📋 Browse Articles

Late-infantile neuronal ceroid lipofuscinosis (LINCL), an autosomal recessively inherited lysosomal storage disorder characterized by autofluorescent inclusions and rapid progression of neurodegeneration, is due to CLN2 gene mutations. However, CLN2 mutation analysis has failed to identify some clinically diagnosed "late-infantile" NCL cases. This study was conducted to further characterize genetic heterogeneity in families affected by LINCL. DNA mutations in the CLN1, CLN2, and CLN3 genes that underlie INCL (infantile NCL), LINCL, and JNCL (juvenile NCL), respectively, were studied with molecular analyses. A total of 252 families affected by childhood NCL were studied. Of 109 families clinically diagnosed as having LINCL, 3 were determined to have either INCL or JNCL by identification of mutation(s) in CLN1 or CLN3. Six families diagnosed initially as having JNCL were found to have LINCL based on the finding of mutations in the CLN2 gene. In addition, several novel mutations were identified. Clinical and genetic heterogeneity of LINCL was demonstrated in nine LINCL families studied. Show less

Studies on acetyl phosphate (AcP2-), one of the so-called 'energy-rich' mixed-acid anhydrides, are summarized. Based on stability constants determined by potentiometric pH titrations in aqueous solution, it is shown that the M(AcP) complexes of Ca2+, Mg2+, Mn2+, Cu2+, and Zn2+ are more stable than is expected from the basicity of the phosphate group of AcP2-. This observed stability increase is attributed to an additional interaction of the already phosphate-coordinated metal ion (M2+) with the carbonyl oxygen of the anhydride unit. These conclusions are corroborated by the properties of the complexes of the hydrolysis-stable acetonylphosphonate (AnP2-). The formation degrees of the various six-membered chelates occurring in the M(AcP) and M(AnP) systems are presented and evidence is given that these chelates persist in mixed ligand complexes and that their formation degree is promoted by a low solvent polarity. The biological relevance of these results regarding carbonyl oxygen-metal ion interactions is briefly indicated. Show less

Disheveled blocks the degradation of beta-catenin in response to Wnt signal by interacting with the scaffolding protein, Axin. To define this interaction in detail we undertook a mutational and binding analysis of the murine Axin and Disheveled proteins. The DIX domain of Axin was found to be important for association with Disheveled and two other regions of Axin (between residues 1-168 and 600-810) were identified that can promote the association of Axin and Disheveled. We found that the DIX domain of Disheveled is critical for association with Axin in vivo and for Disheveled activity. The Disheveled DIX domain controlled the ability of Disheveled to induce the accumulation of cytosolic beta-catenin whereas the PDZ domain was not essential to this function. Show less

This study describes the phenotype/genotype analysis of 159 probands with neuronal ceroid lipofuscinosis (37 CLN1, 72 classic CLN2, 10 variant LINCL, and 40 CLN3) collected at the New York State Institute for Basic Research in Developmental Disabilities (IBR). Phenotype/genotype comparison showed that mutations in the CLN1 gene were associated with different phenotypes: infantile, late infantile, and juvenile. Two common mutations (223A-->C and 451C-->T) were found in 26 of 37 CLN1 subjects (64% of alleles examined). A nonsense point mutation, 451C-->T, was the most common in CLN1 subjects with infantile onset at 0-2 years, accounting for 50% of alleles studied. A missense point mutation, 223A-->C, was the most common among CLN1 subjects with juvenile onset older than 4 years, accounting for 45% of alleles examined. Twenty-one other CLN1 mutations were identified in 4 of 37 subjects with infantile onset, 6 of 37 with late-infantile onset, and 6 of 37 with juvenile onset. All CLN1 probands were palmitoyl-protein thioesterase (PPT)-deficient and showed granular osmiophilic deposits (GROD) at the electron microscopic (EM) level. In the group of classic CLN2 (72 probands), two common mutations were found: an intronic 3556G-->C transversion in the invariant AG of 3' splice junction in 55% of probands, and a nonsense mutation 3670C-->T in 30% of probands. Classic late-infantile onset (2-4 years) was found in 68 of 72 (95%) cases, whereas juvenile onset (> 4 years) occurred only in 4 of 72 (5%) cases. All probands had deficiency of tripeptidyl-peptidase I (TPP1) activity and, at the EM level, curvilinear profiles. Ten probands with late-infantile onset did not show mutations in the CLN2 gene, had normal TPP1 activity, and at the EM level had mixed profiles. Further studies are in progress to identify genetic defect(s) in these subjects. The CLN3 group (40 probands) was divided into two categories: classic or typical presentation, and delayed classic or atypical presentation. All CLN3 patients had onset of symptoms after 4 years of age. In 40 probands, the 1.02-kb common deletion was found in one or two alleles of the CLN3 gene. Homozygotes for the common CLN3 deletion showed the classic phenotype. The phenotype in compound heterozygotes was either the classic or the delayed classic or atypical form. Thus, our study indicates that some mutations in the CLN1 and CLN2 genes may be associated with juvenile onset of the disease process and a more benign clinical course. Interfamilial and intrafamilial variations also were found, especially in the speed of becoming blind and neurologically disabled. Show less

