PubMed Health⌕ Search

Biomedical subjects

L G Moss

Publications and source records attributed to L G Moss.

At least 19 recordsLinked to original sources
PubMed2001

Cooperativity between the preproinsulin mRNA untranslated regions is necessary for glucose-stimulated translation.

Glucose regulates proinsulin biosynthesis via stimulation of the translation of the preproinsulin mRNA in pancreatic beta-cells. However, the mechanism by which this occurs has remained unclear. Using recombinant adenoviruses that express the preproinsulin mRNA with defined alterations, the untranslated regions (UTRs) of the preproinsulin mRNA were examined for elements that specifically control translation of the mRNA in rat pancreatic islets. These studies revealed that the preproinsulin 5'-UTR was necessary for glucose stimulation of preproinsulin mRNA translation, whereas the 3'-UTR appeared to suppress translation. However, together the 5'- and 3'-UTRs acted cooperatively to markedly increase glucose-induced proinsulin biosynthesis. In primary hepatocytes the presence of the preproinsulin 3'-UTR led to reduced mRNA levels compared with the presence of the SV40 3'-UTR, consistent with the presence of mRNA stability determinants in the 3'-UTR that stabilize the preproinsulin mRNA in a pancreatic beta-cell-specific manner. Translation of these mRNAs in primary hepatocytes was not stimulated by glucose, indicating that regulated translation of the preproinsulin mRNA occurs in a pancreatic beta-cell-specific manner. Thus, the untranslated regions of the preproinsulin mRNA play crucial roles in regulating insulin production and therefore glucose homeostasis by regulating the translation and the stability of the preproinsulin mRNA.

Animals↗
PubMed2000

Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting.

Because cocaine- and amphetamine-regulated transcript (CART) coexists with alpha-melanocyte stimulating hormone (alpha-MSH) in the arcuate nucleus neurons and we have recently demonstrated that alpha-MSH innervates TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN), we raised the possibility that CART may also be contained in fibers that innervate hypophysiotropic thyrotropin-releasing hormone (TRH) neurons and modulate TRH gene expression. Triple-labeling fluorescent in situ hybridization and immunofluorescence were performed to reveal the morphological relationships between pro-TRH mRNA-containing neurons and CART- and alpha-MSH-immunoreactive (IR) axons. CART-IR axons densely innervated the majority of pro-TRH mRNA-containing neurons in all parvocellular subdivisions of the PVN and established asymmetric synaptic specializations with pro-TRH neurons. However, whereas all alpha-MSH-IR axons in the PVN contained CART-IR, only a portion of CART-IR axons in contact with pro-TRH neurons were immunoreactive for alpha-MSH. In the medial and periventricular parvocellular subdivisions of the PVN, CART was co-contained in approximately 80% of pro-TRH neuronal perikarya, whereas colocalization with pro-TRH was found in <10% of the anterior parvocellular subdivision neurons. In addition, >80% of TRH/CART neurons in the periventricular and medial parvocellular subdivisions accumulated Fluoro-Gold after systemic administration, suggesting that CART may serve as a marker for hypophysiotropic TRH neurons. CART prevented fasting-induced suppression of pro-TRH in the PVN when administered intracerebroventricularly and increased the content of TRH in hypothalamic cell cultures. These studies establish an anatomical association between CART and pro-TRH-producing neurons in the PVN and demonstrate that CART has a stimulatory effect on hypophysiotropic TRH neurons by increasing pro-TRH gene expression and the biosynthesis of TRH.

Animals↗
PubMed1998

Overexpression of perilipin A and B blocks the ability of tumor necrosis factor alpha to increase lipolysis in 3T3-L1 adipocytes.

Perilipins, a family of phosphoproteins, are specifically located at the surface of intracellular lipid (triacylglycerol) droplets, the site of lipolysis. Stimulation of lipolysis in 3T3-L1 adipocytes by tumor necrosis factor alpha (TNF-alpha) is associated with a decrease in total cellular expression of perilipin A and B, consistent with the hypothesis that a decrease in perilipin protein expression is required for TNF-alpha-induced lipolysis. Adenovirus-mediated overexpression of perilipin A or B maintains perilipin protein levels on the lipid droplet and blocks TNF-alpha-induced lipolysis. In contrast, overexpression of perilipin A or perilipin B does not inhibit isoproterenol-stimulated lipolysis and does not alter the isoproterenol-induced migration of perilipins from the lipid droplet. These results provide the first evidence of how perilipin functions and suggest that TNF-alpha regulates lipolysis, in part, by decreasing perilipin protein levels at the lipid droplet surface.

3T3 Cells↗
PubMed1998

Sorting human beta-cells consequent to targeted expression of green fluorescent protein.

