BRCTx is a novel, highly conserved RAD18-interacting protein

The BRCT domain is a highly conserved module found in many proteins that participate in DNA damage checkpoint regulation, DNA repair, and cell cycle control. Here we describe the cloning, characterization, and targeted mutagenesis of Brctx, a novel gene with a BRCT motif. Brctx was found to be expressed ubiquitously in adult tissues and during development, with the highest levels found in testis. Brctx-deficient mice develop normally, show no pathological abnormalities, and are fertile. BRCTx binds to the C terminus of hRAD18 in yeast two-hybrid and immunoprecipitation assays and colocalizes with this protein in the nucleus. Despite this, Brctx-deficient murine embryonic fibroblasts (MEFs) do not show overt sensitivity to DNA-damaging agents. MEFs from Brctx-deficient embryos grow at a similar rate to wild-type MEF CD4/CD8 expressions, and the cell cycle parameters of thymocytes from wild-type and Brctx knockout animals are indistinguishable. Intriguingly, the BRCT domain of BRCTx is responsible for mediating its localization to the nucleus and centrosome in interphase cells. We conclude that, although highly conserved, Brctx is not essential for the above-mentioned processes and may be redundant.     

Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such "membrane-inserted" form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKC(alpha), the C2-V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.     

Effects of intra-peritoneal injection with NH4Cl, urea, or NH4Cl+urea on nitrogen excretion and metabolism in the African lungfish Protopterus dolloi

This study aimed to (1) determine if ammonia (as NH(4)Cl) injected intra-peritoneally into the ureogenic slender African lungfish, Protopterus dolloi, was excreted directly rather than being converted to urea; (2) examine if injected urea was retained in this lungfish, leading to decreases in liver arginine and brain tryptophan levels, as observed during aestivation on land; and (3) elucidate if increase in internal ammonia level would affect urea excretion, when ammonia and urea are injected simultaneously into the fish. Despite being ureogenic, P. dolloi rapidly excreted the excess ammonia as ammonia within the subsequent 12 h after NH(4)Cl was injected into its peritoneal cavity. Injected ammonia was not detoxified into urea through the ornithine-urea cycle, probably because it is energetically intensive to synthesize urea and because food was withheld before and during the experiment. In addition, injected ammonia was likely to stay in extracellular compartments available for direct excretion. At hour 24, only a small amount of ammonia accumulated in the muscle of these fish. In contrast, when urea was injected intra-peritoneally into P. dolloi, only a small percentage (34%) of it was excreted during the subsequent 24-h period. A significant increase in the rate of urea excretion was observed only after 16 h. At hour 24, significant quantities of urea were retained in various tissues of P. dolloi. Injection with urea led to an apparent reduction in endogenous ammonia production, a significant decrease in the hepatic arginine content, and a significantly lower level of brain tryptophan in this lungfish. All three phenomena had been observed previously in aestivating P. dolloi. Hence, it is logical to deduce that urea synthesis and accumulation could be one of the essential factors in initiating and perpetuating aestivation in this lungfish. Through the injection of NH(4)Cl + urea, it was demonstrated that an increase in urea excretion occurred in P. dolloi within the first 12 h post-injection, which was much earlier than that of fish injected with urea alone. These results suggest that urea excretion in P. dolloi is likely to be regulated by the level of internal ammonia in its body.     

Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control

Cortactin is an important molecular scaffold for actin assembly and organization. Novel mechanistic functions of cortactin have emerged with more interacting partners identified, revealing its multifaceted roles in regulating actin cytoskeletal networks that are necessary for endocytosis, cell migration and invasion, adhesion, synaptic organization and cell morphogenesis. These processes are mediated by its multi-domains binding to F-actin and Arp2/3 complex and various SH3 targets. Furthermore, its role in actin remodeling is subjected to regulation by tyrosine and serine/threonine kinases. Elucidating the mechanisms underlying cortactin phosphorylation and its functional consequences would provide new insights to various aspects of cell dynamics control.     

Aggregation of a monoclonal antibody induced by adsorption to stainless steel

Stainless steel is a ubiquitous surface in therapeutic protein production equipment and is also present as the needle in pre‐filled syringe biopharmaceutical products. Stainless steel microparticles can cause the aggregation of a monoclonal antibody (mAb). The initial rate of mAb aggregation was second order in steel surface area and zero order in mAb concentration, generally consistent with a bimolecular surface aggregation being the rate‐limiting step. Polysorbate 20 (PS20) suppressed the aggregation yet was unable to desorb the firmly bound first layer of protein that adsorbs to the stainless steel surface. Also, there was no exchange of mAb from the first adsorbed layer to the bulk phase, suggesting that the aggregation process actually occurs on subsequent adsorption layers. No oxidized Met residues were detected in the mass spectrum of a digest of a highly aggregated mAb, although there was a fourfold increase in carbonyl groups due to protein oxidation. Biotechnol. Bioeng. 2010;105: 121–129. © 2009 Wiley Periodicals, Inc.     

