Focal adhesion kinase and beta1 integrin regulation of Na+, K+, 2Cl- cotransporter in osmosensing ion transporting cells of killifish, Fundulus heteroclitus.

Focal adhesion kinase (FAK), also known as PYK2, is a tyrosine kinase that functions in integrin-mediated signaling in mechanosensitive cells but its role in osmosensing cells is unknown. Antibodies directed against phosphorylated FAK, whose epitopes are conserved among vertebrates, were used to follow phosphorylation patterns in an osmosensing ion secreting epithelium, the killifish (Fundulus heteroclitus) opercular membrane. At the electron microscopic level, a unique combination of integrin beta1, the phosphorylated form of FAK at tyrosine 407 (pY407) and Na(+), K(+), 2Cl(-) cotransporter (NKCC1) were all colocalized only on the basolateral membrane in chloride cells. The three proteins were also coimmunoprecipitated with each other in isotonic conditions, suggesting an osmosensing complex involving the three proteins. Only FAK pY407 was sensitive to hypotonic shock and became dephosphorylated with hypotonic shock, while FAK pY576 in the apical membrane and pY861 in cell-cell adhesions were insensitive to hypotonicity. NKCC1 contributes to NaCl secretion in seawater and previous reports showed that hypotonic shock (-60 mOsm/kg) rapidly inhibits Cl(-) secretion. These results indicate that chloride cells respond to hypotonic shock using integrin beta1 as an osmosensor that is connected to dephosphorylation of FAK pY407 which leads to NKCC1 deactivation in the basolateral membrane and the inhibition of NaCl secretion by these epithelial cells.

[Result of surgical treatment to early stage peripheral non-small cell lung cancer].

In the lung cancer, the announcement of the definition, the clinical behavior and the treatment result of the central early stage lung cancer, especially in situ lung cancer, have been seen. However, the definition and a clinicopathological concept of the peripheral lung cancer are still uncertain. The so-called small lung cancer of the tumor diameter 20 mm or less are peripheral lung adenocarcinoma. These patients' prognosis is excellent better, in contradiction to the prognosis of the patients with positive for pleural involvement or vessels invasion is worse. We studied the clinicopathological features, the Noguchi's classification, and prognosis of the 101 patients with small lung adenocarcinoma which were performed the operation, and refere about the selection of the operation method including the VATS and the limited operation.

Adrenomedullin (11-26): a novel endogenous hypertensive peptide isolated from bovine adrenal medulla.

Adrenomedullin (AM) is a potent hypotensive peptide originally isolated from pheochromocytoma tissue. Both the ring structure and the C-terminal amide structure of AM are essential for its hypotensive activity. We have developed an RIA which recognizes the ring structure of human AM. Using this RIA, we have characterized the molecular form of AM in bovine adrenal medulla. Gel filtration chromatography revealed that three major peaks of immunoreactive AM existed in the adrenal medulla. The peptide corresponding to Mr 1500 Da was further purified to homogeneity. The peptide was determined to be AM (11-26) which has one intramolecular disulfide bond. Amino acid sequences of bovine AM and its precursor were deduced from the analyses of cDNA encoding bovine AM precursor. The synthetic AM (11-26) produced dose-dependent strong pressor responses in unanesthetized rats in vivo. The hypertensive activity lasted about one minute, and a dose dependent increase in heart rate was also observed. The present data indicate that AM (11-26) is a major component of immunoreactive AM in bovine adrenal medulla and shows pressor activity.

A role for heterologous gap junctions between melanoma and endothelial cells in metastasis.

F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. We found that connexin (Cx) 26 is upregulated in BL6 cells. To examine gap junction formation, we devised a coculture system, in which an opened vein segment was placed at the bottom of a culture dish and then dye-labeled melanoma cells were seeded onto it. Immunohistochemistry indicated that the vein segment preserved the integrity of the endothelial monolayer. In this system, BL6 cells could transfer dye into endothelial cells but F10 cells could not. Transfection with wild-type Cx26 rendered F10 cells competent for coupling with endothelial cells and as spontaneously metastatic as BL6 cells. Conversely, transfection with a dominant-negative form of Cx26 rendered BL6 cells deficient in coupling and less metastatic. In human melanoma lesions, the level of Cx26 expression was low in melanoma cells residing in the basal layer, but significantly upregulated in melanoma cells invading the dermis. The results suggested that Cx26 plays a role in intravasation and extravasation of tumor cells through heterologous gap junction formation with endothelial cells.

