Structure of mouse cytosolic sulfotransferase SULT2A8 provides insight into sulfonation of 7α-hydroxyl bile acids.

Cytosolic sulfotransferases (SULTs) catalyze the transfer of a sulfonate group from the cofactor 3'-phosphoadenosine 5'-phosphosulfate to a hydroxyl (OH) containing substrate and play a critical role in the homeostasis of endogenous compounds, including hormones, neurotransmitters, and bile acids. In human, SULT2A1 sulfonates the 3-OH of bile acids; however, bile acid metabolism in mouse is dependent on a 7α-OH sulfonating SULT2A8 via unknown molecular mechanisms. In this study, the crystal structure of SULT2A8 in complex with adenosine 3',5'-diphosphate and cholic acid was resolved at a resolution of 2.

Male-biased fasting-induced changes in hepatic tauro-cholic acid and plasma cholesterol in Sult2a8-haplodeficient mice.

SULT2A8 is a male-predominant and liver-specific mouse cytosolic sulfotransferase (SULT) that sulfonates 7α-hydroxyl (7α-OH) bile acids in vitro. Sulfonation regulates bile acid homeostasis, which in turn regulates cholesterol and energy metabolism. Using the Sult2a8-heterozygous (HT) mouse model created earlier in our laboratory, we aimed to investigate the physiological role of SULT2A8 in sulfonating 7α-OH bile acids and its impact on energy metabolism in vivo under both fed and energy-deprivation conditions.

Molecular and functional characterization of tumor-induced factor (TIF): Hamster homolog of CXCL3 (GRO) displays tumor suppressive activity.

Previously our lab has created a mouse ovarian xenograft model of copy number variation (CNV)-mediated G protein-coupled receptor (GPCR) MAS-driven tumorigenesis, and RNA profiling identified a putative chemokine tumor-induced factor (Tif). Sequence analysis and chemotactic study suggested that Tif was likely to be a hamster homolog of human GROγ (CXCL3) [IJC 125 (2009): 1316-1327]. In the present study, we report the molecular and functional characterization of the Tif gene.

Identification and characterization of a novel PPARα-regulated and 7α-hydroxyl bile acid-preferring cytosolic sulfotransferase mL-STL (Sult2a8).

PPARα has been known to play a pivotal role in orchestrating lipid, glucose, and amino acid metabolism via transcriptional regulation of its target gene expression during energy deprivation. Recent evidence has also suggested that PPARα is involved in bile acid metabolism, but how PPARα modulates the homeostasis of bile acids during fasting is still not clear. In a mechanistic study aiming to dissect the spectrum of PPARα target genes involved in metabolic response to fasting, we identified a novel mouse gene (herein named mL-STL for mouse liver-sulfotransferase-like) that shared extensive homology with the Sult2a subfamily of a superfamily of cytosolic sulfotransferases, implying its potential function in sulfonation.

Surrogate target cells expressing surface anti-idiotype antibody for the clinical evaluation of an internalizing CD22-specific antibody.

SM03, a chimeric antibody that targets the B-cell restricted antigen CD22, is currently being clinically evaluated for the treatment of lymphomas and other autoimmune diseases in China. SM03 binding to surface CD22 leads to rapid internalization, making the development of an appropriate cell-based bioassay for monitoring changes in SM03 bioactivities during production, purification, storage, and clinical trials difficult. We report herein the development of an anti-idiotype antibody against SM03.

P2Y6 receptor-mediated proinflammatory signaling in human bronchial epithelia.

P2Y receptors are expressed in virtually all epithelia and are responsible for the control of fluid and electrolyte transport. In asthmatic inflammation, the bronchial epithelia are damaged by eosinophil-derived, highly toxic cationic proteins, such as major basic protein (MBP). Consequently, extracellular nucleotides are released into the extracellular space from airway epithelial cells, and act in an autocrine or paracrine fashion to regulate immune functions.

Generation of anti-idiotype scFv for pharmacokinetic measurement in lymphoma patients treated with chimera anti-CD22 antibody SM03.

Pre-clinical and clinical studies of therapeutic antibodies require highly specific reagents to examine their immune responses, bio-distributions, immunogenicity, and pharmacodynamics in patients. Selective antigen-mimicking anti-idiotype antibody facilitates the assessment of therapeutic antibody in the detection, quantitation and characterization of antibody immune responses. Using mouse specific degenerate primer pairs and splenocytic RNA, we generated an idiotype antibody-immunized phage-displayed scFv library in which an anti-idiotype antibody against the therapeutic chimera anti-CD22 antibody SM03 was isolated.

Dynamic localisation of mature microRNAs in Human nucleoli is influenced by exogenous genetic materials.

