Genetic and biochemical characterization of the chromosomal class A beta-lactamases of Raoultella (formerly Klebsiella) planticola and Raoultella ornithinolytica

Enterobacterial strains of Raoultella spp. display a penicillinase-related beta-lactam resistance pattern suggesting the presence of a chromosomal bla gene. From whole-cell DNA of Raoultella planticola strain ATCC 33531(T) and Raoultella ornithinolytica strain ATCC 31898(T), bla genes were cloned and expressed into Escherichia coli. Each gene encoded an Ambler class A beta-lactamase, named PLA-1 and ORN-1 for R. planticola and R. ornithinolytica, respectively. These beta-lactamases (291 amino acids), with the same pI value of 7.8, had a shared amino acid identity of 94%, 37 to 47% identity with the majority of the chromosome-encoded class A beta-lactamases previously described for Enterobacteriaceae, and 66 to 69% identity with the two beta-lactamases LEN-1 and SHV-1 from Klebsiella pneumoniae. However, the highest identity percentage (69 to 71%) was found with the plasmid-mediated beta-lactamase TEM-1. PLA-1, which displayed very strong hydrolytic activity against penicillins, also displayed significant hydrolytic activity against cefepime and, to a lesser extent, against cefotaxime and aztreonam, but there was no hydrolytic activity against ceftazidime. Such a substrate profile suggests that the Raoultella beta-lactamases PLA-1 and ORN-1 should be classified into the group 2be of the beta-lactamase classification of K. Bush, G. A. Jacoby, and A. A. Medeiros (Antimicrob. Agents Chemother. 39:1211-1233, 1995). The highly homologous regions upstream of the bla(PLA-1A) and bla(ORN-1A) genes comprised a nucleotide sequence identical to the -35 region and another one very close to the -10 region of the bla(LEN-1) gene. From now on, as the bla gene sequences of the most frequent Raoultella and Klebsiella species are available, the bla gene amplification method can be used to differentiate these species from each other, which the biochemical tests currently carried out in the clinical laboratory are unable to do.     

The periosteal-cutaneous chimeric medial femoral condyle free flap for subtotal ear reconstruction: A case report

When costal graft is contraindicated or refused by the patient, autologous total/subtotal auricular reconstruction represent a real challenge as limited surgical options has been described. Aim of present report is to offer a novel possible autologous reconstruction of the ear frame using a chimeric free medial femoral condyle (MFC) flap. We present a case of a 29 years old patient who had total loss of the upper 2/3 of the right ear after bombing in Somalia and secondary infected condritis (considered a relative contraindication for costal cartilage graft). The MFC flap was harvested with a chimeric skin paddle (7 × 5 cm), a thin sheet of femoral cortex (6.5 × 8 cm) was used as basal ear frame, while part of the contralateral concha was trimmed as support for the helix, with the periosteal component of the flap wrapping around the whole framework. The chimeric skin paddle assured the retroauricular skin coverage, while the anterior part of the construct was covered by a thinned dermal flap. Postoperative course was uneventful. A defatting procedure of the posterior skin paddle was performed at 2 months post-op. At 6 months post-op, the patient was satisfied with the result, could wear glasses and was socially integrated. This new application of the free chimeric MFC flap, despite being not the primary choice for ear reconstruction, guaranteed satisfactory results in terms of ear shape and infection prevention and may be considered when ordinary cartilage rib reconstruction is refused, contraindicated, or failed.     

Association study between iron-related genes polymorphisms and Parkinson's disease

We have conducted a case-control study in order to test for an association between 8 intragenic polymorphisms of 5 iron-related genes (transferrin, transferrin receptor1, HFE, frataxin and lactoferrin) and Parkinson disease. Comparison of genotypes and allele frequencies did not differ significantly between cases and controls for all studied polymorphisms except the G258S transferrin polymorphism, for which a higher frequency of the G allele was found among cases (p=0.033), particularly among cases with onset older than 60 (p=0.0017) and with negative family history (p=0.022). This finding suggests that genetic variations in the control of iron metabolism may contribute to the pathogenesis of the disease. Received: 23 July 2001, Received in revised form: 8 November 2001, Accepted: 14 November 2001     

