Local targeting of malignant gliomas by the diffusible peptidic vector 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid-substance p

PURPOSE: Malignant glial brain tumors consistently overexpress neurokinin type 1 receptors. In classic seed-based brachytherapy, one to several rigid (125)I seeds are inserted, mainly for the treatment of small low-grade gliomas. The complex geometry of rapidly proliferating high-grade gliomas requires a diffusible system targeting tumor-associated surface structures to saturate the tumor, including its margins. EXPERIMENTAL DESIGN: We developed a new targeting vector by conjugating the chelator 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid to Arg(1) of substance P, generating a radiopharmaceutical with a molecular weight of 1,806 Da and an IC(50) of 0.88 +/- 0.34 nmol/L. Cell biological studies were done with glioblastoma cell lines. neurokinin type-1 receptor (NK1R) autoradiography was done with 58 tumor biopsies. For labeling, (90)Y was mostly used. To reduce the "cross-fire effect" in critically located tumors, (177)Lut and (213)Bi were used instead. In a pilot study, we assessed feasibility, biodistribution, and early and long-term toxicity following i.t. injection of radiolabeled 1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid substance P in 14 glioblastoma and six glioma patients of WHO grades 2 to 3. RESULTS: Autoradiography disclosed overexpression of NK1R in 55 of 58 gliomas of WHO grades 2 to 4. Internalization of the peptidic vector was found to be specific. Clinically, the radiopharmeutical was distributed according to tumor geometry. Only transient toxicity was seen as symptomatic radiogenic edema in one patient (observation period, 7-66 months). Disease stabilization and/or improved neurologic status was observed in 13 of 20 patients. Secondary resection disclosed widespread radiation necrosis with improved demarcation. CONCLUSIONS: Targeted radiotherapy using diffusible peptidic vectors represents an innovative strategy for local control of malignant gliomas, which will be further assessed as a neoadjuvant approach.

Regulation of human liver delta-aminolevulinic acid synthase by bile acids

Aminolevulinic acid synthase 1 (ALAS1) is the rate-limiting enzyme of heme synthesis in the liver and is highly regulated to adapt to the metabolic demand of the hepatocyte. In the present study, we describe human hepatic ALAS1 as a new direct target of the bile acid-activated nuclear receptor farnesoid X receptor (FXR). Experiments in primary human hepatocytes and in human liver slices showed that ALAS1 messenger RNA (mRNA) and activity is increased upon exposure to chenodeoxycholic acid (CDCA), the most potent natural FXR ligand, or the synthetic FXR-specific agonist GW4064. Moreover, overexpression of a constitutively active form of FXR further increased ALAS1 mRNA expression. In agreement with these observations, an FXR response element was identified in the 5' flanking region of human ALAS1 and characterized in reporter gene assays. A highly conserved FXR binding site (IR1) within a 175-bp fragment at -13 kilobases upstream of the transcriptional start site was able to trigger an FXR-specific increase in luciferase activity upon CDCA treatment. Site-directed mutagenesis of IR1 abolished this effect. Binding of FXR/retinoid acid X receptor heterodimers was demonstrated by mobility gel shift experiments. Conclusion: These data strongly support a role of bile acid-activated FXR in the regulation of human ALAS1 and, consequently, hepatic porphyrin and heme synthesis. These data also suggest that elevated endogenous bile acids may precipitate neuropsychiatric attacks in patients with acute hepatic porphyrias.

Rumen-protected choline supplementation in periparturient dairy goats: effects on liver and mammary gland

The current study investigated the effects of supplementing rumen-protected choline (RPC) on metabolic profile, selected liver constituents and transcript levels of selected enzymes, transcription factors and nuclear receptors involved in mammary lipid metabolism in dairy goats. Eight healthy lactating goats were studied: four received no choline supplementation (CTR group) and four received 4g RPC chloride/day (RPC group). The treatment was administered individually starting 4 weeks before expected kidding and continuing for 4 weeks after parturition. In the first month of lactation, milk yield and composition were measured weekly. On days 7, 14, 21 and 27 of lactation, blood samples were collected and analysed for glucose, beta-hydroxybutyrate, non-esterified fatty acids and cholesterol. On day 28 of lactation, samples of liver and mammary gland tissue were obtained. Liver tissue was analysed for total lipid and DNA content; mammary tissue was analysed for transcripts of lipoprotein lipase (LPL), fatty acid synthase (FAS), sterol regulatory binding proteins 1 and 2, peroxisome proliferator-activated receptor gamma and liver X receptor alpha. Milk yield was very similar in the two groups, but R PC goats had lower (P < 0.05) plasma beta-hydroxybutyrate. The total lipid content of liver was unaffected (P = 0.890), but the total lipid/DNA ratio was lower (both P < 0.05) in RPC than CTR animals. Choline had no effect on the expression of the mammary gland transcripts involved in lipid metabolism. The current plasma and liver data indicate that choline has a positive effect on liver lipid metabolism, whereas it appears to have little effect on transcript levels in mammary gland of various proteins involved in lipid metabolism. Nevertheless, the current results were obtained from a limited number of animals, and choline requirement and function in lactating dairy ruminants deserve further investigation.

