Knockout of cyclophilin D in Ppif⁻/⁻ mice increases stability of brain mitochondria against Ca²âº stress.

The mitochondrial peptidyl prolyl isomerase cyclophilin D (CypD) activates permeability transition (PT). To study the role of CypD in this process we compared the functions of brain mitochondria isolated from wild type (BMWT) and CypD knockout (Ppif(-/-)) mice (BMKO) with and without CypD inhibitor Cyclosporin A (CsA) under normal and Ca(2+) stress conditions. Our data demonstrate that BMKO are characterized by higher rates of glutamate/malate-dependent oxidative phosphorylation, higher membrane potential and higher resistance to detrimental Ca(2+) effects than BMWT. Under the elevated Ca(2+) and correspondingly decreased membrane potential the dose response in BMKO shifts to higher Ca(2+) concentrations as compared to BMWT. However, significantly high Ca(2+) levels result in complete loss of membrane potential in BMKO, too. CsA diminishes the loss of membrane potential in BMWT but has no protecting effect in BMKO. The results are in line with the assumption that PT is regulated by CypD under the control of matrix Ca(2+). Due to missing of CypD the BMKO can favor PT only at high Ca(2+) concentrations. It is concluded that CypD sensitizes the brain mitochondria to PT, and its inhibition by CsA or CypD absence improves the complex I-related mitochondrial function and increases mitochondria stability against Ca(2+) stress.

Monoterpenoid agonists of TRPV3.

BACKGROUND AND PURPOSE: Transient receptor potential (TRP) V3 is a thermosensitive ion channel expressed predominantly in the skin and neural tissues. It is activated by warmth and the monoterpene camphor and has been hypothesized to be involved in skin sensitization. A selection of monoterpenoid compounds was tested for TRPV3 activation to establish a structure-function relationship. The related channel TRPM8 is activated by cool temperatures and a number of chemicals, among them the monoterpene (-)-menthol. The overlap of the receptor pharmacology between the two channels was investigated. EXPERIMENTAL APPROACH: Transfected HEK293 cells were superfused with the test substances. Evoked currents were measured in whole cell patch clamp measurements. Dose-response curves for the most potent agonists were obtained in Xenopus laevis oocytes. KEY RESULTS: Six monoterpenes significantly more potent than camphor were identified: 6-tert-butyl-m-cresol, carvacrol, dihydrocarveol, thymol, carveol and (+)-borneol. Their EC(50) is up to 16 times lower than that of camphor. All of these compounds carry a ring-located hydroxyl group and neither activates TRPM8 to a major extent. CONCLUSIONS AND IMPLICATIONS: Terpenoids have long been recognized as medically and pharmacologically active compounds, although their molecular targets have only partially been identified. TRPV3 activation may be responsible for several of the described effects of terpenoids. We show here that TRPV3 is activated by a number of monoterpenes and that a secondary hydroxyl-group is a structural requirement.

High-pressure freezing provides new information on human epidermis: simultaneous protein antigen and lamellar lipid structure preservation. Study on human epidermis by cryoimmobilization.

Current transmission electron microscopy techniques do not permit simultaneous visualization of skin ultrastructure and stratum corneum extracellular lipids. We developed a new procedure, which entails application of high-pressure freezing followed by freeze-substitution with acetone containing uranyl acetate, followed by low temperature embedding in HM20. Electrospray ionization mass spectrometry showed that the amount of lipids lost during preparation was minimal. The ultrastructure of cryoprocessed skin was compared with that of conventionally prepared skin samples. Cryoprocessing, but not conventional processing, enabled visualization of lipid stacks within epidermal lamellar bodies, as well as the extracellular lipid domains of the stratum corneum and the ultrastructure within keratinocytes. Anti-filaggrin immunocytochemistry also showed, e.g., excellent preservation of filaggrin on cryoprocessed samples. Additionally, the cytosol of keratinocytes appeared to be organized in "microdomain"-like areas. Finally, the stratum corneum appeared more compact with smaller intercellular spaces and hence tighter cell-cell interactions, after cryoprocessing, than after conventional tissue preparation for transmission electron microscopy. We conclude here that only cryoprocessing preserves skin in a close to native state.

Localization of ceramide and glucosylceramide in human epidermis by immunogold electron microscopy.

