Characterization of RAFTK, a novel focal adhesion kinase, and its integrin-dependent phosphorylation and activation in megakaryocytes

We have recently isolated a cDNA encoding a novel human intracellular tyrosine kinase, termed RAFTK (for a related adhesion focal tyrosine kinase). The RAFTK cDNA, which encodes a polypeptide of 1,009 amino acids, shares 65% homology to the focal adhesion kinase (FAK), including several consensus motifs. In this report, we describe the biochemical characterization and functional analysis of the RAFTK protein. Coexpression of RAFTK and FAK proteins in megakaryocytic cells and blood platelets was observed. Using a specific antibody to RAFTK and the monoclonal antibody 2A7 to FAK, FAK and RAFTK could be distinguished antigenically. RAFTK had intrinsic tyrosine kinase and autokinase activities. It was phosphorylated on tyrosine in growing cultures of COS cells transfected with the pCDNAIII/flag-RAFTK expression vector containing the RAFTK cDNA ligated with the 8 amino acid flag peptide sequence. Similar to FAK, dephosphorylation of RAFTK was observed when adherent transfected COS cells were detached. Phosphorylation was regained upon replating of these cells on the fibronectincoated dishes. Analysis of tyrosine-phosphorylated RAFTK from adherent transfected COS cells showed that the Src homology 2 (SH2) domains of the Src and Fyn protein kinases as well as the Grb2 adaptor protein were able to specifically associate with RAFTK. Tyrosine phosphorylation of endogenous RAFTK was observed upon fibronectin-induced activation of human megakaryocytic cells. Furthermore, colocalization of RAFTK protein with vinculin, a focal adhesion protein, was observed by confocal microscopy in focal adhesion- like structures in adherent CMK cells and in transfected pCDNAIII/flag- RAFTK COS cells upon fibronectin activation. These data suggest that RAFTK is a novel member of the FAK family, that it localizes to focal adhesion-like structures in CMK megakaryocytic cells, that it participates in integrinmediated signaling pathways in megakaryocytes, and that it is able to associate with the tyrosine kinases Src and Fyn as well as the adaptor protein Grb2 via SH2-phosphotyrosine interactions.     

Detection of Trichomonas vaginalis in pregnant women with the InPouch TV culture system.

Trichomonas vaginalis causes a common genitourinary infection which is frequently asymptomatic. At present, pregnant women are not usually screened for the infection unless they are symptomatic. In the present study, we screened and obtained samples for culture from all pregnant women attending a prenatal clinic with the InPouch TV culture system and compared results with those of standard culture in Diamond's medium and slide wet mount examination. The InPouch TV culture system was as reliable as Diamond's medium in detecting T. vaginalis and may be useful and effective in a pregnancy clinic setting.     

Parental 'expressed emotion' as a predictor of schizophrenic relapse

Fifty-seven schizophrenic patients were initially assessed after admission to hospital, at which time their parents completed a Camberwell Family Interview, generating "expressed emotion" (EE) scores. Relapse over the next nine months was not predicted by household EE status or by individual EE scales. Multivariate analyses suggested that a poor course after hospitalization was best predicted by a poor course before admission and by living in a one-parent household. High household EE status was a predictor only in one multivariate analysis after course of illness and one-parent household status had been entered, suggesting an interaction effect. We query the causal proposition linking high EE and relapse and suggest instead that a poor illness course may elicit high EE in relatives, particularly in one-parent households, and, thus, may make the principal contribution to the proposed link.     

Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase

Recently, 2-halogenated deoxyadenosine analogs (F, Cl, and Br) have been shown to have antitumor activity. These analogs are phosphorylated by cells and are believed to exert their cytotoxic action at the nucleoside triphosphate level. In this work the interaction of these nucleoside triphosphate analogs with potential targets, such as DNA polymerase alpha, beta, and gamma, DNA primase, and ribonucleotide reductase was examined in detail. All of these compounds competitively inhibited the incorporation of dAMP into DNA by DNA polymerase alpha, beta, or gamma. F-dATP was able to completely substitute for dATP using DNA polymerase alpha and gamma, but not with DNA polymerase beta. Cl-dATP and Br-dATP substituted poorly for dATP using DNA polymerase alpha and beta. Extension of a 32P-labeled primer by DNA polymerase alpha, beta, or gamma on a single-stranded M13 template showed that these compounds were incorporated into the 3' end of the growing DNA chain and that elongation beyond the incorporated analogs was significantly retarded for Cl-dATP and Br-dATP using either DNA polymerase alpha or beta. DNA primase using poly(dC) as template was inhibited by these compounds at a concentration 4 to 5 times greater than that required for 2-F-araATP. The 2-halogenated dATP analogs were potent inhibitors of ADP reduction by ribonucleotide reductase. In conclusion, the cytotoxic action of 2-Cl-deoxyadenosine and 2-Br-deoxyadenosine may partially be mediated through the mechanism of "self-potentiation," by depression of the deoxynucleoside triphosphate pools due to inhibition of ribonucleotide reductase, which would facilitate their incorporation into DNA and result in the inhibition of DNA synthesis.     

Human fibroblast tissue factor is inhibited by lipoprotein-associated coagulation inhibitor and placental anticoagulant protein but not by apolipoprotein A-II

Studies of proteins that inhibit tissue factor activity have generally been conducted using either an extracted tissue homogenate ("thromboplastin") or tissue factor protein reconstituted into phospholipid vesicles rather than with tissue factor expressed in cell membranes (its physiological environment). In the present study, a human fibroblast cell strain was used to evaluate the effects of lipoprotein associated coagulation inhibitor (LACI), placental anticoagulant protein (PAP), and apolipoprotein A-II (apo A-II) on human tissue factor in cell membranes. LACI was tested from 7.8 to 500 pmol/L on fibroblasts cultured at cell densities ranging from 3,500 to 9,925 cells/well, and caused a progressive inhibition of tissue factor activity. PAP was tested from 3.9 nmol/L to 1 mumol/L at cell densities ranging from 4,500 to 15,400 cells/well and caused up to 83% inhibition of tissue factor activity. Inhibition by these proteins appeared to be influenced by cell density as well as whether the cells were intact or disrupted. Apo A-II, up to 1 mumol/L, did not inhibit the tissue factor activity of intact or disrupted fibroblasts at any cell density examined even though it did inhibit the activity of tissue factor in phospholipid vesicles. Of these inhibitors of tissue factor-dependent activation of factor X, LACI was the most effective in suppressing the generation of factor Xa activity. The effects obtained with apo A-II are clearly dependent on the nature of the tissue factor preparation with which it is tested. The disparity between the inhibitory effect of apo A-II on the activity of tissue factor reconstituted into lipid vesicles and the absence of effect on the activity of tissue factor remaining in cell membranes serves to reemphasize the necessity of reexamining results obtained with model systems using as nearly physiological reagents as possible.     

Nonlinearity and facilitation in phosphoinositide signaling studied by the use of caged inositol trisphosphate in Xenopus oocytes

The phosphoinositide signaling pathway, which mediates neurotransmitter responses, was studied in Xenopus oocytes by recording membrane currents evoked using lightflash photolysis of caged inositol trisphosphate (caged IP3) to produce rapid and reproducible transients in intracellular IP3 levels. Photolysis of caged IP3 evoked currents which were carried largely by chloride ions and depended upon intracellular, but not extracellular, calcium. A given light flash evoked larger responses when the amount of caged IP3 loaded into the oocyte was increased, and illumination of the vegetal hemisphere gave larger responses than the animal. Long (10 sec) light exposures produced oscillatory currents, resembling responses to serotonin and other agonists, which became larger, more transient, and of shorter latency as the light intensity was increased. Brief (ca. 100 msec) flashes evoked a single "spike" of current. The caged IP3 response showed a threshold, in that light flashes had to be greater than a certain intensity and duration before currents could be detected. Associated with this, sub- and suprathreshold light flashes caused a long-lasting (seconds or minutes) potentiation of responses to subsequent test flashes. The lightflash response was also potentiated by a preceding intracellular injection of IP3 and by extracellular application of an agonist thought to induce IP3 liberation. However, intracellular injections of calcium depressed the response. We conclude that the liberation of calcium from intracellular stores varies nonlinearly with the intracellular level of IP3. This phenomenon may explain earlier observations, including the long latency of currents evoked by low doses of agonists such as acetylcholine and serotonin, and the nonlinear facilitation seen between these agonists. Further, it suggests a mechanism for "chemical integration," which may be important in the functioning of neurons and other cells which use IP3 as an intracellular messenger.     