Plasma leptin and mRNA expression of lipogenesis and lipolysis-related factors in bovine adipose tissue around parturition

The objective was to study changes in plasma leptin concentration parallel to changes in the gene expression of lipogenic- and lipolytic-related genes in adipose tissue of dairy cows around parturition. Subcutaneous fat biopsies were taken from 27 dairy cows in week 8 antepartum (a.p.), on day 1 postpartum (p.p.) and in week 5 p.p. Blood samples were assayed for concentrations of leptin and non-esterified fatty acids (NEFA). Subcutaneous adipose tissue was analysed for mRNA abundance by real-time qRT-PCR encoding for leptin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), hormones-sensitive lipase (HSL), perilipin (PLIN), lipoprotein lipase (LPL), acyl-CoA synthase long-chain family member 1 (ACSL1), acetyl-CoA carboxylase (ACC), fatty acid synthase (FASN) and glycerol-3-phosphate dehydrogenase 2 (GPD2). Body weight and body condition score of the cows were lower after parturition than before parturition. The calculated energy balance was negative in week 1 and 5 p.p., with higher negative energy balance in week 1 p.p. compared with that in week 5 p.p. On day 1 p.p., highest concentrations of NEFA (353.3 mumol/l) were detected compared with the other biopsy time-points (210.6 and 107.7 mumol/l, in week 8 a.p., and week 5 p.p. respectively). Reduced plasma concentrations of leptin during p.p. when compared with a.p. would favour increasing metabolic efficiency and energy conservation for mammary function and reconstitution of body reserves. Lower mRNA abundance of ACC and FASN expression on day 1 p.p. compared with other biopsy time-points suggests an attenuation of fatty acid synthesis in subcutaneous adipose tissue shortly after parturition. Gene expression of AdipoR1, AdipoR2, HSL, PLIN, LPL, ACSL1 and GPD2 was unchanged over time.

Zoledronic acid efficacy and safety over five years in postmenopausal osteoporosis

In a 5-year study involving 119 postmenopausal women, zoledronic acid 4 mg given once-yearly for 2, 3 or 5 years was well tolerated with no evidence of excessive bone turnover reduction or any safety signals. BMD increased significantly. Bone turnover markers decreased from baseline and were maintained within premenopausal reference ranges. INTRODUCTION: After completion of the core study, two consecutive, 2-year, open-label extensions investigated the efficacy and safety of zoledronic acid 4 mg over 5 years in postmenopausal osteoporosis. METHODS: In the core study, patients received 1 to 4 mg zoledronic acid or placebo. In the first extension, most patients received 4 mg per year and then patients entered the second extension and received 4 mg per year or calcium only. Patients were divided into three subgroups according to years of active treatment received (2, 3 or 5 years). Changes in BMD and bone turnover markers (bone ALP and CTX-I) were assessed. RESULTS: All subgroups showed substantial increases in BMD and decreases in bone markers. By the end of the core study, 37.5% of patients revealed a suboptimal reduction (< 30%) of bone ALP levels. After subsequent study drug administration during the extensions, there was no evidence of progressive reduction of bone turnover markers. Furthermore, increased marker levels after treatment discontinuation demonstrates preservation of bone remodelling capacity. CONCLUSIONS: This study showed that zoledronic acid 4 mg once-yearly was well tolerated and effective in reducing biomarkers over 5 years. Detailed analysis of bone marker changes, however, suggests that this drug regimen causes insufficient reduction of remodelling activity in one third of patients.