Ceramides and glucosylceramides are pivotal molecules in multiple biologic processes such as apoptosis, signal transduction, and mitogenesis. In addition, ceramides are major structural components of the epidermal permeability barrier. The barrier ceramides derive mainly from the enzymatic hydrolysis of glucosylceramides. Recently, anti-ceramide and anti-glucosylceramide anti-sera have become available that react specifically with several epidermal ceramides and glucosylceramides, respectively. Here we demonstrate the detection of two epidermal covalently bound omega-hydroxy ceramides and one covalently bound omega-hydroxy glucosylceramide species by thin-layer chromatography immunostaining. Moreover, we show the ultrastructural distribution of ceramides and glucosylceramides in human epidermis by immunoelectron microscopy on cryoprocessed skin samples. In basal epidermal cells and dermal fibroblasts ceramide was found: (i) at the nuclear envelope; (ii) at the inner and outer mitochondrial membrane; (iii) at the Golgi apparatus and the endoplasmic reticulum; and (iv) at the plasma membrane. The labeling density was highest in mitochondria and at the inner nuclear membrane, suggesting an important role for ceramides at these sites. In the upper epidermis, ceramides were localized: (i) in lamellar bodies; (ii) in trans-Golgi network-like structures; (iii) at the cornified envelope; and (viii) within the intercellular space of the stratum corneum, which is in line with the known analytical data. Glucosylceramides were detected within lamellar bodies and in trans-Golgi network-like structures of the stratum granulosum. The localization of glucosylceramides at the cornified envelope of the first corneocyte layer provides further proof for the existence of covalently bound glucosylceramides in normal human epidermis.

Modification of Cys-418 of pyruvate formate-lyase by methacrylic acid, based on its radical mechanism.

The recently determined crystal structure of pyruvate formate-lyase (PFL) suggested a new view of the mechanism of this glycyl radical enzyme, namely that intermediary thiyl radicals of Cys-418 and Cys-419 participate in different ways [Becker, A. et al. (1999) Nat. Struct. Biol. 6, 969-975]. We report here a suicide reaction of PFL that occurs with the substrate-analog methacrylate with retention of the protein radical (K(I)=0.42 mM, k(i)=0.14 min(-1)). Using [1-(14)C]methacrylate (synthesized via acetone cyanhydrin), the reaction end-product was identified by peptide mapping and cocrystallization experiments as S-(2-carboxy-(2S)-propyl) substituted Cys-418. The stereoselectivity of the observed Michael addition reaction is compatible with a radical mechanism that involves Cys-418 thiyl as nucleophile and Cys-419 as H-atom donor, thus supporting the functional assignments of these catalytic amino acid residues derived from the protein structure.

Large-conductance K+ channel openers induce death of human glioma cells.

Large-conductance Ca(2+)-activated K(+) channels (BKCa channels) are highly expressed in human glioma cells. It has been reported that BK(Ca) channels are present in the inner mitochondrial membrane of the human glioma cell line LN229. In the present study we investigated whether BK(Ca)-channel openers, such as CGS7181 (ethyl 2-hydroxy-1-[[(4-methylphenyl)amino]oxo]-6-trifluoromethyl-1H-indole-3-carboxylate) and CGS7184 (ethyl 1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate), affect the functioning of LN229 glioma cell mitochondria in situ. In the micromolar concentration range CGS7181 and CGS7184 induced glioma cell death. Morphological and cytometric analyses confirmed that both substances trigger the glioma cell death. This effect was not inhibited by the pan-caspase inhibitor z-VAD-fmk. Lack of DNA laddering, PARP cleavage, and caspase 3 activation suggested that glioma cell death was not of the apoptotic type. We examined the effect of CGS7184 on mitochondrial membrane potential and mitochondrial respiration. Potassium channel opener CGS7184 increased cell respiration and induced mitochondrial membrane depolarization. The latter was dependent on the presence of Ca(2+) in the external medium. It was shown that CGS7184 induced an increase of cytosolic Ca(2+) concentration due to endoplasmic reticulum store depletion. In conclusion, our results show that CGS7181 and CGS7184 induce glioma cell death by increasing the cytosolic calcium concentration followed by activation of calpains.

Biosynthesis, processing, and targeting of sphingolipid activator protein (SAP )precursor in cultured human fibroblasts. Mannose 6-phosphate receptor-independent endocytosis of SAP precursor.

