Study of erodable paint properties involved in antifouling activity.

To produce ecological marine paints, it is necessary to understand the phenomena involved in antifouling activity. Due to the multivariable components which have to be taken into account and due to their analytical intricacy, only studies based on selected properties are conceivable. In this study, four properties have been chosen, viz. erosion, biocide release, roughness and the physicochemical characteristics of the film surface. A principal-component analysis (PCA) of the experimental data has shown that, among the selected properties, only erosion affected antifouling efficiency. A more detailed investigation of erosion by quantifying global hydration and hydrolysis of immersed paints revealed the difficulty in linking the chemical structure of binders to the final erosion properties. Biocide release from paints, quantified by chromatographic methods coupled with UV detection, was inferior to the doses stated by the paint producers. These observations allowed the conceiving of formulations with reduced amounts of active molecules. The development of erodable, biodegradable binders associated with non toxic compounds is a promising way to obtain efficient antifouling paints compatible with existing, preventive systems.

HLA-DMA alleles are possible new markers of rheumatoid arthritis: study of a Corsican group.

The HLA-DMA gene, along with the HLA-DMB gene, encodes the not classical class II molecule. This molecule catalyzes the class-II-associated invariant-chain peptide (CLIP)-antigen peptide exchange in classical class II molecule peptide-binding groove. As such, the DM heterodimer is an antigen presentation regulator and may be linked to immune system deficiencies such as those observed in autoimmune diseases. The study of DMA gene polymorphism seems be a reasonable approach to provide an answer to this question. Thanks to PCR-derived methods, the relationship between DMA gene polymorphism and rheumatoid arthritis (RA) was demonstrated in the present study. The DMA*0101 allele was observed to confer a significant predisposition to RA while the DMA*0102 allele significantly protected from this disease. Polymorphism experiments with the HLA-DRB1 gene revealed that this relationship between DMA polymorphism and RA is not a consequence of a linkage disequilibrium with the HLA-DRB1 alleles implicated in this pathology. The study of the DMA gene could therefore prove to be very useful in the early diagnosis of RA.

Analysis of the polymorphism of the tumour necrosis factor (TNF) gene and promoter and of circulating TNF-alpha levels in heart-transplant patients suffering or not suffering from severe rejection.

Plasma TNF-alpha levels are generally higher in heart-graft patients who experience a rejection episode than in those who do not. Because the TNF gene and its promoter are polymorphic, we studied the relationships between genetic variability at the TNF locus, the occurrence of graft rejection and TNF-alpha plasma levels in 62 heart-transplant patients in order to investigate inter-individual differences in plasma TNF-alpha levels after allogeneic stimulation. TNF-alpha was immunoenzymatically measured in blood specimens collected on the same day as endomyocardial biopsy. After PCR amplification of DNA, NcoI and AspHI polymorphisms were characterized by their restriction profiles, TNFa microsatellites by electrophoretic separation on acrylamide and the promoter region by sequencing. Plasma levels and molecular genetic results were compared to the grade of heart graft rejection established according to pathological criteria. In our study, allograft rejection was associated neither with NcoI or AspHI polymorphism nor with nucleotide changes in the TNF-A promote. We observed low TNF-alpha levels in n1/n1 homozygous patients and in subjects with G-->A at position--308 of the promoter sequence. Concerning the polymorphism of the TNFa microsatellite, our results might suggest an association with graft rejection but we have to be very careful in drawing conclusions because of the small size of the sample.

Implication of HLA-DMA alleles in corsican IDDM.

The HLA-DM molecule catalyses the CLIP/antigen peptide exchange in the classical class II peptide-binding groove. As such, DM is an antigen presentation regulator and may be linked to autoimmune diseases. Using PCR derived methods, a relationship was revealed between DM gene polymorphism and IDDM, in a Corsican population. The DMA*0101 allele was observed to confer a significant predisposition to this autoimmune disease while the DMA*0102 allele protected significantly. Experiments examining polymorphism of the HLA-DRB1 gene established that these relationships are not a consequence of linkage disequilibrium with HLA-DRB1 alleles implicated in this pathology. The study of the DMA gene could therefore be an additional tool for early IDDM diagnosis in the Corsican population.