Simultaneous determination of 3-hydroxyanthranilic and cinnabarinic acid by high-performance liquid chromatography with photometric or electrochemical detection

A convenient and rapid method for the simultaneous determination by HPLC of 3-hydroxyanthranilic acid and the dimer derived by its oxidation, cinnabarinic acid, is described. Buffers or biological samples containing these two Trp metabolites were acidified to pH 2.0 and extracted with ethyl acetate with recoveries of 96.5 +/- 0.5 and 93.4 +/- 3.7% for 3-hydroxyanthranilic and cinnabarinic acid, respectively. The two compounds were separated on a reversed-phase (C18) column combined with ion-pair chromatography and detected photometrically or electrochemically. The method was applied successfully to biological systems in which formation of either 3-hydroxyanthranilic or cinnabarinic acid had been described previously. Thus, interferon-gamma-treated human peripheral blood mononuclear cells formed and released significant amounts of 3-hydroxyanthranilic acid into the culture medium and mouse liver nuclear fraction possessed high "cinnabarinic acid synthase" activity. In contrast, addition of 3-hydroxyanthranilic acid to human erythrocytes resulted in only marginal formation of cinnabarinic acid. We conclude that the method described is specific, sensitive, and suitable for the detection of the two Trp metabolites in biological systems.

Symptoms with acid and nonacid reflux may be produced by different mechanisms

Twenty-four-hour multichannel intraluminal impedance and pH (MII-pH) esophageal monitoring detects both acid and nonacid gastroesophageal reflux episodes. The MII-pH catheter contains six impedance segments placed 3, 5, 7, 9, 15, and 17 cm above the lower esophageal sphincter (LES). A pH electrode at 5 cm above the LES identifies the type of reflux, i.e. acid or nonacid. Patients with acid and nonacid reflux exhibit typical and atypical symptoms often within 5 min following a reflux episode. The aim of this study is to compare the timing of symptoms after reflux episodes in patients with acid and nonacid reflux. Methods include a review of 70 MII-pH tracings (42 females, mean age 40, range 18-85 years) either on (50 points) or off (20 points) acid suppression therapy. Typical (heartburn, regurgitation) and atypical (cough) symptoms with acid or nonacid reflux episodes detected by impedance were analyzed. Symptoms were considered positive with acid reflux if there was a pH drop to <4, plus an MII detected a reflux episode and with nonacid reflux if pH remained >4 and MII detected a reflux episode. The timing of the symptom after each reflux episode was recorded. Symptom perception occurred significantly sooner after acid versus nonacid reflux (P < 0.05). Acid reflux episodes are more likely to be perceived in the first 2 min following the reflux episode. Patients with acid reflux are likely to perceive symptoms earlier, and symptoms with acid and nonacid reflux may be produced by different mechanisms.

Liver fat content and lipid metabolism in dairy cows during early lactation and during a mid-lactation feed restriction.

During the transition period, the lipid metabolism of dairy cows is markedly affected by energy status. Fatty liver is one of the main health disorders after parturition. The aim of this study was to evaluate the effects of a negative energy balance (NEB) at 2 stages in lactation [NEB at the onset of lactation postpartum (p.p.) and a deliberately induced NEB by feed restriction near 100 d in milk] on liver triglyceride content and parameters of lipid metabolism in plasma and liver based on mRNA abundance of associated genes. Fifty multiparous dairy cows were studied from wk 3 antepartum to approximately wk 17 p.p. in 2 periods. According to their energy balance in period 1 (parturition to wk 12 p.p.), cows were allocated to a control (CON; n=25) or a restriction group (RES; 70% of energy requirements; n=25) for 3 wk in mid lactation starting at around 100 d in milk (period 2). Liver triglyceride (TG) content, plasma nonesterified fatty acids (NEFA), and β-hydroxybutyrate were highest in wk 1 p.p. and decreased thereafter. During period 2, feed restriction did not affect liver TG and β-hydroxybutyrate concentration, whereas NEFA concentration was increased in RES cows as compared with CON cows. Hepatic mRNA abundances of tumor necrosis factor α, ATP citrate lyase, mitochondrial glycerol-3-phosphate acyltransferase, and glycerol-3-phosphate dehydrogenase 2 were not altered by lactational and energy status during both experimental periods. The expression of fatty acid synthase was higher in period 2 compared with period 1, but did not differ between RES and CON groups. The mRNA abundance of acetyl-coenzyme A-carboxylase showed a tendency toward higher expression during period 2 compared with period 1. The solute carrier family 27 (fatty acid transporter), member 1 (SLC27A1) was upregulated in wk 1 p.p. and also during feed restriction in RES cows. In conclusion, the present study shows that a NEB has different effects on hepatic lipid metabolism and TG concentration in the liver of dairy cows at early and later lactation. Therefore, the homeorhetic adaptations during the periparturient period trigger excessive responses in metabolism, whereas during the homeostatic control of endocrine and metabolic systems after established lactation, as during the period of feed restriction in the present study, organs are well adapted to metabolic and environmental changes.

Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ). PGC-1 enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1 levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1 -mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1 and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1 enhances lipogenesis in skeletal muscle through liver X receptor -dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1 and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1 coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo.

Long-term elevation of β-hydroxybutyrate in dairy cows through infusion: effects on feed intake, milk production, and metabolism.

Elevation of ketone bodies in dairy cows frequently occurs in early lactation, usually concomitantly with a lack of energy and glucose. The objective of this study was to induce an elevated plasma β-hydroxybutyrate (BHBA) concentration over 48 h in mid-lactating dairy cows (i.e., during a period of positive energy balance and normal glucose plasma concentrations). Effects of BHBA infusion on feed intake, metabolism, and performance were investigated. Thirteen cows were randomly assigned to 1 of 2 infusion groups, including an intravenous infusion with Na-dl-β-OH-butyrate (1.7 mol/L) to achieve a plasma concentration of 1.5 to 2.0 mmol/L of BHBA (HyperB; n=5), or an infusion of 0.9% saline solution (control; n=8). Blood was sampled before and hourly during the 48 h of infusion. In the liver, mRNA transcripts related to gluconeogenesis (pyruvate carboxylase, glucose 6-phosphatase, mitochondrial phosphoenolpyruvate carboxykinase), phosphofructokinase, pyruvate dehydrogenase complex, and fatty acid synthesis (acetyl-coenzyme A carboxylase, fatty acid synthase) were measured by real-time PCR. Glyceraldehyde-3-phosphate dehydrogenase and ubiquitin were used as housekeeping genes. Changes (difference between before and after 48-h infusion) during the infusion period were evaluated by ANOVA with treatment as fixed effect, and area under the curve of variables was calculated on the second day of experiment. The plasma BHBA concentration in HyperB cows was 1.74 ± 0.02 mmol/L (mean ± SE) compared with 0.59 ± 0.02 mmol/L for control cows. The change in feed intake, milk yield, and energy corrected milk did not differ between the 2 experimental groups. Infusion of BHBA reduced the plasma glucose concentration (3.47 ± 0.11 mmol/L) in HyperB compared with control cows (4.11 ± 0.08 mmol/L). Plasma glucagon concentration in HyperB was lower than the control group. All other variables measured in plasma were not affected by treatment. In the liver, changes in mRNA abundance for the selected genes were similar between 2 groups. Results demonstrate that intravenous infusion of BHBA decreased plasma glucose concentration in dairy cows, but this decrease could not be explained by alterations in insulin concentrations or key enzymes related to gluconeogenesis. Declined glucose concentration is likely functionally related to decreased plasma glucagon concentration.

N-terminal modifications improve the receptor affinity and pharmacokinetics of radiolabeled peptidic gastrin-releasing peptide receptor antagonists: examples of 68Ga- and 64Cu-labeled peptides for PET imaging

