Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis

The pyrazinamidase from Mycobacterium smegmatis was purified to homogeneity to yield a product of approximately 50 kDa. The deduced amino-terminal amino acid sequence of this polypeptide was used to design an oligonucleotide probe for screening a DNA library of M. smegmatis. An open reading frame, designated pzaA, which encodes a polypeptide of 49.3 kDa containing motifs conserved in several amidases was identified. Targeted knockout of the pzaA gene by homologous recombination yielded a mutant, pzaA::aph, with a more-than-threefold-reduced level of pyrazinamidase activity, suggesting that this gene encodes the major pyrazinamidase of M. smegmatis. Recombinant forms of the M. smegmatis PzaA and the Mycobacterium tuberculosis pyrazinamidase/nicotinamidase (PncA) were produced in Escherichia coli and were partially purified and compared in terms of their kinetics of nicotinamidase and pyrazinamidase activity. The comparable Km values obtained from this study suggested that the unique specificity of pyrazinamide (PZA) for M. tuberculosis was not based on an unusually high PZA-specific activity of the PncA protein. Overexpression of pzaA conferred PZA susceptibility on M. smegmatis by reducing the MIC of this drug to 150 micrograms/ml.     

Quantitation of vancomycin and its crystalline degradation product (CDP-1) in human serum by high performance liquid chromatography

Mantle cell lymphoma (MCL) patients represent a difficult problem, sometimes to establish the diagnosis but mostly because of their refractoriness to standard lymphoma treatments. Which treatments to apply and to whom is not yet defined. In this study, we attempted to analyze the clinical features, to identify the major prognostic factors, and to evaluate the outcome of 121 MCL patients treated in our institution between 1979 and 1997. Clinical data, treatment modalities, and International Prognostic Index (IPI) score were evaluated. Median age was 63 years. Patients usually presented with advanced stage disease (87%), disseminated lymph nodes (57%), bone marrow involvement (79%), but with a good performance status (PS) (81%). Lymphocytosis >4000/microl and/or peripheral blood involvement was present in 36% of cases, and gastrointestinal disease in 18%. The t(11;14)(q13;q32) and/or bcl-1 rearrangement was detected in 47/57 studied cases. Median overall survival (OS) was 3.12 years and a longer survival was significantly associated with younger age (<70 years), good PS (<2), localized disease (stage I-II), fewer than two extra-nodal sites, absence of spleen or peripheral blood involvement, normal serum LDH and beta2-microglobulin levels, and hemoglobin level greater than 12 g/dl. However, the IPI failed to identify patients with longer OS and in a multiparametric analysis, only older age, hemoglobin less than 12 g/dl, poor PS, and blood involvement were associated with a poorer outcome. Treatment modalities had no impact on survival with 75% of patients relapsing or progressing. Our data showed that the poor outcome of MCL patients is mainly related to adverse patient characteristics, a highly disseminated tumor, and some unknown parameters associated with the refractoriness to standard therapy.     

Spontaneous thrombosis of a basilar artery traumatic aneurysm in a child

Traumatic aneurysms are rare and occur most commonly in young adults; however, the relative frequency in the pediatric population is high, owing to the low prevalence of congenital saccular aneurysms in children. Traumatic aneurysms typically involve the anterior circulation, and spontaneous thrombosis is uncommon; hence, surgery is usually necessary. We present a case of a posttraumatic aneurysm in a child that occurred after a fall from a large height and that spontaneously thrombosed.     

Characterisation of amorphous carbon coatings for magnetic storage devices via AFM‐Nanoscratching devices. AFM‐Nanoscratching an amorphen Kohlenstoffschichten für die Speichertechnologie

The mechanical properties of ultra‐thin amorphous carbon films used as protective coatings for magnetic storage devices were investigated by means of atomic force microscopy (AFM). Diamond‐tipped cantilevers were used in order to generate scratches with residual scratch depths of only a few Angstroms and even below. The presented method simulates mechanical strains at the head‐disk interface. A driftcompensating image subtracting technique allows the visualisation of these ultra‐shallow scratches and enables the mechanical characterisation of only few Nanometer films widely independent from the hardness of the substrate. The scratch resistance as it is defined here correlates well with the mass density and the sp³ content of the investigated films.     

Possible functions of peptide antibiotics during growth of producer organisms: bacitracin and metal (II) ion transport

The inhibitory effect of bacitracin upon growth of the producer strain Bacillus licheniformis ATCC 10716 was dependent upon the presence of several different metal (II) ions, particularly Mn (II), Co (II), or Zn (II) ions. This supports our previous suggestion that the normal function of bacitracin during growth of the producer organism may be to promote the uptake of several divalent metal ions. Due to the striking similarity between the antimicrobial effect of bacitracin towards susceptible organisms and the effect of bacitracin towards the producer organisms B. licheniformis ATCC 10716, the possibility that the antimicrobial effect of bacitracin may be an induction of uptake of toxic amounts of metal ions is discussed. The possibility that peptide antibiotics may normally participate in ion transport during growth of producer organisms is also discussed.     

