Cell Endoplasmic Reticulum Research Articles

Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments

Macrophages that express representative endoplasmic reticulum (ER) molecules tagged with green fluorescence protein were generated to assess the recruitment of ER molecules to Leishmania parasitophorous vacuoles (PVs). More than 90% of PVs harbouring Leishmania pifanoi or Leishmania donovani parasites recruited calnexin, to their PV membrane. An equivalent proportion of PVs also recruited the membrane-associated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), Sec22b. Both ER molecules appeared to be recruited very early in the formation of nascent PVs. Electron microscopy analysis of infected Sec22b/YFP expressing cells confirmed that Sec22b was recruited to Leishmania PVs. In contrast to PVs, it was found that no more than 20% of phagosomes that harboured Zymosan particles recruited calnexin or Sec22b to their limiting phagosomal membrane. The retrograde pathway that ricin employs to access the cell cytosol was exploited to gain further insight into ER-PV interactions. Ricin was delivered to PVs in infected cells incubated with ricin. Incubation of cells with brefeldin A blocked the transfer of ricin to PVs. This implied that molecules that traffic to the ER are transferred to PVs. Moreover the results show that PVs are hybrid compartments that are composed of both host ER and endocytic pathway components.

Laser Biomodulation on L 929 Cell Culture

The aim of the present study was to analyze the effects of photobiomodulation using a 904-nm diode laser at two energy densities (6 J/cm(2) and 50 mJ/cm(2)) on L929 fibroblast cells. Low-power laser irradiation (LPLI) is a non-pharmacological resource that induces important in vitro photobiomodulation on cell cultures and tissues. Irradiation was performed for three days at 24-h intervals. After each interval, the cells were stained with MitoTracker Orange and DioC6 dyes to assess the photobiomodulatory effects of irradiation on mitochondrial activity and changes in the endoplasmic reticulum. The MTT assay [3-(4.5-dimethylthiazol-2-yl)-2.5 diphenyltetrazolium bromide] was used to evaluate cell proliferation. The fluorescence microscopy assessment of mitochondria and endoplasmic reticulum in cells irradiated with 6 J/cm(2) and 50 mJ/cm(2) demonstrated intense mitochondrial activity, which was confirmed by DioC6 staining. Reticular activity was observed stemming from increased protein synthesis. Photobiomodulation with 50 mJ/cm(2) was slightly higher than with 6 J/cm(2), as demonstrated by fluorescence microscopy results. Photobiomodulation was also time-dependent, with better results 72-h after irradiation.

Human liver mitochondrial cytochrome P450 2D6 – individual variations and implications in drug metabolism

Constitutively expressed human cytochrome P450 2D6 (CYP2D6; EC 1.14.14.1) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however, we have identified this enzyme in the mitochondria of human liver samples and found that extensive inter-individual variability exists with respect to the level of the mitochondrial enzyme. Metabolic assays using 7-methoxy-4-aminomethylcoumarin as a substrate show that the human liver mitochondrial enzyme is capable of oxidizing this substrate and that the catalytic activity is supported by mitochondrial electron transfer proteins. In the present study, we show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the endoplasmic reticulum and mitochondria. In vitro mitochondrial import studies using both N-terminal deletions and point mutations suggest that the mitochondrial targeting signal is localized between residues 23-33 and that the positively-charged residues at positions 24, 25, 26, 28 and 32 are required for mitochondrial targeting. The importance of the positively-charged residues was confirmed by transient transfection of a CYP2D6 mitochondrial targeting signal mutant in COS-7 cells. Both the mitochondria and the microsomes from a CYP2D6 stable expression cell line contain the enzyme and both fractions exhibit bufuralol 1'-hydroxylation activity, which is completely inhibited by CYP2D6 inhibitory antibody. Overall, these results suggest that the targeting of CYP2D6 to mitochondria could be an important physiological process that has significance in xenobiotic metabolism.