Ovarian stimulation for in-vitro fertilization combining administration of gonadotrophins and blockade of the pituitary with D-Trp6-LHRH microcapsules: pilot studies with two protocols

In women undergoing in-vitro fertilization and embryo transfer(TVF-ET), a total of 408IVF cycles were stimulated using humanmenopausal gonadotrophin (HMG) or pure follicle stimulatinghormone (FSH) plus HMG in combination with a single injectionof D-Trp⁶-LHRH microcapsules in order to enhance the ovarianresponse to gonadotrophins and to avoid spontaneous LH surges.Sixty-seven pregnancies were achieved. Two protocols were employed.In protocol 1 (‘blocking protocol’, n = 268), thepituitary was first inhibited with a full dose (3.75 mg) ofD-Trp⁶-LHRH in microcapsules and ovarian stimulation was startedafter the hypogonadotrophic hypogonadal state was ascertained(Ej >50 pg/ml). In protocol 2 (‘flareup protocol’,n = 140), the treatment with D–Trp⁶LHRH microcapsules(half-dose = 1.80 mg) and the ovarian stimulation with gonadotrophinswere started at the same time. Higher doses of gonadotrophinswere needed (39.5 11.2 ampoules FSH and/or HMG) in protocol1, in which the pituitary was blocked prior to and during thestimulation, than in protocol 2 (209 ampoules) where the exogenousgonadotrophin stimulation appeared to be augmented by the initialagonistic effect of the injection of D-Trp⁶LHRH microcapsules.In patients with purely tubal infertility, under 38 years oldand no male factor, the results obtained with protocols 1 and2 were similar in terms of pregnancy rate per cycle or per embryotransfer: 22.6 versus 20.5% and 28.3 versus 27.4%, respectively.However, considering the cost benefit, ‘flare-up’protocols appeared to be a better choice and could be recommended.     

Pharmacokinetic advantage of intra-arterial hepatic oxaliplatin administration: comparative results with cisplatin using a rabbit VX2 tumor model

The aim of this study was to compare intra-arterial hepatic administration (IAH) versus i.v. administration of oxaliplatin and cisplatin in a VX2 tumor model in rabbits. VX2 tumors were implanted in the livers of White New Zealand female rabbits and 2 weeks later they received either cisplatin (4 mg/kg) or oxaliplatin (6 mg/kg) administered by IAH or i.v. Platinum pharmacokinetic parameters were measured by atomic absorption spectrometry at baseline, 2, 5 10, 20, 40 and 60 min, and then at 2, 4, 6 and 24 h after drug administration. Animals were sacrificed 24 h after drug administration to measure platinum concentrations in various tissues. After IAH oxaliplatin administration, we observed a significant decrease for total and filterable platinum in the Cmax compared with i.v. administration (12.4 versus 18.2 microg/l; p=0.02 and 11.2 versus 17.3 microg/l; p=0.02, respectively). Significant differences in various tissue concentrations were reported when comparing IAH and i.v. administration of oxaliplatin with IAH administration offering an advantage over i.v. administration. No differences in pharmacokinetic parameters or platinum tissue accumulation were apparent between the IAH and i.v. administration with cisplatin. We conclude that there is a significant pharmacokinetic advantage to using oxaliplatin for locoregional IAH chemotherapy compared with i.v. administration.     

Genetic predictors of inflammation in the risk of occupational asthma in young apprentices

