Implication of the GRB2-associated phosphoprotein SLP-76 in T cell receptor-mediated interleukin 2 production.

Recently we described the molecular cloning of SLP-76, a hematopoietic cell-specific 76-kD protein that was first identified through its association with GST/Grb2 fusion proteins. The primary sequence of SLP-76 predicts a protein of 533 amino acids comprising an amino-terminal region with numerous potential tyrosine phosphorylation sites, a central region rich in proline residues, and a single carboxy-terminal SH2 domain. Here we demonstrate formally that Grb2 associates with unphosphorylated SLP-76 and map the Grb2 binding site on SLP-76 undergoes rapid tyrosine phosphorylation and associates with tyrosine phosphoproteins of 36, 62, and 130 kD. In vitro experiments show that the SH2 domain of SLP-76 associates with the 62- and 130-kD proteins and additionally with a serine/threonine kinase. Finally, we demonstrate that transient overexpression of SLP-76 results in dramatically enhanced TCR-mediated induction of nuclear factor of activated T cells (NFAT) and interleukin (IL) 2 promoter activity; and we provide evidence that a functional SLP-76 SH2 domain is required for this effect. Our data document the in vivo associations of SLP-76 with several proteins that potentially participate in T cell activation and implicate SLP-76 itself as an important molecule in TCR-mediated IL-2 production.

Inhibition of adipogenesis by Wnt signaling.

Wnts are secreted signaling proteins that regulate developmental processes. Here we show that Wnt signaling, likely mediated by Wnt-10b, is a molecular switch that governs adipogenesis. Wnt signaling maintains preadipocytes in an undifferentiated state through inhibition of the adipogenic transcription factors CCAAT/enhancer binding protein alpha (C/EBPalpha) and peroxisome proliferator- activated receptor gamma (PPARgamma). When Wnt signaling in preadipocytes is prevented by overexpression of Axin or dominant-negative TCF4, these cells differentiate into adipocytes. Disruption of Wnt signaling also causes transdifferentiation of myoblasts into adipocytes in vitro, highlighting the importance of this pathway not only in adipocyte differentiation but also in mesodermal cell fate determination.

Rescue of signaling by a chimeric protein containing the cytoplasmic domain of CD45.

Surface expression of the CD45 tyrosine phosphatase is essential for the T cell antigen receptor (TCR) to couple optimally with its second messenger pathways. CD45 may be required to dephosphorylate a TCR-activated protein tyrosine kinase, which then transduces an activation signal from the TCR. A chimeric molecule that contained extracellular and transmembrane sequences from an allele of a major histocompatibility class I molecule and cytoplasmic sequences of CD45 restored TCR signaling in a CD45-deficient mutant T cell line. Thus, expression of the complex extracellular domain of CD45 is not required for the TCR to couple to its signaling machinery.

Prevalence of headshaking within the equine population in the UK.

REASON FOR PERFORMING STUDY: Headshaking in horses has been reported to be most commonly due to idiopathic neuropathic facial pain (trigeminal-mediated headshaking). The prevalence of headshaking in horses in the UK is unknown. OBJECTIVES: To estimate owner-reported prevalence of headshaking in horses in the UK and to report their case background and disease characteristics, as reported by owners. STUDY DESIGN: Cross-sectional web based owner questionnaire. METHODS: The questionnaire was advertised online via social media, horse forums, veterinary websites and equestrian magazines from 17th June 2016, until >1000 responses had been obtained. All UK horse owners were eligible to complete the questionnaire, however only one questionnaire could be completed per owner. RESULTS: The estimated prevalence of owner-reported headshaking in the sample population of horses (n = 1014), within the last year, was 4.6% (95% confidence interval 3.5-6.1), whereas 6.2% (95% confidence interval 4.9-7.9) of horses were reported by their owners to have shown signs of headshaking at any time-point since ownership. There was no association of sex or breed. Nineteen percent of headshaking horses were reported to show headshaking at rest. Fewer than one-third (30.2%, n = 19) of headshaking horses had been examined by a veterinarian for headshaking. Of horses seen by a veterinarian, the cause for headshaking remained unknown in the majority of cases (57.9% responses) and trigeminal-mediated headshaking was reported as a diagnosis in just one case. MAIN LIMITATIONS: The accuracy in data reporting by horse owners was not verified in this study. There may be a potential for bias towards over-reporting due to the nature of survey participation. CONCLUSIONS: Within this sample, owner-reported prevalence of signs of headshaking within the last year, in horses in the UK was 4.6%. Over two-thirds of owners of headshaking horses did not seek veterinary intervention for headshaking. Trigeminal-mediated headshaking was rarely reported by owners as a diagnosis.

