Effects of 1-mo bolus subcutaneous administration of exendin-4 in type 2 diabetes

A gut insulinotropic peptide, glucagon-like peptide-1 (GLP-1), when given continuously subcutaneously, has been shown to be an effective agent to treat type 2 diabetes. Because of inactivation by dipeptidyl peptidase IV (DPP IV), it has a very short half-life (90-120 s), hence the need for continuous administration. Exendin-4 is an agonist of the GLP-1 receptor. It is not a substrate for DPP IV, and we previously demonstrated that intravenous administration has potent insulinotropic properties in type 2 diabetic volunteers. We evaluated the efficacy of bolus subcutaneous exendin-4 in insulin-naive type 2 diabetic volunteers. Ten patients aged 44-72 yr with mean fasting glucose levels of 11.4 +/- 0.9 mmol/l were enrolled, and daily or twice-daily bolus subcutaneous exendin-4 was self-administered for 1 mo. Glycosylated hemoglobin, multiple daily capillary blood glucose, beta-cell sensitivity to glucose, and peripheral tissue sensitivity to insulin were compared before and after treatment. The greatest decline in capillary blood glucose was seen before bed, with a drop from 15.5 to 9.2 mmol/l (P < 0.0001). Glycosylated hemoglobin improved significantly with treatment, from 9.1 to 8.3% (P = 0.009). beta-Cell sensitivity to glucose was improved, as assessed by C-peptide levels during a hyperglycemic clamp. No significant adverse effects were noted or reported. Our data suggest that, even with this short duration of therapy, exendin-4 treatment had a significant effect on glucose homeostasis.     

The Drosophila SNR1 (SNF5/INI1) subunit directs essential developmental functions of the Brahma chromatin remodeling complex

The Drosophila melanogaster Brahma (Brm) complex, a counterpart of the Saccharomyces cerevisiae SWI/SNF ATP-dependent chromatin remodeling complex, is important for proper development by maintaining specific gene expression patterns. The SNR1 subunit is strongly conserved with yeast SNF5 and mammalian INI1 and is required for full activity of the Brm complex. We identified a temperature-sensitive allele of snr1 caused by a single amino acid substitution in the conserved repeat 2 region, implicated in a variety of protein-protein interactions. Genetic analyses of snr1(E1) reveal that it functions as an antimorph and that snr1 has critical roles in tissue patterning and growth control. Temperature shifts show that snr1 is continuously required, with essential functions in embryogenesis, pupal stages, and adults. Allele-specific genetic interactions between snr1(E1) and mutations in genes encoding other members of the Brm complex suggest that snr1(E1) mutant phenotypes result from reduced Brm complex function. Consistent with this view, SNR1(E1) is stably associated with other components of the Brm complex at the restrictive temperature. SNR1 can establish direct contacts through the conserved repeat 2 region with the SET domain of the homeotic regulator Trithorax (TRX), and SNR1(E1) is partially defective for functional TRX association. As truncating mutations of INI1 are strongly correlated with aggressive cancers, our results support the view that SNR1, and specifically the repeat 2 region, has a critical role in mediating cell growth control functions of the metazoan SWI/SNF complexes.     

Standardization of the physicochemical parameters to assess in vitro the beta-hematin inhibitory activity of antimalarial drugs

Intraerythrocytic plasmodia form hemozoin as a detoxification product of hemoglobin-derived heme. An identical substance, beta-hematin (BH), can be obtained in vitro from hematin at acidic pH. Quinoline-antimalarials inhibit BH formation. Standardization of test conditions is essential for studying the interaction of compounds with this process and screening potential inhibitors. A spectrophotometric microassay of heme polymerization inhibitory activity (HPIA) (Basilico et al., Journal of Antimicrobial Chemotherapy 42, 55-60, 1998) previously reported was used to investigate the effect of pH and salt concentration on BH formation. The yield of BH formation decreased with pH. Moreover, under conditions used in the above HPIA assay (18 h, 37 degrees C, pH = 2.7), several salts including chloride and phosphate inhibited the process. Aminoquinoline drugs formulated as salts (chloroquine-phosphate, primaquine-diphosphate), but not chloroquine-base, also inhibited the reaction. Interference by salts was highest at low pH and decreased at higher pH (pH 4). Here, we describe different assay conditions that eliminate these problems (BHIA, beta-hematin inhibitory activity). By replacing hematin with hemin as the porphyrin and NaOH solution with DMSO as solvent, the formation of BH was independent of pH up to pH 5.1. No interference by salts was observed over the pH range 2.7-5.1. Dose-dependent inhibition of BH formation was obtained with chloroquine-base, chloroquine-phosphate, and chloroquine-sulfate at pH 5.1. Primaquine was not inhibitory. The final product, characterized by solubility in DMSO, consists of pure BH by FT-IR spectroscopy. The BHIA assay (hemin in DMSO, acetate buffer pH 5 +/- 0.1, 18 h at 37 degrees C) is designed to screen for those molecules forming pi-pi interactions with hematin and thus inhibiting beta-hematin formation.     

Characterization of the active site of group B streptococcal hyaluronan lyase

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.     

