A regional outbreak of S. Typhimurium in Denmark and identification of the source using MLVA typing.

In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all S. Typhimurium isolates are currently sub-typed using phage typing, antibiogram typing, and pulsed-field gel electrophoresis (PFGE). However, the discriminatory ability of PFGE is not always high enough to discriminate within certain phage types, and it is not always possible to separate unrelated and related isolates. We have therefore applied multiple locus variable number of tandem repeats analysis (MLVA) for surveillance typing of S. Typhimurium since 2004. In May and June 2005, an outbreak with 26 cases of S. Typhimurium infection was identified by MLVA. The isolates were fully sensitive and had one of the most frequently occurring Danish phage types (DT12) and PFGE types. S. Typhimurium DT12 isolates from routine surveillance of animals and food were typed using MLVA and PFGE for comparison with the human isolates. The typing results revealed that an isolate from a pig herd and its corresponding slaughterhouse located in the same geographic region as the outbreak had the same PFGE and MLVA type as the human isolates. In contrast, all other DT12 isolates investigated, which had the same PFGE profile, had different MLVA types. The conclusion that the pig herd was the source of the human infections was supported by patient information, and pork from the herd stopped entering the market on 29 June. MLVA may contribute significantly to both surveillance and outbreak investigations of S. Typhimurium, as without MLVA typing this outbreak would not have been found nor its origin traced.

A regional outbreak of S. Typhimurium in Denmark and identification of the source using MLVA typing.

In Denmark, as part of the national laboratory-based surveillance system of human enteric infections, all S. Typhimurium isolates are currently sub-typed using phage typing, antibiogram typing, and pulsed-field gel electrophoresis (PFGE). However, the discriminatory ability of PFGE is not always high enough to discriminate within certain phage types, and it is not always possible to separate unrelated and related isolates. We have therefore applied multiple locus variable number of tandem repeats analysis (MLVA) for surveillance typing of S. Typhimurium since 2004. In May and June 2005, an outbreak with 26 cases of S. Typhimurium infection was identified by MLVA. The isolates were fully sensitive and had one of the most frequently occurring Danish phage types (DT12) and PFGE types. S. Typhimurium DT12 isolates from routine surveillance of animals and food were typed using MLVA and PFGE for comparison with the human isolates. The typing results revealed that an isolate from a pig herd and its corresponding slaughterhouse located in the same geographic region as the outbreak had the same PFGE and MLVA type as the human isolates. In contrast, all other DT12 isolates investigated, which had the same PFGE profile, had different MLVA types. The conclusion that the pig herd was the source of the human infections was supported by patient information, and pork from the herd stopped entering the market on 29 June. MLVA may contribute significantly to both surveillance and outbreak investigations of S. Typhimurium, as without MLVA typing this outbreak would not have been found nor its origin traced.

The interaction of adeninylalkylcobalamins with ribonucleotide reductase.

Several structural analogs of adenosylcobalamin, containing 2, 3, 4, 5 and 6 methylene carbons instead of the ribofuranose moiety, have been synthesized and their interaction with ribonucleotide reductase from Lactobacillus leichmannii has been investigated. Kinetic studies of the inhibition of the reductase by these analogs showed that the adeninylalkylcobalamins with 4, 5 and 6 carbons interposed between the adenine moiety and the cobalt atom are potent inhibitors of ribonucleotide reduction. The stronger interaction between adeninylpentylcobalamin and the enzyme than that between adenosylcobalamin and the enzyme suggests that the more flexible acyclic analog of adenosine requires fewer adjustments of the protein upon binding.

Caveolin expression and localization in human keratinocytes suggest a role in lamellar granule biogenesis.

