Epidermal growth factor stimulates tyrosine phosphorylation of phospholipase C-II independently of receptor internalization and extracellular calcium.

Epidermal growth factor (EGF) rapidly stimulates the formation of inositol 1,4,5-trisphosphate in a variety of cell types. Previously we have found that in intact cells stimulation of phospholipase C (PLC) activity by EGF is correlated with the retention of increased amounts of PLC activity by a phosphotyrosine immunoaffinity matrix, suggesting that the EGF-receptor tyrosine kinase phosphorylates PLC. We now define parameters of the mechanism by which EGF addition to A-431 cells stimulates phosphotyrosine immunoisolation of PLC activity and demonstrate that EGF addition to A-431 cells increases tyrosine phosphorylation of PLC. EGF rapidly and reversibly stimulated the anti-phosphotyrosine recovery of increased PLC activity when cells were treated with growth factor at 3 degrees C, indicating that receptor internalization is not required and that the phosphorylation event occurs prior to formation of inositol 1,4,5-trisphosphate. Also, the EGF stimulation of anti-phosphotyrosine recovery of PLC activity occurred in the absence of extracellular Ca2+. Stimulation of PLC activity in intact cells by other agonists, such as bradykinin or ATP, did not result in increased anti-phosphotyrosine recovery of PLC activity, suggesting two separate mechanisms exist in A-431 cells for hormone-stimulated formation of inositol phosphates. Finally, using monoclonal antibodies that specifically recognize three distinct PLC isozymes, we show that an approximately 145-kDa PLC isozyme (PLC-II) is present in A-431 cells and that EGF treatment of A-431 cells stimulates phosphorylation of PLC-II on both tyrosine and serine residues.     

Chromosome 13 deletion and hypodiploidy on conventional cytogenetics are robust prognostic factors in Korean multiple myeloma patients: web-based multicenter registry study

This study was designed to evaluate the prevalence of chromosomal abnormalities and to identify the specific abnormalities associated with poor prognosis. A total of 2,474 patients whose conventional cytogenetics were available at the time of diagnosis were evaluated via a nationwide registry. Normal metaphase cytogenetics was observed in 2,012 patients (81.3%). Among the 462 patients with chromosomal abnormalities, there were 161 (34.8%) patients with hyperdiploidy, 197 (42.6%) with pseudodiploidy, 79 (17.1%) with hypodiploidy, and 25 (5.5%) with near-tetraploidy. Deletion 13 (Δ13) in metaphase was observed in 167 patients (6.8%). Fluorescent in situ hybridization (FISH) was carried out in 967 patients (39.1%), and 66 (13.7%) out of 482 and 63 (10.3%) out of 611 patients were positive for t(4;14) and del(17p), respectively. With a median follow-up duration of 25.1 months, the median overall survival (OS) was 51.2 months (95% confidence interval, 46.5–55.9 months). In univariate analysis, the following four chromosomal abnormalities were significantly associated with a poor survival outcome: Δ13, hypodiploidy, del(13q) in FISH, and del(17p) in FISH. In the subsequent multivariate analysis, in which del(13q) and del(17p) in FISH were excluded due to a relatively low number of patients, Δ13 and hypodiploid status were independently associated with a poor survival outcome after adjusting for important clinical factors, including age, sex, performance, beta2-microglobulin, albumin, and lactate dehydrogenase (LDH). Using conventional metaphase cytogenetics, we confirmed that both Δ13 and hypodiploid status were robust poor prognostic factors. The metaphase karyotyping should remain the primary cytogenetic tool and an essential investigation for risk stratification in newly diagnosed multiple myeloma patients.     

