Arrayed water-in-oil droplet bilayers for membrane transport analysis.

The water-in-oil droplet bilayer is a simple and useful lipid bilayer system for membrane transport analysis. The droplet interface bilayer is readily formed by the contact of two water-in-oil droplets enwrapped by a phospholipid monolayer. However, the size of individual droplets with femtoliter volumes in a high-throughput manner is difficult to control, resulting in low sensitivity and throughput of membrane transport analysis. To overcome this drawback, in this study, we developed a novel micro-device in which a large number of droplet interface bilayers (>500) are formed at a time by using femtoliter-sized droplet arrays immobilized on a hydrophobic/hydrophilic substrate. The droplet volume was controllable from 3.5 to 350 fL by changing the hydrophobic/hydrophilic pattern on the device, allowing high-throughput analysis of membrane transport mechanisms including membrane permeability to solutes (e.g., ions or small molecules) with or without the aid of transport proteins. Thus, this novel platform broadens the versatility of water-in-oil droplet bilayers and will pave the way for novel analytical and pharmacological applications such as drug screening.

AURKA is one of the downstream targets of MAPK1/ERK2 in pancreatic cancer.

DUSP6/MKP-3, a specific inhibitor of MAPK1/ERK2, frequently loses its expression in primary pancreatic cancer tissues. This evidence suggests that constitutive activation of MAPK1 synergistically induced by frequent mutation of KRAS2 and the loss of function of DUSP6 plays key roles in pancreatic carcinogenesis and progression. By profiling of gene expressions associated with downregulation of MAPK1 induced by exogenous overexpression of DUSP6 in pancreatic cancer cells, we found that AURKA/STK15, the gene encoding Aurora-A kinase, which plays key roles in cellular mitosis, was among the downregulated genes along with its related genes, which included AURKB, TPX2 and CENPA. An association of expression and promoter activity of AURKA with MAPK activity was verified. Knockdown of ETS2 resulted in a reduction of AURKA expression. These results indicate that AURKA is a direct target of the MAPK pathway and that its overexpression in pancreatic cancer is induced by hyperactivation of the pathway, at least via ETS2.

Rho family GTPases regulate VEGF-stimulated endothelial cell motility.

Migration of endothelial cells induced by vascular endothelial growth factor (VEGF) is a critical step in angiogenesis. Stimulation of motility by growth factors such as VEGF requires interaction with the signal transduction pathways activated by the extracellular matrix (ECM). Here we demonstrate that the Rac GTPase is the critical intersection activated by type 1 collagen ECM and VEGF during stimulation of endothelial cell motility. To analyze the role of the Rho family GTPases in VEGF-stimulated endothelial cell chemotaxis and ECM-stimulated haptotaxis, we transduced the respective fusion proteins in human foreskin dermal endothelial cells using a Tat peptide from the human immunodeficiency virus Tat protein. VEGF signaling required Rac activation during chemotaxis, and Rac and Cdc42 were activated during haptotaxis on type I collagen. Similar to VEGF, Rac activation induced an increase in endothelial cell stress fiber and focal adhesion. Surprisingly, Rho activation was not present in collagen-induced haptotaxis or stimulation of chemotaxis by VEGF, although Rho induced stress fibers and focal adhesions similar to Rac activation. The result of constitutive Rho activation was an inhibition of haptotaxis. Thus, Rac is required and sufficient for the activation of endothelial cell haptotaxis and VEGF-stimulated chemotaxis.

Angiogenesis as a predictor of long-term survival for 377 Japanese patients with breast cancer.

