Enhancement of crystallization with nucleotide ligands identified by dye-ligand affinity chromatography

Ligands interacting with Mycobacterium tuberculosis recombinant proteins were identified through use of the ability of Cibacron Blue F3GA dye to interact with nucleoside/nucleotide binding proteins, and the effects of these ligands on crystallization were examined. Co-crystallization with ligands enhanced crystallization and enabled X-ray diffraction data to be collected to a resolution of at least 2.7 Å for 5 of 10 proteins tested. Additionally, clues about individual proteins’ functions were obtained from their interactions with each of a panel of ligands

Use of complementary and alternative medicines by a sample of Turkish women for infertility enhancement: a descriptive study

<p>Abstract</p> <p>Background</p> <p>Infertility patients are a vulnerable group that often seeks a non-medical solution for their failure to conceive. World-wide, women use CAM for productive health, but only a limited number of studies report on CAM use to enhance fertility. Little is known about traditional and religious forms of therapies that are used in relation to conventional medicine in Turkey. We investigated the prevalence and types of complementary and alternative medicine (CAM) used by infertile Turkish women for fertility enhancement.</p> <p>Methods</p> <p>A face-to-face questionnaire inquiring demographic information and types of CAM used for fertility enhancement were completed by hundred infertility patients admitted to a primary care family planning centre in Van, Turkey between January and July 2009.</p> <p>Results</p> <p>The vast majority of infertile women had used CAM at least once for infertility. CAM use included religious interventions, herbal products and recommendations of traditional "hodja's" (faith healers). Of these women, 87.8% were abused in the last 12 months, 36.6% felt not being supported by her partner and 80.5% had never spoken with a physician about CAM.</p> <p>Conclusions</p> <p>Infertile Turkish women use complementary medicine frequently for fertility enhancement and are in need of information about CAM. Religious and traditional therapies are used as an adjunct to, rather than a substitute for, conventional medical therapy. Physicians need to approach fertility patients with sensitivity and should be able to council their patients about CAM accordingly.</p

Transduction of Brain Dopamine Neurons by Adenoviral Vectors Is Modulated by CAR Expression: Rationale for Tropism Modified Vectors in PD Gene Therapy

Gene-based therapy is a new paradigm for the treatment of Parkinson disease (PD) and offers considerable promise for precise targeting and flexibility to impact multiple pathobiological processes for which small molecule agents are not available. Some success has been achieved utilizing adeno-associated virus for this approach, but it is likely that the characteristics of this vector system will ultimately create barriers to progress in clinical therapy. Adenovirus (Ad) vector overcomes limitations in payload size and targeting. The cellular tropism of Ad serotype 5 (Ad5)-based vectors is regulated by the Ad attachment protein binding to its primary cellular receptor, the coxsackie and adenovirus receptor (CAR). Many clinically relevant tissues are refractory to Ad5 infection due to negligible CAR levels but can be targeted by tropism-modified, CAR-independent forms of Ad. Our objective was to evaluate the role of CAR protein in transduction of dopamine (DA) neurons in vivo.Ad5 was delivered to the substantia nigra (SN) in wild type (wt) and CAR transgenic animals. Cellular tropism was assessed by immunohistochemistry (IHC) in the SN and striatal terminals. CAR expression was assessed by western blot and IHC. We found in wt animals, Ad5 results in robust transgene expression in astrocytes and other non-neuronal cells but poor infection of DA neurons. In contrast, in transgenic animals, Ad5 infects SNc neurons resulting in expression of transduced protein in their striatal terminals. Western blot showed low CAR expression in the ventral midbrain of wt animals compared to transgenic animals. Interestingly, hCAR protein localizes with markers of post-synaptic structures, suggesting synapses are the point of entry into dopaminergic neurons in transgenic animals.These findings demonstrate that CAR deficiency limits infection of wild type DA neurons by Ad5 and provide a rationale for the development of tropism-modified, CAR-independent Ad-vectors for use in gene therapy of human PD

Designer Gene Delivery Vectors: Molecular Engineering and Evolution of Adeno-Associated Viral Vectors for Enhanced Gene Transfer

