The evaluation of the oxidative state of native-LDL: three methods compared

LDL-oxidation is considered a contributing factor to the development of atherosclerotic lesions. However, to utilise the oxidative state of LDL as a marker of cardiovascular risk, reliable analytical methods for its detection must be defined. We have compared three methods for their capacity to evaluate the difference in the oxidation state of isolated LDL subjected to either dialysis (D-LDL) or gel filtration (F-LDL) to remove EDTA. Their susceptibility to oxidation promoted by Cu(2+) was monitored by following the time course of conjugated diene (CD) and lipid hydroperoxide (ROOH) accumulation. The relative electrophoretic mobility (REM) of the same LDL samples was evaluated by capillary electrophoresis. As measured by all three methods, F-LDL are less prone to oxidation than D-LDL when added with CuSO(4). REM of F-LDL and D-LDL significantly differs already before the addition of the metal catalyst, whereas CD and ROOH contents become significantly different only after it. Besides confirming that a rapid centrifugation followed by gel filtration is a more convenient procedure than dialysis to remove EDTA during LDL isolation, our study suggests the REM of isolated-LDL as the biochemical marker of choice in the evaluation of its oxidative state.     

Infectivity analysis of two variable DNA B components of<Emphasis Type="Italic">Mungbean yellow mosaic virus-Vigna</Emphasis> in<Emphasis Type="Italic">Vigna mungo</Emphasis> and<Emphasis Type="Italic">Vigna radiata</Emphasis>

Mungbean yellow mosaic virus-Vigna (MYMV-Vig), aBegomovirus that causes yellow mosaic disease, was cloned from field-infected blackgram (Vigna mungo). One DNA A clone (KA30) and five different DNA B clones (KA21, KA22, KA27, KA28 and KA34) were obtained. The sequence identity in the 150-nt common region (CR) between DNA A and DNA B was highest (95%) for KA22 DNA B and lowest (85·6%) for KA27 DNA B. The Rep-binding domain had three complete 11 -nt (5’-TGTATCGGTGT-3′) iterons in KA22 DNA B (and KA21, KA28 and KA34), while the first iteron in KA27 DNA B (5’-ATCGGTGT-3’) had a 3-nt deletion. KA27 DNA B, which exhibited 93·9% CR sequence identity to the mungbean-infecting MYMV, also shared the 3-nt deletion in the first iteron besides having an 18-nt insertion between the third iteron and the conserved nonanucleotide. MYMV was found to be closely related to KA27 DNA B in amino acid sequence identity of BV1 (94·1%) and BC1 (97·6%) proteins and in the organization of nuclear localization signal (NLS), nuclear export signal (NES) and phosphorylation sites. Agroinoculation of blackgram (V. mungo) and mungbean (V. radiata) with partial dimers of KA27 and KA22 DNA Bs along with DNA A caused distinctly different symptoms. KA22 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in blackgram. In contrast, KA27 DNA B caused more intense yellow mosaic symptoms with high viral DNA titre in mungbean. Thus, DNA B of MYMV-Vig is an important determinant of host-range betweenV. mungo andV. radiata.     

Unexpected intracellular localization of the AMD-associated cystatin C variant

Cystatin C is abundantly expressed by the retinal pigment epithelium (RPE) of the eye. Targeting of cystatin C to the Golgi apparatus and processing through the secretory pathway of RPE cells are dependent upon a 26-amino acid signal sequence of precursor cystatin C. A variant with an alanine (A) to threonine (T) mutation in the penultimate amino acid of the signal sequence (A25T) was recently correlated with increased risk of developing exudative age-related macular degeneration. The biochemical consequence of the A25T mutation upon targeting of the protein is reported here. Targeting and trafficking of full-length mutant (A25T) precursor cystatin C-enhanced green fluorescent protein fusion protein were studied in living, cultured retinal pigment epithelial and HeLa cells. Confocal microscopy studies were substantiated by immunodetection. In striking contrast to wild-type precursor cystatin C fusion protein conspicuously targeted to the Golgi apparatus, the threonine variant was associated principally with mitochondria. Some diffuse fluorescence was also observed throughout the cytoplasm and nucleus (but not nucleoli). Secretion of fusion protein derived from the threonine variant was reduced by approximately 50% compared with that of the wild-type cystatin C fusion protein. Expression of the variant fusion protein did not appear to impair expression or secretion of endogenous cystatin C.     

