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PubMed1992

A quantitative immunocytochemical method for the measurement of islet cell cytoplasmic antibodies.

A quantitative immunocytochemical method for the measurement of islet cell cytoplasmic antibodies has been developed. The method employs human or rat pancreas, a protein A-peroxidase/diaminobenzidine secondary antibody system and an independent measurement of islet total and exocrine mean integrated absorbance by scanning microdensitometry. Specific islet cell cytoplasmic antibody binding (islet total-exocrine mean integrated absorbance) was dependent on serum dilution and substrate reaction time. The detection limit was approximately 5 JDF units. Specific islet cell cytoplasmic antibody binding values with human and rat pancreas were similar. Specific islet cell cytoplasmic antibody binding (human pancreas) was greater (p less than 0.001) in sera from patients with newly diagnosed insulin dependent diabetes mellitus (0.119 +/- 0.086, n = 29) compared to normal sera (0.003 +/- 0.008, n = 29). Thus, the method has been validated and may be useful for measuring the blocking effect of potential antigens on specific islet cell cytoplasmic antibody.

Adolescent↗
PubMed1998

Functional interactions of cytoplasmic domains of the skeletal muscle Ca2+ release channel.

The skeletal muscle Ca(2+) release channel (RYR1) is a homotetramer with subunits of 565 kD. Although the channel part of this protein is probably formed by the C-terminal one fifth of the protein, most of the regulation of channel activity is likely to arise from intermolecular and intramolecular interactions of its large cytoplasmic domain. This cytoplasmic region of the protein is thought to contain binding sites for a variety of modulators, including the t-tubule voltage sensor, and mutations in some cases of malignant hyperthermia and central core disease. Regulation of channel activity must, therefore, involve long-distance allosteric modulation arising from changes in both intersubunit and intrasubunit interactions within the cytoplasmic domain of the RYR1 tetramer. Biochemical studies are beginning to elucidate some of the sites within the cytoplasmic domains important for modulting channel activity in the membrane-spanning domain. This review summarizes these findings and presents a working model for the regulation of the channel by the interactions of its cytoplasmic domains.

Journal Article↗
PubMed1999

Dual roles of p82, the clam CPEB homolog, in cytoplasmic polyadenylation and translational masking.

In the transcriptionally inert maturing oocyte and early embryo, control of gene expression is largely mediated by regulated changes in translational activity of maternal mRNAs. Some mRNAs are activated in response to poly(A) tail lengthening; in other cases activation results from de-repression of the inactive or masked mRNA. The 3' UTR cis-acting elements that direct these changes are defined, principally in Xenopus and mouse, and the study of their trans-acting binding factors is just beginning to shed light on the mechanism and regulation of cytoplasmic polyadenylation and translational masking. In the marine invertebrate, Spisula solidissima, the timing of activation of three abundant mRNAs (encoding cyclin A and B and the small subunit of ribonucleotide reductase, RR) in fertilized oocytes correlates with their cytoplasmic polyadenylation. However, in vitro, mRNA-specific unmasking occurs in the absence of polyadenylation. In Walker et al. (in this issue) we showed that p82, a protein defined as selectively binding the 3' UTR masking elements, is a homolog of Xenopus CPEB (cytoplasmic polyadenylation element binding protein). In functional studies reported here, the elements that support polyadenylation in clam egg lysates include multiple U-rich CPE-like motifs as well as the nuclear polyadenylation signal AAUAAA. This represents the first detailed analysis of invertebrate cis-acting cytoplasmic polyadenylation signals. Polyadenylation activity correlates with p82 binding in wild-type and CPE-mutant RR 3' UTR RNAs. Moreover, since anti-p82 antibodies specifically neutralize polyadenylation in egg lysates, we conclude that clam p82 is a functional homolog of Xenopus CPEB, and plays a positive role in polyadenylation. Anti-p82 antibodies also result in specific translational activation of masked mRNAs in oocyte lysates, lending support to our original model of clam p82 as a translational repressor. We propose therefore that clam p82/CPEB has dual functions in masking and cytoplasmic polyadenylation.

