Binding of the complement inhibitor C4bp to serogroup B Neisseria meningitidis

Neisseria meningitidis (meningococcus) is an important cause of meningitis and sepsis. Currently, there is no effective vaccine against serogroup B meningococcal infection. Host defense against neisseriae requires the complement system (C) as indicated by the fact that individuals deficient in properdin or late C components (C6-9) have an increased susceptibility to recurrent neisserial infections. Because the classical pathway (CP) is required to initiate efficient complement activation on neisseriae, meningococci should be able to evade it to cause disease. To test this hypothesis, we studied the interactions of meningococci with the major CP inhibitor C4b-binding protein (C4bp). We tested C4bp binding to wild-type group B meningococcus strain (H44/76) and to 11 isogenic mutants thereof that differed in capsule expression, lipo-oligosaccharide sialylation, and/or expression of either porin (Por) A or PorB3. All strains expressing PorA bound radiolabeled C4bp, whereas the strains lacking PorA bound significantly less C4bp. Increased binding was observed under hypotonic conditions. Deleting PorB3 did not influence C4bp binding, but the presence of polysialic acid capsule reduced C4bp binding by 50%. Bound C4bp remained functionally active in that it promoted the inactivation of C4b by factor I. PorA-expressing strains were also more resistant to C lysis than PorA-negative strains in a serum bactericidal assay. Binding of C4bp thus helps Neisseria meningitidis to escape CP complement activation.     

The autophagy-associated Atg8 gene family operates both under favourable growth conditions and under starvation stresses in Arabidopsis plants

Arabidopsis plants possess a family of nine AtAtg8 gene homologues of the yeast autophagy-associated Apg8/Aut7 gene. To gain insight into how these genes function in plants, first, the expression patterns of five AtAtg8 homologues were analysed in young Arabidopsis plants grown under favourable growth conditions or following exposure to prolonged darkness or sugar starvation. Promoters, plus the entire coding regions (exons and introns) of the AtAtg8 genes, were fused to the beta-glucuronidase reporter gene and transformed into Arabidopsis plants. In all plants, grown under favourable growth conditions, beta-glucuronidase staining was much more significant in roots than in shoots. Different genes showed distinct spatial and temporal expression patterns in roots. In some transgenic plants, beta-glucuronidase staining in leaves was induced by prolonged darkness or sugar starvation. Next, Arabidopsis plants were transformed with chimeric gene-encoding Atg8f protein fused to N-terminal green fluorescent protein and C-terminal haemagglutinin epitope tags. Analysis of these plants showed that, under favourable growth conditions, the Atg8f protein is efficiently processed and is localized to autophagosome-resembling structures, both in the cytosol and in the central vacuole, in a similar manner to its processing and localization under starvation stresses. Moreover, treatment with a cocktail of proteasome inhibitors did not prevent the turnover of this protein, implying that its turnover takes place in the vacuoles, as occurs in yeasts. The results suggest that, in plants, the cellular processes involving the Atg8 genes function efficiently in young, non-senescing tissues, both under favourable growth conditions and under starvation stresses.     

Toward an artificial cell based on gene expression in vesicles

We present a new experimental approach to build an artificial cell using the translation machinery of a cell-free expression system as the hardware and a DNA synthetic genome as the software. This approach, inspired by the self-replicating automata of von Neumann, uses cytoplasmic extracts, encapsulated in phospholipid vesicles, to assemble custom-made genetic circuits to develop the functions of a minimal cell. Although this approach can find applications, especially in biotechnology, the primary goal is to understand how a DNA algorithm can be designed to build an operating system that has some of the properties of life. We provide insights on this cell-free approach as well as new results to transform step by step a long-lived vesicle bioreactor into an artificial cell. We show how the green fluorescent protein can be anchored to the membrane and we give indications of a possible insertion mechanism of integral membrane proteins. With vesicles composed of different phospholipids, the fusion protein alpha-hemolysin-eGFP can be expressed to reveal patterns on the membrane. The specific degradation complex ClpXP from E. coli is introduced to create a sink for the synthesized proteins. Perspectives and subsequent limitations of this approach are discussed.     

Seasonal changes in brain melatonin concentration in the three-spined stickleback (Gasterosteus aculeatus): towards an endocrine calendar

Pineal organ and its hormone melatonin (N-acetyl-5-methoxytryptamine) is likely involved in timing and synchronisation of many internal processes, such as reproduction, with annual changes in environmental cues, i.e., photoperiod and water temperature. The seasonal changes in melatonin profile in stickleback brains related to the following reproductive phases were examined, and the link between melatonin concentrations and the stages of spawning cycle was analysed. Two wild populations of sticklebacks were exposed to annual environmental changes in their natural habitats. Brains, gonads, kidneys and livers were collected over 2 years. Melatonin was measured using RIA and the indices, gonadosomatic (GSI), nephrosomatic (NSI) and hepatosomatic (HSI), were calculated. The role of melatonin, as a component of internal calendar engaged in the control of seasonal breeding in this species, is discussed. The extremely high melatonin levels observed in early spring (March) and autumn (October) seem to mark out a time frame for spawning in sticklebacks. The seasonal pattern of melatonin production and identified development stages of gonads suggests the potential inhibitory effect of the hormone on stickleback reproduction in shortening photoperiod and stimulatory effect in lengthening photoperiod.     