Lamellar granules are sphingolipid-enriched organelles, probably intimately related to the tubulo-vesicular elements of the trans-Golgi network, that deliver the precursors of stratum corneum barrier lipids to the extracellular compartment. Caveolins are cholesterol-binding scaffolding proteins that facilitate the assembly of cholesterol- and sphingolipid-enriched membrane domains known as caveolae. Similarities in the composition of lamellar granules and caveolae suggest that caveolins could be involved in lamellar granule assembly, trafficking, and/or function. In order to explore this relationship, we have examined the expression of caveolins in epidermis, keratinocyte cultures, and an isolated lamellar granule fraction using immunolabeling, immunoblotting, and northern blotting. Several antibodies show immunolocalization of caveolin-1 in the basal layer of human epidermis, with a decline in the suprabasal layers and a reemergence of expression at the stratum granulosum/stratum corneum junction. Two of three caveolin-2 antibodies show little basal staining, but strong signal throughout the rest of the epidermis, whereas a third shows a pattern like caveolin-1. An antibody against caveolin-3 shows a strong signal at the stratum granulosum/stratum corneum interface. Caveolins partially colocalize with glucocerebrosidase, an enzyme known to be critical for remodeling of extruded lamellar granule contents, with AE17, a previously described lamellar-granule-associated antibody, and with glucosylceramides, a major lipid component of lamellar granules. Caveolin-1 protein is present in undifferentiated low-calcium-grown keratinocyte cultures, decreases upon induction of differentiation, and then rises to levels above those seen in undifferentiated cultures, consistent with the immunofluorescence findings. Caveolin-1 mRNA expression parallels that of the protein. Caveolin-2 mRNA and protein expression were unchanged over the course of culture differentiation. Keratinocyte caveolin-1 mRNA expression is not induced by an increase in medium calcium level and is markedly reduced by phorbol-ester-mediated protein kinase C induction. Caveolin-1 is enriched in an isolated lamellar granule fraction that is also enriched, as we have previously described, in lysosomal acid lipase and glucocerebrosidase, and localizes to structures consistent with lamellar granules on immunoelectron microscopy. The differentiation-dependent expression of caveolin-1, the colocalization of caveolins with putative lamellar-granule-associated antigens, their enrichment in isolated lamellar granules, and their presence in lamellar-granule-like structures on immunoelectron microscopy, along with their known structural role in the assembly of glycosphingolipid- and cholesterol-enriched domains in other cell types, suggest that caveolins may play a role in lamellar granule assembly, trafficking, and/or function.

Description of extended pre-harvest pig Salmonella surveillance-and-control programme and its estimated effect on food safety related to pork.

Salmonella in pork can be combated during pre- or post-harvest. For large slaughterhouses, post-harvest measures like decontamination might be cost-effective while this is less likely with small-to-medium sized slaughterhouses. In this study, pre-harvest measures might be more relevant. We describe an extended surveillance-and-control programme for Salmonella in finisher pigs, which, to establish equivalence to the Swedish control programme, is intended for implementation on the Danish island, Bornholm. The effect of the programme on food safety was estimated by analysing Salmonella data from pig carcasses originating from herds that would have qualified for the programme during 2006-2008. Food safety was interpreted as prevalence of Salmonella on carcasses as well as the estimated number of human cases of salmonellosis related to pork produced within the programme. Data from the Danish Salmonella programme were obtained from Bornholm. We used a simulation model developed to estimate the number of human cases based on the prevalence of Salmonella on carcass swabs. Herds are only accepted in the programme if they have one or less seropositive sample within the previous 6 months. In this way, the Salmonella load is kept to a minimum. The programme is not yet in operation and pigs that qualify for the programme are currently mixed at slaughter with those that do not qualify. Therefore, we had to assess the impact on the carcass prevalence indirectly. The prevalence of Salmonella in carcass swabs among qualifying herds was 0.46% for the 3 years as a whole, with 2006 as the year with highest prevalence. According to the simulation the expected number of human cases relating to pork produced within the programme was below 10. When the programme is in operation, an extra effect of separating pigs within the programme from those outside is expected to lower the prevalence of Salmonella even further.

Cloning and expression of cDNA encoding human lysosomal acid lipase/cholesteryl ester hydrolase. Similarities to gastric and lingual lipases.

Molecular cloning of a full-length cDNA for human lysosomal acid lipase/cholesteryl ester hydrolase (EC 3.1.1.13) reveals that it is structurally related to previously described enteric acid lipases, but lacks significant homology with any characterized neutral lipases. The lysosomal enzyme catalyzes the deacylation of triacylglyceryl and cholesteryl ester core lipids of endocytosed low density lipoproteins; this activity is deficient in patients with Wolman disease and cholesteryl ester storage disease. Its amino acid sequence, as deduced from the 2.6-kilobase cDNA nucleotide sequence, is 58 and 57% identical to those of human gastric lipase and rat lingual lipase, respectively, both of which are involved in the preduodenal breakdown of ingested triglycerides. Notable differences in the primary structure of the lysosomal lipase that may account for discrete catalytic and transport properties include the presence of 3 new cysteine residues, in addition to the 3 that are conserved in this lipase gene family, and of two additional potential N-linked glycosylation sites. Transfection of the cDNA into Cos-1 cells resulted in the expression of acid lipase activity with the substrate range of the native enzyme at a level that was greater than 40 times the endogenous activity.

p-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside coupled to albumin. A model compound recognized by the fibroblast lysosomal enzyme uptake system. 1. Chemical synthesis and characterization.

