D-amino acid residue in a defensin-like peptide from platypus venom: effect on structure and chromatographic properties

The recent discovery that the natriuretic peptide OvCNPb (Ornithorhynchus venom C-type natriuretic peptide B) from platypus (Ornithorynchus anatinus) venom contains a D-amino acid residue suggested that other D-amino-acid-containing peptides might be present in the venom. In the present study, we show that DLP-2 (defensin-like peptide-2), a 42-amino-acid residue polypeptide in the platypus venom, also contains a D-amino acid residue, D-methionine, at position 2, while DLP-4, which has an identical amino acid sequence, has all amino acids in the L-form. These findings were supported further by the detection of isomerase activity in the platypus gland venom extract that converts DLP-4 into DLP-2. In the light of this new information, the tertiary structure of DLP-2 was recalculated using a new structural template with D-Met(2). The structure of DLP-4 was also determined in order to evaluate the effect of a D-amino acid at position 2 on the structure and possibly to explain the large retention time difference observed for the two molecules in reverse-phase HPLC. The solution structures of the DLP-2 and DLP-4 are very similar to each other and to the earlier reported structure of DLP-2, which assumed that all amino acids were in the L-form. Our results suggest that the incorporation of the D-amino acid at position 2 has minimal effect on the overall fold in solution.

Cardiotrophin-like cytokine/cytokine-like factor 1 is an essential trophic factor for lumbar and facial motoneurons in vivo

The ciliary neurotrophic factor alpha-receptor(CNTFRalpha) is required for motoneuron survival during development, but the relevant ligand(s) has not been determined. One candidate is the heterodimer formed by cardiotrophin-like cytokine (CLC) and cytokine-like factor 1 (CLF). CLC/CLF binds to CNTFRalpha and enhances the survival of developing motoneurons in vitro; whether this novel trophic factor plays a role in neural development in vivo has not been tested. We examined motor and sensory neurons in embryonic chicks treated with CLC and in mice with a targeted deletion of the clf gene. Treatment with CLC increased the number of lumbar spinal cord motoneurons that survived the cell death period in chicks. However, this effect was regionally specific, because brachial and thoracic motoneurons were unaffected. Similarly, newborn clf -/- mice exhibited a significant reduction in lumbar motoneurons, with no change in the brachial or thoracic cord. Clf deletion also affected brainstem motor nuclei in a regionally specific manner; the number of motoneurons in the facial but not hypoglossal nucleus was significantly reduced. Sensory neurons of the dorsal root ganglia were not affected by either CLC treatment or clf gene deletion. Finally, mRNA for both clc and clf was found in skeletal muscle fibers of embryonic mice during the motoneuron cell death period. These findings support the view that CLC/CLF is a target-derived factor required for the survival of specific pools of motoneurons. The in vivo actions of CLC and CLF can account for many of the effects of CNTFRalpha on developing motoneurons.

Suppression and overexpression of adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) influences zebrafish embryo development A - Possible role for AHCYL1 in inositol phospholipid signaling

Adenosylhomocysteine hydrolase-like protein 1 (AHCYL1) is a novel intracellular protein with similar to 50% protein identity to adenosyl homocysteine hydrolase (AHCY), an important enzyme for metabolizing S-adenosyl-L-homocysteine, the by-product of S-adenosyl-L-homomethionine-dependent methylation. AHCYL1 binds to the inositol 1,4,5-trisphosphate receptor, suggesting that AHCYL1 is involved in intracellular calcium release. We identified two zebrafish AHCYL1 orthologs(zAHCYL1A and -B) by bioinformatics and reverse transcription-PCR. Unlike the ubiquitously present AHCY genes, AHCYL1 genes were only detected in segmented animals, and AHCYL1 proteins were highly conserved among species. Phylogenic analysis suggested that the AHCYL1 gene diverged early from AHCY and evolved independently. Quantitative reverse transcription-PCR showed that zAHCYL1A and -B mRNA expression was regulated differently from the other AHCY-like protein zAHCYL2 and zAHCY during zebrafish embryogenesis. Injection of morpholino antisense oligonucleotides against zAHCYL1A and -B into zebrafish embryos inhibited zAHCYL1A and -B mRNA translation specifically and induced ventralized morphologies. Conversely, human and zebrafish AHCYL1A mRNA injection into zebrafish embryos induced dorsalized morphologies that were similar to those obtained by depleting intracellular calcium with thapsigargin. Human AHCY mRNA injection showed little effect on the embryos. These data suggest that AHCYL1 has a different function from AHCY and plays an important role in embryogenesis by modulating inositol 1,4,5-trisphosphate receptor function for the intracellular calcium release.

Solution structure and characterization of the LGR8 receptor binding surface of insulin-like peptide 3

Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development. Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein- coupled receptor family. To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/ insulin-like fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include Arg(B16) and Val(B19), with His(B12) and Arg(B20) playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these residues. Together, these amino acids combine with the previously identified critical residue, Trp(B27), to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists and antagonists of this receptor.

Impaired memory and olfactory performance in NaSi-1 sulphate transporter deficient mice

In the present study, NaSi-l sulphate transporter knock-out (Nas1-/-) mice, an animal model of hyposulphataernia, were examined for spatial memory and learning in a Morris water maze, and for olfactory function in a cookie test. The Nas1-/- mice displayed significantly (P < 0.05) increased latencies to find an escape platform in the reversal teaming trials at 2 days but not 1 day after the last acquisition trial in a Morris water maze test. suggesting that Nas1-/- mice may have proactive memory interference. While the wild-type (Ncis1+/+) mice showed a significant (P < 0.02) decrease in time to locate a hidden food reward over four trials after overnight fasting, Nas1-/- mice did not change their performance, resulting in significantly (P < 0.05) higher latencies when compared to their Nas1+/+ littermates. There were no significant differences between Nas1-/- and Nas1+/+ mice in the cookie test after moderate food deprivation. In addition, both Nas1-/- and Nas1+/+ mice displayed similar escape latencies in the acquisition phase of the Morris water maze test, suggesting that learning, motivation, vision and motor skills required for the task may not be affected in Nas1-/- mice. This is the first study to demonstrate an impairment in memory and olfactory performance in the hyposulphataemic Nas1-/- mouse. (c) 2004 Elsevier B.V. All rights reserved.

