Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide

Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.     

Fungal beta-tubulin, expressed as a fusion protein, binds benzimidazole and phenylcarbamate fungicides

Benzimidazoles are important antitubulin agents used in veterinary medicine and plant disease control. Resistance is a practical problem correlated with single amino acid changes in beta-tubulin and is often linked to greater sensitivity to phenylcarbamates. This negative cross-resistance creates opportunities for durable antiresistance strategies. Attempts to understand the molecular basis of benzimidazole resistance have been hampered by the inability to purify tubulin from filamentous fungi. We have overcome some of these problems by expressing beta-tubulin as a fusion with a maltose binding protein. This fusion protein is soluble, and we confirm for the first time using a gel filtration assay that benzimidazoles indeed bind to beta-tubulin. This binding is reduced by the mutation Glu198-->Gly198, which also confers resistance. Binding of phenylcarbamates is the complete opposite, reflecting their biological activity and the negative cross-resistance. This suggests that the fungicide binding sites fold correctly in the fusion protein.     

Age-dependent inaccuracy of asthma death certification in Northern England, 1991-1992

OBJECTIVE: To test the usefulness of a commercial DNA hybridization assay for the detection of high-risk (HR) human papillomavirus (HPV) types in archival cervical smears and to compare the sensitivity with that of polymerase chain reaction (PCR) using consensus primers. STUDY DESIGN: Stained material was scraped from archival slides and the pellet volume noted. DNA was extracted using silica/guanidinium isothiocyanate and the quality checked by amplification of the beta-globin gene. HR-HPV DNA was detected using a commercial hybrid capture assay (HCA) and the results compared with an in-house amplification system with consensus primers. RESULTS: Of 156 archival smears stored for 12-13 years, 20 were positive by HCA using an HR probe cocktail. Ninety-eight were also tested by PCR, and 35 were positive. The percentage of HPV-positive samples increased with the increasing size of the pellet. HR-HCA detected more positives in samples with high grade squamous intraepithelial lesion (moderate/severe dyskaryosis). CONCLUSION: Both hybridization by HCA and amplification by PCR could be used to detect genital HPV in archival smears. The general primers PCR detected more positives than HR-HCA but included HPV 6/11. While variation in sample size and prolonged storage may reduce the quality of DNA, the use of archival material for longitudinal studies of HPV presence is potentially worthwhile.     

Monocrotaline pyrrole induces apoptosis in pulmonary artery endothelial cells

Eosinophilic myocarditis followed by fibrosis of the cardiac muscle was observed in addition to peripheral blood eosinophilia in CBA/J mice infected with Toxocara canis. The infected mice were used as an experimental model of eosinophilic endomyocarditis associated with hypereosinophilic syndrome. Effects of in vivo treatment with MoAbs to adhesion molecules on eosinophilic myocarditis were examined using this experimental model. Expressions of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on endothelial cells of capillaries in myocardium were increased 1 and 2 weeks after infection. Infiltration of very late antigen (VLA)-4+ and/or CD11a+ cells into the cardiac muscles was also observed 1 and 2 weeks after infection. Infiltration of eosinophils into the heart was significantly suppressed by anti-CD18 MoAb and anti-VLA-4 MoAb, and focal fibrosis of the cardiac muscle was also significantly suppressed by combined administration of anti-CD18 and anti-ICAM-1 MoAbs. These results indicate that adhesion molecules may play important roles in eosinophilic myocarditis, and that blockade of interaction between adhesion molecules and their ligands may help to control it.     

Clinical significance of echocontrast enchancement in neurovascular diagnosis. Review of experience following a year of use

AIM OF THE STUDY: To evaluate the potential and limitations of echocontrast enhancement using Levovist in a non selected consecutive cohort of neurological patients with insufficient native ultrasound investigations. METHODS: In 91 patients an indication for echocontrast application was seen after an insufficient extracranial (n = 17), transtemporal (n = 54), and transforaminal (n = 20) Doppler- und color-coded Duplex sonography. Levovist was injected at a concentration of 400 mg/ml and 200-400 mg/ml for the transcranial and extracranial approach, respectively. The effect of the echocontrast enhancement was assessed semiquantitatively with respect to signal enhancement, imaging quality, and diagnostic confidence. RESULTS: In a total of 83 patients (91%) the signal enhancement led to a moderate to high imaging quality allowing to reach 67 definite neurovascular diagnoses (74%). In subgroup analysis, the amount of sufficiently confident examinations was significantly higher for the transtemporal and transforaminal (both 80%) than for the extracranial approach (47%). The latter was mostly due to artificial signals derived from adjacent neck vessels. CONCLUSION: Levovist constitutes a safe and highly effective diagnostic tool especially for the transtemporal and transforaminal neurosonographical imaging. By means of a differentiated application of echocontrast agents, its cost-effectiveness can be increased and the need for other potential invasive and expansive neuroimaging methods can be further reduced.     

