Intracellular Localization of Mycoplasma bovis in the Bronchiolar Epithelium of Experimentally Infected Calves.

Lung tissues from calves infected experimentally with Mycoplasma bovis were examined by immunohistochemistry and electron microscopy. All inoculated calves had dark red areas of consolidation affecting both left and right lungs, which were characterized microscopically by subacute purulent bronchiolitis with hyperplasia of the surrounding lymphoid tissue. Immunohistochemically, M. bovis antigen was detected on the surface and inside the cytoplasm of bronchiolar epithelial cells in the pneumonic foci. The antigen was also found in the cytoplasm of phagocytes at the margin of bronchiolar exudates. Electron microscopically, numerous organisms were demonstrated in the immunohistochemically-positive sites. These findings suggest that M. bovis organisms adhere to the bronchiolar epithelium and at least some of them invade the epithelium.

Effects of biotin on glucotoxicity or lipotoxicity in rat pancreatic islets.

Biotin (vitamin H) plays an important role as a cofactor in glucose or lipid metabolism. We showed that biotin potentiated glucose-induced insulin release in isolated rat islets, while biotin alone did not affect insulin release. Coculture with biotin in islets for 48 hours significantly enhanced glucose-induced insulin release or islet insulin content. Similarly, preproinsulin or pancreatic/duodenal homeobox-1 (PDX-1) mRNA was also enhanced in islets cultured with biotin for 48 hours. Furthermore, we measured effects of biotin on beta-cell function under glucotoxic or lipotoxic states. In islets cultured with high glucose or palmitate for 48 hours, glucose-induced insulin release or islet insulin content deteriorated. Coculture with biotin significantly restored glucose-induced insulin release or islet insulin content together with the restoration of preproinsulin or PDX-1 mRNA. We conclude that biotin exerts its beneficial effects on beta-cell dysfunction induced by glucose or free fatty acids probably through the enhancement of insulin biosynthesis.

Effects of bezafibrate on beta-cell function of rat pancreatic islets.

Bezafibrate is an activator of peroxisome proliferator-activated receptors (PPAR) alpha. The present study was performed to investigate the effects of bezafibrate and the PPAR alpha activator, 4-Cholro-6-(2.3-xylidino)-2-pyrimidin-ylthio acetic acid (WY14643), on the beta-cell function of rat pancreatic islets in vitro. In islets cultured with 300 microM bezafibrate or WY14643 for 8 h, a low glucose concentration induced insulin release and increased the levels of mRNA for PPAR alpha, acyl CoA oxidase, carnitine palmitoyl transferase-1, pyruvate dehydrogenase E1 alpha or pyruvate carboxylase. In contrast, after a 48-h culture period, a high glucose concentration induced insulin release and islet insulin content, but decreased the levels of mRNA for glucose transporter-2 (GLUT-2), preproinsulin or pancreatic/duodenal homeobox-1. Diazoxide, the KATP channel opener, restored these responses. We conclude that bezafibrate enhances insulin release through the activation of PPAR alpha gene expression during a short culture period, whereas it may contribute to beta-cell dysfunction through the mechanism of "excessive stimulation" during longer culture periods.

The role of cyclic AMP in the pathogenesis of glucose desensitization in rat pancreatic islets.

Adenosine-3',5'-cyclic monophosphate (cyclic AMP) promotes exocytosis of insulin in pancreatic beta cells. This study was performed to investigate the role of cyclic AMP in the pathogenesis of glucose desensitization in rat pancreatic islets. In islets cultured with high glucose for 48 hours, 27 mmol/L glucose-induced insulin release was markedly impaired, while 3.3 mmol/L glucose-or arginine-induced insulin release was enhanced, indicating glucose desensitization. Islet cyclic AMP content was 190% enhanced in high glucose-culture islets for 48 hours. In islets cultured with dibutyryl-cyclic AMP (dbcAMP) or 3-isobutyl methy-xanthine (IBMX), islet insulin content or 27 mmol/L glucose-induced insulin release was deteriorated. In contrast, 3.3 mmol/L glucose- or arginine-induced insulin release was increased, which was similar to glucose-desensitized islets. Wash-out of dbc AMP for the last 24 hours of the 48-hour culture period restored impaired high glucose-induced insulin release in the same manner as wash-out of high glucose. Diazoxide, the KATP channel opener, also restored impaired high glucose-induced insulin release from dbcAMP-cultured islets. The data suggest that enhancement of cyclic AMP in high glucose-culture islets may be one of the pathogenesis of glucose desensitization.

