Identification of G2607A mutation in EGFR gene with a significative rate in Moroccan patients with Bladder Cancer.

The epidermal growth factor receptor (EGFR) is involved in the regulation of several cellular processes and in the development of many human cancers. Somatic mutations of EGFR at tyrosine kinase domain have been associated with clinical response to tyrosine kinase inhibitors (TKIs) in lung cancer patients. In this study, we evaluated the frequency of point mutations in EGFR for future use of TKI in clinical treatment of bladder cancer. A total, 50 Moroccan patient specimens with bladder cancer and 48 healthy controls were analysed for EGFR mutations in the region delimiting exons 18-21 by PCR amplification and direct sequencing. Our results showed the absence of mutations in the EGFR kinase domain in these exons in all analysed specimens. However, sequence analysis of the EGFR-TK domain, revealed the presence of (G2607A) polymorphism at exon 20. Statistical analysis showed significant difference in the frequencies of G2607A polymorphism between cancer cases and healthy controls (p=0.0001) and the frequencies of the GG and GA/AA genotypes among the cancer cases were 28% and 72%, respectively. Moreover, allelic frequencies of G2607A polymorphism showed significant difference between cancer cases and healthy controls (p=0.0025). Data analysis showed no significant association between G2607A polymorphism and patients' age, clinical stage and tumor grade (p > 0.05). However, a significant difference was found between this polymorphism and patients' sex that could be a sampling bias due to the very limited number of women with bladder cancer. Our findings highlight that, mutations in EGFR kinase domain is a rare event in bladder cancer, suggesting, that treatment of bladder cancer patients with TKI may not be effective. However, the EGFR G2607A polymorphism in exon 20 is frequent in bladder cancer cases and must be further explored for its relevance in the treatment of this disease.

Validation of a modified algorithm for the identification of yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS).

Optimising antifungal treatment requires the fast and species-specific identification of yeast isolates. We evaluated a modified protocol for the rapid identification of clinical yeast isolates using matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) technology. First, we evaluated a simplified extraction procedure using 54 clinical yeast isolates. Second, we validated a new protocol with this simplified extraction procedure and lower identification threshold by analysing 167 isolates with either MALDI-TOF or conventional identification techniques. MALDI-TOF analysis with both the standard and short extraction procedure yielded identical identification results, although the log-scores were lower with the latter. With the modified protocol, 163/167 (97.6%) isolates showed a correct identification as compared to conventional identification techniques. A total of 135 out of the 163 (82.8%) correct identifications showed log-scores above 1.7, which we considered as the minimum log-score for secure species identification. The rapid identification of clinical yeast isolates is crucial in patient management. The MALDI-TOF technique using a short extraction procedure can be an alternative for the labourious standard procedure, although the log-scores will be lower. The identification of clinical yeast isolates with the modified protocol is a practical and accurate alternative for conventional identification techniques. If the isolate shows a log-score below 1.7, the standard extraction procedure should be used.

[Desmoid tumors of the abdominal wall: three cases]. / Les tumeurs desmoïdes de la paroi abdominale. A propos de trois cas.

Desmoids tumors are rare. They often develop from the fascia and muscles of the abdominal wall. They are considered as benign, but endowed with local aggressiveness. Treatment is primarily surgical. Complete resection with large safety margins and sometimes complex reconstruction is necessary to reduce the risk of local reccurrence. WE report three cases of histology proven desmoids tumors of the abdominal wall treated between 2000 and 2007. Etiologic factors, diagnosis, surgical management and adjuvant therapy in case of incomplete resection or reccurrence are discussed.

The protein tyrosine phosphatase interacting protein 51 (PTPIP51) is required for the differentiation of photoreceptors.

