Establishment of a doxycycline-regulated cell line with inducible,doubly-stable expression of the wild-type p53 gene from p53-deleted hepatocellular carcinoma cells

p53 is important in the development of hepatocellular carcinoma (HCC) and in therapeutic approaches, but the mechanism whereby it inhibits HCC growth is still unclear. The aim of the present study was to establish a HCC cell system in which p53 levels can be regulated. Full-length wild-type p53 cDNA obtained by PCR was cloned into a retroviral response vector controlled by the tetracycline responsive element (RevTRE-p53). The regulatory vectors RevTet-Off and RevTRE-p53 were transfected into a packaging cell line, PT67. Hep3B cells in which the p53 gene was deleted were infected with RevTet-Off viral particles from the PT67. Three G418-resistant cell clones with high luciferase expression and low background were infected with RevTRE-p53. By screening dozens of RevTRE-p53-infected clones with hygromycin we identified the one with the highest expression of p53 and the lowest background after doxycycline treatment. The results showed that p53 expression in this cell clone could be simply turned on or off by removing or adding doxycycline. Furthermore, it was found that the level of p53 protein was negatively and sensitively related to the doxycycline concentration. In conclusion, we have established a HCC cell line in which p53 expression can be switched on or off and regulated in a dose- and time-dependent manner.     

Detecting substitution-rate heterogeneity among regions of a nucleotide sequence

Likelihood-ratio statistics are proposed to test for heterogeneity in nucleotide substitution rate among regions of a DNA sequence. The tests examine three-sequence phylogenies, and two specific tests are proposed: a test to detect rate heterogeneity among genic regions within a sequence, over all evolutionary lineages; and a test to detect rate heterogeneity among regions in a specific evolutionary lineage. Simulations examine the ability of tests to detect a single region that varies in nucleotide substitution rate relative to the remainder of the sequence. A 50-bp region with a fivefold substitution-rate increase can be detected > or = 90% of the time when it is found in all three lineages of the phylogeny, and a 50-bp region of fivefold rate increase can be detected with approximately 70% power when it is found in only one evolutionary lineage. Simulation also examines the effect of transition- and transversion-rate differences. The tests are applied to published DNA sequences. While the tests are powerful, significant results can be difficult to interpret biologically.      

Predictors of mortality of patients newly diagnosed with clinical type 2 diabetes: a 5-year follow up study

Background

At diabetes diagnosis major decisions about life-style changes and treatments are made based on characteristics measured shortly after diagnosis. The predictive value for mortality of these early characteristics is widely unknown. We examined the predictive value of patient characteristics measured shortly after diabetes diagnosis for 5-year all-cause and cardiovascular mortality with special reference to self-rated general health.

Methods

Data were from a population-based sample of 1,323 persons newly diagnosed with clinical diabetes and aged 40 years or over. Possible predictors of mortality were investigated in Cox regression models.

Results

Multivariately patients who rated their health less than excellent experienced increased all-cause and cardiovascular mortality. These end-points also increased with sedentary life-style, relatively young age at diagnosis and presence of cardiovascular disease (CVD) at diagnosis. Further predictors of all-cause mortality were male sex, low body mass index and cancer, while cardiovascular mortality increased with urinary albumin concentration.

Conclusions

We found that patients who rated their health as less than excellent had increased 5-year mortality, similar to that of patients with prevalent CVD, even when biochemical, clinical and life-style variables were controlled for. This finding could motivate doctors to discuss perceptions of health with newly diagnosed diabetic patients and be attentive to patients with suboptimal health ratings. Our findings also confirm that life-style changes and optimizing treatment are particularly relevant for relatively young and inactive patients and those who already have CVD or (micro)albuminuria at the time of diabetes diagnosis.     

Durable remission of diabetes after bariatric surgery: What is the underlying pathway?

Background: In the past, type 2 diabetes mellitus (DM) was regarded as a progressive, incurable disease for which palliative therapy could not, over the long term, prevent the associated amputations, blindness, renal failure, and early mortality. This is no longer true. Full and durable remission of type 2 DM, with major decreases in morbidity and mortality, is now achieved regularly with several types of surgery that reduce contact between food and the foregut.Objectives: The aims of this article are to review the impact of bariatric surgery on obesity, remission of DM, and obesity-related morbidity and mortality, and the possible mechanisms for this advance.Methods: This article is based on our 2 meta-analyses of the literature published through April 30, 2006, as well as the most significant reports in the bariatric surgical literature that have been published in English since April 30, 2006. The studies included in our second meta-analysis provided the details of the methodology for the present literature review, including the levels of evidence.Results: Results of our 2 meta-analyses were published previously. Briefly, the analyses revealed that the clinical and laboratory manifestations of type 2 DM resolved or improved in most of the patients who underwent bariatric surgery; the responses were greatest in the patients who lost the most excess body weight; and the improvements were maintained for ≥2 years. The studies reported that intestinal operations such as gastric bypass reduced contact between food and the foregut, produced full and durable remission of DM, reduced mortality, and reversed other comorbidities associated with severe obesity (eg, asthma, gastroesophageal reflux, hypertension, stress incontinence). Insulin levels decreased markedly after surgery, as did glycosylated hemoglobin (A1C) and fasting blood glucose levels. Although these effects were initially attributed to weight loss, the rapid reversal of DM within a matter of days after surgery suggest that bariatric surgery changes the signaling mechanism of the gut with pancreatic islet cells, muscles, fat, the liver, and other organs.Conclusions: Bariatric surgery has opened new vistas, producing durable full remission of type 2 DM—a breakthrough previously considered impossible—with normalization of A1C levels over time and discontinuation of all antidiabetes medication for many patients. These advances create new opportunities for exploring the mechanisms of type 2 DM and its control through pharmaceutical approaches. DM is no longer an irreversible, incurable, or hopeless disease.     