Saturated non-esterified fatty acids stimulate de novo diacylglycerol synthesis and protein kinase c activity in cultured aortic smooth muscle cells.

AIMS/HYPOTHESIS: Insulin resistance is linked with a cluster of multiple risk factors and excessive acceleration of atherosclerosis. The underlying mechanism is not, however, fully understood. METHODS: To determine the link between insulin resistance and altered vascular function, we focused on the effect of various non-esterified fatty acids on diacylglycerol-protein kinase C pathway and mitogen-activated protein kinase activity in cultured aortic smooth muscle cells. RESULTS: Incubation of the cells with saturated non-esterified fatty acids (200 micromol/l) for 24 h, such as palmitate or stearate, induced a significant increase in diacylglycerol concentrations by about fivefold or eightfold, respectively, whereas oleate induced a slight increase in diacylglycerol concentrations by 1.8-fold and arachidonate induced none. In addition, the increased diacylglycerol concentrations induced by palmitate were completely restored to control concentrations by triacsin C, acyl-CoA synthetase inhibitor. These results suggest that saturated non-esterified fatty acids may increase diacylglycerol concentrations through de novo pathway by stepwise acylation. In parallel with the increased diacylglycerol, incubation of the cells with saturated non-esterified fatty acids significantly induced the activation of protein kinase C and mitogen-activated protein kinase. The palmitate-induced increase in mitogen-activated protein kinase activity was restored to control concentrations by GF109203X (5 x 10(-7) mol/l), a specific protein kinase C inhibitor, suggesting a protein kinase C-dependent activation of mitogen-activated protein kinase. CONCLUSION/INTERPRETATION: Saturated non-esterified fatty acids induced an increase in de novo diacylglycerol synthesis and subsequent activation of protein kinase C and mitogen-activated protein kinase in cultured aortic smooth muscle cells. This could contribute to the altered vascular functions in the insulin resistant state.

Effects of free fatty acids on beta-cell functions: a possible involvement of peroxisome proliferator-activated receptors alpha or pancreatic/duodenal homeobox.

It is well known that acute administration of fatty acids enhances insulin release from beta cells, although chronic exposure to fatty acids inhibits insulin release (lipotoxicity). The mechanism for these reciprocal effects of fatty acids on insulin release remains to be elucidated. The present study was performed to investigate the effects of fatty acids on gene expression related to glucose metabolism or insulin biosynthesis. In islets cultured with palmitate for 8 hours, glucose-induced insulin release was enhanced together with increment of pyruvate carboxylase (PC) mRNA or peroxisome proliferator-activated receptors (PPAR)alpha. In contrast, by extending the culture period up to 48 hours, glucose-induced insulin release or islet insulin content was significantly impaired by the coexistence of palmitate. Concomitantly, PC, PPARalpha, GLUT-2, glucokinase (GK), preproinsulin, or pancreatic/duodenal homeobox-1 (PDX-1) mRNA were significantly suppressed in those islets cultured for 48 hours with palmitate. These data may imply that during short-term culture period palmitate promotes PPARalpha gene expression, which enhances PC mRNA expression leading to the enhancement of insulin release, whereas during long-term culture period, palmitate rather inhibits PPARalpha mRNA, which reduces PC mRNA expression. Furthermore, palmitate reduces GLUT-2, GK, or preproinsulin mRNA expression probably through the inhibition of PDX-1 mRNA.

