Optical recordings of membrane potential using genetically targeted voltage-sensitive fluorescent proteins

Optical imaging of electrical activity using voltage-sensitive dyes has been envisaged for many years as a powerful method to investigate multineuronal representation of information processing in brain tissue. This article describes the advent of novel genetically targeted voltage-sensitive fluorescent proteins. This new class of membrane voltage sensors overcomes previous limitations related to the nonselective staining of membranes associated with conventional voltage-sensitive dyes. Here, we discuss the methodology, applications, and potential advantages of this novel technique.     

Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia

Bone-marrow minimal residual disease (MRD) causes relapse after chemotherapy in patients with acute myelogenous leukemia (AML). We postulate that the drug resistance is induced by the attachment of very late antigen (VLA)-4 on leukemic cells to fibronectin on bone-marrow stromal cells. We found that VLA-4-positive cells acquired resistance to anoikis (loss of anchorage) or drug-induced apoptosis through the phosphatidylinositol-3-kinase (PI-3K)/AKT/Bcl-2 signaling pathway, which is activated by the interaction of VLA-4 and fibronectin. This resistance was negated by VLA-4-specific antibodies. In a mouse model of MRD, we achieved a 100% survival rate by combining VLA-4-specific antibodies and cytosine arabinoside (AraC), whereas AraC alone prolonged survival only slightly. In addition, overall survival at 5 years was 100% for 10 VLA-4-negative patients and 44.4% for 15 VLA-4-positive patients. Thus, the interaction between VLA-4 on leukemic cells and fibronectin on stromal cells may be crucial in bone marrow MRD and AML prognosis.     

Differential mechanism and site of action of CCK on the pancreatic secretion and growth in rats

Recent studies demonstrated that cholecystokinin (CCK) at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway. This study examined whether chemical ablation of afferent vagal fibers influences pancreatic growth and secretion in rats. Bilateral subdiaphragmatic vagal trunks were exposed, and capsaicin solution was applied. Pancreatic wet weight and pancreatic secretion and growth in response to endogenous and exogenous CCK were examined 7 days after capsaicin treatment. Perivagal application of capsaicin increased plasma CCK levels and significantly increased pancreatic wet weight compared with those in the control rats. Oral administration of CCK-1 receptor antagonist loxiglumide prevented the increase in pancreatic wet weight after capsaicin treatment. In addition, continuous intraduodenal infusion of trypsin prevented the increase in plasma CCK levels and pancreatic wet weight after capsaicin treatment. There were no significant differences in the expression levels of CCK-1 receptor mRNA and protein in the pancreas in capsaicin-treated and control rats. Intraduodenal administration of camostat or intravenous infusion of CCK-8 stimulated pancreatic secretion in control rats but not in capsaicin-treated rats. In contrast, repeated oral administrations of camostat or intraperitoneal injections of CCK-8 significantly increased pancreatic wet weight in both capsaicin-treated and control rats. Present results suggest that perivagal application of capsaicin stimulates pancreatic growth via an increase in endogenous CCK and that exogenous and endogenous CCK stimulate pancreatic growth not via vagal afferent fibers but directly in rats.     

Functions of malonate decarboxylase subunits from Pseudomonas putida

Malonate decarboxylase from Pseudomonasputida is composed of five subunits, alpha, beta, gamma, delta, and epsilon. Two subunits, delta and epsilon, have been identified as an acyl-carrier protein (ACP) and malonyl-CoA:ACP transacylase, respectively. Functions of the other three subunits have not been identified, because recombinant subunits expressed in Escherichia coi formed inclusion bodies. To resolve this problem, we used a coexpression system with GroEL/ES from E. coli, and obtained active recombinant subunits. Enzymatic analysis of the purified recombinant subunits showed that the alpha subunit was an acetyl-S-ACP:malonate ACP transferase and that the betagamma-subunit complex was a malonyl-S-ACP decarboxylase.     

Increased production of antioxidative sesaminol glucosides from sesame oil cake through fermentation by Bacillus circulans strain YUS-2

Bacillus circulans strain YUS-2 was isolated as the strongest antioxidant-producer in fermentation of sesame oil cake (SOC, defatted residue yielded from sesame seed oil production). Two major strong antioxidants from fermented SOC were purified and identified as known sesaminol triglucoside and sesaminol diglucoside, however, our results demonstrated that the fermentation process with B. circulans YUS-2 was highly effective to gain the extraction efficiency of the sesaminol glucosides.     