Regional differences in stratum corneum reactivity to surfactants. Quantitative assessment using the corneosurfametry bioassay

A highly sensitive and specific chemiluminescent immunoassay (CLIA) was developed for quantification of growth hormone (GH) in salmonid species. The CLIA for salmon GH was performed using the sandwich method with anti-GH IgG as the first antibody and chemiluminescent acridinium ester-labelled specific anti-GH F(ab')2 as the second antibody. The measurable range of salmon GH in the CLIA was 39-1250 pg/mL using a short assay (1 day) protocol and 3.9-125 pg/mL in a longer (2-day) assay. The dilution curve in the CLIA of serum from masu salmon (Oncorhynchus masou) was parallel to the standard curve of recombinant chum salmon (Oncorhynchus keta) GH. Seasonal changes of serum GH levels were measured in 1 year-old masu salmon cultivated in a pond from March to November. Their serum GH levels increased during smoltification from March to April, achieved a maximum level of 21 ng/mL in August, and then declined gradually to 11 ng/mL in October.     

Human mesotheliomas contain the simian virus-40 regulatory region and large tumor antigen DNA sequences

BACKGROUND: A cohort (20%) of patients with mesothelioma will not have an exposure to asbestos. Recently, a DNA tumor virus (simian virus 40) has been shown to cause hamster mesotheliomas; we previously described simian virus 40-like DNA amino terminus sequences in 29 of 48 mesotheliomas. We analyzed an additional 42 mesotheliomas to determine (1) whether our initial observations were durable and (2) the extent to which the simian virus 40 genome is present in mesotheliomas. METHODS: Genomic DNA was extracted from snap frozen mesothelioma tumor samples and from the simian virus 40-induced hamster mesothelioma tumor H9A. Polymerase chain reaction primers were used to amplify various simian virus 40 large T-antigen regions including a 105-base pair amino terminus fragment, a 281-base pair carboxyl terminus fragment, and a 310-base pair fragment of the enhancer promoter region. Endonuclease digestions and Southern blotting were used to verify the expected product. RESULTS: Thirty of the 42 (71%) samples amplified T-antigen amino sequences, and specificity was verified by Southern hybridization. Sixteen of 42 samples (38%) amplified the appropriate size fragment for the carboxyl terminus, and digestion with BsaB1 matched that of H9A. Twenty-two of 42 samples (52%) amplified simian virus 40 regulatory sequences and Fok1 digestion matched that of the hamster control tumor. Sequence analysis (4 patients) revealed 100% homology with the regulatory region of simian virus 40 strain 776. CONCLUSIONS: These data suggest an association between the simian virus 40 virus and human mesothelioma that could be exploited for diagnostic/therapeutic options including early detection and potential vaccination strategies.     

Neoadjuvant chemotherapy and bladder-sparing surgery for invasive bladder cancer: ten-year outcome

PURPOSE: To evaluate the 10-year outcome of patients with invasive (T2-3N0M0, staged according to the tumor, node, metastasis system) bladder cancer who responded completely to a combination of methotrexate, vinblastine, adriamycin, and cisplatin (MVAC) chemotherapy followed by bladder-sparing surgery. PATIENTS AND METHODS: Of 111 surgical candidates who received neoadjuvant MVAC, 60 (54%) achieved a complete clinical response (T0) on transurethral resection (TUR) of the primary tumor site. Of these, 28 requested follow-up with TUR alone, 15 had a partial cystectomy, and 17 elected a radical cystectomy. The patients were followed up for a median of 10 years (range, 8 to 13 years). RESULTS: Of 43 patients who had bladder-sparing surgery, 32 (74%) are alive, which includes 25 (58%) with an intact functioning bladder. Twenty-four patients (56%) developed bladder tumor recurrences from 5 to 96 months, which were invasive in 13 (30%) and superficial in 11 (26%). Thirteen patients required a salvage cystectomy, of whom 6 died, which includes 4 (9%) from a new invasive neoplasm. Of the 17 patients who had radical cystectomy, 11 (65%) are alive. CONCLUSION: The majority of patients with invasive bladder tumors who achieve T0 status after neoadjuvant MVAC chemotherapy preserve their bladders for up to 10 years with bladder-sparing surgery. The bladder remains at risk for new invasive tumors. Cystectomy salvages the majority, but not all, of relapsing patients.