Tissue Wet Weight Research Articles

The Effect of Supplemental Ascorbate on Defersirox Iron Chelation In the Iron-Loaded Gerbil

AbstractAbstract 2059 Introduction:Iron dextran injections are often used to induce iron overload in rodents, for the purposes of assessing iron chelation therapy. In gerbils, we have previously described that deferasirox therapy preferentially clears hepatocellular iron when compared with reticuloendothelial stores. Ascorbate deficiency, which is common in humans with iron overload, produces similar profound disparities between reticuloendothelial and parenchymal iron stores. We postulated that iron-induced ascorbate deficiency might be exaggerating reticuloendothelial iron retention in gerbils receiving deferasirox therapy. This study examined the effect of supplemental ascorbate on spontaneous iron loss and deferasirox chelation efficiency in the iron-dextran loaded gerbil. Methods:48 female gerbils underwent iron dextran loading at 200 mg/kg/week for 10 weeks. Sixteen animals were sacrificed at 11 weeks to characterize iron loading; eight were on standard rodent chow and eight had chow supplemented with 2250 ppm of ascorbate. 32 additional animals that were not ascorbate supplemented during iron loading transitioned into the chelation phase. Half were subsequently placed on ascorbate supplemented chow and both groups were assigned to receive either deferasirox 100 mg/kg/day five days per week or sham chelation. Animals received iron chelation for twelve weeks. Liver histology was assessed using H & E and Prussian blue stains. Iron loading was ranked and graded on a five-point scale by an experienced pathologist screened to the treatment arm. Iron quantitation in liver and heart was performed by atomic absorption. Results:Table 1 one summarizes the findings. During iron dextran loading, ascorbate supplementation lowered wet weight liver iron concentration but not liver iron content suggesting primarily changes in tissue water content. Spontaneous iron losses were insignificant, regardless of ascorbate therapy. Deferasirox lowered liver iron content 56% (4.7% per week) in animals without ascorbate supplementation and 48.3% (4.0% per week) with ascorbate supplementation (p=NS).Cardiac iron loading, unloading and redistribution were completely unaffected by ascorbate supplementation. Spontaneous iron redistribution was large (1.9% – 2.3% per week). Deferasirox chelation did not lower cardiac iron to a greater degree than spontaneous cardiac iron redistribution.Histologic grading paralleled tissue wet weight iron concentrations. Ascorbate treatment lowered the rank and absolute iron score in liver during iron loading (p=0.003) and there was a trend toward lower iron scoring in sham treated animals (p=0.13). Ascorbate had no effect on histological score or relative compartment distributions of iron in deferasirox chelated animals (p=0.5).Ascorbate supplementation was sufficient to increase total plasma ascorbate levels from 25 ± 12.2 uM to 38.4 ± 11 uM at 10 weeks (p=0.03). In the liver, ascorbate increased from 1203 ± 212 nmol/g of tissue to 1515 ± 194 nmol/g of tissue (p=0.01) with supplementation. No significant change in total ascorbate was observed in the heart. Discussion:We hypothesized that ascorbate supplementation might improve reticuloendothelial iron accessibility to deferasirox by facilitating redox cycling. Although gerbils synthesize their own ascorbate, supplementation was able to raise both serum and hepatic total ascorbate levels. However, increasing ascorbate did not change either the amount or distribution of tissue iron in deferasirox-treated animals. Disclosures:Nick:Novartis: Employment. Wood:Novartis: Research Funding; Ferrokin Biosciences: Consultancy.

Brown adipose tissue: is it affected by intermittent hypoxia?

BackgroundIntermittent hypoxia (IH), a model of sleep apnea, produces weight loss in animals. We hypothesized that changes in brown adipose tissue (BAT) function are involved in such phenomenon. We investigated the effect of IH, during 35 days, on body weight, brown adipose tissue wet weight (BATww) and total protein concentration (TPC) of BAT.MethodsWe exposed Balb/c mice to 35 days of IH (n = 12) or sham intermittent hypoxia (SIH; n = 12), alternating 30 seconds of progressive hypoxia to a nadir of 6%, followed by 30 seconds of normoxia. During 8 hours, the rodents underwent a total of 480 cycles of hypoxia/reoxygenation, equivalent to an apnea index of 60/hour. BAT was dissected and weighed while wet. Protein was measured using the Lowry protein assay.ResultsBody weight was significantly reduced in animals exposed to IH, at day 35, from 24.4 ± 3.3 to 20.2 ± 2.2 g (p = 0.0004), while in the SIH group it increased from 23.3 ± 3.81 to 24.1 ± 2.96 g (p = 0.23). BATww was also lower in IH than in SIH group (p = 0.00003). TPC of BAT, however, was similar in IH (204.4 ± 44.3 μg/100 μL) and SIH groups (213.2 ± 78.7 μg/100 μL; p = 0.74) and correlated neither with body weight nor with BATww. TPC appeared to be unaffected by exposure to IH also in multivariate analysis, adjusting for body weight and BATww. The correlation between body weight and BATww is significant (rho= 0.63) for the whole sample. When IH and SIH groups are tested separately, the correlations are no longer significant (rho= 0.48 and 0.05, respectively).ConclusionIH during 35 days in a mice model of sleep apnea causes weight loss, BATww reduction, and no change in TPC of BATww. The mechanisms of weight loss under IH demands further investigation.