Sphingolipid activator proteins (SAPs) are essential cofactors for the lysosomal degradation of glycosphingolipids with short oligosaccharide chains by acidic exohydrolases. SAP-A, -B, -C, and -D derive from proteolysis of a 73-kDa glycoprotein, the SAP precursor. In the present publication, we studied the intracellular transport and the endocytosis of SAP precursor in human skin fibroblasts. Our data indicate that SAP precursor bears phosphate residues on noncomplex carbohydrate chains linked to the SAP-C and the SAP-D domain and sulfate residues on complex carbohydrate chains located within the SAP-A, -C, and possibly the SAP-D domain. Treatment of fibroblasts with either bafilomycin A1 or 3-methyladenine indicates that proteolytic cleavage of SAP precursor begins as early as in the late endosomes. To determine whether targeting of SAP precursor depends on mannose 6-phosphate residues, we analyzed the processing of SAP precursor in I-cell disease fibroblasts. In these cells nearly normal amounts of newly synthesized SAP-C were found, although secretion of SAP precursor was enhanced 2-3-fold. Moreover, SAP-C could be localized to lysosomal structures by indirect immunofluorescence in normal and in I-cell disease fibroblasts. Mannose 6-phosphate was not found to interfere significantly with endocytosis of SAP precursor. Normal fibroblasts internalized SAP precursor secreted from I-cells nearly as efficiently as the protein secreted from normal cells. To our surprise, deglycosylated SAP precursor was taken up by mannose 6-phosphate receptor double knock out mouse fibroblasts more efficiently than the glycosylated protein. We propose that intracellular targeting of SAP precursor to lysosomes is only partially dependent on mannose 6-phosphate residues, whereas its endocytosis occurs in a carbohydrate-independent manner.

In vitro characterization of anti-glucosylceramide rabbit antisera.

Glucosylceramides (GlcCer) are biosynthetic precursors of glycosphingolipids. They are widely distributed in biological systems where they exhibit numerous biological functions. Studies on the localization of glucosylceramides in different tissues have used biochemical methods only since specific antibodies against GlcCer were not previously available. We have characterized two commercially available rabbit antisera which were prepared against GlcCer of plant origin (1-O-(beta-D-glucopyranosyl)-N-acyl-4-hydroxysphinganine; GlcCer-3) or human origin (1-O-(beta-D-glucopyranosyl)-N-acyl-sphingosine; GlcCer-2) and claimed to be specific for GlcCer. The antisera were also able to detect specifically GlcCer species in crude lipid extracts from human epidermis after separation by thin-layer chromatography. The reagents are sensitive since both antisera reacted at dilutions higher than 1:500 with their homologous antigen in the nanogram range in thin layer immunostaining or dot-blot assays. The antisera are specific for GlcCer although they did not differentiate between GlcCer-2 and GlcCer-3 containing sphingosine or 4-hydroxysphinganine. The antisera also reacted with N-stearoyl-DL-dihydroglucocere-broside indicating that the naturally occurring structural variations in the amino alcohol moiety are not determining the specificity. No crossreactivity was observed with other mono- or diglycosylceramides (galactosylceramides, lactosyl-ceramide), free ceramides or structurally unrelated lipids (cholesterol, sphingomyelin, or phospholipids). Therefore, the glycosylmoiety seems to represent the major antigenic determinant. Finally, the antisera also proved to be useful for the immunohistochemical localization of GlcCer in human epidermis by which earlier biochemical data on the distribution of GlcCer in the various epidermal layers were confirmed.

Occurrence of two molecular forms of human acid sphingomyelinase.

Human acid sphingomyelinase (ASM) hydrolyses sphingomyelin to ceramide and phosphocholine. Metabolic studies on COS-1 cells transfected with ASM cDNA revealed the occurrence of an enzymically inactive precursor which is differentially processed to two predominant native glycoprotein forms: a 70 kDa polypeptide corresponding to human urinary protein and a 57 kDa form. Formation of these potentially active forms was shown to be restricted to distinct compartments. Maturation of the ASM precursor to a predominant 70 kDa form occurs exclusively inside acidic organelles, whereas variable amounts of 57 kDa ASM are detectable immediately after biosynthesis. Metabolic labelling of transfected COS-1 cells with [32P]Pi further suggests that this form obviously does not carry oligomannosylphosphate residues, in contrast with the mature lysosomal ASM. In order to verify that this early form of active ASM results from co-post-translational proteolysis of the ASM precursor and not from the use of different translation-initiation sites on the ASM mRNA, appropriate 5'-mutagenized cDNA constructs were transiently expressed. These results clearly indicate that the first potential in-frame AUG is exclusively used for translation initiation in vivo and that deletion of the proposed signal sequence for endoplasmic reticulum import completely eliminates the ability of the translation product to enter the vacuolar apparatus. As there are two different subcellular sites of maturation of the ASM precursor, and intracellular targeting of the two processed forms appears to be different, the two ASM proteins may contribute to distinct physiological functions.

Processing of human acid sphingomyelinase in normal and I-cell fibroblasts.

