Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.     

Expression of human and mouse adenine nucleotide translocase (ANT) isoform genes in adipogenesis

Adenine nucleotide translocases (ANTs) are mitochondrial proteins encoded by nuclear DNA that catalyze the exchange of ATP generated in the mitochondria for ADP produced in cytosol. There are four ANT isoforms in humans (hANT1-4) and three in mice (mANT1, mANT2 and mANT4), all encoded by distinct genes. The aim of this study was to quantify expression of ANT isoform genes during the adipogenesis of mouse 3T3-L1 and human Simpson–Golabi–Behmel syndrome (SGBS)-derived preadipocytes. We also studied the effects of the adipogenesis regulators, insulin and rosiglitazone, on ANT isoform expression in differentiated adipocytes and examined the expression of ANT isoforms in subcutaneous and visceral white adipose tissue (WAT) from mice and humans. We found that adipogenesis was associated with an increase in the expression of ANT isoforms, specifically mANT2 in mouse 3T3-L1 cells and hANT3 in human SGBS cells. These changes could be involved in the increases in oxidative metabolism and decreases in lactate production observed during differentiation. Insulin and rosiglitazone induced mANT2 gene expression in mature 3T3-L1 cells and hANT2 and hANT3 gene expression in SGBS adipocytes. Furthermore, human WAT expressed greater amounts of hANT3 than hANT2, and the expression of both of these isoforms was greater in subcutaneous WAT than in visceral WAT. Finally, inhibition of ANT activity by atractyloside or bongkrekic acid impaired proper adipocyte differentiation. These results suggest that changes in the expression of ANT isoforms may be involved in adipogenesis in both human and mouse WAT.     

Ryanodine受体结构和药理学性质

Ｒｙａｎｏｄｉｎｅ受体（ＲｙＲ）是存在于细胞内钙库膜上的一种钙释放通道。在哺乳类动物中,ＲｙＲ存在三种亚型,即骨骼肌型（ＲｙＲ１）、心肌型（ＲｙＲ２）和脑型（ＲｙＲ３）,它们分别由ｒｙｒ１、ｒｙｒ２和ｒｙｒ３基因编码。非哺乳类脊椎动物的ＲｙＲ有另外三种亚型,即同时存在于骨骼肌中的αＲｙＲ和βＲｙＲ,以及存在于心肌中的另一亚型。前两者在氨基酸序列上分别与ＲｙＲ１和ＲｙＲ３有较高的同源性。哺乳类和非哺     

Molecular cloning and characterization of mouse cardiac junctate isoforms.

Junctate is a newly identified integral ER/SR membrane calcium binding protein, which is an alternative splicing form of the same gene generating aspartyl beta-hydroxylase and junctin. Screening a mouse heart cDNA library using canine junctin cDNA as a probe yielded three complete mouse heart cDNAs. One of the cDNAs is homologous to the previously reported human junctate. The three mouse junctate proteins are composed of 270, 259, and 215 amino acids (we named them junctate-1, -2, and -3). The apparent molecular masses of the mouse junctates in SDS-PAGE were in the range between 40 and 53 kDa. Northern and Western blot analyses indicate that mouse junctates are expressed in heart, brain, spleen, lung, liver, kidney, and stomach, but not in skeletal muscle. The apparent molecular weights of junctates from heart and brain were somewhat different from those from the other tissues tested, suggesting that there are tissue-specific expression patterns of the different junctate isoforms. Immunohistochemical studies showed that junctates were expressed both in ventricular and atrial tissues. This is the first study that shows the presence of 3 distinct cardiac junctate isoforms expressed in various mammalian tissues.     

Complete cDNA sequence,expression, alternative splicing,and genomic organization of the mouse Nfat5 gene

Members of the NFAT (nuclear factors of activated T cells) gene family have been investigated in numerous organisms, including man and mouse. All NFATs may be synthesized in several isoforms differing in amino or carboxy termini due to 5' and 3' alternative splicing of the corresponding mRNA. Recently, we mapped the murine Nfat5 gene to chromosome 8D. In the present paper we describe for the first time the complete sequence and primary structure of murine Nfat5, two new spliced isoforms, and the expression of murine Nfat5 in embryonic and adult mouse tissues.     

