Cloning and expression in Pichia pastoris of an Irpex lacteus rhamnogalacturonan hydrolase tolerant to acetylated rhamnogalacturonan.

In order to produce a recombinant rhamnogalacturonase from the basidiomycete Irpex lacteus using a molecular approach, PCR primers were designed based on a sequence alignment of four known ascomycete rhamnogalacturonases. Using 5' and 3' rapid amplification of cDNA ends (RACE) experiments, a 1,437-bp full-length cDNA containing an open reading frame of 1,329 bp was isolated. The corresponding putative protein sequence is of 443 amino acids and contains a secretion signal sequence of 22 amino acids. The theoretical mass of this protein is 44.6 kDa with a theoretical isoelectric point of 6.2. The amino acid sequence shared not only significant identities with ascomycete and basidiomycete putative rhamnogalacturonases but also complete similarity with peptides obtained from a recently purified rhamnogalacturonase from I. lacteus. The recombinant protein was successfully expressed in active form in Pichia pastoris. SDS-PAGE assay demonstrated that the recombinant enzyme was secreted in the culture medium and had a molar mass of 56 kDa. This recombinant rhamnogalacturonan hydrolase exhibited a pH optimum between 4.5 and 5 and a temperature optimum between 40°C and 50°C, which correspond to that of the native rhamnogalacturonase from I. lacteus. The study of its specificity through reaction products analysis showed that it was highly tolerant to the presence of acetyl groups on its substrate, even more than the native enzyme.

Fludarabine-mediated suppression of the excision repair enzyme ERCC1 contributes to the cytotoxic synergy with the DNA minor groove crosslinking agent SJG-136 (NSC 694501) in chronic lymphocytic leukaemia cells.

In this study, we set out to establish whether fludarabine could enhance the DNA interstrand crosslinking capacity of SJG-136 in primary human chronic lymphocytic leukaemia (CLL) cells and thereby offer a rationale for its clinical use in combination with SJG-136. SJG-136 rapidly induced DNA crosslinking in primary CLL cells which was concentration-dependent. Further, the level of crosslinking correlated with sensitivity to SJG-136-induced apoptosis (P=0.001) and higher levels of crosslinking were induced by the combination of SJG-136 and fludarabine (P=0.002). All of the samples tested (n=40) demonstrated synergy between SJG-136 and fludarabine (mean combination index (CI)=0.54+/-0.2) and this was even retained in samples derived from patients with fludarabine resistance (mean CI=0.62+/-0.3). Transcription of the excision repair enzyme, ERCC1, was consistently increased (20/20) in response to SJG-136 (P<0.0001). In contrast, fludarabine suppressed ERCC1 transcription (P=0.04) and inhibited SJG-136-induced ERCC1 transcription when used in combination (P=0.001). Importantly, the ability of fludarabine to suppress ERCC1 transcription correlated with the degree of synergy observed between SJG-136 and fludarabine (r(2)=0.28; P=0.017) offering a mechanistic rationale for the synergistic interaction. The data presented here provides a clear indication that this combination of drugs may have clinical utility as salvage therapy in drug-resistant CLL.

Defense Gene Expression Analysis of Arabidopsis thaliana Parasitized by Orobanche ramosa.

ABSTRACT The infection of Arabidopsis thaliana roots with the obligate parasite Orobanche ramosa represents a useful model for a study of the molecular events involved in the host plant response to a parasitic plant attack. To avoid analysis problems due to the subterranean development of O. ramosa, we developed two in vitro co-culture systems: O. ramosa seedlings infesting Arabidopsis plantlet roots and callus tissues. We were then able to investigate the expression patterns of some host plant genes selected among genes known to be involved in metabolic pathways and resistance mechanisms activated during several plant-pathogen interactions including ethylene, isoprenoid, phenylpropanoid, and jasmonate biosynthesis pathways, oxidative stress responses, and pathogenesis-related proteins. Molecular analyses were carried out using polymerase chain reaction amplification methods allowing semiquantitative evaluation of transcript accumulation during early (first hours) and late (15 days) stages of infestation, in whole roots or parts close to the parasite attachment site. In A. thaliana, O. ramosa induced most of the general response signaling pathways in a transient manner even before its attachment to A. thaliana roots. However, no salicylic acid-dependent defense is observed because no activation of systemic acquired resistance markers is detectable, whereas genes, co-regulated by jasmonate and ethylene, do display enhanced expression.

Host-root exudates increase gene expression of asparagine synthetase in the roots of a hemiparasitic plant Triphysaria versicolor (Scrophulariaceae).