BackgroundThe influence of genetic predictors of inflammation and atopy on occupational asthma in apprentices is not known.ObjectivesTo assess the influence of genetic polymorphisms of IL4RA, IL13, TNFA, IL1A, and IL5 on the decline of lung function and bronchial hyperresponsiveness in a prospective follow-up study of baker/pastry maker and hairdresser apprentices.MethodsA total of 351 apprentices were included in the study. We performed skin testing, spirometry, fractional exhaled nitric oxide measurement, and methacholine hyperreactivity testing at the initial visit and during and at the end of the 18-month training period. Gene variants of IL4RA, IL13, TNFA, IL1A, and IL5 were determined in DNA from nasal lavage.ResultsIL13 R130Q/IL4RA S478P or IL13 R130Q//IL4RA Q551R were significant predictors of the decrease of forced expiratory volume and forced vital capacity (P ≤ .006). Genotype GG of TNFAG308A was associated with bronchial hyperresponsiveness in the whole population and in nonatopic individuals (90.63% vs 9.38%; odds ratio, 3.78; 95% confidence interval, 1.10-12.83). TNFA GA and IL5 CC and TNFA GA and IL1A CC were 2 epistatic predictors of exhaled nitrogen monoxide decrease during follow-up (P = .02 and P = .004, respectively). The association with TNFA GA and IL1A CC was the most significant in nonatopic bakers (P < .001).ConclusionWe evidenced a predicting influence of IL13/IL4RA and TNFA in the early exposure to allergens and irritants that precedes occupational asthma. The significance of the associations in the absence of atopy suggests an influence of the genetics predictors related to inflammatory pathways.     

Detection of the van alphabet and identification of enterococci and staphylococci at the species level by multiplex PCR

A multiplex PCR assay was developed for detection of the six types of glycopeptide resistance characterized in enterococci and for identification of Enterococcus faecium, Enterococcus faecalis, Staphylococcus aureus, and Staphylococcus epidermidis at the species level. Primers targeting the genes vanA, vanB, vanC, vanD, vanE, vanG, and ddl of E. faecium and E. faecalis and nuc of S. aureus and a chromosomal portion specific to S. epidermidis were designed to allow amplification of fragments with various sizes. This specific and sensitive technique allows detection of glycopeptide-resistant strains, in particular methicillin-resistant S. aureus, that may escape phenotype-based automated rapid methods.     

Hereditary breast cancer and family cancer syndromes

Hereditary breast cancer (HBC) shows extant clinical and genetic heterogeneity. Clinically one finds the onset of breast cancer at an early age, an excess of bilaterality, and patterns of multiple primary cancer such as combinations of breast and ovarian carcinoma in the hereditary breast-ovarian cancer (HBOC) syndrome. In addition to HBOC, one sees a variety of putative breast cancer-prone genotypes inclusive of hereditary site-specific breast cancer, and the Li-Frameni (SBLA) syndrome that is characterized by cancers involving all three germinal layers including sarcomas, brain tumors, leukemia, lymphoma, and adrenal cortical carcinoma in addition to often markedly early-onset breast cancer. Breast cancer is also associated with autosomal dominantly inherited Cowden's disease and autosomal recessively inherited ataxia-telangiectasia. Examples of pedigrees depicting clincal examples of these several HBC syndromes are presented in order to describe HBC's heterogeneity. The recent identification of the BRCA1 gene in early-onset hereditary sitespecific breast cancer and the HBOC syndrome has led to new challenges for the genetic counselor. We review genetic counseling, which embraces surveillance and management recommendations that are responsive to the natural history of HBC and address the concept for future development of centers of expertise for HBC in the interest of improving cancer control.

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Resumen El cáncer mamario hereditario (CMH) exhibe una gran heterogeneidad clínica y genética. Desde el punto de vista clínico, se observa el comienzo del cáncer mamario en una edad temprana, una tasa considerable de bilateralidad, y patrones de múltiples cánceres primarios, tal como la combinación de carcinomas mamario y ovárico en el síndrome del cáncer de seno-ovario hereditarios (CSOH). Además del CSOH, se puede observar una variedad de genotipos putativos propensos al cáncer, incluyendo el cáncer mamario hereditario de ubicación específica y el síndrome de Li-Fraumeni, que se caracteriza por cánceres que afectan a todas las tres capas germinales, incluyendo sarcomas, tumores cerebrales, leucemia, linfoma y carcinoma adrenocortical, además de un notorio comienzo precoz del cáncer mamario. El cáncer mamario también se asocia con la enfermedad de Cowden hereditaria y autosómica dominante y con la ataxia-telangiectasia autosómicamente recesiva. Se presentan ejemplos de pedigríes que ilustran diversos síndromes de CMH, con el objeto de demostrar la heterogeneidad del CMH. La reciente identificación del gen BRCA1 en el cáncer mamario hereditario, de ubicación específica y de comienzo temprano, y el sindrome CSOH, ha significado nuevos desafíos para el consejero genético. En este artículo hacemos una revisión de la consejería genética que se refiere a la vigilancia y a las recomendaciones sobre manejo que corresponda a la historia natural del CMH, y enfocamos el concepto en cuanto al desarrollo de centros de especializados en CMH, con el propósito de mejorar el control del cáncer.

