Aspirin-like molecules that covalently inactivate cyclooxygenase-2

Many of aspirin's therapeutic effects arise from its acetylation of cyclooxygenase-2 (COX-2), whereas its antithrombotic and ulcerogenic effects result from its acetylation of COX-1. Here, aspirin-like molecules were designed that preferentially acetylate and irreversibly inactivate COX-2. The most potent of these compounds was o-(acetoxyphenyl)hept-2-ynyl sulfide (APHS). Relative to aspirin, APHS was 60 times as reactive against COX-2 and 100 times as selective for its inhibition; it also inhibited COX-2 in cultured macrophages and colon cancer cells and in the rat air pouch in vivo. Such compounds may lead to the development of aspirin-like drugs for the treatment or prevention of immunological and proliferative diseases without gastrointestinal or hematologic side effects.     

Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo

The mechanism by which an elongated polyglutamine sequence causes neurodegeneration in Huntington's disease (HD) is unknown. In this study, we show that the proteolytic cleavage of a GST-huntingtin fusion protein leads to the formation of insoluble high molecular weight protein aggregates only when the polyglutamine expansion is in the pathogenic range. Electron micrographs of these aggregates revealed a fibrillar or ribbon-like morphology, reminiscent of scrapie prions and beta-amyloid fibrils in Alzheimer's disease. Subcellular fractionation and ultrastructural techniques showed the in vivo presence of these structures in the brains of mice transgenic for the HD mutation. Our in vitro model will aid in an eventual understanding of the molecular pathology of HD and the development of preventative strategies.     

Mastectomy

We report a case of interpleural misplacement of an epidural catheter possibly caused by inappropriate angle of the Tuohy needle. A 71-year-old man was scheduled for left lower lobectomy of the lung with general and epidural anesthesia. A 18-gauge Tuohy needle was introduced into the Th5-Th6 interspace with a right paramedian approach. The direction of the epidural needle was at an angle of about 30 degrees from the skin directed cephalad. The needle was advanced 8 cm from the skin, where loss-of-resistance feeling was evident, and an epidural catheter was easily inserted 5 cm beyond the needle tip. Administration of 7 ml of 1.5% lidocaine given 20 minutes before skin incision did not alter arterial blood pressure or heart rate. Thoracotomy was performed via the fifth intercostal space. The surgeon then found the epidural catheter to be in the left pleural cavity. The catheter was immediately withdrawn. It is, therefore, necessary to employ the appropriate angle of the Tuohy needle on attempting epidural anesthesia to avoid the complication that we experienced.     

Interleukin-10 reduces circulating levels of serum cytokines in experimental pancreatitis

Over the past few years, evidence has accumulated that implicates proinflammatory cytokines as the mediators responsible for the escalation of acute pancreatitis into a multisystem disease. It has been shown that the degree of serum cytokine elevation, particularly the macrophage-derived cytokines interleukin-1, interleukin-6, and tumor necrosis factor-alpha, correlates with the severity and outcome of acute pancreatitis. Interleukin-10 is an anti-inflammatory cytokine that inhibits cytokine production from the macrophage. The aim of this study was to determine whether interleukin-10 would decrease both the severity of acute pancreatitis and the level of circulating proinflammatory cytokines. Ninety female mice were divided into three equal groups. Group 1 (controls) received intraperitoneal saline solution. Groups 2 and 3 received intraperitoneal cerulein (50 mg/kg/hr) for 7 hours. In addition, group 3 was given 1500 units of intraperitoneal interleukin-10, beginning 1 hour after the induction of acute pancreatitis and every 3 hours thereafter. Animals were killed at 3-hour intervals. Blood samples were obtained for serum amylase and cytokine determinations (interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha). Pancreata were dissected free and fixed in formalin for blinded histologic scoring. Interleukin-10 reduced the serum levels of interleukin-1beta, interleukin-6, tumor necrosis factor-alpha, and amylase in comparison to untreated animals with pancreatitis (P < 0.05). Pancreatic edema, necrosis, and inflammatory cell infiltrate were also reduced in those animals given interleukin-10 (P <0.05). Histologic score, serum cytokines, and amylase levels are elevated during acute pancreatitis. Interleukin-10 given therapeutically, that is, after the onset of acute pancreatitis, lessened the severity of disease, probably through inhibition of the macrophage. This was associated with a decrease in circulating cytokine levels.     

