNK-1 carbohydrate. The primary structure deduced from the cDNA sequence predicted a type II transmembrane protein of 334 amino acids. Human GlcAT-P was 98.2% identical with rat GlcAT-P in amino acid sequence, the exception being the length of the cytoplasmic tail. Northern blot analysis indicated that human GlcAT-P is expressed mainly in the brain. There is a single copy of the human GlcAT-P gene (HGMW-approved symbol B3GAT1), and it was mapped to chromosome 11q25.     

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.     

Disinfection of microorganisms by use of electrochemically regenerated periodate

A new method for disinfection of microorganisms by electrochemically regenerated periodate was developed. Oxidation of iodate to periodate was observed at 1.25 V versus a silver/silver chloride electrode in a cyclic voltammogram of potassium iodate. When 1.25 V was applied in 1.0 mM potassium iodate, approximately 4-log inactivation of Escherichia coli was observed in 30 min.     

Age-related changes in cardiac expression of VEGF and its angiogenic receptor KDR in stroke-prone spontaneously hypertensive rats

We examined the age-related changes in cardiac expression of angiogenic molecules during the development of cardiac remodeling in stroke-prone spontaneously hypertensive rats (SHRSP) in comparison with those in Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR). Vascular endothelial growth factor (VEGF) was highly upregulated in SHRSP aged 20 weeks compared with the same age of WKY, but it was downregulated at 40 weeks. On the other hand, KDR, an angiogenic receptor of VEGF, and endothelial nitric oxide synthase, which is important in the VEGF-mediated angiogenic pathway, were markedly downregulated in SHRSP from 20 weeks of age. Such age-related changes in their expression levels seen in SHRSP were quite different from those in SHR. In both SHR and SHRSP, transforming growth factor-₁ (TGF-₁) expression was increased with age, although SHRSP showed more marked upregulation. Cardiac remodeling in SHRSP was characterized by decreased coronary capillary density, cardiomyocyte hypertrophy, and cardiac fibrosis. We conclude that, in addition to overexpression of TGF-₁, which appears to play a pivotal role in promoting cardiac hypertrophy and fibrosis, a defect of the VEGF-KDR system could result in impaired physiologic coronary angiogenesis in SHRSP, contributing to cardiac deteroration associated with myocardial ischemia in this malignant hypertensive model.     

Changes in PKB/Akt and calcineurin signaling during recovery in atrophied soleus muscle induced by unloading

Protein kinase B [PKB, also known as Akt (PKB/Akt)] and calcineurin (CaN) are postulated to play important roles in integrating intracellular signaling in skeletal muscle in response to disuse and increased muscle loading. These experiments investigated changes in signal transduction of the downstream pathways of PKB/Akt and CaN during recovery following disuse-induced muscle atrophy. A 10-day period of hindlimb unloading (HLU) via tail suspension (male rats) was used to produce soleus muscle atrophy. Muscle recovery was achieved by returning animals to normal ambulation for 3-10 days. HLU resulted in significant muscle atrophy and a slow-to-fast fiber transition as revealed by appearance of type IId/x and IIb myosin heavy chain (MHC) isoforms. Muscle mass in HLU animals recovered to control (Con) levels after 10 days of reloading, but the fast-to-slow shift in muscle MHC was incomplete, as indicated by the continued presence of type IId/x MHC. Ten days of HLU resulted in a significant decrease (-43%) in muscle levels of phosphorylated PKB/Akt. In contrast, muscle levels of phosphorylated PKB/Akt were greater (+56%) in HLU than in Con animals early after the onset of reloading (3 days). Soleus levels of phosphorylated p70S6K were significantly higher (+26%) in HLU animals after 3 days of muscle reloading. Muscle levels of phosphorylated PKB/Akt and phosphorylated p70S6K returned to Con levels by day 10 of recovery. Moreover, muscle CaN levels were significantly higher than Con levels after 10 days of muscle reloading. These findings are consistent with the hypothesis that PKB/Akt and its downstream mediators are active in the regrowth of muscle mass during the early periods of recovery from muscle atrophy. Our data support the concept that CaN is involved in muscle remodeling during the later phases of recovery from disuse muscle atrophy.