Analysis of the kefA2 mutation suggests that KefA is a cation-specific channel involved in osmotic adaptation in Escherichia coli

Mechanosensitive channels play an essential role in the regulation of turgor pressure in bacteria. In Escherichia coli, there are multiple mechanosensitive channels that have been characterized genetically: MscL, YggB and KefA. In this report, we describe the cloning of the kefA gene, the organization of the KefA protein and the phenotype of a missense mutation, kefA, which affects the KefA mechanosensitive channel. The altered function of the channel is manifest through increased sensitivity to K+ during growth at low osmolarity and complete inhibition of growth in media containing high K+ concentrations (0.6 M) in the presence of betaine or proline. Growth in high Na+ medium (0.6 M NaCl plus 20 mM K+) is normal. Analysis of the cytoplasmic pools shows that the mutant cannot regulate the K+ content of the cytoplasm when grown in high K+ medium. However, regulation of pools of amino acids is essentially normal and the mutant can accumulate high pools of proline during growth inhibition. The mutant shows increased sensitivity to acid hypo-osmotic shock (transition from neutral to acid pH combined with a reduction in osmolarity). The data are consistent with abnormal regulation of KefA in the presence of high K+ concentrations and either betaine or proline.     

Proteasome subunit Rpn1 binds ubiquitin-like protein domains

The yeast protein Rad23 belongs to a diverse family of proteins that contain an amino-terminal ubiquitin-like (UBL) domain. This domain mediates the binding of Rad23 to proteasomes, which in turn promotes DNA repair and modulates protein degradation, possibly by delivering ubiquitinylated cargo to proteasomes. Here we show that Rad23 binds proteasomes by directly interacting with the base subcomplex of the regulatory particle of the proteasome. A component of the base, Rpn1, specifically recognizes the UBL domain of Rad23 through its leucine-rich-repeat-like (LRR-like) domain. A second UBL protein, Dsk2, competes with Rad23 for proteasome binding, which suggests that the LRR-like domain of Rpn1 may participate in the recognition of several ligands of the proteasome. We propose that the LRR domain of Rpn1 may be positioned in the base to allow the cargo proteins carried by Rad23 to be presented to the proteasomal ATPases for unfolding. We also report that, contrary to expectation, the base subunit Rpn10 does not mediate the binding of UBL proteins to the proteasome in yeast, although it can apparently contribute to the binding of ubiquitin chains by intact proteasomes.     

Lack of mannose-binding lectin-A enhances survival in a mouse model of acute septic peritonitis

The mannose-binding lectin (MBL) (also known as the mannose-binding protein) is a serum protein that plays a role as an "ante-antibody" in innate immunity. In man, MBL is encoded by a single gene, whereas in mice there are two homologous proteins, MBL-A and MBL-C. In order to evaluate the relative roles of these two forms of MBL, we created MBL-A null mice that were MBL-C sufficient. We found MBL-A null mice had enhanced survival in a septic peritonitis model compared to wild-type mice and complement 3 null mice at 24 h, 48 h and 10 d (P < 0.05). Reconstitution of these mice with human MBL reversed the phenotype. Surviving mice had significantly decreased TNF-alpha and IL-6 levels in the blood and peritoneal cavity (P < 0.01). In vitro studies indicate that bacteria opsonized with MBL-A-deficient serum induced significantly less cytokine by peritoneal macrophages compared to those with wild-type serum. Our results indicate that MBL-A is a modulator of inflammation in vivo and in vitro in the mouse and that the role of MBL may extend beyond its role as an opsonin.     

Regulating heart development: the role of Nf1

Neurofibromatosis type 1 (NF1) is one of the most common human genetic disorders and is associated with significant morbidity and mortality. The gene responsible for this disorder, NF1, encodes neurofibromin, which can function to down-regulate ras activity. Mutations that inactivate NF7 result in elevated levels of ras signaling and increased cell proliferation in some tissues. NF7 functions as a tumor suppressor gene; patients inherit one mutated copy and are believed to acquire a "second hit" in tissues that go on to form benign or malignant tumors. NF7 is expressed widely, yet certain tissues are more susceptible to growth dysregulation in NF1 patients. Cardiovascular defects also contribute to NF1, though the cause remains unclear. In a recent study, we used tissue-specific gene inactivation in mice to study the role of neurofibromin in heart development. A further understanding of neurofibromin function will help to elucidate the pathophysiology of NF1 and will also lead to a better understanding of cell cycle regulation and ras pathways in specific cell types. Finally, we comment on how similar genetic strategies can be used in mice to study the role of additional signaling pathways involved in heart development.     

Quantitative determination of the enantiomers of methadone and its metabolite (EDDP) in human saliva by enantioselective liquid chromatography with mass spectrometric detection

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been developed and validated for the simultaneous determination of saliva concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC-MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized alpha(1)-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min and a temperature of 25 degrees C. Under these conditions, enantioselective separations were observed for methadone (alpha=1.30) and EDDP (alpha=1.17) within 15 min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0-600.0 ng/ml, and for EDDP from 0.5 to 15.0 ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5 ng/ml and for EDDP 0.5 ng/ml. Acceptable intra- and inter-day precision of the method (CVs<4.0%) and accuracy (CVs<4.0%) were obtained. These findings demonstrate the accuracy and precision of the method used to successfully analyze saliva obtained from patients enrolled in a methadone-maintenance program.     

Daily and yearly burnout symptoms in Israeli shift work residents

Burnout is a syndrome of physical and emotional exhaustion that develops among individuals who are open to public demands. In view of their heavy work load and sleep deprivation, we decided to evaluate the impact of long working hours on burnout and psychological status among a sample of residents during the first 2 years of their residency. Seventy-eight residents participated in the study, all residents completed self-administered questionnaires, and their sleep-wake cycle was monitored by a wrist-worn actigraph for a period of 5-7 days. The questionnaires included a short form suitable for Experience Sampling Method (ESM), and a longer background Questionnaire. The results revealed that sleep duration, Work Load and the interaction between them, explain the Negative Mood the day after the night shift. However, positive mood, and fatigue were not affected by sleep duration or workload. In general, after one year of residency, residents become more stressed, less involved in the job, and had a high level of burnout and psychosomatic symptoms. However, after the second year, the burnout symptoms were almost the same as at the beginning except for the level of stress that remained high. Sleep duration was unrelated to the burnout symptoms.     

Association of ErbB2 Ser1113 phosphorylation with epidermal growth factor receptor co-expression and poor prognosis in human breast cancer

The carboxyterminal domain of the epidermal growth factor receptor (EGFR) – a putative binding site for the ubiquitin ligase Cbl – is the site of serine phosphorylation events which are essential for ligand-dependent EGFR desensitization and degradation. Using a monoclonal antibody (aPS¹¹¹³) which selectively recognizes the homologous phosphorylated domain in the ErbB2 oncoprotein, we show here that wild-type ErbB2 becomes Ser¹¹¹³-phosphorylated following treatment of 3T3 cells with growth factors or tyrosine phosphatase inhibitors. In EGFR-overexpressing A431 cells, ligand-inducible aPS¹¹¹³ immunoreactivity declines more rapidly than other detectable phosphorylation events and is followed by EGFR downregulation. Analysis of 65 ErbB2-expressing primary breast cancers reveals a highly significant relationship between Ser¹¹¹³ phosphorylation and EGFR overexpression (p < 0.0001) as well as an association with poor prognosis (p = 0.005). We submit that ErbB2 Ser¹¹¹³ phosphorylation status represents a novel and informative biomarker of cancer cell biology and tumor behavior.