Attenuation of store‐operated Ca2+ entry and enhanced expression of TRPC channels in caudal artery smooth muscle from Type 2 diabetic Goto‐Kakizaki rats

1. Previously, we found that Ca(2+) entry from the extracellular space via alpha(1)-adrenoceptor-activated, Ca(2+)-permeable channels, but not voltage-gated Ca(2+) channels, is impaired in endothelium-denuded caudal artery smooth muscle from Type 2 diabetic Goto-Kakizaki (GK) rats. In the present study, we investigated the impairment of Ca(2+) entry mechanisms via Ca(2+)-permeable channels from the extracellular space in response to alpha(1)-adrenoceptor stimulation (cirazoline) in endothelium-denuded caudal artery strips isolated from GK rats. 2. The contraction of caudal artery strips from GK rats in response to the sarcoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid (10 micromol/L), which causes depletion of Ca(2+) stores and subsequent store-operated Ca(2+) (SOC) entry, was significantly depressed compared with that of Wistar rats (maximal force 0.023 +/- 0.004 vs 0.058 +/- 0.005 mN/mg tissue wet weight, respectively). These results suggest that receptor-activated Ca(2+) entry through SOC channels is impaired in caudal artery smooth muscle in GK rats. 3. The classic transient receptor potential (TRPC) channels, which constitute SOC and receptor-operated cation channels, play an important role in Ca(2+) regulation. Therefore, we investigated the mRNA and protein expression of TRPC channels in caudal artery smooth muscle from Wistar and GK rats using reverse transcription-polymerase chain reaction and immunoblotting. 4. Expression of TRPC1, TRPC3 and TRPC6 mRNA and protein was found in Wistar rats. However, in GK rats, in addition to the expression of these TRPC channels, mRNA and protein expression of TRPC4 was found. The expression of TRPC1 and TRPC6, but not TRPC3, was increased approximately twofold in GK rats compared with Wistar rats. 5. These results suggest that changes in TRPC channel expression may be responsible, in part, for the dysfunction of receptor-mediated Ca(2+) entry in caudal artery smooth muscle of GK rats.

Efficacy of Tyrosine Hydroxylase gene modified neural stem cells derived from bone marrow on Parkinson's disease – a rat model study

The aim of this study is to determine the efficacy of injecting adult bone marrow derived stem cells (BMSCs) transfected with a pEGFP-C2 plasmid containing the gene for Tyrosine Hydroxylase (TH) into the lateral ventricle for treating rats with Parkinson's Disease (PD) induced by injections into the Substantia Nigra pars compacta (SNc) with 6-hydroxydopamine (6-OHDA), a potent and selective neurotoxin for catecholamine expressing neurons. BMSCs were obtained from the femur of rats; transfected with plasmid constructed with TH and green fluorescent protein (GFP) (with about 85% co-transfection efficiency rate) and then cultured with neuronal differentiation media. Eighty rats were injected into the SNc with 6-OHDA and tested behaviorally to verify the model was induced. Then, 12 PD rats were injected into the anterior horn of the lateral ventricle with x10 5 cells, while 12 more rats were given saline as control. We found that 10 days after transplantation there was a significant (P < 0.01) reduction in Apomorphine induced rotations in rats receiving transplanted cells. Also, combined SNc and Striatal dopamine contents (μg/g wet tissue weight) in transplanted rats were greater than controls (0.19 ± 0.06 vs 0.63 ± 0.14 P < 0.01). Immunohistological examination found GFP expression, indicating the presence of transplanted cells within the brain, some of which had migrated through the nerve fibers along the ventricular wall. We feel this study shows the efficacy of genetically engineered BMSCs in the treatment of a rat model of PD. However, future experiments are needed to determine the mechanisms.