Adiponectin Profile in Asian Patients Undergoing Coronary Revascularization and Its Association With Plaque Vulnerability: IDEAS‐ADIPO Study

Despite potent insulin‐sensitizing, anti‐inflammatory, and antiatherogenic effects in animal studies, the relationship between serum adiponectin level and coronary artery disease in patients remains unclear. We determined the adiponectin profile in a cohort of multiethnic Asian patients with coronary artery disease, and the association between serum adiponectin level and culprit lesion necrotic core (NC) content. Ninety‐four Asian patients (BMI, 25.3 ± 3.7 kg/m²) undergoing percutaneous coronary intervention were recruited. The serum adiponectin level was measured (n = 94), and the baseline virtual histology intravascular ultrasound examination was analyzed (n = 88). The median level of adiponectin was 3.7 µg/ml (interquartile range, 2.8–4.5 µg/ml). The serum adiponectin level was below 10 µg/ml in 90 patients (95.7%) and below 6 µg/ml in 80 patients (85.1%). There was a significant association between ethnicity and serum adiponectin level (P = 0.048). The median adiponectin level was highest among the Chinese, followed by the Malay and the Indians. Serum adiponectin levels were positively associated with culprit lesion NC content. A 1‐µg/ml increase in log adiponectin was associated with a 3.04% (95% confidence interval: 0.33–5.44) increase in culprit lesion NC content. This association remains significant after adjusting for age, sex, ethnicity, low‐density lipoprotein cholesterol, high‐density lipoprotein cholesterol, and procedural indication. We found a low serum level of adiponectin in Asian patients and a significant ethnic effect on serum adiponectin level. Increased serum adiponectin levels were independently associated with increased culprit lesion NC burden, suggesting a role for adiponectin in modulating coronary plaque vulnerability.     

Sterically Stabilized Phospholipid Micelles Reduce Activity of a Candidate Antimicrobial Wound Healing Adjunct

KSLW is an antimicrobial decapeptide, presumed to associate with micelles. Linear polymeric chains of hydrophobic phospholipids tend to form micelles, spontaneously, and function as efficient drug-stabilizing delivery systems. Our goal was to examine whether association of a cationic decapeptide with sterically stabilized nanomicelles (SSMs), improves stability and antimicrobial effect in vivo, using an impaired healing model. KSLW solutions were prepared in either saline or 12?mM SSM. Bilateral circular excisional wounds were created on the backs of SKH-1 mice followed by intradermal delivery of peptide solutions. Bacterial assays were conducted to assess bioactivity of KSLW in different formulations. Fluorescence analyses demonstrated an optimum lipid:peptide ratio for loading KSLW in PEGylated phospholipid micelles to be 15:1. Stressed animals treated with KSLW?CSSM preparations demonstrated no differences in microbial load at post-operative time points. In vitro assays against Staphylococcus epidermidis confirmed diminished activity of KSLW in SSM solution. The loss of KSLW antimicrobial activity may be based on electrostatic interactions with the anionic surface of SSM, which interfere with the peptide??s interaction with bacterial membranes. This study emphasizes the importance of antimicrobial peptide charge, size, and bioactivity, when designing delivery systems for wound healing agents.     

Performances, meat quality and boar taint of castrates and entire male pigs fed a standard and a raw potato starch-enriched diet

In Europe there is increasing concern about the common practice of surgical castration of piglets without anaesthesia. One possible alternative to completely avoid castration is entire male pig production. Thus, the objective of the study was to compare the growth performance, carcass characteristics, organ weights, meat quality traits, fat score and boar taint compounds in the adipose tissue of group-penned entire male pigs and castrates. Furthermore, the effect of raw potato starch (RPS) fed for 7 days prior to slaughter was determined. Pigs (n = 36) were blocked by BW into 12 blocks (3 littermates/block) and assigned to three experimental groups: surgical castrates (C); entire males (EM); and entire males offered RPS (30 g RPS/100 g diet) for 7 days prior to slaughter (EM+). Pigs had ad libitum access to the feed from 22 to 107 kg, individual feed intake was recorded daily and BW once a week. Entire males grew slower (EM: 771, EM+: 776 v. C: 830 g/day; P < 0.01), consumed less feed (EM: 1.87, EM+: 1.89 v. C: 2.23 kg/day; P < 0.01) and were more efficient (feed conversion ratio: EM: 2.42, EM+: 2.44 v. C: 2.69 kg/kg; P < 0.01) than C. Compared to C, carcass dressing percentage was lower (EM: 79.4, EM+: 79.4 v. C: 81.6%; P < 0.01) and percentage of valuable cuts was higher (EM: 57.3, EM+: 56.5 v. 52.6%; P < 0.01) in entire males. The hearts (EM: 426, EM+: 425 v. C: 378 g), kidneys (EM: 387, EM+: 378 v. C: 311 g), bulbourethral (EM: 200, EM+: 195 v. C: 7 g) and salivary glands (EM: 99, EM+: 94 v. C: 42 g) were heavier (P < 0.001) in entire males than in C. Meat quality traits did not (P > 0.05) differ among experimental groups but the adipose tissue was more unsaturated in entire males than in C as indicated by the higher fat scores (EM: 69.1, EM+: 67.2 v. C: 63.6; P < 0.01). Feeding RPS reduced (P = 0.04) the skatole tissue concentrations (expressed in μg/g lipid) in EM+ (0.22) compared to EM (0.85), whereas androstenone and indole levels were not (P 0.60) affected (EM: 1.7 and 0.10, EM+: 2.0 and 0.09, respectively). Although the current results confirmed the high efficiency of entire males compared to castrates, the observed high androstenone levels represent a major challenge to implement entire males production.