Reproducible two-dimensional capillary electrophoresis analysis of Barrett's esophagus tissues

We have constructed a high-speed, two-dimensional capillary electrophoresis system with a compact and high-sensitivity fluorescence detector. This instrument is used for the rapid and reproducible separations of Barrett's esophagus tissue homogenates. Proteins and biogenic amines are labeled with the fluorogenic reagent 3-(2-furoyl)quinoline-2-carboxaldehyde. Labeled biomolecules are separated sequentially in two capillaries. The first capillary employs capillary sieving electrophoresis using a replaceable sieving matrix. Fractions are successively transferred to a second capillary where they undergo additional separation by micellar electrokinetic capillary chromatography. The comprehensive two-dimensional separation requires 60 min. Within-day migration time reproducibility is better than 1% in both dimensions for the 50 most intense features. Between-day migration time precision is 1.3% for CSE and better than 0.6% for MECC. Biopsies were obtained from the squamous epithelium in the proximal tubular esophagus, Barrett's epithelium from the distal esophagus, and fundus region of the stomach from each of three Barrett's esophagus patients with informed consent. We identified 18 features from the homogenate profiles as biogenic amines and amino acids. For each of the patients, Barrett's biopsies had more than 5 times the levels of phenylalanine and alanine as compared to squamous tissues. The patient with high-grade dysplasia shows the highest concentrations for 13 of the amino acids across all tissue types. Concentrations of glycine are 40 times higher in squamous biopsies compared to Barrett's and fundal biopsies from the patient with high-grade dysplasia. These results suggest that two-dimensional capillary electrophoresis may be of value for the rapid characterization of endoscopic and surgical biopsies.     

Pharmacokinetics of human recombinant tissue-type plasminogen activator, administered intra-abdominally, in a rat peritonitis model

Human recombinant tissue-type plasminogen activator (rtPA), administered intraperitoneally, may promote intra-abdominal fibrinolysis in peritonitis, thereby preventing adhesion and abscess formation. The pharmacokinetics of a single intraperitoneal dose of 0.5 or 2.0 mg/ml human rtPA were assessed in rats with fecal peritonitis and related to their endogenous tPA and plasminogen activator inhibitor (PAI) activity. Endogenous PAI activity was strongly elevated both in peritoneal fluid and in plasma, whereas tPA activity was only slightly increased in the peritoneal fluid. Both doses of rtPA significantly enhanced fibrinolytic activity of the peritoneal fluid up to 24 h in a dose-dependent manner. However, tPA activity was lower than calculated from the tPA antigen levels and the specific activity of rtPA in the fluid of rats with peritonitis. Plasma tPA activity was low at both doses. A single dose of 0.5 or 2.0 mg/ml rtPA can raise fibrinolytic activity in the abdominal cavity up to 24 h. High endogenous peritoneal PAI levels may adversely effect intra-abdominal use of human rtPA in peritonitis.     

Patellar fractures in posterior stabilized knee arthroplasties

In the present study, a significantly higher rate of lateral release was demonstrated in the patients with patellar fractures despite the relative high overall lateral release rate (82%). Although the complication of patellar fractures in total knee arthroplasty has a multifactorial etiology, these findings implicate lateral retinacular release as a significant factor. It is unclear whether the lateral retinacular release was a direct result of the surgical technique or whether it was necessitated by the anatomy of the patellofemoral articulation in this component system. Lateral release may continue to be performed as needed during total knee arthroplasty to maintain optimal patellofemoral mechanics. However, if it is performed, an attempt should be made to preserve the lateral superior genicular artery.     