We have developed a simple synthesis for a conjugate of albumin and isothio-nitrophenyl alpha-D-mannopyranoside to study the requirements of the fibroblast lysosomal enzyme recognition system. p-aminophenyl 6-phospho-alpha-D-mannopyranoside was prepared in two ways: (1) phosphorylation of isothio-nitrophenyl alpha-D-mannopyranoside and subsequent reduction of the nitro group by catalytic hydrogenation and (2) direct phosphorylation of p-aminophenyl alpha-D-mannopyranoside. Mannosides were phosphorylated in a reaction with phosphoryl chloride, pyridine, and water at 0 degrees C for 1 h, by a procedure selective for primary hydroxyl groups. Purified p-a minophenyl 6-phospho-alpha-D-mannopyranoside was characterized by chromatographic, enzymatic, and 13C nuclear magnetic resonance spectroscopic methods. Isothio-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside and the p-isothiocyanatophenyl glycosides of alpha-mannose, alpha-glucose, alpha- and beta-galactose, and alpha-L-fucose were formed by reaction of the respective p-aminophenyl glycosides with thiophosgene. Incubation of the p-isothiocyanatophenyl glycosides with bovine serum albumin at pH 8.5, 25 degrees C, for 18 h generally resulted in the coupling, primarily through lysine residues, of up to 20-30 mol of glycoside per mol of protein. Biological properties of the conjugates in the fibroblast lysosomal enzyme recognition system are described in the accompanying paper.

Recognition and receptor-mediated endocytosis of the lysosomal acid lipase secreted by cultured human fibroblasts.

We have studied the recognition and uptake of acid lipase by human fibroblasts in order to determine requirements for localization and function of the enzyme in lysosomes. Our approach was based on evidence that a number of acid hydrolases involved in mucopolysaccharide metabolism are secreted from cultured fibroblasts and endocytosed by a phosphomannosyl-dependent, receptor-mediated process. Acid fatty acid ester hydrolase activity secreted from human fibroblasts was separable into two major forms by hydrophobic chromatography. The dominant form from normal cells was deficient in fibroblasts from patients with Wolman's disease, an inherited disorder of lysosomal cholesteryl ester and triglyceride metabolism. The time- and temperature-dependent, saturable uptake of this enzyme by fibroblasts was competitively inhibited by mannose 6-phosphate and was destroyed by pretreatment of the enzyme with phosphatase. Internalized lipase had a half-life of 1 day. Application of the enzyme to Wolman's disease fibroblasts reduced cholesteryl ester storage; this effect was blocked by ammonium chloride, a general inhibitor of lysosomal hydrolysis. Our results indicate that extracellular acid lipase is transported to fibroblast lysosomes by the same receptor-mediated process that functions in the packaging of several lysosomal glycosidases.

Human lysosomal acid lipase/cholesteryl ester hydrolase. Purification and properties of the form secreted by fibroblasts in microcarrier culture.

