The basis examination of leukocyte-platelet aggregates with CD45 gating as a novel platelet activation marker.

INTRODUCTION: Platelet activation in circulation is considered to be associated with thrombosis and inflammation; thus, sensitive and easy-to-use markers are necessary. In this study, we established a simple and rapid protocol to clinically examine leukocyte-platelet aggregate formation associated with activated platelets in circulation. METHODS: Whole blood was stained with PC5-conjugated anti-CD45 monoclonal antibody and fluorescent isothiocyanate-conjugated anti-CD41 monoclonal antibody for leukocyte-platelet aggregate analysis. For platelet activation, 5 µm thrombin receptor-activated peptide (TRAP) or 2 µg/mL collagen was added. Samples were analyzed by EPICS XL (Beckman Coulter, Miami, FL, USA). Monocytes, neutrophils, and lymphocytes were gated based on differences in CD45 fluorescence intensity and side scatter. For each gate, the percentage (%) of platelets expressing CD41 was analyzed. Same drawing sample was stained with anti-CD62P monoclonal antibody. Platelet CD62P expression was then analyzed with gating for platelet cell population. RESULTS: We analyzed leukocyte-platelet aggregates and platelet CD62P expression in 18 healthy individuals. Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, increased when platelets were activated by platelet agonists. Monocyte-platelet aggregates and neutrophil-platelet aggregates also increased over time with mild platelet activation. CONCLUSION: Leukocyte-platelet aggregates, mainly monocyte-platelet aggregates, appear to be a sensitive marker of platelet activation in circulation.

Effect of κ-opioid receptor agonist on the growth of non-small cell lung cancer (NSCLC) cells.

BACKGROUND: It is becoming increasingly recognised that opioids are responsible for tumour growth. However, the effects of opioids on tumour growth have been controversial. METHODS: The effects of κ-opioid receptor (KOR) agonist on the growth of non-small cell lung cancer (NSCLC) cells were assessed by a cell proliferation assay. Western blotting was performed to ascertain the mechanism by which treatment with KOR agonist suppresses tumour growth. RESULTS: Addition of the selective KOR agonist U50,488H to gefitinib-sensitive (HCC827) and gefitinib-resistant (H1975) NSCLC cells produced a concentration-dependent decrease in their growth. These effects were abolished by co-treatment with the selective KOR antagonist nor-BNI. Furthermore, the growth-inhibitory effect of gefitinib in HCC827 cells was further enhanced by co-treatment with U50,488H. With regard to the inhibition of tumour growth, the addition of U50, 488H to H1975 cells produced a concentration-dependent decrease in phosphorylated-glycogen synthase kinase 3ß (p-GSK3ß). CONCLUSION: The present results showed that stimulation of KOR reduces the growth of gefitinib-resistant NSCLC cells through the activation of GSK3ß.

Toxicological evaluation of L-proline in a 90-day feeding study with Fischer 344 rats.

L-proline (L-Pro) is a non-essential amino acid, and has become widely used as supplements and health foods, recently. A subchronic oral toxicity study of L-Pro was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.625%, 1.25%, 2.5% and 5.0% of L-Pro for 90 days. No treatment-related clinical signs and mortality were noted. We observed no clear treatment-related effects with regard to body weight, food intake or urinalysis data. The average daily water intakes of the treated female groups were significantly increased compared to the controls. The hematology (red blood cell parameter) and serum biochemistry (glucose, blood urea nitrogen, creatinine or uric acid) of the treated male and/or female groups were lower than those of the control groups. However, these changes were lacked dose-dependence, and no abnormalities were found in corresponding pathological findings. In conclusion, the no-observed-adverse-effect-level (NOAEL) for L-Pro was determined to be a dietary dose of 5.0% (2772.9 mg/kg body weight/day for males and 3009.3mg/kg body weight/day for females) under the present experimental conditions.

Toxic effects of l-aspartic acid at high dose levels on kidneys and salivary glands in Fischer 344 rats detected in a 90-day feeding study.