Tyrosine phosphorylation of Grb2-associated proteins correlates with phospholipase C gamma 1 activation in T cells.

Ligation of the T-cell antigen receptor (TCR) results in the rapid activation of several protein tyrosine kinases, with the subsequent phosphorylation of numerous cellular proteins. We investigated the requirement for tyrosine phosphorylation of proteins which bind the Grb2 SH2 domain in TCR-mediated signal transduction by transfecting the Jurkat T-cell line with a cDNA encoding a chimeric protein designed to dephosphorylate these molecules. Stimulation of the TCR on cells expressing this engineered enzyme fails to result in sustained tyrosine phosphorylation of a 36-kDa protein likely to be the recently cloned pp36/Lnk. Interestingly, TCR ligation of the transfected cells also fails to induce soluble inositol phosphate production and intracellular calcium mobilization, although receptor-mediated tyrosine phosphorylation of phospholipase C gamma 1 still occurs. TCR-mediated Ras and mitogen-activated protein kinase activation remain intact in cells expressing the engineered phosphatase. These data demonstrate that tyrosine phosphorylation of a protein(s) which binds the SH2 domain of Grb2 correlates with phospholipase C gamma 1 activation and suggest that such a phosphoprotein(s) plays a critical role in coupling the TCR with the phosphatidylinositol second-messenger pathway.

Glycogen synthase kinase 3 is an insulin-regulated C/EBPalpha kinase.

CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcription factor involved in creating and maintaining the adipocyte phenotype. We have shown previously that insulin stimulates dephosphorylation of C/EBPalpha in 3T3-L1 adipocytes. Studies to identify the insulin-sensitive sites of phosphorylation reveal that a C/EBPalpha peptide (amino acids H215 to K250) is phosphorylated on T222, T226, and S230 in vivo. The context of these phosphoamino acids implicates glycogen synthase kinase 3 (GSK3), whose activity is known to be repressed in response to insulin, as a potential kinase for phosphorylation of T222 and T226. Accordingly, GSK3 phosphorylates the predicted region of C/EBPalpha on threonine in vitro, and GSK3 uses C/EBPalpha as a substrate in vivo. In addition, the effect of pharmacological agents on GSK3 activity correlates with regulation of C/EBPalpha phosphorylation. Treatment of 3T3-L1 adipocytes with the phosphatidylinositol 3-kinase inhibitor wortmannin results in phosphorylation of C/EBPalpha, whereas treatment with the GSK3 inhibitor lithium results in dephosphorylation of C/EBPalpha. Collectively, these data indicate that insulin stimulates dephosphorylation of C/EBPalpha on T222 and T226 through inactivation of GSK3. Since dephosphorylation of C/EBPalpha in response to lithium is blocked by okadaic acid, strong candidates for the T222 and T226 phosphatase are protein phosphatases 1 and 2a. Treatment of adipocytes with insulin alters the protease accessibility of widespread sites within the N terminus of C/EBPalpha, consistent with phosphorylation causing profound conformational changes. Finally, phosphorylation of C/EBPalpha and other substrates by GSK3 may be required for adipogenesis, since treatment of differentiating preadipocytes with lithium inhibits their conversion to adipocytes.

Antimicrobial selection and dosing in the treatment of wounds in the United Kingdom.

REASONS FOR PERFORMING STUDY: Antimicrobial stewardship within the veterinary profession is recognised by governing and professional bodies as being important; the attitudes and behaviour of veterinary surgeons merit investigation. OBJECTIVES: To investigate levels of protected antimicrobial use and accuracy of antimicrobial dosing in a common clinical scenario in equine practice. STUDY DESIGN: Retrospective cohort study. METHODS: Antimicrobial use was evaluated retrospectively in 113 cases subsequently referred to a single referral hospital for the treatment of limb wounds over a 20-month period. Antimicrobial classification (first-line, alternative or protected) was made according to guidelines produced by the British Equine Veterinary Association. These guidelines also served as the reference for recommended dose rates. RESULTS: Systemic antimicrobials were administered prior to referral in 94/113 (83.2%) horses, of which 8 (8.5%) received the protected third or fourth generation cephalosporins or fluoroquinolones. Forty-eight of 87 (55.2%) horses for which complete dosing data were available received antimicrobials at ≤90% of the recommended dose. Practitioners who held a postgraduate clinical qualification or worked in purely equine practice were no more or less likely to use protected antimicrobials (P = 0.06 and P = 0.64, respectively) or administer inadequate doses (P = 0.75 and P = 0.85, respectively). Veterinary surgeons with more experience were less likely to use protected antimicrobials (P<0.001); however, with the small case numbers, this finding should be interpreted with caution. Heavier horses were more likely to be under-dosed (P<0.002). CONCLUSIONS: This study highlights the administration of certain classes of antimicrobials in situations where their use is unlikely to be justified. If these findings reflect more general attitudes and behaviour then greater awareness of, and compliance with, recommendations for responsible antimicrobial use are required among equine practitioners. Bodyweight ought to be measured or estimated using validated objective techniques prior to systemic medications being administered.