Lysosomal acid lipase was purified to near homogeneity in a yield of 25-30% from secretions of human fibroblasts grown on microcarriers in spinner culture. Ammonium chloride was added to the serum-free medium to stimulate production of extracellular enzyme and minimize modifications, including proteolytic processing and destruction of the mannose 6-phosphate recognition marker, that have been associated with packaging and maturation of acid hydrolases in lysosomes. Chromatography of secretions by decyl-agarose, hydroxylapatite, phenylboronate-agarose, and gel filtration resulted in greater than 1500-fold purification of the lipase, representing a 10,000-fold increase above the specific activity of intracellular enzyme. The apparent molecular weight of approximately 49,000, estimated for the lipase by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, was similar to that determined for the native enzyme by gel filtration (Mr approximately 47,000). By contrast, a smaller molecular weight (Mr approximately 41,000) was estimated for the intracellular enzyme. The purified enzyme was susceptible to hydrolysis by endo-beta-N-acetylglucosaminidase H, which resulted in at least two new forms, reduced in apparent molecular weight by approximately 4,000-6,000. Treatment with the endoglycosidase did not alter the catalytic activity or heat stability of the acid lipase. However, the treated enzyme was no longer internalized by fibroblasts via the mannose 6-phosphate receptor and thereby had lost the capacity to correct cholesteryl ester accumulation in cultured lipase-deficient cells. Acid fatty acyl hydrolase activity for cholesteryl oleate, triolein, and methylumbelliferyl oleate co-purified. All three esters were hydrolyzed optimally at pH 4.0, but the pH profile was altered by addition of salts or albumin to the phospholipid-bile salt substrate mixtures. In a series of saturated fatty acyl esters of 4-methylumbelliferone, a derivative with an intermediate chain length (9 carbons) was the best substrate and was hydrolyzed at a rate comparable to that of the oleate ester at pH 4. The optimal pH for hydrolysis of the intermediate and shorter chain length esters was higher by about 2 pH units than that for the longer chain esters (pH approximately 4). The activity of the purified lipase was stimulated by several different proteins. The relationship of this effect to the possible requirement for a natural activator substance has not been determined.(ABSTRACT TRUNCATED AT 400 WORDS)

p-Isothiocyanatophenyl 6-phospho-alpha-D-mannopyranoside coupled to albumin. A model compound recognized by the fibroblast lysosomal enzyme uptake system. 2. Biological properties.

A conjugate of p-aminophenyl 6-phospho-alpha-D-mannopyranoside and bovine serum albumin was shown to interact with the uptake system for lysosomal enzymes in cultured human diploid fibroblasts. Radioiodinated conjugate containing 20 mol of mannose 6-phosphate/mol of albumin was taken up by the cells and degraded to trichloroacetic acid soluble fragments which were released into the medium. Unlabeled conjugate, mannose 6-phosphate, and a lysosomal enzyme, L-iduronidase, inhibited the uptake of the 125I-labeled conjugate (Ki = 2 X 10(-8), 5 X 10(-6), and 1.5 X 10(-9) M, respectively). Conversely, the uptake of L-iduronidase was competitively inhibited by the mannose 6-phosphate conjugate as well as by free mannose 6-phosphate; however, higher concentrations of these compounds were required (Ki = 10(-6) and 5 X 10(-5) M, respectively). These results suggest that although L-iduronidase and the conjugate are bound to the same receptor by mannose 6-phosphate residues, the uptake of the enzyme involves some additional structure that is not shared by the conjugate. Internalization of the radiolabeled mannose 6-phosphate albumin conjugate was observed only in human diploid fibroblast strains. An SV-40 transformed line of human fibroblasts as well as three permanent rodent fibroblast lines (CHO, NRK, and L cells) failed to take up the conjugate, presumably because they were deficient in receptors or in the ability to internalize receptor-conjugate complexes.

Induction of ceramide glucosyltransferase activity in cultured human keratinocytes. Correlation with culture differentiation.

Ceramides are the major component of the extracellular lipids that comprise the epidermal permeability barrier. They are derived from glucosylceramides (GlcCer) upon their extrusion from lamellar granules into the extracellular space in the upper layers of the epidermis. To better understand the regulation of the unique pathway for ceramide production in epidermis, we have studied the activity of the enzyme responsible for GlcCer synthesis, ceramide glucosyltransferase (CerGlc transferase), during keratinocyte culture differentiation. Human keratinocyte cultures were expanded in low calcium keratinocyte growth medium (KGM) and then switched to either normal calcium KGM (nKGM) or "complete" Dulbecco's modified Eagle's medium/Ham's F-12 (3:1) supplemented with 10% fetal bovine serum (cDMEM). At 7 and 10 days after the medium switch, electron microscopy revealed that cDMEM cultures were more fully differentiated morphologically and contained numerous lamellar granules. The GlcCer/DNA content of cDMEM cultures increased to 6 times that of day 0 cultures and was nearly 4 times greater than that of nKGM cultures, whereas the total lipid/DNA content of cDMEM cultures increased to only 1.8 times that of day 0 cultures and was approximately 1.2 times that of nKGM cultures. CerGlc transferase activity/DNA increased 6 times in cDMEM cultures but <1.5 times in nKGM cultures. By contrast, beta-glucocerebrosidase activity, which is responsible for the conversion of GlcCer to ceramide, increased to a similar extent in both differentiating culture systems. Treatment of cultures with the reversible CerGlc transferase inhibitor, DL-threo-1-phenyl-2-(palmitoylamino)-3-morpholino-1-propanol, prevented the increase of GlcCer in cDMEM cultures, and blocked conversion of exogenously added ceramide to GlcCer. A low level of CerGlc transferase activity, relative to that in differentiated keratinocytes, was detected in cultures of other human cell types. These results indicate that CerGlc transferase activity is induced during epidermal differentiation and that regulation of this enzyme may be an important determinant of the specialized production and compartmentalization of epidermal sphingolipids.