Design of peptides, proteins, and peptidomimetics in chi space

Peptide and protein biological activities depend on their three dimensionals structures in the free state and when interacting with their receptors/acceptors. The backbone conformations such as alpha-helix, beta-sheet, beta-turn, and so forth provide critical templates for the three-dimensional structure, but the overall shape and intrinsic stereoelectronic properties of the peptide or protein important for molecular recognition, signal transduction, enzymatic specificity, immunomodulation, and other biological effects depend on arrangement of the side chain groups in three-dimensional chi space (their chi 1, chi 2, etc. torsional angles). In this paper we explore approaches to the de novo design of polypeptides and peptidomimetics with biased or specific conformational/topographical properties in chi space. We consider computational and experimental methods that can be used to examine the effects of specific structural modifications in constraining side chain groups of amino acid residues and their similarities in chi space to the natural amino acids to evaluate what sort of mimetics are likely to mimic normal amino acids. We then examine some of the asymmetric synthetic methods that are being developed to obtain the amino acid mimetics. Finally, we consider selected examples in the literature where these specialized amino acids have been incorporated in biologically active peptides and the specific insights they have provided regarding the topographical requirements for bioactive peptide potency, selectivity, and other biochemical and pharmacological properties. Constraints in chi space show great promise as useful tools in peptide, protein, and peptidomimetic de novo design of structures and pharmacophores with specific stereostructural, biochemical and biological properties.     

Tuberculin skin testing of hospitalized patients

OBJECTIVES: We sought to define the prevalence of tuberculin skin test (TST) positivity in a group of newly hospitalized patients, to identify risk factors for positive tests, and to examine the impact of testing on infection control practices. DESIGN: Unblinded cohort study over 5 days in July 1992. SETTING: A 1,000-bed university-affiliated hospital. PATIENTS: All patients admitted (excluding obstetric patients and newborns) were interviewed. Patients without a history of tuberculosis (TB) or a positive TST were offered a TST with Candida and tetanus controls. RESULTS: Of 346 patients offered the test, 21 (6%) had a prior history of TB or a positive TST, and 36 (10%) declined to participate; 279 of the remaining 289 completed the study. Anergy was demonstrated in 94 (33.7%) of 279 patients. New positive TSTs were identified in 19 (10.3%) of 185 nonanergic patients. Of the 19 TST-positive patients, 6 (32%) had infiltrates on chest radiographs and were evaluated for active TB. One patient was treated empirically for active TB, and five received isoniazid prophylaxis. Risk factors for a new positive TST included age (odds ratio [OR], 1.56 per decade of life; P = .021), African American race (OR, 4.81; P = .008), alcohol abuse (OR, 5.53; P = .005), and peptic ulcer disease (OR, 4.53; P = .017). Risk factors for anergy included admission to a surgical service (OR, 2.1; P = .006), current use of steroids (OR, 2.65; P = .005), and human immunodeficiency virus (HIV) infection (OR, undefined; P = .034). CONCLUSIONS: Despite a high rate of anergy, routine tuberculin skin testing identified a substantial number of patients with TB infection who might otherwise have gone unrecognized.     

Development of survival responsiveness to brain-derived neurotrophic factor, neurotrophin 3 and neurotrophin 4/5, but not to nerve growth factor, in cultured motoneurons from chick embryo spinal cord

During embryonic development, most neuronal populations undergo a process usually referred to as naturally occurring neuronal death. For motoneurons (MTNs) of the lumbar spinal cord of chick embryos, this process takes place in a well defined period of time, between embryonic days 6 and 10 (E6-E10). Neurotrophins (NTs) are the best characterized family of neurotrophic factors and exert their effects through activation of their specific Trk receptors. In vitro and in vivo studies have demonstrated that rodent motoneurons survive in response to BDNF, NT3, and NT4/5. In contrast, the trophic dependencies of chicken motoneurons have been difficult to elucidate, and various apparently conflicting reports have been published. In the present study, we describe how freshly isolated motoneurons from E5.5 chick embryos did not respond to any neurotrophin in vitro. Yet, because motoneurons were maintained alive in culture in the presence of muscle extract, they developed a delayed specific survival response to BDNF, NT3, and NT4/5 that is clearly dose-dependent, reaching saturation at doses of 100 pg/ml. This trophic response correlated with increasing expression of the corresponding functional receptors TrkB and TrkC. Moreover, TrkB receptor is able to become autophosphorylated and to activate classical intracellular signaling pathways such as the extracellular signal-regulated protein kinase when it is stimulated with its cognate ligand BDNF. Therefore, our results reconcile the reported differences between in vivo and in vitro studies on the ability of chicken MTNs to respond to some members of the neurotrophin family of trophic factors.