Age-Related Reference Intervals of the Main Biochemical and Hematological Parameters in C57BL/6J, 129SV/EV and C3H/HeJ Mouse Strains

Background

Although the mouse is the animal model most widely used to study the pathogenesis and treatment of human diseases, reference values for biochemical parameters are scanty or lacking for the most frequently used strains. We therefore evaluated these parameters in the C57BL/6J, 129SV/EV and C3H/HeJ mice.

Methodology/Principal Findings

We measured by dry chemistry 26 analytes relative to electrolyte balance, lipoprotein metabolism, and muscle/heart, liver, kidney and pancreas functions, and by automated blood counter 5 hematological parameters in 30 animals (15 male and 15 female) of each mouse strain at three age ranges: 1–2 months, 3–8 months and 9–12 months. Whole blood was collected from the retro-orbital sinus. We used quality control procedures to investigate analytical imprecision and inaccuracy. Reference values were calculated by non parametric methods (median and 2.5^th and 97.5^th percentiles). The Mann-Whitney and Kruskal-Wallis tests were used for between-group comparisons. Median levels of GLU, LDH, Chol and BUN were higher, and LPS, AST, ALP and CHE were lower in males than in females (p range: 0.05–0.001). Inter-strain differences were observed for: (1) GLU, t-Bil, K⁺, Ca⁺⁺, PO₄ ⁻ (p<0.05) and for TAG, Chol, AST, Fe⁺⁺ (p<0.001) in 4–8 month-old animals; (2) for CK, Crea, Mg⁺⁺, Na⁺⁺, K⁺, Cl⁻ (p<0.05) and BUN (p<0.001) in 2- and in 10–12 month-old mice; and (3) for WBC, RBC, HGB, HCT and PLT (p<0.05) during the 1 year life span.

Conclusion/Significance

Our results indicate that metabolic variations in C57BL/6J, 129SV/EV and C3H/HeJ mice after therapeutic intervention should be evaluated against gender- and age-dependent reference intervals.     

Purinoceptor-mediated calcium signaling in primary neuron-glia trigeminal cultures

Receptors for extracellular nucleotides (the P2X-calcium channels and the phospholipase C-coupled P2Y receptors) play key roles in pain signaling, but little is known on their function in trigeminal ganglia, whose hyperactivation leads to the development of migraine pain. Here we characterize calcium signaling via P2X(3) and P2Y receptors in primary mouse neuron-glia trigeminal cultures. Comparison with intact ganglion showed that, in dissociated cultures, sensory neurons retain, at least in part, their physical relationships with satellite glia. RT-PCR indicated expression of P2X(2)/P2X(3) (confirmed by immunocytochemistry) and of all cloned P2Y receptors. Single-cell calcium imaging with subtype-selective P2-agonists/antagonists revealed presence of functional neuronal P2X(3), as well as of ADP-sensitive P2Y(1,12,13) and UTP-activated P2Y(2)/P2Y(4) receptors on both neurons and glia. Calcium responses were much higher in glia, that also responded to UDP, suggesting functional P2Y(6) receptors. To study whether trigeminal ganglia P2 receptors are modulated upon treatment with pro-inflammatory agents, cultures were acutely (up to 3 min) or chronically (24 h) exposed to bradykinin. This resulted in potentiation of algogenic P2X(3) receptor-mediated calcium responses followed by their down-regulation at 24 h. At this exposure time, P2Y receptors responses in satellite glia were instead upregulated, suggesting a complex modulation of P2 receptors in pain signaling.     

Biogeography of sulfate-reducing prokaryotes in river floodplains

In this study, a large-scale field survey was conducted to describe the biogeography of sulfate-reducing prokaryotes (SRPs) in river floodplains. Fingerprints obtained with three methods, i.e. 16S rRNA gene-based oligonucleotide microarray, dsrB-based denaturing gradient gel electrophoresis (DGGE) and polar lipid-derived fatty acid (PLFA) analyses, were used as a proxy to describe the SRPs community diversity. Each set of profiles was subjected to a combined multivariate/correlation analysis in order to compare SRP community profiles and to highlight the environmental variables influencing the SRPs distribution along environmental gradients. Floodplain soils harbored distinct SRP communities displaying biogeographic patterns. Nearly all profiles from the tidal sites consistently separated from the nontidal sites, independently from the screening method and the multivariate statistics used. The distribution of the microarray/DGGE/PLFA-based fingerprints in the principal component plots could be correlated to eight soil variables, i.e. soil organic matter, total nitrogen, total phosphorous and total potassium, and extractable ammonium, nitrate, phosphate and sulfate, as well as seven pore water variables, i.e. phosphate, sulfate, sulfide, chloride, sodium, potassium and magnesium ions. Indication of a salinity- and plant nutrient-dependent distribution of SRPs related to Desulfosarcina, Desulfomonile and Desulfobacter was suggested by microarray, DGGE and PLFA analyses.     

Ubiquitin over-expression promotes E6AP autodegradation and reactivation of the p53/MDM2 pathway in HeLa cells

It has been established that intracellular ubiquitin pools are subject to regulatory constrains. Less certain is the mechanism by which the pool of conjugated ubiquitin shift in parallel with total ubiquitin, and how this type of regulation affects the flux of substrates through the pathway. In this study we demonstrate that ubiquitin over-expression promotes the destabilization of the ubiquitin protein ligase E6AP, by a mechanism involving self-ubiquitination, and the stabilization of p53. These results represent the very first evidence that the levels of a ubiquitin ligase can be regulated in vivo by ubiquitin abundance, supporting the idea that a strict interrelationship between pathway component activities and ubiquitin pool size exists. Interestingly, ubiquitin-induced p53 accumulation did not induce cell-cycle arrest, suggesting that although fluctuations of the intracellular ubiquitin content may actively modulate the level of regulatory proteins, this event is not per se sufficient to elicit a cellular response in terms of proliferation.     

