Heparan Sulfate 2-O-Sulfotransferase Is Required for Triglyceride-rich Lipoprotein Clearance

Hepatic clearance of triglyceride-rich lipoproteins depends on heparan sulfate and low density lipoprotein receptors expressed on the basal membrane of hepatocytes. Binding and uptake of the lipoproteins by way of heparan sulfate depends on the degree of sulfation of the chains based on accumulation of plasma triglycerides and delayed clearance of triglyceride-rich lipoproteins in mice bearing a hepatocyte-specific alteration of N-acetylglucosamine (GlcNAc) N-deacetylase-N-sulfotransferase 1 (Ndst1) (MacArthur, J. M., Bishop, J. R., Stanford, K. I., Wang, L., Bensadoun, A., Witztum, J. L., and Esko, J. D. (2007) J. Clin. Invest. 117, 153–164). Inactivation of Ndst1 led to decreased overall sulfation of heparan sulfate due to coupling of uronyl 2-O-sulfation and glucosaminyl 6-O-sulfation to initial N-deacetylation and N-sulfation of GlcNAc residues. To determine whether lipoprotein clearance depends on 2-O-and 6-O-sulfation, we evaluated plasma triglyceride levels in mice containing loxP-flanked conditional alleles of uronyl 2-O-sulfotransferase (Hs2st^f/f) and glucosaminyl 6-O-sulfotransferase-1 (Hs6st1^f/f) and the bacterial Cre recombinase expressed in hepatocytes from the rat albumin (Alb) promoter. We show that Hs2st^f/fAlbCre⁺ mice accumulated plasma triglycerides and exhibited delayed clearance of intestinally derived chylomicrons and injected human very low density lipoproteins to the same extent as observed in Ndst1^f/fAlbCre⁺ mice. In contrast, Hs6st1^f/fAlbCre⁺ mice did not exhibit any changes in plasma triglycerides. Chemically modified heparins lacking N-sulfate and 2-O-sulfate groups did not block very low density lipoprotein binding and uptake in isolated hepatocytes, whereas heparin lacking 6-O-sulfate groups was as active as unaltered heparin. Our findings show that plasma lipoprotein clearance depends on specific subclasses of sulfate groups and not on overall charge of the chains.     

Structural Basis for the cAMP-dependent Gating in the Human HCN4 Channel

Hyperpolarization-activated cAMP-regulated (HCN) channels play important physiological roles in both cardiovascular and central nervous systems. Among the four HCN isoforms, HCN2 and HCN4 show high expression levels in the human heart, with HCN4 being the major cardiac isoform. The previously published crystal structure of the mouse HCN2 (mHCN2) C-terminal fragment, including the C-linker and the cyclic-nucleotide binding domain (CNBD), has provided many insights into cAMP-dependent gating in HCN channels. However, structures of other mammalian HCN channel isoforms have been lacking. Here we used a combination of approaches including structural biology, biochemistry, and electrophysiology to study cAMP-dependent gating in HCN4 channel. First we solved the crystal structure of the C-terminal fragment of human HCN4 (hHCN4) channel at 2.4 Å. Overall we observed a high similarity between mHCN2 and hHCN4 crystal structures. Functional comparison between two isoforms revealed that compared with mHCN2, the hHCN4 protein exhibited marked different contributions to channel function, such as a ∼3-fold reduction in the response to cAMP. Guided by structural differences in the loop region between β4 and β5 strands, we identified residues that could partially account for the differences in response to cAMP between mHCN2 and hHCN4 proteins. Moreover, upon cAMP binding, the hHCN4 C-terminal protein exerts a much prolonged effect in channel deactivation that could have significant physiological contributions.     

Microfibril Structure Masks Fibrillin-2 in Postnatal Tissues

Fibrillin microfibrils are polymeric structures present in connective tissues. The importance of fibrillin microfibrils to connective tissue function has been demonstrated by the multiple genetic disorders caused by mutations in fibrillins and in microfibril-associated molecules. However, knowledge of microfibril structure is limited, largely due to their insolubility. Most previous studies have focused on how fibrillin-1 is organized within microfibril polymers. In this study, an immunochemical approach was used to circumvent the insolubility of microfibrils to determine the role of fibrillin-2 in postnatal microfibril structure. Results obtained from studies of wild type and fibrillin-1 null tissues, using monoclonal and polyclonal antibodies with defined epitopes, demonstrated that N-terminal fibrillin-2 epitopes are masked in postnatal microfibrils and can be revealed by enzymatic digestion or by genetic ablation of Fbn1. From these studies, we conclude that fetal fibrillin polymers form an inner core within postnatal microfibrils and that microfibril structure evolves as growth and development proceed into the postnatal period. Furthermore, documentation of a novel cryptic site present in EGF4 in fibrillin-1 underscores the molecular complexity and tissue-specific differences in microfibril structure.     

Analysis of mouse liver membrane proteins using multidimensional separations and tandem mass spectrometry

In the field of proteomic investigation, the analysis of membrane proteins still faces many technical challenges. A fundamental question in this puzzle is how to maintain a proper solvent environment to allow the hydrophobic proteins to remain solubilized. We propose that the denaturation of membrane proteins in a highly concentrated urea solution enables them to be ionized such that ionic exchange chromatography can be employed to separate them. The membrane proteins prepared from the mouse liver were dissolved in 6M guanidine hydrochloride, 20mM Tris-HCl, pH 9.0, and loaded onto a tandem chromatography apparatus coupled with Q-Sepharose FF and Sephacryl S-200HR. These columns were able to adsorb 97.87% of the membrane protein preparations. Using a linear NaCl (0-1.0M) gradient, the bound proteins were eluted out at 0.1-1.0M NaCl, and examined by SDS-PAGE. Furthermore the protein bands underwent excision and digestion with trypsin, followed by reverse-phase chromatography for the separation of the digested peptides and ionic-trap mass spectrometry for the identification of the proteins. From the SDS-PAGE gels, the overlap between proteins from neighboring bands was only 21.34%, indicating that the anionic-size exclusion coupling chromatography efficiently separated these membrane proteins. Of a total of 392 proteins identified, 306 were membrane proteins or membrane-associated proteins. Based on the calculation of hydrophobicity, the GRAVY scores of 83 proteins are greater than, or equal to, 0.00. Taking all of this evidence together, our results revealed that this approach is satisfactory for studies on the membrane proteome from the mouse liver.     