Homology study of two polyhydroxyalkanoate (PHA) synthases from Pseudomonas aureofaciens.

Recently, we have cloned and analyzed two polyhydroxyalkanoate (PHA) synthase genes (phaC1 and phaC2 in the pha cluster) from Pseudomonas aureofaciens. In this report, the deduced amino acid (AA) sequences of PHA synthase 1 and PHA synthase 2 from P. aureofaciens are compared with those from three other bacterial strains (Pseudomonas sp. 61-3, P. oleovorans and P. aeruginosa) containing the homologous pha cluster. The level of homology of either PHA synthase 1 or PHA synthase 2 was high with each enzyme from these three bacterial strains. Furthermore, multialignment of PHA synthase AA sequences implied that both enzymes of PHA synthase 1 and PHA synthase 2 were highly conserved in the four strains including P. aureofaciens.

High glucose level and free fatty acid stimulate reactive oxygen species production through protein kinase C--dependent activation of NAD(P)H oxidase in cultured vascular cells.

Recent studies have revealed that vascular cells can produce reactive oxygen species (ROS) through NAD(P)H oxidase, which may be involved in vascular injury. However, the pathological role of vascular NAD(P)H oxidase in diabetes or in the insulin-resistant state remains unknown. In this study, we examined the effect of high glucose level and free fatty acid (FFA) (palmitate) on ROS production in cultured aortic smooth muscle cells (SMCs) and endothelial cells (ECs) using electron spin resonance spectroscopy. Exposure of cultured SMCs or ECs to a high glucose level (400 mg/dl) for 72 h significantly increased the free radical production compared with low glucose level exposure (100 mg/dl). Treatment of the cells for 3 h with phorbol myristic acid (PMA), a protein kinase C (PKC) activator, also increased free radical production. This increase was restored to the control value by diphenylene iodonium, a NAD(P)H oxidase inhibitor, suggesting ROS production through PKC-dependent activation of NAD(P)H oxidase. The increase in free radical production by high glucose level exposure was completely restored by both diphenylene iodonium and GF109203X, a PKC-specific inhibitor. Exposure to palmitate (200 micromol/l) also increased free radical production, which was concomitant with increases in diacylglycerol level and PKC activity. Again, this increase was restored to the control value by both diphenylene iodonium and GF109203X. The present results suggest that both high glucose level and palmitate may stimulate ROS production through PKC-dependent activation of NAD(P)H oxidase in both vascular SMCs and ECs. This finding may be involved in the excessive acceleration of atherosclerosis in patients with diabetes and insulin resistance syndrome.

Retinal expression, regulation, and functional bioactivity of prostacyclin-stimulating factor.

Prostacyclin-stimulating factor (PSF) acts on vascular endothelial cells to stimulate the synthesis of the vasodilatory molecule prostacyclin (PGI2). We have examined the expression, regulation, and hemodynamic bioactivity of PSF both in whole retina and in cultured cells derived from this tissue. PSF was expressed in all retinal cell types examined in vitro, but immunohistochemical analysis revealed PSF mainly associated with retinal vessels. PSF expression was constitutive in retinal pericytes (RPCs) but could be modulated in bovine retinal capillary endothelial cells (RECs) by cell confluency, hypoxia, serum starvation, high glucose concentrations, or inversely by soluble factors present in early vs. late retinopathy, such as TGF-beta, VEGF, or bFGF. In addition, RPC-conditioned media dramatically increased REC PGI2 production, a response inhibited by blocking PSF with a specific antisense oligodeoxynucleotide (ODN). In vivo, PGI2 increased retinal blood flow (RBF) in control and diabetic animals. Furthermore, the early drop in RBF during the initial weeks after inducing diabetes in rats, as well as the later increase in RBF, both correlated with levels of retinal PSF. RBF also responded to treatment with RPC-conditioned media, and this effect could be partially blocked using the antisense PSF ODN. We conclude that PSF expressed by ocular cells can induce PGI2, retinal vascular dilation, and increased retinal blood flow, and that alterations in retinal PSF expression may explain the biphasic changes in RBF observed in diabetes.