ERK activation and nuclear signaling induced by the loss of cell/matrix adhesion stimulates anchorage-independent growth of ovarian cancer cells

Ovarian cancer metastasis involves the sloughing of epithelial cells from the ovary into the peritoneal cavity, where the cells can survive and proliferate in peritoneal ascites under anchorage-independent conditions. For normal epithelial cells and fibroblasts, cell adhesion to the extracellular matrix is required to prevent apoptosis and for proper activation and nuclear signaling of the ERK MAP kinase. The mechanisms of ERK regulation by adhesion have been determined by our lab and others. In this report, we elucidate a novel means of ERK regulation by cellular adhesion in ovarian cancer cells. We demonstrate that ERK and its activator MEK are robustly stimulated after cell detachment from a substratum in several ovarian cancer cell lines, but not a benign ovarian cell line, independent of serum and FAK or PAK activity. MEK and ERK activation was sustained for 48 h after detachment, while activation by serum or growth factors in adherent cells was transient. Re-attachment of suspended ovarian cells to fibronectin restored basal levels of MEK and ERK activity. ERK activity in suspended cells was dynamically controlled through an autocrine stimulatory pathway and prevalent phosphatase activity. Suspended cells demonstrated higher levels of ERK nuclear signaling to Elk1 compared to adherent cells. Inhibition of ERK activation with the MEK inhibitor U0126 had minor effects on adherent cell growth, but greatly decreased growth in soft agar. These data demonstrate a unique regulation of ERK by cellular adhesion and suggest a mechanism by which ERK may regulate anchorage-independent growth of metastatic ovarian cancer cells.     

Thermal Inactivation of Salmonella Serotypes in Red Meat as Affected by Fat Content

Survival curves of a cocktail of eight serotypes of Salmonella in ground beef and pork meat of different levels of fat (4% to 28%), at temperatures that ranged from 58°C to 65°C, were examined. Asymptotic D-values (D-values for large times) and initial D-values (D-values for small times, near zero) were estimated by identifying regions where the survival curves were linear, and performing linear regressions on data within the identified regions. The initial lag D-values increase with increasing fat levels for both beef and pork. The relationship of the asymptotic D-values with fat levels and temperature is complex, and definitive conclusions could not be made. It appears that, for ground beef, asymptotic D-values increase with increasing fat levels, but this was not the case for ground pork. The shapes of the survival curves were concave, convex, and sigmoidal, and depended upon the temperature, where for the lower temperatures studied (58°C and 60°C) the curves exhibited tailing. The Gompertz function was found to provide a good fit to the data over the range of temperatures and fat levels studied. These results, particularly for beef, suggest the importance of determining the shape of the survival curves (concave, convex or sigmoidal) when estimating times needed to obtain an adequate margin of safety for thermal processes of red meat.     

EGFR Tyrosine 845 Phosphorylation-Dependent Proliferation and Transformation of Breast Cancer Cells Require Activation of p38 MAPK

Phosphorylation of epidermal growth factor receptor (EGFR) on tyrosine 845 by c-Src has been shown to be important for cell proliferation and migration in several model systems. This cross talk between EGFR and Src family kinases (SFKs) is one mechanism for resistance to EGFR inhibitors both in cell models and in the clinic. Here, we show that phosphorylation of tyrosine 845 on EGFR is required for proliferation and transformation using several cell models of breast cancer. Overexpression of EGFR-Y845F or treating cells with the SFK inhibitor dasatinib abrogated tyrosine 845 phosphorylation, yet had little to no effect on other EGFR phosphorylation sites or EGFR kinase activity. Abrogation of Y845 phosphorylation inhibited cell proliferation and transformation, even though extracellular signal-regulated kinase (ERK) and Akt remained active under these conditions. Importantly, cotransfection of mitogen-activated protein kinase (MAPK) kinase 3 and p38 MAPK restored cell proliferation in the absence of EGFR tyrosine 845 phosphorylation. Taken together, these data demonstrate a novel role for p38 MAPK signaling downstream of EGFR tyrosine 845 phosphorylation in the regulation of breast cancer cell proliferation and transformation and implicate SFK inhibitors as a potential therapeutic mechanism for overcoming EGFR tyrosine kinase inhibitor resistance in breast cancer.     

Heat inactivation of Salmonella typhimurium DT104 in beef as affected by fat content

The heat resistance of an eight-strain cocktail of Salmonella typhimurium DT104 was determined at 58-65 degrees C in beef containing 7, 12, 18 or 24% fat. Inoculated beef was packaged in bags completely immersed in a circulating water bath and held at 58, 60, 62.5 and 65 degrees C for a predetermined length of time. The surviving cell population was enumerated by spiral plating heat-treated samples onto tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. Preliminary studies on thermal inactivation of the Salmonellae isolates in chicken broth indicated no correlation between heat resistance and origin of the isolates. While linear survival curves were observed in chicken broth, inactivation kinetics in beef showed deviations from the first order kinetics, represented by an initial lag period or shoulder before any death occurred with time. Overall, increased fat levels in beef resulted in longer lag periods and lower D-values, suggesting that the lag periods must be taken into account and added to the D-values for calculating the time required at a specific temperature for achieving a specific lethality for Salm. typhimurium DT104 in beef. Thermal death times from this study will assist the retail food industry to design cooking regimes that ensure safety of beef contaminated with Salm. typhimurium DT104.