Comparison of two β-glucosidases for the enzymatic synthesis of β-(1-6)-β-(1-3)-gluco-oligosaccharides

A domain of epiglucan was synthesized by beta-glucosidases. Two beta-glucosidases, an extracellular beta-glucosidase derived from Sclerotinia sclerotiorum grown on xylose, and a commercial lyophilized preparation of beta-glucosidase from Aspergillus niger, were used to synthesize gluco-oligosaccharides from cellobiose and, specially, beta-(1-6) branched beta-(1-3) gluco-oligosaccharides, corresponding to the structure of epiglucan. Gentiobiose, cellotriose, cellotetraose, beta-Glc-(1-3)-beta-Glc-(1-4)-Glc, beta-Glc-(1-6)-beta-Glc-(1-4)-Glc and beta-Glc-(1-6)-beta-Glc-(1-3)-Glc were synthesized from cellobiose by both enzymes. The latter compound was preferentially synthesized by the beta-glycosidase from Sclerotinia sclerotiorum. Under the best conditions, only 7 g l(-1) of beta-Glc-(1-6)-beta-Glc-(1-3)-Glc was synthesized by the beta-glycosidase from Aspergillus niger compared to 20 g l(-1) synthesized with beta-glycosidase from Sclerotinia sclerotiorum.     

Fine needle aspiration cytologic diagnosis of toxoplasma lymphadenitis. A case report with detection of a Toxoplasma bradycyst in a Papanicolaou-stained smear

BACKGROUND: Fine needle aspiration (FNA) cytologic diagnosis of toxoplasmic lymphadenitis with demonstration of a tissue cyst containing bradyzoites has been very rarely reported. CASE: A 17-year-old female presented with a mobile, painless, 2-cm-diameter swelling over the right suprascapular area. Clinical diagnosis was lipoma. FNA smears showed features of reactive lymphoid hyperplasia, including tingible body macrophages and groups of epithelioid histiocytes. A Toxoplasma cyst with bradyzoites was also demonstrated in a Papanicolaou-stained smear. Following FNA cytodiagnosis, serologic tests revealed a high titer of IgG and the presence of IgM-specific antibodies to Toxoplasma gondii, indicating active/recent disease. CONCLUSION: FNA cytology is a valuable tool for the diagnosis of toxoplasmic lymphadenitis. Papanicolaou stain is appropriate for demonstration of the parasite. Serology is an excellent adjunct in clinching the diagnosis.     

Cadmium effects on lipid metabolism of rape (Brassica napus L.)

Treatment of rape seedlings with increasing CdCl₂ concentrations in the culture medium resulted in a cadmium accumulation within plant tissues, which increased with external metal dose; such accumulation was more important in roots than in leaves. Biomass production was severely inhibited, even at low cadmium concentration. In leaves, quantities of chloroplastic lipids, monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfolipids (SL) and phosphatidylglycerol (PG) decreased sharply under metallic treatment. However, contents of extrachloroplastic lipids, mainly phosphatidylcholine (PC) and phosphatidylethanolamine (PE) increased significantly. In contrast to leaves, contents of root phospholipids decreased. Likewise, levels of tri-unsaturated fatty acids: linolenic (C18:3) and hexadecatrienoïc (C16:3) dropped in leaves of treated seedlings as compared to those of controls, suggesting that heavy metals induced an alteration in the fatty acid desaturation process or a stimulation of their peroxidation. Also, trans palmitoleic acid (C16:1-trans) level in PG decreased considerably. In roots, there was a slight decrease in C18:3 level, with a concomitant increase in the C18:2 percentage. Radioactive labelling of leaf lipids with (1-¹⁴C) acetate allowed to show that fatty acid biosynthesis was noticeably altered at the highest cadmium dose used (50 μM). Biosynthesis of tri-unsaturated fatty acids was also inhibited which may explain the decline in non-labelled lipid contents. Results showed that metallic ion seems to affect selectively chloroplastic membranes due to an inhibition of polyunsaturated fatty acid biosynthesis. Moreover, a lipid peroxidation occurred in our case because of the spectacular increase of malondialdehyde (MDA) content observed in cadmium treated leaves. To cite this article: N. Ben Youssef et al., C. R. Biologies 328 (2005).     

