IGF-I attenuates diabetes-induced cardiac contractile dysfunction in ventricular myocytes

Diabetic cardiomyopathy is characterized by impaired ventricular contraction and altered function of insulin-like growth factor I (IGF-I), a key factor for cardiac growth and function. Endogenous IGF-I has been shown to alleviate diabetic cardiomyopathy. This study was designed to evaluate exogenous IGF-I treatment on the development of diabetic cardiomyopathy. Adult rats were divided into four groups: control, control + IGF-I, diabetic, and diabetic + IGF-I. Streptozotocin (STZ; 55 mg/kg) was used to induce experimental diabetes immediately followed by a 7-wk IGF-I (3 mg. kg(-1). day(-1) ip) treatment. Mechanical properties were assessed in ventricular myocytes including peak shortening (PS), time-to-PS (TPS), time-to-90% relengthening (TR(90)) and maximal velocities of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) transients were evaluated as Ca(2+)-induced Ca(2+) release and Ca(2+) clearing constant. Levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), phospholamban (PLB), and glucose transporter (GLUT4) were assessed by Western blot. STZ caused significant weight loss and elevated blood glucose, demonstrating the diabetic status. The diabetic state is associated with reduced serum IGF-I levels, which were restored by IGF-I treatment. Diabetic myocytes showed reduced PS and +/-dL/dt as well as prolonged TPS, TR(90), and intracellular Ca(2+) clearing compared with control. IGF-I treatment prevented the diabetes-induced abnormalities in PS, +/-dL/dt, TR(90), and Ca(2+) clearing but not TPS. The levels of SERCA and GLUT4, but not PLB, were significantly reduced in diabetic hearts compared with controls. IGF-I treatment restored the diabetes-induced decline in SERCA, whereas it had no effect on GLUT4 and PLB levels. These results suggest that exogenous IGF-I treatment may ameliorate contractile disturbances in cardiomyocytes from diabetic animals and could provide therapeutic potential in the treatment of diabetic cardiomyopathy.     

Fusion with HDEL protects cell wall invertase from early degradation when N-glycosylation is inhibited

Previous data obtained in different suspension-cultured plant cells have clearly illustrated that N-glycans are absolutely required for transport of glycoproteins to the extracellular compartment, regardless of their oligosaccharide structure [see Lerouge et al. (1998) Plant Mol. Biol. 38: 31 for review]. In the present study the role of N-glycosylation in the transport of glycoproteins to the cell surface was studied in BY2 tobacco cells using both endogenous and recombinant cell wall invertases as markers. When synthesized without their N-glycans, both invertases were very rapidly degraded. This degradation did not occur in an acidic compartment and was brefeldin A-insensitive. Therefore, it most probably represents a pre-Golgi event. However, the low efficiency of specific inhibitors did not favor a strong contribution of proteasomes in this proteolysis. In contrast, addition of a C-terminal His-Asp-Glu-Leu (HDEL) extension prevented arrival of these non-glycosylated glycoproteins in the compartment where they are degraded. These results argue for the presence of an endoplasmic reticulum (ER) domain specialized in protein degradation. Consistent with our results and the well-known stabilization of recombinant proteins retained in the ER, the addition of an ER retention signal to a protein would prevent its targeting to an ER domain devoted to degradation.     

Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures

Structural analysis of the N-glycosylation of alfalfa proteins was investigated in order to evaluate the capacity of this plant to perform this biologically important post-translational modification. We show that, in alfalfa, N-linked glycans are processed into a large variety of mature oligosaccharides having core-xylose and core alpha(1,3)-fucose, as well as terminal Lewis(a) epitopes. In contrast, expression of the C5-1 monoclonal antibody in alfalfa plants results in the production of plant-derived IgG1 which is N-glycosylated by a predominant glycan having a alpha(1,3)-fucose and a beta(1,2)-xylose attached to a GlcNAc2Man3GlcNAc2 core. Since this core is common to plant and mammal N-linked glycans, it therefore appears that alfalfa plants have the ability to produce recombinant IgG1 having a N-glycosylation that is suitable for in vitro or in vivo glycan remodelling into a human-compatible plantibody. For instance, as proof of concept, in vitro galactosylation of the alfalfa-derived C5-1 mAb resulted in a homogenous plantibody harbouring terminal beta(1,4)-galactose residues as observed in the mammalian IgG.     