3' Untranslated Regions↗
PubMed1994

Clustering of pseudouridine residues around the peptidyltransferase center of yeast cytoplasmic and mitochondrial ribosomes.

Analysis of the high molecular weight RNAs of the larger ribosomal subunit of Saccharomyces cerevisiae cytoplasm and mitochondria by a new method [Bakin, A., & Ofengand, J. (1993) Biochemistry 32, 9754-9762] has for the first time located all of the pseudouridine residues present in these two RNAs. Thirty pseudouridines were found in the cytoplasmic RNA, and one was found in the mitochondrial RNA. The 30 cytoplasmic RNA pseudouridines were clustered in three regions of the RNA known to be at or near the peptidyltransferase center. The single pseudouridine in yeast mitochondrial rRNA at position 2819 was also located at the peptidyltransferase center. The localization of pseudouridines at or near the peptidyltransferase center in both cytoplasmic and mitochondrial ribosomes implies a functional role for pseudouridine in peptide bond formation. A correlation was shown to exist between the locations of the pseudouridines determined in this work and the positions of the methylated nucleotides (both 2'-OCH3 and base-methylated) determined previously by others. In addition, this work has tentatively identified the locations of two previously unknown ribothymidine residues, at positions 955 and 2920 in the cytoplasmic rRNA.

Base Sequence↗
PubMed1997

Interaction of the cytoplasmic tail of CTLA-4 (CD152) with a clathrin-associated protein is negatively regulated by tyrosine phosphorylation.

CTLA-4 (CD152), high-avidity receptor for CD80 and CD86, is a powerful regulator of T cell activation. While CTLA-4 functions at the cell surface, it is primarily localized in intracellular vesicles and cycles to the cell surface. The CTLA-4 cytoplasmic domain contains sequences that direct its intracellular localization and regulate its signaling. Here we demonstrate that effector molecules involved in receptor trafficking and signaling interact with distinct, but overlapping, sequences in the CTLA-4 cytoplasmic domain. Using the yeast two-hybrid method, we demonstrate association of the mu2 subunit of AP-2, the clathrin-associated complex found in plasma membrane-associated coated pits, with the cytoplasmic tail of CTLA-4, but not CD28. The mu1 subunit of AP-1, found in Golgi-associated coated pits, associated with neither CTLA-4 nor CD28. Sequences required for interaction of mu2 and CTLA-4 were localized to residues, 161TTGVY in CTLA-4; this sequence is N-terminal to, but overlaps with, a previously identified SH2 binding motif, 165YVKM, involved in CTLA-4 signaling. Mu2 interacted preferentially with CTLA-4 when residue 165Y was nonphosphorylated, whereas a PI3 kinase SH2 domain interacted preferentially when 165Y was phosphorylated. In co-transfection experiments, both tyrosine residues in the cytoplasmic tail of CTLA-4 (165Y and 182Y) were phosphorylated by the T lymphocyte-associated tyrosine kinase, p56lck. Thus, phosphorylation of CTLA-4 residue 165Y may reciprocally regulate signaling and trafficking of CTLA-4 by determining which effector molecules bind to its cytoplasmic tail.

Abatacept↗
PubMed1999

Mechanism of interaction of different classes of cationic antimicrobial peptides with planar bilayers and with the cytoplasmic membrane of Escherichia coli.

