BID: a novel BH3 domain-only death agonist

The BCL-2 family of proteins consists of both antagonists (e.g., BCL-2) and agonists (e.g., BAX) that regulate apoptosis and compete through dimerization. The BH1 and BH2 domains of BCL-2 are required to heterodimerize with BAX and to repress cell death; conversely, the BH3 domain of BAX is required to heterodimerize with BCL-2 and to promote cell death. To extend this pathway, we used interactive cloning to identify Bid, which encodes a novel death agonist that heterodimerizes with either agonists (BAX) or antagonists (BCL-2). BID possesses only the BH3 domain, lacks a carboxy-terminal signal-anchor segment, and is found in both cytosolic and membrane locations. BID counters the protective effect of BCL-2. Moreover, expression of BID, without another death stimulus, induces ICE-like proteases and apoptosis. Mutagenesis revealed that an intact BH3 domain of BID was required to bind the BH1 domain of either BCL-2 or BAX. A BH3 mutant of BID that still heterodimerized with BCL-2 failed to promote apoptosis, dissociating these activities. In contrast, the only BID BH3 mutant that retained death promoting activity interacted with BAX, but not BCL-2. This BH3-only molecule supports BH3 as a death domain and favors a model in which BID represents a death ligand for the membrane-bound receptor BAX.     

Intensive postremission chemotherapy in adults with acute myeloid leukemia. Cancer and Leukemia Group B

BACKGROUND: About 65 percent of previously untreated adults with primary acute myeloid leukemia (AML) enter complete remission when treated with cytarabine and an anthracycline. However, such responses are rarely durable when conventional postremission therapy is administered. Uncontrolled trials have suggested that intensive postremission therapy may prolong these complete remissions. METHODS: We treated 1088 adults with newly diagnosed AML with three days of daunorubicin and seven days of cytarabine and randomly assigned patients who had a complete remission to receive four courses of cytarabine at one of three doses: 100 mg per square meter of body-surface area per day for five days by continuous infusion, 400 mg per square meter per day for five days by continuous infusion, or 3 g per square meter in a 3-hour infusion every 12 hours (twice daily) on days 1, 3, and 5. All patients then received four courses of monthly maintenance treatment. RESULTS: Of the 693 patients who had a complete remission, 596 were randomly assigned to receive postremission cytarabine. After a median follow-up of 52 months, the disease-free survival rates in the three treatment groups were significantly different (P = 0.003). Relative to the 100-mg group, the hazard ratios were 0.67 for the 3-g group (95 percent confidence interval, 0.53 to 0.86) and 0.75 for the 400-mg group (95 percent confidence interval, 0.60 to 0.94). The probability of remaining in continuous complete remission after four years for patients 60 years of age or younger was 24 percent in the 100-mg group, 29 percent in the 400-mg group, and 44 percent in the 3-g group (P = 0.002). In contrast, for patients older than 60, the probability of remaining disease-free after four years was 16 percent or less in each of the three postremission cytarabine groups. CONCLUSIONS: These data support the concept of a dose-response effect for cytarabine in patients with AML who are 60 years of age or younger. The results with the high-dose schedule in this age group are comparable to those reported in similar patients who have undergone allogeneic bone marrow transplantation during a first remission.     

Muscle mass and fat mass in relation to bone mineral density in very old men and women: the Framingham Heart Study

Aim of the study was investigate the cross-sectional relationship between body composition and bone mineral density (BMD) in very old men and women. The study sample consisted of 504 women and 285 men, aged 72-93 yr, participating in examination 22 (1992-1993) of the Framingham Heart Study. Total body BMD, regional BMD, and soft-tissue body composition was measured by dual-energy X-ray absorptiometry. Both muscle mass and percentage body fat were positively associated with total body BMD in women. After adjustment for age, physical activity, smoking status, estrogen use, and thiazide use, BMD increased with increasing tertile of muscle mass (p = 0.007) and with increasing tertile of percentage body fat (p = 0.0001) in women. In men muscle mass, not percentage body fat, was positively associated with BMD. After adjustment for potential confounders, BMD remained associated with muscle mass only (p = 0.02). These results were similar for leg BMD and arm BMD. The study suggests that the influence of muscle and fat mass on bone mineral density is different between very old men and women.     