Prostaglandin I2 analogs inhibit proinflammatory cytokine production and T cell stimulatory function of dendritic cells

Signaling through the PGI(2) receptor (IP) has been shown to inhibit inflammatory responses in mouse models of respiratory syncytial viral infection and OVA-induced allergic responses. However, little is known about the cell types that mediate the anti-inflammatory function of PGI(2.) In this study, we determined that PGI(2) analogs modulate dendritic cell (DC) cytokine production, maturation, and function. We report that PGI(2) analogs (iloprost, cicaprost, treprostinil) differentially modulate the response of murine bone marrow-derived DC (BMDC) to LPS in an IP-dependent manner. The PGI(2) analogs decreased BMDC production of proinflammatory cytokines (IL-12, TNF-alpha, IL-1alpha, IL-6) and chemokines (MIP-1alpha, MCP-1) and increased the production of the anti-inflammatory cytokine IL-10 by BMDCs. The modulatory effect was associated with IP-dependent up-regulation of intracellular cAMP and down-regulation of NF-kappaB activity. Iloprost and cicaprost also suppressed LPS-induced expression of CD86, CD40, and MHC class II molecules by BMDCs and inhibited the ability of BMDCs to stimulate Ag-specific CD4 T cell proliferation and production of IL-5 and IL-13. These findings suggest that PGI(2) signaling through the IP may exert anti-inflammatory effects by acting on DC.     

Structure shows that a glycosaminoglycan and protein recognition site in factor H is perturbed by age-related macular degeneration-linked single nucleotide polymorphism

A common single nucleotide polymorphism in the factor H gene predisposes to age-related macular degeneration. Factor H blocks the alternative pathway of complement on self-surfaces bearing specific polyanions, including the glycosaminoglycan chains of proteoglycans. Factor H also binds C-reactive protein, potentially contributing to noninflammatory apoptotic processes. The at risk sequence contains His (rather than Tyr) at position 402 (384 in the mature protein), in the seventh of the 20 complement control protein (CCP) modules (CCP7) of factor H. We expressed both His(402) and Tyr(402) variants of CCP7, CCP7,8, and CCP6-8. We determined structures of His(402) and Tyr(402) CCP7 and showed them to be nearly identical. The side chains of His/Tyr(402) have similar, solvent-exposed orientations far from interfaces with CCP6 and -8. Tyr(402) CCP7 bound significantly more tightly than His(402) CCP7 to a heparin affinity column as well as to defined-length sulfated heparin oligosaccharides employed in gel mobility shift assays. This observation is consistent with the position of the 402 side chain on the edge of one of two glycosaminoglycan-binding surface patches on CCP7 that we inferred on the basis of chemical shift perturbation studies with a sulfated heparin tetrasaccharide. According to surface plasmon resonance measurements, Tyr(402) CCP6-8 binds significantly more tightly than His(402) CCP6-8 to immobilized C-reactive protein. The data support a causal link between H402Y and age-related macular degeneration in which variation at position 402 modulates the response of factor H to age-related changes in the glycosaminoglycan composition and apoptotic activity of the macula.     

Monitoring Texas Wood Ducks With a Cooperative Nest-Box Program

ABSTRACT We analyzed 8 years of data from the Texas Cooperative Nest Box Program initiated in 1988 by Texas Parks and Wildlife, USA, as a means of involving private cooperators in statewide wood duck (Aix sponsa) management. Cooperators operated ≤9 boxes and most reported nest-box data for only 1 year (56.5%) and 2 years (20.6%) over the 8-year life of the project. Mean nest-box use differed among ecological regions of the state (F=4.23, df=6, P= 0.001) but did not exceed 30% in any region. Mean nest-box success ranged between 74–91% across ecological regions during 1988–1995, but annual estimates of nest-box success lacked precision (CVs >30%) in most years for all regions. Our project was unsuccessful as a management tool for monitoring wood duck demographics. Future efforts should focus on improving the amount and quality of data collected from box-nesting wood ducks.     

A Hierarchical Analysis of Habitat Selection by Raccoons in Northern Indiana

Abstract: Although numerous studies have examined habitat use by raccoons (Procyon lotor), information regarding seasonal habitat selection related to resource availability in agricultural landscapes is lacking for this species. Additionally, few studies using radiotelemetry have investigated habitat selection at multiple spatial scales or core-use areas by raccoons. We examined seasonal habitat selection of 55 (31 M, 24 F) adult raccoons at 3 hierarchical orders defined by the movement behavior of this species (second-order home range, second-order core-use area, and third-order home range) in northern Indiana, USA, from May 2003 to June 2005. Using compositional analysis, we assessed whether habitat selection differed from random and ranked habitat types in order of selection during the crop growing period (season 1) and corn maturation period (season 2), which represented substantial shifts in resource availability to raccoons. Habitat rankings differed across hierarchical orders, between seasons within hierarchical orders, and between sexes within seasons; however, seasonal and intersexual patterns of habitat selection were not consistent across hierarchical orders of spatial scale. When nonrandom utilization was detected, both sexes consistently selected forest cover over other available habitats. Seasonal differences in habitat selection were most evident at the core-area scale, where raccoon selection of agricultural lands was highest during the maturation season when corn was available as a direct food source. Habitat use did not differ from availability for either sex in either season at the third-order scale. The selection of forest cover across both seasons and all spatial orders suggested that raccoon distribution and abundance in fragmented landscapes is likely dependent on the availability and distribution of forest cover, or habitats associated with forest (i.e., water), within the landscape. The lack of consistency in habitat selection across hierarchical scales further exemplifies the need to examine multiple biological scales in habitat-selection studies.