Hematopoietic malignancies demonstrate loss-of-function mutations of BAX

The BCL-2 gene family regulates the susceptibility to apoptotic cell death in many cell types during embryonic development and normal tissue homeostasis. Deregulated expression of anti-apoptotic BCL-2 can be a primary aberration that promotes malignancy and also confers resistance to chemotherapeutic agents. Recently, studies of Bax-deficient mice have indicated that the pro-apoptotic BAX molecule can function as a tumor suppressor. Consequently, we examined human hematopoietic malignancies and found that approximately 21% of lines possessed mutations in BAX, perhaps most commonly in the acute lymphoblastic leukemia subset. Approximately half were nucleotide insertions or deletions within a deoxyguanosine (G8) tract, resulting in a proximal frame shift and loss of immunodetectable BAX protein. Other BAX mutants bore single amino acid substitutions within BH1 or BH3 domains, demonstrated altered patterns of protein dimerization, and had lost death-promoting activity. Thus, mutations in the pro-apoptotic molecule BAX that confer resistance to apoptosis are also found in malignancies.     

Ethical care at the end of life

In treating dying patients, who by virtue of their physical and emotional situation are frail and vulnerable, physicians must meet a high standard of professional, ethical care. Such a standard is based upon a philosophy of care that recognizes the patients' inherent worth as human beings and their uniqueness as individuals. The ethical and virtuous physician will practice in accordance with the principles of biomedical ethics that form the foundations of thought and treatment approaches in this area and will seek to do the best for the patient and the family. "Doing the best" includes respecting autonomy through gentle truth-telling, helping the patient and family to set treatment goals, and providing for symptom control, continuing attentive care and accompaniment throughout the course of the illness. Total care includes physical, emotional and spiritual aspects, is sensitive to cultural values and is best provided by an interdisciplinary team. Practices of symptom control in routine care and in crisis situations, as well as the cessation and non-initiation of treatment, will have as their goals the relief and comfort of the patient. The ethical physician will not act with the intention of bringing about the death of the patient, whether by ordering medication in excess of that required for symptom control, administering a lethal injection or any other means.     

Sites important for PLCbeta2 activation by the G protein betagamma subunit map to the sides of the beta propeller structure

The betagamma subunits of the heterotrimeric GTP-binding proteins (G proteins) that couple heptahelical, plasma membrane-bound receptors to intracellular effector enzymes or ion channels directly regulate several types of effectors, including phospholipase Cbeta and adenylyl cyclase. The beta subunit is made up of two structurally different regions: an N-terminal alpha helix followed by a toroidal structure made up of 7 blades, each of which is a twisted beta sheet composed of four anti-parallel beta strands (Wall, M. A., Coleman, D. E., Lee, E., I?iguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319). We have previously shown that sites for activation of PLCbeta2, PLCbeta3, and adenylyl cyclase II overlap on the "top" surface of the propeller, where Galpha also binds (Li, Y., Sternweis, P. M., Charnecki, S., Smith, T. F., Gilman, A. G., Neer, E. J., and Kozasa, T. (1998) J. Biol. Chem. 273, 16265-16272). The present study was undertaken to identify the regions on the side of the torus that might be important for effector interactions. We made mutations in each of the outer beta strands of the G protein beta1 propeller, as well as mutations in the loops that connect the outer strands to the adjacent beta strands. Our results suggest that activation of PLCbeta2 involves residues in the outer strands of blades 2, 6, and 7 of the propeller. We tested three of the mutations that most severely affected PLCbeta2 activity against two forms of adenylyl cyclase (ACI and ACII). Both inhibition of ACI and activation of ACII were unaffected by these mutations, suggesting that if ACI and ACII contact the outer strands, the sites of contact are different from those for PLCbeta2. We propose that distinct sets of contacts along the sides of the propeller will define the specificity of the interaction of betagamma with effectors.     

Hemophilia. A new approach to an old disease

The history of hemophilia diagnosis and therapy has been a turbulent one. We are coming full circle, back to the use of genetics as the main diagnostic tool for this disease. Therapeutically, the retroviruses that ravaged one generation of hemophiliac patients now may participate in the cure for the next generation. The hemophilia community hopes that the future of hemophilia care will follow a course guided by this modified quote from James Russell Lowell: "New times demand new measures, and men [and women]. As the world advances and in time outgrows the laws that in our fathers' [and mothers'] days were the best, doubtless after us some purer scheme will be shaped out by wiser man [and women] than we, made wiser by the steady growth of truth."     

Ultraviolet vision in a passeriform bird: from receptor spectral sensitivity to overall spectral sensitivity in Leiothrix lutea

A survey of recent results out of spectrophotometric, microspectrophotometric, and behavioral tests concerning the UV vision of the passeriform bird Leiothrix lutea is presented. In the spectrophotometric study it was shown that the ocular media of Leiothrix' eyes are highly transparent to the near UV with lambda T50 at 320 nm. The comparison of the microspectrophotometric and the behavioral data showed a good fit between the peaks of the four single cones' effective sensitivity spectra and the four peaks in the behavioral spectral sensitivity function. The relation further suggests that the behavioral function might be described as the "over-envelope" of the single cone sensitivities. Leiothrix lutea possesses a genuine UV cone type and reveals it's highest sensitivity in the behavioral test to UV light.     