Structural requirements for Arabidopsis beta1,2-xylosyltransferase activity and targeting to the Golgi

Characterization of a beta1,2-xylosyltransferase from Arabidopsis thaliana (AtXylT) was carried out by expression in Sf9 insect cells using a baculovirus vector system. Serial deletions at both the N- and C-terminal ends proved that integrity of a large domain located between amino acid 31 and the C-terminal lumenal region is required for AtXylT activity expression. The influence of N-glycosylation on AtXylT activity has been evaluated using either tunicamycin or mutagenesis of potential N-glycosylation sites. AtXylT is glycosylated on two of its three potential N-glycosylation sites (Asn51, Asn301, Asn478) and the occupancy of at least one of these two sites (Asn51 and Asn301) is necessary for AtXylT stability and activity. Contribution of the N-terminal part of AtXylT in targeting and intracellular distribution of this protein was studied by expression of variably truncated, GFP-tagged AtXylT forms in tobacco cells using confocal and electron microscopy. These studies have shown that the transmembrane domain of AtXylT and its short flanking amino acid sequences are sufficient to specifically localize a reporter protein to the medial Golgi cisternae in tobacco cells. This study is the first detailed characterization of a plant glycosyltransferase at the molecular level.     

The insect immune protein hemolin is expressed during oogenesis and embryogenesis

Hemolin is the most abundant bacteria-induced proteins in Hyalophora cecropia hemolymph. Its structural features, both at the protein and gene level, ascribe this molecule to the immunoglobulin gene superfamily (IgSF) with particular homology to neural cell adhesion molecules. An increasing number of evidence suggest a role in immune recognition and in cell adhesion events. Hemolin is also developmentally regulated as suggested by changes in its concentration during larval and pupal ecdysis (Trenczek, T., 1998. Endogenous defense mechanisms of insects. Zoology 101, 298-315; Lanz-Mendoza, H., Faye, I., 1999. Physiological aspects of the immunoglobulin superfamily in invertebrates. Dev. Comp. Immunol. 23, 359-374). In the present study the expression of hemolin was investigated in oogenesis and in early embryogenesis. Our results reveal that hemolin is expressed in follicles and in epidermal and neural tissues of embryos.     

Interactions of the nuclear matrix-associated steroid receptor binding factor with its DNA binding element in the c-myc gene promoter

Steroid receptor binding factor (RBF) was originally isolated from avian oviduct nuclear matrix. When bound to avian genomic DNA, RBF generates saturable high-affinity binding sites for the avian progesterone receptor (PR). Recent studies have shown that RBF binds to a 54 bp element in the 5'-flanking region of the progesterone-regulated avian c-myc gene, and nuclear matrix-like attachment sites flank the RBF element [Lauber et al. (1997) J. Biol. Chem. 272, 24657-24665]. In this paper, electrophoretic mobility shift assays (EMSAs) and S1 nuclease treatment are used to demonstrate that the RBF-maltose binding protei (MBP) fusion protein binds to single-stranded DNA of its element. Only the N-terminal domain of RBF binds the RBF DNA element as demonstrated by southwestern blot analyses, and by competition EMSAs between RBF-MBP and the N-terminal domain. Mass spectrometric analysis of the C-terminal domain of RBF demonstrates its potential to form noncovalent protein-protein interactions via a potential leucine-isoleucine zipperlike structure, suggesting a homo- and/or possible heterodimer structure in solution. These data support that the nuclear matrix binding site (acceptor site) for PR in the c-myc gene promoter is composed of RBF dimers bound to a specific single-stranded DNA element. The dimers of RBF are generated by C-terminal leucine zipper and the DNA binding occurs at the N-terminal parallel beta-sheet DNA binding motif. This complex is flanked by nuclear matrix attachment sites.     

