Sphingosine 1-Phosphate Produced by Sphingosine Kinase 2 Intrinsically Controls Platelet Aggregation In Vitro and In Vivo.

Rationale: Platelets are known to play a crucial role in hemostasis. Sphingosine kinases (Sphk) 1 and 2 catalyze the conversion of sphingosine to the bioactive metabolite sphingosine 1-phosphate (S1P). Although platelets are able to secrete S1P on activation, little is known about a potential intrinsic effect of S1P on platelet function.

The direct renin inhibitor aliskiren localizes and persists in rat kidneys.

The aims of this study were to 1) determine whether renal localization of aliskiren and its antihypertensive and renoprotective effects persist after administration of the drug is stopped and 2) define the renal localization of aliskiren by light microscopy autoradiography. Hypertensive double transgenic rats (dTGR) overexpressing genes for human renin and angiotensinogen were treated with aliskiren (3 mg·kg(-1)·day(-1) sc; osmotic minipumps) or enalapril (18 mg/l in drinking water). After a 2-wk treatment, dTGR were assigned to either continued treatment with aliskiren ("continued"), or to cessation of their respective treatment ("stopped") for a 3-wk washout.

Blockade of the activin receptor IIb activates functional brown adipogenesis and thermogenesis by inducing mitochondrial oxidative metabolism.

Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue.

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells.

Preclinical evaluation of potential nilotinib cardiotoxicity.

In vitro, concentrations ≥ 10 μM of nilotinib were needed to induce markers of cytotoxicity, apoptosis, and endoplasmic reticulum stress in both neonatal rat ventricular myocytes, a putative target tissue, and non-target heart fibroblasts, indicating a lack of cardiomyocyte-specific nilotinib toxicity in vitro. In rats, oral nilotinib treatment at 80 mg/kg for 4 weeks induced increased heart weight; however, this was not associated with relevant histopathological changes or effects on heart function. Thus, nilotinib at and above clinically relevant concentrations (4.

Imatinib does not induce cardiotoxicity at clinically relevant concentrations in preclinical studies.

Cytotoxic concentrations of imatinib mesylate (10-50 microM) were required to trigger markers of apoptosis and endoplasmic reticulum stress response in neonatal rat ventricular myocytes and fibroblasts, with no significant differences observed between c-Abl silenced and nonsilenced cells. In mice, oral or intraperitoneal imatinib treatment did not induce cardiovascular pathology or heart failure. In rats, high doses of oral imatinib did result in some cardiac hypertrophy.

Effects of aliskiren on blood pressure, albuminuria, and (pro)renin receptor expression in diabetic TG(mRen-2)27 rats.

The aim of this study was to explore the effects of the renin inhibitor aliskiren in streptozotocin-diabetic TG(mRen-2)27 rats. Furthermore, we investigated in vitro the effect of aliskiren on the interactions between renin and the (pro)renin receptor and between aliskiren and prorenin. Aliskiren distributed extensively to the kidneys of normotensive (non)diabetic rats, localizing in the glomeruli and vessel walls after 2 hours exposure.

Brain penetration of the oral immunomodulatory drug FTY720 and its phosphorylation in the central nervous system during experimental autoimmune encephalomyelitis: consequences for mode of action in multiple sclerosis.

FTY720 [2-amino-2-[2-(4-octylphenyl) ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS).

Cell proliferation measurement in cecum and colon of rats using scanned images and fully automated image analysis: validation of method.

The purpose of this study was to establish and validate fully automatic measurement of cell proliferation on scanned images of rat cecum and colon. Tissue slides were taken from a 4-week mechanistic study and processed for BrdU immunohistochemistry. Four sections of the cecum and colon per slide were scanned with the Zeiss MIRAX SCAN and transferred to the Definiens eCognition Analyst LS5.

Penetration of ASM 981 in canine skin: a comparative study.

ASM 981 has been developed for topical treatment of inflammatory skin diseases. It specifically inhibits the production and release of pro-inflammatory cytokines. We measured the skin penetration of ASM 981 in canine skin and compared penetration in living and frozen skin.

Morphological and morphometric analysis of paclitaxel and docetaxel-induced peripheral neuropathy in rats.

