Position-dependent effects of polylysine on Sec protein transport

The bacterial Sec protein translocation system catalyzes the transport of unfolded precursor proteins across the cytoplasmic membrane. Using a recently developed real time fluorescence-based transport assay, the effects of the number and distribution of positive charges on the transport time and transport efficiency of proOmpA were examined. As expected, an increase in the number of lysine residues generally increased transport time and decreased transport efficiency. However, the observed effects were highly dependent on the polylysine position in the mature domain. In addition, a string of consecutive positive charges generally had a more significant effect on transport time and efficiency than separating the charges into two or more charged segments. Thirty positive charges distributed throughout the mature domain resulted in effects similar to 10 consecutive charges near the N terminus of the mature domain. These data support a model in which the local effects of positive charge on the translocation kinetics dominate over total thermodynamic constraints. The rapid translocation kinetics of some highly charged proOmpA mutants suggest that the charge is partially shielded from the electric field gradient during transport, possibly by the co-migration of counter ions. The transport times of precursors with multiple positively charged sequences, or "pause sites," were fairly well predicted by a local effect model. However, the kinetic profile predicted by this local effect model was not observed. Instead, the transport kinetics observed for precursors with multiple polylysine segments support a model in which translocation through the SecYEG pore is not the rate-limiting step of transport.     

Impact of Nosema (Microsporidia) infection and fumagillin treatment on Lygus lineolaris (Hemiptera: Miridae)

A consistent supply of healthy tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) (Hemiptera: Miridae), is necessary for the development of novel management strategies targeting this pest. After being in culture for several years, a substantial portion of a tarnished plant bug colony was found to be infected with a Nosema (Microsporidia) species. Studies were subsequently undertaken to evaluate the impact of Nosema infection on tarnished plant bug productivity and to test the efficacy of fumagillin to treat this infection. Using buffalo black stain, infections could not be reliably detected in adult tarnished plant bugs until adults were 6-8days post eclosion. Nosema infections reduced adult longevity and fecundity. Maximum fecundity was restored using a concentration of 16.8ppm fumagillin while maximum longevity for females was at a concentration of 33.6ppm fumagillin incorporated into the tarnished plant bug diet. Minimum infection scores were obtained at 67.2ppm, the highest concentration tested. A field survey of tarnished plant bugs in Mississippi found Nosema infections in 3% of wild tarnished plant bugs.     

Large cargo transport by nuclear pores: implications for the spatial organization of FG‐nucleoporins

Nuclear pore complexes (NPCs) mediate cargo traffic between the nucleus and the cytoplasm of eukaryotic cells. Nuclear transport receptors (NTRs) carry cargos through NPCs by transiently binding to phenylalanine‐glycine (FG) repeats on intrinsically disordered polypeptides decorating the NPCs. Major impediments to understand the transport mechanism are the thousands of FG binding sites on each NPC, whose spatial distribution is unknown, and multiple binding sites per NTR, which leads to multivalent interactions. Using single molecule fluorescence microscopy, we show that multiple NTR molecules are required for efficient transport of a large cargo, while a single NTR promotes binding to the NPC but not transport. Particle trajectories and theoretical modelling reveal a crucial role for multivalent NTR interactions with the FG network and indicate a non‐uniform FG repeat distribution. A quantitative model is developed wherein the cytoplasmic side of the pore is characterized by a low effective concentration of free FG repeats and a weak FG‐NTR affinity, and the centrally located dense permeability barrier is overcome by multivalent interactions, which provide the affinity necessary to permeate the barrier.     

A top-down proteomics approach for differentiating thermal resistant strains of Enterobacter sakazakii

Thermal tolerance has been identified as an important factor relevant to the pathogenicity of Enterobacter sakazakii in human neonates. To identify a biomarker specific for this phenotypic trait, intact protein expression profiles of 12 strains of E. sakazakii were obtained using liquid chromatography mass spectrometry. Proteins were extracted from the bacterial cells, separated by reversed-phase liquid chromatography and mass analyzed. At the end of the chromatography run, the uncharged masses of the multiply charged proteins were determined via automated software routines. The resulting data provided an accurate mass expression profile of the proteins found in the individual strains. From the individual expression profiles, it was possible to identify unique proteins corresponding to strains with thermal resistance. One protein found only in the thermal tolerant strains was sequenced and identified as homologous to a hypothetical protein found in the thermal tolerant bacteria, Methylobacillus flagellatus KT. The protein sequence of this protein was then used to reverse-engineer PCR primers for the gene sequence associated with the protein. In all cases, only thermal tolerant strains of E. sakazakii produced amplified PCR products, demonstrating the specificity of this biomarker.     