UNLABELLED Gastrin-releasing peptide receptors (GRPrs) are overexpressed on a variety of human cancers, providing the opportunity for peptide receptor targeting via radiolabeled bombesin-based peptides. As part of our ongoing investigations into the development of improved GRPr antagonists, this study aimed at verifying whether and how N-terminal modulations improve the affinity and pharmacokinetics of radiolabeled GRPr antagonists. METHODS The potent GRPr antagonist MJ9, Pip-d-Phe-Gln-Trp-Ala-Val-Gly-His-Sta-Leu-NH(2) (Pip, 4-amino-1-carboxymethyl-piperidine), was conjugated to 1,4,7-triazacyclononane, 1-glutaric acid-4,7 acetic acid (NODAGA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) and radiolabeled with (68)Ga and (64)Cu. The GRPr affinity of the corresponding metalloconjugates was determined using (125)I-Tyr(4)-BN as a radioligand. The labeling efficiency of (68)Ga(3+) was compared between NODAGA-MJ9 and NOTA-MJ9 in acetate buffer, at room temperature and at 95°C. The (68)Ga and (64)Cu conjugates were further evaluated in vivo in PC3 tumor xenografts by biodistribution and PET imaging studies. RESULTS The half maximum inhibitory concentrations of all the metalloconjugates are in the high picomolar-low nanomolar range, and these are the most affine-radiolabeled GRPr antagonists we have studied so far in our laboratory. NODAGA-MJ9 incorporates (68)Ga(3+) nearly quantitatively (>98%) at room temperature within 10 min and at much lower peptide concentrations (1.4 × 10(-6) M) than NOTA-MJ9, for which the labeling yield was approximately 45% under the same conditions and increased to 75% at 95°C for 5 min. Biodistribution studies showed high and specific tumor uptake, with a maximum of 23.3 ± 2.0 percentage injected activity per gram of tissue (%IA/g) for (68)Ga-NOTA-MJ9 and 16.7 ± 2.0 %IA/g for (68)Ga-NODAGA-MJ9 at 1 h after injection. The acquisition of PET images with the (64)Cu-MJ9 conjugates at later time points clearly showed the efficient clearance of the accumulated activity from the background already at 4 h after injection, whereas tumor uptake still remained high. The high pancreas uptake for all radiotracers at 1 h after injection was rapidly washed out, resulting in an increased tumor-to-pancreas ratio at later time points. CONCLUSION We have developed 2 GRPr antagonistic radioligands, which are improved in terms of binding affinity and overall biodistribution profile. Their promising in vivo pharmacokinetic performance may contribute to the improvement of the diagnostic imaging of tumors overexpressing GRPr.

The Chloroplast-Localized Phospholipases D a4 and a5 Regulate Herbivore-Induced Direct and Indirect Defenses in Rice

The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDα4 and OsPLDα5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLDα4 and -α5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice.

Mycophenolate acid inhibits endothelial NAD(P)H oxidase activity and superoxide formation by a Rac1-dependent mechanism

Endothelial dysfunction precedes hypertension and atherosclerosis and predicts cardiac allograft vasculopathy and death in heart transplant recipients. Endothelial overproduction of reactive oxygen species, such as superoxide anions produced by NAD(P)H oxidase, induces endothelial dysfunction. Because immunosuppressive drugs have been associated with increased reactive oxygen species production and endothelial dysfunction, we sought to elucidate the underlying mechanisms. Reactive oxygen species, release of superoxide anions, and NAD(P)H oxidase activity were studied in human umbilical vein endothelial cells and in polymorphonuclear neutrophils. Gp91ds-tat was used to specifically block NAD(P)H oxidase. Transcriptional activation of different subunits of NAD(P)H oxidase was assessed by real-time RT-PCR. Rac1 subunit translocation and activation were studied by membrane fractionation and pull-down assays. Calcineurin inhibitors significantly increased endothelial superoxide anions production because of NAD(P)H oxidase, whereas mycophenolate acid (MPA) blocked it. MPA also attenuated the respiratory burst induced by neutrophil NAD(P)H oxidase. Because transcriptional activation of NAD(P)H oxidase was not affected, but addition of guanosine restored endothelial superoxide anions formation after MPA treatment, we speculate that the inhibitory effect of MPA was mediated by depletion of cellular guanosine triphosphate content. This prevented activation of Rac1 and, thus, of endothelial NAD(P)H oxidase. Because all heart transplant recipients are at risk for cardiac allograft vasculopathy development, these differential effects of immunosuppressants on endothelial oxidative stress should be considered in the choice of immunosuppressive drugs.

Adaptation of canine distemper virus to canine footpad keratinocytes modifies polymerase activity and fusogenicity through amino acid substitutions in the P/V/C and H proteins

The wild-type canine distemper virus (CDV) strain A75/17 induces a non-cytocidal infection in cultures of canine footpad keratinocytes (CFKs) but produces very little progeny virus. After only three passages in CFKs, the virus produced 100-fold more progeny and induced a limited cytopathic effect. Sequence analysis of the CFK-adapted virus revealed only three amino acid differences, of which one was located in each the P/V/C, M and H proteins. In order to assess which amino acid changes were responsible for the increase of infectious virus production and altered phenotype of infection, we generated a series of recombinant viruses. Their analysis showed that the altered P/V/C proteins were responsible for the higher levels of virus progeny formation and that the amino acid change in the cytoplasmic tail of the H protein was the major determinant of cytopathogenicity.