A subchronic oral toxicity study of l-aspartic acid (l-Asp) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0%, 0.05%, 1.25%, 2.5% and 5.0% concentrations for 90 days. Serum biochemistry showed treatment-related decreases of blood urea nitrogen, creatinine and uric acid levels in both sexes. In addition, incidences of urinary ketone and protein were significantly increased in treated both sexes, while relative kidney weight was significantly increased in the 5.0% male rat, and regenerative renal tubules with tubular dilation were histopathologically observed in male rats of the 2.5% or greater groups. The observed renal injury was confirmed not to be due to accumulation of alpha2u-globulin. Acinar cell hypertrophy of salivary glands was histopathologically evident in male and female rats of the 2.5% or greater groups. The present results indicate that l-Asp causes toxic effects on kidneys and possibly salivary glands at high dose levels in male and female Fischer 344 rats. Such toxic effects were observed only in animals given 2.5% and/or higher doses of l-Asp. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Asp is 1.25% (696.6 mg/kg body weight/day for males and 715.2 mg/kg body weight/day for females) under the present experimental conditions.

Effects of tetrabromobisphenol A, brominated flame retardant, in ICR mice after prenatal and postnatal exposure.

Tetrabromobisphenol A (TBBPA), brominated flame retardant, is produced in the largest amounts globally for use in plastics or building materials. TBBPA has been detected in sediment, air at the dismantling plant or human serum samples. In the present study, we examined the effects of prenatal and postnatal exposure to TBBPA in mice. TBBPA (99.1% pure) in diet was administered to pregnant ICR mice at doses of 0% (control), 0.01%, 0.1% or 1.0% from gestational day 0 to weaning at postnatal day 27. The average daily food intake and body weight of dams showed no significant differences between the control and treated groups. There were no dose-related effects on reproductive data. Serum concentrations of total-cholesterol and liver weights of treated dams and offspring were higher than those of the control mice. Histological findings in treated dams or offspring showed the increase of focal necrosis of hepatocytes and inflammatory cell infiltration in the liver, and increase of dilation or atrophy of renal tubules and cyst in the kidney. TBBPA was developed as a new, safe class of flame retardant and was not highly toxic. However, the present data suggested that TBBPA caused a lipid metabolic disorder and hepatic or kidney lesion, under these conditions.

Impact of glycerol gateway molecule in adipocytes.

Glycerol is one of the essential nutrients in the mammalian body. Glycerol released from adipocytes is delivered to the liver and used for gluconeogenesis. The molecular mechanism of glycerol transport across the cell membrane remains unclear. AQPadipose, which we identified in human adipose cDNA project and later found to be human AQP7, is expressed in adipose tissue, and upregulated during fasting. AQP7 belongs to the aquaglyceroporin subfamily to permealize glycerol as well as water. Loss of function mutation of AQP7 in human caused disturbance of normal rise of plasma glycerol. Disruption of AQP7 gene in mice resulted in profound hypoglycemia during prolonged fasting because of impaired glycerol supply to the liver. In obesity, AQP7 is overexpressed in visceral fat,accompanied by portal hyperglycerolemia and systemic hyperglycemia. Considered together, these works indicate that AQP7 functions as a glycerol gateway molecule in adipocytes.

Suppression of TGF-beta signaling by conophylline via upregulation of c-Jun expression.

In the course of screening for inhibitors of transforming-growth factor-beta (TGF-beta) functions we found that conophylline, a vinca alkaloid, inhibited TGF-beta-induced apoptosis in rat hepatoma cells. Because conophylline also inhibited TGF-b-induced promoter activity in mink lung cells, we studied the mechanism of the inhibition in this cell line. Conophylline did not inhibit nuclear translocation of Smad2. Instead, we found that conophylline increased the expression of c-Jun, which had been earlier shown to interact with the corepressor TGIF to suppress the transcriptional activity dependent on Smad2. Conophylline attenuated the interaction between the Smad2 complex and p300 but enhanced that between the Smad2 complex and TGIF. In cells overexpressing c-Jun, suppression of promoter activity induced by TGF-beta and the enhancement of the association of the Smad2 complex with TGIF were also observed. Thus, our data suggest that inhibition of TGF-beta-induced promoter activity by conophylline can be attributed to its potency in modulating the interaction of downstream transcriptional factors via upregulation of c-Jun expression.

Beneficial effects of tea catechins on diet-induced obesity: stimulation of lipid catabolism in the liver.

