Modulation of cardiac connexin-43 by omega-3 fatty acid ethyl-ester supplementation demonstrated in spontaneously diabetic rats.

Previous data suggest that type 1 diabetes mellitus leads to the deterioration of myocardial intercellular communication mediated by connexin-43 (Cx43) channels. We therefore aimed to explore Cx43, PKC signaling and ultrastructure in non-treated and omega-3 fatty acid (omega-3) treated spontaneously diabetic Goto-Kakizaki (GK) rats considered as type 2 diabetes model. Four-week-old GK and non-diabetic Wistar-Clea rats were fed omega-3 (200 mg/kg/day) for 2 months and compared with untreated rats. Real-time PCR and immunoblotting were performed to determine Cx43, PKC-epsilon and PKC-delta expression. In situ Cx43 was examined by immunohistochemistry and subcellular alterations by electron microscopy. Omega-3 intake reduced blood glucose, triglycerides, and cholesterol in diabetic rats and this was associated with improved integrity of cardiomyocytes and capillaries in the heart. Myocardial Cx43 mRNA and protein levels were higher in diabetic versus non-diabetic rats and were further enhanced by omega-3. The ratio of phosphorylated (functional) to non-phosphorylated Cx43 was lower in diabetic compared to non-diabetic rats but was increased by omega-3, in part due to up-regulation of PKC-epsilon. In addition, pro-apoptotic PKC-delta expression was decreased. In conclusion, spontaneously diabetic rats at an early stage of disease benefit from omega-3 intake due to its hypoglycemic effect, upregulation of myocardial Cx43, and preservation of cardiovascular ultrastructure. These findings indicates that supplementation of omega-3 may be beneficial also in the management of diabetes in humans.

Symbolic approach to verification and control of deterministic/probabilistic Boolean networks.

A Boolean network (BN) is well known as one of the models of biological networks such as gene regulatory networks, and has been extensively studied. In this study, for a BN, the verification/control problems are discussed. First, a probabilistic model including both synchronous and asynchronous Boolean dynamics is derived. This model can be generalised as a probabilistic BN. Next, a solution method of the verification/control problems is proposed, based on a probabilistic model checker PRISM. Finally, the PRISM-based method is applied to an apoptosis network and a WNT5A network. The proposed approach provides us an easy and convenient tool for analysis and control of biological networks.

Expression of (pro)renin receptor in the human brain and pituitary, and co-localisation with arginine vasopressin and oxytocin in the hypothalamus.

(Pro)renin receptor [(P)RR], a specific receptor for renin and prorenin, is a 350 amino acid protein with a single transmembrane domain. In the present study, the expression of (P)RR in the human brain and pituitary, and its co-localisation with arginine vasopressin and oxytocin in the human hypothalamus were studied by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Human brain and pituitary tissues were obtained at autopsy from the subjects without neurological or endocrinological disorders. The antiserum against (P)RR was raised in a rabbit by injecting the peptide fragment of human (P)RR corresponding to 224-237 amino acids conjugated with bovine serum albumin. Quantitative RT-PCR showed that (P)RR mRNA was widely expressed in every region of brain examined and pituitary, with the highest expression levels found in the pituitary and frontal lobe. Immunocytochemistry showed that (P)RR was expressed in the paraventricular and supraoptic nuclei of human hypothalami, and in anterior pituitary cells. Immunostaining of serial sections showed that (P)RR was co-localised with arginine vasopressin and oxytocin in the magnocellular neurones of the paraventricular and supraoptic nuclei. The preabsorption of the antibody by the antigen peptide abolished the immunostaining of (P)RR in the human hypothalamus. The present study has shown that (P)RR mRNA is widely expressed in the human brain and pituitary, consistent with the hypothesis that (P)RR is related to the various brain functions, such as cognitive function and brain development. Co-localisation of (P)RR with vasopressin in the hypothalamus raised the possibility that (P)RR may be related to the central control of water-electrolyte metabolism and blood pressure.

Correlative association between circadian expression of mousePer2 gene and the proliferation of the neural stem cells.

We examined the circadian expression of mousePeriod (mPer) genes (mPer1 and mPer2) and the proliferation of the neural stem cells in vitro. The neural stem cells from the ganglionic eminence of embryonic mice were expanded by the neurosphere method and then treated with epidermal growth factor (EGF) to stimulate their mitotic activity. The time courses of the proliferation were examined by WST-8 assay and bromodeoxyuridine (BrdU) incorporation assay and the expression of mPer1 and mPer2 genes was examined by RT-PCR and immunocytochemistry. We have found that EGF treatment elicited the circadian change in both the increase in viable cell number and DNA synthesis activity of the neural stem cells. Also, the gene expression of mPer2, but not mPer1, changed rhythmically with a period of 24 h and correlated negatively with the DNA synthesis activity rhythm. Furthermore, the treatment with an antisense oligonucleotide against mPer2 increased the DNA synthesis activity of the neural stem cells. These results suggest that mPer2 might periodically suppress the proliferation of neural stem cells.