Comparative Analysis Between Pseudomonas aeruginosa Genotypes and Severity of Symptoms in Patients with Unilateral or Bilateral Otitis Externa

Random amplified polymorphic DNA (RAPD) analysis was done on 32 isolates of Pseudomonas aeruginosa. These isolates were obtained from 22 patients who presented to the emergency room in a major medical center in Beirut, Lebanon, during a 5-month period with the diagnosis of either unilateral or bilateral otitis externa. Patients had yellowish to greenish discharge, moderate to severe external auditory canal swelling, moderate to severe pain, and periauricular cellulitis. None of these patients had intrinsic predisposing factors. An ear swab was obtained from both ears of patients, cultured on trypticase soy agar. P. aeruginosa was identified on the basis of pyocyanine production and API identification kits. RAPD analysis was done by using two primers (10 mer and 21 mer primers) and appropriate PCR conditions on extracted DNA. Our data have shown 23 RAPD patterns (A–W) distributed among the 32 P. aeruginosa isolates. RAPD patterns were reproducible. Twenty of 32 isolates were recovered from 10 patients with bilateral otitis externa. The remaining 12 of 32 isolates were recovered from 12 different patients with unilateral otitis externa. Eleven RAPD patterns (A,B,C,D,E,F,H,I,R,U,V) were associated with severe clinical symptoms, including severe pain, severe external auditory canal swelling, periauricular cellulitis, and a yellowish discharge. The remaining RAPD patterns were not associated with severe infections. This denotes a possible association between certain genotypes and severity of symptoms. Received: 6 July 2000 / Accepted: 5 September 2000     

A Novel Simple Method for Determining CYP2D6 Gene Copy Number and Identifying Allele(s) with Duplication/Multiplication

Background

Cytochrome P450 2D6 (CYP2D6) gene duplication and multiplication can result in ultrarapid drug metabolism and therapeutic failure or excessive response in patients. Long range polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequencing are usually used for genotyping CYP2D6 duplication/multiplications and identification, but are labor intensive, time consuming, and costly.

Methods

We developed a simple allele quantification-based Pyrosequencing genotyping method that facilitates CYP2D6 copy number variation (CNV) genotyping while also identifying allele-specific CYP2D6 CNV in heterozygous samples. Most routine assays do not identify the allele containing a CNV. A total of 237 clinical and Coriell DNA samples with different known CYP2D6 gene copy numbers were genotyped for CYP2D6 *2, *3, *4, *6, *10, *17, *41 polymorphisms and CNV determination.

Results

The CYP2D6 gene allele quantification/identification were determined simultaneously with CYP2D6*2, *3, *4, *6, *10, *17, *41 genotyping. We determined the exact CYP2D6 gene copy number, identified which allele had the duplication or multiplication, and assigned the correct phenotype and activity score for all samples.

Conclusions

Our method can efficiently identify the duplicated CYP2D6 allele in heterozygous samples, determine its copy number in a fraction of time compared to conventional methods and prevent incorrect ultrarapid phenotype calls. It also greatly reduces the cost, effort and time associated with CYP2D6 CNV genotyping.     

Atorvastatin prevents <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> cytoadherence and endothelial damage

Background

The adhesion of Plasmodium falciparum parasitized red blood cell (PRBC) to human endothelial cells (EC) induces inflammatory processes, coagulation cascades, oxidative stress and apoptosis. These pathological processes are suspected to be responsible for the blood-brain-barrier and other organs' endothelial dysfunctions observed in fatal cases of malaria. Atorvastatin, a drug that belongs to the lowering cholesterol molecule family of statins, has been shown to ameliorate endothelial functions and is widely used in patients with cardiovascular disorders.

Methods

The effect of this compound on PRBC induced endothelial impairments was assessed using endothelial co-culture models.

Results

Atorvastatin pre-treatment of EC was found to reduce the expression of adhesion molecules and P. falciparum cytoadherence, to protect cells against PRBC-induced apoptosis and to enhance endothelial monolayer integrity during co-incubation with parasites.

Conclusions

These results might suggest a potential interest use of atorvastatin as a protective treatment to interfere with the pathophysiological cascades leading to severe malaria.