Angiogenesis. as assessed by microvessels, has been a common prognostic indicator for breast cancer in the last decade. However, the significance of angiogenesis remains controversial. This is a retrospective study of 377 Japanese patients selected from 663 breast cancer patients operated on between 1971 and 1987. To evaluate an objective method to quantify microvessel density in angiogenesis, we employed average microvessel count (AMC) per square millimeter. We investigated five factors: angiogenesis, lymph-node status (n), clinical tumor size (T), histological grade (HG). and tumor necrosis (TN), followed for a median of 10 years. Sixty-seven patients (17.8%) had recurrence and 54 patients (14.3%) died of breast cancer. Univariate analysis showed that n, T, HG. and AMC (P = 0.0020) were significantly predictive of 20-year relapse-free survival (RFS). n, T, and HG were significantly associated with 20-year overall survival (OS) but AMC was borderline significant (P = 0.0630). Multivariate analysis for RFS and OS showed that n. T. HG, and AMC (P < 0.0001, P = 0.0033, respectively) were all significant and independent prognostic factors. When stratified by T or n, a significant impact of AMC on RFS or OS was seen both in patients with T2 and T3 carcinomas or in node-negative patients, but not in T1 or node-positive patients. Thus, we can confirm angiogenesis as a significant independent prognostic factor associated with longterm survival in Japanese breast cancer patients, especially in node-negative patients and in patients with T2 and T3 carcinomas.

Rac regulates vascular endothelial growth factor stimulated motility.

During angiogenesis endothelial cells migrate towards a chemotactic stimulus. Understanding the mechanism of endothelial cell migration is critical to the therapeutic manipulation of angiogenesis and ultimately cancer prevention. Vascular endothelial growth factor (VEGF) is a potent chemotactic stimulus of endothelial cells during angiogenesis. The endothelial cell signal transduction pathway of VEGF represents a potential target for cancer therapy, but the mechanisms of post-receptor signal transduction including the roles of rho family GTPases in regulating the cytoskeletal effects of VEGF in endothelial cells are not understood. Here we analyze the mechanisms of cell migration in the mouse brain endothelial cell line (bEND3). Stable transfectants containing a tetracycline repressible expression vector were used to induce expression of Rac mutants. Endothelial cell haptotaxis was stimulated by constitutively active V12Rac on collagen and vitronectin coated supports, and chemotaxis was further stimulated by VEGF. Osteopontin coated supports were the most stimulatory to bEND3 haptotaxis, but VEGF was not effective in further increasing migration on osteopontin coated supports. Haptotaxis on support coated with collagen, vitronectin, and to a lesser degree osteopontin was inhibited by N17 Rac. N17 Rac expression blocked stimulation of endothelial cell chemotaxis by VEGF. As part of the chemotactic stimulation, VEGF caused a loss of actin organization at areas of cell-cell contact and increased stress fiber expression in endothelial cells which were directed towards pores in the transwell membrane. N17 Rac prevented the stimulation of cell-cell contact disruption and the stress fiber stimulation by VEGF. These data demonstrate two pathways of regulating endothelial cell motility, one in which Rac is activated by matrix/integrin stimulation and is a crucial modulator of endothelial cell haptotaxis. The other pathway, in the presence of osteopontin, is Rac independent. VEGF stimulated chemotaxis, is critically dependent on Rac activation. Osteopontin was a potent matrix activator of motility, and perhaps one explanation for the absence of a VEGF plus osteopontin effect is that osteopontin stimulated motility was inhibitory to the Rac pathway.

Rac is essential in the transformation of endothelial cells by polyoma middle T.

Expression of the Polyoma Middle T (PyMT) antigen in endothelial cells results in single-step transformation to hemangioma producing malignant cells. To study the mechanism of PyMT transformation, we used the PyMT induced mouse brain endothelial cell line, bEND.3, expressing constitutively active and dominant negative mutants of the small GTPase Rac. The bEND.3 cell phenotype of tumorigenesis, loss of normal growth control and formation of cysts rather than capillary tubes in fibrin gels was reversed by expression of dominant negative Rac. The mechanism of N17 Rac action in blocking the endothelial cell transformant, PyMT, did not involve effects of Rac on the actin cytoskeleton since this component of the bEND.3 cell phenotype was not affected. Furthermore, the PyMT induced activation of the plasminogen activator (PA)/plasmin system was not affected by Rac inhibition. Inhibition of the downstream effectors of Rac, phosphatidylinositol 3-kinase (PI3-K) and p70S6k, which are known to be constitutively activated by PyMT transformation, inhibited bEND.3 cell proliferation and cyst formation in fibrin gels even in cells expressing V12 constitutively active Rac, but they did not restore capillary tube formation. These results demonstrate that middle T antigen induced endothelial cell transformation requires signal transduction by Rac. The downstream Rac effectors, P13-K and p70S6k, mediate PyMT/Rac effects on cell proliferation and cyst formation, but other unknown effectors of PyMT are required for the cytoskeletal changes and activation of the PA/plasmin system.