Proliferation and differentiation of retinal progenitor cells (RPCs) are tightly controlled by extrinsic cues and distinct combinations of transcription factors leading to the generation of retinal cell type diversity. In this context, we investigated the role of the protein tyrosine phosphatase interacting protein 51 (PTPIP51) in the differentiation of RPCs. The expression pattern of PTPIP51 was analyzed by immunostaining during post-natal retinal development in the rat. Ex vivo electroporation has been used to silence or misexpress PTPIP51 in post-natal retinal explants, and the retinal phenotype was investigated after 3-7days in vitro (div). PTPIP51 expression in the retina started postnatally and was maintained throughout adulthood, especially in retinal ganglion cells and in the inner segment of photoreceptor cells. Silencing of Ptpip51 expression in postnatal retina failed to modify the commitment of late RPCs in the different lineages but severely impaired the final differentiation of photoreceptors, observed by a decrease in the fraction of Rhodopsin-positive cells after 7div. By contrast, misexpression of PTPIP51 in early or late RPCs failed to modify the differentiation of the RPCs. Our data demonstrate that PTPIP51 is implicated in the differentiation process of immature photoreceptors. Because PTPIP51 is specifically localized in the inner segment, PTPIP51 may contribute to the complex stage of maturation of the apical segment of these cells.

The rat beta 3-adrenergic receptor gene contains an intron.

We report that the rat beta 3-adrenergic receptor (beta 3-AR) gene has an intron. The intron starts with an in-frame stop codon with the result that unspliced transcripts will encode a C-terminal truncated protein. The reported protein sequences of mouse and human beta 3-AR were both deduced from genomic DNA sequences. Given the heterogeneity at the C-termini of the otherwise highly similar rat, mouse and human sequences, we discuss the intriguing possibility that the beta 3-AR gene of the latter two species also contain an intron near the extremity of the open reading frame. A beta-adrenergic receptor (beta-AR) cDNA we have cloned from rat colonic tissue which has a sequence essentially identical to that previously reported for the rat adipose beta 3-AR cDNA [(1991) J. Chem. 266, 24053], encodes the spliced version of the beta 3-AR.

Molecular cloning of a human beta 3-adrenergic receptor cDNA.

We report the molecular cloning of a beta 3-adrenergic receptor [beta 3-AR] cDNA from human brown adipose tissue. The cDNA-encoded protein is identical to the previously cloned beta 3-AR but with 6 additional amino acids at the C-terminus. The C-terminus is shared by the beta 3 receptors expressed in human neuroblastoma cells [SK-N-MC] [Mol. Pharmacol. 42 (1992) 964-970]. Furthermore, using a polymerase chain reaction strategy we have cloned and sequenced the beta 3-AR introns. Sequence analysis demonstrates that the human beta 3-AR gene comprises at least 3 exons and 2 introns and that the most abundant beta 3-AR transcripts encode a protein with an exon 3-derived C-terminus. Interestingly, although a similar organization has been found in rodent genes, the rat beta 3-AR transcripts encode a receptor with an exon 2-derived C-terminus.

Expression of tachykinin NK2 receptor mRNA in human brain.

Tachykinin NK2 receptors have been suggested to play an important role in the central nervous system. This study, using reverse transcription-polymerase chain reaction revealed a detectable expression of NK2 receptor mRNA in various human brain regions, including the caudate nucleus, the putamen, the hippocampus, the substantia nigra and the cerebral cortex. The distribution of NK2 receptor expression in the cortex revealed a major expression in frontal and temporal cortex compared to occipital and parietal areas. These results provide a molecular basis for considering a role of NK2 receptors in human pathophysiology.

Multivariate statistical analysis of flat vowel spectra with a view to characterizing dysphonic voices.

The aim of this article is to show how dysphonic voices can be characterized by means of a multivariate statistical analysis of flat vowel spectra. The spectral contour was obtained by means of a wavelet transform of the logarithmic magnitude spectrum, which was subsequently flattened to remove interspeaker variability related to the excitation and vocal tract filter functions. The results of the statistical analysis of flat spectra were the following. Firstly, principal components analysis produced markers that separated noisy from clean spectra. Secondly, the heuristic search for harmonic peaks or interharmonic dips could be omitted. Thirdly, conventional spectral markers of noise appeared as special instances of the markers that were derived statistically. Fourthly, the levels of visually assigned hoarseness and the first two principal components were significantly correlated. The assignment of different levels of (visual) hoarseness to different vowel timbres could be explained by the variability associated with the spectral contour.