ATP-mediated cell–cell signaling in the organ of Corti: the role of connexin channels

Connexin 26 (Cx26) and connexin 30 (Cx30) form hemichannels that release ATP from the endolymphatic surface of cochlear supporting and epithelial cells and also form gap junction (GJ) channels that allow the concomitant intercellular diffusion of Ca²⁺ mobilizing second messengers. Released ATP in turn activates G-protein coupled P2Y₂ and P2Y₄ receptors, PLC-dependent generation of IP₃, release of Ca²⁺ from intracellular stores, instigating the regenerative propagation of intercellular Ca²⁺ signals (ICS). The range of ICS propagation is sensitive to the concentration of extracellular divalent cations and activity of ectonucleotidases. Here, the expression patterns of Cx26 and Cx30 were characterized in postnatal cochlear tissues obtained from mice aged between P5 and P6. The expression gradient along the longitudinal axis of the cochlea, decreasing from the basal to the apical cochlear turn (CT), was more pronounced in outer sulcus (OS) cells than in inner sulcus (IS) cells. GJ-mediated dye coupling was maximal in OS cells of the basal CT, inhibited by the nonselective connexin channel blocker carbenoxolone (CBX) and absent in hair cells. Photostimulating OS cells with caged inositol (3,4,5) tri-phosphate (IP₃) resulted in transfer of ICS in the lateral direction, from OS cells to IS cells across the hair cell region (HCR) of medial and basal CTs. ICS transfer in the opposite (medial) direction, from IS cells photostimulated with caged IP₃ to OS cells, occurred mostly in the basal CT. In addition, OS cells displayed impressive rhythmic activity with oscillations of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) coordinated by the propagation of Ca²⁺ wavefronts sweeping repeatedly through the same tissue area along the coiling axis of the cochlea. Oscillations evoked by uncaging IP₃ or by applying ATP differed greatly, by as much as one order of magnitude, in frequency and waveform rise time. ICS evoked by direct application of ATP propagated along convoluted cellular paths in the OS, which often branched and changed dynamically over time. Potential implications of these findings are discussed in the context of developmental regulation and cochlear pathophysiology.     

Easy Production of a Glycolipid Analogue Using Animal Cells in Culture

A glycolipid analogue, GM4‐type ganglioside, was obtained by a combination of chemical synthesis and biosynthetic processes in animal cells with dodecyl β‐D ‐galactoside (Gal C12) as primer. The primer was conveniently prepared in two steps: glycosylation, followed by deacetylation. The primer was introduced to mouse melanoma B16 cells to serve as substrate for cellular, enzyme‐catalyzed glycosylation. Incubation of the cells in the presence of the primer resulted in sialylation of the galactose residue to afford a GM4 analogue that was released from the cells to the culture medium. The strategy of preparation of the GM4 analogue described in this study is a viable alternative to the existing methods. The saccharide‐primer method is fast, convenient, not requiring expensive enzymes and glycosyl donors, and highly stereoselective.     

LPS下调小鼠肝脏、肠道羧酸酯酶表达

目的:研究细菌脂多糖(LPS)对小鼠肝脏、肠道羧酸酯酶表达及酶活性的影响。方法:小鼠经腹腔注射5.0mg/kg的LPS,对照组给予生理盐水,注射后24h处死小鼠,取肝脏和肠道组织。用qRT-PCR技术检测小鼠肝脏、肠道组织羧酸酯酶1和2(mCES1和mCES2)mRNA水平;Westernblot技术检测小鼠肝脏、肠道组织mCES1和mCES2蛋白表达水平。用分光光度计检测小鼠肝脏、肠道组织羧酸酯酶总活性。结果:LPS显著降低小鼠肝脏、肠道组织羧酸酯酶1和2的mRNA水平(P<0.05),同时LPS也显著降低小鼠肝脏、肠道组织羧酸酯酶的蛋白表达水平及酶活性(P<0.05)。结论:LPS造成的炎症状态可显著降低小鼠肝脏、肠道组织羧酸酯酶的表达及酶活性。     

Immunoexpression of Survivin in non-neoplastic lymphoid tissues and malignant lymphomas using a new monoclonal antibody reactive on paraffin sections

Survivin is a member of the inhibitor of apoptosis gene family, which is also implicated in mitosis regulation. Most reports in the literature impute poor prognosis to neoplasms with overexpression of this protein. The purpose of the present study is to validate and compare the immunohistochemical reactivity of malignant lymphomas and reactive lymphoid tissue using a new mouse monoclonal antibody to Survivin produced in our laboratory, 6-78. Survivin was detected by immunohistochemistry on tissue microarrays. It was shown that the antibody anti-Survivin 6-78 reliably stains formalin-fixed, paraffin-embedded reactive and neoplastic lymphoid tissues, mostly in a nuclear pattern. We confirmed using this novel antibody that Survivin immunostaining has a tendency to be lower in reactive lymphoid tissues and low-grade B cell lymphomas than in aggressive lymphomas. This antibody may represent a useful tool for standardizing the study of the immunoexpression of Survivin in neoplasms.