Tissue preservation biases in stable isotopes of fishes and molluscs from Patagonian lakes

Field work commonly involves preserving samples for later use; however, most preservation methods distort stable‐isotope (SI) signatures that are of interest to ecologists. Although preservation of muscle samples with table salt and rubbing alcohol affected the SI (δ¹³C and δ¹⁵N) of important consumers in Patagonian lakes (molluscs and fishes), variation among individuals and lakes generally exceeded that among preservation treatments. Mathematical corrections for these preservation biases are provided, and a potentially bias‐free preservation by air‐drying is suggested.     

System-wide studies of N-lysine acetylation in Rhodopseudomonas palustris reveal substrate specificity of protein acetyltransferases

N-lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.     

Identification of small molecule proliferating cell nuclear antigen (PCNA) inhibitor that disrupts interactions with PIP-box proteins and inhibits DNA replication

We have discovered that 3,3′,5-triiodothyronine (T3) inhibits binding of a PIP-box sequence peptide to proliferating cell nuclear antigen (PCNA) protein by competing for the same binding site, as evidenced by the co-crystal structure of the PCNA-T3 complex at 2.1 Å resolution. Based on this observation, we have designed a novel, non-peptide small molecule PCNA inhibitor, T2 amino alcohol (T2AA), a T3 derivative that lacks thyroid hormone activity. T2AA inhibited interaction of PCNA/PIP-box peptide with an IC₅₀ of ∼1 μm and also PCNA and full-length p21 protein, the tightest PCNA ligand protein known to date. T2AA abolished interaction of PCNA and DNA polymerase δ in cellular chromatin. De novo DNA synthesis was inhibited by T2AA, and the cells were arrested in S-phase. T2AA inhibited growth of cancer cells with induction of early apoptosis. Concurrently, Chk1 and RPA32 in the chromatin are phosphorylated, suggesting that T2AA causes DNA replication stress by stalling DNA replication forks. T2AA significantly inhibited translesion DNA synthesis on a cisplatin-cross-linked template in cells. When cells were treated with a combination of cisplatin and T2AA, a significant increase in phospho(Ser¹³⁹)histone H2AX induction and cell growth inhibition was observed.     

Stapling mimics noncovalent interactions of γ-carboxyglutamates in conantokins, peptidic antagonists of N-methyl-D-aspartic acid receptors

Conantokins are short peptides derived from the venoms of marine cone snails that act as antagonists of the N-methyl-D-aspartate (NMDA) receptor family of excitatory glutamate receptors. These peptides contain γ-carboxyglutamic acid residues typically spaced at i,i+4 and/or i,i+7 intervals, which by chelating divalent cations induce and stabilize helical conformation of the peptide. Introduction of a dicarba bridge (or a staple) can covalently stabilize peptide helicity and improve its pharmacological properties. To test the hypothesis that stapling can effectively replace γ-carboxyglutamic acid residues in stabilizing the helical conformation of conantokins, we designed, synthesized, and characterized several stapled analogs of conantokin G (conG), with varying connectivities in terms of staple length and location along the face of the α-helix. NMR studies confirmed that the ring-closing metathesis reaction yielded a single product with the Z configuration of the olefinic bond. Based on circular dichroism and molecular modeling, the stapled analogs exhibited significantly enhanced helicity compared with the native peptide in a metal-free environment. Stapling i,i+4 was benign with respect to effects on in vitro and in vivo pharmacological properties. One analog, namely conG[11-15,S(i,i+4)S(8)], blocked NR2B-containing NMDA receptors with IC(50) = 0.7 μm and provided significant protection in the 6-Hz psychomotor model of pharmacoresistant epilepsy in mice. Remarkably, unlike native conG, conG[11-15,S(i,i+4)S(8)] produced no behavioral motor toxicity. Our results extend the applications of peptide stapling to helical peptides with extracellular targets and provide a means for engineering conantokins with improved pharmacological properties.     

Sometimes it takes two to tango: contributions of dimerization to functions of human α-defensin HNP1 peptide

Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association. To gain additional insights into the functional significance of dimerization, we compared wild type HNP1 to dimerization-impaired, N-methylated HNP1 monomers and to disulfide-tethered obligate HNP1 dimers. The structural effects of these modifications were confirmed by x-ray crystallographic analyses. Like the previously studied W26A mutation, N-methylation of Ile-20 dramatically reduced the ability of HNP1 to kill Staphylococcus aureus, inhibit LF, and bind gp120. Importantly, this modification had minimal effect on the ability of HNP1 to kill Escherichia coli. The W26A and MeIle-20 mutations impaired defensin activity synergistically. N-terminal covalent tethering rescued the ability of W26A-HNP1 to inhibit LF but failed to restore its defective killing of S. aureus. Surface plasmon resonance studies revealed that Trp-26 mediated the association of monomers and canonical dimers of HNP1 to immobilized HNP1, LF, and gp120, and also indicated a possible mode of tetramerization of HNP1 mediated by Ile-20 and Leu-25. This study demonstrates that dimerization contributes to some but not all of the many and varied activities of HNP1.     

