Anti- and pro-oxidant factors and endothelial dysfunction in chronic cigarette smokers with coronary heart disease.

BACKGROUND: Endothelial dysfunction in cigarette smokers has been ascribed to increased oxidative damage. The aims of the present study were to compare the endothelial function of normotensive smokers with that of non-smokers and to examine its relation to some parameters representative of oxidative damage and of antioxidant capacity. METHODS: We investigated 32 chronic smokers (15-30 cigarettes daily) affected by coronary heart disease, ranging from acute myocardial infarction to instable angina pectoris, and 28 matched non-smokers without any definite risk factors. All subjects underwent assessment of nitric oxide (NO)-dependent endothelial function, measured as brachial artery vasodilatation in response to reactive ischemia, using a standardized echographic method. Plasma and urinary levels of NO were also measured in all subjects, as were urinary 15-isoprostane F(2t), plasma serum lipids, homocysteine (Hcy), ascorbic acid, retinol, tocopherol, and alpha- and beta-carotene (by high-performance liquid chromatography). RESULTS: Smokers showed a significantly lower NO-mediated vasodilatation response (3.50% vs. 6.18%, p<0.001) and higher levels of urinary NO metabolites and 15-isoprostane F(2t). They also had higher levels of Hcy (p<0.001); these values were significantly and inversely related to NO serum levels (r=-0.512, p<0.001). Moreover, smokers had a significant and corresponding reduction in circulating levels of ascorbic acid, tocopherol, and alpha- and beta-carotene. CONCLUSIONS: The present study shows a clear relation between endothelial dysfunction (NO production impairment) and cigarette smoking, especially in the presence of high levels of LDL-cholesterol. It also defines some markers of both oxidative damage and antioxidant protective capacity in this condition. The monitoring of these factors may be advisable in order to assess the amount of endothelial damage.

Thioredoxin-dependent regulation of photosynthetic glyceraldehyde-3-phosphate dehydrogenase: autonomous vs. CP12-dependent mechanisms.

Regulation of the Calvin-Benson cycle under varying light/dark conditions is a common property of oxygenic photosynthetic organisms and photosynthetic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is one of the targets of this complex regulatory system. In cyanobacteria and most algae, photosynthetic GAPDH is a homotetramer of GapA subunits which do not contain regulatory domains. In these organisms, dark-inhibition of the Calvin-Benson cycle involves the formation of a kinetically inhibited supramolecular complex between GAPDH, the regulatory peptide CP12 and phosphoribulokinase. Conditions prevailing in the dark, i.e. oxidation of thioredoxins and low NADP(H)/NAD(H) ratio promote aggregation. Although this regulatory system has been inherited in higher plants, these phototrophs contain in addition a second type of GAPDH subunits (GapB) resulting from the fusion of GapA with the C-terminal half of CP12. Heterotetrameric A(2)B(2)-GAPDH constitutes the major photosynthetic GAPDH isoform of higher plants chloroplasts and coexists with CP12 and A(4)-GAPDH. GapB subunits of A(2)B(2)-GAPDH have inherited from CP12 a regulatory domain (CTE for C-terminal extension) which makes the enzyme sensitive to thioredoxins and pyridine nucleotides, resembling the GAPDH/CP12/PRK system. The two systems are similar in other respects: oxidizing conditions and low NADP(H)/NAD(H) ratios promote aggregation of A(2)B(2)-GAPDH into strongly inactivated A(8)B(8)-GAPDH hexadecamers, and both CP12 and CTE specifically affect the NADPH-dependent activity of GAPDH. The alternative, lower activity with NADH is always unaffected. Based on the crystal structure of spinach A(4)-GAPDH and the analysis of site-specific mutants, a model of the autonomous (CP12-independent) regulatory mechanism of A(2)B(2)-GAPDH is proposed. Both CP12 and CTE seem to regulate different photosynthetic GAPDH isoforms according to a common and ancient molecular mechanism.

Pro-oxidant and antioxidant factors in acute intermittent porphyria: family studies.