Role of the C Terminus of the Photoreceptor ABCA4 Transporter in Protein Folding, Function, and Retinal Degenerative Diseases

ABCA4 is an ATP-binding cassette transporter that is expressed in rod and cone photoreceptor cells and implicated in the removal of retinal derivatives from outer segments following photoexcitation. Mutations in the ABCA4 gene are responsible for a number of related retinal degenerative diseases, including Stargardt macular degeneration, cone-rod dystrophy, retinitis pigmentosa, and age-related macular degeneration. In order to determine the role of the C terminus of ABCA4 in protein structure and function and understand mechanisms by which C-terminal mutations cause retinal degenerative diseases, we have expressed and purified a series of deletion and substitution mutants of ABCA4 and ABCA1 in HEK 293T cells for analysis of their cellular localization and biochemical properties. Removal of the C-terminal 30 amino acids of ABCA4, including a conserved VFVNFA motif, resulted in a loss in N-retinylidene-phosphatidylethanolamine substrate binding, ATP photoaffinity labeling, and retinal-stimulated ATPase activity. This mutant was also retained in the endoplasmic reticulum of cells. Replacement of the VFVNFA motif with alanine residues also resulted in loss in function and cellular mislocalization. In contrast, C-terminal deletion mutants that retain the VFVNFA motif were functionally active and localized to intracellular vesicles similar to wild-type ABCA4. Our studies indicated that the VFVNFA motif is required for the proper folding of ABCA4 into a functionally active protein. This motif also contributes to the efficient folding of ABCA1 into an active protein. Our results provide a molecular based rationale for the disease phenotype displayed by individuals with mutations in the C terminus of ABCA4.

Solution Structure of Human ζ-COP: Direct Evidences for Structural Similarity between COP I and Clathrin-Adaptor Coats

COP-I-coated vesicles are protein and lipid carriers that mediate intra-Golgi transport and transport from the cis-Golgi complex to the endoplasmic reticulum in cells. The coatomer of the vesicles coat is comprised of seven subunits: α-COP, ɛ-COP, β′-COP, β-COP, γ-COP, δ-COP, and ζ-COP. Here we report the solution structure of a truncated form (residues 1–149; ζ-COP149) of human ζ-COP (total 177 residues). It is the first three-dimensional structure of a “core” subunit of the COP I F-subcomplex. The structure of ζ-COP149 mainly consists of a disordered N-terminal tail, a five-stranded antiparallel β-sheet, a two-stranded antiparallel β-sheet, and five α-helices. The global folding of ζ-COP149 is very similar to the crystal structures of AP1-σ1 and AP2-σ2, directly demonstrating the structural similarity between the “core” subunits of the COP I F-subcomplex and adaptor protein complexes. Through structural comparison and mutagenesis study, we have also demonstrated that the heterodimers of ζ-COP149 and γ-COP have packing interfaces and relative subunit orientations similar to those of AP2-σ2 and AP2-α heterodimers. These results provide direct evidence supporting the previous proposal that the COP I F-subcomplex and adaptor protein complexes have similar tertiary and quaternary structures.

The endoplasmic reticulum stress response in immunity and autoimmunity

Many exogenous sources of stress can lead to cell death. In recent years, endogenous cellular sources of stress have also been identified, including the stress that arises from the accumulation of unfolded proteins within a cell's endoplasmic reticulum (ER). To counterbalance this type of ER stress, higher eukaryotic cells possess a three-pronged signal-transduction pathway termed the unfolded-protein response (UPR). This Review focuses on the role of the UPR in the mammalian immune system and how manipulation of this complex signalling pathway may be of therapeutic benefit in human disease.