A Small Novel A-Kinase Anchoring Protein (AKAP) That Localizes Specifically Protein Kinase A-Regulatory Subunit I (PKA-RI) to the Plasma Membrane

Protein kinase A-anchoring proteins (AKAPs) provide spatio-temporal specificity for the omnipotent cAMP-dependent protein kinase (PKA) via high affinity interactions with PKA regulatory subunits (PKA-RI, RII). Many PKA-RII-AKAP complexes are heavily tethered to cellular substructures, whereas PKA-RI-AKAP complexes have remained largely undiscovered. Here, using a cAMP affinity-based chemical proteomics strategy in human heart and platelets, we uncovered a novel, ubiquitously expressed AKAP, termed small membrane (sm)AKAP due to its specific localization at the plasma membrane via potential myristoylation/palmitoylation anchors. In vitro binding studies revealed specificity of smAKAP for PKA-RI (K_d = 7 nm) over PKA-RII (K_d = 53 nm) subunits, co-expression of smAKAP with the four PKA R subunits revealed an even more exclusive specificity of smAKAP for PKA-RIα/β in the cellular context. Applying the singlet oxygen-generating electron microscopy probe miniSOG indicated that smAKAP is tethered to the plasma membrane and is particularly dense at cell-cell junctions and within filopodia. Our preliminary functional characterization of smAKAP provides evidence that, like PKA-RII, PKA-RI can be tightly tethered by a novel repertoire of AKAPs, providing a new perspective on spatio-temporal control of cAMP signaling.     

Divide to conquer: a complex pattern of biodiversity depicted by vertebrate components in the Brazilian Atlantic Forest

The identification of northern and southern components in different vertebrate species led researchers to accept a two‐component hypothesis for the Brazilian Atlantic forest (BAF). Nevertheless, neither a formal proposal nor a meta‐analysis to confirm this coincidence was ever made. Our main objective here was therefore to systematically test in how many vertebrate components the BAF could be divided by analysing existing empirical data. We used two approaches: (1) mapping and comparing the proposed areas of vertebrate endemism in the BAF and (2) analysing studies mentioning spatial subdivisions in distinct forest‐dependent vertebrates within the biome, by the use of panbiogeography. The four large‐scale endemism area components together with the six small‐scale panbiogeographical ones allowed the definition of three BAF greater regions, subdivided into nine vertebrate components, latitudinally and longitudinally organized. Empirical time estimates of the diversification events within the BAF were also reviewed. Diversification of these vertebrates occurred not only in the Pleistocene but also throughout the Miocene. Our results confirm the BAF's complex history, both in space and time. We propose that future research should be small‐scale and focused in the vertebrate components identified herein. Given the BAF's heterogeneity, studying via sections will be much more useful in identifying the BAF's historical biogeography. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, ??, ??–??.     

EVALUATION OF MICRODIETS VERSUS FROZEN COPEPODSON GROWTH, SURVIVAL AND SELECTED CHEMICALCOMPOSITION OF LARGE YELLOW CROAKERPSEUDOSCIAENA CROCEA LARVAE

Three newly developed microdiets (MDs: Diet 1, Diet 2 and Diet 3), one co–feeding diet (Diet 4, combinedDiet 3 with frozen copepods) and frozen copepods (Diet 5) were tested for weaning large yellow croaker (Pseudosciaenacrocea) larvae from 25 to 60 days after hatching (DAH). The highest final specific growth rate (SGR, 7.97%/d) and sur-vival (40.0%) were observed in fish fed Diet 1, while those were the lowest in fish fed Diet 5 (4.15%/d and 20.4%, re-spectively). The survival of larvae fed Diet 4 showed no significant difference (P>0.05) from that of larvae fed Diet 1.However, SGR of larvae fed Diet 4 was significantly lower (P<0.05) than that of larvae fed Diet 1. Among the MDs,Diet 1 was proved to be superior to Diet 2 and Diet 3 for both SGR and survival. For all treatments, biomass in each tankwas observed a similar trend as SGR. Whole-body protein was not significantly different (P>0.05) among fish fed theMDs, which was lower than that of fish fed Diet 4, but higher than that of fish fed Diet 5. Fish fed the MDs had signifi-cantly higher (P<0.05) lipid deposition (10.6%—13.3%) than fish fed either Diet 4 (7.6%) or Diet 5 (7.2%). DietaryC20:4n-6 (AA) level was significantly lower (P<0.05) in Diet 5 than that in the MDs. However, the AA level was sig-nificantly higher (P<0.05) in fish fed either Diet 4 or Diet 5 than that in fish fed the MDs. The exceeded C22:6n-3 (DHA)levels in fish compared to their corresponding diets were found in those fed either Diet 4 or Diet 5. Inversely, C20:5n-3(EPA) and DHA levels decreased in fish fed the MDs relative to their proportions in MDs. These results showed that fishfed either Diet 4 or Diet 5 had higher efficiency of AA, DHA and EPA deposition than fish fed the MDs. This study sug-gested that large yellow croaker larvae could be successfully weaned by MD. Future research should be based on for-mula of Diet 1, since it was superior to Diet 2 and Diet 3. In addition, changes of digestive enzymes and digestive tractof large yellow croaker larvae fed different diets need to be investigated for an overall evaluation.     

Identification of an active dimeric intermediate populated during the unfolding process of the cambialistic superoxide dismutase from Streptococcus mutans

Superoxide dismutases are enzymes that protect biological systems against oxidative damage caused by superoxide radicals. In this paper, a detailed characterization is presented on the stability of SmSOD, the dimeric cambialistic superoxide dismutase from the dental pathogenic microorganism Streptococcus mutans, towards temperature and guanidine hydrochloride. Thermal and chemical denaturations were investigated by means of circular dichroism, fourth-derivative UV spectroscopy and fluorescence measurements. Data indicate that SmSOD is endowed with a significant thermostability and that both its thermal and guanidine hydrochloride-induced unfolding processes occur through a three-state model, characterized by a catalytically active dimeric intermediate species. To our knowledge, SmSOD is the smallest known dimeric protein that populates a well-structured active dimeric rather than a monomeric intermediate during unfolding processes.