Response to a standard behavioral weight loss intervention by age of onset of obesity.

BACKGROUND: The purpose of this study was to examine weight loss, physical activity, fitness and diet changes in response to a standard behavioral weight loss intervention in adults with self-reported juvenile onset (n = 61) or adult onset (n = 116) obesity. METHODS: Participants (n = 177; 43.0 ± 8.6 years; body mass index [BMI] = 33.0 ± 3.4 kg m-2) engaged in an 18-month standard behavioral weight loss intervention. Participants were randomized into three different intervention groups as part of the larger parent trial. BMI, physical activity, fitness and diet were assessed at baseline, 6, 12 and 18 months. Separate adjusted mixed models were constructed using SAS version 9.4 (SAS Institute, Cary, NC). RESULTS: There was significant weight loss, increased physical activity, improved fitness and reduced caloric intake over time (p < 0.001). There were no significant differences in these outcome variables by obesity onset group. However, there was a significant group by time interaction for fitness (p = 0.001), with the adult onset making significantly greater gains in fitness from baseline to 6 months (p < 0.001); however, this difference was no longer present at 12 or 18 months. CONCLUSIONS: With the exception of fitness at 6 months, weight loss, physical activity and diet did not differ between juvenile onset and adult onset participants, suggesting that those with juvenile onset obesity are equally responsive to a standard behavioral weight loss intervention in adulthood.

Effect of daytime protein restriction on nutrient intakes of free-living Parkinson's disease patients.

Studies have shown that severe daytime restriction of dietary protein improves the efficacy of L-dopa and reduces response fluctuations in some Parkinson's disease (PD) patients. This study investigated the nutritional adequacy of the daytime restricted-protein diet. Eleven free-living PD patients suffering from unpredictable response fluctuations to L-dopa were counseled to limit protein intake to approximately 10 g before 1700. Three sets of 6-d food records obtained during the 8-wk study showed that while on the test diet, mean intakes of most nutrients remained above the recommended nutrient intakes, although significant decreases occurred in protein, calcium, iron, phosphorus, riboflavin, and niacin intakes. The impact of the test diet on nutritional status as evaluated by changes in body weight and serum prealbumin was small. We conclude that healthy and highly motivated patients can maintain adequate intakes of most nutrients while restricting daytime protein intake. However, nutrient intakes might be compromised in patients whose regular diets are marginally adequate.

Bradykinin receptor antagonists containing N-substituted amino acids: in vitro and in vivo B(2) and B(1) receptor antagonist activity.

We report a systematic probing of the structural requirements of the bradykinin (BK) type 2 (B(2)) receptor for antagonist activity by incorporating N-alkyl-amino acid residues at positions 7 and 8 of a potent antagonist sequence. Compound 1 (D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-N-Chg (8)-Arg(9), CP-0597)(1,2) is a potent (pA(2) = 9.3, rat uterus; pK(i) = 9.62, binding, human receptor clone) B(2) receptor antagonist devoid of in vitro B(1) antagonist activity (rabbit aorta). Compound 1 exhibits high potency (ED(50) = 29.2 pmol/kg/min, iv, rabbit) and duration of action when tested in models for in vivo B(2) antagonist activity. Although devoid of activity in a classic B(1) isolated tissue assay, B(1) antagonist activity for 1 was demonstrated in vivo, in a LPS-treated, inducible BK(1) receptor rabbit blood pressure model (ED(50) = 1.7 nmol/kg/min). D-Arg(0) of 1 can be formally replaced by an achiral arginine surrogate, without significant loss in antagonist potency on rat uterus (compound 11, B(2) pA(2) = 9.1). Antagonist 13 (Hyp(2), Nchg(8)), pK(i) = 10.2, and agonist 4 (N-methylcyclohexyl-Gly(8)), pK(i) = 10.1, also exhibited substantial binding to guinea pig ileum membrane receptors as well as a human B(2) receptor clone. Very minor structural changes in the N-alkyl amino acid residues in positions 7 and 8 can modify the activity of this class of compounds from being extremely potent antagonists to tight binding partial or full agonists. These studies have resulted in a series of compounds containing inexpensive amino acid residues but which produce broad spectrum BK receptor blocking potency and exceptional in vivo duration of action.