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the presence of autofluorescent lipopigment in neurons and other cell types. The childhood onset types display autosomal recessive inheritance. Naturally occurring animal NCLs have been described in many species including mouse, sheep and dog. In the last decade major advances have occurred in the molecular genetic analysis of the NCLs. Six disease gene loci have been mapped, and five disease genes have been isolated. Two of these encode lysosomal enzymes: CLN1 encodes palmitoyl-protein thioesterase (PPT), and CLN2 encodes tripeptidyl peptidase 1 (TPP1). The remaining three, CLN3, CLN5 and CLN8 encode putative membrane proteins of unknown function. The murine orthologue of CLN8 causes motor neuron degeneration (mnd), a mouse model of NCL. These advances have revolutionized diagnosis and classification, but a unified theory of pathogenesis and effective treatment remain elusive. Show less

A family-focused psychosocial intervention for stroke survivors is described and illustrated with case studies. It is designed to improve functional recovery through four specific pathways: increased knowledge, efficacy, and control through stroke education; optimized social support; increased network cohesion; and improved problem-solving abilities. Rationales for these pathways are presented and methods of implementing them discussed. Show less

The calculated rate of urea production [U(p); mmol urea/(h. kg(0. 75))], based on urinary urea-N (UUN) excretion and changes in total body urea-N, was compared with the calculated total body V(max) of carbamoyl phosphate synthetase (CPS-1) of 24 neonatal piglets from four treatments as follows: 6 h baseline control (n = 8), 18 h of alanine intravenously (IV) at 50% of resting energy expenditure (REE; n = 4), 36 h of alanine IV at 50% of REE (n = 6), or 36 h of glucose IV at 50% of REE (n = 6). The following significant increases from baseline were seen in piglets infused with alanine for 36 h: 1) UUN excretion [10.6 +/- 5.9 mg N/(h. kg(0.75)) to 53.2 +/- 11.1]; 2) BUN concentrations (9.1 +/- 3.0 mmol urea N/L to 51.2 +/- 7.0); 3) calculated urea production [0.34 +/- 0.21 mmol urea/(h. kg(0.75)) to 2.39 +/- 0.53]; and 4) CPS-1 V(max) [2.0 +/- 0.81 mmol citrulline/(h. kg (0.75)) to 4.4 +/- 1.5], (P < 0.05). With the exception of CPS-1 activity, significant decreases from baseline were seen in these values in piglets infused with glucose for 36 h (P < 0.05). Comparison of calculated urea production with calculated total body CPS-1 V(max) at baseline, 18 or 36 h after the start of infusion of alanine or glucose revealed a positive relationship (slope = 0.263; P < 0.002). At all enzyme activities, infusion of alanine resulted in a significant increase in the rate of urea production compared with controls (P < 0.001). Total body CPS-1 activity varied from 1.8 to 5.8 times that of urea production, suggesting that CPS-1 did not limit urea production. Show less