[Culture of retinal capillary endothelial cells]. / Culture de cellules endothéliales de capillaires rétiniens.

The culture of retinal capillary endothelial cells involves certain problems concerning contamination by pericytes, the maintenance of differentiation and the duration of culture viability. A procedure for the isolation and culture of capillary endothelial cells from bovine retina which overcomes these difficulties, is described. Microvessel fragments isolated by mechanical dispersion and filtration techniques adhere strongly to dishes coated with extracellular matrix produced by bovine corneal endothelial cells. The first migrating cells emerge from the original microvessel fragments two days after plating. This technique and subsequent cloning provides migrating and proliferating cells derived only from the retinal capillaries and uncontaminated by other cell types such as pericytes. Endothelial cells were grown on gelatin coated dishes in a serum supplemented medium (10% calf serum). Cell proliferation was significantly enhanced by the addition of basic fibroblast growth factor (1 ng/ml) to the culture medium. In these culture conditions, retinal capillary endothelial cells can be repeatedly passing without the loss of their principal morphological characteristics and some of the differentiated properties of endothelial cells. Primary cultures and subcultures, at least up to the 8th passage, formed a monolayer of small, elongated, tightly-packed, contact inhibited cells which expressed Factor VIII-related antigen. Ultrastructural examination by transmission electron microscopy of confluent bovine retinal capillary endothelial cells showed many tight junctions and Webel Palade granules. These studies provide new means for the isolation and culture of retinal capillary endothelial cells and presents evidence for growth factor requirements for the ability of cells to be repeatedly passing.

[Clinical heterogeneity of Townes-Brocks syndrome]. / Hétérogénéité clinique du syndrome de Townes-Brocks.

BACKGROUND: Townes-Brocks syndrome (TBS) is a rare autosomal dominant entity mainly characterized by ano-rectal, ear and extremities abnormalities with variable clinical expression. CASE REPORTS: The first case had ear and extremities, but not anorectal, abnormalities; a Pierre-Robin sequence with esophageal atresia was also observed. The second case had the classical triad of abnormalities also associated with tetralogy of Fallot which has been only once reported in the literature. CONCLUSIONS: Both cases are other examples of the frequent clinical variability observed in this syndrome explaining diagnostic difficulties in the absence of a specific marker.

[Choice of a central analgesic for anesthesia with spontaneous respiration]. / Choix d'un analgésique central pour l'anesthésie avec respiration spontanée.

The ventilatory effects of tramadol (T) and nefopam (N) are evaluated in anesthetized patients with enflurane in a closed circuit breathing system and compared with the effects of pentazocine (P). The following parameters tidal volume (VT), minute ventilation (V), CO2 (capnometry) occlusion pressure (OP), ventilatory response to hypercarbia are recorded after 30 minutes of anaesthesia, before and after repeated injections of the analgesics, P: 15 mg, N: 40 mg, T: 100 mg are injected I.V., and analgesics administration is repeated at 30 minutes interval, so that the patients receive a total P dose of 30 mg, a total N dose of 60 mg and a total T dose of 200 mg. The administration of 15 mg of P induces a change in VT (-24%), ventilatory frequency (-40%), OP (-18) OP only returns to basal values after a second dose. The ventilatory response to hypercarbie is indeed satisfying (increase of 61% in V). After N and T, ventilatory frequency is not disturbed. V increases of 16% and 11% respectively after the first injection, and of 31% and 2% after the second injection. OP increases by 39% and 56% respectively after the first injection and gets better over time with nefopam (+ 58%), 30 mg of P. 20 mg of N and 100 mg of T are equivalent for analgesia.(ABSTRACT TRUNCATED AT 250 WORDS)

[Management of late-onset sepsis due to coagulase-negative staphylococci in neonatal intensive care units]. / Actualités sur la prise en charge des infections néonatales nosocomiales tardives à staphylocoque à coagulase négative.