OBJECTIVE: Obesity has increased at an alarming rate in recent years and is now a worldwide health problem. We investigated the effects of long-term feeding with tea catechins, which are naturally occurring polyphenolic compounds widely consumed in Asian countries, on the development of obesity in C57BL/6J mice. DESIGN: We measured body weight, adipose tissue mass and liver fat content in mice fed diets containing either low-fat (5% triglyceride (TG)), high-fat (30% TG), or high-fat supplemented with 0.1-0.5% (w/w) tea catechins for 11 months. The beta-oxidation activities and related mRNA levels were measured after 1 month of feeding. RESULTS: Supplementation with tea catechins resulted in a significant reduction of high-fat diet-induced body weight gain, visceral and liver fat accumulation, and the development of hyperinsulinemia and hyperleptinemia. Feeding with tea catechins for 1 month significantly increased acyl-CoA oxidase and medium chain acyl-CoA dehydrogenase mRNA expression as well as beta-oxidation activity in the liver. CONCLUSION: The stimulation of hepatic lipid metabolism might be a factor responsible for the anti-obesity effects of tea catechins. The present results suggest that long-term consumption of tea catechins is beneficial for the suppression of diet-induced obesity, and it may reduce the risk of associated diseases including diabetes and coronary heart disease.

Effects of soy protein diet on the expression of adipose genes and plasma adiponectin.

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.

Chronic toxicity of thiabendazole (TBZ) in CD-1 mice.

Male and female CD-1 mice (50 mice per group) were administered thiabendazole (TBZ) in diet at levels of 0 (control), 0.031, 0.125 and 0.5% for 78 weeks. A life time study was terminated after 78 weeks due to enhanced strain specific mortality. There were no significant differences in mortality between the control and treated groups. Mean body weights of high-dose groups showed significant decreases compared with the controls. The bladder weights of male and female mice of the 0.5% group were significantly higher than those of the control mice. Gross findings in treated mice included the renal atrophy, hydronephrosis, calculi in renal pelvis and/or bladder and ovarian atrophy. Microscopic findings in the kidneys of treated mice included the nephrosis, hydronephrosis or hyperplasia of transitional epithelium of renal pelvis or papilla. In the bladder of treated mice, hyperplasia or squamous metaplasia of transitional epithelium and one transitional cell papilloma were observed. Dose-dependent decreases in the incidence of spontaneous lesion in the male or female reproductive system were recognized. It is concluded that TBZ is not carcinogenic to CD-1 mice of both sexes. However, caution should be exercised in the long-term application of high TBZ doses.

Thiabendazole induces urinary tract toxicity in male ICR mice.

Male ICR mice were administered thiabendazole (TBZ) in the diet at concentration of 0 (control), 0.8, 1.2 and 1.6% for 44 weeks. The mortality was 10, 6, 40 or 90% in control, 0.8, 1.2 or 1.6% TBZ group, respectively. In dead mice, the gross findings included the abnormalities of kidney such as atrophy, hydronephrosis or swelling in 2, 67, 95 or 96% of the 0, 0.8, 1.2 or 1.6% TBZ group, respectively. In surviving mice at the end of study, the right kidney weight of treated groups was significantly lower than that of control group. The urinary bladder weight of treated groups was significantly higher than that of control group. Gross findings in treated mice included the renal atrophy, hydronephrosis, calculi in renal pelvis or urinary bladder and thickening of the bladder wall. Microscopic findings in the kidneys of treated mice included nephrosis, hydronephrosis and hyperplasia of transitional epithelium of renal pelvis and/or papilla. In the urinary bladder, hyperplasia or squamous metaplasia of transitional epithelium were found in treated mice. Administration of TBZ in the diet for 44 weeks results in nephrosis and calculus formation in the renal pelvis and urinary bladder of male ICR mice, and is associated with hyperplasia of transitional epithelium of renal pelvis or urinary bladder.

Goitrogenic effects of green tea extract catechins by dietary administration in rats.

