First non-alpha-amino acid guanidines acting as efficient NO precursors upon oxidation by NO-synthase II or activated mouse macrophages

A study of the oxidation of a series of guanidines related to L-arginine (L-Arg) and of various alkyl- and arylguanidines, by recombinant NO-synthase II (NOS II), led us to the discovery of the first non-alpha-amino acid guanidine substrate of NOS, acting as an efficient NO precursor. This compound, 3-(trifluoromethyl)propylguanidine, 4, led to a rate of NO formation (k(cat) = 220 +/- 50 min(-1)) only 2 times lower than that of L-Arg. Formation of 1 mol of NO upon NOS II-catalyzed oxidation of 4 occurred with consumption of 2.9 mol of NADPH, which corresponds to a 52% coupling between electron transfer and oxygenation of its guanidine function. Its oxidation by activated mouse macrophages in an L-Arg-free medium resulted in NO(2)(-) formation that was inhibited by classical NOS inhibitors with a rate only 2-3 times lower than that observed with L-Arg itself. These results open the way toward the research of selective, stable guanidine substrates of NOS that could be interesting, new NO donors after in situ oxidation by a given NOS isoform.     

A novel improved method for Aspergillus nidulans transformation

We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods.     

Seasonal variation in whole blood cytokine production after LPS stimulation in normal individuals

We examined seasonal differences in whole blood cytokine production after endotoxin (LPS) stimulation in 17 healthy individuals from an urban area having normal sleep/wakefulness pattern. We used 500 pg/ml of LPS for incubation period of 4 h to stimulate 100 microl of whole blood of the same subjects in June, September, February, and March. We found no differences in the circulating total WBCs and differentials including monocytes between different seasons. We found during September (autumn) a reduced pro-inflammatory cytokine production in terms of TNF-alpha and IL-6 production compared to the other seasons. We also found a reduced anti-inflammatory cytokine production in June (summer) and September (autumn) in terms of IL-10, TNF-RI and TNF-RII compared to February (winter) and March (spring). Our results suggest that in early summer there is a predominating pro-inflammatory cytokine response which is counterbalanced early in autumn. These results may have significant implications in the determination of reference values, in exploration of immune response and inflammatory disease prevalence between different seasons, in determining LPS tolerance (immunoparalysis) and planning clinical trials and immunomodulary therapies. However, the effect of dark/light exposure differences on the circadian periodicity in the responsiveness of immune cells during different seasons should be further investigated.     

Carriage of Neisseria meningitidis and Neisseria lactamica in northern Greece

In response to an increase in the number of cases of invasive meningococcal disease (IMD) in northern regions of Greece, a survey was carried out to determine if there was an increase in carriage of Neisseria meningitidis, particularly in areas where there have been increases in immigrant populations from neighbouring countries. The second objective was to determine if there was an increase in the serogroup C:2a:P1.5,2 a phenotype associated with recent outbreaks or changes in antibiotic sensitivities. As carriage of Neisseria lactamica is associated with development of natural immunity to IMD, the third objective was to determine the carriage rate of N. lactamica in this population. Among 3167 individuals tested, meningococci were isolated from 334 (10.5%). Compared with our previous studies, the proportion of meningococcal carriers was significantly increased among children in secondary education (11.3%) (chi2=9.67, P<0.005) and military recruits (37.4%) (chi2=21.11, P<0.000). Only 5/334 (1.5%) isolates expressed the phenotype associated with the increase in IMD in Greece. N. lactamica was isolated from 146/3167 (4.6%) participants. It was isolated from 71/987 (7.2%) children attending primary or nursery schools; however, the highest proportion of carriers (11.3%) was found in the boarding school for young Albanian men. In the 21-59-year age range, the majority of N. lactamica isolates (22/25, 88%) were from women, probably due to closer or more prolonged contact with children in the primary school age range. Smoking was significantly associated with isolation of meningococci from men but not from women. Penicillin-insensitive strains (25/334, 7.5%) were identified in all four regions examined; the majority (14/25, 56%) were obtained from military personnel. We conclude that there was a higher proportion of carriers in the population of northern Greece; however, the increase in carriage rate was not associated with the influx of immigrants from neighbouring countries, and there was not a higher incidence of the C:2a:P1.5,2 strain responsible for increased disease activity in Greece in either the immigrant or local populations.     

