NMR structure and mutagenesis of the FADD (Mort1) death-effector domain

When activated, membrane-bound receptors for Fas and tumour-necrosis factor initiate programmed cell death by recruiting the death domain of the adaptor protein FADD to the membrane. FADD then activates caspase 8 (also known as FLICE or MACH) through an interaction between the death-effector domains of FADD and caspase 8. This ultimately leads to the apoptotic response. Death-effector domains and homologous protein modules known as caspase-recruitment domains have been found in several proteins and are important regulators of caspase (FLICE) activity and of apoptosis. Here we describe the solution structure of a soluble, biologically active mutant of the FADD death-effector domain. The structure consists of six antiparallel, amphipathic alpha-helices and resembles the overall fold of the death domains of Fas and p75. Despite this structural similarity, mutations that inhibit protein-protein interactions involving the Fas death domain have no effect when introduced into the FADD death-effector domain. Instead, a hydrophobic region of the FADD death-effector domain that is not present in the death domains is vital for binding to FLICE and for apoptotic activity.     

Phylogenetic relationship within Fusarium sambucinum Fuckel sensu lato, determined from ribosomal RNA sequences

The importance of abnormalities of function and epididymis structure in the etiology of male infertility is still not well understood. We studied 52 individuals distributed in five age groups: fetuses, children, adolescents, adults and the elderly. The region of the body of epididymis was obtained by autopsy and immediately fixed by immersion in a solution of 10% buffered formaldehyde, embedded in paraffin and histologically prepared. The samples were observed under an optic microscope. Test-points were counted in 12 random microscopic fields with the M42 test-system. The following stereological parameters were determined: ductal area, volumetric densities (Vv) of the duct, smooth muscle, connective tissue, epithelial duct and blood vessels. The main results distinguished by those whose averages were statistically significant (p < 0.05), showed that the ductal area is 9.7 times greater in the adolescent/adult/elderly group than the children's group. The Vv of the lumen of the epididymis duct occupies 11.7% of the epididymis body in the fetal period, 5.3% in the child and in individuals after puberty this figures reaches more than 15%. The Vv of smooth muscle occupies 28.3% of the body of the epididymis in the fetus and 35.9% in children, but after puberty this figures stays around 22%. The Vv of the connective tissue occupies 26% in prenatal life, 37% in children, and after puberty these figures range from 21 to 27.5%. Comparing the results of the adult group with that of the elderly group there is an increase in the volumetric density of the connective tissue by 18.1%. In conclusion, the epididymal duct area and the Vv of the ductal lumen, smooth muscle and connective tissue were significant comparing the different groups. However, the quantitative relative differences of the duct's epithelium and the blood vessels were not significant comparing these groups. The study of quantitative aspects of the normal human epididymis can increase our knowledge about male fertility.     

Activity of quinolones in the Ames Salmonella TA102 mutagenicity test and other bacterial genotoxicity assays

Eight quinolones were examined for their bacterial mutagenicity in the Ames Salmonella TA102 assay and for their effects in other bacterial genotoxicity assays. In the quantitative Ames plate incorporation assay, all eight quinolones induced His+ deletion reversion in Salmonella tester strain TA102, with maximum reversion observed at about two to eight times the MIC. The quinolones also induced the SOS response. At quinolone concentrations close to the MIC, SOS cell filamentation gene sulA was induced in sulA::lacZ fusion strain Escherichia coli PQ37. RecA-mediated cleavage of lambda repressor in lambda::lacZ fusion strain E. coli BR513 was measurable at about 10 times the MIC, though no induction occurred at 20 micrograms of nalidixic or oxolinic acid per ml. Genotoxicity of quinolones also was observed in the Bacillus subtilis DNA repair assay, in which the mutant strain M45 (recA) was more susceptible to quinolones than its parent strain, H17 (rec+). The results from these analyses indicate that quinolones induce SOS functions and are mutagenic in bacteria; these properties correspond to their antimicrobial activities.     

Apoptosis as a cause of death in measles virus-infected cells

To determine the mechanism of measles virus-induced cell death, we studied the infection of Vero cells and monocytic cell lines with wild-type (Chicago-1) and vaccine (Edmonston) strains of measles virus. DNA fragmentation indicative of apoptosis was apparent by flow cytometry, agarose gel electrophoresis, and electron microscopy. Within syncytia, DNA strand breaks were demonstrated by end labeling with terminal transferase and then by visualization.     

Shear and extensional flow of reinforced plastics in injection molding. I. Effects of temperature and shear rate with bulk molding compound

Capillary flow studies on bulk molding compound (BMC) using an instrumented injection-molding machine are reported. The significance of extensional flow effects with fiber-reinforced materials is emphasized. The extensional flow behavior in converging dies is modeled, and a means of evaluating both extensional and shear viscosity from capillary flow data is proposed. Methods of correcting results for the effect of deformation heating are discussed. The shear and extensional flow behavior of BMC in the temperature region 18 to 58°C can be fitted to a simplified Arrhenius Law.     