Resonance Raman and semiempirical electronic structure studies of an odd-electron dinickel tetraiminoethylenedimacrocycle complex.

Resonance Raman studies of Ni2TIED3+ (TIED = tetraiminoethylenedimacrocycle) reveal that many modes couple to the intense electronic transition centered at 725 nm, a feature that is nominally similar to the intense delocalized intervalence absorption bands observed in the same region for Fe2(TIED)L4(5+) and Ru2(TIED)L4(5+) (L is any of several axial ligands). Time-dependent spectral modeling of the Raman and absorption spectra for the nickel compound was undertaken to understand the electronic transition. We were unable to model the Raman and absorption spectra successfully with a single electronic transition, suggesting that the absorption band is made up of two overlapping transitions. Semiempirical electronic structure calculations corroborate the suggestion. Additionally, these calculations indicate that the transitions are in fact ligand-localized transitions, with little metal involvement and no charge-transfer character. Furthermore, the ground-state electronic structure is best described as an identical pair of NiII centers bridged by a radical anion rather than a three-site mixed-valence assembly. Previous EPR studies (McAuley and Xu, Inorg. Chem. 1992, 31, 5549) had indicated primarily ligand character for the radical. The assignments are consistent with the resonance Raman results where the dominant modes coupled to the transitions are assigned as totally symmetric bridge vibrations.

Lipid metabolism in xanthomatous skin of hypercholesterolemic rabbits.

The authors studied xanthomatous skin in cholesterol-fed rabbits for changes in lipid content and in activities of enzymes regulating intracellular lipid content. After 80 days of hypercholesterolemic diet, xanthomas were widespread and changes in lipid metabolism were marked. In both tissue homogenates and cell membrane pellets, unesterified cholesterol and phospholipids increased 2-fold to 6-fold, and cholesteryl esters increased about 30-fold. Tissue triglycerides, however, decreased to half the levels found in control skin. Cholesterol esterification rates, measured by activity of acyl coenzyme A: cholesterol acyltransferase, increased moderately to markedly; hydrolase activity against 4-methylumbelliferyl oleate also increased at both acid and neutral pH, but hydrolase activity against cholesterol oleate increased only at acid pH. Thus, hypercholesterolemia caused striking increases in intracellular cholesterol esterification rates, increases in lipase activity at both neutral and acid pH, and increases in cholesteryl ester hydrolase activity at acid pH. Increases in cholesterol-esterifying activity uniformly exceeded increases in cholesteryl ester hydrolytic activity in congruence with net accumulation of cholesteryl ester. Skin xanthoma grade, however, had no consistent relation to the cholesterol esterification rates. Instead, the enzyme data suggested that marked abnormalities of lipid metabolism are diffusely distributed through dermal tissue as a precondition for the focal emergence of xanthomas.

Studies on the mechanism of adenosylcobalamin-dependent ribonucleotide reduction by the use of analogs of the coenzyme.