Gene delivery vectors based on adeno-associated virus (AAV) are highly promising due to several desirable features of this parent virus, including a lack of pathogenicity, efficient infection of dividing and non-dividing cells, and sustained maintenance of the viral genome. However, several problems should be addressed to enhance the utility of AAV vectors, particularly those based on AAV2, the best characterized AAV serotype. First, altering viral tropism would be advantageous for broadening its utility in various tissue or cell types. In response to this need, vector pseudotyping, mosaic capsids, and targeting ligand insertion into the capsid have shown promise for altering AAV specificity. In addition, library selection and directed evolution have recently emerged as promising approaches to modulate AAV tropism despite limited knowledge of viral structure–function relationships. Second, pre-existing immunity to AAV must be addressed for successful clinical application of AAV vectors. “Shielding” polymers, site-directed mutagenesis, and alternative AAV serotypes have shown success in avoiding immune neutralization. Furthermore, directed evolution of the AAV capsid is a high throughput approach that has yielded vectors with substantial resistance to neutralizing antibodies. Molecular engineering and directed evolution of AAV vectors therefore offer promise for generating ‘designer’ gene delivery vectors with enhanced properties

Protein A-immobilized microporous polyhydroxyethylmethacrylate affinity membranes for selective sorption of human-immunoglobulin-G from human plasma

WOS: 000087993600004PubMed: 10903036Microporous membranes made of poly(2-hydroxyethylmethacrylate) [poly(HEMA)] carrying protein A were used for selective sorption of human-IgG from human plasma. Poly(HEMA) membranes were prepared by a photo-polymerization technique, and activated by cyanogen bromide (CNBr) in an alkaline medium (pH 11.5). Bioligand protein A was then immobilized by covalent binding onto these CNBr-activated membranes. The amount of immobilized protein A was controlled by changing pH and the initial concentrations of CNBr and protein A. The non-specific adsorption of protein A on the plain poly(HEMA) membranes was 2.9 mu g cm(-2). Maximum protein A immobilization was observed at pH 9.5. Up to 180 mu g cm(-2) was immobilized on the CNBr-activated poly(HEMA) membranes. The maximum adsorption of human-IgG on the protein A-immobilized poly(HEMA) membranes was observed at pH 8.0. The non-specific adsorption of human-IgG onto the plain poly(HEMA) membranes was low (about 4.4 mu g cm(-2)). Higher human-IgG adsorption values (up to 394 mu g cm(-2)) were obtained in which the protein A-immobilized poly(HEMA) membranes were used. Much higher amounts of human-Igg (up to 489 mu g cm(-2)) were adsorbed from human plasma. Up to 91 % of the adsorbed human-IgG was desorbed by using 0.1 M aminoacetic acid as elution agent. The adsorption-desorption cycle was repeated ten times using the same polymeric membranes. There was no remarkable reduction in the adsorption capacity of the protein A-immobilized poly(HEMA) membranes

Removal of cadmium(II) ions from human plasma by thionein modified pHEMA based membranes

WOS: 000088276300002Microporous poly(2-hydroxyethylmethacrylate) [pHEMA] membranes carrying Cibacron Blue F3GA and/or thionein were prepared and used for cadmium(II) removal from human plasma. pHEMA membranes were produced by a photopolymerization of HEMA in the presence of azobisisobutyronitrile. The reactive dye-ligand Cibacron Blue F3GA was then covalently attached onto the pHEMA membranes. The maximum dye-loading was 1.07 mu mol/cm(2). Then, thionein was adsorbed onto the Cibacron Blue F3GA-carrying membranes. The maximum amount of thionein adsorbed was 0.92 mu mol/cm(2). The aqueous swelling properties and contact angle lair underwater) of the pHEMA membrane were not observed to change following derivatisation with Cibacron Blue F3GA. These hydrophilic membranes (contact angle value: 45.3 degrees) with a swelling ratio of 58% (w/w), and carrying Cibacron Blue F3GA and/or thionein were used in the Cd(II) removal studies. The maximum amounts of Cd(II) removed from human plasma by Cibacron Blue F3GA-attached and Cibacron Blue F3GA-attached/thionein adsorbed pHEMA membranes were of 1.72 mu mol used as pectively. Cd(II) ions could be repeatedly adsorbed and desorbed with both types of these novel metallo-peptide affinity pHEMA membranes without noticeable loss in their Cd(II) adsorption capacity. (C) 2000 Elsevier Science B.V. All rights reserved