Chemically defined medium and <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Background  

C. elegans has been established as a powerful genetic system. Use of a chemically defined medium (C. elegans Maintenance Medium (CeMM)) now allows standardization and systematic manipulation of the nutrients that animals receive. Liquid cultivation allows automated culturing and experimentation and should be of use in large-scale growth and screening of animals.     

Effects of electron-beam irradiation on buccal-cell DNA

Buccal cells were collected from 29 participants, by use of mouthwash rinses, and were split into equal aliquots, with one aliquot irradiated by electron-beam (E-beam) irradiation equivalent to the sterilizing dosage used by the U.S. Postal Service and the other left untreated. Aliquots were extracted and tested for DNA yields (e.g., TaqMan assay for quantifying human genomic DNA), genomic integrity, and amplification-based analysis of genetic variants (e.g., single-nucleotide polymorphisms [SNPs] and single tandem repeats [STRs]). Irradiated aliquots had lower median DNA yields (3.7 microg/aliquot) than untreated aliquots (7.6 microg/aliquot) (P<.0005) and were more likely to have smaller maximum DNA fragment size, on the basis of genomic integrity gels, than untreated aliquots (P<.0005). Irradiated aliquots showed poorer PCR amplification of a 989-bp beta-globin target (97% for weak amplification and 3% for no amplification) than untreated aliquots (7% for weak amplification and 0% for no amplification) (P<.0005), but 536-bp and 268-bp beta-globin targets were amplified from all aliquots. There was no detectable irradiation effect on SNP assays, but there was a significant trend for decreased detection of longer STRs (P=.01) in irradiated versus untreated aliquots. We conclude that E-beam irradiation reduced the yield and quality of buccal-cell specimens, and, although irradiated buccal-cell specimens may retain sufficient DNA integrity for some amplified analyses of many common genomic targets, assays that target longer DNA fragments (>989 bp) or require whole-genome amplification may be compromised.     

Site-specific antibodies against hydrophilic domains of subunit III of bovine heart cytochrome c oxidase affect enzyme function

Antibodies were raised against conserved amino acid sequences in four extramembranous portions of subunit III (sIII) from beef cytochrome c oxidase (COX) and the role of these domains in the functional activities of the enzyme was investigated. The binding of one antipeptide antibody corresponding to an externally exposed (facing the intermembrane space) domain of COX sIII (amino acids 180-189 in the primary sequence) exhibited a 30-50% stimulation of electron transfer activity in both detergent-dispersed COX and COX incorporated into phospholipid vesicles (COV). Antibody binding to two different matrix-faced domains (amino acids 57-66 and 148-159 in the sequence) resulted in small stimulations (10-25%) of COX electron transfer activity. The remaining antipeptide antibody (against amino acids 119-128) had no effect on electron transfer activity of COX in detergent solution, but exhibited a slight inhibition of activity (15%) in COV. The mechanism of antibody-induced stimulation of COX electron transfer activity was determined to be an increase in the maximum velocity of the enzyme and not due to a change in the apparent K(m) of cytochrome c interaction with COX as determined by steady state kinetic assays. Antibody binding to COX in COV increased the respiratory control ratio (an indicator of endogenous proton permeability) of COV, but had no effect on the vectorial proton pumping activity of COV. These results suggest that these conserved, hydrophilic domains of COX sIII are conformationally linked to the electron transfer function of the enzyme in subunits I and II and that sIII may serve as a regulatory subunit for COX electron transfer and proton pumping activities.     

Redox-dependent structural changes in archaeal and bacterial Rieske-type [2Fe-2S] clusters

Proteins containing Rieske-type [2Fe-2S] clusters play important roles in many biological electron transfer reactions. Typically, [2Fe-2S] clusters are not directly involved in the catalytic transformation of substrate, but rather supply electrons to the active site. We report herein X-ray absorption spectroscopic (XAS) data that directly demonstrate an average increase in the iron-histidine bond length of at least 0.1 A upon reduction of two distantly related Rieske-type clusters in archaeal Rieske ferredoxin from Sulfolobus solfataricus strain P-1 and bacterial anthranilate dioxygenases from Acinetobacter sp. strain ADP1. This localized redox-dependent structural change may fine tune the protein-protein interaction (in the case of ARF) or the interdomain interaction (in AntDO) to facilitate rapid electron transfer between a lower potential Rieske-type cluster and its redox partners, thereby regulating overall oxygenase reactions in the cells.     