Rho-A is critical for osteoclast podosome organization, motility, and bone resorption.

Rho plays a regulatory role in the formation of actin stress fibers and focal adhesions, and it is also involved in integrin-mediated signaling events. To study the role of Rho in alpha(v)beta(3)/gelsolin-dependent signaling, the HIV-Tat peptide, hemagglutinin (HA)-tagged Rho(Val-14) (constitutively active) and Rho(Asn-19) (dominant negative) were transduced into avian osteoclasts. Protein transduction by HA-Tat was highly efficient, and 90-100% of the cells were transduced with HA-tagged proteins. We demonstrate here that Rho(Val-14) transduction (100 nM) stimulated gelsolin-associated phosphatidylinositol 3-kinase activity, podosome assembly, stress fiber formation, osteoclast motility, and bone resorption, mimicking osteoclast stimulation by osteopontin/alpha(v)beta(3.) The effects of Rho(Val-14) transduction stimulation was time-dependent. C3 exoenzyme blocked the effects of Rho(Val-14) and induced podosome disassembly, loss of motility, and inhibition of bone resorption. Transduction of Rho(Asn-19) produced podosome disassembly, and blocked osteopontin stimulation. These data demonstrate that integrin-dependent activation of phosphoinositide synthesis, actin stress fiber formation, podosome reorganization for osteoclast motility, and bone resorption require Rho stimulation.

Performance of a monolithic silica column in a capillary under pressure-driven and electrodriven conditions

A continuous macroporous silica gel network was prepared in a fused-silica capillary and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, the monolithic silica column derivatized to C18 phase (100 microns in diameter, 25 cm in length, silica skeleton size of approximately 2.2 microns) produced plate heights of about 23 and 81 microns at 0.5 mm/s with a pressure drop of 0.4 kg/cm2, and at 4.0 mm/s with 3.6 kg/cm2, respectively, in 90% acetonitrile for hexylbenzene with a k value of 0.7. The separation impedance, E, calculated for the present monolithic silica column was much smaller at a low flow rate than those for particle-packed columns, although higher E values were obtained at a higher flow rate. Considerable dependence of column efficiency on the linear velocity of the mobile phase was observed despite the small size of the silica skeletons. A major source of band broadening in the HPLC mode was found in the A term of the van Deemter equation. The performance of the continuous silica capillary column in the electrodriven mode was much better than that in the pressure-driven mode. Plate heights of 7-8 microns were obtained for alkylbenzenes at 0.7-1.3 mm/s, although the electroosmotic flow was slow. In HPLC and CEC mode, the dependency of plate height on k values of the solutes was observed as seen in open tube chromatography presumably due to the contribution of the large through-pores. Since monolithic silica capillary columns can provide high permeability, the pressure-driven operation at a very low pressure can afford a separation speed similar to CEC at a high electric field.

Sol-gel modification of silicone to induce apatite-forming ability.

Silicones formed with silanol groups on their surfaces were prepared by partial hydrolysis and polycondensation of tetraethoxysilane (TEOS) dispersed in silicones in molecular scale. Although the silanol-modified silicones thus prepared formed no apatite in a simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma within 21 d, they formed a bone-like apatite layer in situ on their surfaces in a solution (1.5 SBF) with ion concentrations 1.5 times the SBF within 7 d. This indicates that the silanol groups formed on the silicones by the present method can induce the apatite nucleation in 1.5 SBF and hence, apatite-coated silicones can be fabricated by a biomimetic process. Apatite-silicone composites thus prepared could exhibit mechanical properties analogous to the natural bone as well as bioactivity.