Characterization of the lacticin 481 operon: the Lactococcus lactis genes lctF, lctE, and lctG encode a putative ABC transporter involved in bacteriocin immunity.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis strains. The genetic determinants of lacticin 481 production are organized as an operon encoded by a 70-kb plasmid. We previously reported the first three genes of this operon, lctA, lctM, and lctT, which are involved in the bacteriocin biosynthesis and export (A. Rincé, A. Dufour, S. Le Pogam, D. Thuault, C. M. Bourgeois, and J.-P. Le Pennec, Appl. Environ. Microbiol. 60:1652-1657, 1994). The operon contains three additional open reading frames: lctF, lctE, and lctG. The hydrophobicity profiles and sequence similarities strongly suggest that the three gene products associate to form an ABC transporter. When the three genes were coexpressed into a lacticin 481-sensitive L. lactis strain, the strain became resistant to the bacteriocin. This protection could not be obtained when any of the three genes was deleted, confirming that lctF, lctE, and lctG are all necessary to provide immunity to lacticin 481. The quantification of the levels of immunity showed that lctF, lctE, and lctG could account for at least 6% and up to 100% of the immunity of the wild-type lacticin 481 producer strain, depending on the gene expression regulation. The lacticin 481 biosynthesis and immunity systems are discussed and compared to other lantibiotic systems.

[HLA-DQ genotyping using a modified technique of PCR-RFLP: application to HLA-DQA1 gene]. / Génotypage HLA-DQ par une technique modifiée de RFLP-PCR: application au gène HLA-DQA1.

Since 1989, several HLA-DQA1 PCR-RFLP genotyping protocols have been published. These methods require complete digestion of the PCR products and determination of the restriction fragments length. The HLA-DQA1 PCR-RFLP genotyping protocol describe here uses one amplification step through PCR, digestion of the PCR-products with 8 restriction endonucleases, and determination of the fragments size after polyacrylamide gel electrophoresis. Five of the enzymes, having no more than one restriction site in each allele (ApaLI, HphI, BsaJI, FokI and MboII), allow distribution of all the genotypes in 19 allelic-combination groups on the base of the digestion pattern: cut/not cut. Three additional enzymes (MnlI, DdeI and RsaI), having at least 2 restriction sites in each allele, are used to assign the genotype in each allelic-combination group on the base of the restriction fragments length observed. Eight of the 13 alleles, 36 of the 91 HLA-DQA1 genotypes could be characterized. Four to 8 samples could be characterized each day, including DNA extraction. The number of endonucleases used could act as internal control of enzymatic activities and the genotyping protocol can include new HLA-DQA1 alleles without modification of the experimental steps. This protocol can be applied easily in a laboratory without specific technical training or specific equipment.

[Polymerase chain reaction, cold probes and clinical diagnosis]. / Réaction de polymérisation en chaîne, sondes froides et diagnostic clinique.

Until 1985 the only way to study a gene was to clone it. Henceforth, the polymerase chain reaction (PCR) is an alternative method for synthesizing millions of copies of a specific DNA sequence. Without the development of non radioactive probes, these technologies would have been reserved for research applications. PCR with non radioactive probes is a powerful tool of molecular diagnosis in routine laboratories (identification of viruses and bacteria, diagnosis of human genetic diseases). PCR is based on Taq DNA polymerase. This enzyme is able to polymerize deoxynucleotide precursors (dNTP) in a temperature range of 75-80 degrees C. A typical PCR reaction is a repetitive series of thermic cycles involving template DNA denaturation, oligonucleotide primer annealing, and extension of the annealed primers by DNA polymerase. This three-step process results in the exponential accumulation of a specific fragment whose termini are defined by the 5' end of the primers. Amplification can be estimated to be 2n, where n is the number of cycles. The first step involves denaturation of double-stranded target DNA by heating the sample to 90-95 degrees C. In the second step, the temperature is lowered to about 5 degrees C below the melting temperature of the primer, assuring the specificity of the primer annealing and thus the specificity of the product. The third step is carried out by raising the temperature of the sample to 70-73 degrees C, the optimal temperature for primer extension, involving very little denaturation of the enzyme during the 25-30 cycles of a PCR reaction. The primers used are designed on the basis of the known DNA sequence and they must flank the sequence targeted. For microorganism typing, a product of 300 to 900pb can be amplified, though a 2 kb product can be synthesized. The choice of the primer sequence is a function of the target and technical requirements, such as a GC content of 50-60%, which gives the optimal annealing temperature of 50-55 degrees C. The molecular composition of the primer must be chosen to prevent the formation of intra-molecular secondary structures and primer dimers. The complementarity between the template and the 3' OH end must be perfect, because Taq DNA polymerase activity is markedly lowered by mismatches and secondary structures. The 5' end can thus modified by extension or base modification without altering the quality of the amplification. The yield of the reaction can be modified by the composition of the PCR medium.(ABSTRACT TRUNCATED AT 400 WORDS)

Positive and negative effects of estradiol-17 beta in the rat uterus.