Dialysate dihydroxyphenylglycol as a window for in situ axoplasmic norepinephrine disposition

To examine basal axoplasmic norepinephrine (NE) kinetics at the in situ cardiac sympathetic nerve ending, we applied a dialysis technique to the heart of anesthetized cats and performed the dialysate sampling with local administration of a pharmacological tool through a dialysis probe. The dialysis probe was implanted in the left ventricular wall, and dihydroxyphenylglycol (DHPG, an index of axoplasmic NE) levels were measured by liquid chromatogram-electrochemical detection. Control dialysate DHPG levels were 161+/-19 pg/ml. Pargyline (monoamine oxidase inhibitor, 1 mM) decreased the dialysate DHPG levels to 38+/-10 pg/ml. Further alpha-methyl-para-tyrosine, omega-conotoxin GVIA, desipramine (NE synthesis, release and uptake blockers) decreased the dialysate DHPG levels to 64+/-19, 106+/-15, 110+/-22 pg/ml, respectively. In contrast, reserpine (vesicle NE transport inhibitor, 10 microM) increased the dialysate DHPG levels to 690+/-42 pg/ml. Thus, NE synthesis, metabolism and recycling (release, uptake and vesicle transport) affected basal intraneuronal NE disposition at the nerve endings. Measurement of DHPG levels through a dialysis probe provides information about basal intraneuronal NE disposition at the cardiac sympathetic nerve endings. Yohimbine (alpha(2)-adrenoreceptor blocker, 10 microM) and U-521 (catechol-O-methyltransferase blocker, 100 microM) did not alter the dialysate DHPG levels. Furthermore, there were no significant differences in the reserpine induced DHPG increment between the presence and absence of desipramine (10 microM) or alpha-methyl-para-tyrosine (100 mg/kg i.p.). These results may be explained by the presence of two axoplasmic pools of NE, filled by NE taken up and synthesized, and by NE overflow from vesicle. The latter pool of NE may be closed to the monoamine oxidase system in the axoplasma.     

Role of TGF-beta in EGF-induced transformation of NRK cells is sustaining high-level EGF-signaling

We have been isolating and analyzing NRK cell mutants, which fail to transform by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. One such mutant, R14, can respond to the growth inhibitory signal of TGF-beta to the same extent as parental NRK but fail to respond to the growth stimulatory signal of EGF. This mutant has a defect in EGF receptor (EGFR) expression. When R14 mutant expressed a high level of EGFR, however, EGF not only induced proliferation in this mutant but also induced transformation without the aid of TGF-beta. These findings suggest that the major role of TGF-beta in this transformation system should be to counteract the ligand-dependent down-regulation of EGFR, thereby sustaining high-level EGF-signaling.     

Arachidonic acid activates mitogen-activated protein (MAP) kinase-activated protein kinase 2 and mediates adhesion of a human breast carcinoma cell line to collagen type IV through a p38 MAP kinase-dependent pathway

Adhesion of metastatic human mammary carcinoma MDA-MB-435 cells to the basement membrane protein collagen type IV can be activated by treatment with arachidonic acid. We initially observed that this arachidonic acid-mediated adhesion was inhibited by the tyrosine kinase inhibitor genistein. Therefore, we examined the role of the mitogen-activated protein (MAP) kinase family tyrosine phosphorylation-regulated pathways in arachidonic acid-stimulated cell adhesion. Arachidonic acid stimulated the phosphorylation of p38, the activation of MAP kinase-activated protein kinase 2 (MAPKAPK2, a downstream substrate of p38), and the phosphorylation of heat shock protein 27 (a downstream substrate of MAP kinase-activated protein kinase 2). Treatment with the p38 inhibitor PD169316 completely and specifically inhibited arachidonic acid-mediated cell adhesion to collagen type IV. p38 activity was specifically associated with arachidonic acid-stimulated adhesion; this was demonstrated by the observation that 12-O-tetradecanoylphorbol 13-acetate-activated cell adhesion was not blocked by inhibiting p38 activity. Extracellular signal-regulated protein kinases (ERKs) 1 and 2 were also activated by arachidonic acid; however, cell adhesion to collagen type IV was not highly sensitive to PD98059, an inhibitor of MAP kinase kinase/ERK kinase 1 (MEK1) that blocks activation of the ERKs. c-Jun NH(2)-terminal kinase was not activated by arachidonic acid treatment of these cells. Together, these data suggest a novel role for p38 MAP kinase in regulating adhesion of breast cancer cells to collagen type IV.