Room-temperature persistent spectral hole burning of Eu(3+) in sodium aluminosilicate glasses.

Persistent spectral hole burning has been observed at 77 K, 180 K, and room temperature for Eu(3+) in sodium aluminosilicate glass melted under a reducing atmosphere. In particular, room-temperature persistent spectral hole burning is reported for the first time to our knowledge in Eu(3+) -doped materials. The persistent hole is accompanied by no antiholes and lasts for 1 h at least. The thermal stability of the hole is greater than that of a persistent hole burned for Eu(3+) in sodium aluminosilicate glass melted in air.

Apatite formation on silica gel in simulated body fluid: effects of structural modification with solvent-exchange.

The prerequisite for glasses and glass-ceramics to bond to living bone is the formation of biologically active bone-like apatite on their surfaces. It has been shown that even a pure silica gel forms the bone-like apatite on its surface in a simulated body fluid. In the present study, pore structure of silica gels prepared by hydrolysis and polycondensation of tetraethoxysilane in an aqueous solution containing polyethylene glycol was modified by 1M HNO3, and 0.1M and 1M NH4OH solution treatments. The three kinds of resultant gels all contained large amounts of silanol groups and trisiloxane rings, but differ greatly in pore structure of nanometre pore size. Irrespective of these differences, all the gels formed the bone-like apatite on their surface in the simulated body fluid. It was speculated that a certain type of structural unit of silanol groups, which is easily formed in the presence of the polyethylene glycol, is effective for the apatite formation.

Effect of aging on the intestinal transport of hydrophilic drugs in the rat small intestine.

The effect of aging on the intestinal transport of hydrophilic drugs (and probe compounds) was investigated in the rat small intestine. Passive transport was suggested to be unchanged with aging from 8 (young) to 54 (old) and further to 101 (very old) weeks old, as shown for D-xylose and urea in single-pass intestinal perfusion (under urethane anesthesia), where steady-state transport across the intestinal membrane into the blood stream was evaluated. The passive transports of cephradine, 5-fluorouracil (5-FU) and L-glucose were also unchanged, though they were compared only between the young and the old. Consistently, the passive uptake in the intestinal everted sacs, where the entry process into the membrane was evaluated for 5-FU, D-xylose, urea and polyethylene glycol (PEG) 900, was unchanged with aging from the young to the very old. The carrier-mediated transport of cephradine was also unchanged with aging from the young to the old in perfusion under anesthesia, though that of D-glucose was declined by about 50% with aging from the young to the old and thereafter remained constant in the very old. In perfusion in unanesthetized rats, age independency in passive transport (examined for cephradine, L-glucose and D-xylose) and an age-dependent decline in D-glucose transport were also observed, suggesting that the findings under anesthesia are not qualitatively distorted. These results suggest that, although carrier-mediated transport may moderately decline with aging, the barrier function of the intestinal membrane to passive permeation of hydrophilic drugs (with molecular weight below 1000) may be unaffected by aging, supporting the suggestion from our previous in vivo studies that age-dependent increases in the orally absorbed fraction may be predicted for incompletely absorbed drugs because of delayed intestinal transit rather than increased intestinal transport (membrane permeability).

Effect of skeleton size on the performance of octadecylsilylated continuous porous silica columns in reversed-phase liquid chromatography.

We prepared continuous porous silica rods that had silica skeletons with sizes of 1.0-1.7 microns and through-pores of 1.5-1.8 microns, and evaluated their performance as a column in reversed-phase liquid chromatography. The mesoporous silica monoliths (mesopore size: 14 or 24 nm) were derivatized to C18 phase by on-column reaction with octadecyldimethyl-N,N-diethylaminosilane. The C18 silica rods gave minimum plate heights of 10-15 microns for aromatic hydrocarbons in 80% methanol and of 20-30 microns for insulin in acetonitrile-water mixtures in the presence of trifluoroacetic acid. The performance of the silica rods at a high flow-rate was much better than that of conventional columns packed with 5 microns C18 silica particles with pores of 12 or 30 nm, especially for high-molecular-mass species. Silica rods with the smaller sized silica skeletons resulted in Van Deemter plots showing a minimum plate height linear velocity of the mobile phase and a smaller dependence of plate height on the linear velocity. Separation impedance of less than 1000 was achieved with the continuous silica columns. The higher performance and lower pressure drop of silica rods at high flow-rates compared with particle-packed columns is provided by the small silica skeletons and large through-pores.

