pH-sensitive expression of calcium-sensing receptor (CaSR) in type-B intercalated cells of the cortical collecting ducts (CCD) in mouse kidney

Background The localization and role of the calcium-sensing receptor (CaSR) along the nephron including the collecting ducts is still open to debate. Methods Using the quantitative, highly sensitive in situ hybridization technique and a double-staining immunohistochemistry technique, we investigated the axial distribution and expression of CaSR along the nephron in mice (C57B/6J) treated for 6 days with acid or alkali diets. Results Under control condition, CaSR was specifically localized in the cortical and medullary thick ascending limb of Henle’s loop (CTAL and MTAL), macula densa (MD), distal convoluted tubule (DCT), and CCD (TALs, MD > DCT, CCD). Along the CCD, CaSR was co-localized with an anion exchanger type 4 (AE4), a marker of the basolateral membrane of type-B intercalated cell (IC-B) in mice. On the contrary, CaSR was not detected either in principal cells (PC) or in type-A intercalated cell (IC-A). CaSR expression levels in IC-B significantly (P < 0.005) decreased when mice were fed NH4Cl (acid) diets and increased when animals were given NaHCO3 (alkali) diets. As expected, cell heights of IC-A and IC-B significantly (P < 0.005) increased in the above experimental conditions. Surprisingly, single infusion (ip) of neomycin, an agonist of CaSR, significantly (P < 0.005) increased urinary Ca excretion without further increasing the hourly urine volume and significantly (P < 0.05) decreased urine pH. Conclusion CaSR, cloned from rat kidney, was localized in the basolateral membrane of IC-B and was more expressed during alkali-loading. Its alkali-sensitive expression may promote urinary alkali secretion for body acid–base balance.

Constitutive expression of hZnT4 zinc transporter in human breast epithelial cells

Zinc is an essential trace element required by all living organisms. An adequate supply of zinc is particularly important in the neonatal period. Zinc is a significant component of breast milk, which is transported across the maternal epithelia during lactation. The mechanisms by which zinc becomes a constituent of breast milk have not been elucidated. The function of the zinc transporter ZnT4 in the transport of zinc into milk during lactation was previously demonstrated by studies of a mouse mutant, the ‘lethal milk’ mouse, where a mutation in the ZnT4 gene decreased the transport of zinc into milk. In the present study, we have investigated the expression of the human orthologue of ZnT4 (hZnT4) in the human breast. We detected hZnT4 mRNA expression in the tissue from the resting and lactating human breast, using reverse-transcriptase PCR. Western-blot analysis using antibodies to peptide sequences of hZnT4 detected a major band of the predicted size of 47 kDa and a minor band of 77 kDa, in extracts from the resting and lactating breast tissues. There was no difference in the hZnT4 expression levels between lactating and resting breasts. The hZnT4 protein was present in the luminal cells of the ducts and alveoli where it had a granular distribution. A cultured human breast epithelial cell line PMC42 was used to investigate the subcellular distribution of hZnT4 and this showed a granular label throughout the cytoplasm, consistent with a vesicular localization. The presence of zinc-containing intracellular vesicles was demonstrated by using the zinc-specific fluorphore Zinquin (ethyl-[2-methyl-8-p-toluenesulphonamido-6-quinolyloxy]acetate). Double labelling indicated that there was no obvious overlap between Zinquin and the hZnT4 protein, suggesting that hZnT4 was not directly involved in the transport of zinc into vesicles. We detected expression of two other members of the hZnT family, hZnT1 and hZnT3, in human breast epithelial cells. We conclude that hZnT4 is constitutively expressed in the human breast and may be one of the several members of the ZnT family involved in the transport of zinc into milk.

Innovation in construction : experiences from a hypocaust system in Australia

Introducing new technologies poses a particular challenge to the players involved in a project. For a successful low energy building, a new design process is required and players must assume new and additional responsibilities. Hypocaust systems, where conditioned or non-conditioned air is passed through ducts within the concrete floor or ceiling of a building prior to its delivery to the rooms, are starting to appear in new buildings in Australia. This paper describes the lessons learned from the early experiences with a hypocaust system, installed in a new building in Melbourne. It concludes that a more cooperative process among all those involved in introducing and using a new 'technology' is essential if the problems described are to be avoided or at least minimized.

Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, Anguilla japonica

Natriuretic peptides are linked to osmoregulation, cardiovascular and volume regulation in fishes. The peptides bind to two guanylyl-cyclase-linked receptors, natriuretic peptide receptor-A (NPR-A) and NPR-B, to elicit their effects. Atrial natriuretic peptide (ANP) binds principally to NPR-A, whereas C-type natriuretic peptide (CNP) binds to NPR-B. The teleost kidney has an important role in the maintenance of fluid and electrolyte balance; therefore, the location of NPR-A and NPR-B in the kidney could provide insights into the functions of natriuretic peptides. This study used homologous, affinity purified, polyclonal antibodies to NPR-A and NPR-B to determine their location in the kidney of the Japanese eel, Anguilla japonica. Kidneys from freshwater and seawater acclimated animals were fixed overnight in 4% paraformaldehyde before being paraffin-embedded and immunostained. NPR-A immunoreactivity was found on the apical membrane of proximal tubule 1 and the vascular endothelium including the glomerular capillaries. In contrast, NPR-B immunoreactivity was located on the smooth muscle of blood vessels including the glomerular afferent and efferent arterioles, and on smooth muscle tissue surrounding the collecting ducts. No difference in the distribution of NPR-A and NPR-B was observed between freshwater and seawater kidneys. Immunoreactivity was not observed in any tissue in which the antibodies had been preabsorbed. In addition, there was no difference in NPR-A and NPR-B mRNA expression between freshwater-acclimated and seawater-acclimated eels. These results suggest that, although utilizing the same second messenger system, ANP and CNP act on different targets within the kidney and presumably elicit different effects.

Introducing new building technologies : lessons from a case study

Hypocaust systems, where conditioned or non-conditioned air is passed through ducts within the concrete floor or ceiling of a building prior to its delivery to the rooms, are starting to appear in new buildings in Australia.

This paper describes the lessons that can be learned from the early experiences with a hypocaust system, installed in a new building in Melbourne. It concludes that a more cooperative process between all those involved in introducing and using a new 'technology' is essential if the problems described are to be avoided or at least minimized.

Biliary intervention augments chemotactic reaction and aggravates cholestatic liver injury in rats

Background
Intervention of the biliary system is frequently done in patients with obstructive jaundice and is associated with significant morbidity and mortality. The pathogenesis is unknown.
Materials and methods
A rat model of bile duct ligation (BDL) for 2 weeks was established in which biliary intervention was feasible by injection of normal saline through an indwelling catheter in the bile ducts. Plasma levels of C-C chemokine MCP-1 and C-X-C chemokine MIP-2 were measured by using ELISA. Blood monocytes, Kupffer cells, and neutrophils in the liver were characterized with antibodies to ED1, ED2, and myeloperoxidase (MPO). Lipid peroxidation was measured by malondialdehyde contents and apoptosis by TUNEL stain of the liver.
Results
Biliary intervention resulted in an increase of plasma MCP-1 and MIP-2 proteins by 1 h, which declined to normal level by 3 h in both sham and BDL rats. The levels in BDL rats were significantly higher than in sham at most points. There was a transient increase of ED1- and ED2-positive cells and MPO-staining cells in sham rat liver by 1 h after intervention. ED2-positive cells increased significantly by 1 h, while ED1- and MPO-positive cells decreased, yet insignificantly after intervention in BDL rats. The cell counts in BDL were constantly higher than in sham. Malondialdehyde increased precipitously in BDL by 3 h and was significantly higher than in sham throughout the study period. Parenchymal liver injury, manifested by elevated ALT, as well as apoptosis and necrosis of liver cells, was significantly increased in BDL rats, but not in sham rats.
Conclusion
Biliary intervention augments chemokine expression, precipitates lipid peroxidation, and aggravates liver injury in cholestatic rats.

So flat, so cute! Robots, superflatness and asian architectural futures

Many depictions of urban futures have a distinctly Asian flavour. There have been numerous visions of highly technological futures whose environments extrapolate present societies into futures technically, culturally and politically dominated by China or Japan, Such futures are portrayed as both exciting and threatening, to the point that the Japanese academic and cultural critic Toshiya Ueno used the term ‘Techno-Orientalism’ to describe the phenomenon. Nevertheless, whether Western interest is Orientalist or not, Asian architects are also increasingly looking to their own contemporary and future cultures for inspiration. This paper will discuss two manifestations of this. The first is Thai architect Sumet Jumsai’s Bank of Asia. Unlike contemporaneous English hightech buildings, with their coldly mechanistic representation of ducts and struts, Jumsai’s Bank of Asia, takes on the anthropomorphic character of Japanese scifi robots. It is endearing, friendly, even cute. The second example is what might be termed superflat architecture, from the term coined by the artist Takashi Murakami to describe an aesthetic of intrinsic flatness, eliminating depth in favour of skin and surface. The emergence of Techno-Cute and Superflat architecture suggest contemporary Asian architectural sensibilities that neither derive their aesthetic qualities solely from tradition nor from Western Modernism or Postmodernism.