Adhesion dependent release of hepatocyte growth factor and interleukin-1 receptor antagonist from human blood granulocytes and monocytes: evidence for the involvement of plasma IgG, complement C3 and beta2 integrin.

OBJECTIVE: Evolving evidence of anti-inflammatory effects is observed in patients with rheumatoid arthritis or ulcerative colitis following periodic adsorptive granulocyte and monocyte (GM) apheresis with a column containing cellulose acetate (CA) beads as apheresis carriers. This study was undertaken to obtain insights into mechanisms of anti-inflammatory actions of adsorptive GM apheresis with CA beads. METHODS: In a series of in-vitro experiments, we investigated the effects of plasma proteins and the leucocytes beta2 integrin (CD18) on granulocyte adsorption to CA beads. RESULTS: Granulocyte adsorption to CA beads required plasma IgG, the complement C3 and was inhibited by an antibody to leucocytes CD18. Further, hepatocyte growth factor (HGF) and interleukin-1 receptor antagonist (IL-1ra) which have strong anti-inflammatory actions were released by granulocytes that adhered to CA beads. CONCLUSIONS: Plasma IgG, C3 derived complement activation fragments and leucocytes CD18 are involved in granulocyte adhesion to CA beads and hence the release of HGF and IL-1ra.

Cellulose acetate beads induce release of interleukin-1 receptor antagonist, but not tumour necrosis factor-alpha or interleukin-1beta in human peripheral blood.

OBJECTIVE: Dramatic improvements in clinical symptoms of rheumatoid arthritis and ulcerative colitis were observed after patients received granulocyte and monocyte adsorptive apheresis with a column containing cellulose acetate (CA) beads as adsorptive carriers. This study was to investigate the effect of CA beads on the generation of anti-inflammatory and pro-inflammatory cytokines in human blood. MATERIALS AND METHODS: We incubated human whole blood with CA beads at 37 degrees C for up to 2 h and measured tumour necrosis factor-alpha (TNF-alpha) interleukin-1beta (IL-1beta) and IL-1 receptor antagonist (IL-1ra) produced by leucocytes. IL-1ra was also measured at the inflow and outflow of a column containing CA beads as leucocyte adsorptive carriers for the treatment of patients with ulcerative colitis. RESULTS: CA beads induced significant release of IL-1ra from leucocytes, but not TNF-alpha or IL-1beta. In contrast, all three cytokines were released when leucocytes were stimulated with lipopolysaccharide. IL-1ra was also markedly elevated in the outflow of the leucocyte apheresis column. CONCLUSIONS: These results indicate that CA beads selectively induce IL-1ra release from leucocytes which should contribute to the anti-inflammatory effect of granulocyte and monocyte adsorptive apheresis with CA beads as apheresis carriers.

Genetics of cognition: outline of a collaborative twin study.

A multidisciplinary collaborative study examining cognition in a large sample of twins is outlined. A common experimental protocol and design is used in The Netherlands, Australia and Japan to measure cognitive ability using traditional IQ measures (i.e., psychometric IQ), processing speed (e.g., reaction time [RT] and inspection time [IT]), and working memory (e.g., spatial span, delayed response [DR] performance). The main aim is to investigate the genetic covariation among these cognitive phenotypes in order to use the correlated biological markers in future linkage and association analyses to detect quantitative-trait loci (QTLs). We outline the study and methodology, and report results from our preliminary analyses that examines the heritability of processing speed and working memory indices, and their phenotypic correlation with IQ. Heritability of Full Scale IQ was 87% in the Netherlands, 83% in Australia, and 71% in Japan. Heritability estimates for processing speed and working memory indices ranged from 33-64%. Associations of IQ with RT and IT (-0.28 to -0.36) replicated previous findings with those of higher cognitive ability showing faster speed of processing. Similarly, significant correlations were indicated between IQ and the spatial span working memory task (storage [0.31], executive processing [0.37]) and the DR working memory task (0.25), with those of higher cognitive ability showing better memory performance. These analyses establish the heritability of the processing speed and working memory measures to be used in our collaborative twin study of cognition, and support the findings that individual differences in processing speed and working memory may underlie individual differences in psychometric IQ.