A series of 17 analogs of 5'-deoxy-5'-adenosylcobalamin(adenosylcobalamin) have been synthesized with modifications in the base or ribose moiety of the nucleoside ligand. These analogs have been examined for their effects on reactions catalyzed by the ribonucleotide reductase of Lactobacillus leichmannii. All the analogs are inhibitors of ATP reduction in the presence of adenosylcobalamin as coenzyme, and hence all are bound to the catalytic site. Only the 3-beta-D-ribofuranosyladenine analog (isoadenosylcobalamin) showed substantial activity as a coenzyme in ATP reduction, giving a rate of 59% of that obtained with the adenosylcobalamin. Lesser rates of reduction were obtained with nebularyl-, 2'-deoxyadenosyl-, tubercidyl-, isopropylideneadenosyl-, L-adenosyl-, and ara-adenosylcobalamin, coenzyme activity decreasing in that order. Other analogs showed no significant coenzyme activity. The rate of hydrogen exchange into water from the 5'-methylene group of the nucleoside ligand appeared to parallel the coenzyme activity in those analogs examined, but only the four cobalamins with highest coenzyme activity (adenosyl, isoadenosyl, nebularyl, 2'-deoxyadenosyl) gave detectable amounts of "active coenzyme B12," THe rapidly formed paramagnetic intermediate of ribonucleotide reduction. The enzyme system produced the slowly formed paramagnetic species characterized by a doublet EPR spectrum only with adenosyl- and isoadenosylcobalamin. By contrast the enzymic degradation of analogs to cob(II)alamin and 5'-deoxynucleoside occurred not only with those analogs active as coenzymes and in the exchange reaction but also with a number of coenzymically inactive analogs, and the rate of degradation was unrelated to the rate of ribonucleotide reduction for those analogs with coenzyme activity.

Intercellular transport of lysosomal acid lipase mediates lipoprotein cholesteryl ester metabolism in a human vascular endothelial cell-fibroblast coculture system.

We present results from studies of human cell culture models to support the premise that the extracellular transport of lysosomal acid lipase has a function in lipoprotein cholesteryl ester metabolism in vascular tissue. Vascular endothelial cells secreted a higher fraction of cellular acid lipase than did smooth muscle cells and fibroblasts. Acid lipase and lysosomal beta-hexosaminidase were secreted at approximately the same rate from the apical and basolateral surface of an endothelial cell monolayer. Stimulation of secretion with NH4Cl did not affect the polarity. We tested for the ability of secreted endothelial lipase to interact with connective tissue cells and influence lipoprotein cholesterol metabolism in a coculture system in which endothelial cells on a micropore filter were suspended above a monolayer of acid lipase-deficient (Wolman disease) fibroblasts. After 5-7 d, acid lipase activity in the fibroblasts reached 10%-20% of the level in normal cells; cholesteryl esters that had accumulated from growth in serum were cleared. Addition of mannose 6-phosphate to the coculture medium blocked acid lipase uptake and cholesterol clearance, indicating that lipase released from endothelial cells was packaged into fibroblast lysosomes by a phosphomannosyl receptor-mediated pathway. Supplementation of the coculture medium with serum was not required for lipase uptake and cholesteryl ester hydrolysis by the fibroblasts, but was necessary for cholesterol clearance. Results from our coculture model suggest that acid lipase may be transported from intact endothelium to cells in the lumen or the wall of a blood vessel. We postulate that delivery of acid hydrolases and lipoproteins to a common endocytic compartment may occur and have an impact on cellular lipoprotein processing.

Mutations at the lysosomal acid cholesteryl ester hydrolase gene locus in Wolman disease.

The genomic sequences encoding the human lysosomal acid lipase/cholesteryl esterase (sterol esterase; EC 3.1.1.13) have been isolated and sequenced, and the information has been used to identify mutations in both alleles of the gene from a patient with Wolman disease, an autosomal recessive lysosomal lipid storage disorder. The genomic locus consists of 10 exons spread over 36 kb. The 5' flanking region is G+C-rich and has characteristics of a "housekeeping" gene promoter. One of the identified mutations involves the insertion of a T residue after position 634, resulting in the appearance of an in-frame translation stop signal 13 codons downstream. The second mutation is a T-to-C transition at nucleotide 638. This results in a leucine-to-proline substitution at amino acid 179 and is predicted to lead to the disruption of the alpha-helical structure in a highly conserved region of the protein. These mutations are each capable of completely disrupting the catalytic function of the lysosomal acid cholesteryl ester hydrolase; their presence can account for the extreme phenotype of the lysosomal lipid storage disorder manifested in members of this patient's family.

Adult Niemann-Pick disease masquerading as sea blue histiocyte syndrome: report of a case confirmed by lipid analysis and enzyme assays.

