Invited overview: conclusions from a review of electrofishing and its harmful effects on fish

Reviews in Fish Biology and Fisheries - These conclusions are extracted from a published review and synthesis of literature on electrofishing and its harmful effects on fish. Although a valuable...     

Hydrologic sources of carbon cycling uncertainty throughout the terrestrial–aquatic continuum

Linking hydrologic interactions with global carbon cycling will reduce the uncertainty associated with scaling-up empirical studies and facilitate the incorporation of terrestrial–aquatic linkages within global and regional change models. Much of the uncertainty in estimates of carbon fluxes associated with precipitation and hydrologic transport results from the extensive spatial and temporal heterogeneity in both intrinsic functioning and anthropogenic modification of hydrological cycles. To better understand this variation we developed a landscape ecological approach to coupled hydrologic–carbon cycling that merges local mechanisms with multiple-scale spatial heterogeneity. This spatially explicit framework is applied to examine variability in hydrologic influences on carbon cycling along a continental scale water availability gradient with an explicit consideration of human sources of variability. Hydrologic variation is an important component of the uncertainty in carbon cycling; accounting for this variation will improve understanding of current conditions and projections of future ecosystem responses to global change.     

Effect of α-actinin on actin viscosity

As determined by analytical ultracentrifugation, purified α-actinin does not form stable complexes with G-actin, myosin, tropomyosin, or the tropomyosintroponin complex. However, α-actinin forms a stable complex with F-actin polymerized either in 100 mM KC1 or in 2mM MgCl₂ without KCl. Viscosity studies confirm that α-actinin interacts as strongly with Mg²⁺-polymerized actin as it does with KCl-polymerized actin.     

Is The Amphibian Tree of Life really fatally flawed?

Wiens (2007 , Q. Rev. Biol. 82, 55–56) recently published a severe critique of Frost et al.'s (2006, Bull. Am. Mus. Nat. Hist. 297, 1–370) monographic study of amphibian systematics, concluding that it is “a disaster” and recommending that readers “simply ignore this study”. Beyond the hyperbole, Wiens raised four general objections that he regarded as “fatal flaws”: (1) the sampling design was insufficient for the generic changes made and taxonomic changes were made without including all type species; (2) the nuclear gene most commonly used in amphibian phylogenetics, RAG‐1, was not included, nor were the morphological characters that had justified the older taxonomy; (3) the analytical method employed is questionable because equally weighted parsimony “assumes that all characters are evolving at equal rates”; and (4) the results were at times “clearly erroneous”, as evidenced by the inferred non‐monophyly of marsupial frogs. In this paper we respond to these criticisms. In brief: (1) the study of Frost et al. did not exist in a vacuum and we discussed our evidence and evidence previously obtained by others that documented the non‐monophyletic taxa that we corrected. Beyond that, we agree that all type species should ideally be included, but inclusion of all potentially relevant type species is not feasible in a study of the magnitude of Frost et al. and we contend that this should not prevent progress in the formulation of phylogenetic hypotheses or their application outside of systematics. (2) Rhodopsin, a gene included by Frost et al. is the nuclear gene that is most commonly used in amphibian systematics, not RAG‐1. Regardless, ignoring a study because of the absence of a single locus strikes us as unsound practice. With respect to previously hypothesized morphological synapomorphies, Frost et al. provided a lengthy review of the published evidence for all groups, and this was used to inform taxonomic decisions. We noted that confirming and reconciling all morphological transformation series published among previous studies needed to be done, and we included evidence from the only published data set at that time to explicitly code morphological characters (including a number of traditionally applied synapomorphies from adult morphology) across the bulk of the diversity of amphibians (Haas, 2003, Cladistics 19, 23–90). Moreover, the phylogenetic results of the Frost et al. study were largely consistent with previous morphological and molecular studies and where they differed, this was discussed with reference to the weight of evidence. (3) The claim that equally weighted parsimony assumes that all characters are evolving at equal rates has been shown to be false in both analytical and simulation studies. (4) The claimed “strong support” for marsupial frog monophyly is questionable. Several studies have also found marsupial frogs to be non‐monophyletic. Wiens et al. (2005, Syst. Biol. 54, 719–748) recovered marsupial frogs as monophyletic, but that result was strongly supported only by Bayesian clade confidence values (which are known to overestimate support) and bootstrap support in his parsimony analysis was < 50%. Further, in a more recent parsimony analysis of an expanded data set that included RAG‐1 and the three traditional morphological synapomorphies of marsupial frogs, Wiens et al. (2006, Am. Nat. 168, 579–596) also found them to be non‐monophyletic. Although we attempted to apply the rule of monophyly to the naming of taxonomic groups, our phylogenetic results are largely consistent with conventional views even if not with the taxonomy current at the time of our writing. Most of our taxonomic changes addressed examples of non‐monophyly that had previously been known or suspected (e.g., the non‐monophyly of traditional Hyperoliidae, Microhylidae, Hemiphractinae, Leptodactylidae, Phrynobatrachus, Ranidae, Rana, Bufo; and the placement of Brachycephalus within “Eleutherodactylus”, and Lineatriton within “Pseudoeurycea”), and it is troubling that Wiens and others, as evidenced by recent publications, continue to perpetuate recognition of non‐monophyletic taxonomic groups that so profoundly misrepresent what is known about amphibian phylogeny. © The Willi Hennig Society 2007.     