Extruding foams from corn starch acetate and native corn starch

Because of the hydrophilic characteristics of native starch foams and the cost of modifying starch, the uses of starch and modified starch foams are hindered. To decrease hydrophilicity and cost of starch foams, native corn starch was blended with starch acetate and extruded. A twin-screw mixing extruder was used to produce the foams. Native starch content, screw speed, and barrel temperature had significant effects on molecular degradation of starches during extrusion. The melting temperature of extruded starch acetate/native starch foam was higher (216 degrees C) than that for starch acetate (193.4 degrees C). Strong peaks in the X-ray diffractograms of extruded starch acetate/native starch foam suggested new crystalline regions were formed. Optimum conditions for high radial expansion ratio, high compressibility, low specific mechanical energy requirement, and low water absorption index were 46.0% native starch content, 163 rpm screw speed, and 148 degrees C barrel temperature.     

Susceptibility to antimicrobial drugs of enterotoxin producing strains of Bacteroides fragilis isolated from different countries

Twenty two Bacteroides fragilis strains isolated from clinical samples in different countries (England, France, the Netherlands, Poland and USA) were used in the experiments. In all strains the presence of enterotoxin (fragilysin) gene was found by PCR with primers 404/407. Drug susceptibility of B. fragilis strains was determined with Etest (MICs for penicillin G, ceftriaxone, amoxicillin/clavulanic acid, imipenem, clindamycin and metronidazole). MICs were estimated in accordance to the NCCLS recommendations (1997). All tested strains were susceptible to imipenem and metronidazole. Twenty one strains were susceptible and one was intermediate susceptible to amoxicillin/clavulanic acid. Fourteen strains were resistant to ceftriaxone and five were found highly resistant to clindamycin. All examined strains were resistant to penicillin G. Four tested strains were simultaneously resistant to penicillin G, ceftriaxone and clindamycin (three French human strains isolated from postoperative wound, peritoneal fluid and bone inflammation, and one strain isolated from a pig).     

Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric role of NADPH-cytochrome P450 reductase in catalytic regioselectivity

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.     

Properties and modulation of alpha human atrial natriuretic peptide (alpha-hANP)-formed ion channels

Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.     

Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity

Cytochrome P450 (P450) 2D6 is a polymorphic human enzyme involved in the oxidation of >50 drugs, most of which contain a basic nitrogen. In confirmation of previous work by others, substitutions at Asp301 decreased rates of substrate oxidation by P450 2D6. An anionic residue (Asp, Glu) at this position was found to be important in proper protein folding and heme incorporation, and positively charged residues were particularly disruptive in bacterial and also in baculovirus expression systems. Truncation of 20 N-terminal amino acids had no significant effect on catalytic activity except to attenuate P450 2D6 interaction with membranes and NADPH-P450 reductase. The truncation of the N-terminus increased the level of bacterial expression of wild-type P450 2D6 (Asp301) but markedly reduced expression of all codon 301 mutants, including Glu301. Reduction of ferric P450 2D6 by NADPH-P450 reductase was enhanced in the presence of the prototypic substrate bufuralol. Bacterial flavodoxin, an NADPH-P450 reductase homolog, binds tightly to P450 2D6 but is inefficient in electron transfer to the heme. These results collectively indicate that the acidic residue at position 301 in P450 2D6 has a structural role in addition to any in substrate binding and that the N-terminus of P450 2D6 is relatively unimportant to catalytic activity beyond a role in facilitating binding to NADPH-P450 reductase.     

Evaluation of the needs of male carriers of mutations in BRCA1 or BRCA2 who have undergone genetic counseling

To date, the concerns of men at risk of inheriting a BRCA1 mutation or a BRCA2 mutation have received little attention. It had been anticipated that few men would be interested in predictive testing when a BRCA mutation was identified in their family. However, these men are often affected emotionally by diagnoses of breast cancer in their relatives and may themselves harbor fears that cancer will develop. Male carriers of BRCA1/2 mutations are at increased risk of development of cancers of several types, including those of the breast and prostate. We conducted an evaluation of the needs and experiences of 59 male carriers of BRCA1/2 mutations followed at either the University of Toronto or Creighton University. We assessed their motivations for seeking genetic counseling and testing, involvement in family discussions of breast and ovarian cancer, risk perception, changes in cancer-screening practices, and overall satisfaction with the genetic-counseling process. The principal motivation for seeking genetic counseling was concern for their daughters. The majority (88%) of men participated in family conversations about breast and ovarian cancer, and 47% participated in conversations about prophylactic surgery. Most men believed that they were at increased risk of development of cancer (prostate, breast, colorectal, and skin cancers). However, fewer than one-half (43%) of the men with no previous diagnosis of cancer stated that their prostate cancer-surveillance practices had changed after they had received genetic test results. More than one-half (55%) had intrusive thoughts about their cancer risk. Although levels of satisfaction were high, practitioners should be aware of (a) potential pressures influencing men to request predictive testing, (b) the difficulties that men encounter in establishing surveillance regimens for breast and prostate cancer, and (c) the general lack of information about men's particular experiences in the medical community.