The separation of multiple forms of housefly 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)-ethane (DDT) dehydrochlorinase from glutathione S-aryltransferase by electrofocusing and electrophoresis

Electrophoresis in a sucrose gradient at pH values between 5 and 8 separated housefly DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] dehydrochlorinase into two major fractions. GSH S-aryltransferase under similar conditions migrated as a single peak of activity. Separation of housefly homogenates or partially purified enzyme preparations by electrofocusing in a natural pH gradient also showed the presence of multiple forms of DDT dehydrochlorinase.     

Understanding Fluctuations in Bobcat Harvest at the Northern Limit of Their Range

ABSTRACT In Quebec, Canada, harvest of bobcats (Lynx rufus) started to decline in 1985 and by 1991, harvest seasons were closed due to concerns of a perceived population decline. Since the closing of harvest season in 1991, the average temperature has increased, snow quantity has decreased, and important changes in agriculture and forest management have occurred. In light of changing conditions, the situation of Quebec bobcats needed reassessment. Thus, we analyzed harvest data to clarify the current status of bobcat populations in Quebec. From 1980 to 1991, bobcat harvest in Quebec was strongly correlated with bobcat harvest in Maine (USA), Nova Scotia (Canada), Ontario (Canada), and Vermont (USA). Extrapolations of harvest in Quebec relative to harvest in Maine, Ontario, Vermont, and Nova Scotia suggested an increase in number of bobcats after 1991. Mass of male and female bobcats before 1991 was less than mass of animals captured after 1991. Percentage of juveniles in the reported harvest before 1991 was higher than after 1991. However, percentage of males and litter sizes in the harvest did not differ before and after 1991. The geographic distribution of bobcats captured has gradually expanded after the closure of the harvest season. Our findings suggest that bobcat populations in Quebec have recovered from the 1985–1991 decline, and that the harvest season for this species could resume. This study also illustrates how managers can rely on data from neighboring jurisdiction to manage species when local harvest data is unavailable.     

The ins and outs of endoplasmic reticulum‐controlled lipid biosynthesis

Endoplasmic reticulum (ER)‐localized enzymes synthesize the vast majority of cellular lipids. The ER therefore has a major influence on cellular lipid biomass and balances the production of different lipid categories, classes, and species. Signals from outside and inside the cell are directed to ER‐localized enzymes, and lipid enzyme activities are defined by the integration of internal, homeostatic, and external information. This allows ER‐localized lipid synthesis to provide the cell with membrane lipids for growth, proliferation, and differentiation‐based changes in morphology and structure, and to maintain membrane homeostasis across the cell. ER enzymes also respond to physiological signals to drive carbohydrates and nutritionally derived lipids into energy‐storing triglycerides. In this review, we highlight some key regulatory mechanisms that control ER‐localized enzyme activities in animal cells. We also discuss how they act in concert to maintain cellular lipid homeostasis, as well as how their dysregulation contributes to human disease.     

Transmembrane extension and oligomerization of the CLIC1 chloride intracellular channel protein upon membrane interaction

Chloride intracellular channel proteins (CLICs) differ from most ion channels as they can exist in both soluble and integral membrane forms. The CLICs are expressed as soluble proteins but can reversibly autoinsert into the membrane to form active ion channels. For CLIC1, the interaction with the lipid bilayer is enhanced under oxidative conditions. At present, little evidence is available characterizing the structure of the putative oligomeric CLIC integral membrane form. Previously, fluorescence resonance energy transfer (FRET) was used to monitor and model the conformational transition within CLIC1 as it interacts with the membrane bilayer. These results revealed a large-scale unfolding between the C- and N-domains of CLIC1 as it interacts with the membrane. In the present study, FRET was used to probe lipid-induced structural changes arising in the vicinity of the putative transmembrane region of CLIC1 (residues 24-46) under oxidative conditions. Intramolecular FRET distances are consistent with the model in which the N-terminal domain inserts into the bilayer as an extended α-helix. Further, intermolecular FRET was performed between fluorescently labeled CLIC1 monomers within membranes. The intermolecular FRET shows that CLIC1 forms oligomers upon oxidation in the presence of the membranes. Fitting the data to symmetric oligomer models of the CLIC1 transmembrane form indicates that the structure is large and most consistent with a model comprising approximately six to eight subunits.     

