Rho-mediated regulation of tight junctions during monocyte migration across the blood-brain barrier in HIV-1 encephalitis (HIVE)

The blood-brain barrier (BBB) is compromised during progressive HIV-1 infection, but how this occurs is incompletely understood. We studied the integrity of tight junctions (TJs) of brain microvascular endothelial cells (BMVECs) in an in vitro BBB system and in human brain tissues with HIV-1 encephalitis (HIVE). A downregulation of TJ proteins, claudin-5 and occludin, paralleled monocyte migration into the brain during HIVE. Because small G proteins (such as Rho) can play a role in BMVEC TJ assembly, an artificial BBB system explored the relationship among TJs, Rho/Rho kinase (RhoK) activation, and transendothelial monocyte migration. Coculture of monocytes with endothelial cells led to Rho activation and phosphorylation of TJ proteins. Rho and RhoK inhibitors blocked migration of infected and uninfected monocytes. The RhoK inhibitor protected BBB integrity and reversed occludin/claudin-5 phosphorylation associated with monocyte migration. BMVEC transfection with a constitutively active mutant of RhoK led to dislocation of occludin from the membrane and loss of BMVEC cell contacts. When dominant-negative RhoK-transfected BMVECs were used in BBB constructs, monocyte migration was reduced by 84%. Thus, loss of TJ integrity was associated with Rho activation caused by monocyte brain migration, suggesting that Rho/RhoK activation in BMVECs could be an underlying cause of BBB impairment during HIVE.     

Elevation of serum high-mobility group box 1 protein during granulocyte colony-stimulating factor-induced peripheral blood stem cell mobilisation

High mobility group box 1 (HMGB1) is a non-histone protein involved in maintaining the architecture of chromatin. HMGB1 also acts extracellularly as a cytokine, in processes such as inflammation, cell migration and stem cell recruitment. The involvement of HMGB1 in granulocyte colony-stimulating factor (G-CSF)-induced mobilisation of haematopoietic stem cells was investigated in 21 healthy donors. G-CSF treatment significantly elevated serum HMGB1 levels, which increased from 1.16 +/- 0.86 ng/ml, before treatment, to 31.1 +/- 5.99 ng/ml, after treatment. These findings suggest HMGB1 may play a role during the mobilisation of stem cells from the bone marrow into the systemic circulation.     

Magnifying pharmacoendoscopy: response of microvessels to epinephrine stimulation in differentiated early gastric cancers

BACKGROUND: Magnifying endoscopy is a promising modality for fine observation of minute surface structures and microvessel architecture in gastric lesions. OBJECTIVE: To observe the response of microvessels to epinephrine stimulation in early gastric cancer tissues and to assess the usefulness of magnifying pharmacoendoscopy for histologic diagnosis. DESIGN: This was a prospective pilot study. SETTING: This study was conducted at an academic hospital. PATIENTS: Twenty-nine patients with differentiated early gastric cancer were enrolled. INTERVENTIONS: Microvessels in both the cancerous lesion and its adjacent non-neoplastic gastric mucosa were observed by magnifying endoscopy before and after focal spray with epinephrine solution (0.05 mg/mL). MAIN OUTCOME MEASUREMENTS AND RESULTS: After epinephrine stimulation, noncancerous gastric mucosa surrounding the cancerous lesion showed a change in color from red to white; no microvessels were evident. On the other hand, all the cancerous lesions examined clearly showed enhancement of tumor microvessels. The rate of detection of tumor microvessels by magnifying pharmacoendoscopy (100%) was significantly higher than that by magnifying endoscopy alone (41.3%). LIMITATIONS: This was small pilot study. CONCLUSIONS: Magnifying pharmacoendoscopy with epinephrine is a powerful tool for assessing tumor vascularity and may contribute to the histologic diagnosis of differentiated early gastric cancers before endoscopic treatment.     

