Embryonic fibroblasts with a gene trap mutation in Ext1 produce short heparan sulfate chains

Mutational defects in either EXT1 or EXT2 genes cause multiple exostoses, an autosomal hereditary human disorder. The EXT1 and EXT2 genes encode glycosyltransferases that play an essential role in heparan sulfate chain elongation. In this study, we have analyzed heparan sulfate synthesized by primary fibroblast cell cultures established from mice with a gene trap mutation in Ext1. The gene trap mutation results in embryonic lethality, and homozygous mice die around embryonic day 14. Metabolic labeling and immunohistochemistry revealed that Ext1 mutant fibroblasts still produced small amounts of heparan sulfate. The domain structure of the mutant heparan sulfate was conserved, and the disaccharide composition was similar to that of wild type heparan sulfate. However, a dramatic difference was seen in the polysaccharide chain length. The average molecular sizes of the heparan sulfate chains from wild type and Ext1 mutant embryonic fibroblasts were estimated to be around 70 and 20 kDa, respectively. These data suggest that not only the sulfation pattern but also the length of the heparan sulfate chains is a critical determinant of normal mouse development.     

CGI-58 interacts with perilipin and is localized to lipid droplets. Possible involvement of CGI-58 mislocalization in Chanarin-Dorfman syndrome

Lipid droplets (LDs) are a class of ubiquitous cellular organelles that are involved in lipid storage and metabolism. Although the mechanisms of the biogenesis of LDs are still unclear, a set of proteins called the PAT domain family have been characterized as factors associating with LDs. Perilipin, a member of this family, is expressed exclusively in the adipose tissue and regulates the breakdown of triacylglycerol in LDs via its phosphorylation. In this study, we used a yeast two-hybrid system to examine the potential function of perilipin. We found direct interaction between perilipin and CGI-58, a deficiency of which correlated with the pathogenesis of Chanarin-Dorfman syndrome (CDS). Endogenous CGI-58 was distributed predominantly on the surface of LDs in differentiated 3T3-L1 cells, and its expression increased during adipocyte differentiation. Overexpressed CGI-58 tagged with GFP gathered at the surface of LDs and colocalized with perilipin. This interaction seems physiologically important because CGI-58 mutants carrying an amino acid substitution identical to that found in CDS lost the ability to be recruited to LDs. These mutations significantly weakened the binding of CGI-58 with perilipin, indicating that the loss of this interaction is involved in the etiology of CDS. Furthermore, we identified CGI-58 as a binding partner of ADRP, another PAT domain protein expressed ubiquitously, by yeast two-hybrid assay. GFP-CGI-58 expressed in non-differentiated 3T3-L1 or CHO-K1 cells was colocalized with ADRP, and the CGI-58 mutants were not recruited to LDs carrying ADRP, indicating that CGI-58 may also cooperate with ADRP.     

Induction of mitochondrial aldehyde dehydrogenase by submergence facilitates oxidation of acetaldehyde during re-aeration in rice

Post-hypoxic injuries in plants are primarily caused by bursts of reactive oxygen species and acetaldehyde. In agreement with previous studies, we found accumulations of acetaldehyde in rice during re-aeration following submergence. During re-aeration, acetaldehyde-oxidizing aldehyde dehydrogenase (ALDH) activity increased, thereby causing the acetaldehyde content to decrease in rice. Interestingly, re-aerated rice plants showed an intense mitochondrial ALDH2a protein induction, even though ALDH2a mRNA was submergence induced and declined upon re-aeration. This suggests that rice ALDH2a mRNA is accumulated in order to quickly metabolize acetaldehyde that is produced upon re-aeration.     

Crystal structure of Enterococcus hirae enolase at 2.8 A resolution

We report the crystal structure of an enolase from Enterococcus hirae, which is the first report of a structure determination among gram-positive bacteria. We isolated the enolase gene and determined the base sequence. The amino acid sequence deduced from the DNA sequence suggests that this enolase is composed of 431 amino acids. The amino acid sequence is very similar to those of enolases from eukaryotic and prokaryotic organisms, being 65% and 50% identical to enolases from Escherichia coli and yeast, respectively. The enolase prepared from E. hirae lysate yielded crystals containing one dimer per asymmetric unit. X-ray diffraction patterns were obtained at 2.8 A resolution on a SPring-8 synchrotron radiation source. Crystals belong to space group I4 with unit cell dimensions of a = b = 153.5 A, c = 90.7 A. The E. hirae, yeast, E. coli and lobster enolase structures are very similar. The E. hirae enolase takes an "Open" conformation. The regions in the structure that differ most from other enolases are loops L4 (132-140) and L3 (244-265). Considering the positions of these loops relative to the active site, they seem to have no direct involvement in function. Our findings show that the three dimensional structure of an important enzyme in the glycolytic pathway is evolutionarily conserved among eukaryotes and prokaryotes, including gram-positive bacteria.     

Postembryonic development of the wing imaginal discs in the female wingless bagworm moth Eumeta variegata (Lepidoptera,Psychidae)

The process of wing disc development and degeneration in the bagworm moth Eumeta variegata was investigated histologically. Morphological differences between two sexes first appear in the penultimate (eighth) larval instar. In the male, wing discs proliferate rapidly in the penultimate larval instar and continue proliferating; a conspicuous peripodial epithelium forms in the last (ninth) larval instar. The hemopoietic organs break down in this stage and disappear completely by the prepupal stage. In the female, in contrast, the wing discs remain as in the previous (seventh) instar, without proliferation of cells inside. No peripodial epithelium forms in the penultimate instar or later. Hemopoietic organs are still attached to the wing discs in the last larval instar and the entire wing discs transform into a plain, thick epidermis in the prepupal period. It is suggested that the hemopoietic organs may prevent the wing discs from developing in E. variegata.     