Incidence of urolithiasis in northeast Thailand.

BACKGROUND: Urolithiasis is a major health problem in the northeast part of Thailand. In this study, we examined the prevalence of renal stone disease and differences of urinary components between stone formers and healthy control subjects in northeastern rural areas of Thailand. METHODS: We selected 3 villages in the rural areas of Khon Kaen province in northeast Thailand. Three hundred and sixty-seven persons were asked questions relating to urolithiasis and were examined by abdominal ultrasound (US). We collected a spot urine sample from stone formers and healthy control subjects from each village. RESULTS: Abnormal findings by US were detected in 39 individuals (10.6%), and included 31 individuals with renal calculi (8.4%), 16 with hydronephrosis (4.4%), and 1 individual with a renal cyst (0.3%). This yielded a disease rate of urinary stones in this study of 16.9%. The male/female ratio was 2/1 and the average age of the individuals examined was 40.3 +/- 13.9 years. However, there was no significant difference between the urinary parameters of stone formers and the healthy control subjects. CONCLUSION: There was a high incidence of renal stone disease in the northeast part of Thailand, but the tendency for hypocitraturia was only found in stone formers.

Comparative Assessment of Intestinal Transport of Hydrophilic Drugs Between Small Intestine and Large Intestine.

Intestinal passive transport of several hydrophilic drugs (and probe compounds) was examined in the large intestine (colon), in comparison with that in the small intestine, in an effort to obtain basic information for developing rational colonic drug delivery strategies. The drugs tested were polyethylene glycol (PEG 900), L-glucose, D-xylose, 5-fluorouracil (5-FU) and urea. In everted intestinal sacs, the uptake of every drug was larger in the small intestine than in the large intestine, although by various extents. The uptake of urea was larger than those of D-xylose and L-glucose in both the small intestine and large intestine and associated with a larger large intestine (LI)/small intestine (SI) uptake ratio. Assuming that passive transport via the paracellular route (or aqueous pore) is predominant for them, the large intestine may have smaller paracellular (or aqueous) pores, restricting the transport of those monosaccharides compared with smaller molecules such as urea by a larger extent in the large intestine than in the small intestine. The passive transport of 5-FU was significantly larger than those of the monosaccharides in both the small intestine and large intestine and associated with a larger LI/SI uptake ratio, even though 5-FU has a molecular weight close to that of the monosaccharides. 5-FU may be transported predominantly by transcellular diffusion, because its oil-to-water partition coefficient is about 200 times larger than those of the monosaccharides. Although transport mechanisms, including transport pathways, are yet to be fully clarified, drugs with physicochemical properties similar to those of 5-FU or urea may be more feasible for colonic drug delivery than those with physicochemical properties similar to those of monosaccharides.

[Usefulness of 99mTc-DTPA renography and diuretic renography in predicting successful stone discharge following outpatient ESWL in patients with a single ureteral stone].

We analyzed the 99mTc-DTPA renogram with and without diuresis to predict the possibility of stone discharge on the outpatient basis by renogram patterns. Between October, 1993 and December, 1995, 99mTc-DTPA renography was performed in 79 patients with a single ureteral stone. The 99mTc-DTPA renogram pattern was classified into the three types of normal function, obstruction and lower function patterns and the complete stone discharge rate was 93, 63 and 25%, respectively. In addition, diuretic renography using Furosemide was performed in patients with an obstruction pattern and the three renogram patterns of return to the normal curve, a diuretic response and no response were obtained; the complete stone discharge rate was 44, 65.3 and 93%, respectively. From this study, patients with a single ureteral stone with a normal pattern on the regular DTPA renogram and patients with no response pattern on the diuretic renogram, even if in such patients an obstructive pattern was seen on the regular DTPA renogram, seem to be a good candidate for obtaining a high rate of a stone discharge with extracorporeal shock wave lithotripsy (ESWL) treatment in the outpatients basis.