Pathologic survey on the Manila clam Ruditapes philippinarum (Adams and Reeve 1850) from Haeju off the western coastal Yellow Sea

Pathologic condition of Manila clam, Ruditapes philippinarum collected from Haeju off the west coast of North Korea in February and March 2007 was investigated in this study. The diagnostics included a protozoan parasite, Perkinsus olseni, the brown ring disease (BRD) caused by bacteria Vibrio tapetis and metazoan parasites Trematod and Cestod. P. olseni infection was examined using histology and Ray’s fluid thioglycollate medium (RFTM) assay along with Choi’s NaOH digestion technique. Trematode and cestode infection was also examined from the histology. A total of 140 clams with 35–45 mm in shell length (2–3 years old) were analyzed. Condition factor (CF), a ratio of the wet tissue weight to the shell dry weight, ranged from 0.445±0.074 (February) to 0.544±0.132 (March). Total body burden of P. olseni (i.e. infection intensity) ranged from 18,644±37,755 (February) to 26,933±80,611 cells/g wet tissue (March) with the prevalence of 76.7–43.3% (March) respectively. Prevalence of trematode and cestode infection in February and March varied 22.5–25.0 and 15.0–22.5% respectively. Infection intensity of V. tapetis ranged 1–2 and the prevalence was 1.4 (February) and 5.7% (March). Prevalence and infection intensity of P. olseni in clams from Haeju was comparatively lower than those of clams reported on the west coast of Korea. BRD and the other metazoan parasite infection were also relatively lower or similar to the condition of the clams reported previously. CF of Haeju clams was also relatively higher than the clams in Gyeonggi Bay and Taean, suggesting that Haeju clams had relatively better pathologic and health condition.

Cyclodextrin overcomes the transport defect in nearly every organ of NPC1 mice leading to excretion of sequestered cholesterol as bile acid

A mutation in NPC1 leads to sequestration of unesterified cholesterol in the late endosomal/lysosomal compartment of every cell culminating in the development of pulmonary, hepatic, and neurodegenerative disease. Acute administration of 2-hydroxypropyl-beta-cyclodextrin (CYCLO) rapidly overcomes this transport defect in both the 7-day-old pup and 49-day-old mature npc1(-/-) mouse, even though this compound is cleared from the body and plasma six times faster in the mature mouse than in the neonatal animal. The liberated cholesterol flows into the cytosolic ester pool, suppresses sterol synthesis, down-regulates SREBP2 and its target genes, and reduces expression of macrophage-associated inflammatory genes. These effects are seen in the liver and brain, as well as in peripheral organs like the spleen and kidney. Only the lung appears to be resistant to these effects. Forty-eight h after CYCLO administration to the 49-day-old animals, fecal acidic, but not neutral, sterol output increases, whole-animal cholesterol burden is reduced, and the hepatic and neurological inflammation is ameliorated. However, lifespan is extended only when the CYCLO is administered to the 7-day-old animals. These studies demonstrate that CYCLO administration acutely reverses the cholesterol transport defect seen in the NPC1 mouse at any age, and this reversal allows the sequestered sterol to be excreted from the body as bile acid.

Abstract 2319: Increased hyaluronan (HA) levels are associated with tumor invasion in murine lymphoma cell lines