Xbp1, a transcriptional repressor of Saccharomyces cerevisiae with homology to Swi4 and Mbp1, is induced by stress and starvation during the mitotic cycle. It is also induced late in the meiotic cycle. Using RNA differential display, we find that genes encoding three cyclins (CLN1, CLN3, and CLB2), CYS3, and SMF2 are downregulated when Xbp1 is overexpressed and that Xbp1 can bind to sequences in their promoters. During meiosis, XBP1 is highly induced and its mRNA appears at the same time as DIT1 mRNA, but its expression remains high for up to 24 h. As such, it represents a new class of meiosis-specific genes. Xbp1-deficient cells are capable of forming viable gametes, although ascus formation is delayed by several hours. Furthermore, Xbp1 target genes are normally repressed late in meiosis, and loss of XBP1 results in their derepression. Interestingly, we find that a deletion of CLN1 also reduces the efficiency of sporulation and delays the meiotic program but that sporulation in a Deltacln1 Deltaxbp1 strain is not further delayed. Thus, CLN1 may be Xbp1's primary target in meiotic cells. We hypothesize that CLN1 plays a role early in the meiotic program but must be repressed, by Xbp1, at later stages to promote efficient sporulation. Show less

The G(1) cyclins of budding yeast drive cell cycle initiation by different mechanisms, but the molecular basis of their specificity is unknown. Here we test the hypothesis that the functional specificity of G(1) cyclins is due to differential subcellular localization. As shown by indirect immunofluorescence and biochemical fractionation, Cln3p localization appears to be primarily nuclear, with the most obvious accumulation of Cln3p to the nuclei of large budded cells. In contrast, Cln2p localizes to the cytoplasm. We were able to shift localization patterns of truncated Cln3p by the addition of nuclear localization and nuclear export signals, and we found that nuclear localization drives a Cln3p-like functional profile, while cytoplasmic localization leads to a partial shift to a Cln2p-like functional profile. Therefore, forcing Cln3p into a Cln2p-like cytoplasmic localization pattern partially alters the functional specificity of Cln3p toward that of Cln2p. These results suggest that there are CLN-dependent cytoplasmic and nuclear events important for cell cycle initiation. This is the first indication of a cytoplasmic function for a cyclin-dependent kinase. The data presented here support the idea that cyclin function is regulated at the level of subcellular localization and that subcellular localization contributes to the functional specificity of Cln2p and Cln3p. Show less

Tricho-rhino-phalangeal syndrome type I (TRPS I, MIM 190350) is a malformation syndrome characterized by craniofacial and skeletal abnormalities and is inherited in an autosomal dominant manner. TRPS I patients have sparse scalp hair, a bulbous tip of the nose, a long flat philtrum, a thin upper vermilion border and protruding ears. Skeletal abnormalities include cone-shaped epiphyses at the phalanges, hip malformations and short stature. We assigned TRPS1 to human chromosome 8q24. It maps proximal of EXT1, which is affected in a subgroup of patients with multiple cartilaginous exostoses and deleted in all patients with TRPS type II (TRPS II, or Langer-Giedion syndrome, MIM 150230; ref.2-5). We have positionally cloned a gene that spans the chromosomal breakpoint of two patients with TRPS I and is deleted in five patients with TRPS I and an interstitial deletion. Northern-blot analyses revealed transcripts of 7 and 10.5 kb. TRPS1has seven exons and an ORF of 3,843 bp. The predicted protein sequence has two potential nuclear localization signals and an unusual combination of different zinc-finger motifs, including IKAROS-like and GATA-binding sequences. We identified six different nonsense mutations in ten unrelated patients. Our findings suggest that haploinsufficiency for this putative transcription factor causes TRPS I. Show less

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA. Show less

Human apolipoprotein A-IV (apoA-IV) transgenic mice fed an atherogenic diet were shown previously to develop less atherosclerosis than control mice. The question arose whether the antiatherogenic effect of human apoA-IV is due to enhancement of reverse cholesterol transport despite no increase in plasma high-density lipoprotein (HDL) cholesterol. We studied male and female mice overexpressing human apoA-IV and their wild-type (WT) controls, all of which were fed a chow diet. Plasma total and HDL cholesterol and total phospholipids were not increased in the transgenic mice, and regression analysis showed no correlation between plasma levels of cholesterol or phospholipids and plasma human apoA-IV. To study reverse cholesterol transport in vivo, the disappearance of cholesterol from a depot of [(3)H]cholesterol-labeled cationized low-density lipoprotein injected into the rectus femoris muscle was compared in high expressers of human apoA-IV and WT controls. The loss of radioactivity and the diminution of the exogenous cholesterol mass were determined on days 8 and 12 after injection. No enhanced loss of radioactivity or cholesterol mass was seen in the transgenic mice even at levels of 2500 mg/dL of human apoA-IV. In some instances, there was even slower loss of exogenous cholesterol (radioactivity and mass) in the transgenic mice. Although [(3)H]cholesterol efflux from cultured human skin fibroblasts and mouse peritoneal macrophages was only approximately 30% higher in the presence of sera from high expressers of human apoA-IV, addition of phosphatidylcholine liposomes enhanced the efflux in both groups to the same extent. Another paradoxical finding was that the cholesterol esterification rate in plasma was 34% to 36% lower in human apoA-IV mice than in WT controls. In conclusion, even though apoA-IV was found previously to be atheroprotective under hypercholesterolemic conditions, high plasma levels of human apoA-IV did not enhance cholesterol mobilization in vivo in normocholesterolemic mice. Show less