Although microRNAs are commonly known to function as a component of RNA-induced silencing complexes in the cytoplasm, they have been detected in other organelles, notably the nucleus and the nucleolus, of mammalian cells. We have conducted a systematic search for miRNAs in HeLa cell nucleoli, and identified 11 abundant miRNAs with a high level of nucleolar accumulation. Through in situ hybridisation, we have localised these miRNAs, including miR-191 and miR-484, in the nucleolus of a diversity of human and rodent cell lines.

Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions.

Construction and characterization of single-chain variable fragment antibody library derived from germline rearranged immunoglobulin variable genes.

Antibody repertoires for library construction are conventionally harvested from mRNAs of immune cells. To examine whether germline rearranged immunoglobulin (Ig) variable region genes could be used as source of antibody repertoire, an immunized phage-displayed scFv library was prepared using splenocytic genomic DNA as template. In addition, a novel frame-shifting PCR (fsPCR) step was introduced to rescue stop codon and to enhance diversity of the complementarity-determining region 3 (CDR3).

The truncated ghrelin receptor polypeptide (GHS-R1b) is localized in the endoplasmic reticulum where it forms heterodimers with ghrelin receptors (GHS-R1a) to attenuate their cell surface expression.

The ghrelin receptor (GHS-R1a) is remarkable amongst G-protein-coupled receptors for its high degree of constitutive activity, and this agonist-independent activity may be important for its physiological function in the control of food intake and body weight. Ghrelin receptors form heterodimers with the truncated ghrelin receptor polypeptide (GHS-R1b), which has a dominant-negative effect on ghrelin receptor function. Here we show that GHS-R1b has an intracellular localization distinct from ghrelin receptors, being primarily localized in the endoplasmic reticulum.

One-step expression and purification of single-chain variable antibody fragment using an improved hexahistidine tag phagemid vector.

Millions of candidate clones are commonly obtained following rounds of phage-displayed antibody library panning, and expression of those selected single-chain variable fragment (scFv) is required for secondary functional screening to identify positive clones. Large scale functional screening is often hampered by the time-consuming and labor-intensive subcloning of those candidate scFv clones into a bacterial expression vector carrying an affinity tag for scFv purification and detection. To overcome the limitations and to develop a multiplex approach, an improved hexahistidine tag phagemid vector was constructed for one-step scFv expression and purification.

Efficient expression of foreign genes in CHO DHFR(-) cells by electroporation.

DHFR-deficient Chinese hamster ovary (CHO DHFR(-)) cells are the most popular mammalian expression system for inducible amplification of transgene. In order to obtain more stable transfected CHO DHFR(-) cell clones, transfection efficiency of electroporation under different conditions were systemically investigated using plasmid pSV-beta-Gal as reporter gene. Transfection efficiency was proportionally increased with pulse duration and number of pulse applied.

Identification and characterization of a novel CXC chemokine in xenograft tumor induced by mas-overexpressing cells.

Overexpressions of G protein-coupled receptor (GPCR) with elevated downstream signaling events have been reported in various tumors. However, the cellular mechanism that GPCR overexpression leads to tumor formation is largely unknown. The orphan GPCR mas was originally isolated from a human epidermoid carcinoma.

The constitutive activity of ghrelin receptors is decreased by co-expression with vasoactive prostanoid receptors when over-expressed in human embryonic kidney 293 cells.

The functional activity of G protein-coupled receptors can be modified by their ability to form oligomeric complexes with G protein-coupled receptors from other receptor families. Emerging evidence suggests that the appetite-regulating hormone ghrelin is a directly acting vasodilator peptide with anti-inflammatory activity, therefore, we have examined the ability of ghrelin receptors to oligomerize with members of the prostanoid receptor family which are also involved in modulating vascular activity and inflammatory responses. Using the techniques of bioluminescence resonance energy transfer and co-immunoprecipitation, we detected the ability of ghrelin receptors to hetero-oligomerize with prostaglandin E2 receptor subtype EP3-I, prostacyclin receptors, and thromboxane A2 (TPalpha) receptors, when transiently over-expressed in human embryonic kidney 293 cells.

Identification and characterization of a novel mouse peroxisome proliferator-activated receptor alpha-regulated and starvation-induced gene, Ppsig.

The peroxisome proliferator-activated receptor alpha (PPARalpha) has been known to play a pivotal role in maintaining the energy balance during fasting; however, the battery of PPARalpha target genes involved in this metabolic response is still not fully characterized. Here, we report the identification and characterization of Ppsig (for PPARalpha-regulated and starvation-induced gene) with unknown biological function from mouse liver. Multiple Ppsig cDNAs which differed in the 3'-untranslated regions were identified.