Molecular and functional determinants of local anesthetic inhibition of NaChBac

In our recent publication, we describe the local anesthetic (LA) inhibition of the prokaryotic voltage gated sodium channel NaChBac. Despite the numerous functional and putative structural differences with the mammalian sodium channels, the data show that LA compounds effectively and reversibly inhibit NaChBac channels in a concentration range similar to resting blockade on eukaryotic Navs. In addition to current reduction, LA application accelerated channel inactivation kinetics of NaChBac which could be accounted for in a simple state-model whereby local anesthetics increase the probability of entering the inactivated state. We have further explored what state (or states) local anesthetic blockade of NaChBac could pertain to eukaryotic sodium channels, and what molecular similarities exist between these disparate channel families. Here we show that the rate of recovery from inactivation remains unaffected in the presence of local anesthetics. Further, we show that two sites that support use-dependent inhibition in eukaryotic channels, do not affect block to the same extent when mutated in NaChBac channels. The data indicate that the molecular determinants and the inherent mechanisms for LA block are likely to be divergent between bacterial and eukaryotic Navs, but future experiments will help define possible similarities.     

Inhibition of rabbit beta-globin synthesis by complementary oligonucleotides: identification of mRNA sites sensitive to inhibition

We tested the effects of a series of synthetic oligonucleotides (hybridons) complementary to the 5' noncoding and coding regions of rabbit beta-globin mRNA on endogenous protein synthesis in a rabbit reticulocyte cell-free translation system. With highly purified hybridons inhibition was completely specific for beta-globin. The sites most sensitive to inhibition are the beginning of the 5' noncoding region and a sequence including the initiation codon and several upstream bases. The region between these was relatively insensitive to inhibition. The sites of maximum sensitivity coincide with known protein binding sites, suggesting that hybridons exert their effects in part by blocking the binding of proteins required for translation. Their effectiveness seems related to the ease with which they are displaced by ribosomes.     

TREATMENT OF RAT PROXIMAL AND DISTAL COLONIC CELLS WITH SODIUM ORTHOVANADATE ENHANCES THEIR ADHESION AND SURVIVAL IN PRIMARY CULTURE

We have studied the effect of sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, on primary cultures of colonocytes and stromal cells. Everted proximal and distal colonic tissue of adult rats were disintegrated by a collagenase/dispase solution for 60 min at 37°C to prepare viable gland fragments and isolated cells. Cell preparations were inoculated onto plastic substratum or cytodex-3 microcarriers in a defined maintenance medium or in 1% fetal calf serum media. Incorporation of sodium orthovanadate (≥50 μm) in these media constantly enhanced the survival (cell enumeration and trypan blue exclusionP<0.05) and the adhesion (up to four-fold by crystal violet staining,P<0.01) of colonocytes (characterized by cytokeratin-18, transforming growth factor-α or alkaline phosphatase expression) and stromal cells. Removal of sodium orthovanadate from culture media restored cellular death processes. Incorporation of 10 mmn-butyric acid did not promote cell adhesion and survival except for distal cells exposed to 2 mm sodium orthovanadate. Besides studies in the regulation of anoikis in primary culture, the model will help to assay the influences of dietary and growth factors on the biology of non-cancerous colonic cells.     

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

Zinc and cadmium hyperaccumulation by Thlaspi caerulescens from metalliferous and nonmetalliferous sites in the Mediterranean area: implications for phytoremediation

Growth, tolerance and zinc and cadmium hyperaccumulation of Thlaspi caerulescens populations from three metal contaminated soils and three normal soils were compared under controlled conditions. Individuals of six populations were cultivated on five soils with increasing concentrations of zinc (50–25000 μg g⁻¹) and cadmium (1–170 μg g⁻¹). There was no mortality of normal soil populations in the four metal-contaminated soils, but plant growth was reduced to half that of populations from metal-contaminated soils. However, in noncontaminated soil, the growth of individuals from normal soils was greater than that of individuals from metal-contaminated soils. Individuals from normal soils concentrated three times more zinc in the aboveground biomass than those from metal-contaminated soils, but the latter accumulated twice as much cadmium. We conclude that populations of T. caerulescens from both normal and metal-contaminated soils are interesting material for phytoextraction of zinc and cadmium, but to optimize the process of phytoextraction it is necessary to combine the extraction potentials of both type of populations.