Triphysaria is a facultative root parasite in the Scrophulariaceae family. Similar to other related parasites, the development of the parasitic life cycle is initiated by molecular signals released from appropriate host roots. Using a differential display, we isolated cDNAs preferentially abundant in T. versicolor roots exposed to Trifolium repens (white clover) root exudates in vitro. Sequence analysis indicated that one of the differentially expressed cDNAs had significant homology to the nitrogen-assimilating enzyme, asparagine synthetase (AS). T. versicolor AS cDNA clones were isolated and placed into three distinct classes on the basis of nucleotide sequence variations. All three classes encoded identical AS proteins. AS was expressed in both roots and shoots of in-vitro-cultured T. versicolor. Steady-state levels of AS mRNA increased in T. versicolor roots several-fold when seedlings were exposed to exudate obtained from hydroponically grown Arabidopsis thaliana roots. Therefore, AS transcript levels increased in response to exudates from two different hosts (Trifolium and Arabidopsis). The T. versicolor AS message levels increased to a similar magnitude when seedlings were incubated in the dark. Interestingly, AS levels were unaffected by treatment with the Striga haustoria inducer 2,6-dimethoxybenzoquinone. The potential role of AS in root parasitism is discussed.

The rbcL gene from the non-photosynthetic parasite Lathraea clandestina is not transcribed by a plastid-encoded RNA polymerase.

In the plastome of the obligate root-parasitic plant, Lathraea clandestina, the rbcL gene has been maintained and is expressed, despite the reduced size and gene content of the plastid genome. Some of the plastid genes involved in translation (e. g. transfer RNAs, ribosomal RNAs and ribosomal proteins) have been sequenced and still appear to code for functional ribosomal components. Indeed, the 16S rRNA and rpl20 genes are expressed whilst other necessary tRNA and ribosomal protein-encoding genes have probably been deleted or truncated. Although obtained by PCR, the four rpo genes for Escherichia coli-like plastid encoded RNA polymerase appear to be pseudogenes. Nevertheless, the rbcL gene, with a "-10, -35" prokaryotic-like promoter, is still transcribed. In contrast to photosynthetic plants, rbcL transcripts in Lathraea are larger in their 5' region and cover the prokaryotic-like promoter. The transcription initiation site is located near the ATG start codon of the atpB pseudogene. Similarity to non-consensus E. coli-like plastid promoters suggests that rbcL transcription is driven by a nuclear-encoded RNA polymerase.

Penetration of pefloxacin and its desmethyl metabolite into the uroepithelium after a 800-mg single oral dose in human patients.

Penetration of pefloxacin into the uroepithelium was studied in 20 patients (10 men and 10 women) receiving a single oral dose of 800 mg. Samples of serum, urine, and uroepithelium were taken 1.8 h (mean) after the dose. Pefloxacin and its active metabolite, norfloxacin, were assayed by liquid chromatography, and the microbiologically active compounds were quantified by a microbiological assay. Both procedures were correlated (r > 0.7); nevertheless, slight differences detected in concentrations depended on the levels of norfloxacin achieved in the biological samples. The serum and tissue concentrations were higher than the concentration of bactericide (4 micrograms.ml-1), except in one case. The uroepithelium concentration of pefloxacin was proportional to the serum concentration (r = 0.79). The urinary concentrations ranged from 1.2 micrograms.ml-1 to 82.4 micrograms.ml-1. The mean norfloxacin/pefloxacin ratios were 3% in serum, 8% in uroepithelium, and 44% in urine. The mean uroepithelium/serum concentration ratios were 1 for pefloxacin and 2.3 for norfloxacin. This result shows that, at a time close to that of the maximum concentration, there is good penetration of pefloxacin and norfloxacin into the uroepithelium.

Divergent evolution of two plastid genes, rbcL and atpB, in a non-photosynthetic parasitic plant.

Plastid DNA (ptDNA) regions for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubiso) (rbcL) and the beta-subunit of ATP synthase (atpB) genes of the holoparasite Lathraea clandestina L. were sequenced. These regions were obtained by cloning either a Bam HI endonuclease generated fragment from the Lathraea ptDNA or polymerase chain reaction (PCR) amplified products. The Lathraea ptDNA contains the entire sequence for the rbcL gene which shares 94.5% homology with the Nicotiana tabacum gene, whereas atpB is maintained as a pseudogene. The intergenic region between divergently transcribed rbcL and atpB genes is shorter (758 bp) in L. clandestina plastid genome in comparison with N. tabacum (823 bp), however they have a noticeable similarity, mainly in the rbcL 5'-upstream region. A low level of the rbcL gene transcription was detected whereas no atpB transcripts were found in Latraea. The plasmid rbcL gene of the hemiparasite Melampyrum pratense and the autotroph Digitalis purpurea both from the Scrophulariaceae were cloned by PCR amplification and then sequenced. The L. clandestina rbcL gene is highly homologous to the M. pratense and D. purpurea genes. The data indicate that the evolution of the plastid atpB-rbcL region was different in parasites from the Scrophulariaceae and Orobanchaceae families.

[Must pefloxacin and norfloxacin be studied separately against bacteria isolated from urinary tract infections by the API-ATB method?]. / Faut-il étudier séparément la péfloxacine et la norfloxacine sur les bactéries isolées d'infections urinaires par la méthode API-ATB?