Development of gap junctional channels and intercellular communication in rat liver during ontogenesis.

BACKGROUND/AIMS: We investigated the expression of connexin (Cx) 32 and 26 subunit proteins of the gap junction (GJ) in the rat liver during ontogenesis to clarify their roles in control of growth and differentiation, and observed their channels in association with development of gap junctional intercellular communication (GJIC). METHODS: The expression of Cx32 and 26 in prenatal and postnatal livers was examined by Western blot and immunofluorescence. GJ channels were investigated not only by double immunofluorescence study but also by immunogold electron microscopy. The spread of lucifer yellow 5 min after its microinjection was examined in the cultured liver tissues. RESULTS: 1) Western blot showed the expression of both Cx from the late stage of gestation and their peak a week after birth. 2) Cx32- or 26-positive plaques were scattered on hepatocytes of the fetal liver and some of them were colocalized; both were increased just after birth. On day 7 after birth, Cx32-positive plaques were present on all hepatocytes within a lobule, and Cx26-positive plaques were distributed in the periportal area. 3) Double-immunogold electron microscopy just after birth showed that most GJ channels were homotypic type of Cx32 or 26, and that few were heterotypic. On day 7 after birth, most channels had the homotypic type of type of Cx32 in the middle and pericentral areas, and there was a heterotypic type of Cx32 and 26 in the periportal area. 4) The dye transfer of lucifer yellow showed a wider spread in the liver tissues on day 7 after birth than on day 1. CONCLUSION: Increased GJ formation and compatibility or incompatibility of GJ channels are closely associated with development of GJIC, and GJIC may develop at cytodifferentiation during ontogenesis.

Comparison of PCR-restriction fragment length polymorphism analysis and PCR-direct sequencing methods for differentiating Helicobacter pylori ureB gene variants.

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the Helicobacter pylori genes is widely used to differentiate strains. However, with this typing method only a single base change at a specific restriction site can be detected. In addition, it is unclear whether the nucleotide base change recognized by RFLP is related to a substitution of encoded amino acid. To examine the validity of the PCR-RFLP method, 933-bp PCR products were obtained from 41 different clinical H. pylori isolates and were digested with Sau3A restriction endonuclease. Furthermore, the nucleotides of the same region in the ureB gene were directly sequenced and compared. PCR-RFLP confirmed that there was genetic diversity within the ureB gene with three distinct types, one being well conserved and the other two being variations. However, the direct sequencing method revealed that there was no difference at the nucleotide level among these RFLP types. Base substitutions recognized by Sau3A occurred in the third-base position and did not change the encoded amino acid. In addition, many nucleotide mutations, which could not be recognized by Sau3A, were frequently found. These results suggest that the PCR-RFLP method provides for an easy typing scheme of isolates, but does not reveal the true extent of genetic diversity. It is proposed that careful observation is required for the interpretation of results when clinical isolates are differentiated.

Shift of Chloride Cell Distribution during Early Life Stages in Seawater-Adapted Killifish, Fundulus heteroclitus.

The shift of chloride cell distribution was investigated during early life stages of seawater-adapted killifish (Fundulus heteroclitus). Chloride cells were detected by immunocytochemistry with an an-tiserum specific for Na(+), K(+)-ATPase in whole-mount preparations and paraffin sections. Chloride cells first appeared in the yolk-sac membrane in the early embryonic stage, followed by their appearance in the body skin in the late embryonic stage. Immunoreactive chloride cells in the yolk-sac membrane and body skin often formed multicellular complexes, as evidenced by the presence of more than one nucleus. The principal site for chloride cell distribution shifted from the yolk-sac membrane and body skin during embryonic stages to the gill and opercular membrane in larval and later developmental stages. Our observations suggest that killifish embryos and newly-hatched larvae could maintain their ion balance through chloride cells present in the yolk-sac membrane and body skin until branchial and opercular chloride cells become functional.

