<p>Ceramide, the backbone of all sphingolipids, is synthesized by a family of ceramide synthases (CerS) that each use acyl CoAs of defined chain length for N-acylation of the sphingoid long chain base. CerS mRNA expression and enzymatic activity do not always correlate with the sphingolipid acyl chain composition of a particular tissue, suggesting post-translational mechanism(s) of regulation of CerS activity. We now demonstrate that CerS activity can be modulated by dimer formation. Under suitable conditions, high Mr CerS complexes can be detected by western blotting, and various CerS co-immunoprecipitate. CerS5 activity is inhibited in a dominant-negative fashion by co-expression with catalytically inactive CerS5, and CerS2 activity is enhanced by co-expression with a catalytically active form of CerS5 or CerS6. In a constitutive heterodimer comprising CerS5 and CerS2, the activity of CerS2 depends on the catalytic activity of CerS5. Finally, CerS dimers are formed upon rapid stimulation of ceramide synthesis by curcumin. Together, these data demonstrate that ceramide synthesis can be regulated by the formation of CerS dimers, and suggest a novel way to generate the acyl chain composition of ceramide (and downstream sphingolipids), which may depend on the interaction of CerS with each other.<p>

Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40-50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter-species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.

BACKGROUND Numerous studies have documented the short-term impact of BRCA1/BRCA2 (BRCA1/2) testing; however, little research has examined the long-term impact of testing. We conducted the first long-term prospective study of psychosocial outcomes in a U.S. sample of women who had BRCA1/2 testing. METHODS Participants were 464 women who underwent genetic testing for BRCA1/2 mutations. Prior to testing, we measured sociodemographics, clinical variables, and cancer specific and general distress. At long-term follow-up (Median = 5.0 years; Range = 3.4-9.1 years), we assessed cancer-specific and genetic testing distress, perceived stress, and perceived cancer risk. We evaluated the impact of BRCA1/2 test result and risk-reducing surgery on long-term psychosocial outcomes. RESULTS Among participants who had been affected with breast or ovarian cancer, BRCA1/2 carriers reported higher genetic testing distress (β = 0.41, P < 0.0001), uncertainty (β = 0.18, P < 0.0001), and perceived stress (β = 0.17, P = 0.005) compared with women who received negative (i.e., uninformative) results. Among women unaffected with breast/ovarian cancer, BRCA1/2 carriers reported higher genetic testing distress (β = 0.39, P < 0.0001) and lower positive testing experiences (β = 0.25, P = 0.008) than women with negative results. Receipt of risk-reducing surgery was associated with lower perceived cancer risk (P < 0.0001). CONCLUSIONS In this first prospective long-term study in a U.S. sample, we found modestly increased distress in BRCA1/2 carriers compared with women who received uninformative or negative test results. Despite this modest increase in distress, we found no evidence of clinically significant dysfunction. IMPACT Although a positive BRCA1/2 result remains salient among carriers years after testing, testing does not seem to impact long-term psychologic dysfunction.

OBJECTIVE Osteoarthritis (OA) pain mechanisms are poorly understood. We used the monosodium iodoacetate (MIA) model of knee OA to characterize changes in excitability during the course of OA in different classes of mechanosensitive afferents projecting to joint-associated tissues, and examine whether these afferent responses and pain behavior are correlated. METHODS Rats were injected intra-articularly with MIA (1mg in 50 μl). Hind-limb weight bearing was studied 3 (MIA3) and 14 (MIA14) days after MIA, followed by deep anesthesia and teased-nerve-fiber recordings. Spontaneous activity (SA) and mechanically evoked responses of A- and C-mechanosensitive fibers (AM and CM respectively, probably nociceptive) innervating tissues associated with the ipsilateral knee joint were examined. RESULTS MIA3 and MIA14 rats exhibited reduced ipsilateral weight bearing. SA (>0.02 impulses/s) occurred in ∼50% of CMs from MIA rats vs 0% in normals. SA firing rates in CMs were significantly higher than normal; decreased weight bearing was correlated with increased CM SA rates. Neither percentages of AMs with SA (20%) nor their firing rates (0-0.01 impulses/s) significantly increased after MIA. In contrast, in MIA rats AMs, but not CMs, exhibited decreased mechanical thresholds and increased firing rates in response to suprathreshold mechanical stimulation. CONCLUSIONS These findings of increased SA firing rate in CMs but not AMs and increased mechanical sensitivity of AMs, but not CMs, have not previously been reported. These are two distinct important physiological mechanisms that may underpin spontaneous pain (CMs) and stimulus-evoked pain (AMs) in OA. Our data contribute to a mechanism-based understanding of OA pain.

Gang violence is a growing public health concern in the United States, and adolescents are influenced by exposure to gang violence. This study explored the influence of exposure to gang violence on adolescent boys' mental health using a multi-method design. A semi-structured interview guide and the Trauma Symptom Checklist for Children were used to collect data from adolescents. Parents, primary caregivers, and community center employees completed the Child Behavior Checklist or Teacher Report Form. Ten adolescent boys, their parents or primary caregivers, and six community center employees participated in the study. Exposure to gang violence was common among these adolescents and they had a variety of reactions. Parents, primary caregivers, and community center employees had differing perceptions of adolescents' exposure to violence and their mental health. Adolescent boys' exposure to gang violence in the community is alarming. These adolescents encountered situations with violence that influenced their mental health.

Exsanguination from hemodialysis vascular sites may cause a rapid death. Due to extensive blood loss at the scene, investigators may initially suspect a homicide or suicide. We reviewed 100 deaths due to hemorrhage from hemodialysis shunt sites. The majority (81%) of these hemorrhages occurred at home and 44% subsequently died at home. Recognition of this medical complication at the scene is important to prevent the dispatch of the crime scene or homicide unit. In these instances, the common causes of kidney failure included hypertensive cardiovascular disease and diabetes mellitus. The manners of death were certified as therapeutic complication (93%), accident (5%), and suicide (2%). These fatal shunt hemorrhages are rapid and large due to their superficial subcutaneous locations and elevated shunt pressures from the arterial-venous anastamosis. The cause of death statement must include the proximate cause of death, which is usually the disease that resulted in kidney failure, if it is known.

<p>In mammals, ceramides are synthesized by a family of six ceramide synthases (CerS), transmembrane proteins located in the endoplasmic reticulum, where each use fatty acyl-CoAs of defined chain length for ceramide synthesis. Little is known about the molecular features of the CerS that determine acyl CoA selectivity. We now explore CerS structure-function relationships by constructing chimeric proteins combining sequences from CerS2, which uses C22-CoA for ceramide synthesis, and CerS5, which uses C16-CoA. CerS2 and 5 are 41% identical and 63% similar. Chimeras containing approximately half of CerS5 (from the N-terminus) and half of CerS2 (from the C-terminus) were catalytically inactive. However, the first 158 residues of CerS5 could be replaced with the equivalent region of CerS2 without affecting specificity of CerS5 towards C16-CoA; likewise, the putative 6th transmembrane domain (at the C-terminus) of CerS5 could be replaced with the corresponding sequence of CerS2 without affecting CerS5 specificity. Remarkably, a chimeric CerS5/2 protein containing the first 158 residues and the last 83 residues of CerS2 displayed specificity towards C16-CoA, and a chimeric CerS2/5 protein containing the first 150 residues and the last 79 residues of CerS5 displayed specificity towards C22-CoA, demonstrating that a minimal region of 150 residues is sufficient for retaining CerS specificity.</p>