The meaning of serum levels of advanced glycosylation end products in diabetic nephropathy.

It has been reported that advanced glycosylation end products (AGEs) play an important role in the development of diabetic complications. To evaluate the relationship between serum AGEs and diabetic nephropathy, we measured serum AGE levels in diabetic patients with normoalbuminuria (N), microalbuminuria (M), overt proteinuria (O), and hemodialysis (HD), non diabetic patients with nephropathy, and age-matched control subjects using the enzyme-linked immunosorbent assay (ELISA). Urine AGE levels were also measured in these subjects except group HD. Serum AGE levels in diabetic patients were not significantly higher than those in the normal subjects. When we compared serum AGE levels among various stages of diabetic nephropathy, groups O and HD had significantly higher serum AGE levels than the other groups. Serum AGE levels in group HD were almost 6-fold higher than those in groups N and M. In contrast, there were no significant differences in urinary AGE levels among any diabetic groups. As for the variables that determine serum AGE levels in diabetic patients, there was no significant correlation between serum AGEs and fasting blood glucose, hemoglobin A1c (HbA1c), or duration of diabetes. In contrast, serum AGEs showed a strong correlation with serum creatinine and an inverse correlation with creatinine clearance. To evaluate the relationship between serum AGEs and oxidative stress in diabetic nephropathy, urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and serum malondialdehyde (MDA), which are biological markers of total oxidative stress in vivo, were also examined. Both urinary 8-OHdG and serum MDA levels were significantly higher in diabetic patients with proteinuria versus those without proteinuria. However, there was no significant correlation between serum AGEs and urinary 8-OHdG or serum MDA levels in diabetic patients. These results suggest that the accumulation of serum AGEs in diabetic nephropathy may be mainly due to decreased removal in the kidney rather than increased production by high glucose levels or oxidative stress.

High incidence of silent myocardial ischemia in elderly patients with non insulin-dependent diabetes mellitus.

The present study was designed to reveal the incidence of silent myocardial ischemia in asymptomatic elderly non-insulin-dependent diabetic (NIDDM) patients (aged over 60 years). As a first step screening, maximal treadmill exercise test was performed. Of 140 patients studied, 54 (38.6%) were unable or not expected to achieve diagnostic levels of exercise during treadmill testing. A positive exercise test was noted in 39 of 86 (45.3%) subjects. As a second step examination, dipyridamole thallium scintigraphy was performed for 93 subjects who exhibited a positive exercise test and could not perform a maximal exercise test. Abnormal perfusion pattern was found in 39 of 93 (41.9%), who were finally considered to have a silent myocardial ischemia. Coronary angiography was performed in 18 subjects with diagnosis of silent myocardial ischemia, who gave their consent. Significant coronary artery stenosis was in fact found in 17 of 18 (94.4%) subjects studied, confirming a very high positive predictive value of this diagnostic procedure. In conclusion, elderly NIDDM patients (aged over 60 years) had an extremely high prevalence (estimated 26.3%) of silent myocardial ischemia. This evidence suggests that early and intensive detection may be needed as a part of routine care for this group.

Chronic sulfonylurea treatment and hyperglycemia aggravate disproportionately elevated plasma proinsulin levels in patients with type 2 diabetes.