Antimicrobial cationic peptides are prevalent throughout nature as part of the intrinsic defenses of most organisms, and have been proposed as a blueprint for the design of novel antimicrobial agents. They are known to interact with membranes, and it has been frequently proposed that this represents their antibacterial target. To see if this was a general mechanism of action, we studied the interaction, with model membranes and the cytoplasmic membrane of Escherichia coli, of 12 peptides representing all 4 structural classes of antimicrobial peptides. Planar lipid bilayer studies indicated that there was considerable variance in the interactions of the peptides with model phospholipid membranes, but generally both high concentrations of peptide and high transmembrane voltages (usually -180 mV) were required to observe conductance events (channels). The channels observed for most peptides varied widely in magnitude and duration. An assay was developed to measure the interaction with the Escherichia coli cytoplasmic membrane employing the membrane potential sensitive dye 3,5-dipropylthiacarbocyanine in the outer membrane barrier-defective E. coli strain DC2. It was demonstrated that individual peptides varied widely in their ability to depolarize the cytoplasmic membrane potential of E. coli, with certain peptides such as the loop peptide bactenecin and the alpha-helical peptide CP26 being unable to cause depolarization at the minimal inhibitory concentration (MIC), and others like gramicidin S causing maximal depolarization below the MIC. We discuss the mechanism of interaction with the cytoplasmic membrane in terms of the model of Matsuzaki et al. [(1998) Biochemistry 37, 15144-15153] and the possibility that the cytoplasmic membrane is not the target for some or even most cationic antimicrobial peptides.

Amino Acid Sequence↗
PubMed2003

The nucleo-mitochondrial conflict in cytoplasmic male sterilities revisited.

Cytoplasmic male sterility (CMS) in plants is a classical example of genomic conflict, opposing maternally-inherited cytoplasmic genes (mitochondrial genes in most cases), which induce male sterility, and nuclear genes, which restore male fertility. In natural populations, this type of sex control leads to gynodioecy, that is, the co-occurrence of female and hermaphroditic individuals within a population. According to theoretical models, two conditions may maintain male sterility in a natural population: (1) female advantage (female plants are reproductively more successful than hermaphrodites on account of their global seed production); (2) the counter-selection of nuclear fertility restorers when the corresponding male-sterility-inducing cytoplasm is lacking. In this review, we re-examine the model of nuclear-mitochondrial conflict in the light of recent experimental results from naturally occurring CMS, alloplasmic CMS (appearing after interspecific crosses resulting from the association of nuclear and cytoplasmic genomes from different species), and CMS plants obtained in the laboratory and carrying mitochondrial mutations. We raise new hypotheses and discuss experimental models that would take physiological interactions between cytoplasmic and nuclear genomes into account.

DNA, Mitochondrial↗
PubMed1997

Mitochondrial DNA variation within P-type cytoplasmic male sterility of Plantago lanceolata L.

MtDNA restriction fragment polymorphisms were found between cytoplasmic male-sterility types P and R of Plantago lanceolata with the homologous probe pPl311 and maize mtDNA fragments derived from the regions of atp1, cox1 and cox2. No mtDNA differences were observed between male- sterile and restored plants with the same cytoplasmic type. The con-sistency of the polymorphisms was studied in 83 plants from 24 natural populations in the Netherlands. Within the R-cytoplasm no mtDNA polymorphisms were found, whereas seven variant mtDNA RFLP patterns were observed within the P-cytoplasm using pPl311. We also report on a putative new cytoplasmic male-sterility type, which showed a unique RFLP pattern supporting its distinct status.

Journal Article↗
PubMed1998

Association of an 80 kDa protein with C-CAM1 cytoplasmic domain correlates with C-CAM1-mediated growth inhibition.

