Differential activation of pp60(c-src) and pp62(c-yes) in human colorectal carcinoma liver metastases

pp60(c-src) and pp62(c-yes) are protein tyrosine kinases whose specific activities are increased in primary colorectal carcinomas. Activity of pp60(c-src) is further increased in colorectal liver metastases. This study was undertaken to compare pp60(c-src) and pp62(c-yes) expression and activity in human colorectal carcinoma liver metastases and to determine the potential prognostic significance of differences in activation of these two kinases. The pp60(c-src) and pp62(c-yes) tyrosine kinase activities and protein levels relative to those in normal colonic mucosa were determined using an immune complex kinase assay and immunoblot analysis in tissue specimens from 22 patients with primary colorectal carcinoma and synchronous metastatic liver disease and from 9 patients with metachronous colorectal carcinoma liver metastases. Of the primary colon tumors, 64% of the tumors contained elevated activities of both pp60(c-src) and pp62(c-yes). For liver metastases, however, only 10% had activation of both tyrosine kinases, 61% had elevated pp60(c-src) activity only, and 23% had elevated pp62(c-yes) activity only. Analysis of synchronous metastases from primary tumors with elevated activities in both kinases demonstrated that in 71% of these patients, the activity of either pp60(c-src) or pp62(c-yes) decreases relative to the primary tumor. Protein levels of pp60(c-src) and pp62(c-yes) in primary carcinomas and metastases remained unchanged from levels in normal colonic mucosa. These results demonstrate that differential regulation of the activities of pp60(c-src) and pp62(c-yes) occurs during tumor progression. Patients with either synchronous or metachronous liver metastases and elevated pp62(c-yes) kinase activity have biologically more aggressive disease and a worse prognosis than patients without elevated pp62(c-yes) activity in their liver metastases (median survival, 13 months versus 30 months, P < 0.005, Wilcoxon signed rank test). Analysis of patients with synchronous liver metastases also demonstrated a worse prognosis for those with elevated pp62(c-yes) kinase activity (P < 0.05, Wilcoxon signed rank test).     

MR images of the hepatocellular carcinoma in Long-Evans cinnamon (LEC) rats

Long-Evans Cinnamon (LEC) rats which have an abnormal copper accumulation in the liver develop hereditary hepatitis and subsequent hepatocellular carcinoma (HCC). We studied the correlation of MR images of the HCCs developed in LEC rats and histopathological features. The HCCs of LEC rats had high intensity on T 1-weighted images and iso-low intensity on T 2*-weighted images. Histopathological examination showed that the HCCs were highly differentiated. Copper concentration in the HCCs was lower than that in the surrounding non-cancerous liver tissues. From these results, we suggest that copper accumulation may not be responsible for the high intensity of HCCs on T 1-weighted images.     

Phosphorylation of growth factor receptor binding protein-2 by pp60c-src tyrosine kinase

A positive influence of magnesium in the prevention and treatment of hyperactivity in children is more and more frequently raised in the literature. The aim of our work was to estimate magnesium contents in children with attention deficit hyperactivity disorder, (ADHD). The investigations comprised 116 children (94 boys and 20 girls), aged 9-12 years, with recognized ADHD. In 68 out of 116 patients examined ADHD occurred with other coexisting disorders specific to the developmental age and in the remaining 48 patients it occurred together with disruptive behaviour. Magnesium levels have been determined in blood serum, red blood cells and in hair with the aid of atomic absorption spectroscopy. Magnesium deficiency was found in 95 per cent of those examined, most frequently in hair (77.6 per cent), in red blood cells (58.6 per cent) and in blood serum (33.6 per cent) of children with ADHD. The conclusion from the investigations is that magnesium deficiency in children with ADHD occurs more frequently than in healthy children. Analysis of the material indicated the correlation between levels of magnesium and the quotient of development to freedom from distractibility.     

The role of pp60c-src in the regulation of calcium entry via store-operated calcium channels

In many cell types, G protein-coupled receptors stimulate a transient Ca2+ release from internal stores followed by a sustained, capacitative Ca2+ entry, which is mediated by store-operated channels (SOCs). Although it is clear that SOCs are activated by depletion of internal Ca2+ stores, the mechanism for this process is not well understood. Previously, we have reported that inhibitors of tyrosine kinase activity block the bradykinin- and thapsigargin-stimulated Ca2+ entry in fibroblasts, suggesting that a tyrosine kinase activity may be involved in relaying the message from the empty internal Ca2+ stores to the plasma membrane Ca2+ channel (Lee, K.-M., Toscas, K., and Villereal, M. L. (1993) J. Biol. Chem. 268, 9945-9948). We also have demonstrated that bradykinin activates the nonreceptor tyrosine kinase c-src (Lee, K.-M., and Villereal, M. L. (1996) Am. J. Physiol. 270, C1430-C1437). We investigated whether c-src plays a role in the regulation of SOCs by monitoring capacitative Ca2+ entry in 3T3-like embryonic fibroblast lines derived from either wild type or src-/src- (Src-) transgenic mice. We report that Ca2+ entry, following store depletion by either bradykinin or thapsigargin, is dramatically lower in Src- fibroblasts than in wild type fibroblasts. The level of capacitative Ca2+ entry in Src- cells is restored to nearly normal levels by transfecting Src- cells with chicken c-src. These data suggest that c-src may play a major role in the regulation of SOCs.     