YouTube as a source of patient information on gallstone disease

AIM:To investigate the quality of YouTube videos on gallstone disease and to assess viewer response according to quality.METHODS:A YouTube search was performed on September 18,2013,using the keywords‘‘gallbladder disease’’,‘‘gallstone disease’’,and‘‘gallstone treatment’’.Three researchers assessed the source,length,number of views,number of likes,and days since upload.The upload source was categorised as physician or hospital(PH),medical website or TV channel,commercial website(CW),or civilian.A usefulness score was devised to assess video quality and to categorise the videos into‘‘very useful’’,‘‘useful’’,‘‘slightly useful’’,or‘‘not useful’’.Videos with misleading content were categorised as‘‘misleading’’.RESULTS:One hundred and thirty-one videos were analysed.Seventy-four videos(56.5%)were misleading,36(27.5%)were slightly useful,15(11.5%)were useful,three(2.3%)were very useful,and three(2.3%)were not useful.The number of mean likes(1.3±1.5vs 17.2±38.0,P=0.007)and number of views(756.3±701.0 vs 8910.7±17094.7,P=0.001)were both significantly lower in the very useful group compared with the misleading group.All three very useful videos were PH videos.Among the 74 misleading videos,64(86.5%)were uploaded by a CW.There was no correlation between usefulness and the number of views,the number of likes,or the length.The"gallstone flush"was the method advocated most frequently by misleading videos(25.7%).CONCLUSION:More than half of the YouTube videos on gallstone disease are misleading.Credible videos uploaded by medical professionals and filtering by the staff of YouTube appear to be necessary.     

Benefits of PEGylation in the early post-transplant period of intraportal islet transplantation as assessed by magnetic resonance imaging of labeled islets

While a few studies have demonstrated the benefit of PEGylation in islet transplantation, most have employed renal subcapsular models and none have performed direct comparisons of islet mass in intraportal islet transplantation using islet magnetic resonance imaging (MRI). In this study, our aim was to demonstrate the benefit of PEGylation in the early post-transplant period of intraportal islet transplantation with a novel algorithm for islet MRI. Islets were PEGylated after ferucarbotran labeling in a rat syngeneic intraportal islet transplantation model followed by comparisons of post-transplant glycemic levels in recipient rats infused with PEGylated (n = 12) and non-PEGylated (n = 13) islets. The total area of hypointense spots and the number of hypointense spots larger than 1.758 mm² of PEGylated and non-PEGylated islets were quantitatively compared. The total area of hypointense spots (P < 0.05) and the number of hypointense spots larger than 1.758 mm² (P < 0.05) were higher in the PEGylated islet group 7 and 14 days post translation (DPT). These results translated into better post-transplant outcomes in the PEGylated islet group 28 DPT. In validation experiments, MRI parameters obtained 1, 7, and 14 DPT predicted normoglycemia 4 wk post-transplantation. We directly demonstrated the benefit of islet PEGylation in protection against nonspecific islet destruction in the early post-transplant period of intraportal islet transplantation using a novel algorithm for islet MRI. This novel algorithm could serve as a useful tool to demonstrate such benefit in future clinical trials of islet transplantation using PEGylated islets.     

An inducible functional peripheral benzodiazepine receptor in mitochondria of steroidogenic granulosa cells.

Granulosa cell lines, transformed by SV40 T-antigen and Ha-ras oncogene, have recently been established that can produce progesterone at levels comparable to those of highly differentiated cultures of primary granulosa cells (1-4). Here, the hypothesis that these cells contain a mitochondrial benzodiazepine receptor, and that stimulation of the receptor can trigger progesterone production in these cells, was tested. The agonist of the peripheral benzodiazepine receptor, Ro5-4864, produced a 3- to 5-fold stimulation (P less than 0.005) of progesterone production both in differentiated granulosa primary cultures and in the oncogene-transformed cell lines. Ro5-2807 (diazepam, Valium) exerts a similar effect on granulosa cell steroidogenesis while the specific agonist of central benzodiazepine receptor Ro15-4513 was without effect. The effects of Ro5-4864 or Ro5-2807 were not additive to those of gonadotropins and cAMP. Intact isolated mitochondria possessed high-affinity binding sites to [3H]-Ro5-4864 (Kd = 3.03 +/- 0.70 nM), which were enriched by 1 order of magnitude in these organelles compared to total cell homogenate. Bound Ro5-4864 could be competitively displaced with 1 microM unlabeled Ro5-4864 and Ro5-2807, but not with specific ligands of central benzodiazepine receptors Ro15-4513 and Ro15-1788. Prolonged elevation of cAMP in these cells caused a 30% (P less than 0.01) rise in the number of receptors. Mitochondria of NIH 3T3 cells contained only 30-40% (P less than 0.001) of the Ro5-4864 binding sites of mitochondria from steroidogenic cells, whereas yeast mitochondria lacked them completely. The existence of functional peripheral benzodiazepine receptors in mitochondria suggests that they may have a physiological role in the mobilization of cholesterol into mitochondria, and in elevating progesterone production in ovarian cells. The modulation of the interaction between benzodiazepine compounds and the gamma-aminobutyric acid receptor by progesterone metabolites suggests new interrelationships between peripheral and central nervous system receptors sensitive to benzodiazepines.     