Proton transfer limits protein translocation rate by the thylakoid DeltapH/Tat machinery

The thylakoid transmembrane DeltapH is the sole energy source driving translocation of precursor proteins by the DeltapH/Tat machinery. Consequently, proton translocation must be coupled to precursor translocation. For the precursor of the 17 kDa protein of the oxygen-evolving complex (pOE17), the protein translocation process is characterized by a steep drop in efficiency at an external pH below 7.0 and above 8.7. As the membrane DeltapH is virtually unaffected from pH 6.5 to 9.2, the loss in import efficiency is a consequence of the titration of multiple residues within the translocation machinery. Transport is retarded by a factor of 2-3 in deuterium oxide (D(2)O) relative to water, strongly suggesting that proton-transfer reactions limit translocation rate. The solvent isotope effect manifests itself after the precursor binds to the membrane, indicating that the rate-limiting step is a later event in the transport process.     

A new genus of rodent from Wallacea (Rodentia: Muridae: Murinae: Rattini), and its implication for biogeography and Indo‐Pacific Rattini systematics

We describe Halmaheramys bokimekot Fabre, Pagès, Musser, Fitriana, Semiadi & Helgen gen. et sp. nov. , a new genus and species of murine rodent from the North Moluccas, and study its phylogenetic placement using both molecular and morphological data. We generated a densely sampled mitochondrial and nuclear DNA data set that included most genera of Indo‐Pacific Murinae, and used probabilistic methodologies to infer their phylogenetic relationships. To reconstruct their biogeographical history, we first dated the topology and then used a Lagrange analysis to infer ancestral geographic areas. Finally, we combined the ancestral area reconstructions with temporal information to compare patterns of murine colonization among Indo‐Pacific archipelagos. We provide a new and comprehensive molecular phylogenetic reconstruction for Indo‐Pacific Murinae, with a focus on the Rattus division. Using previous results and those presented in this study, we define a new Indo‐Pacific group within the Rattus division, composed of Bullimus, Bunomys, Paruromys, Halmaheramys, Sundamys, and Taeromys. Our phylogenetic reconstructions revealed a relatively recent diversification from the Middle Miocene to Plio‐Pleistocene associated with several major dispersal events. We identified two independent Indo‐Pacific dispersal events from both western and eastern Indo‐Pacific archipelagos to the isolated island of Halmahera, which led to the speciations of H. bokimekot gen. et sp. nov. and Rattus morotaiensis Kellogg, 1945. We propose that a Middle Miocene collision between the Halmahera and Sangihe arcs may have been responsible for the arrival of the ancestor of Halmaheramys to eastern Wallacea. Halmaheramys bokimekot gen. et sp. nov. is described in detail, and its systematics and biogeography are documented and illustrated. © 2013 The Linnean Society of London     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

NPY5R antagonism does not augment the weight loss efficacy of orlistat or sibutramine

Objective: Central counter‐regulatory mechanisms, including those related to the orexigenic hormone neuropeptide Y (NPY), may limit the weight loss observed with conventional pharmacological monotherapy. This study evaluated whether blockade of the NPY Y5 receptor (NPY5R) with the selective antagonist MK‐0557 potentiates sibutramine and orlistat weight loss effects. Research Methods and Procedures: Obese patients (497, BMI 30 to 43 kg/m²) were randomized to 1 of 5 treatment arms [placebo, n = 101; sibutramine 10 mg/d, n = 100; MK‐0557 1 mg/d plus sibutramine 10 mg/d, n = 98; orlistat 120 mg TID, n = 99; MK‐0557 1 mg/d plus orlistat 120 mg TID, n = 99] in conjunction with a hypocaloric diet for 24 weeks. The all‐patients‐treated population, imputing missing data using last observation carried forward, was used to assess weight loss from baseline. Results: The study was completed by 71% of patients in placebo, 76% in sibutramine alone, 79% in MK‐0557 + sibutramine, 69% in orlistat alone, and 76% in MK‐0557 + orlistat groups. Least squares (LS) mean difference [95% confidence interval (CI)] in weight change from baseline between MK‐0557 + sibutramine and sibutramine alone was ?0.1 (?1.6, 1.4) kg (p = 0.892) and between MK‐0557 + orlistat and orlistat alone was ?0.9 (?2.4, 0.6) kg (p = 0.250). Sibutramine alone induced a LS mean weight loss of ?5.9 (?6.9, ?4.9) kg vs. ?4.6 (?5.7, ?3.6) kg for orlistat (p = 0.097). There were no serious drug‐related adverse events and MK‐0557 was well tolerated. Discussion: Blockade of the NPY5R with the potent antagonist MK‐0557 did not significantly increase the weight loss efficacy of either orlistat or sibutramine monotherapy.