Image-directed percutaneous biopsies with a biopsy gun

Core tissue for histologic study is believed by many pathologists to be more diagnostic than material from needle aspiration. Recently, a biopsy "gun" has been introduced, which simplifies core biopsies. With this device, 182 biopsies of multiple anatomic sites were performed with ultrasonic, computed tomographic, and fluoroscopic guidance and 18-gauge needles. High-quality histopathologic specimens were obtained in 177 of the biopsies, and diagnostic target tissue was obtained in 167. Only three significant complications occurred: one bleeding complication that required transfusion and two cases of pneumothorax that necessitated placement of chest tubes. The biopsy gun eliminated the disjointed movements of conventional "skinny" needle biopsies, and none of the samples demonstrated significant "crush" artifact or obscuring blood, problems that are commonly associated with manual biopsy techniques. Patient discomfort was decreased with this system compared with that of manual biopsies, and the total procedure time was reduced. Because of these distinct advantages, the authors now use the biopsy gun exclusively for all percutaneous biopsies and recommend that other institutions consider the use of this biopsy method.     

Neisseria sicca osteomyelitis.

Neisseria sicca was identified as the cause of vertebral osteomyelitis in a male patient who had previously suffered a nonpenetrating, traumatic back injury. The identifying characteristics and antimicrobial susceptibility patterns are presented for this rare human pathogen, which heretofore has not been reported as a cause of infection localized to bone.     

Patient and carer satisfaction with ''hospital at home'': quantitative and qualitative results from a randomised controlled trial.

BACKGROUND: 'Hospital At Home' schemes are set to increase in the United Kingdom (UK) in response to the NHS Plan. To date, little detailed work has been done on the acceptability of these schemes to patients and their carers. AIM: To compare Hospital at Home patient and carer satisfaction with hospital care. DESIGN OF STUDY: Pragmatic randomised controlled trial. SETTING: Consecutive patients assessed as suitablefor the Leicester Hospital at Home scheme were randomised to Hospital at Home or one of three acute hospitals in the city. METHOD: Patient satisfaction was assessed two weeks after randomisation, or at discharge if later using a six-item questionnaire. Patients' and carers' views of the services were assessed by semistructured interviews. RESULTS: One hundred and two patients were randomised to Hospital at Home and 97 to hospital. Forty-eight (47%) patients in the Hospital at Home arm and 35 (36%) in the hospital arm completed the satisfaction questionnaire, representing 96% and 85% of those eligible, respectively. Total scores were significantly higher in the Hospital at Home (median = 15) than in the hospital group (median = 12). (P<0.001, Mann-Whitney U-test.) Responses to all six questions favoured Hospital at Home, with all but one of these differences being statistically significant. In the Hospital at Homegroup, 24 patients and 18 of their carers were interviewed; in the hospital group 18 patients and seven of their carers were interviewed. Themes emerging from these interviews were that patients appreciated the more personal care and better communication offered by Hospital at Home and placed great value on staying at home, which was seen to be therapeutic. Patients largely felt safe in Hospital at Home, although some would have felt safer in hospital. Some patients and carers felt that better medical care would have been provided in hospital. Carers felt that the workload imposed by Hospital at Home was no greater than by hospital admission and that the relief from care duties at home would be counterbalanced by the added strain of hospital visiting. CONCLUSIONS: Patient satisfaction was greater with Hospital at Home than with hospital. Reasons included a more personal style of care and a feeling that staying at home was therapeutic. Carers did not feel that Hospital at Home imposed an extra workload.