Brk/PTK6 sustains activated EGFR signaling through inhibiting EGFR degradation and transactivating EGFR.

Epidermal growth factor receptor (EGFR)-mediated cell signaling is critical for mammary epithelial cell growth and survival; however, targeting EGFR has shown no or only minimal therapeutic benefit in patients with breast cancer. Here, we report a novel regulatory mechanism of EGFR signaling that may explain the low response rates. We found that breast tumor kinase (Brk)/protein-tyrosine kinase 6 (PTK6), a nonreceptor protein-tyrosine kinase highly expressed in most human breast tumors, interacted with EGFR and sustained ligand-induced EGFR signaling. We demonstrate that Brk inhibits ligand-induced EGFR degradation through uncoupling activated EGFR from casitas B-lineage lymphoma-mediated EGFR ubiquitination. In addition, upon activation by EGFR, Brk directly phosphorylated Y845 in the EGFR kinase domain, thereby further potentiating EGFR kinase activity. Experimental elevation of Brk conferred resistance of breast cancer cells to cetuximab (an EGFR-blocking antibody)-induced inhibition of cell signaling and proliferation, whereas knockdown of Brk sensitized the cells to cetuximab by inducing apoptosis. Our findings reveal a previously unknown role of Brk in EGFR-targeted therapy.

Nuclear trafficking of the epidermal growth factor receptor family membrane proteins.

Multiple membrane-bound receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR) and ErbB-2, have been reported to be localized in the nucleus, where emerging evidence suggests that they are involved in transcriptional regulation, cell proliferation, DNA repair and chemo- and radio-resistance. Recent studies have shown that endocytosis and endosomal sorting are involved in the nuclear transport of cell surface RTKs. However, the detailed mechanism by which the full-length receptors embedded in the endosomal membrane travel all the way from the cell surface to the early endosomes and pass through the nuclear pore complexes is unknown. This important area has been overlooked for decades, which has hindered progress in our understanding of nuclear RTKs' functions. Here, we discuss the putative mechanisms by which EGFR family RTKs are shuttled into the nucleus. Understanding the trafficking mechanisms as to how RTKs are transported from the cell surface to the nucleus will significantly contribute to understanding the functions of the nuclear RTKs.

The effect of dietary docosahexaenoic acid on the expression of porcine lipid metabolism-related genes.

To study the effect of dietary docosahexaenoic acid (DHA) on the expression of adipocyte determination and differentiation-dependent factor 1 (ADD1) mRNA in pig tissues, weaned, crossbred pigs (30 d of age) were fed either 2% (as-fed basis) tallow or DHA oil for 18 d. Body weight of the pigs was not affected by different dietary fatty acid (FA) compositions. The plasma and liver FA composition reflected the composition of the diet. The adipose tissue and skeletal muscle FA composition only partially reflected the diet, indicating either a slower FA turnover or that a greater proportion of the FA in these tissues is from endogenous FA synthesis. The ADD1 is an important transcription factor that modulates transcription of FA synthase to regulate the endogenous FA synthesis in the liver and adipose tissue. The ADD1 mRNA was decreased (P < 0.05) in the liver of DHA-treated pigs compared with that of the tallow-treated pigs. The diets did not have an effect on the ADD1 mRNA in pig adipose tissue. The ADD1 transcript was not detected in pig skeletal muscle. These results indicate that significant enrichment of liver DHA content inhibits the expression of ADD1 mRNA. Such an effect is similar to that reported in porcine differentiating adipocytes cultured with DHA. The liver and muscle acyl CoA oxidase mRNA concentration was increased (P < 0.05) by DHA oil treatment, suggesting that DHA treatment may increase peroxisomal fatty acid oxidation in these two tissues. Our present observations demonstrate that dietary DHA enrichment not only affects tissue DHA concentration but also mildly modifies the expression of genes related to fatty acid metabolism in the porcine liver and skeletal muscle.

Effect of polyunsaturated fatty acids on the expression of transcription factor adipocyte determination and differentiation-dependent factor 1 and of lipogenic and fatty acid oxidation enzymes in porcine differentiating adipocytes.

