Infection by Mycobacterium tuberculosis promotes human alveolar macrophage apoptosis

The effect of Mycobacterium tuberculosis infection on the viability of healthy (control) human alveolar macrophages was evaluated by staining with ethidium homodimer and calcein to discriminate live from dead cells. Infection with M. tuberculosis H37Ra or H37Rv increased macrophage mortality at 6 days from the control level of 3.8% +/- 0.7% to 28.7% +/- 6.9% or 12.6% +/- 3.1%, respectively (P < 0.001 for comparisons of all conditions). A role for tumor necrosis factor alpha (TNF-alpha) in the M. tuberculosis-induced cytolysis of alveolar macrophages was demonstrated by increased cytotoxicity following the addition of exogenous TNF-alpha to the cultures and by enhancement of macrophage survival when M. tuberculosis-infected alveolar macrophages were treated with pentoxifylline or anti-TNF-alpha antibody. The cytolytic mechanism was determined to be apoptosis by the demonstration of a characteristic internucleosomal ladder of genomic DNA by agarose gel electrophoresis, by finding nuclear fragmentation and condensation by electron microscopy, and by in situ terminal transferase-mediated nick end labeling of fragmented DNA in alveolar macrophages infected with M. tuberculosis in vitro. The latter technique was employed to reveal extensive apoptosis within caseating granulomas from lung tissue samples from clinical tuberculosis cases. The induction of apoptosis in alveolar macrophages by M. tuberculosis may play a role in the macrophage-pathogen interaction of tuberculosis in vivo.     

Characterization of the major bovine brain Go alpha isoforms. Mapping the structural differences between the alpha subunit isoforms identifies a variable region of the protein involved in receptor interactions

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.     

Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK

Growth factors activate mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinases (ERKs) and Jun kinases (JNKs). Although the signaling cascade from growth factor receptors to ERKs is relatively well understood, the pathway leading to JNK activation is more obscure. Activation of JNK by epidermal growth factor (EGF) or nerve growth factor (NGF) was dependent on H-Ras activation, whereas JNK activation by tumor necrosis factor alpha (TNF-alpha) was Ras-independent. Ras activates two protein kinases, Raf-1 and MEK (MAPK, or ERK, kinase) kinase (MEKK). Raf-1 contributes directly to ERK activation but not to JNK activation, whereas MEKK participated in JNK activation but caused ERK activation only after overexpression. These results demonstrate the existence of two distinct Ras-dependent MAPK cascades--one initiated by Raf-1 leading to ERK activation, and the other initiated by MEKK leading to JNK activation.     

Clinical evaluation of the BDProbeTec strand displacement amplification assay for rapid diagnosis of tuberculosis

The reliability of the BDProbeTec MTB Test (Becton Dickinson, Sparks, Md.) for direct detection of Mycobacterium tuberculosis in respiratory specimens was evaluated by comparing results to those of conventional mycobacterial culture, with the BACTEC TB 460 and Middlebrook 7H11 biplates. Patients known to have tuberculosis were excluded from analysis. Of 523 specimens from 277 patients, 53 grew a mycobacterium: 24 specimens of M. tuberculosis and 29 specimens of nontuberculous mycobacteria. After initial testing, 42 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 95.8, 96. 2, 54.8, and 99.8%, respectively. After resolution of discrepancies, 28 specimens were positive by the BDProbeTec, for overall sensitivity, specificity, and positive and negative predictive values of 100, 99.2, 85.7, and 100%, respectively. These same values were 100, 80.8, 93.4, and 100%, respectively, for smear-positive samples and 100, 99.4, 75.0, and 100%, respectively, for smear-negative specimens.     

The presence of cytoplasmic lipid droplets is not sufficient to account for neutral lipid signals in the 1H MR spectra of neutrophils

Stimulation of human peripheral blood neutrophils with lipopolysaccharide (LPS), arachidonic acid (AA) and oleic acid (OA) resulted in significant increases in cytoplasmic lipid droplets. This phenomenon was also observed in enucleated and degranulated cytoplasts prepared from neutrophils stimulated with LPS. In contrast, only LPS and high concentrations of OA (10 microM) produced an increase in the lipid intensities of the MR spectra of neutrophils as determined by COSY cross peak volume measurements. Lipid intensities in cells stimulated with OA (2.5 microM) and AA (2.5 microM) and phorbol myristate acetate (20 nM) were not elevated. LPS stimulation of resting cytoplasts resulted in increased lipid droplets but not MR lipid intensities. These data suggest that while cytoplasmic lipid droplets may correlate with MR lipid intensity under some circumstances, their presence is not sufficient to account for increased neutral lipid signals.     

Neuropeptide Y antibody attenuates 2-deoxy-D-glucose induced feeding in rats

2-deoxy-D-glucose (2-DG) has been shown to induce increased feeding responses in animals. Recent studies suggest the possible involvement of neuropeptide Y (NPY) in 2-DG-induced feeding. The present study examined the effect of immunoneutralization of endogenous NPY on 2-DG-induced feeding. NPY antibody injected into the paraventricular nucleus of the rats significantly attenuated 2-DG-induced feeding, suggesting that hypothalamic NPY may mediate, at least partly, the effect of 2-DG on food intake.     

Interposition grafting for reoperation on the common femoral artery

PURPOSE: This report details our experience with common femoral artery resection and Dacron interposition grafting in the management of vascular reoperations involving the common femoral artery. DESIGN: Retrospective review. SETTING: University teaching hospital. SUBJECTS: Consecutive reoperative patients who had common femoral artery interposition grafting for arteriosclerotic occlusive disease from 1986 to 1997. INTERVENTIONS: Common femoral artery resection and interposition grafting. MAIN OUTCOME MEASURES: Operative morbidity and mortality rates and long-term patency, limb salvage, patient survival, freedom-from-graft-infection, and freedom-from-reoperation rates. RESULTS: Ninety-nine common femoral arteries (16 bilateral) were resected and replaced with Dacron interposition grafts in 83 patients (50 male, 33 female; mean age, 65 years) who had had 237 previous ipsilateral common femoral artery operations (mean, 2.4 operations; range, 1-9 operations). Simultaneous infrainguinal bypass grafts were performed in 52 operations (53%), and 60 operations (61%) were performed in patients who had had previous ipsilateral proximal bypass grafts. Operative mortality was 2%, with a 14% rate of perioperative wound complications. Mean follow-up time was 22 months. One- and 3-year assisted primary patency rates for the interposition grafts were 90% and 77%, respectively. Both 1- and 3-year life-table-determined limb salvage rates were 95%. One- and 3-year life-table-determined freedom-from-reoperation rates were 74% and 43%, respectively. One- and 3-year life-table-determined freedom-from-infection rates were 99% and 92%, respectively. One- and 3-year life-table-determined survival rates were 82% and 73%, respectively. CONCLUSIONS: Common femoral artery resection and Dacron interposition grafting are safe, and they obviate many difficulties associated with reoperative common femoral artery surgery with satisfactory long-term results.