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.     

Comparison of local tumor recurrence following excision with the CO2 laser, Nd:YAG laser, and Argon Beam Coagulator

This study compares the incidence of local tumor recurrence following primary excision with the CO2 laser, Nd:YAG laser (contact), Argon Beam Coagulator, or electrocautery. One hundred eight Fisher 344 rats with R3230AC mammary tumors (1.6 +/- 0.04 [SD] cm diameter) were used. All animals were randomized into groups of similar tumor size. In groups C and CS, excision was performed with a Sharplan 1060 CO2 laser (TEMoo, 25 W, continuous wave [CW], 0.2-mm spot size). Wounds in group CS were "sterilized" (0.5-mm spot size, 25 W, CW) by gently heating the wound without causing blanching or charring. In group N, a 0.4-mm contact Laser Blade and a Cooper 8000 Nd:YAG laser at 20 W CW was used. In groups SA1 and SA2, tumors were excised with the scalpel, and hemostasis and wound "sterilization" were accomplished with the Bard System 6000 Argon Beam Coagulator (ABC) at 40 W and 4 liters/min argon gas flow in SA1 and 12 liters/min in SA2. In group E, excision was accomplished at 40 W blend mode, 10 W spray mode. In group EA, excision was accomplished at 60 W cutting current, and hemostasis was achieved with the ABC. The animals were examined for evidence of recurrence for 34 days postoperatively. Mortalities were excluded from analysis. The incidence of recurrence was 11/14 (79%) in C, 6/16 (38%) in CS, 10/14 (71%) in SA1, 6/13 (46%) in SA2, 6/15 (40%) in N, 7/10 (70%) in EA, and 3/15 (20%) in E. Group E is statistically different (P less than .01) from groups EA, C, and SA1. Group C was different (P less than .01) from groups E, CS, and N. These results demonstrate an inverse relationship between tumor recurrence and local thermal effects at the surgical site. The ABC did not increase tumor recurrence. Contact YAG surgery was similar to CO2 laser excision and "sterilization." An attempt to study the influence of gas flow and pressure on local tumor recurrence and metastases should be made.     

Asthma deaths during sports: report of a 7-year experience

BACKGROUND: Asthma mortality and the mortality of athletes during sports have been described separately in detail in the medical literature. However, asthma has not been reported as a cause of death in competitive athletes. OBJECTIVE: The object of this study was to raise the awareness of physicians, coaches, trainers, and parents that children and adults can have fatal asthma exacerbations during and immediately after participating in sports. METHODS: The Temple Sports Asthma Research Center identified athletes from 1993 until 2000 who died during or after sporting activity by using the nationwide Burrell's Information Service. Once a possible asthma-related sports death was identified, the autopsy report was requested from the coroner or medical examiner, and an attempt was made to contact the family. Contact with the family was limited to information about the death, medical history, sports involvement, and any medication usage by the person who had died. Secondary sources, including news reports, were used to confirm whether the subject died of asthma during or immediately after a sporting activity. RESULTS: Two hundred sixty-three possible cases were identified. Sixty-one deaths met the criteria for study inclusion. White deaths outnumbered black deaths by 2 to 1. Deaths among male subjects predominated. Most subjects were younger than the age of 20 years, with the most prevalent age group being between 10 to 14 years old. Fifty-one percent (18 of 35) of the competitive athletes had their fatal event while participating in organized sport, 14 in a practice situation and 4 deaths during a game or meet setting. Basketball and track were the 2 most frequent activities performed at the time of the fatal event. CONCLUSION: The subjects who had fatal asthma exacerbations were usually white male subjects between the ages of 10 and 20 years. Mild intermittent or persistent asthma by history was commonly identified. Sudden fatal asthma exacerbations occur in both competitive and recreational athletes and can be precipitated by sporting activity.     

Inhibition of chemotaxis of neutrophil leukocytes to interleukin-8 by endotoxins of various bacteria.

The effects of endotoxins from various bacteria (Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, Shigella flexneri, Salmonella typhosa, and Pseudomonas aeruginosa) on chemotaxis of neutrophil leukocytes to formyl peptide and interleukin-8 were tested in an improved chemotaxis assay involving a "sparse-pore" polycarbonate (Nuclepore) membrane in a Boyden-type chamber. The possible chemotactic activity of the endotoxins themselves were tested by the same technique. In addition, the effects of these substances on random motility of neutrophils were tested with a corresponding assay involving similar chambers fitted with membranes of standard pore density. Possible activation of the complement system of serum by each endotoxin was tested with sheep erythrocyte assays and the maximum endotoxin concentration (100 micrograms/ml) used in the chemotaxis and motility assays. All endotoxins inhibited chemotaxis of neutrophils to interleukin-8. No endotoxin affected chemotaxis to formyl peptide or was itself chemotactic for neutrophils. Endotoxin of S. flexneri inhibited random motility of neutrophils, while the others had no such effect. Endotoxins of K. pneumoniae and of P. aeruginosa produced moderate and marked inhibition, respectively, of total complement, as measured by hemolysis of sheep erythrocytes, without affecting the levels of C3c and C4 in these assays. Endotoxins of the other bacteria had no demonstrable effect in any of these assays of complement activation. These results suggest that chemotaxis to interleukin-8 may be mediated by cellular mechanisms different from those involved in chemotaxis to formyl peptide. Furthermore, the presence of these endotoxins could be significant for the suppression of neutrophil accumulation in inflammatory lesions mediated by interleukin-8.     