Expression and immunochemical analysis of rat and human fibroblast growth factor receptor (flg) isoforms.

Potentially 96 splice variants among four genes that code for the human heparin-binding fibroblast growth factor receptor family complicate study of structure, metabolism, and function of single isoforms in mammalian cells. As an alternative, we expressed structural subdomains and isoforms of the flg receptor gene in bacteria and baculoviral-infected insect cells. We developed and characterized a panel of 16 isoform and domain-specific polyclonal and monoclonal antibodies. The panel of antibodies was used to distinguish mature glycosylated ligand-binding and kinase-active and -inactive recombinant isoforms in baculoviral insect cells and transfected mammalian cells and natural isoforms in rat prostate and human liver cells. The results revealed a cell type-specific expression of the flg gene and isoforms that result from combinations of splice variations. Reactive epitopes of monoclonal antibodies against both the three (alpha) and two (beta) immunoglobulin-like disulfide loop extracellular domain isoforms were mapped by cross-reactivity with synthetic polypeptide sequences and deletion mutants expressed in bacteria. The native alpha and beta receptor isoforms differed in display of shared epitopes and suggested that the NH2-terminal Loop I and COOH-terminal Loops II and III of the alpha isoform are interactive. Although the common Loops II and III appear qualitatively sufficient for ligand binding, the results suggest that tertiary relationships among loops in the three and two loop isoforms are distinct and, therefore, the two isoforms may have distinct activities. Spatial models for arrangement of immunoglobulin-like loops in the extracellular domain of the two isoforms are presented.     

Searching the human genome database for novel relaxin- and insulin-like peptides

Summary In recent times, new members of the insulin/relaxin peptide superfamily have been identified by both differential cloning strategies as well as bioinformatic searching of the EST databases. We have used the public and Celera Genomics databases to search for novel members of this peptide family. No new members of the insulin/relaxin family were identified although the human (H3) and mouse (M3) relaxin 3 genes that we recently discovered in the Celera Genomics database were identified in the public database. We were able to confirm that there are no mouse equivalents of human INSL-4 or human gene 1 relaxin. Hence, as the two human relaxin genes (H1 and H2) are localized together with INSL6 and INSL4 on chromosome 9 it is probable that INSL4 and H1 relaxin are the result of a gene duplication which did not occur in non-primates. The discovery of a full relaxin 3 sequences in a new Zebrafish brain EST library, which retains a high homology in both A and B chain peptide sequence with the H3 peptide, indicate that this novel peptide has important conserved functions.     

Myosin 1b Regulates Amino Acid Transport by Associating Transporters with the Apical Plasma Membrane of Kidney Cells

Amino acid transporters (AATers) in the brush border of the apical plasma membrane (APM) of renal proximal tubule (PT) cells mediate amino acid transport (AAT). We found that the membrane-associated class I myosin myosin 1b (Myo1b) localized at the apical brush border membrane of PTs. In opossum kidney (OK) 3B/2 epithelial cells, which are derived from PTs, expressed rat Myo1b-GFP colocalized in patched microvilli with expressed mouse V5-tagged SIT1 (SIT1-V5), which mediates neutral amino acid transport in OK cells. Lentivirus-mediated delivery of opossum Myo1b-specific shRNA resulted in knockdown (kd) of Myo1b expression, less SIT1-V5 at the APM as determined by localization studies, and a decrease in neutral AAT as determined by radioactive uptake assays. Myo1b kd had no effect on Pi transport or noticeable change in microvilli structure as determined by rhodamine phalloidin staining. The studies are the first to define a physiological role for Myo1b, that of regulating renal AAT by modulating the association of AATers with the APM.