Polyunsaturated fatty acids (FA) regulate genes involved in lipid metabolism. The effects of polyunsaturated FA on the transcription factor adipocyte determination and differentiation-dependent factor (ADD) 1 and fatty acid synthase (FAS) mRNA in differentiating porcine adipocytes were measured using a stromal vascular cell culture system. Porcine stromal vascular cells were isolated from subcutaneous adipose tissues and plated in Dulbecco's modified Eagle's medium (DMEM)-nutrient mixture F-12 Ham (F-12) plus fetal bovine serum (100 ml/l) for 24 h. Then cells were differentiated in DMEM-F12 plus insulin, hydrocortisone and transferrin without or with polyunsaturated FA at 6.25, 25.00 or 100.00 microM. The ADD1 mRNA was decreased by 100.00 microM-arachidonic acid, 6.25 to 100.00 microM-docosahexaenoic acid or cis-9,trans-11-conjugated linoleic acid. The polyunsaturated FA reduced the transcription rate of FAS, but not of ADD1. All three polyunsaturated FA accelerated degradation of ADD1 and FAS mRNA to reduce the abundance of ADD1 and FAS mRNA. Results also showed that polyunsaturated FA inhibit the ADD1 expression, not only of mRNA concentration, but also of mature ADD1 protein concentration, suggesting an overall reduction of ADD1 function by polyunsaturated FA. Our present experiments demonstrate that polyunsaturated FA regulate the gene expression of ADD1 and enzymes involved in lipid metabolism in porcine adipocytes.

Coiled coil region of streptokinase gamma-domain is essential for plasminogen activation.

The specific functions of the amino acid residues in the streptokinase (SK) gamma-domain were analyzed by studying the interactions of human plasminogen (HPlg) and SK mutants prepared by charge-to-alanine mutagenesis. SK with mutations of groups of amino acids outside the coiled coil region of SK gamma-domain, SK(K278A,K279A,E281A,K282A), and SK(D360A,R363A) had similar HPlg activator activities as wild-type SK. However, significant changes of the functions of SK with mutations within the coiled coil region were observed. Both SK(D322A,R324A,D325A) and SK(R330A,D331A,K332A,K334A) had decreased amounts of complex formation with microplasminogen and failed to activate HPlg. SK(D328A,R330A) had a 21-fold reduced catalytic efficiency for HPlg activation. The studies of SK with single amino acid mutation to Ala demonstrate that Arg(324), Asp(325), Lys(332), and Lys(334) play important roles in the formation of a HPlg.SK complex. On the other hand, amino acid residues Asp(322), Asp(328), and Arg(330) of SK are involved in the virgin enzyme induction. Potential contact between Lys(332) of SK and Glu(623) of human microplasmin and strong interactions between Asp(328) and Lys(330), Asp(331) and Lys(334), and Asp(322) and Lys(334) of SK are noticed. These interactions are important in maintaining a coiled coil conformation. Therefore, we conclude that the coiled coil region of SK gamma-domain, SK(Leu(314)-Ala(342)), plays very important roles in HPlg activation by participating in virgin enzyme induction and stabilizing the activator complex.

Gastric choristoma of the hypopharynx presenting in an infant: a case report and review of the literature.

Normal gastric mucosa has been found throughout the alimentary tract, ranging from scattered rests of cells to well formed mucosa with submucosal smooth muscle. Many terms have been applied to these collections; however, the term gastric choristoma seems most appropriate. We present a case of hypopharyngeal gastric choristoma presenting with dysphagia and frequent emesis. Our literature review reveals that this is the seventh reported case of gastric choristoma found in the hypopharynx, and the first to present in an infant in the absence of respiratory distress. Excision or CO(2) laser ablation is useful for symptom relief; however, complete removal often requires multiple attempts.

Design of microprism-type symmetric Y-junction waveguides with the full phase compensation method.

Using the full phase compensation method, we derive a set of precise phase compensation formulas to design a three-dimensional microprism-type Y junction. The simulated and traditional results are compared to verify that the proposed structure is better. By taking into account the orthogonality principle to estimate the branching losses, the proposed structure was improved to better than approximately 6-10% over the traditional structure.

