Lipolytic activity from Halobacteria: screening and hydrolase production

Strains of Halobacteria from an Algerian culture collection were screened for their lipolytic activity against p-nitrophenyl butyrate (PNPB) and p-nitrophenyl palmitate (PNPP). Most strains were active on both esters and 12% hydrolyzed olive oil. A strain identified as Natronococcus sp. was further studied. It grew optimally at 3.5 M NaCl, pH 8 and 40 degrees C. An increase in temperature shifted the optimum salt concentration range for growth from a wider range of 2-4 M, obtained at 25-30 degrees C, to a narrower range of 3.5-4 M, obtained at 35-40 degrees C. At 45 degrees C the optimum salt concentration was 2 M. These results show a clear correlation between salt and temperature requirement. The optimum conditions for the production of hydrolytic activity during growth were: 3.5 M NaCl and pH 8 for PNPB hydrolytic activity and 4 M NaCl and pH 7.5 for PNPP hydrolytic activity; both at 40 degrees C. The clear supernatant of cells grown at 4 M NaCl showed olive oil hydrolysis activity (in presence of 4 M NaCl) demonstrating the occurrence of a lipase activity in this strain. To our knowledge, this is the first report of a lipase activity at such high salt concentration.     

Prognostic value of CA 15.3 kinetics for metastatic breast cancer

Up to 80% of breast cancer patients developing metastases have high levels of CA 15.3. We studied the prognostic implications of CA 15.3 kinetics in 119 patients before and at first metastasis by univariate and multivariate statistics. At first metastasis, CA 15.3 was elevated in 82.4% of patients, with a lead time (median 162 days) in 42.0% of them. Kaplan-Meier analysis showed overall survival (median 1477 days) to be significantly related to estrogen receptor (ER) and progesterone receptor (PgR) status (p=0.0001) and tumor size (p=0.025). The interval between diagnosis and first abnormal CA 15.3 (p=0.0001), the CA 15.3 concentration (p=0.013), and the presence or absence of a lead time (p=0.001) also had prognostic value. ER and PgR status (p=0.0005 and p=0.0103, respectively), metastasis-free interval (p=0.0003), existence of a CA 15.3 lead time (p=0.0028), and days from diagnosis to first abnormal CA 15.3 (p=0.0055) entered in the Cox model. After first metastasis (median survival 573 days), ER and PgR status (p=0.0001 and p=0.0004, respectively), existence of a lead time for CA 15.3 (p=0.0138), and the concentration of first abnormal CA 15.3 (p=0.0145) had individual prognostic value. In the Cox model ER status (p=0.0001), nodal status (p=0.0191), existence of a lead time for CA 15.3 (p=0.0033), days from diagnosis to first abnormal CA 15.3 (p=0.0132), and concentration of first abnormal CA 15.3 (p=0.0320) were found to be independent prognostic variables. Compared to a matched historical control group that was not monitored by CA 15.3 assaying (n=140), the study group had a significantly longer survival after the first metastasis (p=0.0005). In conclusion, the kinetics of CA 15.3 before the first metastasis is of prognostic value. When associated with 18-fluorodeoxyglucose imaging, serial CA 15.3 assays may help to implement early treatment of metastases.     

Bioinformatics and molecular approaches to detect NRPS genes involved in the biosynthesis of kurstakin from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Degenerated primers designed for the detection by polymerase chain reaction of nonribosomal peptide synthetases (NRPS) genes involved in the biosynthesis of lipopeptides were used on genomic DNA from a new isolate of Bacillus thuringiensis CIP 110220. Primers dedicated to surfactin and bacillomycin detection amplified sequences corresponding respectively to the surfactin synthetase operon and to a gene belonging to a new NRPS operon identified in the genome of B. thuringiensis serovar pondicheriensis BSCG 4BA1. A bioinformatics analysis of this operon led to the prediction of an NRPS constituted of seven modules beginning with a condensation starter domain and which could be involved in the biosynthesis of a heptalipopeptide similar to kurstakin. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS) performed on whole cells of B. thuringiensis CIP 110220 confirmed the production of kurstakin by this strain. The kurstakin operon was thus used to design a new set of degenerated primers specifically to detect kurstakin genes. These primers were used to screen kurstakin producers in a collection of nine B. thuringiensis strains isolated from different areas in Algeria and two from the Pasteur Institute collection. For eight among the 11 tested strains, the amplified fragment matched with an operon similar to the kurstakin operon and found in the newly sequenced genome of Bacillus cereus or B. thuringiensis serovar pulsiensis, kurstaki, and thuringiensis. Kurstakin production was detected by MALDI-ToF-MS on whole cells for six strains. This production was compared with the spreading of the strains and their antimicrobial activity. Only the spreading can be correlated with the kurstakin production.     