Evidence for kinetically distinct forms of pp60c-src with different Km values for their protein substrate

The biphasic kinetics for phosphorylation of poly(E4Y) by the protein tyrosine kinase pp60c-src were examined. At pH 6.5 substrate inhibition was observed, whereas at pH 8.0 the kinetics were still biphasic, but the enzyme was no longer inhibited. The reaction rate increased in a nonlinear fashion with increasing concentration of substrate. The kinetics were examined from the view that the biphasic kinetics at pH 8.0 were due to two enzymes acting simultaneously on the same substrate. A 55-fold difference in Km values (0.029 versus 1.6 mg/ml) was calculated. The low Km form of the enzyme (0.043 mg/ml) was physically separated from the mixture of kinetic variants by immunoaffinity chromatography, and phosphorylation by protein kinase A resulted in the formation of an enzyme with an intermediate Km (0.3-0.4 mg/ml). The presence of multiple kinetic forms of this tyrosine kinase has important implications in our efforts to understand the role of pp60c-src in human oncology.     

Tight-binding inhibitory sequences against pp60(c-src) identified using a random 15-amino-acid peptide library

A method is presented for matching wedge field dose distributions on different treatment units, simplifying the transfer of patients between machines during machine failure or scheduled downtime and avoiding the need for a full re-plan in most cases. Differences in wedge field characteristics between machines are accounted for and differences in energy are easily accommodated.     

Early effects of pp60(v-src) kinase activation on caveolae

Members of the nonreceptor tyrosine kinase family appear to be targeted to caveolae membrane. We have used a Rat-1 cell expressing a temperature sensitive pp60(v-src) kinase to assess the initial changes that take place in caveolae after kinase activation. Within 24-48 h after cells were shifted to the permissive temperature, a set of caveolae-specific proteins became phosphorylated on tyrosine. During this period there was a decline in the caveolae marker protein, caveolin-1, a loss of invaginated caveolae, and a 70% decline in the sphingomyelin content of the cell. One of the phosphorylated proteins was caveolin-1 but it was associated in coimmunoprecipitation assays with both a 30 kDa and a 27 kDa tyrosine-phosphorylated protein. Finally, the cells changed from having a typical fibroblast morphology to a rounded shape lacking polarity. In light of the recent evidence that diverse signaling events originate from caveolae, pp60(v-src) kinase appears to cause global changes to this membrane domain that might directly contribute to the transformed phenotype.     

Expression of GLUT1 in stratified squamous epithelia and oral carcinoma from humans and rats

Most cells express facilitative glucose transporters. Four isoforms (GLUT1-4) transporting D-glucose across the plasma membrane show a specific tissue distribution, which is the basis for tissue-specific patterns in glucose metabolism. GLUT1 is expressed at high levels in tissue barriers such as the blood-brain barrier, and this isoform has been suggested as an indicator of such barriers. GLUT1 has been found in basal layers of human epidermis where no such tissue barrier is present. To further clarify these issues, we examined the distribution of GLUT1 and GLUT4 in skin, different types of oral mucosa from rat and man, and a human oral carcinoma by indirect immunofluorescence microscopy. The results showed that GLUT1 was expressed in the basal and parabasal layers of the different stratified squamous epithelia, with some variations between keratinized and non-keratinized subtypes. GLUT1 was also expressed in ductal- and myoepithelial cells of minor salivary glands and perineural sheath located in the lamina propra, and furthermore in the cells of an oral carcinoma. GLUT4 was not expressed in any of the tissues examined. This distribution of GLUT1 does not fit with the idea of GLUT1 as a general indicator of tissue barriers. In contrast, our results support the prevailing, but limited knowledge of glucose metabolism in squamous stratified epithelia, a metabolism believed to depend mostly on glycolysis, especially in the basal layers. High-level expression seemed to be confined to keratinocytes without glycogen stores.     