Bradykinin antagonists in human systems: correlation between receptor binding, calcium signalling in isolated cells, and functional activity in isolated ileum.

The determination of the relationship between ligand affinity and bioactivity is important for the understanding of receptor function in biological systems and for drug development. Several physiological and pathophysiological functions of bradykinin (BK) are mediated via the B2 receptor. In this study, we have examined the relationship between B2 receptor (soluble and membrane-bound) binding of BK peptidic antagonists, inhibition of calcium signalling at a cellular level, and in vitro inhibition of ileum contraction. Only human systems were employed in the experiments. Good correlations between the studied activities of BK antagonists were observed for a variety of different peptidic structures. The correlation coefficients (r) were in the range of 0.905 to 0.955. In addition, we analyzed the effect of the C-terminal Arg9 removal from BK and its analogs on B2 receptor binding. The ratios of binding constants (Ki(+Arg)/Ki(-Arg)) for the Arg9 containing compounds and the corresponding des-Arg9 analogs varied from about 10 to 250,000. These ratios strongly depend on the chemical structures of the compounds. The highest ratios were observed for two natural agonist pairs, BK/des-Arg9-BK and Lys0-BK/des-Arg9-Lys0-BK.

Reintubation as an outcome predictor in trauma patients.

STUDY OBJECTIVE: Determine reintubation rate, identify its cause, and detail adverse outcomes from reintubation. DESIGN: Retrospective review of extubation failures in the trauma ICU. SETTING: University hospital and regional trauma center. PATIENTS: Four hundred five patients arriving intubated or requiring intubation during hospitalization after 2,516 traumatic injury admissions over 18 months. INTERVENTIONS: None. RESULTS: Reintubation incidence was 7% (27 times per 405 patients). Comparative mortality of the reintubated group (2/24 = 8%) is similar to overall trauma center mortality (224/2516 = 6.5%), but less than the cohort of patients admitted to the hospital intubated (63/405 = 16%). Reintubated patients had an increased frequency of stridor than reported previously (33%), and an increased tracheostomy rate (62% vs 30%). Stridor was not predictable from injury severity score, Glasgow coma score, age, sex, length of intubation, or place of intubation. Pulmonary complications (atelectasis, tracheobronchitis, pneumonia) developed in half of reintubated patients; stridorous patients did not have an increased rate of pulmonary complications. CONCLUSION: Reintubation in trauma ICU patients does not predict poor outcome.

Complications of anticoagulation for pulmonary embolism in low risk trauma patients.

Trauma patients are at significant risk for deep venous thrombosis (DVT) and pulmonary embolism (PE). Anticoagulation is standard therapy for DVT/PE, but may cause severe complications. We reviewed the course of 70 trauma ICU patients treated over a 28-month period. Thirty-six patients (51.4 percent) were treated by continuous IV heparin and/or oral warfarin. Of these, 13 patients (36 percent) developed complications requiring termination of anticoagulation. These included recurrent PE (four), subdural hematomas (three), hemothorax (two), heparin-induced thrombocytopenia (one), hemorrhagic pericardial effusion (one), retroperitoneal hematoma (one), and sudden unexplained drop in hemoglobin and shock (one). All patients with subdural hematomas had no prior evidence of head injury on brain computed tomography. All patients with recurrent PE received adequate anticoagulation therapy. Age > 55 was associated with increased risk of complications (8 of 13; p = .02:chi 2). Thirty-four other patients (48.6 percent) received inferior vena caval filters with no related complications or deaths. Anticoagulation for DVT/PE should be used selectively in trauma patients and avoided in elderly patients. Such patients should undergo early caval filter placement.

Multichamber gunshot wounds of the heart. The utility of transesophageal echocardiography.

A patient had a gunshot wound to the heart involving three cardiac chambers. Conventional echocardiography failed to identify the intracardiac injuries. The utility of transesophageal echocardiography in a patient with cardiac trauma is described.

Routine initial computed tomography of the chest in blunt torso trauma.