Adamts5, the gene encoding a proteoglycan-degrading metalloprotease, is expressed by specific cell lineages during mouse embryonic development and in adult tissues.

The secreted metalloprotease ADAMTS5 is implicated in destruction of the cartilage proteoglycan aggrecan in arthritis, but its physiological functions are unknown. Its expression profile during embryogenesis and in adult tissues is therefore of considerable interest. β-Galactosidase (β-gal) histochemistry, enabled by a LacZ cassette inserted in the Adamts5 locus, and validated by in situ hybridization with an Adamts5 cRNA probe and ADAMTS5 immunohistochemistry, was used to profile Adamts5 expression during mouse embryogenesis and in adult mouse tissues. Embryonic expression was scarce prior to 11.5 days of gestation (E11.5) and noted only in the floor plate of the developing brain at E9.5. After E11.5 there was continued expression in brain, especially in the choroid plexus, peripheral nerves, dorsal root ganglia, cranial nerve ganglia, spinal and cranial nerves, and neural plexuses of the gut. In addition to nerves, developing limbs have Adamts5 expression in skeletal muscle (from E13.5), tendons (from E16.5), and inter-digital mesenchyme of the developing autopod (E13.5–15.5). In adult tissues, there is constitutive Adamts5 expression in arterial smooth muscle cells, mesothelium lining the peritoneal, pericardial and pleural cavities, smooth muscle cells in bronchi and pancreatic ducts, glomerular mesangial cells in the kidney, dorsal root ganglia, and in Schwann cells of the peripheral and autonomic nervous system. Expression of Adamts5 during neuromuscular development and in smooth muscle cells coincides with the broadly distributed proteoglycan versican, an ADAMTS5 substrate. These observations suggest the major contexts in which developmental and physiological roles could be sought for this protease.

Expression of the male reproduction-related gene (Mar-Mrr) in the spermatic duct of the giant freshwater prawn, Macrobrachium rosenbergii

Phosphorylated sperm proteins are crucial for sperm maturation and capacitation as a priori to their fertilization with eggs. In the freshwater prawn, Macrobrachium rosenbergii, a male reproduction-related protein (Mar-Mrr) was known to be expressed only in the spermatic ducts as a protein with putative phosphorylation and may be involved in sperm capacitation in this species. We investigated further the temporal and spatial expression of the Mar-Mrr gene using RT-PCR and in situ hybridization and the characteristics and fate of the protein using immunblotting and immunocytochemistry. The Mar-Mrr gene was first expressed in 4-week-old post larvae and the protein was produced in epithelial cells lining the spermatic ducts, at the highest level in the proximal region and decreased in the middle and distal parts. The native protein had a MW of 17 kDa and a high degree of serine/threonine phosphorylation. It was transferred from the epithelial cells to become a major protein at the anterior region of the sperm. We suggest that it is involved in sperm capacitation and fertilization in this open thelycal species and this is being investigated.

Characterisation and vascular expression of nitric oxide synthase 3 in amphibians

In mammals, nitric oxide (NO) produced by nitric oxide synthase 3 (NOS3) localised in vascular endothelial cells is an important vasodilator but the presence of NOS3 in the endothelium of amphibians has been concluded to be absent, based on physiological studies. In this study, a nos3 cDNA was sequenced from the toad, Rhinella marina. The open reading frame of R. marina nos3 encoded an 1170 amino acid protein that showed 81 % sequence identity to the recently cloned Xenopus tropicalis nos3. Rhinella marina nos3 mRNA was expressed in a range of tissues and in the dorsal aorta and pulmonary, mesenteric, iliac and gastrocnemius arteries. Furthermore, nos3 mRNA was expressed in the aorta of Xenopus laevis and X. tropicalis. Quantitative real-time PCR showed that removal of the endothelium of the lateral aorta of R. marina significantly reduced the expression of nos3 mRNA compared to control aorta with the endothelium intact. However, in situ hybridisation was not able to detect any nos3 mRNA in the dorsal aorta of R. marina. Immunohistochemistry using a homologous R. marina NOS3 antibody showed immunoreactivity (IR) within the basal region of many endothelial cells of the dorsal aorta and iliac artery. NOS3-IR was also observed in the proximal tubules and collecting ducts of the kidney but not within the capillaries of the glomeruli. This is the first study to demonstrate that vascular endothelial cells of an amphibian express NOS3.