Effects of parent Shamo cocks on the histochemical properties of M. iliotibialis lateralis and M. supracoracoideus on their crossbred broilers.

1. Four Shamo (a Japanese game bird) cocks showing different characteristics in the histochemical properties of M. iliotibialis lateralis (ITL) were crossed with White Rock hens to produce male and female crossbred broilers of the 4 lines (90 d of age). Normal broilers (56 d) were used, for comparison. 2. Histochemical properties of ITL and M. supracoracoideus (SC) were compared among the crossbred lines and normal broilers. Myofibres were divided into Types II R, II I and II W showing high, moderate and low reduced nicotinamide adenine dinucleotide dehydrogenase (NADH-DH) activities, respectively. 3. In the ITL of the crossbred cockerels, the percentage of Type II R and II I fibres decreased and conversely Type II W increased in comparison to those in the Shamo. 4. Sex differences of the histochemical properties were recognised only in the ITL of the crossbred, in which the percentage of Type II R fibres was greater in the male. 5. The different characteristics of the parent Shamo cocks were reproduced only in the different fibre type composition of the ITL muscle in the crossbred cockerels. 6. The histochemical features of fibre type seemed to develop with bird age, particularly subsarcolemmal accumulation of formazan granules (indicating high NADH-DH activity) in Type IIR fibres. 7. Breed, line, sex and age differences in the histochemical properties were demonstrated clearly in ITL but not in SC.

Production of adrenomedullin in human vascular endothelial cells.

To examine the production of adrenomedullin (AM) in human vascular endothelial cells, AM concentrations in cultured endothelial cells derived from the human umbilical vein and the conditioned media of the cells were measured in the present study. The cultured endothelial cells secreted immunoreactive AM (ir-AM) into the medium at a rate of 14.7 +/- 3.0 fmol/10(6) cells/24 h with an intracellular ir-AM of 5.2 +/- 0.8 fmol/l0(6) cells. Analysis by reverse phase high performance-liquid chromatography (HPLC) showed that ir-AM in both the cells and the conditioned medium eluted at the position identical to that of human AM(1-52). Treatment with dexamethasone significantly augmented the secretion of ir-AM from the cells without any effect on the intracellular ir-AM concentration. Northern blot analysis showed not only the presence of the 1.6 kb human AM precursor mRNA in the endothelial cells, but also its increased expression in the dexamethasone-treated cells. Thus, AM was synthesized and secreted by the human endothelial cells of the umbilical vein, and glucocorticoid augmented the AM production. These findings suggest not only the role of AM as a local modulator of the vascular tone but also the possibility that endothelial cells contribute to circulating AM in the human blood.

Inhibition of catecholamine synthesis by proadrenomedullin N-terminal 20 peptide in cultured bovine adrenal medullary cells.

In cultured bovine adrenal medullary cells, proadrenomedullin N-terminal 20 peptide (PAMP), at concentrations > or = 3 microM, inhibited carbachol-induced [14C]catecholamine synthesis from [14C]tyrosine. Carbachol-induced activation of tyrosine hydroxylase was also attenuated by PAMP. These results suggest that PAMP is a novel endogenous peptide that regulates catecholamine synthesis via the suppression of its rate-limiting enzyme in adrenal medullary cells.

Proadrenomedullin N-terminal 20 peptide (PAMP), an endogenous anticholinergic peptide: its exocytotic secretion and inhibition of catecholamine secretion in adrenal medulla.

In cultured bovine adrenal medullary cells, stimulation of nicotinic receptors by carbachol evoked the Ca(2+)-dependent exocytotic cosecretion of proadrenomedullin N-terminal 20 peptide (PAMP) (EC50 = 50.1 microM) and catecholamines (EC50 = 63.0 microM), with the molar ratio of PAMP/catecholamines secreted being equal to the ratio in the cells. Addition of PAMP [1-20]NH2 inhibited carbachol-induced 22Na+ influx via nicotinic receptors (IC50 = 2.5 microM in a noncompetitive manner and thereby reduced carbachol-induced 45Ca2+ influx via voltage-dependent Ca2+ channels (IC50 = 1.0 microM) and catecholamine secretion (IC50 = 1.6 microM). It did not alter high K(+)-induced 45Ca2+ influx via voltage-dependent Ca2+ channels or veratridine-induced 22Na+ influx via voltage-dependent Na+ channels. PAMP seems to be a novel antinicotinic peptide cosecreted with catecholamines by a Ca(2+)-dependent exocytosis in response to nicotinic receptor stimulation.