Decreased expression of C-CAM, a member of the CEA family of immunoglobulin like cell adhesion molecules, occurs in carcinomas of the colon, liver and prostate. Down regulation of C-CAM during the early stages of carcinogenesis in rat liver and human prostate has also been reported. We have recently shown that restoration of the expression of the isoform with long cytoplasmic domain, C-CAM1, leads to suppression of the tumorigenicity of prostatic carcinoma cells in vivo and growth suppression in vitro. These observations suggest that C-CAM1 may play an important role in regulating cell growth in normal tissues. Previous studies have demonstrated that the function of many members of the Ig-supergene family is dependent on interactions with cytoplasmic proteins. In the present study, we have used a bifunctional cross-linker to identify cellular proteins that interact directly with C-CAM1. Immunoblot analysis of WGA bound membrane proteins crosslinked with DSS identified a 180 kDa complex composed of C-CAM and an 80 kDa protein designated CAP-80 (C-CAM Associated Protein). Immunoprecipitation with anti-C-CAM antibodies showed that CAP-80 was co-precipitated with C-CAM from detergent solubilized, WGA-purified proteins. To assess the specificity of CAP-80 binding, the ability of CAP-80 to form stable complexes with C-CAM1 mutants expressed in insect cells was tested. Deletion of the cytoplasmic domain of C-CAM1 abolished complex formation whereas deletion of the extracellular Ig domains had no effect. These results suggest that a CAP-80 homologue (ICAP-80) is present in insect cells and ICAP-80 interacts with the cytoplasmic domain of C-CAM1. Replacement of Tyr488, a residue in the cytoplasmic domain known to be phosphorylated in vivo, with Phe did not diminish the association between C-CAM1 and ICAP-80, suggesting that Tyr488 phosphorylation is not required for association. The ability of various C-CAM1 mutants to associate with ICAP-80 correlated with their growth inhibitory activities, suggesting that ICAP-80/CAP-80 may play an important role in C-CAM1-mediated growth inhibition.

Adenosine Triphosphatases↗
PubMed1993

Characterization of phosphorylation sites in the cytoplasmic domain of the 300 kDa mannose-6-phosphate receptor.

The human 300 kDa mannose-6-phosphate receptor (MPR 300) is phosphorylated in vivo at serine residues of its cytoplasmic domain. Two-dimensional separation can resolve tryptic phosphopeptides into four major species. To identify the kinases involved in MPR 300 phosphorylation and the phosphorylation sites the entire coding sequence of the cytoplasmic tail was expressed in Escherichia coli. The isolated cytoplasmic domain was used as a substrate for four purified serine/threonine kinases [casein kinase II (CK II), protein kinase A (PKA), protein kinase C and Ca2+/calmodulin kinase]. All kinases phosphorylate the cytoplasmic tail exclusively on serine residues. Inhibition studies using synthetic peptides, partial sequencing of isolated tryptic phosphopeptides and co-migration with tryptic phosphopeptides from MPR 300 labelled in vivo showed that (i) PKA phosphorylates the cytoplasmic MPR 300 domain at Ser20 and at a non-identified site, neither of which are phosphorylated in vivo, and that (ii) the two sites phosphorylated by CK II in vivo and in vitro are Ser82 and Ser157. The results indicate that the human MPR 300 is a physiological substrate of either CK II or a related kinase which may play a role in the transport function of MPR 300 and/or interaction with other proteins.

Amino Acid Sequence↗
PubMed1997

Analysis of blood T-cell cytokine expression in B-chronic lymphocytic leukaemia: evidence for increased levels of cytoplasmic IL-4 in resting and activated CD8 T cells.

The cytoplasmic cytokines of purified blood T cells (CD4/CD8) in B-CLL patients (n = 5) and controls (n = 5) were evaluated by flow cytometry. The mean levels of cytoplasmic IL-4 were significantly elevated in resting and activated B-CLL CD8 cells compared to control CD8 cells. IL-4 cytoplasmic levels were comparable for resting B-CLL and control CD4 cells but greater for B-CLL activated CD4 cells. The mean fluorescence intensity of B-CLL CD8 cytoplasmic IL-4 was 4-5-fold greater, indicating higher IL-4 density per CLL CD8 than control CD8 cells. Both CLL CD4 and CD8 cells post-activation had higher levels of cells double positive for cytoplasmic IL-4 and interferon. These data indicate that freshly isolated CD8 and CD4 blood T cells from B-CLL patients have significantly elevated (above control) levels of commitment to expression of IL-4. Since IL-4 has an important modulatory impact on CLL and normal B cells, this observation has implications regarding the biology of B-CLL.

