Conjugation of methotrexate to IgG antibodies and their F(ab)2 fragments and the effect of conjugated methotrexate on tumor growth in vivo

Summary Methotrexate (MTX) was first conjugated to antibovine serum albumin IgG (antiBSA) or its F(ab)₂ fragment to define conditions for retention of drug and antibody activity. With identical drug: protein molar ratios, incorporation in the F(ab)₂ fragment was lower than in intact antiBSA, an observation consistent with analysis of the number of lysine residues (22 in F(ab)₂ compared to 40 in antiBSA). In either case, up to approximately 10 mol MTX could be incorporated per mol protein, with recovery of 70% of the protein. At an incorporation ratio of 6 mol MTX per mol protein, MTX-antiBSA retained 100% of antibody activity and MTX-F(ab)₂antiBSA retained 75%. MTX-antiBSA and MTX-F(ab)₂antiBSA were equally potent in vitro inhibitors of dihydrofolate reductase. Conjugates prepared from antiEL4 IgG (AELG) and from F(ab)₂AELG significantly increased survival in EL4 lymphoma-bearing mice compared with mice receiving equal amounts (5 mg MTX/kg) of free MTX, MTX linked to the F(ab)₂ fragment of normal rabbit IgG, or a simple mixture of MTX and F(ab)₂AELG. MTX-AELG at this dose level produced longer survival than MTX-F(ab)₂AELG (0.0052AELG MTX linked to the F(ab)₂ fragment of AELG - MTX-F(ab)₂antiBSA MTX linked to the F(ab)₂ fragment of antiBSA - MTX-F(ab)₂NRG MTX linked to the F(ab)₂ fragment of NRG - MTX-NRG MTX linked to NRG - NHS N-hydroxysuccinimide - NRG normal rabbit IgG - PBS 0.01 M sodium phosphate (pH 7.1) containing 0.45 M sodium chloride - TAA tumor-associated antigen - t_1/2 half-life     

Improved photosynthetic characteristics correlated with enhanced biomass in a heterotic F1 hybrid of maize (Zea mays L.)

Photosynthesis Research - Heterosis is a phenomenon wherein F1 hybrid often displays phenotypic superiority and surpasses its parents in terms of growth and agronomic traits. Investigations on the...     

Identification of lanthionine synthase C-like protein-1 as a prominent glutathione binding protein expressed in the mammalian central nervous system

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.     

7Li and 23Na NMR studies of transmembrane cation transport mediated by ionophore lasalocid A

Ion transport across phospholipid vesicles was studied by ⁷Li and ²³Na-NMR using an aqueous anionic paramagnetic shift reagent, dysprosium nitrilotriacetate [Dy(NTA)₂]³⁻, mediated by ionophores, lasalocid A and A23187. The intra- and extracellular ⁷Li and ²³Na-NMR signals were well separated (20 Hz) at mM concentration of the shift reagent. The observed data on the rate constant for lithium transport across DPPC vesicles at various concentrations of the ionophores indicated that lasalocid A is a more efficient carrier for lithium ion compared with the sodium ion transport by this ionophore, while A23187 was not specific to either of the ions (Li or Na). ©1998 European Peptide Society and John Wiley & Sons, Ltd.     

<Emphasis Type="Italic">In vitro</Emphasis> germination and micropropagation of <Emphasis Type="Italic">Givotia rottleriformis</Emphasis> Griff.

The propagation of Givotia rottleriformis Griff. is difficult as a result of long seed dormancy associated with poor seed germination. The present study was undertaken to develop a protocol to overcome seed dormancy by culture of zygotic embryo axes and then develop an efficient method for micropropagation of Givotia. Best germination frequency (78.3%) was achieved from mature zygotic embryo axes isolated from acid-scarified fresh seeds when cultured on Murashige and Skoog (MS) medium (half-strength major salts) with 28.9 μM gibberellic acid (GA₃). Efficient plant conversion was achieved by transfer of 10-d-old germinated embryos to MS medium (half-strength major salts) supplemented with 1.2 μM kinetin (KN) and 0.5 μM indole-3-butyric acid (IBA). However, acid scarification of 1-yr-old seeds decreased the germination frequency of zygotic embryo axes in comparison to those obtained from non-acid-scarified seeds which germinated (96.2%) and converted into plants (80.3%) on MS basal (half-strength major salts) medium. Multiple shoot bud induction was achieved by culture of shoot tips derived from in vitro germinated seedlings on MS medium with 0.5 μM thidiazuron for 4 wk, and the shoots elongated after transfer to a secondary medium with 1.2 μM KN. A maximum number of 7.8 shoots per explant with an average shoot length of 3.2 cm was achieved after two subcultures on this medium. The in vitro regenerated shoots rooted (41.5%) on half-strength MS medium with 0.5 μM IBA. The in vitro generated seedlings and micropropagated plants were established in soil with a survival frequency of 70% and 60%, respectively.     

Estimation of Enantiomeric Impurity in Piperidin‐3‐Amine by Chiral HPLC With Precolumn Derivatization

A simple, precise, accurate, robust chiral high‐performance liquid chromatographic (chiral HPLC) method was developed for estimation of (S)‐piperidin‐3‐amine (S‐isomer) in (R)‐piperidin‐3‐amine dihydrochloride (R‐AMP). As AMP is a high‐melting solid and nonchromophoric compound, development of a suitable chiral method is a challenging task. The proposed chiral HPLC‐UV method involves a precolumn derivatization technique with para toluene sulphonyl chloride (PTSC) in the presence of a base to introduce chromophore into analytes. It utilizes chiralpak AD‐H column with a simple mobile phase of 0.1% diethyl amine in ethanol with a 0.5 mL/min flow rate. Analytes were monitored by using a UV detector at 228 nm. The resolution between the two enantiomers was more than 4.0. The developed method was validated as per current International Conference on Harmonization (ICH) guidelines. Chirality 26:775–779, 2014. © 2014 Wiley Periodicals, Inc.