CFTR gene mutations – including three novel nucleotide substitutions – and haplotype background in patients with asthma, disseminated bronchiectasis and chronic obstructive pulmonary disease

In order to investigate the incidence of cystic fibrosis transmembrane conductance regulator (CFTR) gene mutations and unclassified variants in chronic pulmonary disease in children and adults, we studied 20 patients with asthma, 19 with disseminated bronchiectasis (DB) of unknown aetiology, and 12 patients with chronic obstructive pulmonary disease (COPD), and compared the results to 52 subjects from the general Greek population. Analysis of the whole coding region of the CFTR gene and its flanking intronic regions revealed that the proportion of CFTR mutations was 45% in asthma (P<0.05), 26.3% in DB (P>0.05), 16.7% in COPD (P>0.05), compared to 15.4% in the general population. Seventeen different molecular defects involved in disease predisposition were identified in 16 patients. Three potentially disease-causing mutations, T388 M, M1R and V11I, are novel, found so far only in three asthma patients. The hyperactive M470 allele was found more frequently in COPD patients (frequency 70.8%, P<0.01) than in the controls. The study of the TGmTnM470 V polyvariant CFTR allele revealed the presence of CFTR function-modulating haplotypes TG13/T5/M470, TG11/T5/M470, TG12/T5/V470 and TG12/T7, combined with M470 or V470, in six asthma patients, four DB patients (P<0.01), and two COPD patients (P<0.05). These results confirm the involvement of the CFTR gene in asthma, DB and possibly in COPD.     

Correlation of the Insecticidal Activity of the Bacillus thuringiensis A4 Strain Against Bactrocera oleae (Diptera) with the 140-kDa Crystal Polypeptide

The crystals of the soil-isolated Bacillus thuringiensis (Bt) strain A4 consist of two polypeptides with molecular mass of 140 kDa and 32 kDa that exhibit insecticidal activity against adult flies of Bactrocera oleae (Diptera). Plasmid curing applied to this strain resulted in the isolation of several subclones exhibiting alterations in their crystal polypeptides as well as two acrystalliferous subclones. The crystals of subclone 1.1 lacked the 32-kDa polypeptide and consisted uniquely of a 140-kDa polypeptide antigenically related to the parental 140-kDa crystal polypeptide. Additionally, the crystals of this subclone exhibited insecticidal activity against B. oleae equivalent to that of the parental strain. Therefore, the 32-kDa crystal polypeptide is dispensable for insecticidal activity, which appears to be dependent on the presence of the 140-kDa crystal polypeptide. Received: 5 April 2000 / Accepted 2 May 2000     

Migration Behavior of Rodent Granule Neurons in the Presence of Antibody to the 4C5 Antigen

Abstract: We have reported the production of monoclonal antibody 4C5, which recognizes a cell surface antigen, the 4C5 antigen, involved in granule cell migration processes. In the present study, we investigated in a more precise manner the role of the 4C5 antigen in the different types of granule cell migrations that take place during cerebellar development. When cerebellar explant cultures derived from 10-day-old rats were performed for 2 days in the presence of monoclonal antibody 4C5, vertical granule cell migration, occurring in the presence of glia, was not significantly inhibited. In contrast, when monoclonal antibody 4C5 was included in the medium of microexplant cultures derived from 4-day-old mice and maintained for 4 days in vitro, granule cell migrations that occurred both parallel and perpendicular to the neurite bundles that were free of glia were inhibited. Moreover, a stronger inhibitory effect of the antibody was observed on migration perpendicular to the neurite bundles compared with the parallel type of migration. Our results indicate that the 4C5 antigen differentially affects the different developmental stages and types of granule cell migration during rodent cerebellar development.     

Smc5/6 maintains stalled replication forks in a recombination‐competent conformation

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome mis‐segregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination‐dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome‐wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage‐induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination‐competent conformation primed for replication restart.