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Résumé Le cancer du sein héréditaire (CSH) est cliniquement et génétiquement hétérogène. au point de vue clinique, le cancer dbute habituellement à un âge jeune, est souvent bilatéral, et est parfois associé à d'autres cancers primitifs, comme par exemple dans le syndrome de cancer héréditaire du sein et de l'ovaire (SCO). On peut également observer d'autres génotypes présumés susceptibles de donner des cancers du sein héréditaires ainsi que le syndrome Li-Fraumeni (SBLA), caractérisé par l'envahissement des trois couches germinales et comprenant les tumeurs sarcomateuses, les tumeurs du cerveau, les leucémies, les lymphomes, et des cancers des corticosurrénales associées à des cancers du sein à un âge précoce. Le cancer du sein est parfois associé à la maladie de Cowden, une maladie autosomique dominante, et la télangiectasie ataxique, une maladie autosomique récessive. Des exemples de pedigrees de plusieurs types des CSH sont présentés, soulignant l'hétérogénéité de ce syndrome. La plus récente identification du gène BRCA1 et son rôle dans le cancer du sein et le syndrome HBOC est un nouveau challenge pour le généticien d'aujourd'hui. Nous passons en revue nos attitudes de conseil génétique comprenant à la fois les recommandations de surveillance nouveau challenge pour le généticien d'aujourd'hui. Nous passons en revue nos attitudes de conseil génétique comprenant à la fois les recommandations de surveillance et d'attitude thérapeutiques compatibles avec l'histoire naturelle des CSH, et qui ont trait au développement future des Centres spécialisés pour évaluer ces CSH dans le but l'améliorer leur contrôle.

     

AAC(3)-XI,a New Aminoglycoside 3-N-Acetyltransferase from Corynebacterium striatum

Corynebacterium striatum BM4687 was resistant to gentamicin and tobramycin but susceptible to kanamycin A and amikacin, a phenotype distinct among Gram-positive bacteria. Analysis of the entire genome of this strain did not detect any genes for known aminoglycoside resistance enzymes. Yet, annotation of the coding sequences identified 12 putative acetyltransferases or GCN5-related N-acetyltransferases. A total of 11 of these coding sequences were also present in the genomes of other Corynebacterium spp. The 12th coding sequence had 55 to 60% amino acid identity with acetyltransferases in Actinomycetales. The gene was cloned in Escherichia coli, where it conferred resistance to aminoglycosides by acetylation. The protein was purified to homogeneity, and its steady-state kinetic parameters were determined for dibekacin and kanamycin B. The product of the turnover of dibekacin was purified, and its structure was elucidated by high-field nuclear magnetic resonance (NMR), indicating transfer of the acetyl group to the amine at the C-3 position. Due to the unique profile of the reaction, it was designated aminoglycoside 3-N-acetyltransferase type XI.     

Antiphospholipid syndrome: From pathogenesis to novel immunomodulatory therapies

Antiphospholipid syndrome (APS) is characterized by recurrent thrombosis and pregnancy morbidity in association with the persistent presence of circulating antiphospholipid antibodies (aPL). APS remains the most frequent cause of acquired hypercoagulability and recurrent miscarriage. Long-term anticoagulation therapy is the only treatment with proven benefit in the APS. Anticoagulation is not effective in all patients and carries a risk of bleeding. Recent improvements in the understanding of the pathogenic mechanisms have led to the identification of potential targets and future therapies for APS. In contrast to non-specific anticoagulation, the emergence of immunomodulatory drugs provides the possibility of interfering with specific pathogenic pathways. Novel therapies might be used in the future for APS.