The molecular machinery of cell cycle control is known in more detail for budding yeast, Saccharomyces cerevisiae, than for any other eukaryotic organism. In recent years, many elegant experiments on budding yeast have dissected the roles of cyclin molecules (Cln1-3 and Clb1-6) in coordinating the events of DNA synthesis, bud emergence, spindle formation, nuclear division, and cell separation. These experimental clues suggest a mechanism for the principal molecular interactions controlling cyclin synthesis and degradation. Using standard techniques of biochemical kinetics, we convert the mechanism into a set of differential equations, which describe the time courses of three major classes of cyclin-dependent kinase activities. Model in hand, we examine the molecular events controlling "Start" (the commitment step to a new round of chromosome replication, bud formation, and mitosis) and "Finish" (the transition from metaphase to anaphase, when sister chromatids are pulled apart and the bud separates from the mother cell) in wild-type cells and 50 mutants. The model accounts for many details of the physiology, biochemistry, and genetics of cell cycle control in budding yeast. Show less

Hereditary multiple exostoses, a dominantly inherited genetic disorder characterized by multiple cartilaginous tumors, is caused by mutations in members of the EXT gene family, EXT1 or EXT2. The proteins encoded by these genes, EXT1 and EXT2, are endoplasmic reticulum-localized type II transmembrane glycoproteins that possess or are tightly associated with glycosyltransferase activities involved in the polymerization of heparan sulfate. Here, by testing a cell line with a specific defect in EXT1 in in vivo and in vitro assays, we show that EXT2 does not harbor significant glycosyltransferase activity in the absence of EXT1. Instead, it appears that EXT1 and EXT2 form a hetero-oligomeric complex in vivo that leads to the accumulation of both proteins in the Golgi apparatus. Remarkably, the Golgi-localized EXT1/EXT2 complex possesses substantially higher glycosyltransferase activity than EXT1 or EXT2 alone, which suggests that the complex represents the biologically relevant form of the enzyme(s). These findings provide a rationale to explain how inherited mutations in either of the two EXT genes can cause loss of activity, resulting in hereditary multiple exostoses. Show less

Gene expression of activin, activin receptors, and follistatin was investigated in vivo and in vitro using semiquantitative RT-PCR in intestinal epithelial cells. Rat jejunum and the intestinal epithelial cell line IEC-6 expressed mRNA encoding the betaA-subunit of activin, alpha-subunit of inhibin, activin receptors IB and IIA, and follistatin. An epithelial cell isolation study focused along the crypt-villus axis in rat jejunum showed that betaA mRNA levels were eight- to tenfold higher in villus cells than in crypt cells. Immunohistochemistry revealed the expression of activin A in upper villus cells. The human intestinal cell line Caco-2 was used as a differentiation model of enterocytes. Four- to fivefold induction of betaA mRNA was observed in postconfluent Caco-2 cells grown on filter but not in those cells grown on plastic. In contrast, follistatin mRNA was seen to be reduced after reaching confluence. Exogenous activin A dose-dependently suppressed the proliferation and stimulated the expression of apolipoprotein A-IV gene, a differentiation marker, in IEC-6 cells. These results suggest that the activin system is involved in the regulation of such cellular functions as proliferation and differentiation in intestinal epithelial cells. Show less

