Factors influencing detection of quantitative cytomegalovirus antigenemia

Of 20 blood specimens testing positive for cytomegalovirus antigen after immediate processing, 19 (95%) remained positive when kept at room temperature for 24 h before processing. Quantitative antigenemia decreased by an average of 44% after storage. Compared with acetone fixation, formaldehyde fixation showed improved readability, fewer artifacts, and a higher degree of sensitivity.     

Blinded, externally controlled multicenter evaluation of light microscopy and PCR for detection of microsporidia in stool specimens. The Diagnostic Multicenter Study Group on Microsporidia

The quality parameters for the detection of microsporidia in identical sets of 50 stool samples were determined for six laboratories where technicians used light microscopy and for six laboratories where technicians used PCR. The average overall sensitivities were 67% (89% for patient samples only) for the PCR laboratories and 54% (80% for patient samples only) for the light microscopy laboratories. Specificities were 98 and 95%, respectively. Differences in results were most apparent between the individual laboratories rather than between the two major methods used.     

Antimalarial activities of polyhydroxyphenyl and hydroxamic acid derivatives

Several known mammalian ribonucleotide reductase inhibitors featuring a polyhydroxyphenyl and/or hydroxamate moiety as the active group were screened for potency in inhibiting growth of the malaria parasite Plasmodium falciparum. Compounds containing a 2,3- or 3,4-dihydroxyphenyl group as well as benzohydroxamate appear to be the most effective inhibitors of the malaria parasite.     

KIF2beta, a new kinesin superfamily protein in non-neuronal cells, is associated with lysosomes and may be implicated in their centrifugal translocation

Lysosomes concentrate juxtanuclearly in the region around the microtubule-organizing center by interaction with microtubules. Different experimental and physiological conditions can induce these organelles to move to the cell periphery by a mechanism implying a plus-end-directed microtubule-motor protein (a kinesin-like motor). The responsible kinesin-superfamily protein, however, is unknown. We have identified a new mouse isoform of the kinesin superfamily, KIF2beta, an alternatively spliced isoform of the known, neuronal kinesin, KIF2. Developmental expression pattern and cell-type analysis in vivo and in vitro reveal that KIF2beta is abundant at early developmental stages of the hippocampus but is then downregulated in differentiated neuronal cells, and it is mainly or uniquely expressed in non-neuronal cells while KIF2 remains exclusively neuronal. Electron microscopy of mouse fibroblasts and immunofluorescence of KIF2beta-transiently-transfected fibroblasts show KIF2 and KIF2beta primarily associated with lysosomes, and this association can be disrupted by detergent treatment. In KIF2beta-overexpressing cells, lysosomes (labeled with anti-lysosome-associated membrane protein-1) become abnormally large and peripherally located at some distance from their usual perinuclear positions. Overexpression of KIF2 or KIF2beta does not change the size or distribution of early, late and recycling endosomes nor does overexpression of different kinesin superfamily proteins result in changes in lysosome size or positioning. These results implicate KIF2beta as a motor responsible for the peripheral translocation of lysosomes.     

Determination of non-protein-bound plasma 1,25-dihydroxyvitamin D by symmetric (rate) dialysis

The biochemical and pharmacological properties of bioactive peptides and proteins can be altered by conjugation with polymers. This report describes site-specific attachment of insulin to activated carboxyl groups of carboxymethyl dextran (CMD, MW=51000) through the GlyA1 insulin amino group. On average, three or four insulin molecules were grafted to a CMD linear chain. Coupled insulin molecules were properly folded, and the bioactivity of conjugated insulin in the blood glucose depression assay was 9.6 IU/mg, which was only 2.6 times less than that for native insulin. The cell growth study indicated that the CMD-insulin conjugate was as mitogenic as insulin on vascular smooth muscle cells, whereas the starting CMD polymer was not. The insulin receptor binding constant of the conjugate (3.6 x 10[9] M[-1]) compared well with that of native insulin (7.6 x 10[9] M[-1]), indicating that the CMD chain does not present any major constraints to binding. Plasma clearance of CMD-insulin obeyed a two-compartment pharmacokinetic (PK) model with a CMD-insulin conjugate plasma elimination half-life of 114.1 min, which was significantly longer than that of soluble Zn-insulin (12.4 min). In contrast, pharmacodynamic (PD) profiles (blood glucose lowering effects) after intravenous (iv) administration of the conjugate or insulin in rats were not different. Subcutaneous (sc) administration of the conjugate resulted in a significantly prolonged plasma profile with a noncompartmental PK parameter mean residence time (MRT) of 103.5 min which was significantly longer than that of soluble Zn-insulin (40.5 min). This was reflected in the protracted PD effect of sc administered conjugate with time needed to reach minimum glucose concentration Tnadir of 95.7 min, which was significantly longer than that of insulin (62 min). We conclude that the conjugation of insulin to CMD leads to a bioactive conjugate with a delayed sc PD profile showing prolonged response, resembling intermediate acting insulin preparations.     

Analytical methods for the determination of 3-chloro-1,2-propandiol and 2-chloro-1,3-propandiol in hydrolysed vegetable protein, seasonings and food products using gas chromatography/ion trap tandem mass spectrometry

The EC Scientific Committee for Foods and more recently the Food Advisory Committee in the UK have proposed that levels of 3-chloro-1,2-propanediol (3-MCPD) in foods and ingredients should be reduced to the lowest possible. This paper reports on the development of methods for the determination of parts-per-billion (microgram/kg) levels of 3-MCPD in hydrolysed vegetable protein (HVP), flour, bread, meat and starch products using gas chromatography/ion-trap tandem mass spectrometry (GC/ITMS/MS). Mass spectrometer conditions for detecting 3-MCPD and the stable isotope internal standard (3-chloro-1,2-propandiol-d7) were established. Candidate extraction methods were initially evaluated for recovery and repeatability by spiking selected commodities at a level of 100 micrograms/kg. Extracts of ingredients and foods prepared by the candidate extraction methods were examined by GC/ITMS/MS using samples spiked at a level of 25 micrograms/kg. The results showed that detection limits of between 3 and 5 micrograms/kg could be achieved for all commodities.