2113 bacterial strains were isolated from urinary tract infections in 1992 in 133 French pathology laboratories, 2069 strains were tested using the API-ATB method and the UR-14030 system including NFX and an additional test for PFX. Frequencies of susceptible (S), intermediate (I) and resistant (R) strains to PFX and NFX were respectively (%): 83.3; 8.6; 8.1 and 83.1; 9.5; 7; 4. Overall rate of concordance (C), SS, II and RR, between the two antibiotics reached 92.8, minor discrepancies (Dm), SI, IS, RI and IR, 6.3 and major discrepancies (DM), SR and RS, 0.9 (K = 0.82). For Enterobacteriaceae (n = 1830), frequencies of strains S, I and R were: 90.4; 5.9 and 3.7 with PFX and 90.4; 6.1 and 3.5 with NFX. Percentages of C, Dm and DM were 95.1; 4.4 and 0.5 respectively. The Lee test showed that results obtained with NFX and PFX were equivalent (p < 0.001) allowing to consider that the test of NFX is sufficient to conclude for susceptibility or resistance to both antibiotics using the API-ATB method and the UR-14030 system. Dm were probably related in some cases to a low level resistance mechanism and to the difference between the higher breakpoints (4 micrograms/ml for PFX and 8 micrograms/ml for NFX). DM might be due to artefacts related to the bacterial inoculum size or to the antibiotic concentration obtained in the cupules.

[Antibioprevention of recurrent cystitis. A randomized double-blind comparative trial of 2 dosages of pefloxacin]. / Antibioprophylaxie des cystites récidivantes. Essai comparatif randomisé en double-insu de deux modalités posologiques de péfloxacine.

OBJECTIVES: The aim of this randomized double-blind trial was to compare two 400 mg pefloxacin regimens either once-a-week or once-a-month applied for 48 weeks for the prophylaxis of recurrent urinary tract infection in women. METHODS: The main outcome measures were symptomatic and bacteriological reinfections during the period of prophylaxis and rates of reinfections during the three months of surveillance following the end of prophylaxis. 361 women of 18 to 51 years of age suffering from recurrent lower urinary tract infection were randomly allocated to receive pefloxacin 400 mg once-a-week (group A: n = 185) or 400 mg once-a-month (groupe B: n = 176) for 48 weeks. RESULTS: Seventeen of the 185 patients in group A (9.1%) and 52/176 patients in group B (29.5%) experienced at least one reinfection during the period of prophylaxis (p < 0.0001). The rates of reinfection during the three months of surveillance following the end of the treatment were not significantly different between the two groups with 14/101 (13.8%) patients with at least a reinfection in group A and 8/75 patients (10.6%) in group B (p = 0.51). In group A, 49/174 (28.1%) patients reported at least an adverse event compared with 33/169 (19.5%) patients in group B (p = 0.06). CONCLUSIONS: Once-a-week treatment with 400 mg of pefloxacin can be considered as a new effective and well tolerated approach for the prophylaxis of recurrent urinary tract infection in women and does not entail excessive emergence of pefloxacin resistant bacteria, even after 48 weeks of treatment.

[Acute cystitis in women over 50 years of age. Efficacy of pefloxacin with single dose and norfloxacin for 10 days]. / Cystite aiguë chez la femme de plus de 50 ans. Efficacité de la péfloxacine en dose unique et de la norfloxacine prescrite pendant 10 jours.

This multicentre, open, randomized trial, involving 482 patients and conducted by private practitioners, compared the effectiveness and safety of a single 800 mg dose of pefloxacin and of a 10 days' course of norfloxacin 400 mg bid. in the treatment of uncomplicated acute cystitis in women aged over 50 years. Clinical effectiveness was evaluated on days 17-19 and 28-32 respectively, and bacteriological effectiveness on days 15-17 and 26-28 respectively. The median time taken for the symptoms to disappear was 2 days with pefloxacin and 3 days with norfloxacin (P < 0.001). Irrespective of the nature of cystitis and the patients' age, no significant difference could be found in eradication of the pathogens. Undesirable side-effects were recorded in 7.8 percent of patients under pefloxacin and in 8.8 percent of those under norfloxacin (P = 0.68); gastrointestinal disorders were predominant. The acceptability of treatment, as judged by the patients themselves, was regarded as excellent by 55 percent of women treated with single dose pefloxacin and by 37.6 percent of those treated with norfloxacin (P = 0.001).

[Lichen planus: etiopathogenesis]. / Lichen plan: étiopathogénie.

Lichen planus could result from a succession of immunological events: stimulation of Langerhans cells and keratinocytes by foreign antigen--production of interleukin 1 (IL 1)--activation, attraction and multiplication of helper T lymphocytes--production of interferon gamma--appearance of intercellular adhesion molecule-1 (ICAM 1) and HLA DR Antigen on keratinocytes membranes, allowing adhesion of T lymphocytes to keratinocytes and subsequent destruction of the latter.