Interference by Mes [2-(4-morpholino)ethanesulfonic acid] and related buffers with phenolic oxidation by peroxidase

While characterizing the kinetic parameters of apoplastic phenolic oxidation by peroxidase, we found anomalies caused by the Mes [2-(4-morpholino)ethanesulfonic acid] buffer being used. In the presence of Mes, certain phenolics appeared not to be oxidized by peroxidase, yet the oxidant, H(2)O(2), was utilized. This anomaly seems to be due to the recycling of the phenolic substrate. The reaction is relatively inefficient, but at buffer concentrations of 10 mM or greater the recycling effect is nearly 100% with substrate concentrations less than 100 microM. The recycling effect is dependent on substrate structure, occurring with 4'-hydroxyacetophenone but not with 3',5'-dimethoxy-4'-hydroxyacetophenone (acetosyringone). Characterization of the reaction parameters suggests that the phenoxyl radical from the peroxidase reaction interacts with Mes, causing the reduction and regeneration of the phenol. Similar responses occurred with related buffers such as Hepes [4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] and Pipes [piperazine-1,4-bis(2-ethanesulfonic acid)]. Results from this work and other reports in the literature indicate that great care is required in interpreting any results involving these buffers under oxidizing conditions.     

Effect of Vallisneria americana (L.) on community structure and ecosystem function in lake mesocosms

Submerged aquatic vegetation is known as a key structural component and regulator in ecosystems. In this mesocosm study, we examine community- and system-level responses to the presence of Vallisneria americana (L), a deep-rooted macrophyte. Phytoplankton, bacteria and filamentous algal biomasses were significantly lowered in the presence of V. americana. In addition, mesocosms with macrophytes had significantly reduced porewater phosphate and iron, water column dissolved organic carbon and total suspended solids, but elevated sediment redox. All mesocosms were net autotrophic (gross primary production/respiration >1). Compared to the macrophyte treatments, the control mesocosms had lower diel net primary production (NPP) midway through the experiment (d 16), but at the end of the experiment (d 36), the controls had the higher values, presumably due to increased filamentous algae. NPP and NPP/R were constant in the macrophyte treatments, whereas NPP/R increased significantly from middle to end of the experiment in the controls. We show that community and system-level responses to the presence of V. americana have significant consequences on system structure and function.     

Local Infiltrative Analgesia is Equivalent to Fascia Iliaca Block for Perioperative Pain Management for Prophylactic Cephalomedullary Nail Fixation

BackgroundImpending pathologic fractures of the femur due to metastatic bone disease are treated with prophylactic internal fixation to prevent fracture, maintain independence, and improve quality of life. There is limited data to support an optimal perioperative pain regimen.MethodsA proof of concept comparative cohort analysis was performed: 21 patients who received a preoperative fascia iliacus nerve block (FIB) were analyzed retrospectively while 9 patients treated with local infiltrative analgesia (LIA) were analyzed prospectively. Primary outcomes included: visual analog scale (VAS) pain scores, narcotic requirements and hospital length of stay. Patient cohorts were compared via two-sample t-tests and Fischer’s exact tests. Differences in VAS pain scores, length of stay and morphine milligram equivalents (MME) were assessed with Wilcoxon rank sum.ResultsThe LIA group had more patients treated with preoperative narcotics (p=0.042). There were no significant differences between the FIB and LIA groups in MME utilized intraoperatively (30.0 vs 37.5, p=0.79), on POD 0 (38.0 vs 30.0, p=0.93), POD 1 (46.0 vs 55.5, p=0.95) or POD 2 (40.0 vs 60.0 p=0.73). There were no significant differences in analog pain scale at any time point or in hospital length of stay (78 vs 102 hours, p=0.86).ConclusionDespite an increased number of patients being on preoperative narcotics in the LIA group, use of LIA compared with FIB is not associated with an increase in VAS pain scores, morphine milligram equivalents (MME), or length of hospital stay in patients undergoing prophylactic internal fixation of impending pathologic femur fractures.Level of Evidence: III     

Expanded bed adsorption in the purification of monoclonal antibodies: a comparison of process alternatives

Expanded bed adsorption (EBA) was examined as the initial capture/purification step in the purification of monoclonal antibodies from Chinese hamster ovary (CHO) cultures. Two process alternatives each using EBA were compared to a conventional Protein A process without EBA. One alternative used Protein A affinity EBA followed by packed-bed cation and anion-exchange steps. The other alternative used cation-exchange EBA as the capture step followed by packed-bed Protein A and anion-exchange steps. The process using Protein A EBA produced comparable purity (host cell protein, DNA, Protein A, antibody aggregate) to the conventional process. However, the Protein A EBA column showed a significant decrease in dynamic capacity with a limited number of cycles. The process using cation EBA achieved comparable levels of host cell proteins (HCP) and DNA but not antibody aggregate or leached Protein A compared to the conventional process.     

Cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells

Although it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin-Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans-Golgi network with a halftime of approximately 45 min and have an approximate t_1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg-651 were localized predominantly at the apical surface of filter-grown cells, whereas receptors truncated at Leu-723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise loacation of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states. © 1995 Wiley-Liss, Inc.     