Hepatic steatosis and low cardiorespiratory fitness in youth with type 2 diabetes

The purpose of this study was to examine the association between cardiorespiratory fitness, ectopic triglyceride accumulation, and insulin sensitivity among youth with and without type 2 diabetes. Subjects included 137 youth ages 13-18 years including 27 with type 2 diabetes, 97 overweight normoglycemic controls, and 13 healthy weight normoglycemic controls. The primary outcome measure was cardiorespiratory fitness defined as peak oxygen uptake indexed to fat free mass. Secondary outcomes included liver and muscle triglyceride content determined by (1)H-magnetic resonance spectroscopy and insulin sensitivity determined by frequently sampled intravenous glucose tolerance test. Despite similar measures of adiposity, peak oxygen uptake was 11% lower (38.9 ± 7.9 vs. 43.9 ± 6.1 ml/kgFFM/min, P = 0.002) and hepatic triglyceride content was nearly threefold higher (14.4 vs. 5.7%, P = 0.001) in youth with type 2 diabetes relative to overweight controls. In all 137 youth, cardiorespiratory fitness was negatively associated with hepatic triglyceride content (r = -0.22, P = 0.02) and positively associated with insulin sensitivity (r = 0.29, P = 0.002) independent of total body and visceral fat mass. Hepatic triglyceride content was also negatively associated with insulin sensitivity (r = -0.35, P < 0.001), independent of adiposity, sex, age, and peak oxygen uptake. This study demonstrated that low cardiorespiratory fitness and elevated hepatic triglyceride content are features of type 2 diabetes in youth. Furthermore, cardiorespiratory fitness and hepatic triglyceride are associated with insulin sensitivity in youth. Taken together, these data suggest that cardiorespiratory fitness and hepatic steatosis are potential clinical biomarkers for type 2 diabetes among youth.     

Caldimonas hydrothermale sp. nov., a novel thermophilic bacterium isolated from roman hot bath in south Tunisia

A polyphasic approach was used to characterize a bacterium, HAN-85^T, isolated from thermal water in natural thermal spring at Tozeur, an oasis in southwest Tunisia. The novel isolate was thermophilic, strictly aerobic and amylolytic bacterium, which stained Gram negative. Cells were short rods motile by means of a single polar flagellum. Their optimum temperature and pH required for growth were 55°C and pH 7, respectively. Comparative 16S rRNA gene sequence analyses showed that strain HAN-85^T belonged to the genus Caldimonas, with highest sequence similarity to the type strains Caldimonas manganoxidans and Caldimonas taiwanensis. DNA–DNA hybridization measurements revealed low DNA relatedness (35.2–44.5%) between the novel isolate and its closest relative, C. manganoxidans. The major cellular fatty acid components were 16:0, 17:0 cyclo and summed feature 3. The DNA G+C content was 68.3 mol%. Taken together, the results of DNA–DNA hybridization, fatty acids profile, physiological tests and biochemical analyses have allowed the genotypic and phenotypic differentiation of the isolate from currently recognized Caldimonas species. Therefore, we suggest that this isolate is a novel species within the genus Caldimonas and propose that it should be named Caldimonas hydrothermale sp. nov. The type strain is HAN-85^T (=DSM 18497^T =LMG 23755^T). The Gen Bank/Embl/DDBJ accession number for the 16S rRNA gene sequence of strain DSM 18497^T is AM283038.     

Serum HER-2 extracellular domain: relationship with clinicobiological presentation and prognostic value before and after primary treatment in 701 breast cancer patients

PURPOSE: To determine the clinical correlations and prognostic value of serum HER-2 (sHER-2) before and after primary breast cancer treatment. METHODS: sHER-2 from 701 consecutive patients with stage I-III tumors (median follow-up 7.7 years) was assayed by an enzyme-linked immunosorbent assay (Immuno 1, Bayer Diagnostics). RESULTS: The median pretreatment sHER-2 concentration was 8.30 ng/mL (range 3.15-82.00 ng/mL). Forty-seven patients (6.7%) had sHER-2 concentrations >12 ng/mL (cutoff level). Pretreatment sHER-2 correlated positively with CA 15.3 (p=0.0169), pathological tumor size (p=0.0082), number of invaded lymph nodes (pN, p=0.0160) and histological grading (p=0.0086). Kaplan-Meier analyses indicated that pretreatment sHER-2 was of prognostic value for contralateral breast cancer (p=0.0018), metastasis-free survival (MFS) (p=0.0008) - particularly lung (p=0.0082) and liver metastases (p=0.0035) - and overall disease-specific survival (DSS) (p=0.0020). According to pN status, pretreatment sHER-2 was of prognostic value only for pN-positive patients (p=0.0017). When combined with estradiol or progesterone receptor status, patients with elevated sHER-2 and receptor-negative tumors had a significantly shorter DSS (p<0.0001 for both receptors). Post-treatment sHER-2 also had individual prognostic value for MFS (p=0.0144) and DSS (p=0.0212). In multivariate analysis, only sHER-2 after primary treatment was an independent prognostic variable for MFS and DSS (p=0.0078 and p=0.0058, respectively). CONCLUSION: sHER-2 elevation in early breast cancer correlates with the principal criteria of tumor aggressiveness, thus permitting selection of patients with a high risk of visceral metastases and contralateral breast tumors. Post-treatment sHER-2 is an independent prognostic factor enabling to identify patients likely to benefit from aggressive adjuvant treatments.     

Is adjuvant tamoxifen recommended in post-menopausal node-negative breast cancer patients with high estrogen receptor values?

The impact of ER levels on the response to tamoxifen was evaluated in 1,623 postmenopausal primary breast cancer patients treated at our center (median follow-up 8.2 years). In patients receiving adjuvant tamoxifen a significantly longer disease-free survival (DFS) was observed when ER levels were elevated (p<0.00001). Very high ER (>424 fmol/mg protein) appeared to be detrimental in node-negative patients not treated with tamoxifen.