Somatostatin receptor type 5 modulates somatostatin receptor type 2 regulation of adrenocorticotropin secretion

Somatostatin inhibits adrenocorticotropin (ACTH) secretion from pituitary tumor cells. To assess the contribution of somatostatin receptor subtype 5 (SST5) to somatostatin receptor subtype 2 (SST2) action in these cells, we assessed multipathway responses to novel highly monoreceptor-selective peptide agonists and multireceptor agonists, including octreotide and somatostatin-28. Octreotide and somatostatin-28 cell membrane binding affinities correlated with their respective SST2-selective peptide ligand. Although octreotide had similar inhibiting potency (picomolar) for cAMP accumulation and ACTH secretion as an SST2-selective agonist, somatostatin-28 exhibited a higher potency (femtomolar). Baseline spontaneous calcium oscillations assessed by fluorescent confocal microscopy revealed two distinct effects: SST2 activation reduced oscillations at femtomolar concentrations reflected by high inhibiting potency of averaged normalized oscillation amplitude, whereas SST5 activation induces brief oscillation pauses and increased oscillation amplitude. Octreotide exhibits an integrated effect of both receptors; however, somatostatin-28 exhibited a complex response with two separate inhibitory potencies. SST2 internalization was visualized with SST2-selective agonist at lower concentrations than for octreotide or somatostatin-28, whereas SST5 did not internalize. Using monoreceptor-selective peptide agonists, the results indicate that, in AtT-20 cells, SST5 regulates the dominant SST2 action, attenuating SST2 effects on intracellular calcium oscillation and internalization. This may explain superior somatostatin-28 potency and provides a rationale for somatostatin ligand design to treat ACTH-secreting pituitary tumors.     

Structures of APRIL-receptor complexes: like BCMA, TACI employs only a single cysteine-rich domain for high affinity ligand binding

TACI is a member of the tumor necrosis factor receptor superfamily and serves as a key regulator of B cell function. TACI binds two ligands, APRIL and BAFF, with high affinity and contains two cysteine-rich domains (CRDs) in its extracellular region; in contrast, BCMA and BR3, the other known high affinity receptors for APRIL and BAFF, respectively, contain only a single or partial CRD. However, another form of TACI exists wherein the N-terminal CRD is removed by alternative splicing. We find that this shorter form is capable of ligand-induced cell signaling and that the second CRD alone (TACI_d2) contains full affinity for both ligands. Furthermore, we report the solution structure and alanine-scanning mutagenesis of TACI_d2 along with co-crystal structures of APRIL.TACI_d2 and APRIL.BCMA complexes that together reveal the mechanism by which TACI engages high affinity ligand binding through a single CRD, and we highlight sources of ligand-receptor specificity within the APRIL/BAFF system.     

Expression and regulation of lipocalin-type prostaglandin d synthase in rat testis and epididymis

Lipocalin-type prostaglandin D synthase (L-PGDS), a bifunctional protein, is expressed in the male reproductive organs of many species. However, the expression and regulation of L-PGDS in rat are still uncertain. The present study investigated the regionalization and regulation of L-PGDS expression in rat testis and epididymis by in situ hybridization and immunohistochemistry under the conditions of sexual maturation, castration, and ethylene dimethane sulfonate (EDS) treatments. In sexually mature rats, L-PGDS mRNA was weakly expressed only in the testicular peritubular cells, whereas L-PGDS immunostaining was highly detected in the Leydig cells by Day 70 postpartum. During sexual maturation, L-PGDS mRNA expression was highly detected in the caput, corpus, and cauda of the epididymis 70 days after birth. Compared with normal L-PGDS expression in adult epididymis, both L-PGDS mRNA expression and protein immunostaining were significantly reduced in the caput, corpus, and cauda epididymis after castration. Testosterone propionate treatment induced a significant increase of L-PGDS expression in the epididymis of castrated rats. Compared with adult rat epididymis, L-PGDS mRNA and protein expression was down-regulated after EDS treatment. Testosterone propionate treatment could induce an increase of L-PGDS mRNA and protein expression in the epididymis of EDS-treated rats. In conclusion, both castration and EDS treatments caused a significant decrease of L-PGDS expression in the epididymis, whereas testosterone propionate treatment could induce an increase of L-PGDS expression in the epididymis of both castrated and EDS-treated rats, indicating that L-PGDS expression in the rat epididymis can be up-regulated by testosterone.     