Coagulase-negative staphylococci (CoNS) are the most frequent cause of late-onset sepsis (LOS) in neonatal intensive care units (NICUs). Staphylococcus epidermidis is usually considered the most prevalent CoNS in this setting. However, recent reports have identified Staphylococcus capitis, another CoNS, as an emerging cause of bacteremia in NICU wards. S. capitis is the main cause of LOS in several NICUs in France, whereas this species is rarely found in adult patients from the same hospitals. S. capitis isolates from NICU infants share several striking features: they all belong to the same pulsed-field gel electrophoresis type, designated as NRCS-A, which indicates their clonal relatedness; their resistance profile reflects adaptation to antimicrobial agents specifically used in NICUs, including resistance to beta-lactams and aminoglycosides but not to fluoroquinolones, and reduced susceptibility to vancomycin; and they are associated with more severe LOS than those caused by other CoNS. The molecular characterization of NICU S. capitis isolates from several countries has shown that S. capitis NRCS-A strains have disseminated in both Western Europe (France, the United Kingdom, and Belgium) and Australia. The dissemination of such multiresistant strains imposes difficult therapeutic choices on pediatricians. As a consequence of the recent strengthening of the French and European guidelines that regulate the interpretation of clinical vancomycin susceptibility in staphylococci, a non-negligible proportion of NICU CoNS isolates (including S. capitis as well as other CoNS species) that were usually reported as vancomycin-susceptible are now categorized as vancomycin-resistant. In such cases, practitioners are faced with uncomfortable alternatives: the continued use of vancomycin in spite of the pathogen being unambiguously reported as resistant to this molecule and the use of antimicrobial agents such as linezolid or daptomycin that retain an in vitro efficacy against CoNS but whose use in neonates has not received approval by the healthcare authorities. To cope with this emerging challenge, clinical investigations of the relative tolerance and efficacy of vancomycin, linezolid, and daptomycin in NICU infants infected with these newly reported vancomycin-resistant CoNS are urgently needed.

[Neonatal adrenal hemorrhage revealed by jaundice: a case report]. / Hémorragie surrénalienne révélée par un ictère néonatal: à propos d'un cas.

The clinical presentation of adrenal hemorrhage varies, depending on the extent of hemorrhage as well as the amount of adrenal cortex involved by the hemorrhage. We report here a case of neonatal adrenal hemorrhage revealed by late onset of neonatal jaundice. This adrenal hemorrhage most probably resulted from shoulder dystocia. The aim of this work was to focus on the fact that jaundice can be caused by adrenal hemorrhage and to emphasize the crucial importance of abdominal ultrasound in cases of persistent jaundice.

Effectiveness of cell-adsorbed bacteriocin produced by Lactobacillus curvatus CWBI-B28 and selected essential oils to control Listeria monocytogenes in pork meat during cold storage.

AIMS: To study the effectiveness of a combination of cell-adsorbed bacteriocin (CAB; a suspension of producer cells on which maximum bacteriocin has been immobilized by pH adjustments) of a Lactobacillus curvatus strain with oregano or savory essential oil to control Listeria monocytogenes in pork meat at 4 degrees C. METHODS AND RESULTS: The antimicrobial activity of the CAB and six different essential oils was tested by the well diffusion assay against L. monocytogenes M, Escherichia coli 10536 and Salmonella serotype Typhi CWBI-H1. The anti-Listeria activity of the CAB and oregano or savory essential oils was also investigated in pork meat. The results of the well diffusion assay showed that CAB was only inhibitory to L. monocytogenes while savory and oregano essential oils were the most active against the three indicator bacteria. In pork meat, Listeria counts have declined from c. 10(2) CFU g(-1) to below the detectable limit during the first week of storage in samples treated with CAB or oregano essential oil and in those treated with CAB combined with oregano or savory essential oil. However, the counts of L. monocytogenes have increased after the third week of storage in all samples with the exception of those treated with the combination of CAB and oregano essential oil. The combination of CAB with savory essential oil resulted in a 2-week delay of the growth rebound compared with samples treated with CAB alone. CONCLUSIONS: Addition of oregano or savory essential oil exhibited a synergistic effect with CAB to control L. monocytogenes in pork meat during storage at 4 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of CAB with oregano or savory essential oil may be effectively used in meat industry to enhance the safety and stability of meat products.