Molecular and cell models of biological effects of heavy ion radiation

The nitric oxide (NO) production by porcine aortic valve endothelial cells was estimated in cusps incubated at 37 degrees C by measuring their cyclic GMP content and the nitrite levels of the incubation medium. After a stabilization period, incubation for 5 min with acetylcholine, bradykinin, ADP and bovine thrombin resulted in a receptor-mediated increase in cyclic GMP which could be blocked by EGTA, N-omega-nitro-L-arginine methyl ester (L-NAME) and NG-monomethyl-L-arginine (L-NMMA). Incubation with lipopolysaccharide (endotoxin) from E. coli O111:B4 or bovine for 5 h, dose-dependently increased nitrite production as well as cyclic GMP content. The elevated nitrite production was completely abolished in the presence of the protein synthesis inhibitor cycloheximide, was reduced by more than 50% by dexamethasone but was not affected by EGTA. L-NMMA dose-dependently reduced the increased nitrite production and cyclic GMP content. These results suggest that besides the presence of a constitutive NO synthase in porcine aortic valve endothelial cells thrombin, like lipopolysaccharide, triggers the de novo expression of an inducible Ca(2+)-independent NO synthase.     

Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.     

Expression of polysialylated neural cell adhesion molecule by proliferating cells in the subependymal layer of the adult rat, in its rostral extension and in the olfactory bulb

The highly sialylated isoform of the neural cell adhesion molecule is thought to be expressed predominantly in the developing nervous system, where it is implicated in a variety of dynamic events linked to neural morphogenesis. It has become increasingly evident, however, that this "embryonic" neural cell adhesion molecule isoform continues to be expressed in certain adult neuronal systems, and in particular, in those that can undergo structural plasticity. In the present study, we performed light microscopic immunocytochemistry with an antibody specific for polysialylated neural cell adhesion molecule and confirmed our earlier observations [Bonfanti L. et al. (1992) Neuroscience 49, 419-436] showing polysialylated neural cell adhesion molecule-immunoreactive cells in the subependymal layer of the lateral ventricle of the adult rat, a region where cell proliferation continues into the postnatal period. In addition, we used an antibody raised against the proliferating cell nuclear antigen and found that proliferating cells continue to be visible in this area, even in the adult. Double immunolabeling showed that many of these newly generated cells displayed high polysialylated neural cell adhesion molecule immunoreactivity. Cells from a portion of the subependymal layer migrate to the olfactory bulb and contribute to the continual replacement of its granule neurons [Luskin M. B. (1993) Neuron 11, 173-189]. We found polysialylated neural cell adhesion molecule-immunoreactive cells all along the pathway purported to be followed by the newly generated cells to their final destination and in neurons corresponding to granular and periglomerular cells in the olfactory bulb. Our present observations thus support the contention that polysialylation is a feature of neurons capable of dynamic change and may contribute to the molecular mechanisms permitting cell proliferation and migration not only during development but also in the adult.     

Iron(II)/reductant(DH2)-induced activation of dioxygen for the hydroxylation and ketonization of hydrocarbons; mimics for the cytochrome P-450 hydroxylase/reductase system

