Identification of Heterotrophic Nanoflagellates by Restriction Fragment Length Polymorphism Analysis of Small Subunit Ribosomal DNA

Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the 'naked' flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.     

Gene transfer by jet injection into differentiated tissues of living animals and in organ culture

Jet injection can be used to introduce genes into the cells of differentiated tissues of living animals and organ cultures. When a solution of plasmid DNA is jet injected into a selected tissue or organ, cells lying in or near the path of the jet injection are transfected with the DNA and the introduced gene(s) are expressed. Since there is minimal morbidity from each jet injection, multiple injections can be performed at the same or nearby sites. Both mRNA and protein expression from transfected genes can be quantitated using standard methods. In addition, the technique is an efficient means of DNA immunization. Methodology for using jet injection to transfer plasmid DNA into the cells of skin, fat, mammary gland, and muscle are described.     

Quantitative study of enzyme immunocytochemical reactions performed with enzyme conjugates immobilized on nitrocellulose

Summary The nitrocellulose binding assay was used for quantitative studies on the cytochemical reactions for the three enzymes most frequently used in immunocytochemistry. The results show a linear relationship between the amount of enzyme immobilized on nitrocellulose and the amount of the enzyme reaction product. The similar course of the formation of the reaction product after DAB/H₂O₂ staining for peroxidase immobilized on nitrocellulose and for immunoperoxidase labeled cells indicates a linear relationship between the amount of enzyme-coupled antibodies bound to cells and the amount of enzyme reaction product. Furthermore, a mild acid treatment for the abolition of endogeneus peroxidase activity in tissues and cells applicable to immunoperoxidase staining procedures is proposed.In honour of Prof. P. van Duijn     

A putative urinary biosignature for diagnosis and follow-up of tuberculous meningitis in children: outcome of a metabolomics study disclosing host–pathogen responses

Introduction

Tuberculous meningitis (TBM) is a severe manifestation of tuberculosis, presenting with high morbidity and mortality in children. Existing diagnostic methods for TBM are invasive and time-consuming and the need for highly sensitive and selective diagnosis remains high on the TBM agenda.

Objective

Our aim was to exploit metabolomics as an approach to identify metabolites as potential diagnostic predictors for children with TBM through a non-invasive means.

Methods

Urine samples selected for this study were from three paediatric groups: patients with confirmed TBM (n = 12), patients clinically suspected with TBM but later confirmed to be negative (n = 19) and age-matched controls (n = 29). Metabolomics data were generated through gas chromatography–mass spectrometry analysis and important metabolites were identified according to standard statistical procedures used for metabolomics data.

Results

A global metabolite profile that characterized TBM was developed from the data, reflecting the host and microbial responses. Nine different logistic regression models were fitted to selected metabolites for the best combination as predictors for TBM. Four metabolites—methylcitric, 2-ketoglutaric, quinolinic and 4-hydroxyhippuric acids—showed excellent diagnostic ability and provided prognostic insight into our TBM patients.

Conclusions

This study is the first to illustrate holistically the metabolic complexity of TBM and provided proof-of-concept that a biosignature of urinary metabolites can be defined for non-invasive diagnosis and prognosis of paediatric TBM patients. The biosignature should be developed and validated through future prospective studies to generate a medical algorithm for diagnosis in the initial stages of the disease and for monitoring of treatment strategies.

     

Purification and properties of a constitutive beta-lactamase from Pseudomonas aeruginosa strain Dalgleish.

1. The beta-lactamase (penicillin amido-beta-lactamhydrolase EC 3.5.2.6) appeared to be periplasmic rather than truly intracellular, since it was released by freeze-thawing without gross morphological changes in the cell. 2. The partially purified enzyme had pI between 5.0 and 5.5, mol. wt 32 000 and a broad pH vs activity profile with a maximum at pH 8. 3. The cephalosporins tested were hydrolysed less rapidly than most of the penicillins, and the Km values for penicillins were lower than for cephalosporins. However cloxacillin was hydrolysed very slowly although it was strongly bound. The substrate-induced inactivation common to many beta-lactamases was particularly marked with cephaloridine and cloxacillinmthe cloxacillin-induced inactivation was shown to be reversible.     

An iPSC Line from Human Pancreatic Ductal Adenocarcinoma Undergoes Early to Invasive Stages of Pancreatic Cancer Progression

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