Isolation and characterization of integron-containing bacteria without antibiotic selection

The emergence of antibiotic resistance among pathogenic and commensal bacteria has become a serious problem worldwide. The use and overuse of antibiotics in a number of settings are contributing to the development of antibiotic-resistant microorganisms. The class 1 and 2 integrase genes (intI1 and intI2, respectively) were identified in mixed bacterial cultures enriched from bovine feces by growth in buffered peptone water (BPW) followed by integrase-specific PCR. Integrase-positive bacterial colonies from the enrichment cultures were then isolated by using hydrophobic grid membrane filters and integrase-specific gene probes. Bacterial clones isolated by this technique were then confirmed to carry integrons by further testing by PCR and DNA sequencing. Integron-associated antibiotic resistance genes were detected in bacteria such as Escherichia coli, Aeromonas spp., Proteus spp., Morganella morganii, Shewanella spp., and urea-positive Providencia stuartii isolates from bovine fecal samples without the use of selective enrichment media containing antibiotics. Streptomycin and trimethoprim resistance were commonly associated with integrons. The advantages conferred by this methodology are that a wide variety of integron-containing bacteria may be simultaneously cultured in BPW enrichments and culture biases due to antibiotic selection can be avoided. Rapid and efficient identification, isolation, and characterization of antibiotic resistance-associated integrons are possible by this protocol. These methods will facilitate greater understanding of the factors that contribute to the presence and transfer of integron-associated antibiotic resistance genes in bacterial isolates from red meat production animals.

Comparative agonist/antagonist responses in mutant human C5a receptors define the ligand binding site

The C terminus is responsible for all of the agonist activity of C5a at human C5a receptors (C5aRs). In this report we have mapped the ligand binding site on the C5aR using a series of agonist and antagonist peptide mimics of the C terminus of C5a as well as receptors mutated at putative interaction sites ( Ile(116), Arg(175), Arg(206), Glu(199), Asp(282), and Val(286)). Agonist peptide 1 (Phe-Lys-Pro-D-cyclohexylalanine-cyclohexylalanine-D-Arg) can be converted to an antagonist by substituting the bulkier Trp for cyclohexylalanine at position 5 ( peptide 2). Conversely, mutation of C5aR transmembrane residue Ile(116) to the smaller Ala (I116A) makes the receptor respond to peptide 2 as an agonist (Gerber, B. O., Meng, E. C., Dotsch, V., Baranski, T. J., and Bourne, H. R. (2001) J. Biol. Chem. 276, 3394 - 3400). However, a potent cyclic hexapeptide antagonist, Phe-cyclo-[Orn-Pro-D-cyclohexylalanine-Trp-Arg] ( peptide 3), derived from peptide 2 and which binds to the same receptor site, remains a full antagonist at I116AC5aR. This suggests that although the residue at position 5 might bind near to Ile(116), the latter is not essential for either activation or antagonism. Arg(206) and Arg(175) both appear to interact with the C-terminal carboxylate of C5a agonist peptides, suggesting a dynamic binding mechanism that may be a part of a receptor activation switch. Asp(282) has been previously shown to interact with the side chain of the C-terminal Arg residue, and Glu(199) may also interact with this side chain in both C5a and peptide mimics. Using these interactions to orient NMR-derived ligand structures in the binding site of C5aR, a new model of the interaction between peptide antagonists and the C5aR is presented.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single similar to 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')(2) also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to Fc gamma R. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Mammalian L-to-D-amino-acid-residue isomerase from platypus venom

The presence Of D-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian D-amino-acid-residue isomerase(s) responsible for the modification of the all-L-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is similar to 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known L-to-D-amino-acid-residue isomerase in a mammal. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Mini-dose glucagon rescue for mild hypoglycaemia in children with type 1 diabetes: The Brisbane experience

Objectives: To evaluate the use of small doses of glucagon using an insulin syringe in mild or impending hypoglycaemia in children with type 1 diabetes. Methods: Data were collected from patients attending the Paediatric Diabetes Clinic at the Queensland Diabetes Centre at the Mater Hospital, Brisbane in 2002-2004 following the institution of a new protocol for home management of mild or impending hypoglycaemia associated with inability or refusal to take oral carbohydrate. The protocol recommended the use of subcutaneous injections of glucagon using insulin syringes at a dose of two ' units ' (20 mu g) in children 2 years of age or younger, and for older children one unit per year of age up to a maximum of 15 units (150 mu g), with an additional doubled dose given if the blood glucose had not increased in 20 min. Results: Over a 2-year period, 25 children were treated with mini-dose glucagon on a total of 38 occasions. Additional doses were required for recurring hypoglycaemia on 20 (53%) occasions. The child could be managed at home on 32 (84%) of these 38 occasions, with only 6 (16%) children needing hospital treatment. Conclusions: Our study confirmed that small doses of glucagon given subcutaneously with an insulin syringe is a simple, practical and effective home treatment of mild or impending hypoglycaemia due to gastroenteritis or food refusal in children with type 1 diabetes.