Systematic design of full phase compensation microprism-type low-loss bent waveguides.

For traditional microprism-type bent waveguides one must take into consideration only the two outer optical paths to compensate for the phase difference between phase fronts in front of and behind the bent region. We propose a systematic design rule to achieve an optimal phase matching condition by taking account of the whole optical paths for full phase compensation. The simulated results obtained with the fast Fourier transform beam propagation method indicate that the normalized transmitted powers are greater than 95% even though the bent angle is as large as 10 degrees .

Systematic design of microprism-type low-loss step-index bent waveguides.

By taking into account the curved travel path instead of the conventional abrupt optical line path in the bent region of three-dimensional embedded structures, we propose two kinds of microprism-type waveguide bends and systematically derive precise phase compensation formulas. The simulation results obtained with the fast Fourier-transform beam propagation method indicate that the normalized transmitted powers are greater than 95% even though the bent angle is as large as 10 degrees .

Relationship of ceftazidime pharmacokinetic indices with therapeutic outcome in patients with cystic fibrosis.

In this study we investigated the relationships between ceftazidime pharmacokinetic indices and clinical outcome measurements during acute pulmonary exacerbations in patients with cystic fibrosis. Twenty patients received ceftazidime during the study period. On Days 2, 7 and 14 outcome measurements were assessed. Ceftazidime peaks and troughs were calculated as was the percentage of time of the dosing interval the serum concentration/minimum inhibitory concentration ratio exceeded 8, 4 and 1. There were significant differences between Days 2 and 7 and between Days 2 and 14 for the outcome measurements. There were no significant between-day differences for the pharmacokinetic indices. Significant correlations, involving both within and between study days, existed between the ceftazidime pharmacokinetic indices and the clinical outcome measurements. Further investigation of these relationships is warranted.

Acute changes in endothelin-1 after hemodialysis for chronic renal failure.

Endothelin is a recently described, potent renal vascular and systemic vasoconstrictor peptide. To evaluate the response of this peptide to volume contraction, we measured eight baseline and posthemodialysis samples from seven children, aged 14.5 +/- 3 years, with chronic renal failure. Plasma was extracted and endothelin-1 was measured by radioimmunoassay. Dialysis was performed for a 3- to 3 1/2-hour period, and body weight decreased from 38.0 +/- 14.3 to 36.2 +/- 13.8 kg (p < 0.01) during this time. There were no significant changes in heart rate or respiratory rate after dialysis, but blood pressure fell from 127/80 +/- 22/16 to 114/72 +/- 20/21 mm Hg (p = 0.05 for the systolic pressure). Plasma endothelin-1 concentration increased from 1.5 +/- 1.2 pg/ml at baseline to 7.3 +/- 8.9 pg/ml (p = 0.06) after dialysis; the fall in body weight from dialysis correlated with the increase in endothelin (r = -0.75; p = 0.05). Thus volume contraction from hemodialysis is associated with a rise in plasma endothelin-1, which is related to the acute change in body weight.

Relation of elevated plasma endothelin in congenital heart disease to increased pulmonary blood flow.

Endothelin-1 (ET), a potent vasoconstrictor peptide, has been found to be elevated in children with pulmonary hypertension associated with congenital heart defects. To evaluate the effect of pulmonary blood flow on ET concentrations, 5 ml blood samples were obtained peripherally at cardiac catheterization from 35 patients, ages 0.13 to 17 years (median 2). Plasma was extracted and ET measured by radioimmunoassay. Patients were classified into 2 groups based on the presence (group A) or absence (group B) of increased pulmonary blood flow defined as a Qp/Qs > or = 1.5. When the 13 patients (37%) in group A were compared with the 22 patients (63%) in group B there were no significant differences in age, cardiac index, or pulmonary and systemic resistances. ET concentrations were significantly higher in group A patients (median 3.25, range 0 to 16.5 vs median 0, range 0 to 6.35 pg/ml; p < or = 0.05). Pulmonary blood flow and pulmonary artery pressure were also higher in group A patients (p < or = 0.01). When patients within group A were subdivided into those with and without pulmonary hypertension, no difference was present in their ET concentrations (mean/SD: 4.4/4.3 vs 4.0/6.4 pg/ml, p = NS). Thus, ET is elevated in patients with congenital heart disease associated with left-to-right shunts and it appears that this increase is related to increased pulmonary blood flow independent of pulmonary artery pressure.

