Characterization of anticoagulant heparinoids by immunoprofiling

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and–when additional saccharides are present–inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics.     

KCNE3 mutation V17M identified in a patient with lone atrial fibrillation

BACKGROUND: Atrial fibrillation (AF) is the most common cardiac rhythm disorder with a lifetime risk for development of 25% for people aged 40 or older. In this study we aim for the functional assessment of a mutation in KCNE3 identified in a proband with early-onset lone AF. METHODS: Screening of genomic DNA from the proband led to identification of a KCNE3 V17M missense mutation. We heterologously expressed the accessory channel subunit in Xenopus laevis oocytes together with its known interacting potassium channel alpha-subunits. Further, we applied RT-PCR on human total RNA from left and right atria and ventricle. RESULTS: Electrophysiological recordings revealed an increased activity of Kv4.3/KCNE3 and Kv11.1/KCNE3 generated currents by the mutation, thereby conferring susceptibility of mutation carriers to faster cardiac action potential repolarization and thus vulnerability to re-entrant wavelets in the atria and thereby AF. CONCLUSION: Here we report a novel mutation in KCNE3 identified in a proband with early-onset lone AF possibly leading to gain-of-function of several cardiac currents. We suggest abnormalities in the KCNE3 gene as a potential genetic risk factor for initiation and/or maintenance of AF.     

Minimal chemical modification of reductive end of dextran to produce an amphiphilic polysaccharide able to incorporate onto lipid nanocapsules

In order to graft an amphiphilic polysaccharide to lipid nanocapsules, we present here a new method of dextran lipidation. The lipidation strategy is based on the formation of an oxime linkage between the amphiphilic hydroxylamine C16E20ONH2 and the reductive end of a 40 kDa dextran. This chemoselective reaction allows us to control the lipidation site and the number of lipid introduced on the dextran molecule. This new amphiphilic dextran was used to coat the surface of lipid nanocapsules. The coating efficiency was followed by dynamic light scattering and the presence of the polysaccharide was confirmed by (1)H NMR and observed by electronic microscopy.     

Expression of the transcription factor Zfp191 during embryonic development in the mouse

The human zinc finger protein 191 (ZNF191) is a Krüppel-like protein and can specifically interact with the widespread TCAT motif which constitutes the HUMTH01 microsatellite in the tyrosine hydroxylase (TH) gene (encoding the rate-limiting enzyme in the synthesis of catecholamines). Allelic variations of HUMTH01 are known to have a quantitative silencing effect on TH gene expression and to correlate with quantitative and qualitative changes in the binding by ZNF191. This factor has been isolated from bone marrow and promyelocytic leukemia cell lines indicating that ZNF191 also plays a role in hematopoiesis. Thus, ZNF191 could participate in the regulation of several genes implicated in different functions. Moreover, mice that are deficient in Zfp191, the murine homologue of ZNF191, have been shown to be severely retarded in development and to die approximately at embryonic day 7.5. In order to gain further insight into its biological functions, we have analysed the localisation of Zfp191 throughout mouse development. Expression was detected early during embryogenesis in ectodermal, endodermal, mesodermal and extra-embryonic tissues. In particular, Zfp191 was observed in the developing central nervous system. Interestingly, its expression levels were prominent in areas of proliferation such as the subventricular zone. Zfp191 expression pattern during development can account for the phenotypic features of Zfp191(-/-) embryos.     

Genes induced during the early developmental stages of the Cane Toad, Bufo (Chaunus) marinus

Metamorphosis, a critical stage in the development of toads and frogs, involves rapid levels of morphological change. In the current study, we have used microarray analysis to identify shifts in gene expression between tadpole and toadlet stages of the cane toad, Bufo (Chaunus) marinus. Here, we report on nine genes that show the greatest induction during metamorphosis; the gut-associated gastrokine and trefoil factor, blood components haemoglobins alpha/beta, apolipoprotein and serum albumin, a nasal gene olfactomedin, a lens gene gamma-crystallin, and a novel gene with low homology to frog harderin. We present both temporal and spatial expression patterns of these genes identified in developing and adult cane toads. This study extends our knowledge of the molecular basis of toad metamorphosis, and not only offers insights to the genes induced during the general remodelling that occurs but also reveals possible targets for control and manipulation of amphibian pest species, for example, the cane toad in Australia.     