Opposing effects of basic fibroblast growth factor and transforming growth factor-beta on the proliferation of cultured bovine retinal capillary endothelial (BREC) cells.

Transforming Growth Factor-beta (TGF-beta) inhibits the serum and basic fibroblast growth factor (bFGF)-induced proliferation of cultured bovine retinal endothelial capillary (BREC) cells in a dose-dependent manner. The concentration of TGF-beta required to get half-maximal inhibition (ED50) are 10 pg ml-1 in serum and 17 pg ml-1 in the presence of additional bFGF (1 ng ml-1). These TGF-beta ED50 values are greatly increased when BREC cells were seeded at high density: 610 pg ml-1 in serum and 1 ng ml-1 in the presence of additional bFGF. At low initial cell density BREC cells are more sensitive to TGF-beta than aortic bovine arch endothelial (ABAE) cells for which TGF-beta ED50 values are respectively 40 pg ml-1 and 100 pg ml-1 in serum and in the presence of additional bFGF. In contrast, at high cell density BREC cells appeared to be more resistant to TGF-beta inhibition than ABAE cells for which TFG-beta ED50 values are 210 and 300 pg ml-1. Moreover bFGF added at increasing concentrations neutralize totally TGF-beta inhibition of BREC cell proliferation but only partially that of ABAE cell proliferation. Our results suggest a key role of equilibrium TFG-beta bFGF on the proliferation of BREC cells.

Regulation of cyclic adenosine 3',5'-monophosphate-dependent protein kinase activity and regulatory subunit RII beta content by basic fibroblast growth factor (bFGF) during granulosa cell differentiation: possible implication of protein kinase C in bFGF action.

We have previously shown that basic fibroblast growth factor (bFGF) inhibits the FSH-induced differentiation of cultured rat granulosa cells, as manifested by prominent reduction of the LH receptor expression. We now investigate the possible sites and mechanism of action of bFGF. Whereas bFGF decreased the cAMP formation induced by FSH, it enhanced the cAMP production caused by cholera toxin and forskolin, suggesting that bFGF exerted its inhibitory action on cell differentiation at a step to cAMP production. Photoaffinity labeling with 8-azido-[32P]cAMP revealed that bFGF markedly reduced the FSH-induced increase in the level of regulatory subunit RII beta of the cAMP-dependent protein kinase (PKA) type II. In contrast to its striking effect on RII beta expression (70-80% inhibition), bFGF decreased PKA enzymatic activity by only 30%. On the other hand, transforming growth factor-beta (TGF beta) slightly amplified the stimulatory action of FSH and antagonized the bFGF inhibitory effect on both LH receptor expression and RII beta synthesis. We report that the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), which impaired granulosa cell differentiation, also abolished the RII beta synthesis induced by FSH. The activation of PKC by bFGF in granulosa cells was supported by the following findings: (i) bFGF markedly enhanced the production of diacylglycerol (2.3-fold stimulation at 5 min), the intracellular activator of PKC; (ii) bFGF promoted tight association of PKC to cellular membranes, a process that is believed to correlate with the enzyme activation; (iii) bFGF induced the phosphorylation of an endogenous M(r) 78,000/pI 4.7 protein that appears as a specific PKC substrate; (iv) bFGF mimicked the TPA-induced transmodulation of the epidermal growth factor (EGF) receptor, reducing by 36% the 125I-EGF binding on granulosa cells. We conclude that bFGF may exert its repressive action on RII beta synthesis, PKA activity, and granulosa cell differentiation by primarily targeting PKC activation.