Effect of ascorbic acid on plasma alcohol clearance.

The effects of short-term and long-term ascorbic acid supplements on plasma alcohol clearance were studied in 13 clinically healthy male subjects. Two dose levels of alcohol, 0.5 and 0.8 g/kg body weight, were used. Blood samples were taken at zero time, 0.5 hours, then hourly up to 6 hours after alcohol consumption for the measurement of plasma alcohol and ascorbic acid levels, red-cell reduced glutathione level, and plasma alanine aminotransferase activity. At both dosages of alcohol, short-term as well as long-term pretreatment with ascorbic acid significantly enhanced the clearance of plasma alcohol. Although long-term ascorbic acid pretreatment resulted in better alcohol clearance, no significant difference in alcohol clearance was found between short-term and long-term ascorbic acid pretreatment. The two dose levels of alcohol had no significant effect on the red-cell reduced glutathione concentration or plasma alanine aminotransferase activity.

A frame-shift mutation in the cystic fibrosis gene.

Cystic fibrosis (CF) is a common recessive lethal genetic disorder, affecting 1 in 1,600 Caucasians. The disease causes defective regulation of chloride-ion transport in exocrine cells. Although in all CF families the disease is linked to a locus on chromosome 7q31, there is clinical heterogeneity in the severity of the disease and the age at which it is diagnosed. CF is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. A three-nucleotide deletion (delta F508) causing the loss of a phenylalanine residue in the tenth exon of the CFTR gene has been found on 70% of CF chromosomes. We have now characterized a CF family in which neither parent of the affected individual carries the common mutation, and identified a two-nucleotide insertion in the CF allele of the mother. The mutation introduces a termination codon in exon 13 of the CFTR gene at residue 821, and is predicted to result in the production of a severely truncated nonfunctional protein.

The effect of sodium selenite toxicity on tissue distribution of zinc, iron, and copper in rats.

Male Sprague-Dawley rats were used to determine the effects of suptoxic and toxic concentrations of selenite in the drinking water on tissue distribution of zinc (Zn), iron (Fe), and copper (Cu). Se (as sodium selenite) was provided in drinking water at concentrations of 0, 2, 4, and 8 ppm. At 19 d, half of the rats in 4 and 8 ppm Sesupplemented groups were kept on drinking water alone for additional 13 d. All rats were sacrificed at the end of 32 d of experiment. Heart, liver, and kidney were analyzed for the concentrations of Fe, Zn, and Cu by atomic absorption spectrophotometry and of Se by a fluorometric method.Results indicated that rats receiving 4 and 8 ppm Se in drinking water showed a marked reduction in food intake and a reduced growth rate. These adverse effects were quickly reversed when high Se intake was discontinued. Se toxicity caused minimal change in zinc status, reduced tissue iron concentrations and caused a marked increase in copper contents in heart, liver, and kidney. The latter findings were only partly reversed after removal of Se in drinking water. The accumulation of Cu in the tissues of Se-toxic rats provides the evidence of some interaction between Se and Cu.

Ethanol-ascorbate interrelationship in acute and chronic alcoholism in the guinea pig.

The effects of low (200 ppm) and of high (2000 ppm) ascorbic acid, in a nutritionally adequate diet, on blood ethanol levels have been studied in permanently carotid-cannulated, ethanol-infused, unanesthetized guinea pigs. In the acute study, the postinfusion rate of ethanol decline in the blood of animals treated with ascorbic acid was significantly higher when compared with animals treated with fructose, and the rate in the two treated groups was significantly higher than in untreated controls. In the chronic study, animals were infused with sublethal doses of ethanol (30% of the total caloric intake) for 8 weeks. Blood ethanol levels monitored throughout this period showed, at 3 hr postinfusion, a lower concentration in the group on a high ascorbic acid diet. Both experimental groups receiving ethanol lost significantly more body weight in the second week of dieting; but, while the group on high ascorbic acid regained weight steadily thereafter, the group on low ascorbic acid was still 50 g below the controls at the end of the experiment. Liver, kidney, and adrenal ascorbic acid concentrations were lower in the ethanol-treated groups compared to controls. Examination of the liver revealed more fatty metamorphosis or steatosis in the low ascorbic acid group, but there was no evidence of liver fibrosis or cirrhosis. These results demonstrate the feasibility of utilizing the guinea pig for the study of the biochemical and morphological sequelae of alcoholism. They further support the contention that a diet which is nutritionally adequate may no longer be so in the presence of high ethanol intake, and that supplemental vitamin C ingestion may afford protection against ethanol toxicity.

