Benzodiazepine Receptor and Thyroid Hormones: In Vivo and In Vitro Modulation

In rats rendered hyperthyroid by chronic treatment with L-triiodothyronine (T3) hormone there was a 21 and 27% decrease, respectively, in the number of binding sites for [3H]flunitrazepam ([3H]FNZ) and [3H]ethyl-beta-carboline-3-carboxylate ([3H]beta-CCE) without changes in affinity for the two ligands. Two weeks after thyroidectomy there was a 44% increase in [3H]FNZ sites and a 17% increase in [3H]beta-CCE binding sites. In vitro we found that T3 produces a decrease in Bmax and an increase in KD, both changes being characteristic of a mixed type of inhibition. Thyroid status dramatically affected the Ki of T3 in displacing [3H]FNZ from sites on isolated membranes of the cerebral cortex: in hypothyroid rats the Ki value was 0.9 microM, whereas in hyperthyroid rats, it was 83 microM, a 92-fold difference. In control rats, the Ki was 11 microM. These findings are discussed in relation to a possible modulation of benzodiazepine receptors by thyroid hormones.     

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo

Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.     

白藜芦醇对高脂饲养小鼠脂肪组织氧化还原状态及血脂的影响

本研究旨在探索白藜芦醇(RSV)对不同程度肥胖小鼠脂肪氧化应激状态和血脂的影响。高脂日粮(HFD)处理12周的昆明小鼠分为3类:肥胖抵抗(DIO-R)、中体重(Med)和肥胖(DIO),分别饲喂HFD、HFD+0.3 g/kg RSV和HFD+0.6 g/kg RSV日粮18周,并以正常日粮小鼠为对照。结果表明,0.6 g/kg RSV处理可显著降低DIO小鼠体重、腹脂率,显著提高脂肪组织抗氧化能力,改善血脂。0.3 g/kg RSV处理对DIO-R小鼠也有类似趋势,但0.6 g/kg RSV处理引起DIO-R小鼠脂肪组织抗氧化能力下降、血脂紊乱。总之,RSV在不同程度肥胖小鼠具有剂量特异性的氧化应激调控作用。     

Inactivation of Factor VIIa by Antithrombin In Vitro,Ex Vivo and In Vivo: Role of Tissue Factor and Endothelial Cell Protein C Receptor

Recent studies have suggested that antithrombin (AT) could act as a significant physiologic regulator of FVIIa. However, in vitro studies showed that AT could inhibit FVIIa effectively only when it was bound to tissue factor (TF). Circulating blood is known to contain only traces of TF, at best. FVIIa also binds endothelial cell protein C receptor (EPCR), but the role of EPCR on FVIIa inactivation by AT is unknown. The present study was designed to investigate the role of TF and EPCR in inactivation of FVIIa by AT in vivo. Low human TF mice (low TF, ∼1% expression of the mouse TF level) and high human TF mice (HTF, ∼100% of the mouse TF level) were injected with human rFVIIa (120 µg kg⁻¹ body weight) via the tail vein. At varying time intervals following rFVIIa administration, blood was collected to measure FVIIa-AT complex and rFVIIa antigen levels in the plasma. Despite the large difference in TF expression in the mice, HTF mice generated only 40–50% more of FVIIa-AT complex as compared to low TF mice. Increasing the concentration of TF in vivo in HTF mice by LPS injection increased the levels of FVIIa-AT complexes by about 25%. No significant differences were found in FVIIa-AT levels among wild-type, EPCR-deficient, and EPCR-overexpressing mice. The levels of FVIIa-AT complex formed in vitro and ex vivo were much lower than that was found in vivo. In summary, our results suggest that traces of TF that may be present in circulating blood or extravascular TF that is transiently exposed during normal vessel damage contributes to inactivation of FVIIa by AT in circulation. However, TF’s role in AT inactivation of FVIIa appears to be minor and other factor(s) present in plasma, on blood cells or vascular endothelium may play a predominant role in this process.     