Several metal complexes [(FeII(DPAH)2 (DPAH2 = 2,6-dicarboxyl pyridine), FeII(PA)2 (PAH = picolinic acid), FeII(bpy)2(2+), FeII(OPPh3)4(2+), (Cl8TPP)FeIIIX (X = Cl, OH, SCH2Ph) [Cl8TPP = tetrakis (2,6-dichlorophenyl)porphyrin], (TPP) FeIIICl (TPP = tetraphenylporphyrin), and CuI(tpy)2+ (typ = 2,2'-6,2"-terpyridine)] in combination with one of several reductants [DH2; PhNHNHPh (mimic of dihydroflavin), PhNHNH2, ascorbic acid (H2asc), and PhCH2SH (model ligand for cysteine residue)] catalytically activate O2 (1 atm) for the hydroxylation of saturated hydrocarbons (e.g. c-C6H12-->c-C6H11OH). This chemistry closely parallels that of cytochrome P-450 proteins, and both appear to involve a Fenton-like reactive intermediate), [LxFeOOH(DH)]. With cyclohexane as the substrate the dominant product is its ketone (as well as significant amounts of its hydroperoxide). 1,4-Cyclohexadiene (with two double-allylic carbon centers) undergoes dehydrogenation to give benzene, but also yields substantial amounts of phenol via ketonization of an allylic carbon. The 1:1 FeII(bpy)2(2+)/(PhNHNH2 or H2asc), FeII(PA)2/H2asc, and (Cl8TPP)FeIIICl/PhNHNH2 combinations initiate the autoxidation of 1,4-cyclohexadiene with turnover numbers (moles of product per mole of reductant) from 71 to 26, respectively (alpha-tocophenol reduces the turnover numbers by 20-80%). With respect to aerobic biology, the present results indicate that dysfunctional transition metals (degradation products of metalloproteins) in combination with biological reductants activate O2 for reaction with organic substrates. The level of activation is similar to that for Fenton reagents and cytochrome P-450 hydroxylases. Hence, dysfunctional transition metals, reductants, and O2 are a hazardous combination within a biological matrix.     

The testing of bulls for the polledness gene]

Main regularities have been stated concerning the poll gene inheritance by the progeny of breeding bulls mated with cows of various (homozygous and heterozygous) genotypes by the "poll" or "hornedness" genes.     

The role of protein kinase in C ischemic/reperfused preconditioned isolated rat hearts

Protein kinase C (PKC) has been implicated in the preconditioning-induced cardiac protection in ischemic/reperfused myocardium. We studied the effect of PKC inhibition with calphostin C (25, 50, 100, 200, 400, and 800 nM), a potent and specific inhibitor of PKC, in isolated working nonpreconditioned and preconditioned ischemic/reperfused hearts. In the nonpreconditioned groups, all hearts underwent 30 min of normothermic global ischemia followed by 30 min of reperfusion. In the preconditioned groups, hearts were subjected to four cycles of ischemic preconditioning by using 5 min of ischemia followed by 10 min reperfusion, before the induction of 30 min ischemia and reperfusion. At low concentrations of calphostin C (25, 50, and 100 nM), the PKC inhibitor had no effect on the incidence or arrhythmias or postischemic cardiac function in the nonpreconditioned ischemic/reperfused groups. With 200 and 400 nM of calphostin C, a significant increase in postischemic function and a reduction in the incidence of arrhythmias were observed in the nonpreconditioned ischemic/reperfused groups. Increasing the concentration of calphostin C to 800 NM, the recovery of postischemic cardiac function was similar to that of the drug-free control group. In preconditioned hearts, lower concentrations (< 100 nM) of calphostin C did not change the response of the myocardium to ischemia and reperfusion in comparison to the preconditioned drug-free myocardium. Two hundred and 400 nM of the PKC inhibitor further reduced the incidence of ventricular fibrillation (VF) from the preconditioned drug-free value of 50% to 0 (p < 0.05) and 0 (p < 0.05), respectively, indicating that the combination of the two, preconditioning and calphostin C, affords significant additional protection. Increasing the concentration of calphostin C to 800 nM blocked the cardioprotective effect of preconditioning (100% incidence of VF). The recovery of cardiac function was similarly improved at calphostin C doses of 200 and 400 nM and was reduced at 800 nM (p < 0.05). With 200 and 400 nM of calphostin C, both cytosolic and particulate PKC activity were reduced by approximately 40 and 60%, respectively, in both preconditioned and preconditioned/ischemic/reperfused hearts. The highest concentration of calphostin C (800 nM) resulted in almost a complete inhibition of cytosolic (100%) and particulate (85%) PKC activity correlated with the abolition of preconditioning-induced cardiac protection. In conclusion, calphostin C protects the ischemic myocardium obtained from intact animals, provides significant additional protection to preconditioning at moderate doses, and blocks the protective effect of preconditioning at high concentrations. The dual effects of calphostin C appear to be strictly dose and "enzyme inhibition" related.