Highly sensitive recombinant antibodies capable of reliably differentiating heart-type fatty acid binding protein from noncardiac isoforms

During recent times, heart-type fatty acid binding protein (hFABP) has gained increasing credence as a promising cardiac biomarker. This is largely due to its rapid myocardial release and subsequent clearance kinetics, which are superior to those of myoglobin and offer an earlier diagnostic window than the troponins. Realization of its full diagnostic and prognostic potential is dependent on accessibility to robust hFABP-specific assays. Here we describe a rational strategy for generation and screening of hFABP-specific avian-derived recombinant antibodies. These antibodies were confirmed to be exquisitely specific for hFABP, with no cross-reactivity observed in a representative panel of the most homologous non-heart-type FABP isoforms. All of the antibodies tested exhibited single-figure nanomolar affinities, and their analytical potential was demonstrated in a simple inhibition enzyme-linked immunosorbent assay (ELISA) format that returned an impressive limit of quantitation (LOQ) value of 1.9 ng/ml. The cumulative results underline the potential value of these antibodies as enabling reagents for use in a variety of immunodiagnostic configurations.     

Histidine mapping of serine protease: a synergic study by IMAC and molecular modelling

The immobilized metal ion affinity (IMA) interaction of different serine proteases, namely porcine and bovine trypsins and BPN' and Carlsberg subtilisins, was studied on Sepharose-IDA-CuII. Both trypsins were resolved into their different subspecies, whereas the subtilisins appeared as only one species. The use of diethyl pyrocarbonate-modified enzymes demonstrated the contribution of histidine(s) as the sole interacting site(s). The use of different peptidic and chemical inhibitors complexed to the enzymes confirmed the contribution of histidine(s) as the interacting site(s) and further resulted in different chromatographic patterns for the free and complexed serine proteases. Comparison of the chromatographic data for each enzyme with the accessible surface area calculation by molecular modelling on the available crystallographic structure allowed us to hypothesize a map of the surface-accessible histidine on each enzyme.     

Fatty acid composition of phospholipids in seed oils containing unusual acids

In oilseeds whose glycerides carry common fatty acids (groundnut, sweet almond, cotton-seed, soyabean, safflower, sunflower and sesame), the constituent fatty acids of phospholipids were qualitatively but not quantitatively the same. Even when present in quantity in the triglyceride, unusual fatty acids like erucic (mustard, nasturtium, turnip), arachidic (soapnut), petroselinic (coriander) or conjugated trienes (bitter gourd, snake gourd) were totally or substantially absent in the corresponding phospholipids. In all the 14 seed oil phospholipids studied, the main fatty acids were always palmitic (17–43%), oleic (16–57%) and linoleic (10–34%).     

Circadian rhythmicity in the nervous system of the cockroach, Periplaneta americana

Diurnal rhythmicity of nervous activity in Periplaneta americana was investigated, using acetylcholine (ACh) content, acetylcholinesterase (AChE) and spontaneous electrical activities as indices. AChE and electrical activities were maximum at 0 hr and minimum at 12 hr, while ACh showed an opposite rhythm. Central nervous system extract from cockroaches at 12 hr elevated the electrical activity while 0 hr-extract exerted inhibition. Lower concentrations of ACh had an elevatory influence while higher concentrations inhibited the electrical activity. A hypothesis is proposed, suggesting synthetic and releasing phases of ACh in a regular diurnal cycle, to explain the results obtained.     

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.     