CD4-Positive T-Lymphocytes↗
PubMed1997

Transcription induction of the ferric citrate transport genes via the N-terminus of the FecA outer membrane protein, the Ton system and the electrochemical potential of the cytoplasmic membrane.

Ferric citrate induces transcription of the ferric citrate transport genes fecABCDE without entering the cells of Escherichia coli K-12. Point mutants of the outer membrane-receptor protein FecA are affected in induction independent of the FecA transport activity, suggesting that FecA is directly involved in induction. Alignment of FecA with the other ferric siderophore receptors of E. coli reveals an N-terminal extension in FecA that is not found in the receptors whose synthesis is not induced by their cognate ferric siderophores. In this study, we show that excision of the N-terminal region abolished the inducing activity of FecA, but retained its transport activity. Overproduction of the N-terminal FecA fragment inhibited FecA-dependent induction, but not transport. Constitutive expression caused by C-terminally truncated FecR derivatives was not inhibited by the N-terminal FecA fragment. The N-terminal region of FecA was localized in the periplasm, which indicates that FecA probably interacts with FecR, which is involved in signal transduction across the cytoplasmic membrane. Transcription initiation of the fec transport genes required the Ton system, consisting of TonB, ExbB, and ExbD, and was inhibited by carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), which dissipate the electrochemical potential of the cytoplasmic membrane. fec transcription of mutant fecA4, which displays constitutive fec transcription in the absence of TonB, was not affected by CCCP. The data support a model that proposes initiation of fec transport gene transcription by binding of ferric citrate to FecA. The transcription initiation signal is transferred across the outer membrane through the activity of the Ton system at the expense of the electrochemical potential of the cytoplasmic membrane. The N-terminus of FecA interacts in the periplasm with the C-terminus of FecR, through which the signal is transferred across the cytoplasmic membrane into the cytoplasm, where it increases the activity of the sigma factor Fecl, which then directs the RNA polymerase to the fec promoter upstream of fecA.

Amino Acid Sequence↗
PubMed1998

Transport of LDS-751 from the cytoplasmic leaflet of the plasma membrane by the rhodamine-123-selective site of P-glycoprotein.

P-glycoprotein is an ATP-dependent transporter of an extremely wide variety of lipophilic compounds. We showed previously [Shapiro, A. B. & Ling, V. (1997a) Eur. J. Biochem. 250, 130-137] that P-glycoprotein contains two drug transporting sites, dubbed H (for Hoechst 33342-selective) and R (for rhodamine-123-selective), that interact with positive cooperativity. The H site transports 2-[2-(4-ethoxyphenyl)-6-benzimidazolyl]-6-(1-methyl-4-piperazyl)be nzimidazole (Hoechst 33342) from the cytoplasmic leaflet of the plasma membrane to the aqueous extracellular medium [Shapiro, A. B. & Ling, V. (1997b) Eur. J. Biochem. 250, 122-129]. The environment from which the R site transports its substrates is unknown. In this paper, we used the fluorescent DNA dye 2-[4-[4-(dimethylamino)phenyl]-1,3-butadienyl]-3-ethylbenzothiazolium perchlorate (LDS-751), a substrate of the R site, to address this issue. LDS-751 which, like Hoechst 33342, exhibits lipid-dependent fluorescence and slow transleaflet diffusion, allowed us to use the same methodology that we used for the H site to study the location of the R site. As with Hoechst 33342, the specific initial rate of LDS-751 transport by P-glycoprotein-rich, isolated plasma membrane vesicles from CH(R)B30 cells was directly proportional to the amount of membrane-bound LDS-751 and inversely proportional to the concentration of free, aqueous LDS-751. This result demonstrates that the R site of P-glycoprotein transports LDS-751 out of the lipid membrane. The slight decrease, instead of an increase, in the initial rate of active transport of LDS-751 with the amount of time elapsed for slow diffusion of LDS-751 from the cytoplasmic leaflet to the extracellular leaflet indicates that the R site of P-glycoprotein removes LDS-751 from the cytoplasmic leaflet of the plasma membrane. Thus, both known drug-transporting sites of P-glycoprotein remove their substrates from the cytoplasmic leaflet. Since all of the P-glycoprotein substrates we have examined so far are recognized by one or both of the two known drug-transporting sites, these two sites in the cytoplasmic leaflet of the plasma membrane may be able to account for all substrate transport by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