Abstract HA is a glycosaminoglycan component of the mammalian extracellular matrix (ECM) where it has a structural function influencing hydration. HA biosynthesis is carried out by HA synthases (Has) existing three isoforms: Has-1,-2 and −3. The control for HA deposition occurs at catabolism mainly by the action of hyaluronidases (Hyal), being Hyal-1, −2 and −3 the most important ones. Increased levels of HA as well as differential expression of Has/Hyals have been found in the ECM within tumors and have been related with tumor invasion and metastasis. The aim of this work was to analyze the ability of lymphoma cells to produce HA and to evaluate HA levels within the tumors induced by these cells. For this purpose, we used murine lymphoma cells resistant to doxorubicin (LBR-D160), vincristine (LBR-V160), and a sensitive line (LBR-) with dissimilar invasive behaviour in vivo, being LBR-V160 the less aggressive one. First we evaluated endogenous HA surface expression by flow cytometry finding that LBR- presented higher HA expression than LBR-D160 and LBR-V160 (p&amp;lt;0.01 respectively). Secondly, we studied mRNA expression for Has-1, Has-2, Has-3 and Hyal-1, Hyal-2 and Hyal-3 by RT-PCR. We observed that none of the cell lines presented Has-1 and Has-2, but all of them expressed similar levels of Has-3 mRNA. Although we were unable to detect Hyal-1, Hyal-2 was found significantly decreased in LBR- compared with LBR-D160 and LBR-V160 (p&amp;lt;0.05 repectively). Besides, Hyal-3 was similarly expressed in the 3 cell lines. Finally BALB/c mice were injected with tumor cells or vehicle, sacrificed and infiltrated lung, lymph nodes, spleen and liver as well as blood were taken for ELISA measurement of HA. Serum HA levels were increased in LBR- and LBR-D160 injected mice compared with control mice (4.1±1.2 and 3.9±0.8 µg/ml vs. 1.9±0.4 µg/ml; P&amp;lt;0.05), finding no differences in LBR-V160 injected mice (2.5±1.1 µg/ml). In lungs from LBR-D160 injected mice, HA concentration was increased compared with control (0.4±0.1 vs. 0.2±0.1 µg/mg tissue wet weight; P&amp;lt;0.05), while no changes were observed in LBR- or LBR-V160 injected mice. Besides, a slight increase in HA levels was observed in lymph nodes from LBR- and LBR-D160 injected mice compared with control mice (3.1±3.0 and 2.7±2.2 µg/mg tissue vs. 1.0±0.8 µg/mg tissue), while no changes were observed in spleen and liver. Our results showed an association between tumor progression and HA levels in this lymphoma model since mice injected with the cell lines with major invasive capacity (LBR- and LBR-D160) presented increased serum HA levels as well as increased deposition of HA in infiltrated lungs and lymph nodes. In LBR- this may be explained by the ability of this cell line to express more HA than the other ones which could be related with decreased Hyal-2 expression. Further analysis of Has/Hyal balance in the infiltrated organs will contribute to understanding the causes of HA increase in LBR-D160 injected mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2319.

In vitro study on the vaporization ratio of 2-μm laser in human prostatic tissue

In this study, the vaporization ratio of the 2-mum laser in the prostatic tissue with benign prostatic hyperplasia was examined in vitro, to explore a technique to estimate the clearance rate of prostatic tissue during the transurethral vaporesection of the prostate. A total of 9 fresh prostatic tissue specimens were obtained by open surgery and the wet weight of the prostatic tissue were measured immediately after the sample collection. Under the simulated conditions of transurethral vaporesection of the prostate by 2-microm laser, each prostate gland was completely vaporesected into fragments with a diameter of less than 1.0 cm in vitro. After the vaporesection, the whole fragments of prostatic tissue were collected and measured. Then the lost weight of prostatic tissue, the weight of the collected prostatic tissue and the ratio of the lost weight of prostatic tissue to the wet weight of the prostate glandular organ specimen were calculated. The correlation between the weight of collected prostatic tissue and the weight of the whole glandular organ was analyzed. All the experimental procedures were carried out by one operator. Wet weight of the prostatic gland specimen and the weight of the harvested prostatic tissues after the procedure were recorded. With respect to the wet weight of prostate gland specimen, the percentage of the weight of collected prostatic tissue was (34.45 + or - 1.51) %, and the percentage of the lost weight of prostatic tissue was (65.55 + or - 1.51)%. Satisfactory linear relationship was observed between the weight of collected prostatic tissue and the wet weight of prostate gland specimen [y = 3.245 x -6.475 (t=15.097, P=0.000)]. It is concluded that under the simulated conditions of transurethral vaporesection of the prostate by 2-mum laser, the vaporization ratio of prostatic tissue can be calculated on the basis of the weight of collected prostatic tissue, and thereby the clearance of prostatic tissue during the formal operation by 2-microm laser could be quantitatively determined.

Chronic oral administration of Ocimum sanctum Linn. augments cardiac endogenous antioxidants and prevents isoproterenol-induced myocardial necrosis in rats