Apatite-forming ability of silicate ion dissolved from silica gels.

It is known that the prerequisite for glasses and glass-ceramics to bond to living bone is the formation of biologically active bonelike apatite on their surfaces, and a certain type of hydrated silica developed on their surfaces plays an important role in nucleating the apatite. In the present study, the apatite-forming ability of silicate ion dissolved from different silica materials into a simulated body fluid was examined as follows. Polyether sulfone substrates pretreated with O2 plasma were placed in parallel to plates of three kinds of silica gels prepared in different media and silica glass, with a distance of 0.5 mm between them in a simulated body fluid with ion concentrations nearly equal to those of human blood plasma for 4 days, and then soaked in a solution with ion concentrations 1.5 times those of the simulated body fluid for 6 days. After the first soaking, silicon combined with oxygen was detected on the surfaces of the substrates faced to all the silica gels, whereas it was not detected on that faced to the silica glass. After the second soaking, the former formed a bonelike apatite layer on their surfaces, but the latter did not. These results indicate that silicate ion which is dissolved from the silica gels and adsorbed on the substrates has an apatite-forming ability, irrespective of the microstructure of the original silica gels.

[A patient with clinically de novo Ph-positive acute lymphoblastic leukemia had Ph chromosome in all metaphases at the time of hematological remission].

A 58-year-old man was referred to our hospital because of his refractory leukemia. Laboratory examinations showed mild anemia and leukocytosis but no blast was seen in the blood. The patient's bone marrow was hyperplastic and 64.8% of marrow cells were lymphoblastoid cells. They were positive for CD10, CD19, CD34 and HLA-DR antigen. Cytogenetic analysis revealed the Ph chromosome in 17 of 20 metaphases. A Southern blot analysis demonstrated no rearrangement of M-BCR gene. A diagnosis of Ph-positive ALL was made. The patient received chemotherapy and reached a complete remission. At that time, however, his marrow cells had Ph chromosome in 7 of 7 metaphases and rearrangement of m-BCR was positive in PCR analysis. He died of septic shock during the intensive consolidation therapy. Clinically this patient seems to have de novo Ph-positive ALL though his marrow cells had Ph chromosome in all metaphases at the time of complete remission. Recently the rare cases of Ph-positive CML with an m-BCR breakpoint are reported in the literature. This patient may have such a type of CML in blastic phase.

[A case of leiomyoma of the urinary bladder associated with transitional cell carcinoma].

We report a case of leiomyoma of the urinary bladder associated with transitional cell carcinoma. A 60-year-old-male was referred to our hospital because of the complaint of dysuria and for detailed examination of left hydronephrosis. Drip infusion pyelography revealed left uretero-vesico junction stenosis. Flexible cystoscopy revealed benign prostatic hypertrophy and epithelial bladder tumor at the bladder neck and left ureteral orifice. The tumor was histologically diagnosed as TCC (transitional cell carcinoma). M-VAC chemotherapy (methotrexate 30 mg/m2, day 1, 15, 22, vinblastine 3 mg/m2, day 1, 15, 22, adriamycin 30 mg/m2, day 2, cisplatin 70 mg/m2, day 2) was performed as a neoadjuvant chemotherapy. However, since pelvic MRI revealed tumor invasion in to the muscle area, total cystoprostatourethrectomy and ileal conduit were done. Pathological examination of the tumor of left ureteral orifice revealed TCC, G2, INF beta, pT1, ly0, v(-), pN0, PM0. The tumor in the bladder neck was histologically diagnosed as submuscosal type leiomyoma. No cases of leiomyoma of the urinary bladder associated with transitional cell carcinoma have been reported in Japan.