Template-free ZnS nanorod synthesis by microwave irradiation.

We report template-free, microwave-irradiation-assisted growth of ZnS nanorods. Using this facile and high yield technique we could grow nanostructures of approximately 50-100 nm diameter and more than 1 µm in length. Effects of microwave power and irradiation time on the growth process were investigated. It was revealed that the time of refluxing plays a vital role in determining the thickness of the rods. This simple technique using a multimode microwave source may prove to be a potential tool for growing similar nanostructures of other oxide-, sulfide- and selenide-based compound semiconductors.

Correction of sickle cell disease in transgenic mouse models by gene therapy.

Sickle cell disease (SCD) is caused by a single point mutation in the human betaA globin gene that results in the formation of an abnormal hemoglobin [HbS (alpha2betaS2)]. We designed a betaA globin gene variant that prevents HbS polymerization and introduced it into a lentiviral vector we optimized for transfer to hematopoietic stem cells and gene expression in the adult red blood cell lineage. Long-term expression (up to 10 months) was achieved, without preselection, in all transplanted mice with erythroid-specific accumulation of the antisickling protein in up to 52% of total hemoglobin and 99% of circulating red blood cells. In two mouse SCD models, Berkeley and SAD, inhibition of red blood cell dehydration and sickling was achieved with correction of hematological parameters, splenomegaly, and prevention of the characteristic urine concentration defect.

Beta 93 modified hemoglobin: kinetic and conformational consequences.

The reactive sulfhydryl on Cys beta93 in human adult hemoglobin (HbA) has been the focus of much attention. It has purported functional roles such as a transporter of nitric oxide and a detoxifier of super oxide. In addition, it has a proposed role in the allosteric mechanism. The present study addresses the functional and conformational consequences of modifying the beta93 sulfhydryl using either maleimide or disulfide-based reactions. The geminate and bimolecular recombination of CO derivatives of several different beta93-modified Hbs in both solution and sol-gel matrixes provide a window into functional modifications associated with both the R and T states of these proteins. Nanosecond time-resolved visible resonance Raman spectroscopy is used to probe conformational consequences associated with the proximal heme environment. The results show functional and conformational consequences that depend on the specific chemistry used to modify beta93. Maleimide-based modification show the most significant alterations of R-state properties including a consistent pattern of a reduced geminate yield and a loss of the favorable heme-proximal histidine interaction normally seen for liganded R-state HbA. A mechanism based on a displacement of the side chain of Tyr beta145 is explored as a basis for this effect as well as other situations where there is loss of the quaternary enhancement effect. The quaternary enhancement effect refers to the enhancement of ligand binding properties of the alphabeta dimers when they are associated into the R-state tetramer.

Synthesis of aminobenzyltriethylenetetraaminohexaacetic acid: conjugation of the chelator to protein by an alkylamine linkage.

The conjugation of a chelating agent to an antibody as an anchoring site for a radionuclide is the first step in the successful preparation of a radiolabeled antibody for a diagnostic and therapeutic application. The high affinity of the protein bound chelator towards radionuclide ensures a higher selectivity in the delivery of the radionuclide to the targeted tissue. 4-Aminobenzylderivativetriethlenetetraaminohexaacetic acid (TTHA), a hexadentate chelating agent has been now prepared for conjugation with proteins in view of the higher affinity of TTHA metal ions as compared to DTPA. The latent crosslinking potential of alpha-hydroxy aldehydes has been used to conjugate the new chelating agent to proteins through an alkylamine linkage. On incubation of amino benzyl TTHA with glycoladehyde at neutral pH and room temperature, the reagent is converted to oxo ethyl amino benzyl TTHA. On addition of albumin to this reaction mixture, the oxo ethylamino benzyl TTHA generates reversible schiff base adducts with the amino groups of albumin. The reduction of the Schiff base adducts of the chelator with the protein by sodium cyanoborohydride stabilizes the schiff base adducts as stable alkylamine linkages. 4-Thiocyanatobenzyl TTHA has also been prepared and conjugated to albumin through a thiocarbamoyl linkage. Both preparations of TTHA conjugated albumin complexed with 99mTc and 111In, with high affinity and no decomposition of the complex was noticed for at least up to 6 hrs after the preparation. The radiolabels complexed with these TTHA -albumin conjugates could not be 'chased' out by free DTPA. A comparison of the biodistribution of 111In, bound to the TTHA conjugated through an alkylamine and a thiocarbamoyl linkage showed that 111In complexed with alkylamine linked TTHA was retained in blood to a level nearly 17% higher compared to that seen with thicarbamoyl linked TTHA, one hr after the injection into mice. Thus, the alkylamine linkage appears to be more stable under the in vivo conditions. The glycolaldehyde mediated alkylation procedure offers a mild, simple and rapid method for preparation of drug-protein (antibody) conjugates.

Labeling of monoclonal antibodies with radionuclides.

Antibodies, specifically monoclonal antibodies, are potentially very useful and powerful carriers of therapeutic agents to target tissues and diagnostic agents. The loading or charging of antibodies with agents, especially radiotracers, is reviewed here. The choice of radioisotope for immunodetection and/or immunotherapy is based on its availability, half-life, nature of the radiation emitted, and the metabolic pathways of the radionuclide in the body. Most important of all are the derivatization techniques available for labeling the antibody with the given radionuclide. Isotopes of iodine and divalent metal ions are the most commonly used radionuclides. Antibodies labeled with iodine at tyrosine residues are metabolized rapidly in vivo. This leads to the incorporation of metabolized radioactive iodine into various tissues, mainly the thyroid gland and stomach, and to the accumulation of high levels of circulating iodine in the blood, which masks tumor uptake considerably. To overcome these limitations, the use of iodohippurate as an iodine-anchoring molecule to the protein should be considered. When divalent or multivalent metal ions are used as the preferred radionuclide, bifunctional chelating reagents such as ethylenediaminepentaacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA) are first coupled to the protein or antibody. These chelating molecules are attached to the protein by formation of an isopeptide linkage between the carboxylate of the chelating reagent and the amino group of the protein. Several procedures are available to generate the isopeptide linkage. When the anchoring of the chelating agent through isopeptide linkage results in the inactivation of the antibody, periodate oxidation of the carbohydrate moiety of the antibody, followed by reductive coupling of chelator, could be considered as an alternative. There is still a need for better, simpler, and more direct methods for labeling antibodies with radionuclides.