Wistar rats (200-250 g) of either sex were fed with fresh leaf homogenate of Ocimum sanctum by oral gavage in two different doses, 50 mg kg-1(Os 50) and 100 mg kg-1 (Os 100), daily for 30 days. This was followed by isoproterenol administration (85 mg kg-1 s.c. two doses at 24h intervals) in both control and 0. sanctum-fed rats to induce myocardial necrosis. Hearts were isolated for estimation of endogenous myocardial antioxidants (superoxide dismutase (SOD), catalase, reduced glutathione (GSH) and glutathione peroxidase (GPx) and myocardial lipid peroxidation) and light microscopic study. Increased basal myocardial antioxidant SOD (9.3 +/- 1.2 vs 3.7 +/- 0.7 units mg-1 protein; P<0.05) and catalase activities (34.3 +/- 5.4 vs 17.9 +/- 5.1 units mg-1 protein; P< 0.05) were observed in the Os 50 group only without any evidence of cellular injury in both the groups. In control rats, isoproterenol administration caused significant depletion of myocardial SOD (1.7 +/- 0.2 units mg-1 protein) and GPx (104 +/- 2mU mg-1 protein) activities and increase in GSH (551.7 +/- 30.9, microg g-1 wet weight of tissue) level, with evidence of myocardial necrosis. Isoproterenol-induced changes in myocardial SOD, GPx and GSH were prevented by both the doses of 0. sanctum, however cellular injury was minimal only with 50mg kg-1. The results indicate that long-term feeding of 0. sanctum offered significant protection against isoproterenol-induced myocardial necrosis through a unique property of enhancement of endogenous antioxidants.

3H]AL-5848 ([3H]9beta-(+)-Fluprostenol). Carboxylic acid of travoprost (AL-6221), a novel FP prostaglandin to study the pharmacology and autoradiographic localization of the FP receptor.

AL-5848 (5Z,13E)-(9 S,11R,15S)-9,11,15-trihydroxy-5,13-prostadienoic acid) is the carboxylic acid of travoprost (AL-6221), a single (+)-isomer of (+/-)-fluprostenol, an FP-class prostaglandin agonist which lowers intraocular pressure. We have prepared a radioligand from this selective prostaglandin and demonstrated its utility for studying the pharmacology and autoradiographic location of the FP-receptor. Specific [3H]AL-5848 binding (84% of total) was linearly related to bovine corpus luteum tissue concentration and reached equilibrium within 275 min at 23 degrees C. Scatchard analysis of saturation isotherms indicated interaction of [3H]AL-5848 with a single class of high-affinity (dissociation constant, Kd, = 33.8+/-2.9 nM, n = 4) and saturable (Bmax = 37.3+/-3.0 pmol (g wet weight tissue)(-1)) FP receptor-binding sites in bovine corpus luteum. Specific [3H]AL-5848 binding was potently inhibited by the FP-receptor ligands 16-phenoxyPGF2alpha (inhibition constant Ki = 17.3 nM); cloprostenol (Ki = 56.8 nM); 17-phenyl PGF2alpha (Ki = 87.0 nM); AL-5848 (Ki = 52.1 nM); PGF2alpha (Ki = 195 nM); PHXA85 (Ki = 223 nM); (n = 3-11) but very weakly by PGD2, ZK118182, BW245C, PGE2, PGI2 and U-46619. The pharmacology of specific [3H]AL-5848 binding correlated well with the pharmacology of [3H]PGF2alpha binding in the bovine corpus luteum preparation (r = 0.98, n = 14, P<0.0001) and also with functional responses in Swiss 3T3 and rat vascular smooth muscle cells (A7r5) (r = 0.96) expressing FP receptors. Autoradiographic studies revealed high levels of specific FP-receptor binding with [3H]AL-5848 on granulosa cells in the bovine corpus luteum sections, and on longitudinal ciliary muscle, the ciliary process, the iris sphincter and the retina in eye sections from man. These studies show [3H]AL-5848 to be a high-affinity agonist radioligand capable of selectively labelling the FP prostaglandin receptor.

Pharmacological characterization and identification of EP3 prostanoid receptor binding sites in hamster uterus homogenates.

The pharmacological properties of [(3)H]-prostaglandin E(2) ([(3)H]-PGE(2)) binding to washed homogenates of hamster uterus were determined. Scatchard analysis of competition data yielded dissociation constants (K(d)s) of 30.9 +/- 5.6 nM (n = 3) and apparent receptor density (B(max)) of 25.25 +/- 1.89 pmol g(-1) wet weight tissue (74 +/- 8% specific binding). Competition studies yielded the following affinity parameters (K(i)) for various prostanoids: GR63799X = 13 4 nM; PGE(2) = 17 +/- 3 nM; sulprostone = 64 +/- 5 nM; enprostil = 67 +/- 3 nM; misoprostol = 124 +/- 15 nM; cloprostenol = 187 +/- 33 nM; carba-prostacyclin = 260 +/- 167 nM; iloprost = 555 +/- 162 nM; PGF(2 alpha) = 767 +/- 73 nM; PGD(2) > 3560 nM; fluprostenol = 11 790 +/- 2776 nM; RS93520 = 21 558 +/- 14 228 nM. These data closely matched the pharmacological profile of previously described EP(3) receptors such as in bovine corpus luteum (BCLM) and the cloned mammalian EP(3) receptors. The high correlation between the current hamster uterus pharmacology data vs the EP(3) receptor binding in BCLM (r = 0.94; P < 0.0001), vs cloned human EP(3) receptor (r = 0.94, P < 0.0001), vs the cloned mouse EP(3) receptor binding (r = 0.78; P < 0.002), vs cloned rat EP(3) receptor (r = 0.9, P < 0.0004), and vs EP(3) receptor-mediated functional responses (r = 0.72, P < 0.02) substantiated the conclusion that the hamster uterus contains EP(3) receptor binding sites.

