Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na⁺-phosphate cotransporter (NaP_i-IIa) in the brush border membrane (BBM). The activity and localization of NaP_i-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaP_i-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na⁺/H⁺-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaP_i-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaP_i-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1–34) led to internalization of NaP_i-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3–34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl-sn-glycerol or 8-bromo-cAMP induced normal internalization of NaP_i-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaP_i-IIa and, specifically, couples the apical PTHR to PLC. phosphate cotransporter; PDZ protein; parathyroid hormone; proximal tubule     

A comparison of five methods for DNA isolation from liver and rumen flukes to perform ITS-2+ amplification

Five different DNA isolation methods (4 commercial kits and a modification of phenol-chloroform method) were compared for the discrimination of adults of Fasciola hepatica and Dicrocoelium dendriticum (liver flukes), and Calicophoron daubneyi (rumen fluke) collected from sheep in southern Italy. The second internal transcribed spacer (ITS-2) of ribosomal DNA (rDNA) plus flanking 5.8S and 28S sequence (ITS-2+) was amplified by polymerase chain reaction (PCR) from serial diluted DNA templates (6 ng - 60 fg) of each fluke species. Overall, in terms of efficiency in detection limit, the best results were obtained either with phenol-chloroform purification or with QIAamp DNA Mini Kit (Qiagen), but using this latter method, rapid, safe and not expensive, an increased level of sensitivity sufficient to detect small amounts of target-DNA was achieved. In addition, electrophoresis analysis following PCR also showed that ITS-2+ could be useful as a genetic marker for the molecular identification of F. hepatica, D. dendriticum and C. daubneyi in definitive and intermediate hosts. Furthermore, for the first time, the ITS-2 sequence of D. dendriticum was defined.     

Properties and exploitation of oleosins

Oleosins stabilize oil bodies in seeds and other tissues and contain a unique hydrophobic domain which appears to be inserted into the oil matrix as an alpha-helical hairpin. The oleosin proteins may be exploited to stabilize emulsions while the ease of oil body preparation has led to the expression of bioactive proteins as oleosin fusions in molecular farming.     

Cloning and expression of two plant proteins: similar antimicrobial activity of native and recombinant form

Antimicrobial peptides and proteins are being studied with increasing interest because of their broad range antimicrobial activity. Among plant antimicrobial proteins, the wheat seed polypeptides, puroindoline a and puroindoline b, are particularly interesting because of their established antibacterial activity. In this paper we describe different strategies used to clone His tagged and GST tagged puroindolines obtaining 1.5 mg recombinant protein from 1 l culture. The antimicrobial activity of recombinant and native puroindolines was comparable.     

New 1,3,4-thiadiazole derivatives endowed with analgesic and anti-inflammatory activities

Two series of N-[5-oxo-4-(arylsulfonyl)-4,5-dihydro-1,3,4-thiadiazol-2-yl]-amides were synthesized and tested in vivo for their analgesic and anti-inflammatory activities. All the new compounds possess good antalgic action in the acetic acid writhing test and some terms of the series showed also fair anti-inflammatory activity in the carrageenan rat paw edema test. Ulcerogenic and irritative action on the gastrointestinal mucose, in comparison with indomethacin is low.     

Cosegregation of novel mitochondrial 16S rRNA gene mutations with the age-associated T414G variant in human cybrids

Ever increasing evidence has been provided on the accumulation of mutations in the mitochondrial DNA (mtDNA) during the aging process. However, the lack of direct functional consequences of the mutant mtDNA load on the mitochondria-dependent cell metabolism has raised many questions on the physiological importance of the age-related mtDNA variations. In the present work, we have analyzed the bioenergetic properties associated with the age-related T414G mutation of the mtDNA control region in transmitochondrial cybrids. The results show that the T414G mutation does not cause per se any detectable bioenergetic change. Moreover, three mtDNA mutations clustered in the 16S ribosomal RNA gene cosegregated together with the T414G in the same cybrid cell line. Two of them, namely T1843C and A1940G, are novel and associate with a negative bioenergetic phenotype. The results are discussed in the more general context of the complex heterogeneity and the dramatic instability of the mitochondrial genome during cell culture of transmitochondrial cybrids.     

Blood-borne cells involved in arterial repair upon experimental incision injury

We had previously shown that microscopically detectable infiltration of dendritic cells and expression of Hsp47 in tissue lysates occur during repair upon experimental arterial injury. We have further analysed here the cell types involved in the repair process by histology, electron microscopy and immunofluorescence. Rat carotid arteries were subjected to brief crushing and full thickness incision and were analysed up to 21 d thereafter. Adhesion and activation of platelets occurred 3 h after surgery. A neointima had formed 7 d after surgery, where immature cells entered from the lumen and gave rise to cells rich in organelles of the secretory pathway and endowed with bundles of phalloidin-binding microfilaments. Alpha smooth muscle-positive, secretory and contractile smooth muscle cells were found in the neointima 14 and 21 d after injury. Seven to 21 d after surgery, endothelial cells appeared immature and the newly formed tissue contained MHC-II positive, CD43 positive dendritic cells which clustered with lymphocytes, a few macrophages containing apoptotic remnants and cells labelled for Hsp47. Thin elastic fibrils appeared in the neointima 21 d after injury. The results suggest that the response to acute arterial incision injury is mediated by blood borne cells which differentiate along multiple pathways; the process evolves without reaching stabilization within the observed time lapse; the secretion of extracellular matrix is marked by the expression of Hsp47; and the constant presence of dendritic cells clustered with lymphocytes makes these cells candidate to a pivotal role in the tissue response to injury.     

Specific pathogen-free macaques: definition, history, and current production

Specific pathogen-free (SPF) macaque colonies are now requested frequently as a resource for research. Such colonies were originally conceived as a means to cull diseased animals from research-dedicated colonies, with the goal of eliminating debilitating or fatal infectious agents from the colony to improve the reproductive capacity of captive research animals. The initial pathogen of concern was Mycobacterium tuberculosis (M.tb.), recognized for many years as a pathogen of nonhuman primates as well as a human health target. More recently attention has focused on four viral pathogens as the basis for an SPF colony: simian type D retrovirus (SRV), simian immunodeficiency virus (SIV), simian T cell lymphotropic/leukemia virus (STLV), and Cercopithecine herpesvirus 1 (CHV-1). New technologies, breeding, and maintenance schemes have emerged to develop and provide SPF primates for research. In this review we focus on the nonhuman primates (NHPs) most common to North American NHP research facilities, Asian macaques, and the most common current research application of these animals, modeling of human AIDS.