Plasmodium falciparum sexual stage antigens: immunogenicity and cell-mediated responses.

Antibody and cell-mediated immune responses to the transmission-blocking target antigens of Plasmodium falciparum, Pfs 48/45, were determined in infected non-immune patients and in immune individuals from an endemic area. Characterization of the B cell epitopes with monoclonal antibodies showed that there were five regions identifiable but there could be interactions between them causing either competitive or enhancing effects. Sera from infected non-immune patients contained antibodies that would compete with one or more of the mAbs to the different epitopes. Immune responsiveness to purified Pfs 48/45 in P. falciparum-immune adults measured as lymphoproliferation, production of interferon-gamma, or as Pfs 48/45-specific antibody was very limited. This did not appear to be due to MHC class II restriction, to diversity in structure of the parasite antigens or to a failure of immunological memory. The antibody-response data were more consistent with down-regulation of immunity as a result of prolonged exposure to infection.     

Reactivity of microhemagglutination, fluorescent treponemal antibody absorption, Venereal Disease Research Laboratory, and rapid plasma reagin tests in primary syphilis.

Seroreactivity of sera from 109 patients with first-infection primary syphilis was 98.2% in the fluorescent treponemal antibody absorption test, 92.7% in the rapid plasma reagin 18-mm circle card test, 72.5% in the microhemagglutination test (MHA-TP), and 72.5% in the Venereal Disease Research Laboratory test. Seroreactivity of sera from 18 patients with primary syphilis with documented previous infection(s) was 100% in the fluorescent treponemal antibody absorption test, the rapid plasma reagin 18-mm circle card test, and the MHA-TP test and 88.9% in the Venereal Disease Research Laboratory test. The MHA-TP test failed to confirm reactivity in 13 of 79 sera which were reactive in the Venereal Disease Research Laboratory test and in 24 of 101 sera which were reactive in the rapid plasma reagin 18-mm circle card test. Testing another production lot of MHA-TP reagents resulted in even poorer correlation. The reactivity of the MHA-TP test in primary syphilis appeared to vary with the sensitivity of the production lot of reagents.     

H-2b bound to egg lecithin liposomes: biochemical and functional properties.

Purified H-2b and H-2a molecules were bound to egg lecithin liposomes by a detergent dialysis procedure. Analysis of the liposomes indicated that only 30-50% of bound H-2b is oriented with the hydrophilic, antigenic portion of the molecule toward the outside of the liposome. Saturation of the liposomes occurred at a ratio of 64 molecules of egg lecithin per molecule of H-2b. Liposomes containing H-2 molecules were capable of stimulating spleen cells from primed donors to produce specific, alloreactive, cytotoxic T lymphocytes in vitro. Stimulation was dependent on adherent cells present in the responder spleen cells. Optimal stimulation occurred with highly saturated liposomes and at a ratio of 4-8 micrograms of H-2b per 8 X 10(6) responder cells.     

The right to an heir in the era of assisted reproduction

The latest remarkable technological advances in assisted reproduction, which enable cryopreservation of spermatozoa, embryos and ovarian tissue, raise difficult and debatable legal, social, ethical and moral issues concerning the right to posthumous reproduction. Furthermore, reports on the attitudes of the general public and of centres licensed for infertility treatment in the United Kingdom found that the majority of women and centres support the idea of posthumous reproduction. In this paper we review the data published on this issue, and after considering the various aspects, we conclude that each case should be discussed and authorized by a multidisciplinary committee that includes physicians, clergy, psychiatrists, psychologists, sociologists and other appropriate parties. In our opinion, the main principles that should guide this committee would allow posthumous reproduction in the context of marriage when a prior consent exists. For unmarried persons, post-mortem donation of gametes should be done only anonymously, if they are in agreement with existing laws concerning infertility treatments in every country and after appropriate consent and proper counselling. Moreover, any case which involves consanguinity or a possibility of incest should be forbidden, both for ethical and genetic reasons. In a case of pre-existing siblings, they should be consulted and their informed consent should be granted in advance so as to avoid legal problems in the inheritance of property.      

IgD production and other lymphocyte functions in HIV infection: immaturity and activation of B cells at different clinical stages.

T and B cell function, in particular IgD production in vitro, were studied across the spectrum of HIV infection in homosexual men and compared with seronegative homosexual and heterosexual male controls. Proliferation to phytohaemagglutinin (PHA) was reduced most strikingly in symptomatic HIV infection; it was also impaired in HIV seronegative homosexual men and there was no difference between these and asymptomatic HIV seropositives or those with persistent generalized lymphadenopathy (PGL). Spontaneous IgG and IgM production were increased in patients with PGL and Kaposi's sarcoma; pokeweed mitogen (PWM)-induced production of IgG and IgM was reduced in all HIV infected subjects. Spontaneous production of IgD was highest in asymptomatic HIV infection, with raised values also seen in PGL and AIDS with opportunist infection; IgD production was suppressed by PWM in the same groups. These data indicate an increase in circulating immature B cells. Markers of B cell immaturity and polyclonal activation are apparent to differing degrees at different stages of HIV infection.