     

A bioinformatics approach for biomarker identification in radiation-induced lung inflammation from limited proteomics data

Many efforts have been made to discover novel bio-markers for early disease detection in oncology. However, the lack of efficient computational strategies impedes the discovery of disease-specific biomarkers for better understanding and management of treatment outcomes. In this study, we propose a novel graph-based scoring function to rank and identify the most robust biomarkers from limited proteomics data. The proposed method measures the proximity between candidate proteins identified by mass spectrometry (MS) analysis utilizing prior reported knowledge in the literature. Recent advances in mass spectrometry provide new opportunities to identify unique biomarkers from peripheral blood samples in complex treatment modalities such as radiation therapy (radiotherapy), which enables early disease detection, disease progression monitoring, and targeted intervention. Specifically, the dose-limiting role of radiation-induced lung injury known as radiation pneumonitis (RP) in lung cancer patients receiving radiotherapy motivates the search for robust predictive biomarkers. In this case study, plasma from 26 locally advanced non-small cell lung cancer (NSCLC) patients treated with radiotherapy in a longitudinal 3 × 3 matched-control cohort was fractionated using in-line, sequential multiaffinity chromatography. The complex peptide mixtures from endoprotease digestions were analyzed using comparative, high-resolution liquid chromatography (LC)-MS to identify and quantify differential peptide signals. Through analysis of survey mass spectra and annotations of peptides from the tandem spectra, we found candidate proteins that appear to be associated with RP. On the basis of the proposed methodology, α-2-macroglobulin (α2M) was unambiguously ranked as the top candidate protein. As independent validation of this candidate protein, enzyme-linked immunosorbent assay (ELISA) experiments were performed on independent cohort of 20 patients' samples resulting in early significant discrimination between RP and non-RP patients (p = 0.002). These results suggest that the proposed methodology based on longitudinal proteomics analysis and a novel bioinformatics ranking algorithm is a potentially promising approach for the challenging problem of identifying relevant biomarkers in sample-limited clinical applications.     

Evaluation of a duplex real-time PCR assay to detect MRSA from broth culture,human sera seeded with MRSA and from patient's serum

The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient''s serum. One hundred and thirty-five clinical isolates of MRSA strains and different species were utilised in this study. In addition, a pilot study with 9 patients'' serum samples was performed. The sensitivity and specificity values for this assay were 99% and 100% respectively. The detection limit for this method was 1.23×10² CFU/ml from the serum seeded with MRSA cells and the limiting concentration of DNA for detection was 18 fg, which equates to 5.14 genomic DNA copies. In addition, this assay detected MRSA from patient''s serum (7 out of 9) with sensitivity of 77.8%. Overall, the assay was rapid, efficient, sensitive and easy to perform.     

Through scaffolding and catalytic actions nucleoside diphosphate kinase B differentially regulates basal and β-adrenoceptor-stimulated cAMP synthesis

β-adrenoceptors (βAR) play a central role in the regulation of cAMP synthesis and cardiac contractility. Nucleoside diphosphate kinase B (NDPK B) regulates cAMP signalling by complex formation with Gβγ dimers thereby activating and stabilizing heterotrimeric G_s proteins, key transducer of βAR signals into the cell. Here, we explored the requirement of NDPK B for basal and βAR-stimulated cAMP synthesis and analysed the underlying mechanisms by comparing wild-type NDPK B (WT) and its catalytically inactive H118N mutant. Stable overexpression of both WT- and H118N-NDPK B in cardiomyocyte derived H10 cells increased the plasma membrane content of G_s and caveolin-1 and thus enhanced the isoproterenol (ISO)-stimulated cAMP-synthesis by about 2-fold. Conversely, the loss of NDPK B in embryonic fibroblasts from NDPK A/B-depleted mice was associated with a severe reduction in membranous G_s protein and carveolin-1 content causing a marked decrease in basal and ISO-induced cAMP formation. Re-expression of NDPK B, but not of NDPK A, was able to rescue this phenotype. Both, re-expression of WT- and H118N-NDPK B induced the re-appearance of G_s and caveolin-1 at the plasma membrane to a similar extent. Accordingly, WT- and H118N-NDPK B similarly enhanced ISO-induced cAMP formation. In contrast, the catalytically inactive H118N-NDPK B was less potent and less effective in rescuing basal cAMP production. Identical results were obtained in neonatal rat cardiac myocytes after siRNA-induced knockdown and adenoviral re-expression of NDPK B.Our data reveal that NDPK B regulates G_s function by two different mechanisms. The complex formation of NDPK B with G_s is required for the stabilization of the G protein content at the plasma membrane. In addition, the NDPK B-dependent phosphotransfer reaction, which requires the catalytic activity, specifically allows a receptor-independent, basal G_s activation.