Sinusoidal endothelial cell injury by superoxide anion and iron in the Propionibacterium acnes-pretreated and lipopolysaccharide-stimulated rat liver.

AIMS/BACKGROUND: We attempted to measure the generation of superoxide anion, examine its site of release and determine its pathological role in Propionibacterium acnes-lipopolysaccharide-induced liver injury in the rat. METHODS: The P. acnes-pretreated (16 mg/kg i.v.) rat liver was perfused with buffer containing lipopolysaccharide (2.5 microg/ml). Chemiluminescence enhanced with Cypridina luciferin analog, MCLA, and reduction of nitro blue tetrazolium were used for detecting superoxide anion. Leakage of enzymes and release of cytokines into the perfusate, and histological specimens were also examined. RESULTS: Superoxide dismutase-inhibitable chemiluminescence peaked at 30 min of lipopolysaccharide infusion and blue formazan precipitate was histochemically deposited mainly on hepatic macrophages. Purine nucleoside phosphorylase (PNP) activity in the perfusate, as a marker of sinusoidal endothelial cell injury, reached its maximum at 50 min and aspartate aminotransferase (AST) activity, as a marker of hepatocyte injury, reached a plateau at 90 min. Simultaneous treatment with superoxide dismutase and deferoxamine mesylate significantly suppressed the leakage of PNP and AST. Release of tumor necrosis factor-alpha and growth-related oncogene/cytokine-induced neutrophil chemoattractant-1 lagged behind PNP leakage. Light microscopy showed destruction of the sinusoids followed by hepatocyte necrosis. Electron microscopy revealed adherence of hepatic macrophages to sinusoidal endothelial cells. CONCLUSION: These results indicate that superoxide anion released from hepatic macrophages may induce sinusoidal endothelial cell injury via interaction with iron in the P. acnes-lipopolysaccharide-treated liver.

[Novel extracorporeal granulocyte apheresis column inhibits myocardial reperfusion injury: future clinical applications].

OBJECTIVES: Leukocyte infiltration is very important in myocardial ischemia/reperfusion injury. A new extracorporeal circulation therapy using a granulocyte-apheresis column (G-1) has recently been proved to be effective and safe in patients with rheumatoid arthritis and with ulcerative colitis. This study investigated whether this therapy would reduce myocardial ischemia/reperfusion injury. METHODS: Rabbits were subjected to 30 min ischemia followed by 180 min reperfusion with no treatment (control group: n = 12); or treated with extracorporeal blood circulation (2 ml/min) passing through the G-1 (G-1 group: n = 12), or sham column (sham group: n = 12). Infarct size was determined by tetrazolium staining and expressed as a percentage of the area at risk. RESULTS: Infarct size in the G-1 group (15.9 +/- 3.4%) was significantly (p < 0.01) smaller than in the control (34.7 +/- 3.9%) and sham (35.2 +/- 4.1%) groups. Granulocyte emigration into the ischemic myocardium assessed by histopathological score was significantly (p < 0.01) less in the G-1 group (0.58 +/- 0.17) than in the sham group (1.42 +/- 0.26). There was a significant (r = 0.78, p < 0.0001) positive correlation between this score and infarct size. Flow cytometry showed alterations in the adhesion molecule expression on granulocytes during the passage through the G-1, but not sham, columns as a low L-selectin and high Mac-1 form, which is associated with the inability to home to the inflamed site. CONCLUSIONS: Extracorporeal circulation with the G-1 column, a newly-developed clinically applicable therapy, appears to reduce infarct size at least in part by limiting the emigration capacity of granulocytes into the ischemic myocardium.

[Long-term follow-up of the Le Duc-Camey antireflux technique].

BACKGROUND: An effective ureteroileal reimplantation without reflux and obstruction is indispensable for lower urinary reconstruction or diversion using an ileal segment. Based on our earlier experience of afferent nipple valve malfunction of the Kock pouch, we performed Le Duc-Camey antireflux ureteroileal implantation. In the present study, we evaluated the long-term outcome of the Le Duc-Camey technique on the upper urinary tract in patients with a postoperative observation period of 2 years or more. PATIENTS AND METHODS: From 1989 to 1994, 39 patients (73 ureters) underwent Le Duc-Camey technique for prevention of reflux of a Kock pouch. In 28 patients the ileal reservoir was connected to the skin for cutaneous urinary diversion, and in 11 it was connected to the urethra for lower urinary reconstruction. The ureters were implanted into the reservoir through the anterior wall window of the reservoir. In most patients, excretory urography (DIP) was performed once a year after the first year. Retrograde cystography or voiding cystourethrography was performed within 12 months postoperatively. After the first year, retrograde study was performed once during the follow-up period in the majority of the patients. The mean follow-up period was 44.8 months (range 24-71 months). RESULTS: There was no urinary tract dilatation in 68 renal units (93.1%), while slight dilatation was noted in 3 (4.1%), moderate dilatation in 1 (1.4%), and marked dilatation due to stenosis of ureteroileal anastomosis in 1 (1.4%). Retrograde studies revealed absence of reflux in all patients. Another complication that related to this procedure was not observed in any patients. CONCLUSION: We conclude that Le Duc-Camey technique is effective in reflux prevention and protection of the upper urinary tract, and recommend it for use with detubularized ileal reservoir.