Given the crucial role of iron and porphyrins in oxidative cellular damage in the chronic porphyrias, we undertook an extensive study in families with acute porphyrias to evaluate the possible role of similar oxidative damage in these diseases, whose natural history is often also complicated by neoplastic evolution. Four unrelated patients with acute intermittent porphyria (AIP) were studied together with 37 members of four different families. Aminolevulinic acid and porphobilinogen were measured in urine, and porphyrins in urine, plasma and stools. The activity of the congenitally deficient enzyme, porphobilinogen deaminase, and the concentrations of plasma iron, transferrin, ferritin, and various antioxidants (ascorbic acid, retinol, tocopherol, alpha- and beta-carotene, by a personal HPLC method) and the urinary and plasma metabolites of nitrous oxide were also assayed. The results showed no relationship between the observed increase of porphyrin metabolites and the presence of markers of oxidative damage or the decrease of circulating antioxidants: however, when such a decrease was registered, it depended on spontaneous or iatrogenic iron accumulation. We conclude that family screening, recommended for the identification of AIP carriers, must also include evaluation of iron stores with a view to preventing the oxidative damage and in order to forestall the neoplastic evolution of the disease.

A connection between stress and development in the multicellular prokaryote Streptomyces coelicolor A3(2).

Morphological changes leading to aerial mycelium formation and sporulation in the mycelial bacterium Streptomyces coelicolor rely on establishing distinct patterns of gene expression in separate regions of the colony. sigmaH was identified previously as one of three paralogous sigma factors associated with stress responses in S. coelicolor. Here, we show that sigH and the upstream gene prsH (encoding a putative antisigma factor of sigmaH) form an operon transcribed from two developmentally regulated promoters, sigHp1 and sigHp2. While sigHp1 activity is confined to the early phase of growth, transcription of sigHp2 is dramatically induced at the time of aerial hyphae formation. Localization of sigHp2 activity using a transcriptional fusion to the green fluorescent protein reporter gene (sigHp2-egfp) showed that sigHp2 transcription is spatially restricted to sporulating aerial hyphae in wild-type S. coelicolor. However, analysis of mutants unable to form aerial hyphae (bld mutants) showed that sigHp2 transcription and sigmaH protein levels are dramatically upregulated in a bldD mutant, and that the sigHp2-egfp fusion was expressed ectopically in the substrate mycelium in the bldD background. Finally, a protein possessing sigHp2 promoter-binding activity was purified to homogeneity from crude mycelial extracts of S. coelicolor and shown to be BldD. The BldD binding site in the sigHp2 promoter was defined by DNase I footprinting. These data show that expression of sigmaH is subject to temporal and spatial regulation during colony development, that this tissue-specific regulation is mediated directly by the developmental transcription factor BldD and suggest that stress and developmental programmes may be intimately connected in Streptomyces morphogenesis.

Feasibility of a cell kinetic-based adjuvant chemotherapy trial in axillary node-negative breast cancer.

AIMS AND BACKGROUND: Accumulated information on biologic prognostic indicators and predictors of response to different types of treatment in patients with different tumor characteristics has made it possible to design clinical protocols on biologic bases. Among cell proliferation indices, the thymidine labelling index (TLI) has proved to be an independent and consistent prognostic indicator over time. Moreover, experimental and retrospective analyses of clinical studies have revealed a direct relation between TLI and response to chemotherapy. On the basis of the results, a prospective clinical protocol on axillary node-negative breast cancer was activated in Italy in 1989. METHODS: Patients with low TLI tumors were treated with local-regional therapy alone, whereas patients with high TLI tumors were randomized to receive local-regional therapy followed or not by adjuvant chemotherapy consisting of 6 cycles of CMF. RESULTS AND CONCLUSIONS: The present paper reports on the feasibility of a prospective clinical protocol based on a subgroup of patients with specific pathologic (node negative) and biologic (rapidly proliferating) breast cancers. However, patient eligibility was only 11%.

A putative sigma factor from Streptomyces sp. strain A21 can activate the expression of the cryptic operon bgl in Escherichia coli K-12.

