Comparative role of proteins in transport of HCH-isomers in desert locust, Schistocerca gregaria Forskal and bovines

The studies on binding of hexachlorocyclohexane (HCH) with carrier proteins were carried out to establish the role of proteins in the transport of insecticides in insects. Sephadex G-200 column chromatography resolved haemolymph of adult male desert locust, Schistocerca gregaria into three major protein peaks. There was significant binding of gamma-HCH with first protein peak (F1). Two classes of binding sites were observed on first protein peak for gamma-HCH. However low level of binding was observed with the third protein peak (F3) of the haemolymph. Bindings of HCH-isomers (alpha, beta and gamma) with bovine serum albumin (BSA) were not related to their water solubilities. Moderate to low affinities (1.4 -1.84 x 10(6) M(-1)) of HCH-isomers for BSA were observed. The present studies showed that more HCH binds to haemolymph lipoprotein of locust as compared to BSA. This indicates a significant role of haemolymph proteins in the transport of insecticides in insects.     

Lipid-gramicidin interactions using two-dimensional Fourier-transform electron spin resonance.

The application of two-dimensional Fourier-transform electron-spin-resonance (2D-FT-ESR) to the study of lipid/gramicidin A (GA) interactions is reported. It is shown that 2D-FT-ESR spectra provide substantially enhanced spectral resolution to changes in the dynamics and ordering of the bulk lipids (as compared with cw-ESR spectra), that result from addition of GA to membrane vesicles of dipalmitoylphosphatidylcholine (DPPC) in excess water containing 16-PC as the lipid spin label. The agreement between the theory of Lee, Budil, and Freed and experimental results is very good in the liquid crystalline phase. Both the rotational and translational diffusion rates of the bulk lipid are substantially decreased by addition of GA, whereas the ordering is only slightly increased, for a 1:5 ratio of GA to lipid. The slowing effect on the diffusive rates of adding GA in the gel phase is less pronounced. It is suggested that the spectral fits in this phase would be improved with a more detailed dynamic model. No significant evidence is found in the 2D-FT-ESR spectra for a second immobilized component upon addition of GA, which is in contrast to cw-ESR. It is shown from simulations of the observed 2D-FT-ESR spectra that the additional component seen in cw-ESR spectra, and usually attributed to "immobilized" lipid, is inconsistent with its being characterized by increased ordering, according to a model proposed by Ge and Freed, but it would be consistent with the more conventional model of a significantly reduced diffusional rate. This is because the 2D-FT-ESR spectra exhibit a selectivity, favoring components with longer homogeneous relaxation times, T2. The homogeneous linewidths of the 2D-FT-ESR autopeaks appear to broaden as a function of mixing time. This apparent broadening is very likely due to the process of cooperative order director fluctuations (ODF) of the lipids in the vesicle. This real-time observation of ODF is distinct from, but appears in reasonable agreement with, NMR results. It is found that addition of GA to give the 1:5 ratio has only a small effect on the ODF, but there is a significant temperature dependence.     

Insecticide interaction with carrier and neuroproteins.

Binding of alpha-, beta-, gamma-hexachlorocyclohexane, p,p'-DDT and p,p'-DDE with bovine serum albumin (BSA) and locust brain homogenate was studied. Binding affinities of pesticides were higher for the locust brain homogenates than for BSA. Results of uptake by isolated locust brain revealed higher uptake of gamma-HCH than alpha-HCH. gamma-HCH uptake was also higher from locust haemolymph than either from BSA or from buffer.     

Isolation and characterization of the haemolymph lipoprotein of desert locust, Schistocerca gregaria Forskal.

A yellow lipoprotein was isolated and purified from haemolymph of desert locust, Schistocerca gregaria Forskal, by precipitation at low ionic concentration and by gel filtration. Sephadex G-200 resolved haemolymph into five protein peaks. First peak was of lipoprotein with molecular weight of 501,000 +/- 7000 (n = 3) and on electrophoresis, it separated into three bands. Maximum lipids from lipoprotein could be extracted with chloroform: methanol solvent system and were 52% by weight.     

The wmN1 Enhancer Region of the Mouse Myelin Proteolipid Protein Gene (mPlp1) is Indispensable for Expression of an mPlp1-lacZ Transgene in Both the CNS and PNS

The myelin proteolipid protein gene (PLP1) encodes the most abundant protein in CNS myelin. Expression of the gene must be strictly regulated, as evidenced by human X-linked leukodystrophies resulting from variations in PLP1 copy number, including elevated dosages as well as deletions. Recently, we showed that the wmN1 region in human PLP1 (hPLP1) intron 1 is required to promote high levels of an hPLP1-lacZ transgene in mice, using a Cre-lox approach. The current study tests whether loss of the wmN1 region from a related transgene containing mouse Plp1 (mPlp1) DNA produces similar results. In addition, we investigated the effects of loss of another region (ASE) in mPlp1 intron 1. Previous studies have shown that the ASE is required to promote high levels of mPlp1-lacZ expression by transfection analysis, but had no effect when removed from the native gene in mouse. Whether this is due to compensation by another regulatory element in mPlp1 that was not included in the mPlp1-lacZ constructs, or to differences in methodology, is unclear. Two transgenic mouse lines were generated that harbor mPLP(+)Z/FL. The parental transgene utilizes mPlp1 sequences (proximal 2.3 kb of 5?-flanking DNA to the first 37 bp of exon 2) to drive expression of a lacZ reporter cassette. Here we demonstrate that mPLP(+)Z/FL is expressed in oligodendrocytes, oligodendrocyte precursor cells, olfactory ensheathing cells and neurons in brain, and Schwann cells in sciatic nerve. Loss of the wmN1 region from the parental transgene abolished expression, whereas removal of the ASE had no effect.

     

Studies on lipid membranes by two-dimensional Fourier transform ESR: Enhancement of resolution to ordering and dynamics.

The first two-dimensional Fourier-transform electron spin resonance (2D-FT-ESR) studies of nitroxide-labeled lipids in membrane vesicles are reported. The considerable enhancement this experiment provides for extracting rotational and translational diffusion rates, as well as orientational ordering parameters by means of ESR spectroscopy, is demonstrated. The 2D spectral analysis is achieved using theoretical simulations that are fit to experiments by an efficient and automated nonlinear least squares approach. These methods are applied to dispersions of 1-palmitoyl-2oleoyl-sn-glycerophosphatidylcholine (POPC) model membranes utilizing spin labels 1-palmitoyl-2-(16-doxyl stearoyl) phosphatidylcholine and the 3-doxyl derivative of cholestan-3-one (CSL). Generally favorable agreement is obtained between the results obtained by 2D-FT-ESR on vesicles with the previous results on similar systems studied by continuous wave (cw) ESR on aligned samples. The precision in determining the dynamic and ordering parameters is significantly better for 2D-FT-ESR, even though the cw ESR spectra from membrane vesicles are resolved more poorly than those from well aligned samples. Some small differences in results by the two methods are discussed in terms of limitations of the methods and/or theoretical models, as well as possible differences between dynamic molecular structure in vesicles versus aligned membranes. An interesting observation with CSL/POPC, that the apparent homogeneous linewidths seem to increase in "real time," is tentatively attributed to the effects of slow director fluctuations in the membrane vesicles.