We present the clinical, pathologic, and metabolic findings of an adult woman with debilitating coronary artery disease and hepatosplenomegaly who was discovered to have multiorgan infiltration by sea blue histiocytes. A diagnosis of sea blue histiocyte (SBH) syndrome was made and no further workup performed. The patient suffered from progressive heart failure and sepsis following coronary artery bypass surgery and died 9 months after presentation. Tissues examined at autopsy showed pronounced infiltrates of both granular sea blue histiocytes and foamy, vacuolated histiocytes, which were morphologically compatible with Niemann-Pick cells. Ultrastructural examination of these cells revealed lamellar myelin-like figures as described in Niemann-Pick (N-P) disease. Fibroblast enzyme assay studies and liver lipid analyses performed after the patient's death revealed pronounced sphingomyelinase deficiency and a lipid profile diagnostic of N-P disease, type B. This case adds further support to the claim that some cases of apparent SBH syndrome actually represent a type of N-P disease.

Decreased messenger RNA translation in herpesvirus-infected arterial cells: effects on cholesteryl ester hydrolase.

Herpes simplex viruses (HSVs) contain a function that can cause the degradation of host mRNA and mediate the shutoff of host protein synthesis. Previously, we observed that HSV infection causes a 40-fold increase in cholesteryl ester (CE) accretion in arterial smooth muscle cells due, in part, to a substantial decrease in CE hydrolysis. In studies reported herein, we found that HSV infection leads to reduced immunoprecipitable lysosomal (acid) CE hydrolase (ACEH) and beta-galactosidase, another lysosomal enzyme in vascular smooth muscle cells. The HSV-induced reduction was greater with respect to ACEH than beta-galactosidase. To determine whether degradation of host cellular mRNA or inhibition of cellular translation was responsible for decreased CE hydrolysis in HSV-infected smooth muscle cells, we utilized an in vitro translation system that permitted us to compensate for any mRNA degradation during viral infection. Reduced ACEH activity was observed in the total cellular RNA translation products of HSV-infected smooth muscle cells compared to uninfected cells owing to posttranscriptional modification. We conclude that the decrease in CE hydrolysis in HSV-infected smooth muscle cells is caused primarily by decreased ACEH synthesis and activity, which can contribute to CE accretion in these vascular cells.

Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the Golgi.

Although lamellar granules are critical to the formation of the epidermal permeability barrier and are a known marker of late keratinocyte differentiation, very little is known about the physiologic regulators of lamellar granule assembly and extrusion. Ceramide glucosyltransferase (CGT), the enzyme responsible for the synthesis of lamellar granule glucosylceramides (GlcCer; the precursors of the stratum corneum ceramides), is localized to the Golgi apparatus in other cell types. We have found that CGT is induced during keratinocyte culture differentiation coincident with increased GlcCer content and the appearance of lamellar granules. In this study we show that the differentiation-related CGT induction is likely mediated at the transcriptional level. In addition, all-trans retinoic acid, a well-known inhibitor of keratinocyte differentiation, prevents the appearance of lamellar granules and decreases culture CGT activity and GlcCer content without affecting sphingomyelin or total lipid content, indicating a specific inhibition of this enzymatic pathway. These data show a direct relationship between CGT activity and epidermal differentiation, suggesting that regulation of CGT expression is a critical part of epidermal barrier generation. The differentiation dependence of CGT activity, the key role of this Golgi-localized enzyme in epidermal GlcCer synthesis, and our previous finding that ceramides are converted to GlcCer in the Golgi apparatus in keratinocyte cultures, strongly suggest a Golgi origin for lamellar granules. In contrast to CGT, the activity of the lysosomal enzymes acid lipase and glucocerebrosidase is less clearly related to epidermal differentiation and the appearance of lamellar granules, although both enzymes show striking colocalization and enrichment in a subcellular lamellar granule fraction derived from pig epidermis. Acid lipase activity in the lamellar granule fraction was found to contain primarily a small lysosomal form of the enzyme, whereas total acid lipase secreted by keratinocyte cultures was found to contain a mannose-6-phosphorylated large prelysosomal form as well as a small lysosomal form. That secreted acid lipase activity is derived from both prelysosomal and lysosomal compartments suggests there may be multiple pathways by which lysosomal enzymes are secreted from keratinocytes. The combined secretion of lipid and lysosomal enzymes from lamellar granules places these organelles in the category of "dual-function" specialized secretory vesicles described in certain other cell types. Electron microscopic images of lamellar granules show shapes consistent with cross-sections of tubules or buds from tubules in addition to vesicles. These images provide evidence for the involvement of trans-Golgi network tubules and/or buds in lamellar granule synthesis and secretion.