Dry Powder PA-824 Aerosols for Treatment of Tuberculosis in Guinea Pigs

Novel treatments for multidrug-resistant tuberculosis (MDR-TB), extensively drug-resistant tuberculosis (XDR-TB), or latent TB are needed urgently. Recently, we reported the formulation and characterization of the nitroimidazo-oxazine PA-824 for efficient aerosol delivery as dry powder porous particles and the subsequent disposition in guinea pigs after pulmonary administration. The objective of the present study was to evaluate the effects of these PA-824 therapeutic aerosols on the extent of TB infection in the low-inoculum aerosol infection guinea pig model. Four weeks after infection by the pulmonary route, animals received daily treatment for 4 weeks of either a high or a low dose of PA-824 dry powder aerosol. Animals received PA-824 cyclodextrin/lecithin suspensions orally as positive controls, and those receiving placebo particles or no treatment were negative controls. The lungs and spleens of animals receiving the high dose of inhaled PA-824 particles exhibited a lower degree of inflammation (indicated by wet tissue weights), bacterial burden, and tissue damage (indicated by histopathology) than those of untreated or placebo animals. Treatment with oral PA-824 cyclodextrin/lecithin suspension resulted in a more significant reduction in the bacterial burden of lungs and spleen, consistent with a dose that was larger than inhaled doses (eight times the inhaled low dose and four times the inhaled high dose). However, histopathological analysis revealed that the extent of tissue damage was comparable in groups receiving the oral or either inhaled dose. The present studies indicate the potential use of PA-824 dry powder aerosols in the treatment of TB.

Episcleral clearance of sodium fluorescein from a bioerodible sub-tenon's implant in the rat

We quantified episcleral drug clearance of sodium fluorescein (NaFl) in rats to examine the hypothesis that there is rapid clearance of episcleral water soluble drugs, and that this rapid clearance may limit the amount of drug that is able to reach the posterior segment from an episcleral location. 2 mm implants containing either 12 or 22 μg of NaFl were manufactured and in vitro release rates were determined. Implants were placed in the sub-Tenon's space and the amount of drug remaining in the conjunctiva/sclera/choroid complex (CSCC) at various time points was quantified following tissue solubilization and fluorescence quantification using a spectrofluorometer. Kinetics of NaFl clearance was determined in live animals, following euthanasia and in animals in which choroidal non-perfusion had been achieved with indocyanine green-enhanced 810 nm diode laser thrombosis of the choroidal vasculature. Choroidal non-perfusion in these laser-treated rats was verified with Concavalin-A staining of choroidal flatmounts. In vitro, >99% of drug was released by 25 min for the low dose implants, and by 60 min for the high dose implants. In vivo, both implant doses were >99% cleared from the episcleral tissue by 3 h. By 7 h, an average of only 0.14 ± 0.131 ng of NaFl per mg of wet tissue weight (mean ± SD) remained in the CSCC with the low dose implant, and 0.29 ± 0.428 ng of NaFl per mg of wet tissue weight remained in animals with the high dose implant. By comparison, in euthanized animals at 7 h following sub-Tenon's implantation, 432.0 ± 181.40 ng of NaFl per mg of wet tissue weight was in the episcleral tissue of animals with the low dose implant, and of 787.8 ± 409.89 ng of NaFl per mg of wet tissue weight remained in the animals with the high dose implant. In live animals with selective thrombosis of the choroidal vasculature, the difference in the amount of drug remaining in the episcleral tissue as compared to control live animals was not significant at all time points for both implant doses. In conclusion, there is rapid clearance of episcleral NaFl delivered from a bioerodible sub-tenon's implant. The clearance mechanisms are dramatically reduced following euthanasia, suggesting that elimination is occurring via active physiologic mechanisms, rather than by passive diffusion clearance (CL(diff)) ( Pfister et al., 2003). Interestingly, the choroid does not appear to play a prominent role as clearance of episcleral NaFl was not affected by elimination of choroidal blood flow. Further work is needed to delineate the pathways of episcleral drug clearance.