Naturally Induced Humoral Immunity to West Nile Virus Infection in Raptors

West Nile virus (WNV) infection can be fatal to many bird species, including numerous raptors, though population- and ecosystem-level impacts following introduction of the virus to North America have been difficult to document. Raptors occupy a diverse array of habitats worldwide and are important to ecosystems for their role as opportunistic predators. We documented initial (primary) WNV infection and then regularly measured WNV-specific neutralizing antibody titers in 16 resident raptors of seven species, plus one turkey vulture. Most individuals were initially infected and seroconverted between July and September of 2003, though three birds remained seronegative until summer 2006. Many of these birds became clinically ill upon primary infection, with clinical signs ranging from loss of appetite to moderate neurological disease. Naturally induced WNV neutralizing antibody titers remained essentially unchanged in some birds, while eight individuals experienced secondary rises in titer presumably due to additional exposures at 1, 2, or 3 years following primary infection. No birds experienced clinical signs surrounding or following the time of secondary exposure, and therefore antibodies were considered protective. Results of this study have implications for transmission dynamics of WNV and health of raptor populations, as well as the interpretation of serologic data from free-ranging and captive birds. Antibodies in raptors surviving WNV may persist for multiple years and protect against potential adverse effects of subsequent exposures.     

Systematic interpretation of cyclic nucleotide binding studies using KinetXBase

Functional proteomics aims to describe cellular protein networks in depth based on the quantification of molecular interactions. In order to study the interaction of adenosine-3',5'-cyclic monophosphate (cAMP), a general second messenger involved in several intracellular signalling networks, with one of its respective target proteins, the regulatory (R) subunit of cAMP dependent protein kinase (PKA), a number of different methods was employed. These include fluorescence polarisation (FP), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), amplified luminescence proximity homogeneous assay (ALPHA-screen), radioligand binding or activity-based assays. Kinetic, thermodynamic and equilibrium binding data of a variety of cAMP derivatives to several cAMP binding domains were integrated in a single database system, we called KinetXBase, allowing for very distinct data formats. KinetXBase is a practical data handling system for molecular interaction data of any kind, providing a synopsis of data derived from different technologies. This supports ongoing efforts in the bioinformatics community to devise formal concepts for a unified representation of interaction data, in order to enable their exchange and easy comparison. KinetXBase was applied here to analyse complex cAMP binding data and highly site-specific cAMP analogues could be identified. The software package is free for download by academic users.     

The role of prolactin in the regulation of brood care in the cooperatively breeding fish Neolamprologus pulcher

The hormone prolactin (PRL) is important for the regulation of parental care in many species of mammals, birds and fish, and for alloparental care (care directed at nondescendant young) in some mammals and birds. Its significance in alloparental brood care of cooperatively breeding fish has not yet been assessed. Here, we test the role of PRL in brood care behavior of the cooperatively breeding cichlid Neolamprologus pulcher. The expression of PRL mRNA was determined in the pituitary glands of breeders of both sexes, helpers that showed brood care behavior and nonbreeding fish as controls. In addition, PRL levels were experimentally manipulated in male breeders and helpers by intraperitoneal injections of ovine PRL, and the behavior of these test fish was recorded toward standardized clutches. Adult females had higher levels of PRL mRNA than adult males, which was true both for breeders and nonbreeders. Contrary to expectation, there was no positive correlation between PRL and brood care behavior in any category of test fish, and the experimental application of PRL did not change brood care propensity. Interestingly, brood-caring adult females had significantly lower levels of PRL mRNA than adult female nonbreeders, whereas there was no difference between helpers and similar-sized nonbreeding group members. PRL mRNA levels increased with body mass in juveniles, but decreased with body mass in adults. In conclusion, we found no evidence that elevated levels of PRL are directly involved in the regulation of brood care behavior in this species.