Functional roles of the neuropeptide PACAP in brain and pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide implicated in a broad variety of physiological processes. To assess PACAP's function in vivo, we recently generated PACAP knockout mice (PACAP(-/-)) and transgenic mice overexpressing PACAP specifically in the pancreas (PACAP-Tg). In PACAP(-/-) mice, we have demonstrated a marked phenotypic changes including a high early mortality rate, increased novelty-seeking behavior and abnormal explosive jumping in a novel environment, as well as reduced female fertility. In this paper, we reevaluated these phenotypes in terms of the genetic background of the mice. Genetic background appears to modulate critically the magnitude but not the general nature of the PACAP-null phenotype. In PACAP-Tg mice, we have recently demonstrated that enhanced glucose-induced insulin secretion with normal glucose tolerance, amelioration of streptozotocin-induced diabetes with increased beta-cell proliferation, and a trend towards an increase in total islet mass with age. Here we show that PACAP(-/-) mice exhibit significantly impaired glucose-induced insulin secretion but still have normal glucose tolerance. These observations suggest that PACAP may play important roles in and beyond the regulation of insulin release. Taken together, the mutant phenotypes revealed both expected and unexpected roles of PACAP in the brain and pancreatic functions.     

Crystal structure of human L-xylulose reductase holoenzyme: probing the role of Asn107 with site-directed mutagenesis

L-Xylulose reductase (XR), an enzyme in the uronate cycle of glucose metabolism, belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. Among the SDR enzymes, XR shows the highest sequence identity (67%) with mouse lung carbonyl reductase (MLCR), but the two enzymes show different substrate specificities. The crystal structure of human XR in complex with reduced nicotinamide adenine dinucleotide phosphate (NADPH) was determined at 1.96 A resolution by using the molecular replacement method and the structure of MLCR as the search model. Features unique to human XR include electrostatic interactions between the N-terminal residues of subunits related by the P-axis, termed according to SDR convention, and an interaction between the hydroxy group of Ser185 and the pyrophosphate of NADPH. Furthermore, identification of the residues lining the active site of XR (Cys138, Val143, His146, Trp191, and Met200) together with a model structure of XR in complex with L-xylulose, revealed structural differences with other members of the SDR family, which may account for the distinct substrate specificity of XR. The residues comprising a recently proposed catalytic tetrad in the SDR enzymes are conserved in human XR (Asn107, Ser136, Tyr149, and Lys153). To examine the role of Asn107 in the catalytic mechanism of human XR, mutant forms (N107D and N107L) were prepared. The two mutations increased K(m) for the substrate (>26-fold) and K(d) for NADPH (95-fold), but only the N107L mutation significantly decreased k(cat) value. These results suggest that Asn107 plays a critical role in coenzyme binding rather than in the catalytic mechanism.     

Poly(ADP-ribose) polymerase 1 binds to Kaposi's sarcoma-associated herpesvirus (KSHV) terminal repeat sequence and modulates KSHV replication in latency

During latency, Kaposi's sarcoma-associated herpesvirus (KSHV) is thought to replicate once and to be partitioned in synchrony with the cell cycle of the host. In this replication cycle, the KSHV terminal repeat (TR) sequence functions as a replication origin, assisted by the latency-associated nuclear antigen (LANA). Thus, TR seems to function as a cis element for the replication and partitioning of the KSHV genome. Viral replication and partitioning are also likely to require cellular factors that interact with TR in either a LANA-dependent or -independent manner. Here, we sought to identify factors that associate with TR by using a TR DNA column and found that poly(ADP-ribose) polymerase 1 (PARP1) and known replication factors, including ORC2, CDC6, and Mcm7, bound to TR. PARP1 bound directly to a specific region within TR independent of LANA, and LANA was poly(ADP-ribosyl)ated by PARP1. Drugs such as hydroxyurea and niacinamide, which raise or lower PARP activity, respectively, affected the virus copy number in infected cells. Thus, the poly(ADP-ribosyl)ation status of LANA appears to affect the replication and/or maintenance of the viral genome. Drugs that specifically up-regulate PARP activity may lead to the disappearance of latent KSHV.     

Porcine FAD-containing monooxygenase metabolizes lidocaine, bupivacaine and propranolol in vitro

Lidocaine, bupivacaine and propranolol are amines that can be expected to act as substrates for FAD-containing monooxygensae (FMO) (EC 1. 14. 13. 8). We found that FMO metabolizes lidocaine, bupivacaine and propranolol. The Km and Vmax values of lidocaine, bupivacaine and propranolol for FMO are 143, 408 and 210 microM, and 145, 119 and 135 nmol/min/mg FMO protein, respectively. The lipophilicity of the drugs decreased in the following order: lidocaine>propranolol>bupivacaine, under our experimental conditions. Furthermore, the metabolic products of FMO were separated by high-performance liquid chromatography and analyzed by gas chromatography-mass spectrometry, and were found to be the N-oxides and N-hydroxylamines of the respective drugs. These findings suggest that lidocaine, bupivacaine and propranolol are substrates for FMO, and the enzymatic toward lidocaine or bupivacaine may be inhibited exclusively and competitively by propranolol.     

Total analysis and purification of cellular proteins binding to cisplatin-damaged DNA using submicron beads

A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)(n), on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.