Dexamethasone acutely suppresses the anabolic SNAT2/SLC38A2 amino acid transporter protein in L6-G8C5 rat skeletal muscle cells.

Chronic metabolic acidosis plays a role in cachexia by enhancing total proteolysis in skeletal muscle. Glucocorticoid also triggers proteolysis and plays a permissive role in the effect of acidosis. The System A amino acid transporter SNAT2/SLC38A2 is ubiquitously expressed in mammalian cells including muscle, performing Na+-dependent active import of neutral amino acids, and is strongly inhibited by low pH. Exposure of rat skeletal muscle cell line L6-G8C5 to low pH rapidly inhibits SNAT2 transport activity and enhances total proteolysis rate. Pharmacological inhibition or silencing of SNAT2 also enhances proteolysis. This study tests the hypothesis that the glucocorticoid dexamethasone (DEX), like low pH, inhibits SNAT2 activity in L6-G8C5 myotubes, thus contributing to total proteolysis. Incubation with 500 nM DEX for 4 h reduced the System A amino acid transport rate to half the rate in control cultures. This inhibition depended on glucocorticoid receptor-mediated gene transcription, but SNAT2 mRNA levels were unaffected by DEX. In contrast, the SNAT2 protein assessed by immunoblotting was significantly depleted. The co-inhibitory effects of DEX and low pH on System A transport activity were additive in stimulating total proteolysis. In keeping with this mechanism, DEX's inhibitory effect on SNAT2 transport activity was significantly blunted by the proteasome inhibitor MG132. Proof of principle was achieved in similar experiments using recombinant expression of a GFP-tagged SNAT2 fusion protein in HEK293A cells. It is concluded that DEX acutely depletes the SNAT2 transporter protein, at least partly through proteasome-dependent degradation of this functionally important transporter.

A blood-based proteomic classifier for the molecular characterization of pulmonary nodules.

Each year, millions of pulmonary nodules are discovered by computed tomography and subsequently biopsied. Because most of these nodules are benign, many patients undergo unnecessary and costly invasive procedures. We present a 13-protein blood-based classifier that differentiates malignant and benign nodules with high confidence, thereby providing a diagnostic tool to avoid invasive biopsy on benign nodules. Using a systems biology strategy, we identified 371 protein candidates and developed a multiple reaction monitoring (MRM) assay for each. The MRM assays were applied in a three-site discovery study (n = 143) on plasma samples from patients with benign and stage IA lung cancer matched for nodule size, age, gender, and clinical site, producing a 13-protein classifier. The classifier was validated on an independent set of plasma samples (n = 104), exhibiting a negative predictive value (NPV) of 90%. Validation performance on samples from a nondiscovery clinical site showed an NPV of 94%, indicating the general effectiveness of the classifier. A pathway analysis demonstrated that the classifier proteins are likely modulated by a few transcription regulators (NF2L2, AHR, MYC, and FOS) that are associated with lung cancer, lung inflammation, and oxidative stress networks. The classifier score was independent of patient nodule size, smoking history, and age, which are risk factors used for clinical management of pulmonary nodules. Thus, this molecular test provides a potential complementary tool to help physicians in lung cancer diagnosis.

An exploratory study on the effect of pain interference and attentional interference on neuromuscular responses during rapid arm flexion movements.

OBJECTIVES: This study examined the effect of pain interference and attentional interference on the anticipatory postural adjustments of trunk muscles in patients with nonspecific chronic low back pain. METHODS: Fifty-nine patients performed rapid flexion movements of the right arm under 6 conditions, namely a control condition and conditions with different attention demands. The latency between the activations of the shoulder and different trunk muscles, as measured with surface electromyography, was used as the outcome. Using repeated measures analysis of variance, attention conditions and group comparisons were tested between those who scored high and low on pain intensity, fear of movement, or pain catastrophizing. RESULTS: There were significant (although minimal) interactive effects but significant and potentially clinically relevant group and attention main effects. The group with the lowest scores showed delayed activity (14 to 29 ms) relative to those with higher scores. One attention-demanding condition delayed (20 to 35 ms) the latencies of some trunk muscles relative to the control condition, namely the one that was the most attention-demanding according to the reaction time results. DISCUSSION: These findings suggest that patients with chronic low back pain, who are characterized by higher scores on some pain-related variables (visual analog scale, Tampa Scale of Kinesiophobia, Pain Catastrophizing Scale), react favorably to protect the spine from further pain and injuries but would be at greater risk of injury when performing a complex physical task requiring more attention demand.

Indoleamine 2,3-dioxygenase overexpression causes kynurenine-modification of proteins, fiber cell apoptosis and cataract formation in the mouse lens.

Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the kynurenine pathway. The kynurenines formed in this pathway chemically modify proteins and cause apoptosis in cells. Evidence suggests that kynurenines and their protein modifications are involved in cataract formation, but this has yet to be directly demonstrated. We generated transgenic (Tg) mouse lines that overexpress human IDO in the lens. Homozygous Tg (homTg) lenses had higher IDO immunoreactivity, approximately 4.5 times greater IDO mRNA, and approximately 8 times higher IDO activity compared to lenses from hemizygous Tg (hemTg) animals. The kynurenine content was threefold higher in homTg than in hemTg but was not detected in wild-type (Wt) lenses. Kynurenine modifications were approximately 2.6 times greater in homTg than in hemTg or Wt. HomTg lenses had vacuoles in the epithelium and cortical fiber cells. Kynurenine modifications coincided with apoptosis in the secondary fiber cells of homTg lenses. Caspase-3 and caspase-9 activities were markedly higher in homTg than in hemTg and Wt. The glutathione content was approximately 36% lower in homTg compared to hemTg and Wt lenses. HomTg animals also developed bilateral cataracts within 3 months of birth. Together these data demonstrate that IDO-mediated production of kynurenines results in defects in fiber cell differentiation and their apoptosis and suggest that IDO activity is kept low in the lens to prevent deleterious effects by kynurenines.

Intracellular adaptation of Brucella abortus.

Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strain's adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.

Social norms information enhances the efficacy of an appearance-based sun protection intervention.

This experiment examined whether the efficacy of an appearance-based sun protection intervention could be enhanced by the addition of social norms information. Southern California college students (N=125, predominantly female) were randomly assigned to either an appearance-based sun protection intervention that consisted of a photograph depicting underlying sun damage to their skin (UV photo) and information about photoaging or to a control condition. Those assigned to the intervention were further randomized to receive information about what one should do to prevent photoaging (injunctive norms information), information about the number of their peers who currently use regular sun protection (descriptive norms information), both injunctive and descriptive norms information, or neither type of norms information. The results demonstrated that those who received the UV photo/photoaging information intervention expressed greater sun protection intentions and subsequently reported greater sun protection behaviors than did controls. Further, the addition of both injunctive and descriptive norms information increased self-reported sun protection behaviors during the subsequent month.

An evaluation of multidimensional fingerprinting in the context of clinical proteomics.

Multidimensional fingerprinting (MDF) utilizes measurable peptide characteristics to identify proteins. In this study, 3-D fingerprinting, namely, parent protein molecular weight, peptide mass, and peptide retention time on RPLC, is used to identify 331 differentially expressed proteins between normal and human colon cancer plasma membrane samples. A false discovery rate (FDR) procedure is introduced to evaluate the performance of MDF on the colon cancer dataset. This evaluation establishes a false protein identification rate below 15% for this dataset. Western blot analysis is performed to validate the differential expression of the MDF-identified protein VDAC1 on the original tissue samples. The limits of MDF are further assessed by a simulation study where key parameters such as database size, query size, and mass accuracy are varied. The results of this simulation study demonstrate that fingerprinting with three dimensions yields low FDR values even for large queries on the complete human proteome without the need for prior peptide sequencing by tandem mass spectrometry. Specifically, when mass accuracy is 10 ppm or lower, full human proteome searches can achieve FDR values of 10% or less.

Stimulation of protein degradation by low pH in L6G8C5 skeletal muscle cells is independent of apoptosis but dependent on differentiation state.

BACKGROUND: In chronic renal failure, metabolic acidosis increases protein degradation (PD) in skeletal muscle, an effect which in vivo requires glucocorticoid (GC). This disorder is poorly understood, but can be studied in vitro using L6G8C5 rat skeletal muscle cells. Two potential confounding factors in studies of PD in culture are apoptosis and dedifferentiation, both of which resemble catabolic states. The aim of this study was to determine the extent to which these factors contribute to the observed effects of acid and GC on PD. METHODS: PD was measured in intact cells by pre-labelling cell protein with [(14)C]phenylalanine. Apoptosis was assessed morphologically by staining DNA with Hoechst 33342, by terminal deoxynucleotide transferase-mediated nick-end labelling and by cell-surface binding of Annexin V. Differentiation was assessed morphologically from myotube fusion and from activity of the marker enzyme creatine phosphokinase (CPK). RESULTS: In undifferentiated myoblasts, pH had no detectable effect on apoptosis provided that serum was present and GC (dexamethasone; 5 nmol/l) decreased apoptosis. In spontaneously fused cultures in 2% serum, inhibition of apoptosis with caspase-3 inhibitor (C3I; Ac-Asp-Met-Gln-Asp-CHO; 50 micro mol/l) only decreased PD by 9% at pH 7.4. In contrast, the proteasome inhibitor MG132 decreased PD by 79%. Acid (pH 7.1) increased PD, with no requirement for GC, and this effect was blocked by MG132, but not by C3I. Differentiation was unaffected by 1-4 days of exposure to acid or GC. However, differentiation to myotubes led to decreased sensitivity of PD to acid. This effect of acid was lost completely in highly fused myotubes, but was partly restored by 500 nmol/l dexamethasone. CONCLUSIONS: Stimulation of PD in these cells by acid and GC is not an artefact of apoptosis or dedifferentiation, but differentiation state does determine whether PD responds spontaneously to acid or (as in vivo) only does so in the presence of GC.