Angiotensinogen gene polymorphism (Met235Thr) influences visceral obesity and insulin resistance in obese Japanese women

To investigate the relationship between angiotensinogen (AGT) Met235Thr polymorphism (M235T) and human obesity, because AGT is regarded as one of the cytokines produced from adipocytes and serum AGT concentrations are reported to be positively correlated with body mass index. One hundred and twenty obese Japanese women (age, 58.8+/-9.4 years; body mass index, 32.2+/-4.9 kg/m(2)) were enrolled. Angiotensinogen genotypes were determined with a fluorescent allele-specific DNA primer assay system. Subjects were divided into M/M, M/T, and T/T groups. Control subjects comprised 146 healthy age-matched women. Clinical characteristics and the effects of diet and exercise therapy for 6 months were compared among the 3 genotypes. The genotype frequencies of AGT M235T polymorphism were in accordance with the Hardy-Weinberg equation (obese: M/M, 6.7%; M/T, 27.5%; T/T, 65.8%; control: M/M, 6.8%; M/T, 21.2%; T/T, 71.9%). The frequency of the T allele did not differ between obese and control subjects (0.80 vs 0.83). As the number of obese women with M/M genotype was only 8, comparisons of the characteristics and outcomes of weight reduction therapy were performed only between subjects with M/T genotype and T/T genotype. In the T/T group, % body fat and waist circumference at baseline were significantly greater than in the M/T group (36.3%+/-4.8% vs 33.8%+/-4.7%, P=.0105; 107.9+/-10.9 vs 102.6+/-7.9 cm, P=.0428, respectively). Before the weight reduction therapy, significantly higher insulin and higher homeostasis model assessment (HOMA-R) were demonstrated in the T/T group than in the M/T group (9.1+/-5.5 microU/mL vs 5.9+/-4.4 microU/mL, P=.0056; 2.3+/-1.4 vs 1.6+/-1.3, P=.0252, respectively). Both systolic and diastolic blood pressure at baseline in the T/T group tended to be higher than those in the M/T group, but the differences were not significant. No genotype-dependent difference in energy expenditure or outcome of weight reduction therapy was observed with respect to AGT M235T polymorphism. After the diet and exercise therapy, the blood pressure in the T/T group tended to be higher than that in the M/T group, but the difference was not significant. We demonstrated that the T/T genotype of the AGT M235T gene polymorphism was positively related to visceral obesity and hyperinsulinemia in obese Japanese women. Blood pressure did not show genotype-specific differences before or after the treatment. Further studies of the association between obesity and this gene polymorphism should contribute to understanding and treating obesity-related diseases.     

Tick troponin I-like molecule is a potent inhibitor for angiogenesis

This is the first report identifying the anti-angiogenesis saliva molecule of the ixodid tick. In our previous study, we identified a troponin I-like molecule (HLTnI) of the ixodid hard tick Haemaphysalis longicornis, a vector for various pathogens. To investigate its potential inhibitory effects for angiogenesis, we expressed and purified recombinant HLTnI in Escherichia coli. In a vascular endothelial growth factor (VEGF) competitive angiogenesis assay, the recombinant HLTnI significantly inhibited the capillary formation of human vascular endothelial cells (HUVEC) in vitro. The inhibition was dose-dependent with an IC(50) of 18.95 nM. These results indicated that HLTnI is a potent angiogenesis inhibitor.     

Inhibition of ganciclovir-resistant human cytomegalovirus replication by Kampo (Japanese herbal medicine)

We examined the effect of Kampo on the replication of ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) in the human embryonic fibroblast cell line MRC-5. Treatment of HCMV-infected cells with Sho-seiryu-to (SST; Xiao-Qing-Long-Tang in Chinese) resulted in the inhibition of viral replication without affecting the cell growth. SST treatment decreased the synthesis of viral DNA, but had no virucidal effect on cell-free HCMV. However, the inhibitory effect of SST on HCMV replication was ablated by anti-interferon-beta (IFN-beta) antibody suggesting that SST inhibits the replication of GCV-resistant HCMV through the induction of IFN-beta. These results suggest that SST is a novel compund with potential as an anti-HCMV.     