Computer tomography (CT) is an effective technique in the initial evaluation of the abdomen and head following blunt trauma. To evaluate the role of CT of the thorax, a prospective study comparing routine early thoracic CT scanning with initial chest roentgenogram (CXR) was carried out on 73 patients with blunt torso trauma undergoing concomitant abdominal CT examination. Initial CXR and CT scans were interpreted independently by radiologists in a blinded fashion. CXR diagnosed more bony injuries than CT, while the CT identified pulmonary contusions and effusions more accurately. Only those contusions diagnosed by CXR proved clinically significant. Patient treatment was changed in one case based on CT findings. In the absence of CXR findings, chest CT scanning frequently identifies abnormalities with limited clinical significance. Although more sensitive, CT of the thorax has a limited role in the initial emergent evaluation of victims of blunt torso trauma.

The pivotal role of transesophageal echocardiography in the management of traumatic thoracic aortic rupture with associated intra-abdominal hemorrhage.

Rupture of the thoracic aorta after blunt trauma, particularly when associated with multiple injuries, presents a major problem of resuscitation and management. Transesophageal color Doppler echocardiography (TEE) during laparotomy played a major role in confirming the diagnosis of thoracic aortic rupture in a patient.

Traumatic disruptions of the thoracic aorta: treatment and outcome.

Of 27 patients admitted to our level I trauma center with acute disruption of the thoracic aorta, two patients died of exsanguination before aortic repair. One patient had massive leakage from the aneurysm after aortography and died during surgery. All patients suffered from multiple injuries. Eighty-three percent of the patients had major operations in addition to the aortic repair. "Clamp and sew" technique was used in 18 patients (75%), two of whom had multiple tears of the aortic arch. Heparin-coated shunts were used in five patients (20.8%), and a cardiopulmonary bypass was performed in one patient who had multiple tears. Three postoperative deaths were related to polytrauma, cardiogenic shock, and sepsis. Paraplegia developed in three patients, two of whom had multiple aortic lesions necessitating longer ischemia time during the repair. Only one patient had complete neurologic deficit at the 1-year follow-up. In our series, neither surgical procedure proved superior. We conclude that the "clamp and sew" technique for repair of the disrupted thoracic aorta may allow for a more favorable outcome.

Isolation of a gastrin releasing peptide receptor from normal rat pancreas.

The presence of a putative GRP receptor on rat pancreatic particulate membranes was demonstrated by covalent cross-linking to 125I-gastrin releasing peptide (GRP), which revealed a radioactive band with Mr = 80-90 kDa on reduced SDS-PAGE. Fresh rat pancreatic membranes contained a GRP receptor which was solubilized with Triton X-100 as assessed by its failure to sediment at 100,000 x g for one hour and its ability to pass through a 0.22 mu filter. When 125I-GRP binding was studied using Sephadex G50 gel filtration chromatography to separate bound from unbound ligand, substantial amounts of 125I-GRP binding were observed in rat crude solubilized pancreatic membranes, but essentially no specific binding was observed until the crude solubilized membranes were fractionated by ammonium sulfate precipitation. Specific 125I-GRP binding was 500, 700 and 1400 fmol/mg protein, respectively, in the 0-25%, 25-50% and 50-80% saturated ammonium sulfate fractions (125I-GRP concentration = 1 nM). Specific binding was temperature dependent, saturable and of high affinity, (KD = 2.3 nM). A unique 70 kDa band was visualized by silver staining of the SDS-PAGE of eluates of GRP(14-27) affinity gel compared with eluates of control affinity gels incubated with the 25-50% (NH4)2SO4 fraction. The lower Mr than that observed with covalent cross-linking may represent the binding subunit of a larger receptor protein. This ligand-affinity isolated protein is thus a good candidate for the GRP receptor, or the binding subunit of it, from normal rat pancreas.

Novel soluble, high-affinity gastrin-releasing peptide binding proteins in Swiss 3T3 fibroblasts.

Swiss 3T3 cells contained substantial amounts of soluble and specific [125I]GRP binders. Like the membrane-associated GRP receptor, they were of high affinity, saturable, bound to GRP(14-27) affinity gels, and exhibited specificity for GRP(14-27) binding. They differed in that acid or freezing destroyed specific binding, specific binding exhibited different time and temperature effects, no detergent was required for their solubilization, ammonium sulfate fractionation yielded different profiles, the M(rs) were lower, GRP(1-16) also blocked binding, and a polyclonal anti-GRP receptor antiserum did not bind on Western blots. The isolated, soluble GRP binding protein(s) rapidly degraded [125I]GRP. These soluble GRP binding proteins may play a role in the regulation of the mitogenic effects of GRP on these cells.