The effects of green tea extract catechins on the rat thyroid were examined in a 13-week feeding study and subsequent 2-,4- and 8-week studies. Commercially available polyphenon-60 (P-60) which contains green tea extract catechins at 66.2% was used as a source of catechins. A basic diet containing different concentrations of P-60 was used for experiments. In the 13-week study, 10 rats of each sex were administered diets containing P-60 at 0 (control), 0.625, 1.25, 2.5 and 5.0%. Goiters were observed in the 13-week test. The mean thyroid weight of rats fed a diet containing 5.0% of P-60 (5.0% group) significantly increased to 444% of the control in males and to 304% of the control in females. Histological examinations of the thyroid of the 5.0% group revealed marked hypertrophy and/or hyperplasia of the follicles, some with depletion of colloid and some with rich colloid, and formation of a fibrous capsule. Slight hypertrophy of follicular cells was observed in male rats fed a diet containing 1.25% of P-60 (1.25% group) and female rats fed a diet containing 2.5% of P-60 (2.5% group). Degree and incidence of thyroid lesions were higher in males than in females in the 1.25, 2.5 and 5.0% groups. In the 2-8-week studies, five rats of each sex were given diets containing 0 (control) and 5.0% of P-60. In the 5.0% group, the mean thyroid weight in males significantly increased to 161% of the control as early as 2 weeks and increased to 357% of the control at 8 weeks. Histologically, these goiters were also associated with follicular cell hypertrophy/hyperplasia as in the 13-week study. The degree and incidence of thyroid lesions were higher in males than in females. These results indicate that dietary administration of the green tea extract catechins at high doses induced goiters in rats, and this may be due to antithyroid effects of catechins. In the 13-week study, the no-observed effect level (NOEL) of green tea extract catechins for F344 rats based on histological changes of the thyroid was considered to be 0.625% in males and 1.25% in females in the diet, respectively.

Teratogenicity of thujaplicin in ICR mice.

Beta-thujaplicin (TP) was studied by in vitro and in vivo tests for teratogenicity using ICR mice. In the in vitro study, TP (0, 3.125, 6.25, 12.5 microg/ml medium) dissolved in dimethyl sulfoxide (DMSO) was administered to cultured embryos on 9 day of gestation. After 24 hr of exposure to TP, the embryos were examined for developmental parameters and external anomalies. Growth retardation and embryos with facial dysplasia or hydrocyst of the tail tip were observed among the embryos given 12.5 microg/ml. In the in vivo study, TP (0, 420, 560, 750 or 1000 mg/kg) dissolved in olive oil was administered orally to pregnant mice on day 9 of gestation. All foetuses were removed from the uterus on day 18 of gestation, and were examined for external and skeletal anomalies. Various types of malformations were observed in the mice given 560 mg/kg or more. The number of litters having foetuses with external or skeletal anomalies increased in proportion to the dose of TP. The regression lines of Y (probit response) on X (log dose) for external anomalies was Y = 4.87X-8.43 . The 1% effective dose (ED1) for the malformation was 190 mg/kg. The present study shows that TP has teratogenic effects on mice.

Human and mouse ISLR (immunoglobulin superfamily containing leucine-rich repeat) genes: genomic structure and tissue expression.

We have previously reported a transcript of the novel gene for human immunoglobulin superfamily containing leucine-rich repeat (ISLR). By additional screening of a human retina cDNA library, we isolated another type of transcript with a 5' UTR different from that of the previously reported type. Genomic sequencing of the ISLR gene revealed that these two types of transcripts, ISLR-1 and ISLR-2, originated from the same gene but are composed of different first exons. Because the entire open reading frame is contained in the second exon, these two transcripts produce the same protein. Radiation hybrid mapping linked the ISLR gene to AFM248yh1, which is in the critical region of Bardet-Biedl syndrome type 4 (BBS4) on chromosome 15. Sequence analysis of the ISLR gene in five BBS4 patients, however, showed no mutations, although a few polymorphic changes were detected. Cloning of the mouse homolog of ISLR (Islr) revealed that the predicted protein consists of 428 amino acids, 86% of which are identical to those of ISLR. The Islr gene was expressed in various mouse tissues, including retina, in which Islr mRNA was detected in the ganglion cell layer, the inner nuclear layer, and the inner segment of the photoreceptor.

[Efficacy of mupirocin in eradicating methicillin-resistant Staphylococcus aureus from nasal discharge in carrying cardiovascular surgical patients].