Studies on the tumor-promoting activity of additives in biomaterials: inhibition of metabolic cooperation by phenolic antioxidants involved in rubber materials.

For the detection of tumor-promoting activities of phenolic antioxidants, the inhibitory activities on the intercellular gap-junctional communication were investigated using the V79 metabolic cooperation (MC) assay. Among eight antioxidants, 4,4'-butylidene-bis(3-methyl-6-tert-butyl-phenol), 2,2'-methylene-bis(4-methyl-6-tert-butylphenol) (MBMBP), and styrenated phenol (SP) showed stronger inhibitory activities than lithocholic acid, which is known to be a tumor promotor. However, 4,4'-thio-bis(3-methyl-6-tert-butylphenol), Irganox 1010, and 1330 did not inhibit at any concentrations. When the single-electron oxidation potentials were compared among antioxidants, the electrochemical ease estimated with the first oxidation potential was correlated with the cytotoxic potentials (r = 0.88), but not with the inhibitory activities in an MC assay. The tumor-promoting activity of MBMBP was also investigated using an in vitro, two-stage Balb/c 3T3 transformation assay. MBMBP did not show initiating activity, but significant promoting activity at concentrations of both 1 and 2.5 micrograms/ml were noted. These concentrations were close to the lowest effective inhibitory concentration (1.3 micrograms/ml) of MBMBP in an MC assay. In conclusion, there is a possibility that the phenolic antioxidants that show inhibitory activities in an MC assay contribute to the enhancement of tumor incidence induced by biomaterials.

Ca(2+)-dependent cosecretion of adrenomedullin and catecholamines mediated by nicotinic receptors in bovine cultured adrenal medullary cells.

Bovine cultured adrenal medullary cells (4 x 10(6)) contained 4266.5 +/- 370.0 fmol of immunoreactive adrenomedullin and 373.4 +/- 32.6 nmol of catecholamines. Nicotinic (but not muscarinic) receptors mediated the Ca(2+)-dependent co-secretion of adrenomedullin and catecholamines, with the molar ratio of adrenomedullin/catecholamines secreted into the medium being equal to the ratio stored in the cells. The concentration-response curve of carbachol for adrenomedullin secretion (EC50 42 microM) was similar to that for catecholamine secretion (EC50 63 microM). Reverse phase HPLC analysis showed that immunoreactive adrenomedullins in the cells and secreted into the medium were both eluted exclusively at the position almost identical to synthetic human adrenomedullin[1-52]NH2.

New vertebral body impactors for posterolateral decompression of burst fracture.

A new type of impactor was developed to decompress the anterior aspect of the vertebral body. These instruments are particularly useful for cases of burst fracture.

Association of viral oncogene-induced changes in gap junctional intercellular communication and morphological transformation in BALB/c3T3 cells.

In order to study the relationship between altered gap junctional intercellular communication (GJIC) and induction of cell transformation by oncogenes, we transfected six viral oncogenes into BALB/c3T3 A31-1-1 cells. BALB/c3T3 cells with v-src, v-ras or polyoma middle T (PyMT) genes grew in soft agar and formed distinct transformed foci in the absence or presence of a vast excess of non-transfected cells. On the other hand, those with v-myc, v-fos or polyoma large T (PyLT) genes expressed less distinctly transformed phenotypes (less transformed morphology, higher saturation density than non-transfected counterparts and less growth in soft agar), and did not form distinct foci in coculture with non-transformed cells. When their homologous GJIC capacities were examined by the microinjection/dye transfer assay, no decrease in GJIC was observed in any of the v-onc-transformed cells. Non-transformed and all v-onc-transformed cell lines expressed similar levels of connexin 43 mRNA. v-myc-, v-fos- and PyLT-transformed cells, but not v-ras-, v-src- and PyMT-transformed cells were able to communicate heterologously with non-transformed cells. Tumor promoting phorbol esters strongly inhibited GJIC of non-transformed and all v-onc-transformed BALB/c3T3 cell lines. In cocultures of v-myc-, v-fos- or PyLT-transformed cells with non-transformed BALB/c3T3 A31-1-1 cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the number of transformed foci. However, when these v-onc-transformed cells were co-cultured with non-transformed BALB/c3T3 A31-1-13 cells (which lose GJIC at growth confluence, as if TPA had been added), no morphologically transformed foci appeared. These results suggest that factors other than GJIC are involved in the suppression of oncogene-transformed cells by surrounding normal counterparts.

