Autosomal Dominant Hypercalciuria in a Mouse Model Due to a Mutation of the Epithelial Calcium Channel,TRPV5

Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5 ^682P/+) and homozygous (Trpv5 ^682P/682P) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and ∼10% of males developed tubulointerstitial nephritis. Trpv5 ^682P/682P mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D₃ concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5 ^682P/+ and Trpv5 ^682P/682P mice consistent with a trafficking defect. In addition, Trpv5^682P/682P mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D_28K, consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria.     

Effects of cimetidine-like drugs on recombinant GABAA receptors

Even though conventional systemic doses of cimetidine and other histamine H(2) antagonists display minimal brain penetration, central nervous system (CNS) effects (including seizures and analgesia) have been reported after administration of these drugs in animals and man. To test the hypothesis that cimetidine-like drugs produce these CNS effects via inhibition of GABA(A) receptors, the actions of these drugs were studied on seven different, precisely-defined rat recombinant GABA(A) receptors using whole-cell patch clamp recordings. The H(2) antagonists famotidine and tiotidine produced competitive and reversible inhibition of GABA-evoked currents in HEK293 cells transfected with various GABA(A) receptor subunits (IC(50) values were between 10-50 microM). In contrast, the H(2) antagonist ranitidine and the cimetidine congener improgan had very weak (if any) effects (IC(50) > 50 microM). Since the concentrations of cimetidine-like drugs required to inhibit GABA(A) receptors in vitro (greater than 50 microM) are considerably higher than those found during analgesia and/or seizures (1-2 microM), the present results suggest that cimetidine-like drugs do not appear to produce seizures or analgesia by directly inhibiting GABA(A) receptors.     

The avian hippocampus, homing in pigeons and the memory representation of large-scale space

The extraordinary navigational ability of homing pigeons providesa unique spatial cognitive system to investigate how the brainis able to represent past experiences as memory. In this paper,we first summarize a large body of lesion data in an attemptto characterize the role of the avian hippocampal formation(HF) in homing. What emerges from this analysis is the criticalimportance of HF for the learning of map-like, spatial representationsof environmental stimuli used for navigation. We then exploresome interesting properties of the homing pigeon HF, using fordiscussion the notion that the homing pigeon HF likely displayssome anatomical or physiological specialization(s), comparedto the laboratory rat, that account for its participation inhoming and the representation of large-scale, environmentalspace. Discussed are the internal connectivity among HF subdivisions,the occurrence of neurogenesis, the presence of rhythmic thetaactivity and the electrophysiological profile of HF neurons.Comparing the characteristics of the homing pigeon HF with thehippocampus of the laboratory rat, two opposing perspectivescan be supported. On the one hand, one could emphasize the subtledifferences in the properties of the homing pigeon HF as possibledeparture points for exploring how the homing pigeon HF maybe adapted for homing and the representation of large-scalespace. Alternatively, one could emphasize the similarities withthe rat hippocampus and suggest that, if homing pigeons representspace in a way different from rats, then the neural specializationsthat would account for the difference must lie outside HF. Onlyfuture research will determine which of these two perspectivesoffers a better approximation of the truth.     

An anion binding site in the active centre of phospholipase C from Bacillus cereus

The bi-Zn2+-enzyme phospholipase C (Bacillus cereus) is readilly inhibited by univalent anions. N.m.r. studies on the 113Cd-substituted enzyme showed the presence of an inert and a perturbable metal, neither of which seemed affected by I-. X-ray crystallographic analysis showed the binding of one I- to the enzyme 4.8 A from the nearest metal (too far for a metal-halide bond). Phospholipase C contains an arginine residue apparently necessary for substrate binding and I- partially protected against inactivation by an arginine reagent. Thus an arginine residue may represent the binding site for univalent anions in the enzyme active centre.     

An HIV-1 tat-autoantigen fusion protein suppresses insulitis in NOD mice

To assess the immunomodulatory activity of the HIV Tat transduction peptide for enhancement of suppression of Type 1 autoimmune diabetes, the 11 amino acid HIV-1 Tat transduction peptide was genetically linked to the major islet autoantigens proinsulin (INS) and glutamic acid decarboxylase (GAD). The Tat-autoantigen fusion proteins were synthesized in Escherichia coli and characterized by acrylamide gel separation and immunoblot analysis. Histological examination of pancreatic islets isolated from juvenile NOD mice inoculated orally with the Tat-autoantigen conjugates revealed a significant reduction in islet inflammation (insulitis) in comparison with islets from unimmunized mice. Increased serum IgG1 antibody isotype titers detected in Tat-autoantigen inoculated mice suggest that the transduction peptide-autoantigen fusion proteins stimulate Th2 lymphocyte mediated bystander suppression. The reduction of islet insulitis observed in Tat-autoantigen inoculated mice suggests that the adjuvant effect of the Tat transduction peptide resides in Tat enhanced delivery of linked autoantigens through enterocytes to lymphocytes in the gut-associated lymphoid tissues.     

High-resolution atomic force microscopy of soluble Abeta42 oligomers

Soluble oligomers and protofibrils are widely thought to be the toxic forms of the Abeta42 peptide associated with Alzheimer's disease. We have investigated the structure and formation of these assemblies using a new approach in atomic force microscopy (AFM) that yields high-resolution images of hydrated proteins and allows the structure of the smallest molecular weight (MW) oligomers to be observed and characterized. AFM images of monomers, dimers and other low MW oligomers at early incubation times (< 1h) are consistent with a hairpin structure for the monomeric Abeta42 peptide. The low MW oligomers are relatively compact and have significant order. The most constant dimension of these oligomers is their height (approximately 1-3 nm) above the mica surface; their lateral dimensions (width and length) vary between 5 nm and 10nm. Flat nascent protofibrils with lengths of over 40 nm are observed at short incubation times (< or = 3h); their lateral dimensions of 6-8 nm are consistent with a mass-per-length of 9 kDa/nm previously predicted for the elementary fibril subunit. High MW oligomers with lateral dimensions of 15-25 nm and heights ranging from 2-8 nm are common at high concentrations of Abeta. We show that an inhibitor designed to block the sheet-to-sheet packing in Abeta fibrils is able to cap the heights of these oligomers at approximately 4 nm. The observation of fine structure in the high MW oligomers suggests that they are able to nucleate fibril formation. AFM images obtained as a function of incubation time reveal a sequence of assembly from monomers to soluble oligomers and protofibrils.     

A Mouse Model for Osseous Heteroplasia

GNAS/Gnas encodes G_sα that is mainly biallelically expressed but shows imprinted expression in some tissues. In Albright Hereditary Osteodystrophy (AHO) heterozygous loss of function mutations of GNAS can result in ectopic ossification that tends to be superficial and attributable to haploinsufficiency of biallelically expressed G_sα. Oed-Sml is a point missense mutation in exon 6 of the orthologous mouse locus Gnas. We report here both the late onset ossification and occurrence of benign cutaneous fibroepithelial polyps in Oed-Sml. These phenotypes are seen on both maternal and paternal inheritance of the mutant allele and are therefore due to an effect on biallelically expressed G_sα. The ossification is confined to subcutaneous tissues and so resembles the ossification observed with AHO. Our mouse model is the first with both subcutaneous ossification and fibroepithelial polyps related to G_sα deficiency. It is also the first mouse model described with a clinically relevant phenotype associated with a point mutation in G_sα and may be useful in investigations of the mechanisms of heterotopic bone formation. Together with earlier results, our findings indicate that G_sα signalling pathways play a vital role in repressing ectopic bone formation.