Erythropoietin in hepatocellular carcinoma

Hemopoietic alterations may occur during tumoral diseases, determining anemia. In most cases, serum EPO levels were lower than normal values. Hepatocellular carcinoma (HCC), one of the most frequent malignancies world-wide, is often characterized by mild anemia and increased serum EPO levels. We studied 30 HCC patients and 20 healthy subjects. We found that HCC patients presented higher serum EPO levels than healthy controls. In HCC patients, there was a significant inverse correlation between serum EPO levels and red blood cell count or hemoglobin levels. We postulated that the elevated serum EPO levels observed in these patients may be due to reduced hepatic clearance of EPO, and to the influence of cytokine-mediated inflammatory factors.     

Copper in plasma reflects its status and subsequent toxicity in the liver of LEC rats

Differential treatment by gender has been an ongoing area of concern and uncertainty both in society at large and in clinical research. In this investigation, therapist attributions over the course of therapy for three different couples were coded and analyzed to determine if cause for positive and negative events was assigned differentially to females and males. Additionally, the stability and globality dimensions of the therapist's attributions about the couples were examined for stereotypical gender-related patterns. Results indicate no gender differences in locus of causal attributions but some gender-related patterns in stability and globality dimensions. Implications for both couples therapy and gender bias in couples research are discussed.     

Therapy of hepatocellular carcinoma

The therapeutic modalities in patients with hepatocellular carcinoma (HCC) depend on the number, size and location of the lesions as well as the stage of the underlying liver disease and the physical condition of the patient. In patients with small and solitary lesions, resection, liver transplantation and in some cases percutaneous ethanol injection (PEI) can be curative. In more advanced stages of the disease with larger or multiple lesions, PEI and/or transarterial chemotherapy with or without embolization (TACE or TAC) can slow the progression of the disease. In disseminated disease, a radiotherapeutic approach can be taken in selected cases. The therapeutic strategy in patients with HCCs should be individualized, frequently involving a combination of therapeutic modalities. In contrast to the earlier dismal prognosis, for most HCC patients there is today a therapeutic strategy that results in prolongation of life and in some cases even cure.     

Removal and efflux of copper from Cu-metallothionein as Cu/tetrathiomolybdate complex in LEC rats

Tetrathiomolybdate (TTM) removes copper (Cu) accumulating in a form bound to metallothionein (MT) in the liver of LEC rats (Long-Evans rats with a cinnamon-like coat color). The first step in the removal of Cu from Cu-MT has been shown to form a complex between MT and TTM through (MT)-S-Cu-S-(TTM) bridges (referred to as MT/TTM complex). Additional TTM was demonstrated to remove Cu from MT/TTM complex as the second step to form Cu/TTM complex by liberating MT. The Cu/TTM complex binds specifically to albumin in serum and to high molecular weight proteins in the absence of albumin, and is assumed to be a form of Cu for efflux by the treatment with TTM.     

Anorexic humans and rats.

Based on an article by S. Schachter (see record 1971-24450-001) that draws parallel findings for obese humans and rats, a case is made for the parallel features of humans with anorexia nervosa and rats made aphagic through destruction of the lateral hypothalamus (LTH). The etiology of anorexia nervosa may in part be a function of pathology, either functional or structural, in the LTH. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Metabolism and activation of cyclopenta polycyclic aromatic hydrocarbons in isolated human lymphocytes, HL-60 cells and exposed rats

The metabolism of radiolabelled benz(j)aceanthrylene (B(j)A) was studied in suspensions of isolated human peripheral mononuclear blood cells (lymphocytes), using high performance liquid chromatography (HPLC). The only known metabolite found after 24 h exposure to 30 microg/ml (120 microM) B(j)A, was B(j)A-1,2-dihydrodiol, representing approximately 35% of the total metabolites formed. B(j)A, benz(l)aceanthrylene (B(l)A) and benzo(a)pyrene (B(a)P) all caused DNA adducts in human lymphocytes, as well as in the human promyelocytic cell line HL-60 cells, as measured by the 32P-postlabelling technique (30 microg/ml, 24 h). The total DNA adduct levels in human lymphocytes exposed to B(j)A, B(l)A or B(a)P were 0.13 +/- 0.03, 1.10 +/- 0.62 and 0.37 +/- 0.10 fmol/microg DNA, respectively, and similar levels were obtained in HL-60 cells (0.18 +/- 0.14, 0.97 +/- 0.35 and 0.29 +/- 0.17 fmol/microg DNA, respectively). For each compound, the human lymphocytes and HL-60 cells in addition showed similar DNA adduct patterns. Cells exposed to B(j)A revealed only one DNA adduct, which, judged by its TLC properties, resulted from B(j)A-1,2-epoxide. As measured by the alkaline filter elution technique, only low levels of single strand DNA breaks (SSB) were observed in both human lymphocytes and HL-60 cells after exposure to B(j)A, B(l)A or B(a)P (24 h, 30 microg/ml). By adding cytosine-1-beta-D-arabinofuranoside (Are C) and hydroxyurea (HU) 1 h prior to analysis to prevent strand break rejoining, some increase in SSB (2-3 times) was observed in the lymphocytes. Co-incubation of human lymphocytes with liver microsomes from PCB-treated rats for 1 h and exposure to B(j)A or B(l)A, increased the DNA adduct levels in the lymphocytes to 12.3 and 37.8 fmol/microg DNA, respectively. A large increase in SSB was also observed, whereas no such increase was observed after co-incubation with human liver microsomes. In vivo exposure of rats to 30 mg/kg B(j)A (i.p.) for 24 h revealed one major DNA adduct in lymphocytes and lung tissue (only one of three rats showed an adduct in liver tissue), apparently resulting from B(j)A-1,2-epoxide. The total DNA adduct level in the lymphocytes was 0.09 fmol/microg DNA, and in lung tissue between 0.10 and 0.62 fmol/microg DNA. Overall, the present data suggests that oxidation at the cyclopenta-ring is an important activation pathway for B(j)A in rats as well as in humans.     