Regulation of Activation and Processing of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by a Complex Electrostatic Interaction between the Regulatory Domain and Cytoplasmic Loop 3

NEG2, a short C-terminal segment (817–838) of the unique regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, has been reported to regulate CFTR gating in response to cAMP-dependent R domain phosphorylation. The underlying mechanism, however, is unclear. Here, Lys-946 of cytoplasmic loop 3 (CL3) is proposed as counter-ion of Asp-835, Asp-836, or Glu-838 of NEG2 to prevent the channel activation by PKA. Arg-764 or Arg-766 of the Ser-768 phosphorylation site of the R domain is proposed to promote the channel activation possibly by weakening the putative CL3-NEG2 electrostatic attraction. First, not only D835A, D836A, and E838A but also K946A reduced the PKA-dependent CFTR activation. Second, both K946D and D835R/D836R/E838R mutants were activated by ATP and curcumin to a different extent. Third, R764A and R766A mutants enhanced the PKA-dependent activation. However, it is very exciting that D835R/D836R/E838R and K946D/H950D and H950R exhibited normal channel processing and activity whereas D835R/D836R/E838R/K946D/H950D was fractionally misprocessed and silent in response to forskolin. Further, D836R and E838R played a critical role in the asymmetric electrostatic regulation of CFTR processing, and Ser-768 phosphorylation may not be involved. Thus, a complex interfacial interaction among CL3, NEG2, and the Ser-768 phosphorylation site may be responsible for the asymmetric electrostatic regulation of CFTR activation and processing.     

Binding of the factor IX gamma-carboxyglutamic acid domain to the vitamin K-dependent gamma-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41). In addition, we characterized the effect of the Gla domain and carboxylation on propeptide and substrate binding. For the propeptide of factor IX (proFIX18), FIXproGla41, and carboxylated FIXproGla41, the Kd values were 50, 2.5, and 19.7 nM and the koff values were 273 x 10(-5), 9 x 10(-5), and 37 x 10(-5) s(-1), respectively. The koff of proFIX18 is reduced 3-fold by FLEEL and 9-fold by the Gla domain (residues 1-46) of FIX. The pre-steady state rate constants for carboxylation of FIXproGla41 was 0.02 s(-1) in enzyme excess and 0.016 s(-1) in substrate excess. The steady state rate in substrate excess is 4.5 x 10(-4) s(-1). These results demonstrate the following. 1) The pre-steady state carboxylation rate constant of FIXproGla41 is significantly slower than that of FLEEL. 2) The Gla domain plays an allosteric role in substrate-enzyme interactions. 3) Carboxylation reduces the allosteric effect. 4) The similarity between the steady state carboxylation rate constant and product dissociation rate constant suggests that product release is rate-limiting. 5) The increased dissociation rate after carboxylation contributes to the release of product.     

The putative vitamin K-dependent gamma-glutamyl carboxylase internal propeptide appears to be the propeptide binding site

The vitamin K-dependent gamma-glutamyl carboxylase binds an 18-amino acid sequence usually attached as a propeptide to its substrates. Price and Williamson (Protein Sci. (1993) 2, 1997-1998) noticed that residues 495-513 of the carboxylase shares similarity with the propeptide. They suggested that this internal propeptide could bind intramolecularly to the propeptide binding site of carboxylase, thereby preventing carboxylation of substrates lacking a propeptide recognition sequence. To test Price's hypothesis, we created nine mutant enzyme species that have single or double mutations within this putative internal propeptide. The apparent K(d) values of these mutant enzymes for human factor IX propeptide varied from 0.5- to 287-fold when compared with that of wild type enzyme. These results are consistent with the internal propeptide hypothesis but could also be explained by these residues participating in propeptide binding site per se. To distinguish between the two alternative hypotheses, we measured the dissociation rates of propeptides from each of the mutant enzymes. Changes in an internal propeptide should not affect the dissociation rates, but changes to a propeptide binding site may affect the dissociation rate. We found that dissociation rates varied in a manner consistent with the apparent K(d) values measured above. Furthermore, kinetic studies using propeptide-containing substrates demonstrated a correlation between the affinity for propeptide and V(max). Taken together, our results indicated that these mutations affected the propeptide binding site rather than a competitive inhibitory internal propeptide sequence. These results agree with our previous observations, indicating that residues in this region are involved in propeptide binding.     

Immunoaffinity purification of the calpains

A monoclonal antibody to the small subunit common to both mu- and m-calpains can be used in an immunoaffinity column to purify either mu- or m-calpain in a proteolytically active form. Extracts in 150 mM NaCl, pH 7.5, are loaded onto a column containing the anti-28-kDa antibody; the column is washed with 500 mM NaCl, pH 7.5, and the bound calpain is eluted with 150 mM NaCl, 50 mM Tris-HCl, pH 9.5, and 1 mM EDTA. These elution conditions do not affect the proteolytic activity of either mu- or m-calpain. It is most efficient to reduce the volume and to remove any proteolytic activity from crude extracts by using successive phenyl Sepharose and ion-exchange columns before loading onto the immunoaffinity column. The column purifies m-calpain more effectively than mu-calpain; m-calpain is greater than 90% pure after a single pass through this column, whereas mu-calpain can be purified to >70% purity. The epitope for the monoclonal antibody is between amino acids 92 and 104 (numbers for human calpain) in the 28-kDa subunit. Evidently, this area is shielded in the calpain molecule in a way that affects binding of the antibody to the native molecule.