The use of radioactive zinc oxide nanoparticles in determination of their tissue concentrations following intravenous administration in mice

The increasing uses of zinc oxide nanoparticles (ZnONPs) in coatings, paints, personal care products and many other products increase the possibility of the body's exposure to ZnONPs. Accurate and quantitative profiling on the tissue distribution and body clearance of ZnONPs, which is an important factor to clarify the acute and chronic safety concerns of ZnONPs, is interfered by the abundance of the body's endogenous zinc moiety. In this report, radioactive zinc oxide nanoparticles (R-ZnONPs) generated from neutron activation were employed for the in vivo bio-distribution studies using mice as the animal model. Gamma-ray emitting radioactive R-ZnONPs were produced from neutron activation. Zeta potentials of the ZnONPs before and after the neutron irradiation remained about the same, and R-ZnONPs largely remained its original nano-particulate form after neutron irradiation. After intravenous administration into ICR mice, R-ZnONPs exhibited a primary retention in lung (43.6% injected dose (ID)/g tissue wet weight) for the first hour and began to be translocated to intestinal tract for feces excretion at a later stage. This type of labeling free and radioactive nanoparticles retains the surface property and can be a convenient protocol for studying bio-distribution of nanoparticles in pristine chemical form.

Gender- and age-dependent changes in nucleoside levels in the cerebral cortex and white matter of the human brain

Nucleosides are neuromodulators that participate in various neuronal functions in the brain. In previous studies, we described regional differences in the concentrations of nucleosides and their derivatives in the human brain. To better understand the functions of nucleosides in the central nervous system, we investigated gender- and age-dependent changes in the levels of nucleosides and their metabolites. The concentrations of uridine, inosine, guanosine and adenosine as well as uracil, hypoxanthine and xanthine were measured in the frontal cortex and white matter of post- mortem brain tissue samples of middle-aged and old men as well as women. The average in vivo concentrations calculated from the 40 samples investigated (regardless of anatomical locations, gender or age; mean ± S.E.M.) were as follows (pmol/mg wet tissue weight): 9.7 ± 0.8 adenosine, 85.8 ± 3.9 inosine, 14.3 ± 0.9 guanosine, 37.3 ± 1.8 uridine, 8.9 ± 0.6 uracil, 63.3 ± 2.1 hypoxanthine and 38.7 ± 1.5 xanthine. We conclude that concentration differences between uridine, inosine, guanosine and adenosine in the frontal cortex and cerebral white matter suggest that nucleoside metabolism is altered with aging and regulated differently between men and women.

Selective Binding of Glutathione Conjugates of Fatty Acid Derivatives by Plant Glutathione Transferases

Proteomic studies with Arabidopsis thaliana have revealed that the plant-specific Tau (U) class glutathione transferases (GSTs) are selectively retained by S-hexylglutathione affinity supports. Overexpression of members of the Arabidopsis GST superfamily in Escherichia coli showed that 25 of the complement of 28 GSTUs caused the aberrant accumulation of acylated glutathione thioesters in vivo, a perturbation that was not observed with other GST classes. Each GSTU caused a specific group of fatty acyl derivatives to accumulate, which varied in chain length (C(6) to C(18)), additional oxygen content (0 or 1), and desaturation (0 or 1). Thioesters bound tightly to recombinant GSTs (K(d) approximately 1 microm), explaining their accumulation. Transient expression of GSTUs in Nicotiana benthamiana followed by recovery by Strep-tag affinity chromatography allowed the respective plant ligands to be extracted and characterized. Again, each GST showed a distinct profile of recovered metabolites, notably glutathionylated oxophytodienoic acid and related oxygenated fatty acids. Similarly, the expression of the major Tau protein GSTU19 in the endogenous host Arabidopsis led to the selective binding of the glutathionylated oxophytodienoic acid-glutathione conjugate, with the enzyme able to catalyze the conjugation reaction. Additional ligands identified in planta included other fatty acid derivatives including divinyl ethers and glutathionylated chlorogenic acid. The strong and specific retention of various oxygenated fatty acids by each GSTU and the conservation in binding observed in the different hosts suggest that these proteins have selective roles in binding and conjugating these unstable metabolites in vivo.