A Stressful Situation

CELL BIOLOGYWhen unfolded proteins amass, they stress the cell's endoplasmic reticulum. This ER stress is now being linked to diabetes, cancer, neurodegeneration, and other ills. ([Read more][1].) [1]: http://www.sciencemag.org/cgi/content/full/313/5793/1564

Isolation and characterization of a novel v-SNARE family protein that interacts with a calcium-dependent protein kinase from the common ice plant, Mesembryanthemum crystallinum

McCPK1 (Mesembryanthemum crystallinum calcium-dependent protein kinase 1) mRNA expression is transiently salinity- and dehydrationstress responsive. The enzyme also undergoes dynamic subcellular localization changes in response to these same stresses. Using the yeast-two hybrid system, we have isolated and characterized a M. crystallinum CPK1 Adaptor Protein 2 (McCAP2). We show that McCPK1 interacts with the C-terminal, coiled-coil containing region of McCAP2 in the yeast two-hybrid system. This interaction was confirmed in vitro between the purified recombinant forms of each of the proteins and in vivo by coimmunoprecipitation experiments from plant extracts. McCAP2, however, was not a substrate for McCPK1. Computational threading analysis suggested that McCAP2 is a member of a novel family of proteins with unknown function also found in rice and Arabidopsis. These proteins contain coiled-coil spectrin repeat domains present in the syntaxin super-family that participate in vesicular and protein trafficking. Consistent with the interaction data, subcellular localization and fractionation studies showed that McCAP2 colocalizes with McCPK1 to vesicular structures located on the actin cytoskeleton and within the endoplasmic reticulum in cells subjected to low humidity stress. McCAP2 also colocalizes with AtVTIl1a, an Arabidopsis v-SNARE [vesicle-soluble N-ethyl maleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] present in the trans-Golgi network (TGN) and prevacuolar compartments (PVCs). Both interaction and subcellular localization studies suggest that McCAP2 may possibly serve as an adaptor protein responsible for vesicle-mediated trafficking of McCPK1 to or from the plasma membrane along actin microfilaments of the cytoskeleton.

Red Grape Juice Polyphenols Alter Cholesterol Homeostasis and Increase LDL-Receptor Activity in Human Cells In Vitro

Red grape juice (RGJ) polyphenols have been shown to reduce circulating levels of LDL cholesterol and to increase LDL receptor activity. To explore the effect of RGJ-derived polyphenols on intracellular cholesterol homeostasis, human hepatocarcinoma HepG2 and promyelocytic HL-60 cell lines were incubated in serum-free medium, with or without LDL, in the presence or absence of RGJ. In the presence of LDL, RGJ increased both the activity and cell surface expression of the LDL receptor, and increased the cell total cholesterol content. In cells exposed to LDL, RGJ also increased levels of the active form of sterol regulatory element-binding protein-1 and mRNA expression of the LDL receptor and hydroxymethylglutaryl-CoA reductase. In contrast, RGJ caused a marked reduction in the expression of CYP7A1, apolipoprotein B, ABCA1, and ABCG5. Experiments using the acyl-CoA cholesterol acyltransferase inhibitor S-58035 indicated that no measurable free cholesterol from endocytosed LDL reaches the endoplasmic reticulum in cells treated with RGJ. Finally, fluorescence microscopy revealed that in RGJ-treated cells, DiI-labeled LDL did not colocalize with CD63, a protein localized at steady state in the internal vesicles of late endosomes. These results indicate that RGJ polyphenols disrupt or delay LDL trafficking through the endocytic pathway, thus preventing LDL cholesterol from exerting regulatory effects on intracellular lipid homeostasis.