Estrogens could act as effectors or inhibitors of protein synthesis in the rat uterus, depending on the doses given to animals. A single injection of estradiol-17 beta to immature female rats led to the increase in protein synthesis and in enzyme activities involved in DNA synthesis. Four injections, given once daily, resulted in the inhibition of enzyme activity and synthesis of all proteins but one. The 105 kD protein which showed a gradual increase with the duration of estrogen treatment could be responsible for the negative action of estrogens on uterine growth.

[Stimulation and selective inhibition of protein synthesis by 17 beta-estradiol, in the uterus of immature rats]. / Stimulation et inhibition sélective de la synthèse protéique par le 17 beta-oestradiol, dans l'utérus de la ratte immature.

The in vitro translation of RNAs extracted from immature female rat uteri 24 hrs. after one or four daily injections of 17 beta-oestradiol was carried out. The proteins labelled with (35S) methionine were separated by polyacrylamide gel electrophoresis and the resulting autoradiograms were scanned. The densitometric profile of uterine proteins obtained after a single injection of oestrogen showed a considerable increase in the majority of them, compared with controls. Following four injections of 17 beta-oestradiol the synthesis of all but one of the uterine proteins was observed. The Mr of the protein induced by repeated administration of oestrogens was about 105 kdaltons. This protein could possibly be involved in the inhibition of protein synthesis observed in the rat uterus after reiterated injections of 17 beta-oestradiol to female rats.

Some characteristics of fetal and adult isoenzymes of thymidine kinase in human breast cancers.

Two isoenzymes of Thymidine Kinase were isolated from 90 human breast cancers by ion exchange chromatography. One was the enzyme present in adult tissues (TK-A), the second corresponded with the enzyme found in fetal tissues (TK-F). The presence of both isoenzymes was observed in the 90 tumors investigated. The activity of TK-A varied in the same range as in normal tissues, while the activity of TK-F varied to a larger extent and was higher than that of TK-A in a majority of tumors. Moreover, TK-F activity was independent of the presence or of the level of estradiol and progesterone receptors. Both isoenzymes were further characterized and compared.

Stimulation by estradiol-17 beta of thymidine kinase activity in the rat uterus.

The action of estradiol-17 beta (E2) on thymidine kinase (TK) activity was studied in uteri from immature female rats. It was demonstrated that a single injection of E2 highly stimulated the enzyme activity which reached its maximum level 24 h after hormone administration. Physiological amounts of E2 were efficient and changes in TK activity were observed exclusively in uterus and liver. A single injection of Tamoxifen produced the same effect as E2 but repeated administration resulted in the complete inhibition of enzyme activity. Using antibiotics it was demonstrated that E2 induced the synthesis of new enzyme molecules rather than an increase in enzyme activity. This statement was corroborated by the fact that after hormone administration the increase in TK activity was preceded by an increase in RNA-polymerase activity and followed by that in DNA-polymerase alpha activity. Moreover, the separation of TK isoenzymes on DEAE-Sephadex and the use of d-CTP as inhibitor of the adult isozyme suggested that E2 induced the "fetal" form of the enzyme. In addition, it was demonstrated that TK activity in uteri from ovariectomized adult female rats was enhanced by E2 administration, and that the increase was due to the stimulation of the fetal isoenzyme. It was suggested that TK could be used as a marker of the action of estrogens and antiestrogens in target organs.

[Induction, by 17 beta-estradiol, of thymidine kinase activity in uteri of rats]. / Induction, par le 17 beta-oestradiol, de l'activité de la thymidine kinase dans l'utérus de la ratte.

In uteri from adult female rats, Thymidine Kinase activity varied during ovarian cycle and was maximum at metestrus. Subcutaneous injections of 5, 10 or 25 ng of estradiol-17 beta to immature female rats, resulted in a 3, 5 or 10 fold increase of enzyme activity. Moreover, Thymidine Kinase activity was decreased by injection of medroxyprogesterone alone or associated with estradiol-17 beta. From these results it seemed that Thymidine Kinase activity in rats uteri was specifically induced by estradiol-17 beta.