Risk stratification for in-hospital mortality in spontaneous intracerebral haemorrhage: a Classification and Regression Tree analysis

BACKGROUND: Risk stratification for mortality in intracerebral haemorrhage (ICH) helps guide care, but existing clinical prediction rules are too cumbersome for clinical practice because of their complexity. AIM: To develop a simple decision tree model of in-hospital mortality risk stratification for ICH patients. METHODS: We collected information on spontaneous ICH patients hospitalized in a teaching hospital in Japan from August, 1998 to December, 2001 (n = 374). All variables were abstracted from data available at the time of initial evaluation. A prediction rule for in-hospital mortality was developed by the Classification and Regression Tree (CART) methodology. The accuracy of the model was evaluated using the area under receiver-operator characteristic curve. RESULTS: Overall in-hospital mortality rate was 20.2%. The CART methodology identified four groups for mortality risk, varying from low (2.1%) to high (58.9%). Level of consciousness (coma) was the best single predictor for mortality, followed by high ICH volume (cut-off 10.4 ml), and then age (cut-off 75 years). The accuracy of our CART model (0.86) exceeded that of a multivariate logistic regression model (0.81). DISCUSSION: ICH patients can easily be stratified for mortality risk, based on three predictors available on admission. This simple decision tree model provides clinicians with a reliable and practical tool.     

Yellow flowers generated by expression of the aurone biosynthetic pathway

Flower color is most often conferred by colored flavonoid pigments. Aurone flavonoids confer a bright yellow color on flowers such as snapdragon (Antirrhinum majus) and dahlia (Dahlia variabilis). A. majus aureusidin synthase (AmAS1) was identified as the key enzyme that catalyzes aurone biosynthesis from chalcones, but transgenic flowers overexpressing AmAS1 gene failed to produce aurones. Here, we report that chalcone 4'-O-glucosyltransferase (4'CGT) is essential for aurone biosynthesis and yellow coloration in vivo. Coexpression of the Am4'CGT and AmAS1 genes was sufficient for the accumulation of aureusidin 6-O-glucoside in transgenic flowers (Torenia hybrida). Furthermore, their coexpression combined with down-regulation of anthocyanin biosynthesis by RNA interference (RNAi) resulted in yellow flowers. An Am4'CGT-GFP chimeric protein localized in the cytoplasm, whereas the AmAS1(N1-60)-RFP chimeric protein was localized to the vacuole. We therefore conclude that chalcones are 4'-O-glucosylated in the cytoplasm, their 4'-O-glucosides transported to the vacuole, and therein enzymatically converted to aurone 6-O-glucosides. This metabolic pathway is unique among the known examples of flavonoid, including anthocyanin biosynthesis because, for all other compounds, the carbon backbone is completed before transport to the vacuole. Our findings herein not only demonstrate the biochemical basis of aurone biosynthesis but also open the way to engineering yellow flowers for major ornamental species lacking this color variant.     

Pamidronate induced anti-proliferative, apoptotic, and anti-migratory effects in hepatocellular carcinoma

BACKGROUND/AIMS: The small GTPase of Ras and Rho families are widely involved in human tumorgenesis and metastasis. It has recently been reported that pamidronate inhibits the mevalonate pathway, which is required for the prenylation of the small GTPase. We demonstrated a possible beneficial use of pamidronate in the treatment of hepatocellular carcinoma (HCC). METHODS: The effect of pamidronate on cell proliferation was analyzed with five hepatoma cell lines using MTT assay. Apoptosis was evaluated by staining with DAPI and a histon ELISA assay. A cell migration assay was performed using the Modified Boyden Chamber. To analyze anti-proliferation effect of pamidronate in vivo, tumor volumes were monitored with the intraperitoneal injection of pamidronate after subcutaneous inoculation of PLC/PRF/5 cells into nude mice. RESULTS: Pamidronate inhibited cell growth for all hepatoma cell lines. The amount of membrane associated Ras and phosphorylated extracellular signal-regulated kinase 2 (ERK 2) were reduced after pamidronate treatment. Pamidronate increased apoptosis and cleavage of Caspase-3, and -9. Pamidronate suppressed membrane associated RhoA and cell motility. In vivo, tumor volumes were significantly suppressed by pamidronate at three weeks (P<0.03). CONCLUSIONS: We conclude that pamidronate has therapeutic potential in inducing anti-proliferative, apoptotic, and anti-migratory effects in HCC.