We have devised a sensitive method for the isolation and structural analysis of glycosaminoglycans from two genetically tractable model organisms, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. We detected chondroitin/chondroitin sulfate- and heparan sulfate-derived disaccharides in both organisms. Chondroitinase digestion of glycosaminoglycans from adult Drosophila produced both nonsulfated and 4-O-sulfated unsaturated disaccharides, whereas only unsulfated forms were detected in C. elegans. Heparin lyases released disaccharides bearing N-, 2-O-, and 6-O-sulfated species, including mono-, di-, and trisulfated forms. We observed tissue- and stage-specific differences in both chondroitin sulfate and heparan sulfate composition in Drosophila. We have also applied these methods toward the analysis of tout-velu, an EXT-related gene in Drosophila that controls the tissue distribution of the growth factor Hedgehog. The proteins encoded by the vertebrate tumor suppressor genes EXT1 and 2, show heparan sulfate co-polymerase activity, and it has been proposed that tout-velu affects Hedgehog activity via its role in heparan sulfate biosynthesis. Analysis of total glycosaminoglycans from tout-velu mutant larvae show marked reductions in heparan sulfate but not chondroitin sulfate, consistent with its proposed function as a heparan sulfate co-polymerase. Show less

We examined the effect of ileal infusions of lipid emulsion on mRNA levels and biosynthesis of apolipoprotein A-IV (apo A-IV) in jejunal Thiry-Vella fistulas in rats. The rats were surgically prepared with jejunal Thiry-Vella fistulas; after recovery they were deprived of food, equipped with ileal infusion cannulas, then given 8 hr ileal infusions of fatty acid/monoglyceride emulsions. Mucosal synthesis and transcript levels of apo A-IV in the Thiry-Vella loop were then measured. Lipid infusion produced a two-fold stimulation in incorporation of 3H-leucine into apo A-IV-specific protein, but had no significant effect on apo A-IV mRNA levels. These results support the hypothesis that a lipid-elicited, distal gut-derived, systemic signal stimulates the production of apo A-IV by a post-transcriptional mechanism. Show less

Mitogen-activated protein kinases (MAPKs) are specifically phosphorylated and activated by the MAPK kinases, phosphorylate various targets such as MAPK-activated protein kinases and transcription factors, and are inactivated by specific phosphatases. Recently, docking interactions via the non-catalytic regions of MAPKs have been suggested to be important in regulating these reactions. Here we identify docking sites in MAPKs and in MAPK-interacting enzymes. A docking domain in extracellular-signal-regulated kinase (ERK), a MAPK, serves as a common site for binding to the MAPK kinase MEK1, the MAPK-activated protein kinase MNK1 and the MAPK phosphatase MKP3. Two aspartic acids in this domain are essential for docking, one of which is mutated in the sevenmaker mutant of Drosophila ERK/Rolled. A corresponding domain in the MAPKs p38 and JNK/SAPK also serves as a common docking site for their MEKs, MAPK-activated protein kinases and MKPs. These docking interactions increase the efficiency of the enzymatic reactions. These findings reveal a hitherto unidentified docking motif in MAPKs that is used in common for recognition of their activators, substrates and regulators. Show less

Although the CLN3 gene for Batten disease, the most common inherited neurovisceral storage disease of childhood, was identified in 1995, the function of the corresponding protein still remains elusive. A key to understanding the pathology of this devastating disease will be to elucidate the function of CLN3 at the molecular level. CLN3 has proven difficult to study, as it is predicted to be a membrane protein, and is of apparently low abundance in cells. Different groups have reported differing subcellular localization of CLN3. The purpose of this review is to critically examine the various cell biological approaches undertaken to localize CLN3 and to piece together a potential function for CLN3 in neuronal cells. The most likely conclusion of this is that CLN3 is a lysosomal/endosomal protein that is trafficked through the endoplasmic reticulum (ER) and Golgi. Furthermore, studies are required to confirm whether CLN3 has a potential role in the recycling of synaptic vesicles through the endosome/lysosome. Show less