Immunoreactive endomorphin 2 is generated extracellularly in rat isolated L4,5 dorsal root ganglia by DPP-IV

Background and aims The gene(s) encoding for endomorphin precursor(s) is/are still unknown. We have raised the possibility of and did find some evidence for a potential de novo biosynthetic route starting from Tyr-Pro precursor. To pursue further this possibility we measured the generation of immunoreactive endomorphin-2 (E2-IR) in adult rat isolated L4,5 dorsal root ganglia. Results and conclusions In rat isolated dorsal root ganglia the combination of presumed biosynthetic precursor of endomorphin 2 (E2), Tyr-Pro with the dipeptidyl peptidase IV (DPP-IV) inhibitor Ile-Pro-Ile generated 1.60 ± 0.37 pg/mg Wet Tissue Weight_30min E2-IR in the bathing fluid ( n = 4) with an 8-fold increase upon depolarization whereas the tissue content was low (0.50 ± 0.08 pg/mg_WTW). Substance P, as determined by ELISA in the pilot experiments, was found almost exclusively within the tissues. It is concluded that E2-IR was generated extracellularly by a membrane-bound DPP-IV, which was switched to “synthase” mode by the hydrolase inhibitor Ile-Pro-Ile. DPP-IV was depolarization-sensitive in “synthase” functional mode.

Distribution of persistent organochlorine pollutants in maternal and foetal tissues: Data from an Italian polluted urban area

This study was designed to evaluate both levels and distributions of organochlorine pollutants in placenta and other maternal–foetal tissues, from women resident in an Italian highly industrialized urban area with demonstrated PCB pollution. To this end, 30 polychlorinated biphenyl congeners (PCBs), p,p′-dichlorodiphenyltrichloroethane (p,p′-DDT), p,p′-dichlorodiphenyldichloroethylene (p,p′-DDE) and hexachlorobenzene (HCB), were determined in placenta, both maternal and cord blood serum and adipose tissue of Caucasian pregnant women ( n = 70) undergoing to programmed caesarean delivery. Median placental concentrations of PCBs, p,p′-DDE and HCB (0.89 ng g −1, 0.63 ng g −1 and 0.19 ng g −1 on wet tissue weight basis, respectively), were lower than previously published data. p,p′-DDT could be detected in most of adipose tissue samples, but very rarely in the other compartments. Distributions of investigated compounds in biological matrices showed significant relationships, confirming their transplacental transfer. It is worthy of note that HCB tended to be transferred more easily from maternal to foetal tissues. Maternal serum levels of these pollutants can allow a reliable estimation of organochlorine compound body burden, and can be useful markers of prenatal exposure, given their relationships with placental and umbilical cord serum levels.

Isolation, purification and identification of HSPgp96-peptide complex from human osteosarcom tissues

0bjective To derive and purify HSPgp96-peptide complex from human osteosarcoma tissues.Methods Three-step purification of HSPgp96 and Western-blot were used to derive.purify and ceaify HSPgp96-peptide complex from 15 osteosarcoma tissues,and advanced Bradford's method was used to calculate the final protein recovery.Results The HSPgp96-peptide complex had a high purity and specificity.The final protein recovery was 30-40μg/g tumor tissue(wet weight).Conclusion Three-step purification of HSPgp96 is simple and has a high reproducibility.HSPgp96-peptide complex obtained by this method has the merits of high purity,and high specificity. Key words: Osteosarcoma; HSP; Protein purification

Quantification of endogenous α- and γ-endorphins in rat brain by liquid chromatography–tandem mass spectrometry

Quantification of α- and γ-endorphins in rat brain using liquid chromatography–electrospray ionization–tandem mass spectrometry is described. [D-Ala 2]-γ-endorphin is used as an internal standard. The precursor-to-product ion MRM transitions for α-endorphin, γ-endorphin, and [D-Ala 2]-γ-endorphin were m/ z 873.6 → 429.6; 929.6 → 542.3; 936.6 → 542.3, respectively. The method was validated in terms of linearity, specificity, sensitivity, recovery, precision, and accuracy. The assay was linear over a concentration range of 0.1–200 ng/mL with the limit-of-detection of 0.03 ng/mL and limit-of-quantification of 0.1 ng/mL. The endogenous concentrations of α- and γ-endorphins in rat brains were 13.8 ± 0.57 (mean ± SD; n = 5) and 2.5 ± 0.43 ng/g of wet tissue weight, respectively.