Methicillin-resistant Staphylococcus aureus from nasal discharge was identified in 37 (2.5%) cardiovascular patients operated between 1995 and 1997; 25 male and 12 female, ranging from 1 to 83 years (mean 63); 2 were excluded because of Arbekacin or Isodine-gel treatment. The first 17 were treated with Vancomycin inhalation (V group) and eradication was considered to have been achieved when 3 consecutive negative cultures were obtained; the subsequent 18 were treated with Mupirocin (M group) and eradication was determined by 1 negative culture. In post-eradication electively operated 13 V and 15 M, postoperative MRSA infection was observed in one M (wound infection); the interval from the first nasal culture to the operation was 68 +/- 58 in V and 32 +/- 12 days in M, respectively (p < 0.05). In the remaining 7 who had to undergo emergency surgery while waiting for eradication because of progression of symptoms (2 V) or prior to instituting treatment (2 V, and 3 M), postoperative MRSA infection was observed in 2 M (both pneumonia). No deaths from infection were observed. Though the time required for conversion of the nasal culture was longer in V (13 +/- 20) than in M (7 +/- 1 days) differences were not significant. Mupirocin is easier to use, eradication can be achieved generally within a week.

Plastic surgical reconstruction of left main coronary artery.

OBJECTIVE(S): To report the early and mid-term results of surgical plasty of the left main coronary artery in 12 patients operated upon between 1993 and 1997. METHODS: The anterior approach was used in all patients. Saphenous vein (n = 4) of glutaraldehyde treated autologous pericardium (n = 8) were used as patch material. Additional coronary artery bypass grafting was performed in 7 patients, the first 3 as a safety back up, and for coexisting stenosis of other coronary branches not revascularized by the plastic procedure in the remaining 4. RESULTS: Pathologic specimens of the left main coronary artery in 5 revealed atheroma in 3 and myxomatous intimal thickening in 2. The left main coronary artery was widely patent angiographically in all patients prior to discharge. Six patients consented to angiographic restudy 5-40 months after the procedure and revealed excellent results in 5. One patient upon whom autologous pericardial patch had been used underwent percutaneous coronary angioplasty for restenosis of the left main coronary artery and a new lesion of the proximal left anterior descending branch 5 months after the operation. There were no late deaths nor other cardiac events. All patients were in CCS class 1 at their last follow-up. CONCLUSIONS: Surgical angioplasty of the left main coronary artery could be used to revascularize the left heart safely in patients with a discrete localized lesion of the left main coronary artery and is particularly useful in the face of unavailability of other conduits.

Cyanoglycosylation products of 17-O-acetyl-testosterone.

17-O-Acetyl testosterone, which has no susceptible hydroxyl or carboxyl group for glycosylation, was glycosylated with 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranosyl bromide in the presence of a mixed catalyst, Hg(CN)2 and HgBr2, in benzene-nitromethane. Reaction occurred on the alpha, beta-unsaturated ketone on the six-membered A-ring to give six 3-O-glycosides, each bearing a cyano group at the 3- or 5-position of the aglycon, and a 3-O-glycoside bearing a CONH2 group at the 3-position. Structural analyses of these products were carried out by various NMR (1H, 13C NMR, 1H-1H and 1H-13C COSY, HMBC, and DEPT), FABMS and X-ray analyses. The mechanisms of the formations of the products are discussed. It was determined that mercuric cyanide was essential as a catalyst for the progress of the cyanoglycosylation.

Mouse myocilin (Myoc) gene expression in ocular tissues.

Human myocilin is identical to TIGR (trabecular meshwork inducible glucocorticoid response) which is responsible for the pathogenesis of juvenile-onset primary open angle glaucoma (GLCIA). We have isolated cDNA for mouse myocilin (Myoc) and investigated mouse myocilin gene expression in ocular tissues with in situ RNA hybridization. Hybridization signals were observed in the iris, ciliary body, trabecular meshwork, sclera, and retina in the mouse eye. The marked signals were seen in trabecular meshwork cells and the anterior portion of sclera. These findings suggest that myocilin mutation could affect the capacity of aqueous outflow and cause elevation of the intraocular pressure which is involved in the pathogenesis of glaucoma.

[Emergent operation after percutaneous transluminal coronary rotational atherectomy (ROTABLATOR)].

Emergent operations were performed in seven patients after percutaneous transluminal coronary rotational atherectomy (PTCRA). The causes of the emergent operations were coronary rupture in three patients, acute coronary occlusion in two patients, perforation of the ascending aorta in one patient and impossible weaning from IABP in one patient. IABP was used preoperatively in all patients. Coronary artery bypass grafting was performed in all patients. Ruptured sites of the coronary arteries were closed and perforated site of the ascending aorta was repaired. Two patients died due to cardiac failure but five patients were recovered. Coronary artery rupture was main complication after PTCRA. It is important to recognize the difference between the complication after PTCRA and that after conventional PTCA.