The effect of magnesium depletion on thyroid function in rats.

The effects of dietary magnesium (Mg) depletion on thyroid function were studied in young male rats. The rats were fed a semipurified diet containing either 12 ppm Mg (deficient rats) or 662 ppm Mg (control rats) for 14 to 28 days. Results showed that the Mg-deficient rats had decreased body weight gain, lowered concentrations of plasma thyroxine (T4) and Mg, but increased weight of the thyroid gland when expressed in proportion to the body weight (milligrams/100 g). There was no difference in the accumulation (uptake) of 131I, 24 hours after Na131I injection, between the Mg-deficient and Mg-supplemented rats. The protein-bound 131I (PB131I) level and the ratio of PB131I to total 131I in plasma was significantly reduced in Mg-deficient rats. Serum thyroid-stimulating hormone (TSH) levels after thyrotropin-releasing hormone injection (TRH, 50 ng/100 g body weight) increased fivefold at 30 minutes, but declined to near the basal level at 2 hours in both groups. No consistent difference in TSH response was observed between the two treatments. Serum T4 response to TRH challenge was significantly reduced in Mg-deficient as compared to Mg-adequate rats at all time intervals. The reduction of T4 level could be due to an impaired T4 synthesis or release in Mg-deficient rats.

Cysteine feeding affects urinary zinc excretion in normal and ethanol-treated rats.

The effects of L-cysteine feeding on urinary zinc excretion were studied in normal and ethanol-treated rats. In rats fed 3% cysteine X HCl for 1-10 weeks, a three-fold increase of urinary zinc excretion was observed. That effect was variable but was significant throughout the experimental weeks. Despite the excessive zinc loss, cysteine-fed animals showed no evidence of zinc depletion as judged by zinc concentrations in plasma, hair, spleen, pancreas and muscle. Furthermore, zinc contents in liver, kidney and tibia were significantly higher in cysteine-fed rats than in their controls. In rats consuming 20% ethanol for 5 months, urinary excretion of magnesium was markedly elevated and of zinc, only slightly elevated. When rats that drank either 20% ethanol or an isocaloric sucrose solution were fed the cysteine-fortified diet, elevations of urinary zinc excretion were similar. Cysteine intake apparently did not affect urinary excretion of copper and magnesium. Diets supplemented with DL-ethionine or L-cystine, but not inorganic sulfate, methionine or ascorbic acid, induced minor elevation of urinary zinc output. Those findings suggest that cysteine has a specific role in zinc metabolism.

Impairment of ascorbic acid synthesis in liver extracts of magnesium-deficient rats.

The effect of feeding a magnesium (Mg)-deficient diet for 9-34 days to weanling and young male rats on urinary and tissue ascorbate levels were studied. The concentrations of ascorbic acid in the liver and kidney were significantly reduced in the rats receiving a Mg-deficient diet as compared to those receiving a Mg-supplemented diet. The response to trichloro-2-methyl-2-propanol stimulation of urinary ascorbic acid was found to be considerably suppressed by dietary deficiency of Mg, suggesting that the decrease was not due to feed intake. In in vitro studies, the enzymatic synthesis of the vitamin from glucuronolactone or gulonolactone by liver extracts from Mg-deficient rats was significantly decreased as compared with Mg-supplemented rats. These results suggest that Mg-deficient rats have a reduced capacity to synthesize ascorbate which in turn produces a decrease in ascorbic acid concentrations in the liver.