Pancreatic islets of Langerhans are composed of four major endocrine cell types with a smaller number of nonendocrine cells. To study the molecular constituents and function of just one subpopulation of islet cells, it is necessary to sort them from the other cell types. While rat beta-cells can be sorted by autofluorescence-activated flow cytometry, this has not proved possible on a routine and reproducible basis for human beta-cells. In the present study, we have selectively labeled human beta-cells with green fluorescent protein (GFP), allowing for their sorting by flow cytometry. Human islet cells were infected with replication-defective (attenuated) recombinant adenovirus expressing GFP driven by the rat insulin I promoter (Ad-RIP-GFP) for targeted expression in beta-cells, or beta-galactosidase driven by the promiscuous cytomegalovirus (CMV) promoter (Ad-CMV-beta-gal) as control. Whereas the majority of islet cells can be infected by adenovirus, as shown by control infection with Ad-CMV-beta-gal, increased fluorescence after infection with Ad-RIP-GFP was limited to insulin-containing beta-cells. Infection of islet cells with Ad-RIP-GFP resulted reproducibly in the appearance of a population of intensely fluorescent cells, when analyzed by flow cytometry. These cells were sorted using a fluorescence-activated cell sorter (FACS) and shown by immunofluorescence to consist of >95% beta-cells. The targeted expression of GFP thus allows for preparation of human beta-cells purified close to homogeneity. This method should be readily applicable in any laboratory with FACS capability.

Adenoviridae↗
PubMed1997

Individual beta cells within the intact islet differentially respond to glucose.

Insulin production by the pancreatic islet is tightly coupled to the concentration of blood glucose. The mechanism by which glucose controls proinsulin biosynthesis in beta cells is poorly understood. Analysis of insulin gene expression in individual cells within whole, living islets using adenovirus gene transfer and direct observation of insulin promoter-directed green fluorescent protein activity indicates that beta cells are functionally heterogeneous. An increase in glucose concentration not only stimulates expression within individual beta cells, but unexpectedly acts to increase the total number of positive cells. The net islet response to a given glucose stimulus reflects an integrated action of beta cells with individually differing behaviors. This additional level of functional complexity may provide new insights into the pathophysiology and treatment of diabetes mellitus.

Animals↗
PubMed1996

Requirements for green fluorescent protein detection in transgenic zebrafish embryos.

We have generated transgenic (Tg) lines of zebrafish in which the green fluorescent protein (GFP)-encoding gfp cDNA is driven by the Xenopus laevis ef1 alpha enhancer/promoter; Tg embryos from most of these lines show detectable fluorescence throughout their body. We have investigated the copy number of the Tg genes in fluorescent and non-fluorescent lines, in order to determine how this affects the production of detectable levels of GFP in the zebrafish embryo. Additionally, we have injected purified recombinant GFP into embryos to determine the intracellular GFP concentration required for detection, both when all of the cells in the embryo contain GFP and when only a few do.

Animals↗
PubMed1996

The molecular structure of green fluorescent protein.

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.

Animals↗
PubMed1994

Two distinct class A helix-loop-helix transcription factors, E2A and BETA1, form separate DNA binding complexes on the insulin gene E box.

Mutations in the RIPE3a element have shown it to be crucial for efficient tissue-specific expression of the insulin gene. In order to isolate factors binding to this element, we used a labeled RIPE3 probe to screen an expression library derived from a hamster insulinoma cell line. We isolated a clone encoding beta-cell E-box transcriptional activator1 (BETA 1). This clone is a member of the class A subfamily of the helix-loop-helix superfamily of transcriptional activators, as determined both by sequence analysis and by functional association with a class B member (myogenin). This clone is related to, but distinct from, other clones isolated from the same library which are also capable of binding RIPE3a. Analysis showed these additional clones to be the hamster homologs of E12 and E47 (German, M. S., Blaner, M. A., Nelson, C., Moss, L. G., and Rutter, W. J. (1991) Mol. Endocrinol. 5, 292-299). Antibodies were raised against BETA 1 and against a common epitope of E12 and E47 to determine which proteins were contained in the native RIPE3a binding complex. Using these antibodies, we were able to separate the complex into major and minor fractions which contained either E12/47 or BETA 1, respectively. Thus, these two gene products are found in separate fractions of the tissue-specific binding activity and are therefore both likely to be important in insulin gene regulation.

Amino Acid Sequence↗
PubMed1992

Hepatocyte nuclear factor 1 alpha is expressed in a hamster insulinoma line and transactivates the rat insulin I gene.