We prepared the specific antibodies for EXT1 and EXT2, hereditary multiple exostoses (HME) gene products, and characterized their expression, subcellular localization, and protein association among EXT members. Biochemical analyses indicate that EXT1 and EXT2 can associate and form homo/hetero-oligomers in vivo with or without HME-linked mutations, EXT1 (R340C) and EXT2 (D227N), when exogenously expressed in COS-7 cells. An immunocytochemical analysis showed that both EXT1 and EXT2 localized in Golgi apparatus, irrespective of HME mutations. An immunohistochemical analysis on developing bones further showed that both EXT1 and EXT2 were concomitantly expressed in hypertrophic chondrocytes of forelimb bones from 1-day-old neonatal mouse, but down-regulated in maturing chondrocytes of developing cartilage from 21-day-old mouse. Taken together with the recent finding that EXTs encode for the glycosyltransferase required for the synthesis of heparan sulfate [Lind, T., Tufaro, F., McCormick, C., Lindahl, U., and Lindholt, K. (1998) J. Biol. Chem. 273, 26265-26268], our results implied a molecular basis that a HME-linked mutation found in EXT genes could interfere the physiological function(s) of EXT homo/hetero-oligomers as glycosyltransferases in the developing bones of HME patients. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the formation of exostoses, which are cartilage-capped bony protuberances mainly located on long bones. Two genes, EXT1 and EXT2, and at least one other unidentified gene, are known to be involved in the formation of exostoses. To date, 49 different EXT1 and 25 different EXT2 mutations have been found in EXT patients, and there is evidence that mutations in these two genes are responsible for over 70% of the EXT cases. Among the 49 EXT1 mutations there are 9 nonsense, 21 frameshift, and 5 splice site mutations; 2 in-frame deletions of 1 and 5 amino acids respectively; and 12 missense mutations. For EXT2, 8 nonsense, 11 frameshift, 3 splice site and 3 missense mutations are described. The majority of these mutations are mutations causing loss of function, which is consistent with the presumed tumor suppressor function of the EXT genes. Show less

Phospholipid transfer protein gene knock-out (Pltp KO) mice have defective transfer of very low density lipoprotein (VLDL) phospholipids into high density lipoprotein (HDL) and markedly decreased HDL levels (Jiang et al. 1999. J. Clin. Invest. 103: 907-914). These animals also accumulated VLDL- and LDL-sized lipoproteins on a high saturated fat diet. The goals of this study were to further characterize the abnormal lipoproteins of Pltp KO mice and to determine the mechanisms responsible for low HDL levels. A lipoprotein fraction enriched in lamellar structures was isolated from the low density lipoprotein (LDL) region and was shown to be phospholipid- and free cholesterol-rich and to have apoA-IV (55%) and apoE (25%) as major apolipoproteins. The lamellar lipoproteins accumulating in these mice probably represent surface material derived from triglyceride-rich lipoproteins (TRL). The HDL was found to be protein-rich (primarily apoA-I) and specifically depleted in phosphatidylcholine (PC) (28% in wild-type mice (WT) vs. 15% in Pltp KO mice, P < 0.001). Unexpectedly, turnover studies using autologous HDL revealed a profound 4-fold increase in the catabolism of HDL protein and cholesteryl ester in Pltp KO mice compared to wild-type, with minor differences in synthesis rates. In contrast, injection of WT mouse HDL into Pltp KO mice showed only a 2-fold increase in fractional catabolism. Reminiscent of the defect in Tangier disease, the failure of transfer of PC from TRL into the HDL fraction results in dramatic hypercatabolism of HDL. These results suggest that defective phospholipid transfer from TRL into HDL, arising from decreased lipolysis or decreased PLTP activity, could lead to hypoalphalipoproteinemia characterized by hypercatabolism of HDL protein. lipoprotein levels, due to hypercatabolism, and accumulate apoA-IV-rich lamellar lipoproteins. Show less