Dual effect of endothelin 1 on angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens.

Angiotensin and endothelin are vasoactive peptides with neuromodulatory effect, however their interactions in facilitating neurotransmission are largely unknown. In the present study, effort was made to examine how endothelin 1 modulates angiotensin II-potentiated purinergic neurotransmission in prostatic rat vas deferens. Both peptides facilitated field-stimulated muscle contraction in a concentration-dependent manner with Kd values of 16.

A temporal study on the histopathological, biochemical and molecular responses of CCl(4)-induced hepatotoxicity in Cyp2e1-null mice.

Previous study using Cyp2e1-null mice showed that Cyp2e1 is required in CCl(4)-induced liver injury at 24h, what remains unclear are the temporal changes in liver damage and the spectrum of genes involved in this process. We investigated the time-dependent liver changes that occurred at morphological, histopathological, biochemical and molecular levels in both Cyp2e1(+/+) and Cyp2e1(-/-) mice after treating with either corn oil or CCl(4) (1 ml/kg) for 2, 6, 12, 24 and 48 h. A pale orange colored liver, indicative of fatty infiltration, was observed in Cyp2e1(+/+) mice treated with CCl(4) for 24 and 48 h, while the Cyp2e1(+/+) mice treated with corn oil and Cyp2e1(-/-) mice treated with either corn oil or CCl(4) showed normal reddish brown colored liver.

Mechanism of non-capacitative Ca2+ influx in response to bradykinin in vascular endothelial cells.

Bradykinin is a potent vasoactive nonapeptide. It elicits a rise in cytosolic Ca(2+) (Ca(2+))(i) in endothelial cells, resulting in Ca(2+)-dependent synthesis and release of endothelial vasodilators. In the present study, we investigated the mechanism of bradykinin-induced Ca(2+) influx in primary cultured rat aortic endothelial cells and in a mouse heart microvessel endothelial cell line (H5V).

Identification and characterization of surrogate peptide ligand for orphan G protein-coupled receptor mas using phage-displayed peptide library.

In the present study, a phage-displayed random peptide library was used to identify surrogate peptide ligands for orphan GPCR mas. Sequence analysis of the isolated phage clones indicated a selective enrichment of some peptide sequences. Moreover, multiple alignments of the isolated phage clones gave two conserved peptide motifs from which we synthesized peptide MBP7 for further evaluation.

Cross-reactivity of antibody against SARS-coronavirus nucleocapsid protein with IL-11.

Infection of SARS-associated coronavirus (SARS-CoV) induced a strong anti-nucleocapsid (anti-N) antibody response. However, the pathophysiological significance of the anti-N antibodies in SARS pathogenesis is largely unknown. To profile the anti-N antibodies, a phage-displayed scFv library was prepared from mice immunized with heat-inactivated SARS-CoV-infected Vero E6 cell lysate.

Requirement of PPARalpha in maintaining phospholipid and triacylglycerol homeostasis during energy deprivation.

The peroxisome proliferator-activated receptor alpha (PPARalpha) has been implicated as a key control of fatty acid catabolism during the cellular fasting. However, little is known regarding changes of individual fatty acids in hepatic triacylglycerol (TG) and phospholipid (PL) as a result of starvation. In the present work, the effects of 72 h fasting on hepatic TG and PL fatty acid profiles in PPARalpha-null (KO) mice and their wild-type (WT) counterparts were investigated.

Immunohistochemical colocalization of type II angiotensin receptors with somatostatin in rat pancreas.

Earlier studies indicate that binding sites of type II angiotensin (AT2) receptors are detected all over the pancreas, as well as in the pancreatic exocrine cell line AR4-2J. However, lack of corresponding functional AT2 receptor responses can be detected in the exocrine pancreas. The aim of present study is to determine the protein expression of AT2 receptors in the pancreas by probing with an AT2 receptor-specific antibody, and to examine the role of AT2 receptors in the regulation of pancreatic endocrine hormone release.

Application of fluorescent differential display and peroxisome proliferator-activated receptor (PPAR) alpha-null mice to analyze PPAR target genes.

In conclusion, we have applied the fluorescent differential method and the PPAR alpha-null mouse model for the rapid isolation of expression tags of PPAR alpha target genes that are involved in the action of peroxisome proliferators and in the regulation of lipid homeostasis under energy deprivation. Identification of a wide spectrum of PPAR alpha target genes will provide new insights into the diverse cellular pathways regulated by these receptor, and this information will be critical for understanding the complicated biological interactions among members of the PPAR alpha target genes. With the recent technological advancement, a newer method, such as DNA microarray, has emerged in the identification of differential gene expressions.