Neural agrin controls maturation of the excitation-contraction coupling mechanism in human myotubes developing in vitro

The aim of this study was to elucidate the mechanisms responsible for the effects of innervation on the maturation of excitation-contraction coupling apparatus in human skeletal muscle. For this purpose, we compared the establishment of the excitation-contraction coupling mechanism in myotubes differentiated in four different experimental paradigms: 1) aneurally cultured, 2) cocultured with fetal rat spinal cord explants, 3) aneurally cultured in medium conditioned by cocultures, and 4) aneurally cultured in medium supplemented with purified recombinant chick neural agrin. Ca(2+) imaging indicated that coculturing human muscle cells with rat spinal cord explants increased the fraction of cells showing a functional excitation-contraction coupling mechanism. The effect of spinal cord explants was mimicked by treatment with medium conditioned by cocultures or by addition of 1 nM of recombinant neural agrin to the medium. The treatment with neural agrin increased the number of human muscle cells in which functional ryanodine receptors (RyRs) and dihydropyridine-sensitive L-type Ca(2+) channels were detectable. Our data are consistent with the hypothesis that agrin, released from neurons, controls the maturation of the excitation-contraction coupling mechanism and that this effect is due to modulation of both RyRs and L-type Ca(2+) channels. Thus, a novel role for neural agrin in skeletal muscle maturation is proposed.     

Overcoming hERG issues for brain-penetrating cathepsin S inhibitors: 2-cyanopyrimidines. Part 2

We describe here orally active and brain-penetrant cathepsin S selective inhibitors, which are virtually devoid of hERG K(+) channel affinity, yet exhibit nanomolar potency against cathepsin S and over 100-fold selectivity to cathepsin L. The new non-peptidic inhibitors are based on a 2-cyanopyrimidine scaffold bearing a spiro[3.5]non-6-yl-methyl amine at the 4-position. The brain-penetrating cathepsin S inhibitors demonstrate potential clinical utility for the treatment of multiple sclerosis and neuropathic pain.     

The problem of bone lamellation: An attempt to explain different proposed models

Collagen texture and osteocyte distribution were analyzed in human woven‐ and lamellar‐bone using scanning and transmission electron microscopy. We provide data substantiating the concept that lamellar bone is made up of an alternation of dense‐acellular lamellae and loose‐cellular lamellae, all exhibiting an interwoven texture of collagen fibers. An attempt is also made to explain how the present findings might conform to those of authors whose models propose orderly, geometric arrangements of collagen fibers inside bony lamellae. Such a comparison is possible because the present investigation analyzes split loose lamellae and tangentially‐sectioned dense lamellae. It emerged that only loose lamellae can be dissected, revealing a loose interwoven collagen texture and halved osteocyte lacunae. Dense lamellae cannot be split because of their compactness. The analysis of tangentially sectioned dense lamellae demonstrates that they consist of a network of interwoven collagen fiber bundles. Inside each bundle, collagen fibers run parallel to each other but change direction where they enter adjacent bundles, at angles as described by other authors whose TEM investigations were performed at a much higher magnification than those of the present study. Consequently, what these authors consider to be a lamella are, instead, bundles of collagen fibers inside a lamella. There is discussion of the role played by the manner of osteocyte‐recruitment in the deposition of lamellar‐ and woven‐bone and how the presence of these cells is crucial for collagen spatial arrangement in bone tissues. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

In Vivo Detection of Human TRPV6-Rich Tumors with Anti-Cancer Peptides Derived from Soricidin

Soricidin is a 54-amino acid peptide found in the paralytic venom of the northern short-tailed shrew (Blarina brevicauda) and has been found to inhibit the transient receptor potential of vallinoid type 6 (TRPV6) calcium channels. We report that two shorter peptides, SOR-C13 and SOR-C27, derived from the C-terminus of soricidin, are high-affinity antagonists of human TRPV6 channels that are up-regulated in a number of cancers. Herein, we report molecular imaging methods that demonstrate the in vivo diagnostic potential of SOR-C13 and SOR-C27 to target tumor sites in mice bearing ovarian or prostate tumors. Our results suggest that these novel peptides may provide an avenue to deliver diagnostic and therapeutic reagents directly to TRPV6-rich tumors and, as such, have potential applications for a range of carcinomas including ovarian, breast, thyroid, prostate and colon, as well as certain leukemia''s and lymphomas.     

The bone and the kidney

Renal tubular diseases may present with osteopenia, osteoporosis or osteomalacia, as a result of significant derangements in body electrolytes. In case of insufficient synthesis of calcitriol, as in renal failure, the more complex picture of renal osteodystrophy may develop. Hypothetically, also disturbed renal production of BMP-7 and Klotho could cause bone disease. However, the acknowledgment that osteocytes are capable of producing FGF23, a phosphaturic hormone at the same time modulating renal synthesis of calcitriol, indicates that it is also bone that can influence renal function. Importantly, a feed-back mechanism exists between FGF23 and calcitriol synthesis, while Klotho, produced by the kidney, determines activity and selectivity of FGF23. Identification of human diseases linked to disturbed production of FGF23 and Klotho underlines the importance of this new bone-kidney axis. Kidney and bone communicate reciprocally to regulate the sophisticated machinery responsible for divalent ions homeostasis and for osseous or extraosseous mineralisation processes.