[The usefulness of three-dimensional helical CT for the detection of abnormalities of the auditory ossicles].

To evaluate the usefulness of three-dimensional (3D) helical CT for the detection of abnormalities of the auditory ossicles, 3D helical CT of the middle ear was performed in seven patients with hearing disorder. It revealed that 4 patients had congenital deficiency of the auditory ossicles, 2 patients with chronic otitis media had shortening of the incus and one patient with head injury had doubtful fracture of the incus. This study indicated that 3D helical CT of the middle ear can represent the auditory ossicles objectively and can offer detailed diagnosis.

The first observation of O2- generation at real time in vivo from non-Kupffer sinusoidal cells in perfused rat liver during acute ethanol intoxication.

Infusion of ethanol or phorbol myristate acetate (PMA) into the perfused rat liver immediately produces O2- which was detected directly by infusion of a Cypridina luciferin analogue, MCLA as a chemiluminescence reagent. The MCLA photon emission was inhibitable by SOD. Generation of O2- in the liver was further verified by nitroblue tetrazolium, formazan precipitate formation. Ethanol-induced O2- generation was unaffected by gadolinium chloride (GdCl3), an inhibitor of kupffer cells, while PMA induced O2- generation was completely abolished by GdCl3. Since PMA is a known stimulator of phagocytic cells including Kupffer cells, the results indicate, for the first time that ethanol stimulates a non-Kupffer cell population, probably liver sinusoid endothelial cell to produce O2-.

Induction by 5-aza-2'-deoxycytidine, an inhibitor of DNA methylation, of Le(y) antigen, apoptosis and differentiation in human lung cancer cells.

We recently developed a monoclonal antibody directed to carbohydrate antigen Le(y), BM-1/JIMRO, and found that expression of Le(y) antigen defined by BM-1/JIMRO was associated with the process of apoptosis, but not with cell proliferation or necrosis. In the present experiments, we examined with BM-1/JIMRO the effects of various differentiation inducers on the growth and expression of Le(y) antigen in human lung cancer A549 cells. We found that a specific inhibitor of methylation of DNA, 5-aza-2' deoxycytidine (ADC), could markedly induce expression of Le(y) antigen in association with induction of apoptosis and differentiation in the A549 cells. These results suggest that hypomethylation of DNA is involved in the molecular mechanisms of induction of Le(y) antigen, apoptosis and differentiation in the cells.

[Evaluation of AMI-25 enhanced MR imaging and enhanced helical CT for liver tumors].

To evaluate the diagnostic capability of superparamagnetic iron oxide enhanced MRI and enhanced helical CT, 17 patients with histologically or clinically diagnosed hepatocellular carcinoma or metastatic liver tumor and a total of 208 nodules were studied. The pulse sequences used were SE 500/20 (T1WI), SE 2000/20 (PDWI) and SE 2000/70 (T2WI). Contrast medium was administrated intravenously for more than 30 minutes at a dose of 10 mumoles Fe/kg. Enhanced MR images were obtained 30-90 minutes after the injection of contrast medium. With helical CT, 133 nodules were detected. With unenhanced MRI, 62 were detected on T1WI, 60 on PDWI and 77 on T2WI. On the other hand, with enhanced MRI, 165 were detected on T1WI, 190 on PDWI and 186 on T2WI. Enhanced MRI revealed the most nodules among these images (P < 0.05), and was especially effective in detecting small nodules less than 10 mm in diameter (P < 0.05). In conclusion, it was considered that superparamagnetic iron oxide enhanced MRI was useful for the detection of hepatic nodules and that the most adequate pulse sequence was PDWI.

Blueberry juice: preliminary evaluation as an oral contrast agent in gastrointestinal MR imaging.