Latent membrane protein 2A (LMP2A) is expressed in latent Epstein-Barr virus (EBV) infection. LMP2A functions to downregulate B-cell signal transduction and viral reactivation from latency in EBV-immortalized B cells in vitro, and acts to provide B cells with both a survival and developmental signal in vivo. Identification of proteins associated with LMP2A is important for elucidation of the mechanism that LMP2A employs to regulate B-cell signal transduction and EBV latency. LMP2A is constitutively tyrosine phosphorylated and is associated with protein tyrosine kinases such as Lyn and Syk when specific LMP2A tyrosines are phosphorylated. The amino-terminal domain of LMP2A includes multiple proline-rich regions, which may provide binding sites for proteins containing SH3 or WW domains. In this study, we demonstrate that four cellular proteins bind specifically to two PPPPY (PY) motifs present within the LMP2A amino-terminal domain. Protein microsequence analysis determined that three of these proteins were AIP4, WWP2/AIP2, and Nedd4. All of these proteins are members of the Nedd4-like ubiquitin-protein ligases family and have conserved domains including the C2, WW, and ubiquitin-protein ligase domain. The mutation of both PY motifs completely abolished binding activity of these proteins to LMP2A and the interaction of AIP4 and WWP2 with LMP2A was confirmed in cell lines expressing LMP2A, WWP2, and AIP4. Furthermore, a reduction in the level of Lyn and the rapid turnover of LMP2A and Lyn were observed in LMP2A-expressing cells. These findings suggest that LMP2A recruits Nedd4-like ubiquitin-protein ligases and B-cell signal transduction molecules, resulting in the degradation of LMP2A and Lyn by a ubiquitin-dependent mechanism. This provides a new means by which LMP2A may modulate B-cell signal transduction. Show less

We have cloned a cardiovascular-restricted basic helix-loop-helix factor that interacts with arylhydrocarbon receptor nuclear translocator (ARNT) in a yeast two-hybrid screen. Cardiovascular helix-loop-helix factor 1 (CHF1) is distantly related to the hairy family of transcriptional repressors. We analyzed its expression pattern during mouse embryo development. At day 8.5, the expression of CHF1 is first detected in the primitive ventricle of the primordial heart tube and persists throughout gestation. In rat hearts, this expression is down-regulated after birth, concurrent with terminal differentiation of cardiomyocytes. In the developing vasculature, CHF1 first appears in the dorsal aorta at day 9.0, which precedes the reported expression of smooth muscle cell markers, and persists into adulthood. In an in vitro system of smooth muscle cell differentiation, CHF1 mRNA was barely detectable in undifferentiated cells but was induced highly in differentiated smooth muscle cells. To determine whether CHF1 might affect the function of ARNT, we performed transfection studies. Co-transfection of CHF1 inhibited ARNT/EPAS1-dependent transcription by 85%, and this inhibition is dose-dependent. In electrophoretic mobility studies, CHF1 inhibited the binding of the ARNT/EPAS1 heterodimer to its target site. Our data suggest that CHF1 functions as a transcriptional repressor and may play an important role in cardiovascular development. Show less

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response was abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. Beta-arrestin-1 transfection also induced a significantly decrease in GIP-stimulated cAMP production, and this effect was greater with cotransfection of both GRK2 and beta-arrestin-1 than with either alone. In betaTC3 cells, expression of GRK2 or beta-arrestin-1 attenuated GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion was not affected. GRK2 and beta-arrestin-1 messenger RNAs were identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells. Overexpression of GRK2 enhanced agonist-induced GIPR phosphorylation, but receptor endocytosis was not affected by cotransfection with GRKs or beta-arrestin-1. These results suggest a potential role for GRK2/beta-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR. Show less

The Wnt signaling pathway is essential for development and organogenesis. Wnt signaling stabilizes beta-catenin, which accumulates in the cytoplasm, binds to 1-cell factor (TCF; also known as lymphocyte enhancer-binding factor, LEF) and then upregulates downstream genes. Mutations in CTNNB1 (encoding beta-catenin) or APC (adenomatous polyposis coli) have been reported in human neoplasms including colon cancers and hepatocellular carcinomas (HCCs). Because HCC5 tend to show accumulation of beta-catenin more often than mutations in CTNNB1, we looked for mutations in AXIN1, encoding a key factor for Wnt signaling, in 6 HCC cell lines and 100 primary HCC5. Among the 4 cell lines and 87 HCC5 in which we did not detect CTNNB1 mutations, we identified AXIN1 mutations in 3 cell lines and 6 mutations in 5 of the primary HCCs. In cell lines containing mutations in either gene, we observed increased DNA binding of TCF associated with beta-catenin in nuclei. Adenovirus mediated gene transfer of wild-type AXINI induced apoptosis in hepatocellular and colorectal cancer cells that had accumulated beta-catenin as a consequence of either APC, CTNNB1 or AXIN1 mutation, suggesting that axin may be an effective therapeutic molecule for suppressing growth of hepatocellular and colorectal cancers. Show less