Bimodal targeting of human cytochrome P450 2D6 to mitochondria and microsomes: A pharmacogenomic approach for identifying genetic variants defective in mitochondrial targeting

Cytochrome P450 2D6 (CYP2D6) is responsible for the metabolism of approximately 25% of drugs in common clinical use. It is widely accepted that CYP2D6 is localized in the endoplasmic reticulum of cells; however recent studies in our laboratory have identified this enzyme in the mitochondria of human liver samples, and demonstrated extensive inter-individual variability in the mitochondrial levels. Our results show that CYP2D6 contains an N-terminal chimeric signal that mediates its bimodal targeting to the ER and mitochondria. The mitochondrial targeting signal and protein kinase A mediated phosphorylation are both required for mitochondrial import in isolated mitochondria as well as in intact COS cells. Metabolic studies show that the mitochondrial enzyme is active in drug metabolism and that the level of enzymatic activity varies significantly between individuals. Screening of the liver samples by RT-PCR has also enabled us to identify several variant forms with mutations involving the ER targeting signal, the proline rich domain, as well as the PKA-specific phosphorylation site. These mutations affect the efficiency of mitochondrial targeting thus providing a valuable system to study the role of mitochondrial CYP2D6 in modulating both pharmacological efficacies and adverse toxic effects. (Supported by NIH grant GM34883 and MSTP grant 5T32GM007170).

Temperature sensitive secretion of mutant myocilins

Recent studies have demonstrated that glaucoma-causing mutant myocilin proteins are misfolded and retained in the endoplasmic reticulum of cells. We showed previously that P370L mutant myocilin is poorly secreted at 37 °C and prolonged expression of the protein in differentiated human trabecular meshwork cells results in abnormal morphology and cell killing. Culturing cells at a lower temperature, a condition known to facilitate protein folding, enhances secretion and reverses the cytotoxic effects. We wanted to determine if temperature sensitive secretion is a general property of myocilin missense mutants. Wild-type or mutant forms of myocilin were transiently expressed in HEK 293 cells cultured at either 37 or 30 °C and protein secretion was assessed by immunoblotting. Of 15 myocilin missense mutants tested, representing a range in severity of associated glaucoma phenotypes, 14 displayed increased secretion at 30 °C. The sole exception was K423E, which is associated with an unusual mode of glaucoma inheritance. Generally, there is an inverse relationship between the degree of mutant myocilin secretion at 30 °C and the severity of the associated glaucoma phenotype. Mutants that show abundant secretion at 30 °C such as T377M, G364V, I499F and D380A are associated with less virulent glaucoma phenotypes, while mutants such as P370L, I477N, and Y437H display little secretion at 30 °C and are associated with more virulent glaucoma phenotypes. We conclude that temperature sensitive secretion is a property of most olfactomedin-domain myocilin mutants. The correlation between temperature sensitive secretion and glaucoma phenotype likely reflects the intrinsic susceptibility to misfolding of individual mutant proteins. These results support the hypothesis that myocilin-induced glaucoma is a protein conformational disease. Facilitating mutant protein folding could be a new approach to development of therapies for this disease.

Polycystin-2 accelerates Ca 2+ release from intracellular stores in Caenorhabditis elegans

Polycystin-2, a member of the TRP family of calcium channels, is encoded by the human PKD2 gene. Mutations in that gene can lead to swelling of nephrons into the fluid-filled cysts of polycystic kidney disease. In addition to expression in tubular epithelial cells, human polycystin-2 is found in muscle and neuronal cells, but its cell biological function has been unclear. A homologue in Caenorhabditis elegans is necessary for male mating behavior. We compared the behavior, calcium signaling mechanisms, and electrophysiology of wild-type and pkd-2 knockout C. elegans. In addition to characterizing PKD-2-mediated aggregation and mating behaviors, we found that polycystin-2 is an intracellular Ca 2+ release channel that is required for the normal pattern of Ca 2+ responses involving IP 3 and ryanodine receptor-mediated Ca 2+ release from intracellular stores. Activity of polycystin-2 creates brief cytosolic Ca 2+ transients with increased amplitude and decreased duration. Polycystin-2, along with the IP 3 and ryanodine receptors, acts as a major calcium-release channel in the endoplasmic reticulum in cells where rapid calcium signaling is required, and polycystin-2 activity is essential in those excitable cells for rapid responses to stimuli.

