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.     

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.     

Activities and sources of beta-lactamase in sputum from patients with bronchiectasis.

beta-Lactamase activity was measured in secretions from patients with bronchiectasis. Of 28 sputum samples, 23 contained measurable amounts of activity; values were significantly higher (P less than 0.01) in purulent samples than in mucoid or mucopurulent samples. beta-Lactamase activity was usually present in saliva collected before and between sputum expectorations, although values for sputum were higher than for either group of saliva samples (P less than 0.025 and P less than 0.005, respectively). This difference suggests that at least part of sputum beta-lactamase activity originates in the bronchial tree. Detailed microbiological study of a further eight specimens (seven were beta-lactamase positive) led to the isolation of Haemophilus influenzae from six, although only two of these isolates were beta-lactamase positive. Several other beta-lactamase-producing organisms were also isolated, including Staphylococcus aureus (n = 3), Escherichia coli (n = 1), Proteus spp. (n = 1), and Bacteroides spp. (n = 3). Size-exclusion high-performance liquid chromatography of the sputum showed several peaks of beta-lactamase activity which usually coeluted in fractions similar to those of their beta-lactamase-positive isolates. Therefore, sources of sputum beta-lactamases are often bacteria not considered truly pathogenic or not isolated during routine bacteriological assessment. These observations should be considered when embarking on antimicrobial therapy in bronchiectatic patients and suggest that increased dosages of penicillins are indicated.     

Comparison between testosterone oenanthate-induced azoospermia and oligozoospermia in a male contraceptive study. IV. Suppression of endogenous testicular and adrenal androgens

Administration of supraphysiological doses of testosterone to normal men causes inhibition of spermatogenesis, but while most become azoospermic, 30-55% maintain a low rate of spermatogenesis. We have investigated whether there are differences in endogenous androgen production, of testicular and adrenal origin, which may be related to the degree of suppression of spermatogenesis. Thirty-three healthy Caucasian men were given weekly i.m. injections of 200 mg testosterone oenanthate (TE), 18 became azoospermic, while 15 remained oligozoospermic. Urinary excretion of epitestosterone, a specific testicular product, was reduced to <10% of pretreatment values, with no differences between the groups. Similar results were obtained for other markers of testicular steroidogenesis. Urinary and plasma adrenal androgens were also reduced during TE treatment: a statistically significant decrease in both (P < 0.001 and P < 0.05 respectively) was seen in the azoospermic but not oligozoospermic responders. These results suggest that testicular steroidogenesis is decreased to <10% by the administration of supraphysiological doses of exogenous testosterone. Differences in the degree of ongoing steroidogenesis in the testis do not appear to account for incomplete suppression of spermatogenesis, thus differences in androgen metabolism may underlie this heterogeneous response. A small but significant reduction in secretion of adrenal androgens was also detectable, the relevance of which is unclear.      

The effect ofin vitro andin vivo dexamethasone on human neutrophil function

There is a significant fall in PMN chemotaxis to the peptide FMLP in response to increasing concentrations of dexamethasonein vitro. The response fell in a dose related manner from a control value of 53.7 SE±9.6 cells per high power field (cpf) to 47.3 SE±8.1 at 10^–6 M (p<0.05) and 24.7±8.9 at 10^–3 M (p<0.025). A similar response was observed for the chemoattractants zymosan activated serum and the sol phase of purulent sputum. The effect was independent of protein synthesis or the period of incubation. Twelve milligrams of dexamethasone taken daily by 6 healthy volunteers resulted in a significant (p<0.025) reduction in the chemotactic response of PMN to 10^–8 M FMLP (from 29.5±1.55 to 13.7±1.8 cpf) which was apparent within 2 hours of taking the first dose. This effect was sustained for the three days on which dexamethasone was taken but returned to normal 7 days after the last dose had been administered.Dexamethasone therapy had no effect on unstimulated PMN superoxide anion production eitherin vitro orin vivo.Thein vivo effect on neutrophil function occurred at mean serum dexamethasone concentrations of 1.26 (±0.28)×10^–7 M on day 1, 1.44 (±0.15)×10^–7 M on day 2 and 1.31 (±0.13)×10^–7 M on day 3. Thus we conclude that dexamethasone concentration which inhibit PMN chemotaxisin vivo are much lower than those required to exert the same effectin vitro.     

STXBP1: the second synaptic vesicle release gene involved in epilepsy

De novo mutations in the gene encoding STXBP1 (MUNC18‐1) cause early infantile epileptic encephalopathy
Saitsu et al. (2008)
Nature Genetics 40: 782–788