Induction of Antiviral Activity In Vivo and In Vitro by Human Placenta Ribonucleic Acid Treated with Nitrous Acid

Induction of antiviral activity and interferon by human placenta ribonucleic acid deaminated with sodium nitrite (NO₂-RNA) was studied in vitro and in vivo. (1) Viral multiplication in diploid cells from human kidney (HK cells) was depressed by pretreatment with NO₂-RNA, but not by pretreatment with the original placenta RNA. (2) NO₂-RNA showed an interferon-inducing activity in rabbits and mice. (3) NO₂-RNA sedimenting in 18 S and 28 S regions showed a higher antiviral activity than that sedimenting in 4 S region.     

Regulation of Adipose Tissue Inflammation and Insulin Resistance by MAPK Phosphatase 5

Obesity and metabolic disorders such as insulin resistance and type 2 diabetes have become a major threat to public health globally. The mechanisms that lead to insulin resistance in type 2 diabetes have not been well understood. In this study, we show that mice deficient in MAPK phosphatase 5 (MKP5) develop insulin resistance spontaneously at an early stage of life and glucose intolerance at a later age. Increased macrophage infiltration in white adipose tissue of young MKP5-deficient mice correlates with the development of insulin resistance. Glucose intolerance in MKP5-deficient mice is accompanied by significantly increased visceral adipose weight, reduced AKT activation, enhanced p38 activity, and increased inflammation in visceral adipose tissue when compared with wild-type (WT) mice. Deficiency of MKP5 resulted in increased inflammatory activation in macrophages. These findings thus demonstrate that MKP5 critically controls inflammation in white adipose tissue and the development of metabolic disorders.     

In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

BackgroundSchistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.Conclusions/SignificanceOur data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.     

Neuroprotection by the N-Methyl-d-Aspartate Receptor Antagonist CGP 40116: In Vivo and In Vitro Studies

Abstract: The goal of this study was to evaluate the effects of a novel competitive N -methyl- d -aspartate (NMDA) receptor antagonist, d -( E )-2-amino-4-methyl-5-phosphono-3-pentoic acid (CGP 40116), on neuronal damage in vivo and in vitro. We studied 20 rabbits that underwent a 2-h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries followed by 4 h of reperfusion. Ten minutes after occlusion the animals were treated with either normal saline (n = 7) or CGP 40116 at two different doses (20 mg/kg, n = 6; 40 mg/kg, n = 7) administered over a 5-min period. Somatosensory evoked potentials were used to confirm adequate ischemia and neuronal injury was assessed by histopathology and magnetic resonance imaging. CGP 40116 decreased cortical ischemic neuronal damage by 74 and 77% (control, 37.8%± 13.1%; CGP 20 mg/kg, 9.9 ± 3.6%; CGP 40 mg/kg, 8.7 ± 3.7%; p < 0.01) and reduced cortical ischemic edema by 52 and 35% (control, 42.3 ± 10.4%; CGP 20 mg/kg, 20.1 ± 6.7%; CGP 40 mg/kg, 27.5 ± 13.3%; p < 0.05) but did not protect against striatal injury. We performed a second study using primary cell cultures from mouse neocortex to determine the effects of CGP 40116 on neuronal death induced by a 10-min exposure to 500 µ M NMDA or by 45 min of oxygen-glucose deprivation (OGD). Our results demonstrate that CGP 40116 was effective at attenuating neuronal death in a concentration-dependent manner (ED₅₀ of 3.2 µ M against NMDA toxicity and 23.1 µ M against OGD) as measured by lactate dehydrogenase levels 24 h after the insult. The neuroprotective effects of CGP 40116 in vivo and in vitro suggest it may be of potential clinical therapeutic value.     