Repeat measurement of case-control data: corrections for measurement error in a study of ischaemic stroke and haemostatic factors

BACKGROUND: Haemostatic factors are suspected to be involved in the aetiology of cerebrovascular events. METHODS: In a case-control study of 105 cases of transient ischaemic attack and minor ischaemic stroke, and 241 controls, data were available on levels of the haemostatic factors-von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI), tissue plasminogen activator (TPA) and factor VII (FVII). These are subject to measurement error and within-person fluctuation of true levels, which may bias relative risk estimates. For all subjects, two determinations were performed on the same blood sample, which allowed estimation of pure measurement error. For estimation of within-person fluctuation, levels were measured from a repeat blood sample on 81 of the controls one year later. RESULTS: The pure measurement error accounted for a very small proportion of the total variation in all cases. Uncorrected for within-person fluctuation, the odds ratio estimates associated with exceeding the median of vWF, PAI, TPA and FVII respectively were 1.88, 0.87, 1.30 and 0.93. After correction for within-person fluctuation odds ratios were 3.56, 0.80, 1.41 and 0.91. Because the PAI determination was not robust to storage conditions, it was estimated that 75% of the variation in this factor was within-person rather than between-persons. Thus, estimates of relative risk relation to PAI cannot be regarded as reliable in this study. CONCLUSIONS: It is likely that elevated levels of vWF are associated with increased risk of ischaemic stroke, but interpretation must be tentative, due to relatively large within-person fluctuation of vWF levels.     

Evaluation of the European Spine Phantom in a multi-centre clinical trial

Transurethral resection of the prostate is the most common method of relieving urinary outflow obstruction secondary to prostatic enlargement. However, this procedure can be responsible for various complications, including irrigant-fluid absorption and blood loss, both of which are strongly dependent on operation duration time. To reduce the latter, a new resection device has been designed for transurethral prostatectomy. The device basically consists of a rotating cutting loop controlled externally, with three degrees of freedom, to fit the adenoma shape. Its performance is assessed in vitro by drilling conical and semi-ellipsoidal cavities in agar gel models. The mean difference between the calculated and obtained cavity volumes is 3% (SD = 0.9%). The volume cutting rate, found to be independent of the type of cavity drilled, is equal to 2.9 +/- 0.3 cm3 min-1. The advantages of this motorised resection device prototype are reduction in operation duration and accuracy of the resected volume. In vivo resection of a 20 cm3 adenoma in less than 15 min can be expected.     

Evidence for a requirement for both phospholipid and phosphotyrosine binding via the Shc phosphotyrosine-binding domain in vivo

The adapter protein Shc is a critical component of mitogenic signaling pathways initiated by a number of receptors. Shc can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosine-binding (PTB) domain, and a role for the PTB domain in phosphotyrosine-mediated signaling has been well documented. The structure of the Shc PTB domain demonstrated a striking homology to the structures of pleckstrin homology domains, which suggested acidic phospholipids as a second ligand for the Shc PTB domain. Here we demonstrate that Shc binding via its PTB domain to acidic phospholipids is as critical as binding to phosphotyrosine for leading to Shc phosphorylation. Through structure-based, targeted mutagenesis of the Shc PTB domain, we first identified the residues within the PTB domain critical for phospholipid binding in vitro. In vivo, the PTB domain was essential for localization of Shc to the membrane, as mutant Shc proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane preceded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylation of Shc following receptor activation. Thus, Shc, through its interaction with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor via a single PTB domain.