Studies of human liver bilirubin-glycosyl transferase. Bilirubin UDP-xylosyl and UDP-glucuronyl transferase activities in diseased human livers.

The activity of bilirubin UDP-xylosyl transferase as well as UDP-glucuronyl transferase in liver biopsy specimens of 3 control subjects, 42 cases with liver disease and 5 cases with Gilbert's syndrome was measured. Normal values of these enzyme levels were determined to be 142--302 U/kg protein for the former and 260--400 U/kg protein for the latter. Both enzyme levels in acute hepatitis in convalescence and chronic hepatitis were nearly in the normal range. In the cirrhotic liver they tended to a small decrease and patients with Gilbert's syndrome demonstrated significantly decreased enzyme levels. These enzyme levels were only correlated with serum unconjugated bilirubin concentration, but not with the other liver function tests. Finally, both enzyme activities were exactly correlated with each other.     

Discovery of a bicyclo[4.3.0]nonane derivative DS88790512 as a potent,selective, and orally bioavailable blocker of transient receptor potential canonical 6 (TRPC6)

In this study, we aimed to synthesize a novel blocker of transient receptor potential canonical 6 (TRPC6). The sp² carbon atoms of the aminoindane skeleton of the known inhibitor were replaced with sp³ carbon atoms to increase the molecular complexity, measured by fraction sp³ (Fsp³). The representative compound, a bicyclo[4.3.0]nonane derivative DS88790512, inhibited TRPC6 with an IC₅₀ value of 11?nM. Notably, DS88790512 exhibited excellent selectivity against hERG and hNa_V1.5 channels, and was identified as an orally bioavailable compound.     

Identification of dermcidin in human gestational tissue and characterization of its proteolytic activity

Dermcidin (DCD) is a gene for an antimicrobial peptide DCD-1 in human sweat glands. It has become evident that the gene products of DCD exhibit a wide range of biological functions. In addition to its antimicrobial function, it is reported to be a neuronal survival factor, a putative oncogene in breast cancer and a proteolysis-inducing factor (PIF) that induces skeletal muscle proteolysis to cause cancer cachexia. Here we identified DCD in human placental tissue and determined its previously uncharacterized proteolytic activity. We also show that recombinant DCD induced an invasive phenotype in a human choriocarcinoma cell line JAR in vitro. This work suggests that DCD might participate in the regulation of placental function by means of modulating the proteolytic cascades on the trophoblastic cell surface, and might be involved in the pathophysiology of pregnancy-related disorders, as well as cancer and neuronal diseases.     

Isolation of a class IV chitin synthase gene from a zygomycete fungus, Rhizopus oligosporus

We found the presence of DNA sequence which shows sequence similarity to the class IV chitin synthase gene (CHS3) of Saccharomyces cerevisiae in the genome of 14 Rhizopus species which belong to zygomycetes. We cloned a gene (chs3), which might correspond to one of these homologous sequences, from Rhizopus oligosporus by low stringency plaque hybridization probed with CHS3. The deduced amino acid sequence of this gene showed highest similarity to the class IV chitin synthase of Neurospora crassa (46.7% identity over 1087 amino acids), showing that this gene encodes a class IV chitin synthase. Northern analysis revealed the differential expression pattern of this gene in the asexual life cycle with highest expression in the early stage of asexual spore formation. This is the first report of the isolation and analysis of a class IV chitin synthase gene from zygomycete fungi.     

Motif-specific sampling of phosphoproteomes

Phosphoproteomics, the targeted study of a subfraction of the proteome which is modified by phosphorylation, has become an indispensable tool to study cell signaling dynamics. We described a methodology that linked phosphoproteome and proteome analysis based on Ba2+ binding properties of amino acids. This technology selected motif-specific phosphopeptides independent of the system under analysis. MudPIT (Multidimensional Identification Technology) identified 1037 precipitated phosphopeptides from as little as 250 microg of proteins. To extend coverage of the phosphoproteome, we sampled the nuclear extract of HeLa cells with three values of Ba2+ ions molarity. The presence of more than 70% of identified phosphoproteins was further substantiated by their nonmodified peptides. Upon isoproterenol stimulation of HEK cells, we identified an increasing number of phosphoproteins from MAPK cascades and AKAP signaling hubs. We quantified changes in both protein and phosphorylation levels of 197 phosphoproteins including a critical kinase, MAPK1. Integration of differential phosphorylation of MAPK1 with knowledge bases constructed modules that correlated well with its role as node in cross-talk of canonical pathways.     

Co-expression of soybean glycinins A1aB1b and A3B4 enhances their accumulation levels in transgenic rice seed

The soybean major storage protein glycinin is encoded by five genes, which are divided into two subfamilies. Expression of A3B4 glycinin in transgenic rice seed reached about 1.5% of total seed protein, even if expressed under the control of strong endosperm-specific promoters. In contrast, expression of A1aB1b glycinin reached about 4% of total seed protein. Co-expression of the two proteins doubled accumulation levels of both A1aB1b and A3B4 glycinins. This increase can be largely accounted for by their aggregation with rice glutelins, self-assembly and inter-glycinin interactions, resulting in the enrichment of globulin and glutelin fractions and a concomitant reduction of the prolamin fraction. Immunoelectron microscopy indicated that the synthesized A1aB1b glycinin was predominantly deposited in protein body-II (PB-II) storage vacuoles, whereas A3B4 glycinin is targeted to both PB-II and endoplasmic reticulum (ER)-derived protein body-I (PB-I) storage structures. Co-expression with A1aB1b facilitated targeting of A3B4 glycinin into PB-II by sequestration with A1aB1b, resulting in an increase in the accumulation of A3B4 glycinin.     

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia

Background

Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)₄-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4.

Methodology/Principal Findings

PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity.

Conclusions/Significance

Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.