It is established that disproportionately elevated plasma proinsulin levels occur in patients with Type 2 diabetes. In the present study, multivariate analysis was performed to determine what factors contributed to the disproportionately elevated plasma proinsulin levels in Japanese patients with Type 2 diabetes (n=276). Results from univariate analysis showed that both fasting proinsulin/C-peptide ratio and proinsulin/IRI ratio were approximately 2-fold higher in patients with Type 2 diabetes than those in healthy nondiabetic subjects (n=45). In patients with Type 2 diabetes, both proinsulin/C-peptide ratio and proinsulin/IRI ratio were significantly positively correlated with fasting plasma glucose level (FPG) and HbA1c. Neither proinsulin/C-peptide ratio nor proinsulin/IRI ratio was significantly correlated with BMI. Sulfonylurea-treated subjects had a significant elevation in both proinsulin/C-peptide ratio and proinsulin/IRI ratio compared with diet-treated subjects, whereas nonsulfonylurea hypoglycemic agent-treated subjects did not. Multivariate analysis confirmed that sulfonylurea treatment and FPG were significant determinants of both fasting proinsulin/C-peptide ratio (P=0.006 and P=0.030, respectively) and proinsulin/IRI ratio (P=0.003 and P=0.016, respectively) in patients with Type 2 diabetes. These results imply that disproportionate hyperproinsulinemia may reflect an excessive overwork of beta cells under chronic sulfonylurea treatment as well as hyperglycemia.

Oxidative stress measurement by in vivo electron spin resonance spectroscopy in rats with streptozotocin-induced diabetes.

Enhanced oxidative stress in diabetic patients may contribute to the pathogenesis of diabetic angiopathy. We have recently developed a method to determine the electron spin resonance (ESR, electron paramagnetic resonance; EPR) of reactive oxygen species and free radicals in vivo, using the nitroxide derivative, carbamoyl-PROXYL as a probe. In this study, diabetes was induced in Wistar rats by streptozotocin (STZ) injection (65 mg/kg, body weight, intravenously). Two, 4, and 8 weeks later, the animals received carbamoyl-PROXYL (300 nmol/g, intravenously), and ESR was measured at the upper abdominal level at a frequency of 300 MHz. The intensity of the carbamoyl-PROXYL ESR signal decreased gradually after the injection, and the spin clearance rate was determined over the first 5 min. At all time points, the spin clearance rate was significantly greater in the diabetic rats than in control rats. Moreover, the spin clearance rate in the diabetic rats was significantly correlated with urinary malondialdehyde (MDA) levels, which serve as a marker for lipid peroxidation. Daily treatment with 4 units neutral protamin Hagedorn (NPH) insulin for 4 weeks reduced the spin clearance rate in the diabetic rats. Simultaneous injection of carbamoyl-PROXYL and superoxide dismutase reduced the spin clearance rate in the diabetic rats in a dose-dependent manner. Injection of the antioxidant alpha-tocopherol (40 mg/kg, intraperitoneally) for 2 weeks restored the spin clearance rate in the diabetic rats without concomitant glycaemic restoration. These results suggest that a diabetic state enhances the generation of free radicals in vivo, and that both glycaemic control and antioxidant treatment can reduce this oxidative stress. Non-invasive in vivo ESR measurement may be useful for evaluating oxidative stress in diabetes.

Subcutaneous or visceral adipose tissue expression of the PPARgamma gene is not altered in the fatty (fa/fa) Zucker rat.

We cloned 537 basepairs (bp) of rat partial peroxisome proliferator-activated receptor gamma2 (PPARgamma2) cDNA and examined the effect of fasting or obesity on the expression of two isoforms of rat PPARgamma, gamma1 and gamma2, in either subcutaneous or mesenteric adipose tissue specimens using an RNase A protection assay. In Wistar rats, expression of both isoforms was dramatically reduced after 48 hours of fasting in the two fat tissue specimens. In comparing genetically obese (fa/fa) Zucker rats and lean control rats, no significant difference was observed in expression of the two isoforms in either type of adipose tissue. From these findings, we conclude that the adipose tissue level of rat PPARgamma depends on nutritional deprivation but is not closely associated with either obesity or insulin resistance in obese Zucker rats.

Reduced expression of a novel peptide, prostacyclin-stimulating factor, in the kidneys of streptozotocin-induced diabetic rats.

