EGF and its related growth factors mediate sodium transport in mpkCCDc14 cells via ErbB2 (neu/HER‐2) receptor

Amiloride‐sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate‐limiting step for Na⁺ absorption. Epidermal growth factor (EGF) is involved in the regulation of Na⁺ transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na⁺ absorption. The aim of the present study was to characterize the EGF regulation of Na⁺ transport in cultured mouse renal collecting duct principal mpkCCD_c14 cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na⁺ transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF‐α, HB‐EGF, and amphiregulin which also belong to the EGF‐related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na⁺ reabsorption. Inhibition of PI3‐kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD_c14 cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague–Dawley rat kidneys. Both acute and long‐term effects of EGF were abolished when cells were treated with tyrphostin AG‐825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na⁺ transport and that EGF biphasically modulates sodium transport in mpkCCD_c14 cells via the ErbB2 receptor. J. Cell. Physiol. 223: 252–259, 2010. © 2009 Wiley‐Liss, Inc.     

ABCD2 is abundant in adipose tissue and opposes the accumulation of dietary erucic acid (C22:1) in fat

The ATP binding cassette transporter, ABCD2 (D2), is a peroxisomal protein whose mRNA has been detected in the adrenal, brain, liver, and fat. Although the role of this transporter in neural tissues has been studied, its function in adipose tissue remains unexplored. The level of immunoreactive D2 in epididymal fat is >50-fold of that found in brain or adrenal. D2 is highly enriched in adipocytes and is upregulated during adipogenesis but is not essential for adipocyte differentiation or lipid accumulation in day 13.5 mouse embryonic fibroblasts isolated from D2-deficient (D2^−/−) mice. Although no differences were appreciated in differentiation percentage, total lipid accumulation was greater in D2^−/− adipocytes compared with the wild type. These results were consistent with in vivo observations in which no significant differences in adiposity or adipocyte diameter between wild-type and D2^−/− mice were observed. D2^−/− adipose tissue showed an increase in the abundance of 20:1 and 22:1 fatty acids. When mice were challenged with a diet enriched in erucic acid (22:1), this lipid accumulated in the adipose tissue in a gene-dosage-dependent manner. In conclusion, D2 is a sterol regulatory element binding protein target gene that is highly abundant in fat and opposes the accumulation of dietary lipids generally absent from the triglyceride storage pool within adipose tissue.     

11-Phenoxyundecyl phosphate as a 2-acetamido-2-deoxy-α-d-glucopyranosyl phosphate acceptor in O-antigen repeating unit assembly of Salmonella arizonae O:59

A synthesis of 11-phenoxyundecyl phosphate and its biochemical transformation (using GlcNAc-P transferase from Salmonella arizonae O:59 membranes catalysing transfer of GlcNc-phosphate from UDP-GlcNAc on lipid-phosphate) into P¹-11-phenoxyundecyl, P²-2-acetamido-2-deoxy-α-d-glucopyranosyl diphosphate are described.     

Transient permeability induced by alkyl derivatives of amphotericin B in lipid membranes

Individual ionic channels were shown to be formed in the brain cholesterol containing phospholipid membranes by two-sided addition of the amphotericin B alkyl derivatives. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to be transient. Existence of different antibiotic assemblies was justified by the kinetic analysis of the membrane conductance decline following the antibiotic washing out. In order to account for the transient characteristics of the induced conductance, it was proposed that the antibiotic oligomers incorporate into the membrane from the aqueous phase, form channels aggregating with cholesterol, and then dissociate in the bilayer into non-active degraded oligomeric or monomeric forms.     

Nonantibody-based recognition: alternative molecules for detection of pathogens

Immunoassays have been well established for many years as the cornerstone of detection technologies. These assays are sensitive, selective and, in general, highly resistant to interference from complex sample matrices when compared with nucleic acid-based tests. However, both antibody- and nucleic acid-based detection systems require a priori knowledge of the target and development of specific reagents; multiplexed assays can become increasingly problematic when attempting to detect a plethora of different targets, the identities of which are unknown. In an effort to circumvent many of the limitations inherent in these conventional assays, other recognition reagents are being explored as alternatives, or indeed as adjuncts, to antibodies for pathogen and toxin detection. This article will review a number of different recognition systems ranging in complexity from small molecules, such as nucleic-acid aptamers, carbohydrates and peptides, to systems as highly complicated as whole cells and organisms. All of these alternative systems have tremendous potential to achieve superior sensitivity, selectivity, and stability, but are also subject to their own limitations, which are also discussed. In short, while in its infancy, this field holds great promise for the development of rapid, fieldable assays that are highly complementary to existing antibody- and nucleic acid-based technologies.     

Decreases in plasma membrane Ca2+‐ATPase in brain synaptic membrane rafts from aged rats

Precise regulation of free intracellular Ca²⁺ concentrations [Ca²⁺]_i is critical for normal neuronal function, and alterations in Ca²⁺ homeostasis are associated with brain aging and neurodegenerative diseases. One of the most important proteins controlling [Ca²⁺]_i is the plasma membrane Ca²⁺‐ATPase (PMCA), the high‐affinity transporter that fine tunes the cytosolic nanomolar levels of Ca²⁺. We previously found that PMCA protein in synaptic plasma membranes (SPMs) is decreased with advancing age and the decrease in enzyme activity is much greater than that in protein levels. In this study, we isolated raft and non‐raft fractions from rat brain SPMs and used quantitative mass spectrometry to show that the specialized lipid microdomains in SPMs, the rafts, contain 60% of total PMCA, comprised all four isoforms. The raft PMCA pool had the highest specific activity and this decreased progressively with age. The reduction in PMCA protein could not account for the dramatic activity loss. Addition of excess calmodulin to the assay did not restore PMCA activity to that in young brains. Analysis of the major raft lipids revealed a slight age‐related increase in cholesterol levels and such increases might enhance membrane lipid order and prevent further loss of PMCA activity.     

Printed glycan array: antibodies as probed in undiluted serum and effects of dilution

Using printed glycan array (PGA) we compared the results of antibody profiling in undiluted, moderately (1:15) and highly (1:100) diluted human blood serum. Undiluted serum is suitable for studying blood as a tissue in its native state, whereas to study the serum of newborns or small animals one usually has to dilute the starting material in order to have sufficient volume for PGA experimentation. The PGA used in this study allows for the use of whole serum without modifications to the protocol, and the background is surprisingly low. Antibodies profiles observed in undiluted serum versus 1:15 dilution were similar, with only a limited number of new signals identified in the undiluted serum. However, unexpected irregularities were found when IgG and IgM are measured separately, namely, at a 1:15 dilution more intensive IgG signals for many glycans are observed. We believe that in conditions of moderate dilution IgG and IgM antibodies can compete with each other for antigen and as a result, the higher affinity anti-glycan IgGs give rise to more intense signals. Therefore depending on the purpose, different dilutions of serum will be optimal: in competitive 1:15 conditions the observed IgG/IgM ratio corresponds to their titer, whereas at 1:100 dilution the measured ratio corresponds to real molar concentration of IgG and IgM.     

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.