Antagonistic Activity against Helicobacter Infection In Vitro and In Vivo by the Human Lactobacillus acidophilus Strain LB

The purpose of the present study was to examine the activity of the human Lactobacillus acidophilus strain LB, which secretes an antibacterial substance(s) against Helicobacter pylori in vitro and in vivo. The spent culture supernatant (SCS) of the strain LB (LB-SCS) dramatically decreased the viability of H. pylori in vitro independent of pH and lactic acid levels. Adhesion of H. pylori to the cultured human mucosecreting HT29-MTX cells decreased in parallel with the viability of H. pylori. In conventional mice, oral treatment with the LB-SCS protected against infection with Helicobacter felis. Indeed, at both 8 and 49 days post-LB-SCS treatment (29 and 70 days postinfection), inhibition of stomach colonization by H. felis was observed, and no evidence of gastric histopathological lesions was found. LB-SCS treatment inhibits the H. pylori urease activity in vitro and in H. pylori that remained associated with the cultured human mucosecreting HT29-MTX cells. Moreover, a decrease in urease activity was detected in the stomach of the mice infected with H. felis and treated with LB-SCS.     

Effects of Lead In Vivo and In Vitro on GABAergic Neurochemistry

Abstract: Alterations in aspects of neurotransmission utilizing -γ-aminobutyric acid (GABA) are associated with in vivo exposure of rats to lead at doses that do not produce convulsions, but sensitize animals to convulsant agents. These effects are observed regionally and include: decreased GABA levels in cerebellum; increased activity of glutamate decarboxylase (GAD) in caudate; and decreased GABA release (both resting and K⁺-stimulated) in cortex, caudate, cerebellum and substantia nigra. Sodium-dependent uptake of GABA by synaptosomes of cerebellum, substantia nigra and caudate was also affected: in these regions, affinity (K_m) was increased and maximal velocity (V_max) was reduced. Sodium-independent binding of GABA to synaptic membranes was increased in cerebellum, but was observed only when tissue was Tritonized and prepared without freezing and washing. No effects on GAD or on GABA uptake, release, or binding were observed when lead was added to brain tissue in vitro in concentrations as high as 100 μM. The results suggest that lead may produce chronic inhibition of presynaptic GABAergic function, notably in the cerebellum, which is associated with supersensitivity of postsynaptic GABA receptors. Failure of lead to affect GABAergic function in vitro may indicate that these effects are secondary to another neurotoxic action of lead in the CNS or are consequent to a nonneuronal metabolic action of lead.     

Effects of Propyzamide on Tobacco Cell Microtubules In Vivo and In Vitro

Treatment with propyzamide at 2 ? 10^-6 M or at higher concentrationsarrested the cell cycleat metaphase in tobacco BY-2 cells. Metaphasecells having disorganized spindle microtubulesand scatteredchromosomes began to appear within several minutes of the additionof propyzamide. Within 30 min, disrupted spindle microtubulesand dispersed chromosomes were seenin all metaphase cells. Propyzamideat 2 ? 10^-6 M or at higher concentrations also disrupted corticalmicrotubules, but disruption of cortical microtubules requiredmore time than disruption of spindle microtubules. The effectof propyzamide on microtubules was found to be readily reversible.The cells arrested at metaphase by 2 ? 10^-6 M propyzamide resumedmitosis within 2 h from the termination of treatment with propyzamide.Spindle microtubules reappeared within 15 min from the terminationof treatment with propyzamide, and the cortical microtubuleswithin 1 h. Tubulin was isolated from tobacco BY-2 cells bycolumn chromatography on ethyl Nphenylcarbamate-Sepharose 4B.On incubation with EGTA, Mg²⁺ and DMSO, the purified tobaccotubulin polymerized into microtubules. Propyzamide at 1 ? 10^-4M completely inhibitedthe polymerization of tobacco tubulin,but did not inhibit polymerization of bovine braintubulin. Tobaccotubulin was adsorbed onto a column of propyzamide-analogue-linkedSepharose 4B and then purified by chromatography on this column. (Received February 15, 1988; Accepted June 29, 1988)     

TBK1 at the Crossroads of Inflammation and Energy Homeostasis in Adipose Tissue

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Ornithine Decarboxylase Activity in In Vivo and In Vitro Models of Cerebral Ischemia