The biosynthesis of acid sphingomyelinase in normal and I-cell disease fibroblasts was investigated by metabolic labeling with [35S]methionine and immunoprecipitation followed by polyacrylamide gel electrophoresis and fluorography. Two major polypeptides with apparent molecular masses of 75 and 72 kDa (peptide chains of 64 and 61 kDa, respectively) and a minor one with molecular mass of 57 kDa (peptide chain of 47 kDa) were found intracellularly soon after pulse labeling. The 75-kDa form is assumed to be the propropolypeptide of sphingomyelinase which is converted into the precursor form of 72 kDa. The precursor is subjected to two distinct processing events. A minor part is already cleaved in the endoplasmic reticulum-Golgi complex yielding the beta-endo-N-acetylglucosaminidase H-resistant form of 57 kDa; whereas, the major part of the precursor is processed within 4 h to a 70-kDa mature beta-endo-N-acetylglucosaminidase H-sensitive form of sphingomyelinase, which is subsequently converted into a polypeptide with molecular mass of 52 kDa within a chase of about 20 h. Both the precursor (72 kDa) as well as its early cleavage product of 57 kDa are secreted into the culture medium to a minor extent. Intracellular transport of sphingomyelinase into lysosomes depends on the phosphomannosyl specific receptor by following criteria: (i) about 80% of newly synthesized precursor was secreted in NH4Cl-treated fibroblasts as well as in I-cells, (ii) the maturation of sphingomyelinase was inhibited in normal fibroblasts exposed to NH4Cl as well as in I-cell fibroblasts, and (iii) the [32P]phosphate associated with oligosaccharides was cleavable by beta-endo-N-acetylglucosaminidase H. However, endocytosis of radiolabeled extracellular precursor by fibroblasts was not prevented by the addition of mannose 6-phosphate, whereas uptake of arylsulfatase A and beta-hexosaminidase was almost completely blocked under these conditions. This indicates that endocytosis of acid sphingomyelinase by cultured fibroblasts might be mediated by an alternative pathway.

Sphingolipid activator proteins are required for epidermal permeability barrier formation.

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.

Mouse anti-ceramide antiserum: a specific tool for the detection of endogenous ceramide.

Ceramide is a pivotal molecule in signal transduction and an essential structural component of the epidermal permeability barrier. The epidermis is marked by a high concentration of ceramide and by a unique spectrum of ceramide species: Besides the two ceramide structures commonly found in mammalian tissue, N-acylsphingosine and N-2-hydroxyacyl-sphingosine, six additional ceramides differing in the grade of hydroxylation of either the sphingosine base or the fatty acid have been identified in the epidermis. Here we report on the characterization of an IgM-enriched polyclonal mouse serum against ceramide. In dot blot assays with purified epidermal lipids the antiserum bound to a similar extent to N-acyl-sphingosine (ceramide 2), N-acyl-4-hydroxysphinganine (ceramide 3), and N-(2-hydroxyacyl)-sphingosine (ceramide 5), whereas no specific reaction was detected with glycosylceramides, sphingomyelin, free sphingosine, phospholipids, or cholesterol. In contrast, a monoclonal IgM antibody, also claimed to be specific for ceramide, was shown to bind specifically to sphingomyelin and therefore was not further investigated. In thin-layer chromatography immunostaining with purified lipids a strong and highly reproducible reaction of the antiserum with ceramide 2 and ceramide 5 was observed, whereas the reaction with ceramide 1 and ceramide 3 was weaker and more variable. Ceramide 2 and ceramide 5 were detected in the nanomolar range at serum dilutions of up to 1:100 by dot blot and thin-layer immunostaining. In thin-layer chromatography immunostaining of crude lipid extracts from human epidermis, the antiserum also reacted with N-(2-hydroxyacyl)-4-hydroxysphinganine (ceramide 6) and N-(2-hydroxyacyl)-6-hydroxysphingosine (ceramide 7). Furthermore, the suitability of the antiserum for the detection of endogenous ceramide by immunolight microscopy was demonstrated on cryoprocessed human skin tissue. Double immunofluorescence labeling experiments with the anti-ceramide antiserum and the recently described anti-glucosylceramide antiserum (Brade et al., 2000, Glycobiology 10, 629) showed that both lipids are concentrated in separate epidermal sites. Whereas anti-ceramide stained the dermal and basal epidermal cells as well as the corneocytes, anti-glucosylceramide staining was concentrated in the stratum granulosum. In conclusion, the specificity and sensitivity of the reagent will enable studies on the subcellular distribution and biological functions of endogenous ceramide.