Application of transcatheter valves for aortic valve replacement in pediatric patients: A case series

• Diabetic patients are frequently affected by coronary artery disease (CAD) and are at increased risk of CAD‐related adverse events, even after drug‐eluting stent (DES) implantation. If currently available DES have similar safety and efficacy in diabetic and nondiabetic patients is still debated.
• This prospective, multicenter registry showed similar 3‐year outcome in patients undergoing different DES implantation, although diabetic patients, especially those requiring insulin treatment, had significantly higher risk of adverse events than nondiabetic patients.
• Specific efforts to improve the performance of DES in diabetic patients are mandatory to adequately address the unsolved issue of diabetic patients affected by CAD.

     

Intrathecal beta-funaltrexamine antagonizes intracerebroventricular beta-endorphin- but not morphine-induced analgesia in mice

We have reported previously that beta-endorphin and morphine administered supraspinally produce analgesia by activating different descending pain inhibitory systems in rats. The descending system activated by beta-endorphin involves a spinal endorphinergic system whereas the descending system activated by morphine does not. To determine if this differential action of intraventricular beta-endorphin and morphine also occurs in mice, the effects of pretreatment with intrathecal (i.t.) and i.c.v. beta-funaltrexamine (beta-FNA) on analgesic response induced by i.c.v. and i.t. beta-endorphin and morphine were studied in mice. beta-FNA (2.5 micrograms) was injected i.t. or i.c.v. 24 hr before beta-endorphin or morphine administration and hot-plate and tail-flick responses were measured. Intrathecal beta-FNA attenuated i.c.v. beta-endorphin- but not i.c.v. morphine-induced analgesia. On the other hand, i.t. beta-FNA blocked both i.t. beta-endorphin- and morphine-induced analgesia, but was more effective in blocking the effects of i.t. morphine than beta-endorphin. At the supraspinal sites, beta-FNA administered i.c.v. was found to antagonize i.c.v. morphine-induced analgesia but not i.c.v. beta-endorphin-induced analgesia. The present results in mice are consistent with previous studies in rats and indicate that beta-endorphin and morphine activate different supraspinal opioid receptors. Also, analgesia produced by these two opioids resulted from activation of different descending pain inhibitory systems. The spinal endorphinergic system was involved in the production of i.c.v. beta-endorphin-, but not morphine-induced analgesia.     

Adenosine 3',5'-monophosphate suppresses metastatic spread in nude mice of steroidogenic rat granulosa cells transformed by simian virus-40 and Ha-ras oncogene.

8-Bromo-cAMP and substances elevating cAMP levels within cells, such as forskolin, cholera toxin, and Bordetella pertussis-invasive adenylate cyclase (BPAC), suppress the growth of cultured granulosa cells cotransfected by simian virus-40 (SV40) DNA and Ha-ras oncogene concomitantly with the induction of steroidogenesis and without affecting oncogene expression. We, therefore, tested the hypothesis that cAMP can modulate tumorigenesis and metastatic spread of these cells in vivo. The cotransfected cells induced rapid development of tumors when injected sc in nude mice. Tumor development was faster in less differentiated cotransfected cells originating from preantral ovarian follicles than in those obtained from highly differentiated transformed cells originating from preovulatory follicles. Cells transfected by SV40 DNA alone produced only slow-growing small tumors. Metastatic lesions of cotransfected cells were most abundant in lung and less frequent in ovaries, kidney, and spleen. No metastatic lesions were found in the liver. However, metastatic spread was dramatically suppressed when cotransfected cells injected into nude mice were pretreated with the invasive BPAC. In contrast, no suppression of metastases was observed when the cells were pretreated with 8-bromo-cAMP, forskolin, or cholera toxin. Removal of forskolin in cultured cotransfected cells yielded a rapid decrease in cAMP levels. In contrast, high levels of cAMP persist in cell cultures even several hours after 1-h pretreatment and subsequent removal of BPAC from the medium of culture cotransfected cells. It is suggested that the inhibitory effect of BPAC on the metastatic spread of these cells is due to prolonged elevation of cAMP in vivo. The newly established granulosa cell lines transformed by SV40 and the Ha-ras oncogene can serve as a model for further studies of cAMP modulation of carcinogenesis in ovarian malignancies.