The interaction with the cytoplasmic loops of rhodopsin plays a crucial role in arrestin activation and binding.

The binding of arrestin to rhodopsin is initiated by the interaction of arrestin with the phosphorylated rhodopsin C-terminus and/or the cytoplasmic loops, followed by conformational changes that expose an additional high-affinity site on arrestin. Here we use an arrestin mutant (R175E) that binds similarly to phosphorylated and unphosphorylated, wild-type rhodopsin to identify rhodopsin elements other than C-terminus important for arrestin interaction. R175E-arrestin demonstrated greatly reduced binding to unphosphorylated cytoplasmic loop mutants L72A, N73A, P142A and M143A, suggesting that these residues are crucial for high-affinity binding. Interestingly, when these rhodopsin mutants are phosphorylated, R175E-arrestin binding is less severely affected. This effect of phosphorylation on R175E-arrestin binding highlights the co-operative nature of the multi-site interaction between arrestin and the cytoplasmic loops and C-terminus of rhodopsin. However, a combination of any two mutations disrupts the ability of phosphorylation to enhance binding of R175E-arrestin. N73A, P142A and M143A exhibited accelerated rates of dissociation from wild-type arrestin. Using sensitivity to calpain II as an assay, these cytoplasmic loop mutants also demonstrated reduced ability to induce conformational changes in arrestin that correlated with their reduced ability to bind arrestin. These results suggest that arrestin bound to rhodopsin is in a distinct conformation that is co-ordinately regulated by association with the cytoplasmic loops and the C-terminus of rhodopsin.

Amino Acid Substitution↗
PubMed1977

Phalloidin-induced actin polymerization in the cytoplasm of cultured cells interferes with cell locomotion and growth.

Phalloidin, the toxic drug from the mushroom Amanita phalloides, was injected into the cytoplasm of tissue culture cells and the changes in intracellular actin distribution were followed by immunofluorescence microscopy with actin antibody. At low concentrations, phalloidin recruits the non- or less highly polymerized forms of cytoplasmic actin into stable "islands" of aggregated actin polymers and does not interfere with the preexisting thick bundles of microfilaments (stress fibers). Differential focusing shows that these islands of phalloidin-induced actin polymers occur at a level in the cytoplasm that is above the submembranous bundles of microfilaments present on the adhesive side of the cells. The pattern of cytoplasmic microtubules remains unaffected by the injection of phalloidin; however, filamin, a protein usually associated with actin in the cytoplasm, is also recruited into the islands. At higher phalloidin concentrations, contraction of the cell is observed. These results are discussed in the light of previous biochemical studies by Wieland and Faulstich and their coworkers [for a review see Wieland, T. (1977) Naturwissenschaften 64, 303-309] on the in vitro interaction of phalloidin with muscle actin, which have documented that phalloidin reacts stoichiometrically with actin, promotes actin polymerization, and stabilizes actin polymers. In addition, we show that microinjection of phalloidin interferes in a concentration-dependent manner with cell locomotion and cell growth. These results indicate that a well-balanced controlled reversible equilibrium between different polymerization states of actin may be a necessary requirement for cell locomotion and may also influence other cellular functions such as growth.