Protein rotational dynamics investigated with a dual EPR/optical molecular probe. Spin-labeled eosin

An acyl spin-label derivative of 5-aminoeosin (5-SLE) was chemically synthesized and employed in studies of rotational dynamics of the free probe and of the probe when bound noncovalently to bovine serum albumin using the spectroscopic techniques of fluorescence anisotropy decay and electron paramagnetic resonance (EPR) and their long-lifetime counterparts phosphorescence anisotropy decay and saturation transfer EPR. Previous work (Beth, A. H., Cobb, C. E., and J. M. Beechem, 1992. Synthesis and characterization of a combined fluorescence, phosphorescence, and electron paramagnetic resonance probe. Society of Photo-Optical Instrumentation Engineers. Time-Resolved Laser Spectroscopy III. 504-512) has shown that the spin-label moiety only slightly altered the fluorescence and phosphorescence lifetimes and quantum yields of 5-SLE when compared with 5-SLE whose nitroxide had been reduced with ascorbate and with the diamagnetic homolog 5-acetyleosin. In the present work, we have utilized time-resolved fluorescence anisotropy decay and linear EPR spectroscopies to observe and quantitate the psec motions of 5-SLE in solution and the nsec motions of the 5-SLE-bovine serum albumin complex. Time-resolved phosphorescence anisotropy decay and saturation transfer EPR studies have been carried out to observe and quantitate the microseconds motions of the 5-SLE-albumin complex in glycerol/buffer solutions of varying viscosity. These latter studies have enabled a rigorous comparison of rotational correlation times obtained from these complementary techniques to be made with a single probe. The studies described demonstrate that it is possible to employ a single molecular probe to carry out the full range of fluorescence, phosphorescence, EPR, and saturation transfer EPR studies. It is anticipated that "dual" molecular probes of this general type will significantly enhance capabilities for extracting dynamics and structural information from macromolecules and their functional assemblies.     

Evaluation of sensitivity of 10 diagnostic assays for Chlamydia trachomatis by use of a simple laboratory procedure

AIMS: To determine the sensitivity of commercially available diagnostic assays for Chlamydia trachomatis using a simple method. METHODS: Nine commercial assays and an "in-house" polymerase chain reaction (PCR) were evaluated using serial dilutions of a laboratory grown H serovar--four of them using a laboratory grown E serovar. Seven of the assays were further tested using dilutions of several cervical samples known to contain chlamydiae. RESULTS: The most sensitive assays were the MicroTrak direct fluorescent antibody (DFA) test (Syva) and the PCR which detected C trachomatis at a 10(-8) dilution of the H serovar, while the two least sensitive, Clearview (Unipath) and TestPack (Abbott), were positive only at 10(-4) and 10(-3) dilutions, respectively. A range of enzyme immunoassays (EIAs) and a nucleic acid hybridisation test were of intermediate sensitivity. The results with serovar E were consistent with these. When clinical samples were examined, the DFA test detected C trachomatis in dilutions at least 10-fold greater than any other assay. CONCLUSIONS: The range of sensitivity of diagnostic assays determined by the laboratory dilution procedure is very wide. Sensitivity assessed in this way, however, reflects the ability of the assays to detect C trachomatis in large scale clinical trials. The dilution procedure, which is simple to undertake, could therefore be applied by any laboratory before a new diagnostic method is considered for routine use.     

Helicobacter pylori, acid, and omeprazole revisited: bacterial eradication and rebound hypersecretion

Mouse blastocysts were exposed for 24 h to various concentrations of recombinant mouse tumor necrosis factor alpha (TNFalpha) and observed for their capacity to implant in vitro on a fibronectin-coated substrate or to develop in vivo after their transfer into surrogate females. Compared with findings in control blastocysts, exposure to TNFalpha resulted in a significant reduction in the average number of cells in the inner cell mass (ICM) lineage. This effect was associated with a significant increase in the frequency of cells identified as engaged in apoptosis by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling technique. No difference was found in the incidence of nuclear fragmentation between control and TNFalpha-exposed blastocysts. When TNFalpha-pretreated blastocysts were allowed to implant in vitro, significantly fewer embryos were able to maintain a structured ICM cluster at the center of the trophectoderm outgrowth. Although no difference was found in the average surface area of the outgrowths, implants derived from TNFalpha-treated blastocysts contained significantly fewer nuclei than implants from control embryos. After transfer into recipient mice, TNFalpha-pretreated blastocysts implanted at about the same rate as control embryos, but a significantly higher rate of resorption was found among fetuses after exposure to the cytokine. In addition, the weight of the surviving fetuses was significantly lower than for control fetuses. These data indicate that the impact of TNFalpha on blastocysts is specifically aimed at the ICM lineage and that TNFalpha decreases the ability of embryos to differentiate into fetuses after implantation.