Systematic mutational analysis previously identified two primary regulatory elements within a minienhancer (-247 to -198) of the rat insulin I promoter that are critical for transcriptional activity. The Far box (-241 to -232) and the FLAT element (-222 to -208) synergistically upregulate transcription and, together, are sufficient to confer tissue-specific and glucose-responsive transcriptional activity on a heterologous promoter. Detailed analysis of the FLAT element further revealed that, in addition to the positive regulatory activity it mediates in tandem with the Far box, it is a site for negative regulatory control. A portion of the FLAT element bears considerable sequence similarity to the consensus binding site for hepatocyte nuclear factor 1 alpha (HNF1 alpha; LF-B1) a liver-enriched homeodomain-containing transcription factor. Here we show that the HNF1-like site within the FLAT element exhibited positive transcriptional activity in both HepG2 and HIT cells and bound similar, but distinguishable, nuclear protein complexes in the respective nuclear extracts. Screening of a hamster insulinoma cDNA library with a PCR-derived probe encompassing the DNA-binding domain of rat HNF1 alpha resulted in isolation of a hamster HNF1 alpha (hHNF1 alpha) cDNA homolog. Specific antiserum identified the HNF1 alpha protein as one component of a specific FLAT-binding complex in HIT nuclear extracts. Expression of the hHNF1 alpha cDNA in COS cells resulted in transactivation of reporter constructs containing multimerized segments of the rat insulin I minienhancer. Thus, HNF1 alpha, one component of a DNA-binding complex involved in transcriptional regulation of the rat insulin I gene, may play a significant role in nonhepatic as well as hepatic gene transcription.

Amino Acid Sequence↗
PubMed1992

The insulin and islet amyloid polypeptide genes contain similar cell-specific promoter elements that bind identical beta-cell nuclear complexes.

The pancreatic beta cell makes several unique gene products, including insulin, islet amyloid polypeptide (IAPP), and beta-cell-specific glucokinase (beta GK). The functions of isolated portions of the insulin, IAPP, and beta GK promoters were studied by using transient expression and DNA binding assays. A short portion (-247 to -197 bp) of the rat insulin I gene, the FF minienhancer, contains three interacting transcriptional regulatory elements. The FF minienhancer binds at least two nuclear complexes with limited tissue distribution. Sequences similar to that of the FF minienhancer are present in the 5' flanking DNA of the human IAPP and rat beta GK genes and also the rat insulin II and mouse insulin I and II genes. Similar minienhancer constructs from the insulin and IAPP genes function as cell-specific transcriptional regulatory elements and compete for binding of the same nuclear factors, while the beta GK construct competes for protein binding but functions poorly as a minienhancer. These observations suggest that the patterns of expression of the beta-cell-specific genes result in part from sharing the same transcriptional regulators.

Amyloid↗
PubMed1992

Cloning of a novel alpha 1-subunit of the voltage-dependent calcium channel from the beta-cell.

To study the molecular regulation of voltage-dependent Ca2+ channels (VDCCs) in the beta-cell, we have cloned a cDNA for the alpha 1-subunit from a hamster insulin-secreting cell line (HIT-T15). The cDNA (HCa3a) encodes a 1610-amino acid protein with four repeating membrane domains and an overall structure characteristic of other alpha 1-subunits. Although the cDNA shows a high degree of sequence homology (97%) with a rat brain alpha 1-subunit (RB alpha 1), the C-terminal 15 amino acids of HCa3a share no similarity with any cloned alpha 1 protein. High stringency Northern blot analysis revealed a single transcript of approximately 8.6 kilobases in HIT cells and hamster pancreas. A similarly sized species was detected in hamster brain, heart, and skeletal muscle. Using polymerase chain reaction and a primer set unique to HCa3a, this alpha 1 isoform was found to be expressed in islet cell lines derived from rat, mouse, and hamster. The HIT cell alpha 1-subunit is also expressed in discrete regions of the rat central nervous system, including the cortex, cerebellum, hypothalamus, and brain stem. The expression of two alpha 1 isoforms (HCa3a and cardiac) in the HIT cell underscores the possible complexity of VDCCs in the regulation of beta-cell signal transduction. With its widespread tissue distribution, HCa3a does not conform to the current classification system used for L-type VDCCs; this suggests that an alternative system of classification is required.

Amino Acid Sequence↗
PubMed1991

Two related helix-loop-helix proteins participate in separate cell-specific complexes that bind the insulin enhancer.