Protection and Potentiation of 1-Methyl-4-Phenylpyridinium-Induced Toxicity by Cytochrome P450 Inhibitors and Inducer May Be Due to the Altered Uptake of the Toxin

Abstract: Earlier studies from our laboratory have demonstrated that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity could be modulated by inhibitors and inducer of cytochrome P450 (P450) in an in vitro model consisting of sagittal slices of mouse brain. To understand the molecular mechanisms underlying the role of P450 on MPTP toxicity, it was undertaken to study the effect of the modulators of P450 on the toxicity of the metabolite of MPTP, namely, 1-methyl-4-phenylpyridinium ion (MPP⁺). Incubation of mouse brain slices with various concentrations of MPP⁺ (1–100 µ M ) resulted in dose-dependent inhibition of mitochondrial enzyme NADH-dehydrogenase (NADH-DH) and leakage of the cytosolic enzyme lactate dehydrogenase from the slice into the medium. MPP⁺-induced toxicity was abolished by pretreatment of the slices with inhibitors of monoamine oxidase (MAO; pargyline and deprenyl) or inhibitors of P450 (piperonyl butoxide or SKF-525A) or dopamine uptake blocker (GBR-12909), as measured by the activity of NADH-DH in slices and leakage of lactate dehydrogenase from the slice into the medium. Slices prepared from mice pretreated with phenobarbital (an inducer of P450) potentiated the toxic effects of MPP⁺. Pretreatment of slices with MAO-inhibitor, P450 inhibitors, or dopamine uptake blocker attenuated the uptake of MPP⁺ into the slices. In contrast, MPP⁺ uptake was significantly increased in slices prepared from phenobarbital-pretreated mice. Thus, both MAO and P450 inhibitors abolish the toxicity of MPP⁺ in the sagittal slices of mouse brain by altering the uptake of the toxin into the slices.     

Host Range of an Insecticidal Crystal Protein of Bacillus thuringiensis subsp. kurstaki Produced in Escherichia coli

A crylA(c)-like gene of Bacillus thuringiensis subsp. kurstaki strain HD1 was over-expressed in Escherichia coli from a multicopy plasmid. Biological toxicity tests conducted on the larvae of three lepidopteran insects showed that the host range of transgenic E. coli HB 101 (pRT 200) was a subset of the host range of B. thuringiensis kurstaki HD1. Both were toxic to the larvae of Helicoverpa armigera (Gram pod borer) and Bombyx mori (Silkworm). However. though the sporecrystal formulation of HDl was toxic to the larvae of Phthorimaea operculella (Potato tuber moth). the transgenic E. coli was not. Product of St toxin gene other than crylA(c) present In HD1 may be responsible for Its toxicity to the larvae of P. opercuiella.     

Effect of glutaraldehyde on hemoglobin: functional aspects and M?ssbauer parameters

Glutaraldehyde is widely used for the cross-linking of hemoglobin for blood substitute research or for technological purposes. The effects of this reagent on the biochemical properties of hemoglobin were correlated with M?ssbauer data. Human hemoglobin was cross-linked by glutaraldehyde as soluble polymers and insoluble particles. Effects of cross-linking on oxygen affinity, oxidation-reduction potential, autoxidation kinetics, and thermal stability were studied. Stability of cross-linked hemoglobin was specifically studied by M?ssbauer spectroscopy. Oxygen affinity is increased, redox potential is decreased, autoxidation rates are increased, and stability towards thermal denaturation is increased. The regeneration of partially denatured hemoglobin by glutaraldehyde cross-linking is shown. Effects of cross-linking on biochemical properties are explained by the hypothesis of the opening of the heme pocket on the distal-histidine side and the concomitant charge transfer from the iron to the oxygen.     

Separation and purification of IgG1 and IgG2 from IgG-Cohn-II fraction of human plasma by pseudobioaffinity chromatography on histidyl-AH-sepharose.

L-histidine coupled to aminohexyl-sepharose (H-AH) has been used as an affinity sorbent to separate IgG from human plasma. Two subclasses IgG1 and IgG2 were specifically bound to histidyl-AH-sepharose at pH 7.4 and eluted using 0.2 M and 1M NaCl. The specificity of the two subclasses were determined by immunoelectrophoresis. Quantitative determination of IgG1, IgG2 was carried out using radial immunodiffusion technique.