To clarify the role of neural factors in the regulation of apolipoprotein (apo) A-IV expression in the small intestine, we investigated the effect of neural blockers on mRNA levels of apo A-IV in rat small intestine. Either ganglionic blocker (hexamethonium), cholinergic blocker (atropine) or beta-adrenergic blocker (propranolol) was infused intravenously to unrestrained conscious rats for 8 h, and then total RNA was isolated from the small intestine and analyzed using Northern hybridization. Apo A-IV mRNA levels in the ileum were significantly lower in hexamethonium- or atropine-infused rats than in saline- (control) or propranolol-infused rats. Immunoblot analysis showed no difference in plasma apo A-IV concentrations between hexamethonium- and saline-infused groups. The lower mRNA levels of apo A-IV in the ileum of hexamethonium-infused rats were observed even in bile-drained rats, indicating that the lower expression was not due to any changes in bile availability. The ileal apo A-IV mRNA levels were significantly higher in rats infused with lipid emulsion into the ileum than in rats infused with glucose-saline, and the concomitant infusion of intravenous hexamethonium did not affect the higher levels of apo A-IV mRNA. These results suggest that the basal expression of the ileal A-IV gene is at least partially regulated in a site-specific manner by cholinergic neurons. Show less

Cell division in Schizosaccharomyces pombe is achieved through the use of a medially positioned actomyosin ring. A division septum is formed centripetally, concomitant with actomyosin ring constriction. Genetic screens have identified mutations in a number of genes that affect actomyosin ring or septum assembly. These cytokinesis-defective mutants, however, undergo multiple S and M phases and die as elongated cells with multiple nuclei. Recently, we have shown that a mutant allele of the S. pombe drc1(+)/cps1(+) gene, which encodes a 1,3-(beta)-glucan synthase subunit, is defective in cytokinesis but displays a novel phenotype. drc1-191/cps1-191 cells are capable of assembling actomyosin rings and completing mitosis, but are incapable of assembling the division septum, causing them to arrest as binucleate cells with a stable actomyosin ring. Each nucleus in arrested cps1-191 cells is able to undergo S phase but these G(2) nuclei are significantly delayed for entry into the M phase. In this study we have investigated the mechanism that causes cps1-191 to block with two G(2) nuclei. We show that the inability of cps1-191 mutants to proceed through multiple mitotic cycles is not related to a defect in cell growth. Rather, the failure to complete some aspect of cytokinesis may prevent the G(2)/M transition of the two interphase-G(2) nuclei. The G(2)/M transition defect of cps1-191 mutants is suppressed by a mutation in the wee1 gene and also by the dominant cdc2 allele cdc2-1w, but not the cdc2-3w allele. Transient depolymerization of all F-actin structures also allowed a significant proportion of the cps1-191 cells to undergo a second round of mitosis. We conclude that an F-actin and Wee1p dependent checkpoint blocks G(2)/M transition until previous cytokinesis is completed. Show less

Many transcription factors, particularly those involved in the control of cell growth, are unstable proteins destroyed by ubiquitin-mediated proteolysis. In a previous study of sequences targeting the transcription factor Myc for destruction, we observed that the region in Myc signaling ubiquitin-mediated proteolysis overlaps closely with the region in Myc that activates transcription. Here, we present evidence that the overlap of these two activities is not unique to Myc, but reflects a more general phenomenon. We show that a similar overlap of activation domains and destruction elements occurs in other unstable transcription factors and report a close correlation between the ability of an acidic activation domain to activate transcription and to signal proteolysis. We also show that destruction elements from yeast cyclins, when tethered to a DNA-binding domain, activate transcription. The intimate overlap of activation domains and destruction elements reveals an unexpected convergence of two very different processes and suggests that transcription factors may be destroyed because of their ability to activate transcription. Show less

The first artery and vein of the vertebrate embryo assemble in the trunk by migration and coalescence of angioblasts to form endothelial tubes. The gridlock (grl) mutation in zebrafish selectively perturbs assembly of the artery (the aorta). Here it is shown that grl encodes a basic helix-loop-helix (bHLH) protein belonging to the Hairy/Enhancer of the split family of bHLH proteins. The grl gene is expressed in lateral plate mesoderm before vessel formation, and thereafter in the aorta and not in the vein. These results suggest that the arterial endothelial identity is established even before the onset of blood flow and implicate the grl gene in assignment of vessel-specific cell fate. Show less