Streptomyces sp A21 is a cellulolytic strain isolated from soil which was assigned to the genus Streptomyces on the basis of distinctive morphological features. A genomic library of A21 DNA has been constructed and transformed into Escherichia coli K-12 using a high-copy-number vector. One of the recombinant plasmids activates the cryptic bgl operon when inserted into appropriate strains. The complete sequence of the 1629-bp A21 DNA fragment has been determined. The analysis revealed the presence of an ORF whose putative product shows a high degree of similarity to RNA polymerase sigma factors; we therefore designated the gene psfS (Putative sigma factor, Streptomyces). Mapping of the 5' terminus of transcript by primer extension indicated that PsfS induces transcription initiation within the bgl promoter-silencer region.

Presence of antibacterial peptides on the laid egg chorion of the medfly Ceratitis capitata.

Female reproductive accessory glands of the medfly Ceratitis capitata produce a secretion with antibacterial activity mainly ascribed to ceratotoxin peptides. To study whether the secretion from the accessory glands of the female protects the eggs and early larva from microbes, we examined whether ceratotoxins and other accessory gland components could be found on the egg surface. This was found to be the case; a water-soluble material with the same protein and antibacterial pattern as that of the accessory gland secretion was recovered from the laid egg surface and was observed as electrondense, clustered droplets over the outer exochorion. Such material showed the same electrophoretic pattern in both mated and virgin females. These findings indicate that the accessory gland secretion is spread, at oviposition, onto the eggs producing an antibacterial coating, irrespective of fertilization. This is the first report of antimicrobial components recovered from a material layered on insect laid eggs.

Restriction enzyme and DNA hybridization analysis of cellulolytic Streptomyces isolates of different origin.

Streptomyces rochei A2 endoglucanase (eglS) and beta-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgs1 probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis and genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.

Molecular characterization of ceratotoxin C, a novel antibacterial female-specific peptide of the ceratotoxin family from the medfly Ceratitis capitata.

Ceratotoxins A and B are antibacterial peptides produced by the sexually mature females of Ceratitis capitata. The gene expression is restricted to the female reproductive accessory glands, and is not affected by bacterial infection, but is enhanced by mating. We report here the purification and the amino acid sequence of ceratotoxin C, a novel member of the ceratotoxin family, the cloning of its cDNA and the analysis of its expression. Ceratotoxin C is coordinately expressed with the other members of the ceratotoxin family. Its antibacterial activity is directed against both Gram-negative and Gram-positive bacterial strains but it is lower than that of ceratotoxin A. We demonstrate in the genome of C. capitata the presence of at least three ceratotoxin genes which express, in the female accessory glands, a set of peptides presumably involved in the protection of the genital tract during fertilization.

The novel antibacterial peptide ceratotoxin A alters permeability of the inner and outer membrane of Escherichia coli K-12.

Ceratotoxins are antibacterial 3-kDa amphiphilic peptides isolated from the female reproductive apparatus of the medfly Ceratitis capitata. The antibacterial activity of a chemically synthesized ceratotoxin A (ctx A) has been investigated. Ctx A was mainly active against Gram-negative organisms, and it had a lytic effect on nongrowing Escherichia coli K-12. Data showed that ctx A alters both the outer and the inner membrane of E.coli K-12 cells.

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of beta-glucosidase activity in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli. Two clones containing a common fragment encoded a polypeptide of 58,000 Da. Cloned beta-glucosidase, expressed in E. coli, showed activity against natural beta-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids (M(r) 53,896) which showed significant homology with beta-glucosidases from glycosyl hydrolase family 1.

Acid maltase deficiency in childhood. Early diagnosis and clinical follow-up of late-onset glycogen storage disease type II.

