Structure, antihyperglycemic activity and cellular actions of a novel diglycated human insulin

Human insulin was glycated under hyperglycemic reducing conditions and a novel diglycated form (M(r) 6135.1 Da) was purified by RP-HPLC. Endoproteinase Glu-C digestion combined with mass spectrometry and automated Edman degradation localized glycation to Gly(1) and Phe(1) of the insulin A- and B-chains, respectively. Intraperitoneal (i.p.) administration of diglycated insulin to mice alone or in combination with glucose (7 nmol/kg) resulted in a 43-61% and 11-34% reduction in glucose lowering activity, respectively, compared with native insulin. Consistent with these findings, diglycated insulin (10(-9) to 10(-7) mol/liter) was 22-38% less effective (P < 0.001) than native insulin in stimulating glucose uptake, glucose oxidation and glycogen production in isolated mouse abdominal muscle.     

Singular contributions of fish neuroendocrinology to mammalian regulatory peptide research

During the past 20 years, several bioactive peptides have been identified in teleost fishes that subsequently have been shown to play important regulatory roles in mammalian physiology. The urophysis, corpuscles of Stannius and Brockmann body are anatomical structures particular to fish that have no obvious counterpart in mammals. Extracts and/or cDNA libraries prepared from these tissues have been used to identify for the first time urotensin II (U-II), urotensin-I (U-I), stanniocalcin and glucagon-like peptide-1 (GLP-1). Although U-II and U-I were originally regarded as exclusively the products of the teleost urophysis, the peptides have a wide phylogenetic distribution across the vertebrate lineage, including mammals. U-II is localized to motor neurones in the human spinal cord and is a potent vasoconstrictor that may be implicated in the pathogenesis of heart failure. The human ortholog of urotensin-I is urocortin which is synthesized in selected regions of the brain and is the endogenous ligand for the CRF type 2 receptor. Urocortin is believed to important in mediating the effects of stress on appetite. Stanniocalcin is involved in maintaining calcium and phosphate homeostasis in teleost fish. An ortholog of stanniocalcin has a widespread distribution in mammalian tissues and is postulated to regulate renal phosphate excretion and to protect neurons against damage during cerebral ischemia. The biological actions and therapeutic potential of GLP-1 in humans are now fully appreciated but the peptide was first identified as a domain in a preproglucagon cDNA prepared from anglerfish Brockmann bodies. In contrast to mammalian preproglucagons, GLP-1 is present in anglerfish preproglucagon as the bioactive, truncated sequence [corresponding to human GLP-1(7-37)] rather than the inactive, N-terminally extended form [corresponding to GLP-1(1-37)]. Failure to appreciate the significance of this fact retarded progress in the field for several years.     

Novel 2-imidazoles as potent and selective alpha1A adrenoceptor partial agonists

Novel 2-imidazoles have been identified as potent partial agonists of the α_1A adrenergic receptor, with good selectivity over the α_1B, α_1D and α_2A receptor sub-types. Sulfonamide 23 possessed attractive drug-like properties with respect to physicochemical and ADME properties and wide ligand selectivity.     

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle¹. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions^2-5.Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner^4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes¹², has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.     

A genetic variant in osteoprotegerin is associated with progression of joint destruction in rheumatoid arthritis

Introduction

Progression of joint destruction in rheumatoid arthritis (RA) is partly heritably; 45 to 58% of the variance in joint destruction is estimated to be explained by genetic factors. The binding of RANKL (Receptor Activator for Nuclear Factor κ B Ligand) to RANK results in the activation of TRAF6 (tumor necrosis factor (TNF) receptor associated factor-6), and osteoclast formation ultimately leading to enhanced bone resorption. This bone resorption is inhibited by osteoprotegerin (OPG) which prevents RANKL-RANK interactions. The OPG/RANK/RANKL/TRAF6 pathway plays an important role in bone remodeling. Therefore, we investigated whether genetic variants in OPG, RANK, RANKL and TRAF6 are associated with the rate of joint destruction in RA.

Methods

1,418 patients with 4,885 X-rays of hands and feet derived from four independent data-sets were studied. In each data-set the relative increase of the progression rate per year in the presence of a genotype was assessed. First, explorative analyses were performed on 600 RA-patients from Leiden. 109 SNPs, tagging OPG, RANK, RANKL and TRAF6, were tested. Single nucleotide polymorphisms (SNPs) significantly associated in phase-1 were genotyped in data-sets from Groningen (Netherlands), Sheffield (United Kingdom) and Lund (Switzerland). Data were summarized in an inverse weighted variance meta-analysis. Bonferonni correction for multiple testing was applied.

Results

We found that 33 SNPs were significantly associated with the rate of joint destruction in phase-1. In phase-2, six SNPs in OPG and four SNPs in RANK were associated with progression of joint destruction with P-value <0.05. In the meta-analyses of all four data-sets, RA-patients with the minor allele of OPG-rs1485305 expressed higher rates of joint destruction compared to patients without these risk variants (P = 2.35x10⁻⁴). This variant was also significant after Bonferroni correction.

Conclusions

These results indicate that a genetic variant in OPG is associated with a more severe rate of joint destruction in RA.     

IL-1 as adjuvant. Role of T cells in the augmentation of specific antibody production by recombinant human IL-1 alpha

Human rIL-1 alpha significantly enhanced splenic plaque-forming cells (PFC) to SRBC in vitro and in vivo. A single i.p. injection was sufficient to produce a fivefold or greater increase in the generation of PFC in a primary response. IL-1 treatment resulted in an increased production of Ag-specific PFC, both in vitro and in vivo, in combination with suboptimal doses of Ag. When IL-1 was given with a primary dose of Ag in vivo, an enhanced IgG response occurred. IL-1 enhanced in vivo carrier priming for an anti-hapten PFC response, indicating increased Th activity. Furthermore, T cells from spleens of mice treated with IL-1 provided significantly more help in both carrier (SRBC)- and hapten (TNP)- specific PFC. The enhancement of PFC by IL-1 in vitro occurred even in the presence of an excess of neutralizing anti-IL-2 antibody. These results suggest that IL-1 may enhance T cell-dependent antibody production in part by increasing Th activity, and that the mechanism of IL-1 action in increasing antibody production involves pathways in addition to the induction of IL-2 secretion.