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis, and an increase in putrescine after central nervous system (CNS) injury appears to be involved in neuronal death. Cerebral ischemia and reperfusion trigger an active series of metabolic events, which eventually lead to neuronal death. In the present study, ODC activity was evaluated following transient focal cerebral ischemia and reperfusion in rat. The middle cerebral artery (MCA) was occluded for 2 h in male rats with an intraluminal suture technique. Animals were sacrificed between 3 and 48 h of reperfusion following MCA occlusion, and ODC activity was assayed in cortex and striatum. ODC activity was also estimated in an in vitro ischemia model using primary rat cortical neuron cultures, at 6–24 h reoxygenation following 1 h oxygen-glucose deprivation (OGD). In cortex, following ischemia, ODC activity was increased at 3 h (P < .05), reached peak levels by 6–9 h (P < .001) and returned to sham levels by 48 h reperfusion. In striatum the ODC activity followed a similar time course, but returned to basal levels by 24 h. This suggests that ODC activity is upregulated in rat CNS following transient focal ischemia and its time course of activation is region specific. In vitro, ODC activity showed a significant rise only at 24 h reoxygenation following ischemic insult. The release of lactate dehydrogenase (LDH), an indicator for cell damage, was also significantly elevated after OGD. 0.25 mM -difluoromethylornithine (DFMO) inhibited ischemia-induced ODC activity, whereas a 10-mM dose of DFMO appears to provide some neuroprotection by suppressing both ODC activity and LDH release in neuronal cultures, suggesting the involvement of polyamines in the development of neuronal cell death.     

In Vitro and in Vivo Effects of Nitrofurantoin on Experimental Toxoplasmosis

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.     

The Additive Inflammatory In Vivo and In Vitro Effects of IL-7 and TSLP in Arthritis Underscore the Therapeutic Rationale for Dual Blockade

Introduction

The cytokines interleukin (IL)-7 and thymic stromal lymphopoietin (TSLP) signal through the IL-7R subunit and play proinflammatory roles in experimental arthritis and rheumatoid arthritis (RA). We evaluated the effect of inhibition of IL-7R- and TSLPR-signalling as well as simultaneous inhibition of IL-7R- and TSLPR-signalling in murine experimental arthritis. In addition, the effects of IL-7 and TSLP in human RA dendritic cell (DC)/T-cell co-cultures were studied.

Methods

Arthritis was induced with proteoglycan in wildtype mice (WT) and in mice deficient for the TSLP receptor subunit (TSLPR-/-). Both mice genotypes were treated with anti-IL-7R or phosphate buffered saline. Arthritis severity was assessed and local and circulating cytokines were measured. Autologous CD1c-positive DCs and CD4 T-cells were isolated from peripheral blood of RA patients and were co-cultured in the presence of IL-7, TSLP or both and proliferation and cytokine production were assessed.

Results

Arthritis severity and immunopathology were decreased in WT mice treated with anti-IL-7R, in TSLPR-/- mice, and the most robustly in TSLPR-/- mice treated with anti-IL-7R. This was associated with strongly decreased levels of IL-17, IL-6 and CD40L. In human DC/T-cell co-cultures, TSLP and IL-7 additively increased T-cell proliferation and production of Th17-associated cytokines, chemokines and tissue destruction factors.

Conclusion

TSLP and IL-7 have an additive effect on the production of Th17-cytokines in a human in vitro model, and enhance arthritis in mice linked with enhanced inflammation and immunopathology. As both cytokines signal via the IL-7R, these data urge for IL-7R-targeting to prevent the activity of both cytokines in RA.     

Cellular Origin of Ischemia-Induced Glutamate Release from Brain Tissue In Vivo and In Vitro