Cellular mechanisms underlying carbachol-induced oscillations of calcium-dependent membrane current in smooth muscle cells from mouse anococcygeus

1. At a holding potential of -40 mV, carbachol (50 microM) produced a complex pattern of inward currents in single smooth muscle cells freshly isolated from the mouse anococcygeus. Membrane currents were monitored by the whole-cell configuration of the patch-clamp technique. Previous work has identified the first, transient component as a calcium-activated chloride current (ICl(Ca)) and the second sustained component as a store depletion-operated non-selective cation current (I(DOC)). The object of the present study was to examine the cellular mechanisms underlying the third component, a series of inward current oscillations (I(oscil)) superimposed on I(DOC). 2. Carbachol-induced I(oscil) (amplitude 97 +/- 11 pA; frequency 0.26 +/- 0.02 Hz) was inhibited by the chloride channel blocker anthracene-9-carboxylic acid (A-9-C; 1 mM), and by inclusion of 1 mM EGTA in the patch-pipette filling solution. 3. In calcium-free extracellular medium (plus 1 mM EGTA), carbachol produced an initial burst of oscillatory current which lasted 94 s before decaying to zero; I(oscil) could be restored by re-admission of calcium. The frequency, but not the amplitude, of I(oscil) increased with increasing concentrations of extracellular calcium (0.5-10 mM). 4. Inclusion of the inositol triphosphate (IP3) receptor antagonist heparin (5 mg ml(-1) in the patch-pipette filling solution, or pretreatment of cells with the sarcoplasmic reticulum (SR) calcium ATPase inhibitor cyclopiazonic acid (CPA; 10 microM), prevented the activation of I(oscil) by carbachol. Caffeine (10 mM) activated both ICl(Ca) and I(DOC) and prevented the induction of I(oscil) by carbachol. Caffeine and CPA also abolished I(oscil) in the presence of carbachol, as did both a low (3 microM) and a high (30 microM) concentration of ryanodine. 5. Carbachol-induced I(oscil) was abolished by the general calcium entry blocker SKF 96365 (10 MM) and by Cd2+ (100 microM), but was unaffected by La3+ (400 microM). As found previously, I(DOC) was also blocked by SKF 96365 and Cd2+, but not La3+; the inhibition of I(DOC) preceded the abolition of I(oscil) by 27 s with SKF 96365 and by 30 s with Cd2+. Nifedipine (1 microM) produced a partial inhibition of the carbachol-induced I(oscil) frequency at holding potentials of -20 mV and -60 mV and, in addition, reduced I(DOC) at -60 mV by 18%. 6. It is concluded that carbachol-induced inward current oscillations in mouse anococcygeus cells are due to a calcium-activated chloride current, and reflect oscillatory changes in cytoplasmic calcium ion concentration. These calcium oscillations are derived primarily from the SR stores, but entry of calcium into the cell is necessary for store replenishment and maintenance of the oscillations. Capacitative calcium entry (via I(DOC) appears to be important not only for sustained contraction of this tissue, but also as a route for re-filling of the SR and, therefore, represents an important target for the development of novel and selective drugs.     

Characterization of mutated transforming growth factor-beta s which possess unique biological properties

Transforming growth factor-beta (TGF-beta) is a potent regulator of cell growth and differentiation. On the basis of the crystal structure of TGF-beta 2, we have designed and synthesized two mutant TGF-beta s, TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73). Although both of these molecules inhibited the growth of Mv1Lu mink lung epithelial cells and LS1034 colorectal cancer cells, which are affected equally by TGF-beta 1 and TGF-beta 2, TGF-beta 1 (delta 69-73) was much less potent than TGF-beta 1 or TGF-beta 1 (71 Trp) at inhibiting the growth of LS513 colorectal cancer cells which are growth-inhibited by TGF-beta 1 but not TGF-beta 2. Both TGF-beta 1 (71 Trp) and TGF-beta 1 (delta 69-73) increased levels of mRNAs for fibronectin and plasminogen activator inhibitor with Mv1Lu cells, whereas only TGF-beta 1 (71 Trp) and not TGF-beta 1 (delta 69-73) up-regulated the mRNA level of carcinoembryonic antigen in LS513 cells. The expression level of carcinoembryonic antigen mRNA in LS1034 cells was not altered by either wild-type or mutant TGF-beta s. Receptor labeling experiments demonstrated that TGF-beta 1 (71 Trp) bound with high affinity to the cell-surface receptors of Mv1Lu, LS1034, and LS513 cells while TGF-beta 1 (delta 69-73) bound effectively to the receptors of Mv1Lu and LS1034 cells but much less to the receptors on LS513 cells.(ABSTRACT TRUNCATED AT 250 WORDS)