Simulations of transient membrane behavior in cells subjected to a high-intensity ultrashort electric pulse

A molecular dynamics (MD) scheme is combined with a distributed circuit model for a self-consistent analysis of the transient membrane response for cells subjected to an ultrashort (nanosecond) high-intensity (approximately 0.01-V/nm spatially averaged field) voltage pulse. The dynamical, stochastic, many-body aspects are treated at the molecular level by resorting to a course-grained representation of the membrane lipid molecules. Coupling the Smoluchowski equation to the distributed electrical model for current flow provides the time-dependent transmembrane fields for the MD simulations. A good match between the simulation results and available experimental data is obtained. Predictions include pore formation times of about 5-6 ns. It is also shown that the pore formation process would tend to begin from the anodic side of an electrically stressed membrane. Furthermore, the present simulations demonstrate that ions could facilitate pore formation. This could be of practical importance and have direct relevance to the recent observations of calcium release from the endoplasmic reticulum in cells subjected to such ultrashort, high-intensity pulses.

The role of thiols and disulfides in platelet function.

Disulfide bonds formed in newly synthesized proteins in the endoplasmic reticulum of cells are important for protein structure and stability. Recent research, however, emphasizes a role for thiol-disulfide reactions with disulfide bond rearrangement as a dynamic process in cell and protein function, and in platelet function in particular. Protein disulfide isomerase was found on the platelet surface where it appears to play an important role in the platelet responses of aggregation and secretion, as well as activation of the platelet fibrinogen receptor, the alphaIIbbeta3 integrin. Additionally, sulfhydryl groups in alphaIIbbeta3 have been implicated in the activation of this integrin. Physiologic concentrations of reduced glutathione generate sulfhydryls in alphaIIbbeta3 and potentiate sulfhydryl-dependent reactions in alphaIIbbeta3. Sulfhydryl labeling in alphaIIbbeta3 is inhibited by phenylarsine oxide, a reagent that binds to vicinal thiols. As vicinal thiols are in equilibrium with disulfide bonds, they provide redox-sensitive sites in alphaIIbbeta3 able to respond to external or cytoplasmic reducing equivalents. Furthermore, protein disulfide isomerase and sulfhydryls are now implicated in platelet adhesion by a second platelet integrin, the alpha2beta1 collagen receptor. Most recently, extracellular sulfhydryls in the P2Y12 ADP receptor were found to be required for platelet activation by this receptor. We here provide an overview of this field with a focus on recent developments, and conclude with a working model.

Inhibition of prolyl 4-hydroxylase in vitro and in vivo by members of a novel series of phenanthrolinones

Examples of a novel series of phenanthrolinones are shown to be potent competitive inhibitors of avian prolyl 4-hydroxylase, and of collagen hydroxylation, in embryonic chick tendon cells and human foreskin fibroblasts in vitro and in the oestradiol-stimulated rat uterus in vivo. Two compounds, Compound 1 (1,4-dihydrophenanthrolin-4-one-3-carboxylic acid) and Compound 5 [8-(N-butyl-N-ethylcarbamoyl)-1,4-dihydrophenathrolin-4-one-3-carboxylic acid], with comparable potencies in vivo, were chosen to investigate the effect of the inhibition of the hydroxylation of newly synthesized uterine collagen on the turnover of this protein in vivo. Inhibition of hydroxylation by more than 50% for approx. 8h following single oral doses of the compounds was associated with significant losses of radiolabelled proline and 4-hydroxyproline from collagen during this period. Progressive hydroxylation of collagen over 48h, as the inhibitory action of the compounds declined, was accompanied by a decreased loss of radiolabel from the uterine collagen. Earlier reports indicated that underhydroxylated collagen, accumulating within the endoplasmic reticulum in cells where prolyl 4-hydroxylase is inactivated, is slowly degraded, but is then rapidly hydroxylated and secreted when the activity of prolyl 4-hydroxylase is restored. Taken with the present results, this suggests that the potential use of inhibitors of prolyl 4-hydroxylase to control excessive collagen deposition in pathological fibrosis may be limited by the need to maintain continuous inhibition of collagen hydroxylation so as to facilitate intracellular degradation of the accumulated protein.

Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates

Two closely related enzymes with more than 50% sequence identity have been identified that catalyze the esterification of cholesterol using acyl-CoA substrates, namely acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2. Both are membrane-spanning proteins believed to reside in the endoplasmic reticulum of cells. ACAT2 has been hypothesized to be associated with lipoprotein particle secretion whereas ACAT1 is ubiquitous and may serve a more general role in cellular cholesterol homeostasis. We have prepared and affinity purified rabbit polyclonal antibodies unique to either ACAT enzyme to identify their cellular localization in liver and intestine, the two main lipoprotein-secreting tissues of the body, and for comparison, kidney and adrenal. In the liver, ACAT2 was identified in the rough endoplasmic reticulum of essentially all hepatocytes whereas ACAT1 was confined to cells lining the intercellular spaces among hepatocytes in a pattern typical of Kupffer cells. In the intestine, ACAT2 signal was strongly present in the apical third of the mucosal cells, whereas ACAT1 staining was diffuse throughout the mucosal cell, but with strong signal in goblet cells, Paneth cells, and villus macrophages. In the kidney, ACAT1 immunostaining was specific for the distal tubules and podocytes within the glomerulus. In the adrenal, ACAT1 signal was strongly present in the cells of the cortex, and absent from other adrenal cell types. No ACAT2 signal was identified in the kidney or adrenal. We conclude that only the cells of the liver and intestine that secrete apolipoprotein B-containing lipoproteins contain ACAT2, whereas ACAT1 is present in numerous other cell types. The data clearly suggest separate functions for these two closely related enzymes, with ACAT2 being most closely associated with plasma cholesterol levels.

Scavenger Receptor, Class B, Type I-dependent Stimulation of Cholesterol Esterification by High Density Lipoproteins, Low Density Lipoproteins, and Nonlipoprotein Cholesterol

Scavenger receptor, class B, type I (SR-BI) is a cell surface glycoprotein that mediates selective uptake and efflux of sterols from high density lipoproteins (HDL) to cells. A Chinese hamster ovary cell line that is deficient in functional LDL receptors, but has high expression levels of recombinant SR-BI (ldlA7-SR-BI), was used to examine the effect of SR-BI on the trafficking of sterols between lipoproteins and cells. To monitor the fate of sterols transported by SR-BI into cells, we measured the incorporation of [14C]oleate into cholesterol esters by acyl-CoA:cholesteryl acyltransferase in the endoplasmic reticulum. We show that incubation of ldlA7-SRBI cells with either LDL or HDL resulted in an equally dramatic increase in the formation of [14C]oleate-labeled cholesterol esters. The lipoprotein-stimulated, SR-BI-dependent increase in cholesterol esterification was inhibited by chloroquine. The uptake of sterols and their incorporation into cholesterol esters by SR-BI from LDL was largely a selective process. The addition of free cholesterol to ldlA7-SRBI cells also stimulated cholesterol ester formation in a chloroquine-sensitive fashion. We also show that SR-BI mediates the efflux of endogenously synthesized sterols from the cell membrane. From these studies we conclude that, in the absence of the LDL receptor, overexpression of SR-BI can mediate significant transport of sterols between lipoproteins and the endoplasmic reticulum of cells.