Catalytic activity of the SH2 domain of human pp60c-src; evidence from NMR, mass spectrometry, site-directed mutagenesis and kinetic studies for an inherent phosphatase activity

During solution structural studies it was apparent that the human recombinant pp60c-src SH2 domain (srcSH2, residues 144-249) possessed an inherent phosphatase (Pase) activity. Complexes of U[13C,15N]srcSH2 with unlabeled Ac-pYEEIE (I) were examined using 31P and 1H-detected isotope filtered NMR methods. The presence of a high-affinity complex in equimolar solutions of I and U[13C, 15N]-srcSH2 was demonstrated by chemical shift perturbations, line broadening, and the observation of intermolecular nuclear Overhauser effects from the pY and Ile side-chain protons of I to protons on amino acid residues present in the binding pocket of srcSH2. Solutions containing excess I relative to srcSH2 revealed a slow hydrolysis of I to produce Ac-YEEIE and inorganic phosphate. The hydrolysis rate determined from NMR and HPLC-electrospray ionization mass spectrometry data at 30 degrees C for solutions containing excess I was 0.002-0.003 h-1. srcSH2 also catalyzed the hydrolysis of p-nitrophenyl phosphate (pNPP). Isoelectric focusing gels of a number of mutant srcSH2s demonstrated that this activity comigrated with srcSH2. Km, kcat, and kcat/Km were 3.7 +/- 0.4 mM, 3.1 +/- 0.2 x 10(-2) min-1, and 8.4 +/- 0.4 M-1 min-1, respectively, toward pNPP. The C188A mutant of the srcSH2 domain displayed 15% of the activity displayed by wild-type srcSH2, demonstrating that this residue is not absolutely required for activity. Two additional mutations in the known pY binding site, R178K and R158K, also resulted in decreased pNPPase activity, suggesting that the activity resides in or near this site. The inhibitor profile and pH dependence suggest that this is a novel protein Pase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-sensitivity of X-ray-hypersensitive cells derived from LEC strain rats to DNA-damaging agents

The cross-sensitivity of X-ray-hypersensitive lung fibroblasts from LEC strain (LEC) rats to other DNA-damaging agents was examined. The LEC cells were 2- to 3-fold more sensitive to bleomycin (BLM) that induces DNA double-strand breaks, and to a cross-linking agent, mitomycin C, than the cells from WKAH strain (WKAH) rats, while they were slightly sensitive to alkylating agents, ethyl nitrosourea and N-methyl-N'-nitro-N-nitrosoguanidine, but not to UV-irradiation. Although no difference was observed in the initial yields of DNA double-strand breaks induced by BLM between LEC and WKAH cells, the repair process of DNA double-strand breaks was significantly slower in LEC cells than in WKAH cells.     

Magnetic resonance imaging of Long-Evans cinnamon rats as a new model of hepatocellular carcinoma

A case of recurrent and metastatic nonfunctioning adrenocortical carcinoma in a 40-year-old woman is reported. The patient received 4 laparotomies and 1 thoracotomy for recurrent and metastatic disease after removal of the primary adrenal tumor. She has been alive for over 18 years following multiple surgery for diseases after the first adrenalectomy. In selected patients with recurrence and/or metastasis, repeated surgical resection offers the possibility of a cure or extended palliation.     

Molybdenum and copper kinetics after tetrathiomolybdate injection in LEC rats: specific role of serum albumin

A contest to encourage smokers to quit and to remain nonsmokers for at least six weeks was held in a low income neighbourhood in Montreal. The contest was one of many activities for a multifactorial community-based heart health promotion program. The objective of this article is to describe the intervention and its evaluation and to reflect on its relative successes. Thirty-one persons registered for the contest, seven stopped smoking during the six-week contest, and six persons remained abstinent two weeks after the end of it. Concrete recommendations regarding the contest and implementation of a smoking cessation program are discussed.