Autosomal Recessive Polycystic Kidney Disease Epithelial Cell Model Reveals Multiple Basolateral Epidermal Growth Factor Receptor Sorting Pathways

Sorting and maintenance of the EGF receptor on the basolateral surface of renal epithelial cells is perturbed in polycystic kidney disease and apical expression of receptors contributes to severity of disease. The goal of these studies was to understand the molecular basis for EGF receptor missorting using a well-established mouse model for the autosomal recessive form of the disease. We have discovered that multiple basolateral pathways mediate EGF receptor sorting in renal epithelial cells. The polycystic kidney disease allele in this model, Bicc1, interferes with one specific EGF receptor pathway without affecting overall cell polarity. Furthermore one of the pathways is regulated by a latent basolateral sorting signal that restores EGF receptor polarity in cystic renal epithelial cells via passage through a Rab11-positive subapical compartment. These studies give new insights to possible therapies to reconstitute EGF receptor polarity and function in order to curb disease progression. They also indicate for the first time that the Bicc1 gene that is defective in the mouse model used in these studies regulates cargo-specific protein sorting mediated by the epithelial cell specific clathrin adaptor AP-1B.     

A Genome-Wide Survey of Transgenerational Genetic Effects in Autism

Effects of parental genotype or parent-offspring genetic interaction are well established in model organisms for a variety of traits. However, these transgenerational genetic models are rarely studied in humans. We have utilized an autism case-control study with 735 mother-child pairs to perform genome-wide screening for maternal genetic effects and maternal-offspring genetic interaction. We used simple models of single locus parent-child interaction and identified suggestive results (P<10⁻⁴) that cannot be explained by main effects, but no genome-wide significant signals. Some of these maternal and maternal-child associations were in or adjacent to autism candidate genes including: PCDH9, FOXP1, GABRB3, NRXN1, RELN, MACROD2, FHIT, RORA, CNTN4, CNTNAP2, FAM135B, LAMA1, NFIA, NLGN4X, RAPGEF4, and SDK1. We attempted validation of potential autism association under maternal-specific models using maternal-paternal comparison in family-based GWAS datasets. Our results suggest that further study of parental genetic effects and parent-child interaction in autism is warranted.     

Rabies-Specific Antibodies: Measuring Surrogates of Protection against a Fatal Disease

Antibodies play a central role in prophylaxis against many infectious agents. While neutralization is a primary function of antibodies, the Fc- and complement-dependent activities of these multifunctional proteins may also be critical in their ability to provide protection against most viruses. Protection against viral pathogens in vivo is complex, and while virus neutralization—the ability of antibody to inactivate virus infectivity, often measured in vitro—is important, it is often only a partial contributor in protection. The rapid fluorescent focus inhibition test (RFFIT) remains the “gold standard” assay to measure rabies virus–neutralizing antibodies. In addition to neutralization, the rabies-specific antigen-binding activity of antibodies may be measured through enzyme-linked immunosorbent assays (ELISAs), as well as other available methods. For any disease, in selecting the appropriate assay(s) to use to assess antibody titers, assay validation and how they are interpreted are important considerations—but for a fatal disease like rabies, they are of paramount importance. The innate limitations of a one-dimensional laboratory test for rabies antibody measurement, as well as the validation of the method of choice, must be carefully considered in the selection of an assay method and for the interpretation of results that might be construed as a surrogate of protection.     

PKCtheta is a key player in the development of insulin resistance

Activation of PKCtheta is associated with lipid-induced insulin resistance and PKCtheta knockout mice are protected from the lipid-induced defects. However, the exact mechanism by which PKCtheta contributes to insulin resistance is not known. To investigate whether an increase in PKCtheta expression leads to insulin resistance, C2C12 skeletal muscle cells were transfected with PKCtheta DNA and treated with different concentrations of insulin for 10 min. PKCtheta overexpression induced reduction of IRS-1 protein levels with a decrease in insulin-induced p85 binding to IRS-1, phosphorylation of PKB and its substrates, p70 and GSK3. Pretreatment of these cells with GF-109203X (a non-specific PKC inhibitor, IC50 for PKCtheta = 10 nM) recovered insulin signaling. PKCtheta was found to be expressed in liver and treatment of human hepatoma cells (HepG2) with high insulin and glucose resulted in an increase in PKCtheta expression that correlated with a decrease in IRS-1 protein levels and the development of insulin resistance. Reduction of PKCtheta expression using RNAi technology significantly inhibited the degradation of IRS-1 and enhanced insulin-induced IRS-1 tyrosine phosphorylation, p85 association to IRS-1 and PKB phosphorylation. In conclusion, by overexpressing PKCtheta or using RNAi technology to downregulate PKCtheta, we have demonstrated that PKCtheta has a key role in the development of insulin resistance. These findings suggest that PKCtheta mediates not only insulin resistance in muscle but also in liver, which may contribute to the development of whole body insulin resistance and diabetes.