Actins↗
PubMed1978

Variation in mitochondrial translation products associated with male-sterile cytoplasms in maize.

Mitochondria were isolated under sterile conditions from maize lines with normal cytoplasm and with T, C, and S sources of cytoplasmic male sterility. Protein synthesis by these mitochondria was dependent upon the presence of ADP and an oxidizable substrate or an energy generating system and displayed characteristic sensitivity towards inhibitors of protein synthesis. Approximately 20 polypeptides were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis of the [(35)S]methionine-labeled mitochondrial translation products. Mitochondria from the T and C cytoplasms were characterized by the synthesis of a single additional or variant polypeptide.These observations are discussed with reference to the possible mechanisms of male sterility and the susceptibility of plants with T cytoplasm to Helminthosporium maydis, race T. Our results also suggest a potentially rapid method of classifying sources of cytoplasmic male sterility that could be used in conjunction with existing methods, which depend on patterns of fertility restoration after an extended breeding program.

Journal Article↗
PubMed1982

Mobility of cytoplasmic and membrane-associated actin in living cells.

We have combined fluorescent analogue cytochemistry with fluorescence photobleaching recovery to measure the mobility of fluorescently labeled actin and other labeled test proteins microinjected into living amoebae. Bovine serum albumin, ovalbumin, and ribonuclease A have a cytoplasmic mobility, expressed as a diffusion coefficient, that is 1/2 to 1/3 of that observed in aqueous solution; 90% of the actin has a mobility 1/2 to 1/8 of that of G-actin in aqueous solution, and approximately equal to 10% of the actin has a mobility comparable to that of F-actin in aqueous solution. Therefore, no more than 10% of the actin in the cytoplasm of amoebae can exist as static filaments. Microinjection of phalloidin decreases the diffusion coefficient of the mobile component of cytoplasmic actin, and it also increases the low-mobility fraction to 50% but has no effect on the mobility of labeled ovalbumin. By comparing the mobility of actin in different parts of amoebae and by separating cytoplasm from plasmalemma-ectoplasm, we found the low-mobility fraction of actin to be enriched in the tail, along the plasmalemma-ectoplasm, and in contracted cytoplasm.

Actins↗
PubMed1983

Localization of urinary lactosylceramide in cytoplasmic vesicles of renal tubular cells in homozygous familial hypercholesterolemia.

An average 15-fold increase in lactosylceramide (LacCer) in the sediment of receptor-negative, familial hypercholesterolemic (FH) homozygotes has been reported [Chatterjee, S., Sekerke, C.S. & Kwiterovich, P.O., Jr. (1982) J. Lipid Res. 23, 513-522]. We report here the abnormal urinary excretion of significant numbers of renal tubular cells in eight FH homozygotes. The mean activity of gamma-glutamyltransferase, a marker for renal tubular cells, was twice as high in urinary sediment of FH homozygotes as in normals. Membrane-enclosed cytoplasmic vesicles that stained strongly positive with a fluorescein-labeled antibody against LacCer were found in the renal tubular cells of all homozygotes except two who had undergone a portacaval shunt. These two had normal urinary levels of LacCer, and the cytoplasmic vesicles were vacuolated. In the other six, most of the fluorescent antibody label was intracellular and perinuclear. The cytoplasmic vesicles stained strongly with polychromatic Papanicolaou stain, periodic acid/Schiff reagent, and oil red O. Electron microscopy revealed perinuclear membrane-enclosed lipid and free lipid droplets. When two FH homozygotes, who excreted increased LacCer, underwent plasma exchange, the cytoplasmic vesicles became empty, and the urinary LacCer level decreased into the normal range. We conclude that the increased urinary excretion of LacCer in FH homozygotes occurs in renal tubular cells and that the intracellular locatin of LacCer is within cytoplasmic vesicles. The presence of LacCer within these vesicles can be modulated by treatment with plasma exchange.

Adolescent↗