The experimentally induced neurotoxic effects of paclitaxel and docetaxel have never been compared, since no animal models of docetaxel peripheral neurotoxicity have yet been reported. In this experiment, we examined the effect of the chronic administration of these two taxanes in the Wistar rat using neurophysiological, neuropathological and morphometrical methods. Our results showed that both paclitaxel and docetaxel induced a significant, equally severe and dose-dependent reduction in nerve conduction velocity.

Imaging of macrophages in soft-tissue infection in rats: relationship between ultrasmall superparamagnetic iron oxide dose and MR signal characteristics.

Purpose: To describe dose-dependent signal intensity (SI) characteristics of experimentally induced soft-tissue abscesses on 1.5-T T1- and T2*-weighted magnetic resonance (MR) images obtained 24 hours after administration of ultrasmall superparamagnetic iron oxide (USPIO) and to describe the relationship between SI and amount of USPIO uptake and macrophage iron content.

Materials And Methods: Local institutional review committee on animal care approved the experiments, which were performed according to the guidelines of the National Institutes of Health and the committee on animal research at our institution.

A comparative immunohistochemical study of spontaneous and chemically induced pheochromocytomas in B6C3F1 mice.

Spontaneously occurring and chemically induced pheochromocytomas are rare in mice. That the mouse pheochromocytoma is a more appropriate animal model than that of the rat for study of human medullary adrenal tumors has been suggested. The expression of phenylethanolamine-N-methyltransferase (PNMT), the enzyme responsible for production of epinephrine from norepinephrine, is common to both mouse and human pheochromocytomas.

Removal of anti-Galalpha1,3Gal xenoantibodies with an injectable polymer.

Preformed and elicited Ab's against the Galalpha1,3Gal terminating carbohydrate chains (alphaGal Ab's) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. alphaGal Ab's are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of alphaGal Ab's might be achieved in vivo by systemic administration of nonimmunogenic polyvalent alphaGal structures with high avidity for alphaGal Ab's.

Combination of low-dose valsartan and enalapril improves endothelial dysfunction and coronary reserve in Nomega-nitro-L-arginine methyl ester-treated spontaneously hypertensive rats.

Combination of nonhypotensive doses of valsartan and enalapril markedly improved survival (+87%) compared with untreated animals (37%) in spontaneously hypertensive rats (SHRs) with endothelial dysfunction. However, the combination had no effect on kidney function, and proteinuria persisted over the 12 weeks of the study. It was hypothesized that the greater survival was due to improvement in endothelial function or coronary vasculature despite blockade of nitric oxide synthase and high blood pressure.

PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration.

PTK787/ZK 222584 (1-[4-chloroanilino]-4-[4-pyridylmethyl] phthalazine succinate) is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases, active in the submicromolar range. It also inhibits other class III kinases, such as the platelet-derived growth factor (PDGF) receptor beta tyrosine kinase, c-Kit, and c-Fms, but at higher concentrations. It is not active against kinases from other receptor families, such as epidermal growth factor receptor, fibroblast growth factor receptor-1, c-Met, and Tie-2, or intracellular kinases such as c-Src, c-Abl, and protein kinase C-alpha.

Induction of apoptosis by the O-hydroxyethyl-D(Ser)(8)-cyclosporine A derivative SDZ IMM 125 in rat hepatocytes.

The immunosuppressive cyclosporine A derivative, O-hydroxyethyl-D(Ser)(8)-cyclosporine (SDZ IMM 125), was examined for its ability to induce apoptosis in rat hepatocytes cultured for 4 or 20 h. Four hours after SDZ IMM 125 treatment, chromatin condensation and fragmentation, and the number of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeled and Annexin V-positive cells increased dose dependently without any observable lactate dehydrogenase leakage. The activity of the cysteine protease, caspase-3, was increased, but not that of caspase-1 and -6.

Mechanisms of cyclosporine A-induced apoptosis in rat hepatocyte primary cultures.

In rat hepatocytes and isolated liver mitochondrial fractions, Cyclosporine A (CsA) is often used as a specific inhibitor of mitochondrial Ca(2+) release and as a specific blocker of mitochondrial membrane potential and permeability transition (MPT), which are all processes involved in the inhibition of apoptosis. However, neither inhibition nor induction of apoptosis by CsA has yet been described in the rat hepatocyte primary culture during incubation for 4 and 20 h. It was the purpose of the present study to examine by means of morphological and biochemical criteria the effects of CsA on apoptosis and to characterize the underlying mechanisms.

Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells.

Intestinal fatty acid-binding protein (I-FABP) is a cytosolic protein expressed at high levels (up to 2% of cytosolic proteins) in the small intestine epithelium. Despite cell transfection studies, its function is still unclear. Indeed, different effects on fatty acid metabolism depending on the cell type and the amount of I-FABP expressed have been reported.

Spontaneous malignant craniopharyngioma in an albino rat.

Craniopharyngioma is a rare neoplasm in the rat, and few cases have been described. These lesions are thought to originate from squamous cell remnants of Rathke's pouch, an evagination of primitive stomatodeum. This neoplasm is usually locally invasive, and neither cranial nor extracranial metastases have been described.

Spontaneous osteosarcoma of the meninges in an albino rat.

A search of the rat tumor literature revealed no papers on naturally occurring primary osteosarcoma of meninges. In humans, this tumor, arising primarily from the meninges, is unusual; only two cases have been reported. We report on a spontaneously occurring osteosarcoma arising from meninges in a 2-year-old female albino rat.

Automated counting of nuclei in series of conventional liver sections to differentiate hypertrophy and hyperplasia.

A method based on image analysis and applicable in pharmacology and toxicology is described that has been designed to meet the statistical requirements for the determination of liver hypertrophy or hyperplasia. An algorithm has been developed to detect and count fluorescent nuclei in Feulgen-stained liver sections (12,500 to 25,000 nuclei or 170 to 350 fields of observation per section within 2-4 hr) by means of fully automatic image analysis with the Leitz Texture Analysis System (TAS) (up to 23 sections in series on the microscope stage). The applicability of this method to conventionally derived, formaldehyde-fixed, tissue sections has been tested on 3-microns sections cut from archived paraplast blocks of already diagnosed materials.

Immunoelectron microscopic localization of cytochrome P-450 isoenzyme CYP4A1 in liver, ileum and kidney of nafenopin treated male rats.

A monoclonal antibody raised against and specific for cytochrome P-450 isoenzyme CYP4A1 was used to investigate the subcellular distribution of this enzyme in the liver, kidney and ileum of nafenopin treated rats by means of immunoelectron microscopy. In the liver and kidney, labelling was restricted to peroxisomes and mitochondria of hepatocytes and proximal tubular epithelial cells whereas in ileum, immunolabelling was exclusively detected in mitochondria of absorptive cells.

Comparative molecular neuroanatomy of cloned GABAA receptor subunits in the rat CNS.

gamma-Aminobutyric acidA (GABAA) receptors in the mammalian central nervous system (CNS) are members of a family of ligand-gated ion channels consisting of heterooligomeric glycoprotein complexes in synaptic and extrasynaptic membranes. Although molecular cloning studies have identified 5 subunits (with approximately 40% amino acid homology) and isoforms thereof (approximately 70% homology), namely alpha 1-6, beta 1-4, gamma 1-3, delta, and rho, the subunit composition and stoichiometry of native receptors are not known. The regional distribution and cellular expression of GABAA receptor messenger RNAs (mRNAs) in the rat CNS have now been investigated by in situ hybridization histochemistry with subunit-specific 35S-labelled oligonucleotide probes on adjacent cryostat sections.

In situ hybridization histochemistry reveals a diversity of GABAA receptor subunit mRNAs in neurons of the rat spinal cord and dorsal root ganglia.

The distribution and relative abundance of gene transcripts for diverse GABAA receptor subunits (alpha 1-3,5, beta 1-3, gamma 2) in neurons of the rat cervical spinal cord and dorsal root ganglia were determined by in situ hybridization histochemistry using 35S-labeled 60mer oligonucleotide probes. The receptor proteins (mapped by benzodiazepine receptor radioautography and immunohistochemistry with [3H]flumazenil and a monoclonal antibody for the beta 2 + beta 3 subunits, respectively) were most abundant in the dorsal horn (layers II and III) and in layer X around the central canal. Although diverse receptor subunit mRNAs were detected (to varying degrees) in neurons throughout layers II-X of the spinal cord, motoneurons in layer IX were particularly strongly labeled.