Effect of a lysyl hydroxylase inhibitor, minoxidil, on ultrastructure and behavior of cultured rabbit subconjunctival fibroblasts

Minoxidil is an inhibitor of lysyl hydroxylase, an enzyme involved in collagen production, and decreases collagen production in vitro. We investigated the in vitro effects of minoxidil on behavior such as proliferation and migration of rabbit subconjunctival fibroblasts (SCFs). The ultrastructural effect of the drug on SCFs was also examined. Proliferation of SCFs and closure of the defect produced in monolayer cultures in the presence or absence of minoxidil was studied. The ultrastructure of SCFs treated with minoxidil was also examined. Minoxidil inhibited SCF proliferation and the closure of the defect produced in monolayer cell sheets. Ultrastructural observations revealed extensive areas of irregularly dilated endoplasmic reticulum in cells treated with minoxidil, indicating the accumulation of protein, probably underhydroxylated collagen precursors, in the cisternae of endoplasmic reticulum. The results indicated that minoxidil attenuated cellular activities of SCFs such as proliferation and migration in vitro. The exact mechanism of the inhibitory effects of minoxidil on these cellular activities is unknown. The findings suggest that the drug might help to prevent bleb scarring after glaucoma filtering surgery.

Regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in permeabilized cells.

We have studied the regulated degradation of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase within the endoplasmic reticulum in cells permeabilized with digitonin. Using Chinese hamster ovary cells transfected with a plasmid encoding HMGal, a chimeric protein containing the membrane domain of HMG-CoA reductase coupled to beta-galactosidase, we have demonstrated mevalonate and sterol-stimulated loss of beta-galactosidase activity. In pulse-chase experiments we have demonstrated mevalonate-stimulated degradation of both HMGal and HMG-CoA reductase. The rate of mevalonate-stimulated degradation observed in permeabilized cells tends to be slightly slower than that observed in intact cells treated with mevalonate and is dependent upon incubation of cells with mevalonate prior to permeabilization. The degradation process measured in this report extends a previous report of HMG-CoA reductase degradation in digitonin-permeabilized cells (Leonard, D. A., and Chen, H. W. (1987) J. Biol. Chem. 262, 7914-7919) by mimicking key physiological features of the in vivo process, including: stimulation by regulatory molecules, specifically mevalonate and sterols; inhibition by cycloheximide; and inhibition by an inhibitor of neutral cysteine proteases.

Effects of organic solvent vehicles on the viability and morphology of isolated perfused porcine skin

Although many organic solvents are known tio be cutaneous irritants, they are commonly utilized as vehicles in percutaneous absorption and toxicity studies. The isolated perfused porcine skin flap (IPPSF) is an alternative animal model that has been used to study percutaneous absoprtion and cutaneous toxicity. The purpose of this study was to evaluate the effect of five organic solvents (ethanol, acetone, dimethyl sulfoxide (DMSO), toluene, and cyclohexane) on biochemical viability parameters, vascular response, and epidermal morphology of the OPPSF. Cumulative glucose utilization (CGU), the ratio of lactate production/glucose utilization (L/CGU ratio),a nd the leakage of lactate dehydrogenase (LDH) were used as biochemical indicators of alterations in glucose metabolism and flap viability. Only ethanol resulted in a statistically sigficantly decrease in the average rate of CGU over the perfusion. All of the solvent treatments resulted in slight in LDH release versus the control. Vascular resistance (VR) was measured to examine the response of the cutaneous vasculature to these solvents,a nd most treatments resulted in a decreased VR in the terminal phases of perfusion. Ethanol was the only solvent to cause an apparent increase in terminal VR. Light micriscopy demonstrated a modelrate increase in intracellular edema in the DMSO, toluene, and acetone flaps. Ultrastructural evaluation showed focal bledding of the nuclear envelope amd vesiculation of the rough endoplasmic reticulum in cells of the stratum basale and styratum spinosum layers with DMSO treatment. The IPPSF allowed the evaluation of subtle biochemical, vascular, and morphological changes associated with non-occlusive topical exposure to these organic solvents. These findings support the necessity of documenting vehicle efefcts which might mask or other wise alter subtle,but potentially important, compound-specific responses.