Prostacyclin (PGI2) produced by vascular endothelial cells (ECs) is a potent vasoactive prostanoid involved in maintenance of vessel wall homeostasis. Reduced PGI2 synthesis by vascular ECs could be a mechanism of pathogenesis in the development of vascular lesions such as diabetic angiopathy. Recently, we purified and cloned a novel bioactive peptide, PGI2-stimulating factor (PSF), which stimulates PGI2 production by vascular ECs. PSF may act on vascular ECs in a paracrine and/or autocrine fashion to regulate PGI2 synthesis. Decreased PSF production in the vessel wall may result in an imbalance of prostanoid synthesis, leading to the development of vascular lesions such as diabetic angiopathy. Our immunohistochemical study demonstrated that PSF is located in vascular resident cells such as vascular smooth muscle cells (SMCs) and ECs, as well as in bronchial SMCs. Moreover, PSF mRNA was found to be expressed in various tissues in Wistar rats, particularly in the kidneys and lungs. The present study demonstrated that streptozotocin (STZ)-induced diabetic rats showed less PSF mRNA expression in the kidneys (PSF mRNA/28S rRNA ratio; STZ versus control; 1.7+/-0.2 versus 2.5+/-0.2, p < 0.05) and reduced immunohistochemical staining for PSF in arteries in the kidney. However, in the lungs, there were no changes in tissue PSF mRNA expression (STZ versus control; 10.9+/-0.9 versus 11.5+/-1.0, NS) or in the extent of PSF staining in bronchial SMCs of STZ-induced diabetic rats. These findings suggest that decreased expression of PSF in renal vessels of STZ-induced diabetic rats may cause an imbalance of prostanoid synthesis, leading to the development and progression of vascular damage in the kidney.

Effect of eicosapentaenoic acid on glucose-induced diacylglycerol synthesis in cultured bovine aortic endothelial cells.

Hyperglycemia has been postulated to increase diacylglycerol (DAG) level through de novo synthesis pathway and subsequently activate protein kinase C (PKC) in vascular cells, possibly leading to vascular dysfunction associated with diabetes. In this study, we examined the effect of eicosapentaenoic acid (EPA) on high glucose-induced increase in DAG level in cultured aortic endothelial cells (ECs). In ECs, total DAG level was significantly increased in the cells cultured with high glucose levels (400 mg/dl) compared with the cells with normal glucose levels (100 mg/dl). The addition of EPA completely prevented high glucose-induced increase in total DAG level. In contrast, other common fatty acids such as palmitate and oleate significantly stimulated DAG syntheisis, although arachidonate did not affect it. High glucose level significantly stimulated the incorporation of 3H-palmitate into DAG, while it did not affect the incorporation of 3H-arachidonate into DAG. The addition of EPA completely prevented the high glucose-induced increase in 3H-palmitate incorporation into DAG, while it did not affect the 3H-arachidonate incorporation. These findings suggest that EPA can prevent high glucose induced-increase in DAG level in ECs, probably by specifically inhibiting de novo synthesis at the step of acylation. EPA may be one of the candidates for clinical agents normalizing activation of DAG-PKC pathway in diabetic vascular tissues and preventing vascular complications associated with diabetes.

Broad spectrum and mode of action of an antibiotic produced by Scytonema sp. TISTR 8208 in a seaweed-type bioreactor.

A photobioreactor was constructed using anchored polyurethane foam strips (1 x 1 x 40 cm) fixed onto a stainless-steel ring to prevent flotation, as a biomass support material (BSM). This type of reactor was named a seaweed-type bioreactor. A filamentous cyanobacterium, Scytonema sp. TISTR 8208, which produces a novel cyclic dodecapeptide antibiotic, was immobilized in seaweed-type photobioreactor and cultivated with air containing 5% CO2 sparged at a gas flow rate of 250 mL/min under illumination at a light intensity of 200 mmol photon m-2 s-1. The antibiotic produced in the seaweed-type photobioreactor was purified by HPLC and examined regarding its spectrum and mode of action. The antibiotic effectively inhibited the growth of Gram-positive bacteria, pathogenic yeasts, and filamentous fungi, but it had only a weak effect on Gram-negative bacteria. Scanning electron micrograph analysis showed that the most characteristic change was swelling of the cells after exposure to the antibiotic. The antibiotic seems to alter the conformation of the microbial cell membrane, thereby changing its permeability, leading to osmotic shock.