Cell-specific expression of the insulin gene is dependent on a conserved 8-basepair sequence, GCCATCTG, present in two copies in the 5' flanking DNA of the rat insulin 1 gene (Nir and Far elements). A protein factor with well characterized binding affinities binds to this sequence and is unique to the nuclei of insulin-producing cells. Using the Nir element as a probe to screen a hamster insulinoma cDNA expression library, we cloned two cDNA inserts that encode two related helix-loop-helix DNA-binding proteins: Syrian hamster Pan-1 (shPan-1) and Syrian hamster Pan-2 (shPan-2). These clones have minimal differences from the previously reported human E47/E12 and rat PAN (rPan) DNA-binding proteins. In vitro translated protein products of both clones bound the insulin gene promoter Nir and far elements as well as the E2 elements of the mu heavy chain and kappa light chain immunoglobulin genes. Treating insulinoma cell nuclear extract with antiserum selectively directed to each of the two shPan proteins demonstrated the presence of each form of shPan in separate DNA-binding complexes, which together form the previously described, cell-specific, Nir element-binding complex. We conclude that shPan-1 and shPan-2 are the hamster homologs of the ubiquitous E47/E12 and rPan proteins, but form parts of distinct DNA-binding complexes apparently found only in the nuclei of insulin-producing cells.

Amino Acid Sequence↗
PubMed1990

Regulation of insulin gene expression by glucose and calcium in transfected primary islet cultures.

To study the regulation of insulin gene expression by physiological regulators, primary cultures of rat islet cells were transfected with portions of the rat insulin I gene 5'-flanking sequence linked to the reporter gene chloramphenicol acetyltransferase (CAT). Incubation of the cells in increasing glucose concentrations led to a parallel increase in both CAT activity and CAT mRNA levels. Pretreatment of the cells with the beta-cell-specific toxin streptozotocin reduced CAT activity 97%. Beta-Cell-specific expression of CAT was also demonstrated by co-staining the transfected cells with antisera to both CAT and insulin. Experiments showing a reduction in the response to glucose in the presence of the calcium channel blocker verapamil suggest that calcium plays a role in the glucose response, possibly via regulation of factors interacting with this limited portion of the insulin gene.

Animals↗
PubMed1990

Molecular cloning of a novel splice variant of the alpha subunit of the mammalian Go protein.

We screened a HIT (hamster insulin-secreting tumor) cell cDNA library constructed in lambda gt11 with a Go-specific oligonucleotide probe and isolated six recombinant phages. The inserts of these phages encoded two forms of alpha o, called here alpha o1 and alpha o2. The deduced amino acid sequence of alpha o1 is identical in all of its 354 amino acids to that reported previously for rat and bovine alpha o; that of alpha o2, also of 354 amino acids, is identical to alpha o1 up to and including amino acid 248 and differs thereafter in 26 amino acids. At the nucleotide level, alpha o1 and alpha o2 are identical up to and including the second base of the codon that specifies amino acid 243 and differs thereafter in 88 nucleotides of the remaining open reading frame and has no similarity to alpha o1 in its 3'-untranslated region. We propose that alpha o1 and alpha o2 result as a consequence of alternative splicing of a single alpha o transcript. Northern analysis with specifically designed oligonucleotides indicates that both forms of alpha o are expressed in normal tissues, e.g. brain. After in vitro transcription and translation, the peptides encoded in the alpha o1 and alpha o2 cDNAs could be ADP-ribosylated by pertussis toxin in the presence of added beta gamma dimers. The count of distinct G proteins keeps increasing.

Adenosine Diphosphate Ribose↗
PubMed1988

Systematic binding analysis of the insulin gene transcription control region: insulin and immunoglobulin enhancers utilize similar transactivators.

The 5' regulatory region (-345 to +1) of the rat insulin I gene (Ins-I) was examined for binding to cellular factors with short oligodeoxynucleotide probes. Over 40 binding species were detected. The binding profiles were specific for each cell type studied. We characterized the factors binding two elements crucial for enhancer activity (the Nir and Far boxes) which bear sequence similarity to the microE1, microE2, and microE3 elements of the immunoglobulin heavy-chain enhancer. The Nir box binds three cellular factors that display preferential affinities for microE1, microE2, or microE3, and the Far box binds two factors related to microE2 or microE3. The insulin gene enhancer was mutated at the Nir box element to reflect the sequences of microE1, microE2, or microE3. Ins-microE2 was fully active, Ins-microE3 was partially active, and Ins-microE1 was inactive. Thus, factors similar or identical to nuclear factor NF-microE1, NF-microE2, or NF-microE3 may play a role in the activity of the insulin gene enhancer.

Animals↗
PubMed1987

Convenient vectors for cloning and sequencing EcoRI and HindIII fragments.

The polylinker regions of plasmid pUC and bacteriophage M13mp vectors have been specifically modified to provide alternative positions for cloning and reexcising EcoRI and HindIII fragments; the EcoRI and HindIII sites have been moved internal to BamHI and Bg/II sites. The location of EcoRI and HindIII sites in these HinEco vectors allows either selective linearization or excision of the cloned fragments at unique flanking sites.

Base Sequence↗