Blood plasma chemistries from wild mourning doves held in captivity

Despite the extensive amount of research conducted on mourning doves (Zenaida macroura), no biochemical reference values exist for this species. Our objective, therefore, was to establish base line clinical chemistry reference values for mourning doves to assist with establishing clinical diagnoses. Wild mourning doves were captured 19 March 1996 to 8 August 1996, and 6 February 1998 to 12 May 1998; blood samples were collected from 382 mourning doves. Plasma biochemical values were established for glucose, sodium, potassium, chloride, enzymatic CO2, albumin, total protein, globulin, calcium, phosphorus, cholesterol, magnesium, aspartate aminotransferase (AST), gamma glutamyl transferase (GGT), lactate dehydrogenase (LDH), and uric acid. These reference values are invaluable for determining diagnosis of diseases of the gastrointestinal, hepatic, renal, cardiovascular, musculoskeletal, and endocrine systems.     

Fluorescent oligonucleotide ligation technology for identification of ras oncogene mutations

A mutation detection strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was developed to detect single nucleotide substitutions in ras oncogenes, a common genetic abnormality in many human cancers. Mutation-specific probes are synthesized for each possible single-base, nonsilent mutation in codons 12, 13, and 61 of H-, K-, and N-ras oncogenes. Mutations are identified by competitive oligonucleotide probe ligation to detect normal and /or mutant genotypes in one reaction. Three probes (one common and two allelic probes) are needed for analysis of each mutation. Probes hybridized to target ras oncogene DNA are joined by a thermostable ligase if there are no mismatches at their junctions; temperature cycling results in a linear increase in product. Common probes are labeled with fluorochromes, and allelic probes each have different lengths. Ligation products are analyzed by denaturing polyacrylamide gel electrophoresis on a fluorescent DNA sequencer. We have applied this technology to identify ras mutations in pancreatic cancers and lung cancers and in patients with myelodysplastic syndromes and leukemias.     

Antibody arrays for high-throughput screening of antibody-antigen interactions

We have developed a novel technique for high-throughput screening of recombinant antibodies, based on the creation of antibody arrays. Our method uses robotic picking and high-density gridding of bacteria containing antibody genes followed by filter-based enzyme-linked immunosorbent assay (ELISA) screening to identify clones that express binding antibody fragments. By eliminating the need for liquid handling, we can thereby screen up to 18,342 different antibody clones at a time and, because the clones are arrayed from master stocks, the same antibodies can be double spotted and screened simultaneously against 15 different antigens. We have used our technique in several different applications, including isolating antibodies against impure proteins and complex antigens, where several rounds of phage display often fail. Our results indicate that antibody arrays can be used to identify differentially expressed proteins.     

Evidence from mutational specificity studies that yeast DNA polymerases delta and epsilon replicate different DNA strands at an intracellular replication fork

Although polymerases delta and epsilon are required for DNA replication in eukaryotic cells, whether each polymerase functions on a separate template strand remains an open question. To begin examining the relative intracellular roles of the two polymerases, we used a plasmid-borne yeast tRNA gene and yeast strains that are mutators due to the elimination of proofreading by DNA polymerases delta or epsilon. Inversion of the tRNA gene to change the sequence of the leading and lagging strand templates altered the specificities of both mutator polymerases, but in opposite directions. That is, the specificity of the polymerase delta mutator with the tRNA gene in one orientation bore similarities to the specificity of the polymerase epsilon mutator with the tRNA gene in the other orientation, and vice versa. We also obtained results consistent with gene orientation having a minor influence on mismatch correction of replication errors occurring in a wild-type strain. However, the data suggest that neither this effect nor differential replication fidelity was responsible for the mutational specificity changes observed in the proofreading-deficient mutants upon gene inversion. Collectively, the data argue that polymerases delta and epsilon each encounter a different template sequence upon inversion of the tRNA gene, and so replicate opposite strands at the plasmid DNA replication fork.