Cloning and sequence analysis of the poly (3-hydroxyalkanoic acid)-synthesis genes of Pseudomonas acidophila.

Pseudomonas acidophila can grow with CO2 as a sole carbon source by the possession of a recombinant plasmid that clones genes that confer chemolithoautotrophic growth ability derived from the H2-oxidizing bacterium Alcaligenes hydrogenophilus. H2-oxidizing bacteria produce poly(3-hydroxybutyric acid) (PHB) from CO2, but recombinant P. acidophila can produce the more useful biopolymer poly(3-hydroxyalkanoic acid) (PHA). In this study, the pha genes of P. acidophila were cloned and a sequence analysis was carried out. A gene library was constructed using the cosmid vector pVK102. A recombinant cosmid carrying the pha genes was selected by the complementation of a PHB-negative mutant of Alcaligenes eutrophus H16. The resulting recombinant cosmid pIK7 contained a 14.8-kb DNA insert. Subcloning was done. and the recombinant plasmid pEH74 was selected by hybridization with the A. eutrophus H16 pha genes. Escherichia coli possessing pEH74 produced PHB, indicating that pEH74 contained the pha genes of P. acidophila. The nucleotide sequences of the PHA-synthesis genes phaA (beta-ketothiolase), phaB (acetoacetyl-CoA reductase), and phaC (PHA synthase) in pEH74 were determined. The homologies of phaA, phaB, and phaC between P. acidophila and A. eutrophus H16 were 64.7, 76.1 and 56.6%, respectively.

Increased mRNA expression of a novel prostacyclin-stimulating factor in human colon cancer.

We recently cloned a prostacyclin (PGl2)-stimulating factor (PSF), which stimulates PGl2 production by cultured vascular endothelial cells. Immunohistochemistry and Northern blot analysis demonstrated that PSF was highly expressed in colon cancer sites compared with normal colon mucosa obtained from the same patient, as well as in cultured adenocarcinoma cell lines compared with cultured normal colon mucosal cell lines. Increased levels of the PSF protein were detected in the culture media of these adenocarcinoma cells compared with levels in the culture media of normal mucosal cells. These results suggest that PSF is closely associated with carcinogenesis of colon mucosa.

High glucose induces alteration of gap junction permeability and phosphorylation of connexin-43 in cultured aortic smooth muscle cells.

Gap junction is thought to have a crucial role in maintaining tissue homeostasis. We examined the effect of a high glucose level on gap junctional intercellular communication (GJIC) activity in cultured vascular smooth muscle cells (VSMCs) using the fluorescent dye transfer method. After a 48-h incubation with 22 mmol/l glucose (high glucose level), GJIC activity of VSMCs was significantly reduced compared with incubation with 5.5 mmol/l glucose (normal glucose level) (P < 0.05). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA; 5 x 10(-8) mol/l), a protein kinase C (PKC) activator, for 1 h also reduced GJIC activity (P < 0.01). In addition, treatment of the cells with calphostin C, a specific PKC inhibitor, for 3 h completely restored the GJIC activity inhibited by the high glucose level. Western blot analysis showed that connexin 43 (Cx43), which is the major functional protein of gap junction, is present in multiphosphorylated forms: a nonphosphorylated form (P0) and phosphorylated forms (P1, P2, and P3). Incubation of VSMCs with a high glucose level significantly increased the density ratio of P3/P0 compared with a normal glucose level (P < 0.05). Similarly, treatment of the cells with TPA significantly increased the P3/P0 ratio compared with controls (P < 0.01). In addition, the increase in the P3/P0 density ratio induced by a high glucose level was restored to the control level by both staurosporine and calphostin C. These results suggest that the high glucose level induced the inhibition of GJIC activity in cultured VSMCs through excessive phosphorylation of Cx43, mediated by PKC activation. This may contribute to the development of the macroangiopathy associated with diabetes.