Prospective evaluation of criteria for microbiological diagnosis of prosthetic-joint infection at revision arthroplasty. The OSIRIS Collaborative Study Group

A prospective study was performed to establish criteria for the microbiological diagnosis of prosthetic joint infection at elective revision arthroplasty. Patients were treated in a multidisciplinary unit dedicated to the management and study of musculoskeletal infection. Standard multiple samples of periprosthetic tissue were obtained at surgery, Gram stained, and cultured by direct and enrichment methods. With reference to histology as the criterion standard, sensitivities, specificities, and likelihood ratios (LRs) were calculated by using different cutoffs for the diagnosis of infection. We performed revisions on 334 patients over a 17-month period, of whom 297 were evaluable. The remaining 37 were excluded because histology results were unavailable or could not be interpreted due to underlying inflammatory joint disease. There were 41 infections, with only 65% of all samples sent from infected patients being culture positive, suggesting low numbers of bacteria in the samples taken. The isolation of an indistinguishable microorganism from three or more independent specimens was highly predictive of infection (sensitivity, 65%; specificity, 99.6%; LR, 168.6), while Gram staining was less useful (sensitivity, 12%; specificity, 98%; LR, 10). A simple mathematical model was developed to predict the performance of the diagnostic test. We recommend that five or six specimens be sent, that the cutoff for a definite diagnosis of infection be three or more operative specimens that yield an indistinguishable organism, and that because of its low level of sensitivity, Gram staining should be abandoned as a diagnostic tool at elective revision arthroplasty.     

Finding intron/exon splice junctions using INFO, INterruption Finder and Organizer

INFO, INterruption Finder and Organizer, has been used to find coding sequence intron-exon splice junctions in human and other DNA by comparing the six conceptual translations of the input DNA sequence with sequences in protein databanks using a similarity matrix and windowing algorithm. Similarities detected both delineate position of the gene and provide clues as to the function of the gene product. In addition to use of a standard similarity matrix and windowing algorithm, INFO uses two novel steps, the MiniLibrary and Reverse Sequence steps, to enhance identification of small exons and to improve precision of junction nucleotide delineation. Exons as small as about 30 bases can be reliably found, and > 90% of junctions are precisely identified when canonical splice junction information is used. With the MiniLibrary and Reverse Sequence steps, INFO parameters need not be optimized by the user. In comparative test runs using 19 human DNA sequences, INFO found 108 of 111 exons, with 0 reported false positives, compared with 111 exons and 51 false positives for BLASTX, 99 exons and 6 false positives for GRAIL II, 77 exons and 24 false positives for GeneMark, 61 exons and 9 false positives for GeneID, and 105 exons and 6 false positives for PROCRUSTES. The correlation coefficient for finding and positioning these 111 exons was greater than 98% for INFO. Comparable results were obtained in test runs of 13 nonhuman DNA sequences. INFO is applicable to DNA from any species, will become more robust as sequence databanks expand, and complements other heuristic approaches.     

NO/cGMP pathway is involved in exocrine secretion from rat pancreatic acinar cells

OBJECTIVE: To identify factors associated with the initiation of breast-feeding in a predominantly Puerto Rican population living in inner-city Hartford, Conn. DESIGN: Retrospective study of 144 Latino women (mean +/- standard deviation age = 26.3 +/- 5.7 years) with children at least 1 year old but younger than 6 years old (mean +/- standard deviation age = 3.0 +/- 1.2 years) at the time of the survey. Women were recruited from agencies sponsoring health programs for mothers and children. They were interviewed in their homes (69%) or at the Hispanic Health Council, Hartford, Conn (31%). SUBJECTS/SETTING: Low-income Latino women who had at least 1 preschooler at the time of the interview. The women lived in inner-city Hartford, and the overwhelming majority were Puerto Rican and received welfare assistance and food stamps. Seventy-eight percent of the women chose to be interviewed in Spanish; the other 22% were interviewed in English. STATISTICAL ANALYSES: Explanatory variables that related to breast-feeding initiation (P < or = .2) in bivariate chi 2 analyses were entered into a multivariate logistic regression model that was reduced using backward stepwise elimination procedures. RESULTS: Multivariate analyses indicated that breast-feeding the previous child, shorter length of maternal residence in the United States, not receiving prenatal bottle-feeding advice, more recent birth, and higher birth weight were positively associated with breast-feeding initiation. A major reason for choosing not to breast-feed was that women felt socially uncomfortable doing it. APPLICATIONS: Breast-feeding initiation was more likely in Latino women who received prenatal breast-feeding counselling and postpartum support. Mothers of low-birth-weight infants and women breast-feeding for the first time may need additional help. These findings can be used by programs like the Special Supplemental Nutrition Program for Women, Infants, and Children to increase breast-feeding initiation.