Fractionation and characterization of kinesin II species in vertebrate brain

Recent research on kinesin motors has outlined the diversity of the superfamily and defined specific cargoes moved by kinesin family (KIF) members. Owing to the difficulty of purifying large amounts of native motors, much of this work has relied on recombinant proteins expressed in vitro. This approach does not allow ready determination of the complement of kinesin motors present in a given tissue, the relative amounts of different motors, or comparison of their native activities. To address these questions, we isolated nucleotide-dependent, microtubule-binding proteins from 13-day chick embryo brain. Proteins were enriched by microtubule affinity purification, then subjected to velocity sedimentation to separate the 20S dynein/dynactin pool from a slower sedimenting KIF containing pool. Analysis of the latter pool by anion exchange chromatography revealed three KIF species: kinesin I (KIF5), kinesin II (KIF3), and KIF1C (Unc104/KIF1). The most abundant species, kinesin I, exhibited the expected long range microtubule gliding activity. By contrast, KIF1C did not move microtubules. Kinesin II, the second most abundant KIF, could be fractionated into two pools, one containing predominantly A/B isoforms and the other containing A/C isoforms. The two motor species had similar activities, powering microtubule gliding at slower speeds and over shorter distances than kinesin I.     

Metabolic effects and the methylenetetrahydrofolate reductase (MTHFR) polymorphism associated with neural tube defects in southern Brazil

BACKGROUND: The importance of metabolic factors in neural tube defects (NTDs) has been the focus of many investigations. Several authors have suggested that abnormalities in homocysteine metabolism, such as hyperhomocysteinemia, folate deficiency, and low vitamin B12, may be responsible for these malformations and that both nutritional factors and genetic abnormalities are associated with them. METHODS: We conducted a case-control study to investigate the influence of biochemical and genetic factors in NTDs in infants in southern Brazil. Levels of folate, vitamin B12, total homocysteine (t-Hcy) and the 677C>T and 1298A>C polymorphisms of the MTHFR gene were analyzed in 41 NTD child-mother pairs and 44 normal child-mother control pairs. RESULTS: Subjects in the case group had a higher mean blood folate level than those in the control group. The level of vitamin B12 was lower in mothers in the NTD group than in control mothers (p = 0.004). The level of t-Hcy was not different in the two groups, but t-Hcy and vitamin B12 were correlated (p = 0.002). There was no difference in the genotype distribution for 677C>T and 1298A>C polymorphisms of MTHFR in the case and control pairs. The level of t-Hcy was correlated with 677TT. CONCLUSIONS: Despite the small sample in this study, we suggest that low vitamin B12 and, consequently, hyperhomocysteinemia are important risk factors for NTDs in our population.     

Expression of Fibroblast growth factor 19 (Fgf19) during chicken embryogenesis and eye development, compared with Fgf15 expression in the mouse

The normal development of eyes relies on proper signaling through Fibroblast growth factor (FGF) receptors, but the source and identity of cognate ligands have remained largely unknown. We have found that Fgf19 is expressed in the developing chicken retina. In situ hybridization discloses dynamic expression patterns for Fgf19 in the optic vesicle, lens primordia and retinal horizontal cells. Overall expression pattern of Fgf19 during chicken embryogenesis was also examined: Fgf19 is expressed in the regions associated with cranial placodes induction, boundary regions of rhombomeres, somites, specific groups of neural cells in midbrain, hindbrain, and those derived from epibranchial placodes, and the apical ectodermal ridge of limb buds. Expression pattern of the Fgf19-orthologous gene Fgf15 was further examined in the mouse developing eye. Fgf15 is expressed in the optic vesicle, a subset of progenitor cells of neural retina, and emerging ganglion and amacrine cells during retinogenesis.     

Colloidal gold-based immunochromatographic assay for detection of botulinum neurotoxin type B

A rapid immunochromatographic assay was developed to detect botulinum neurotoxin type B (BoNT/B). The assay was based on the sandwich format using polyclonal antibody (Pab). The thiophilic gel purified anti-BoNT/B Pab was immobilized to a defined detection zone on a porous nitrocellulose membrane and conjugated to colloidal gold particles that served as a detection reagent. The BoNT/B-containing sample was added to the membrane and allowed to react with Pab-coated particles. The mixture was then passed along the porous membrane by capillary action past the Pab in the detection zone, which will bind the particles that had BoNT/B bound to their surface, giving a red colour within this detection zone with an intensity proportional to BoNT/B concentration. In the absence of BoNT/B, no immunogold was bound to the solid-phase antibody. With this method, 50 ng/ml of BoNT/B was detected in less than 10 min. The assay sensitivity can be increased by silver enhancement to 50 pg/ml. The developed BoNT/B assay also showed no cross reaction to type A neurotoxin (BoNT/A) and type E neurotoxin (BoNT/E).