The uptake and release of D-[3H]aspartate (used as a tracer for endogenous glutamate and aspartate) were studied in cultured glutamatergic neurons (cerebellar granule cells) and astrocytes at normal (5 mM) or high (55 mM) potassium and under conditions of hypoglycemia, anoxia or "ischemia" (combined hypoglycemia and anoxia). In glutamatergic neurons it was found that "ischemic" conditions led to a 2.4-fold increase in the potassium-induced release of D-[3H]aspartate as compared to normal conditions. Hypoglycemia or anoxia alone affected the release only marginally. The ischemia-induced induced increase in the evoked D-[3H]aspartate release was shown to be calcium-dependent. In astrocytes no difference was found in the potassium-induced release between the four conditions and the K+-induced release was not calcium-dependent. The uptake of D-[3H]aspartate was found to be stimulated at high potassium in both glutamatergic neurons (98%) and in astrocytes (70%). This stimulation of D-aspartate uptake, however, was significantly reduced under conditions of anoxia or "ischemia" in both cell types. In glutamatergic neurons (but not in astrocytes) hypoglycemia also decreased the potassium stimulation of D-aspartate uptake. In a previous report it was shown, using the microdialysis technique, that during transient cerebral ischemia in vivo the extracellular glutamate content in hippocampus was increased eightfold. In the present paper it is shown that essentially no increase in extracellular glutamate is seen under ischemia when the perfusion is performed using calcium-free, cobalt-containing perfusion media. The results from the in vitro and in vivo experiments indicate that the glutamate accumulated extracellularly under ischemia in vivo originates from transmitter pools in glutamatergic neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of spironolactone and RU486 on gene expression and cell proliferation after freshwater transfer in the euryhaline killifish

We have explored the possible mechanisms by which mineralocorticoid (MR) and glucocorticoid (GR) receptors regulate the response to freshwater transfer in the gills of the euryhaline killifish Fundulus heteroclitus. Killifish were implanted with RU486 (GR antagonist) or spironolactone (MR antagonist) at doses of 0.1–1.0 mg g⁻¹, and subsequently transferred from 10‰ brackish water to freshwater. Compared to brackish water sham fish, mRNA expression of CFTR and NKCC1 decreased in the gills of sham fish transferred to freshwater, whereas Na⁺,K⁺–ATPase α_1a mRNA expression and α protein abundance, as well as cell proliferation (detected using BrdU) increased. Spironolactone inhibited the normal increase in cell proliferation and Na⁺,K⁺-ATPase expression after freshwater transfer. RU486 increased plasma cortisol levels and may have slightly inhibited Na⁺,K⁺–ATPase activity, but did not change α _1a expression. RU486 had no effect on cell proliferation in the non-lamellar region of the gills, but increased proliferation in the lamellar region. Neither antagonist inhibited the suppression of CFTR or NKCC1 expression after freshwater transfer. Glucocorticoid receptor expression was reduced in all sham and antagonist treatments compared to untreated controls, but no other consistent differences were observed. The effects of spironolactone suggest that MR is important for regulating ion transport in killifish gills after freshwater transfer.     

Antitumor Activity of a Polypyridyl Chelating Ligand: In Vitro and In Vivo Inhibition of Glioma

Glioblastoma multiforme is an extremely aggressive and invasive form of central nervous system tumor commonly treated with the chemotherapeutic drug Temozolomide. Unfortunately, even with treatment, the median survival time is less than 12 months. 2,9-Di-sec-butyl-1,10-phenanthroline (SBP), a phenanthroline-based ligand originally developed to deliver gold-based anticancer drugs, has recently been shown to have significant antitumor activity in its own right. SBP is hypothesized to initiate tumor cell death via interaction with non-DNA targets, and considering most glioblastoma drugs kill tumors through DNA damage processes, SBP was tested as a potential novel drug candidate against glial-based tumors. In vitro studies demonstrated that SBP significantly inhibited the growth of rodent GL-26 and C6 glioma cells, as well as human U-87, and SW1088 glioblastomas/astrocytomas. Furthermore, using a syngeneic glioma model in mice, in vivo administration of SBP significantly reduced tumor volume and increased survival time. There was no significant toxicity toward nontumorigenic primary murine and human astrocytes in vitro, and limited toxicity was observed in ex vivo tissues obtained from noncancerous mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling staining and recovery assays suggest that SBP induces apoptosis in gliomas. This exploratory study suggests SBP is effective in slowing the growth of tumorigenic cells in the brain while exhibiting limited toxicity to normal cells and tissues and should therefore be further investigated for its potential in glioblastoma treatment.     

Caloric Restriction and Diet-Induced Weight Loss Do Not Induce Browning of Human Subcutaneous White Adipose Tissue in Women and Men with Obesity

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