Twelve years of contemporary armor evolution in a threespine stickleback population

Abstract —Loberg Lake, Alaska was colonized by sea-run Gasterosteus aculeatus between 1983 and 1988, after the original stickleback population was exterminated. Annual samples from 1990 to 2001 reveal substantial evolution of lateral plate (armor) phenotypes. The 1990 sample was nearly monomorphic for the complete plate morph, which is monomorphic in local sea-run populations; the low plate morph, which is usually monomorphic in local freshwater populations, was absent. By 2001, the frequency of completes had declined to 11%, and lows had increased to 75%. The partial plate morph and two unusual intermediate plate phenotypes were generally rare, but occurrence of the intermediates was unexpected. These intermediate phenotypes rarely occur in other, presumably older, polymorphic populations. When low morphs first appeared, they averaged 6.8 plates per side, indicating that the ancestral plate number of low morphs is high, and their mean has subsequently declined. Contemporary evolution in this population indicates that threespine stickleback adapt to freshwater habitats within decades after invasion from the ocean, and thus phenotypes in most populations are adapted to current conditions.     

Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues

Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.     

Engineering an APRIL-specific B cell maturation antigen

B cell maturation antigen (BCMA) is a tumor necrosis factor receptor family member whose physiological role remains unclear. BCMA has been implicated as a receptor for both a proliferation-inducing ligand (APRIL) and B cell-activating factor (BAFF), tumor necrosis factor ligands that bind to multiple tumor necrosis factor receptor and have been reported to play a role in autoimmune disease and cancer. The results presented herein provide a dual perspective analysis of BCMA binding to both APRIL and BAFF. First, we characterized the binding affinity of monomeric BCMA for its ligands; BAFF binding affinity (IC50 = 8 +/- 5 microm) is about 1000-fold reduced compared with the high affinity interaction of APRIL (IC50 = 11 +/- 3 nm). Second, shotgun alanine scanning of BCMA was used to map critical residues for either APRIL or BAFF binding. In addition to a previously described "DXL" motif (Gordon, N. C., Pan, B., Hymowitz, S. G., Yin, J., Kelley, R. F., Cochran, A. G., Yan, M., Dixit, V. M., Fairbrother, W. J., and Starovasnik, M. A. (2003) Biochemistry 42, 5977-5983), the alanine scanning results predicted four amino acid positions in BCMA (Tyr13, Ile22, Gln25, and Arg27) that could impart ligand specificity. Substitution of Tyr13 was tolerated for BAFF binding but not APRIL binding. Arg27 was required for high affinity binding to APRIL, whereas substitutions of this residue had minimal effect on affinity for BAFF. Further phage display experiments suggested the single mutations of I22K, Q25D, and R27Y as providing the greatest difference in APRIL versus BAFF binding affinity. Incorporation of the Q25D and R27Y substitutions into BCMA produced a dual specificity variant, since it has comparable binding affinity for both APRIL and BAFF, IC50 = 350 and 700 nm, respectively. Binding of the I22K mutant of monomeric BCMA to BAFF was undetectable (IC50 > 100 microm), but affinity for binding to APRIL was similar to wild-type BCMA. Based on these results, a BCMA-Fc fusion with the single I22K mutation was produced that binds APRIL, IC50 = 12 nm, and has no measurable affinity for BAFF. These results suggest that APRIL is the preferred ligand for BCMA and show that specificity can be further modified through amino acid substitutions.