Regulation of gap-junctional intercellular communication in transformation-sensitive and transformation-resistant BALB/c 3T3 cell variants.

BALB/c 3T3 A31-1-13 cells are highly susceptible to chemical-induced cell transformation and their gap-junctional intercellular communication (GJIC) is decreased when these cells become confluent. On the other hand, A31-1-1 cells do not show such a change in GJIC and are more resistant to chemical induction of transformation. In order to see which phenotypes are dominant, cells of these two types were hybridized and the hybrids were analyzed for their phenotypes for GJIC and induction of cell transformation. These two cell lines were tagged with neor gene or hygromycin-resistance gene respectively, and their hybrids were selected in medium containing G418 and hygromycin. Six independently isolated clones were characterized and all showed a loss of intercellular communication at confluence, suggesting that the phenotype of transformation-sensitive cell line A31-1-13 was dominant. However, when these hybrid cells were exposed to 3-methylcholanthrene, no transformed foci were produced, suggesting that the transformation-resistant phenotype is dominant. These results suggest that the regulation of GJIC and the susceptibility to chemical induction of transformation are genetically separate traits, and indirectly suggest that the loss of GJIC alone cannot explain the high susceptibility of A31-1-13 cells to induction of transformation.

Okadaic acid and phorbol esters: comparative effects of these tumor promoters on cell transformation, intercellular communication and differentiation in vitro.

Okadaic acid is a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter in the mouse skin carcinogenesis system. Here we report on the in vitro activity of okadaic acid in 3 assay systems: BALB/c 3T3 cell transformation, gap junctional intercellular communication (GJIC) in various cell types, and inhibition of induction of differentiation of Friend virus-transformed murine erythroleukemia (MEL) cells. The activity of okadaic acid was compared to that of the phorbol ester tumor promoters TPA and phorbol-12,13-didecanoate (PDD). In a test system involving a 2-week exposure of BALB/c 3T3 cells following 3-methylcholanthrene initiation, okadaic acid at a concentration of 10 ng/ml was equipotent to PDD as a promoter of cell transformation (4.9 and 3.7 foci/dish, respectively). Longer exposures to okadaic acid resulted in cytotoxicity. Okadaic acid-generated as well as PDD-generated transformed foci displayed a selective lack of GJIC between focus cells and surrounding normal cells, i.e., transformed cells communicate among themselves but not with surrounding cells. However, in contrast to TPA, there was no inhibition by okadaic acid, except at toxic doses, of homologous GJIC in BALB/c 3T3 cells or human and mouse keratinocytes. Furthermore, okadaic acid, unlike TPA, did not inhibit MEL cell differentiation. Together, these results indicate that okadaic acid acts as a promoter of cell transformation but that its mechanism of action is different from that of the phorbol ester tumor promoters.

Effects of sodium L-ascorbate, uracil, butylated hydroxyanisole and extracellular pH on junctional intercellular communication of BALB/c 3T3 cells.

To study the mechanism of tumor promotion by different classes of urinary bladder promoters, the effect of sodium L-ascorbate, uracil and butylated hydroxyanisole (BHA) on junctional intercellular communication was examined in cultured BALB/c 3T3 cells using a dye-transfer method. In addition, since administration of sodium L-ascorbate and several other bladder tumor promoters is known to result in increased urinary pH, the effect of pH of the culture medium on intercellular communication was investigated. Results showed that under the present experimental conditions on the BALB/c 3T3 cells, BHA inhibited intercellular communication while sodium L-ascorbate and uracil did not. Intercellular communication was inhibited in proportion to the increase of medium pH after incubation of 4 h. Although further study is necessary to confirm the negative results of sodium L-ascorbate and uracil, these results suggest differences in the promoting mechanism(s) among these agents.