PURPOSE: To evaluate the use of blueberry juice as an oral contrast agent in magnetic resonance (MR) imaging. MATERIALS AND METHODS: MR imaging of phantoms of blueberry juice and of manganese chloride solutions with different manganese concentrations were conducted. Patient trials were conducted to evaluate manganese blood levels and signal intensity changes on MR images of the abdomen. T1- and T2-weighted conventional spin-echo MR imaging was performed at various field strengths. Six radiologists graded image contrast; signal intensities were calculated. RESULTS: T1 and T2 relaxation curves for blueberry juice and manganese chloride showed similar signal intensity profiles as a function of manganese concentration. Blood concentrations of manganese were unchanged before and after an oral dose of blueberry juice that contained 18 mg of manganese. The results of patient studies indicated that the optimal oral dose of manganese in blueberry juice was 3-4 mg/dL. T1 and T2 shortening were apparent at field strengths of 0.2-1.5 T. CONCLUSION: At appropriate concentrations, blueberry juice has the potential to be an effective oral contrast agent for MR imaging.

[A comparison among 123I-IMP SPECT, EEG and MRI in patients with temporal lobe epilepsy].

N-isopropyl-p-[123I]iodoamphetamine (123I-IMP) single photon emission computed tomography (SPECT), electroencephalography (EEG), and magnetic resonance imaging (MRI) were performed in 19 patients with temporal lobe epilepsy during interictal stage. MRI demonstrated abnormal signal in mesial temporal lobe (hippocampus) in 10 of 19 patients and 123I-IMP SPECT showed a hypoperfusion area in 15 of 19 patients. When compared with EEG and MRI data, disagreement of the affected area was observed in 3 cases. In comparison of EEG and 123I-IMP SPECT data, disagreement of the affected area was observed in 6 cases. Although there were no disagreement in comparison of MRI and 123I-IMP SPECT. We made a reprojection data parallel to the hippocampus in 123I-IMP SPECT. These data demonstrated obviously a hypoperfusion area around the hippocampus. In cases within one month from seizure attack, wide hypoperfusion area was showed on 123I-IMP SPECT in comparison of abnormal signal area on MRI. It could be considered that a reprojection data parallel to the hippocampus was useful to know extent of hypoperfusion area in temporal lobe epilepsy.

[Contrast enhancement effect of new oral agent (blueberry juice) for upper abdominal MR imaging].

Manganese is known to affect relaxation times. Blueberry is naturally rich in manganese, and oral contrast effect by its squeezed juice (B. J) was expected. At some appropriate concentration of manganese within daily intake level, B. J worked as positive contrast agent on T1WIs and negative one on T2WIs simultaneously without any side effects. The negative effect on T2WIs could reduce ghost artifacts from motion of high intensity structures. B. J improved confirmation of gastroduodenal tract regardless of field strength and could be adapted for fast SE. We introduced a preliminary examination of B. J as an oral contrast agent for upper abdominal MRI.

[MR imaging findings in patients with epilepsy].

We retrospectively examined the MR imaging (MRI) findings in 144 patients with epilepsy (31 with temporal lobe epilepsy and 113 with other epilepsies). 110 cases (76.4%) showed abnormal findings such as spotty lesions in white matter, hippocampal atrophy and/or signal change, ventricular dilatation and/or deformity, developmental lesions, brain tumors and so on. Hippocampal atrophy and/or signal change was shown in 74.1% of temporal lobe epilepsy, a remarkably high percentage (P < 0.01) compared with the other types of epilepsies (18.1%). This finding means that hippocampal lesions may play a large part in the cause of temporal lobe epilepsy. Investigation of the relationship between clinical term and abnormal findings revealed that the longer the clinical term, the larger the number of hippocampal lesions, regardless of whether it is temporal lobe epilepsy or not. Thus hippocampal lesions may occur as a result of hypoxia accompanied with seizure. Therefore we recommend horizontal and/or vertical sections of hippocampus in MR imaging of all patients with epilepsy. Even though MR findings may reflect some secondary lesions, MRI will shed some light on the proper understanding of epilepsy.

[A case of benign paraganglioma arising in the middle mediastinum; 201Tl-SPECT for differentiation from malignant tumor].

A case of benign paraganglioma arising in the middle mediastinum was reported. 201Tl SPECT showed high accumulation in tumor on early images at 15 min and reduced on late images at 3 hours after infusion. The patient was a 57 year old female. In the contrast enhanced CT, 3 x 4 cm tumor with intensive enhancement was recognized at the right middle mediastinum. Under the radiological images, the tumor was surgically removed. The pathologic diagnosis was a low atypical nonfunctioning aortico-pulmonary paraganglioma. This report was suggested that 201Tl SPECT was useful for differential diagnosis of benign neurogenic mediastinal neoplasms.