Autocrine regulation of bovine retinal capillary endothelial cell (BREC) proliferation by BREC-derived basic fibroblast growth factor.

Fibroblast growth factors (FGFs) are mitogenic for bovine retinal capillary endothelial cells (BREC) seeded at a low density. Seeding BREC cells at a high density greatly reduces their requirement for basic FGF (bFGF) in order to proliferate actively. We show here that monolayers of BREC cells synthesize and release into the culture medium a growth factor, which on the basis of biological activity, heparin affinity, immuno-cross reactivity with anti-bFGF antibodies and mRNA analysis, has been identified as basic fibroblast growth factor. These data indicate that BREC cells are able to synthesize and release bFGF, which can act as a promoting-growth factor for these cells by a para- and/or autocrine mechanism. We suggest thus, that this para- and/or autocrine mechanism involving bFGF may play a key role in preretinal neovascularization, particularly in diabetic patients presenting a proliferative retinopathy.

Protein kinase NII and the regulation of rDNA transcription in mammalian cells.

Transcription of ribosomal RNA genes is generally accepted to correlate with cell growth. Using primary cultures of adult bovine aortic endothelial (ABAE) cells, we have shown that transcription of rDNA in confluent cells falls to 5% of the transcription level in growing cells. Protein kinase NII appears to be a limiting factor to promote rDNA transcription in isolated nuclei of confluent cells. Protein kinase NII was detected by immunocytochemistry in the cytoplasm, nuclei and nucleoli of growing cells while it was no longer present in nucleoli of confluent cells. The kinase activity, in isolated nuclei, was estimated by endogenous phosphorylation of a specific substrate, nucleolin. A 10% residual activity was present in confluent cell nuclei compared to growing cell nuclei. Concomitantly, the transcription 'in vitro' of rDNA in the corresponding nuclei was also highly reduced (by 85%). Addition of exogenous protein kinase NII to confluent cell nuclei induced a strong increase in the phosphorylation of specific proteins including nucleolin. In parallel, the transcription of rDNA was increased by a factor of 5, to nearly the level observed in nuclei prepared from growing cells. These data suggest that, in confluent cells, factors necessary for rDNA transcription machinery are present but inactive in the nucleolus and that the phosphorylation of one or several of these factors (nucleolin, topoisomerase I,...) by protein kinase NII is a key event in the regulation of rDNA transcription.

Duodenal mRNA expression of iron related genes in response to iron loading and iron deficiency in four strains of mice.

BACKGROUND: Although much progress has been made recently in characterising the proteins involved in duodenal iron trafficking, regulation of intestinal iron transport remains poorly understood. It is not known whether the level of mRNA expression of these recently described molecules is genetically regulated. This is of particular interest however as genetic factors are likely to determine differences in iron status among mouse strains and probably also contribute to the phenotypic variability seen with disruption of the haemochromatosis gene. AIMS: To investigate this issue, we examined concomitant variations in duodenal cytochrome b (Dcytb), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hephaestin, stimulator of Fe transport (SFT), HFE, and transferrin receptor 1 (TfR1) transcripts in response to different dietary iron contents in the four mouse strains C57BL/6, DBA/2, CBA, and 129/Sv. SUBJECTS: Six mice of each strain were fed normal levels of dietary iron, six were subjected to the same diet supplemented with 2% carbonyl iron, and six were fed an iron deficient diet. METHODS: Quantification of mRNAs isolated from the duodenum was performed using real time reverse transcription-polymerase chain reaction. RESULTS: There was a significant increase in mRNA expression of Dcytb, DMT1, FPN1, and TfR1 when mice were fed an iron deficient diet, and a significant decrease in mRNA expression of these molecules when mice were fed an iron supplemented diet. Strain to strain differences were observed not only in serum transferrin saturations, with C57BL/6 mice having the lowest values, but also in hepatic iron stores and in duodenal mRNA expression of Dcytb, DMT1, FPN1, hephaestin, HFE, and TfR1. CONCLUSIONS: The results favour some degree of genetic control of mRNA levels of these molecules.