A case is described of late-onset glycogenosis type II presenting with an isolated rise in serum transaminase levels. Histological, histochemical, ultrastructural and biochemical examinations performed on muscle biopsy showed the typical laboratory features of late-onset glycogenosis type II, which was diagnosed more than four years before the first appearance of disease-related signs and symptoms. A heterozygote status for the same defect was also demonstrated by enzyme assays in both parents, thus confirming the autosomal recessive mode of inheritance of the disorder. Even though an elevation in transaminases and other serum enzymes of possible muscle origin has been previously described as a diagnostic clue in some unsuspected muscular diseases in childhood, as far as we know no other patient with a sporadic form of glycogenosis type II has been identified when still completely asymptomatic. The possibility of silent primary metabolic diseases and myopathies should be carefully considered when evaluating children with persistently elevated serum transaminases, even in the absence of suggestive anamnestic, familial and physical findings, in order to obtain an early diagnosis and to provide an appropriate genetic counselling.

Chromosome Polymorphisms among Strains of Hansenula polymorpha (syn. Pichia angusta).

Contour-clamped homogeneous electrophoresis and an embedded-agarose method of sample preparation were combined to carry out an analysis of the chromosome sets of nine strains of Hansenula polymorpha (syn. Pichia angusta). Chromosomal DNA molecules could be separated into a series of bands ranging, approximately, from 650 up to 2,200 kb in size. Polymorphism of the electrophoretic pattern was demonstrated among the strains investigated in this study. Cross-hybridization between H. polymorpha and Saccharomyces cerevisiae ribosomal DNA was also observed.

Size of the Streptococcus mutans GS-5 chromosome as determined by pulsed-field gel electrophoresis.

Rare cutting restriction endonucleases were used to cut the Streptococcus mutans chromosome into large fragments. Restriction enzymes utilizing recognition sites containing 6-, 7-, or 8-base-pair sequences with only G and C nucleotides produced few fragments, most of which were greater than 100 kilobase pairs in size. Addition of the fragments from digests of SmaI, NotI, ApaI, RsrII, and EagI yielded a molecular size for the S. mutans GS-5 genome of 2,819 +/- 60 kilobase pairs.

Transposon-916-like elements in clinical isolates of Enterococcus faecium.

Tetracycline (Tc) resistance was found in nine out of ten clinical isolates of Enterococcus faecium. Conjugative transposons, designated Tn5031, Tn5032 and Tn5033, were present in the chromosome of three isolates. The transposons were similar both structurally and functionally to Tn916 containing the tetM determinant. A large non-conjugative plasmid found in a fourth isolate contained an element homologous to Tn916. The four isolates containing the element showing homology to Tn916 exhibited a substantially higher level of Tc resistance than the remaining five Tc-resistant isolates. Tc-resistance genes which have not been identified are apparently responsible for the low-level Tc resistance in five clinical isolates.

Tn916 insertional inactivation of multiple genes on the chromosome of Streptococcus mutans GS-5.

Streptococcus mutans GS-5 was transformed with the Escherichia coli plasmid pAM150 containing the cloned streptococcal transposon Tn916. Southern blot analyses with the tetracycline-resistant determinant of Tn916 showed that Tn916 was inserted into the chromosome of S. mutans at a variety of different sites. Tn916 insertions resulted in the inactivation of genes that code for various steps in the biosynthesis of several different amino acids. Two auxotrophs which contained a single copy of Tn916 were shown to revert to prototrophy at frequencies of about 10(-8). All of the revertant prototrophs were susceptible to tetracycline, indicating regeneration of the functional gene by excision of Tn916.

Transformation as a tool for studying the epidemiology of tet determinants in Streptococcus pneumoniae.

Transformation of pneumococcus was used to detect homology among tetracycline resistance determinants of clinical isolates of Streptococcus pneumoniae. A strain of pneumococcus containing a mutated tet determinant (tet-3), of class M, integrated into the chromosome was used as a recipient in transformation experiments, where donor DNA was from the tetracycline resistant isolates. 34/34 strains appeared to have tet determinants homologous to tet-3 (i.e. tet M). Still using transformation it was possible to determine that the tet-3 transforming activity of DNA from Tn916 and S. pneumoniae BM6001 was contained in a 5 kb HincII fragment. For this purpose a transformation technique where donor DNA was directly taken from low melting point agarose gels was standardized and used.