Radioenzymatic Assay Research Articles

Association between BanI genotype and increased phospholipase A2 activity in schizophrenia

Phospholipases A2 (PLA2) are a family of key enzymes in the metabolism of membrane phospholipids. Several studies reported on increased blood and brain PLA2 activity in schizophrenia, which suggest a disordered phospholipid metabolism in the disease. In addition, a genetic variant of a cytosolic PLA2 gene has been reported to be associated with schizophrenia. These data indicate that variants of PLA2 encoding genes are plausible candidates for increasing the susceptibility for schizophrenia. In this study, we investigated a possible association between PLA2 activity in platelets and a polymorphic site for BanI in the PLA2 (group 4A) gene on chromosome 1q25. Seventy-five schizophrenic patients (DSM-IV) and 68 healthy controls were recruited and the PCR assays were performed. A radioenzymatic assay for the cytosolic PLA2 activity in platelets was used. The allele A2 and the genotype A2A2 were more frequent in schizophrenic patients than in controls (p<0.005 and p<0.05 respectively). When we assorted the subjects according to their genotypes, we found that PLA2 activity was significantly higher in patients with the A2A2 genotype (29.6+/-5.1 pMol/mg protein/min) than in those with the A1A2 (20.8+/-3.6 pMol/mg protein/min, p<0.001) or A1A1 genotype (15.9+/-5.1 pMol/mg protein/min, p<0.001). Also in controls, carriers of the A2 allele (A1A2 and A2A2) had higher PLA2 activity than the A1A1 group (p=0.004 for both). Our data suggest an association between BanI genotype and PLA2G4A activity in platelets and that the presence of the allele A2 may increase risk for schizophrenia through an increment of PLA2 activity.

Effect of l-Carnitine Infusion and Feed Restriction on Carnitine Status in Lactating Holstein Cows

Previously we determined that abomasal infusion of l-carnitine increased in vitro hepatic fatty acid oxidation, decreased liver lipid accumulation, and supported higher fat-corrected milk yield in feed-restricted lactating cows. The objectives of this study were to examine the effects of supplemental l-carni-tine and amount of feed intake on free carnitine and carnitine ester concentrations in liver, muscle, milk, and plasma of lactating dairy cows. Eight lactating Holstein cows (132±36 d in milk) were used in a replicated 4×4 Latin square design with 14-d periods to test factorial combinations of water or l-carnitine infusion (20g/d; d 5 to 14) and ad libitum or restricted (50% of previous 5-d intake; d 10 to 14) dry matter intake. Plasma was obtained 3 times daily on d 4, 8, and 12; milk samples were collected on d 8, 9, 13, and 14. Liver and muscle were biopsied on d 14 of each period. Free carnitine, short-chain acylcarnitine, and long-chain acylcarnitine concentrations were determined using a radioenzymatic assay coupled with ion exchange chromatography. Abomasal l-carnitine infusion increased total carnitine in plasma on d 8 and d 12. All liver carnitine fractions were increased by carnitine infusion. Feed restriction elevated concentrations of free carnitine, long-chain acylcarnitine, and total carnitine in liver tissue from carnitine-infused cows but not in those infused with water. In muscle, acid-soluble carnitine, long-chain acylcarnitine, and total carnitine concentrations were increased by carnitine infusion and feed restriction without significant interaction. Feed restriction increased free carnitine concentrations in muscle from water-infused cows but not in carnitine-infused cows. Carnitine infusion increased the concentration of each milk carnitine fraction as well as milk carnitine output on d 8 to 9. On d 13 to 14, all carnitine fractions except short-chain acylcarnitine were increased in milk from water-infused, feed-restricted cows, whereas all fractions were increased in carnitine-infused, feed-restricted cows. Carnitine infusion increased total carnitine in plasma, liver, muscle, and milk during feed restriction, whereas feed restriction alone increased carnitine concentrations in muscle and milk but not in liver. Liver carnitine concentrations might limit hepatic fatty acid oxidation capacity in dairy cows during the periparturient period; therefore, supplemental l-carnitine might decrease liver lipid accumulation in periparturient cows.

The determination of l-carnitine in several food samples

We designed this study to re-validate some methodological parameters of the radio-enzymatic assay, including phenomena of residual, non-carnitine radioactivity present in some assay mixtures of food samples. The second part of the paper presents l-carnitine concentrations (total-, free- and acyl-carnitine) in a wide range of food samples of animal and plant origin. Due to an appropriate procedure of sample preparation and an elimination of influencing factors in the biological samples, more homogeneous and reliable results were obtained and smaller variances between the sample duplicates were obtained. For total and free carnitine, the inter-assay variances of the method were 6.5% and 7.2%, respectively. The lower limit of detection was 0.11 μmol carnitine/l of assay mixture. The highest carnitine contents were found in muscle from kangaroo (6370 mg/kg DM (dry matter)) and horse (4230 mg/kg DM), followed by different cattle meats (3320–1870 mg/kg DM). The carnitine content in the tissues from pigs and fowls ranged from 135 to 830 mg/kg DM. The carnitine content in most dairy products ranged, approximately, between animal tissues and plant-derived food with concentrations ranging from 8 to 530 mg/kg DM. The method was found to be easily and rapidly usable for the determination of l-carnitine in complex biological samples.

Preserved Norepinephrine Reuptake but Reduced Sympathetic Nerve Endings in Hypertrophic Volume-Overloaded Rat Hearts

Background In congestive heart failure (CHF), an activation of the cardiac sympathetic nervous system results in depleted cardiac norepinephrine (NE) stores. The underlying regulatory mechanisms are discussed controversially and were investigated in the present study in CHF resulting from volume overload. Methods and Results Aorto-caval shunt (AVS) was performed in rats. Plasma NE levels were determined by radioenzymatic assay, left ventricular NE by high-performance liquid chromatography, endothelin-1 by enzyme-linked immunosorbent assay. Tyrosine-hydroxylase (TH)– and nerve growth factor (NGF)–mRNA was determined by Northern blot analysis and ribonuclease-assay. Cardiac [ 3H]-NE uptake was measured in isolated perfused hearts. Glyoxylic acid–induced histofluorescence was used to quantify cardiac sympathetic nerves. Compared with sham-operated animals (SH), AVS rats were characterized by depleted cardiac NE stores and enhanced NE plasma levels. Neither TH-mRNA levels in stellate ganglia, nor cardiac [ 3H]-NE-uptake were reduced in AVS. The left ventricular density of sympathetic nerves was markedly decreased. Gene expression of myocardial NGF (a positive regulator of NE reuptake and cardiac sympathetic nerve density) and left ventricular endothelin-1 (a negative regulator of NE reuptake and positive regulator of cardiac NGF expression) were unchanged. Conclusion In volume-overloaded hypertrophic hearts, depletion of cardiac NE stores is caused by a reduction of the sympathetic nerve density, whereas cardiac NE reuptake is preserved.

Serum TK levels in CLL identify Binet stage A patients within biologically defined prognostic subgroups most likely to undergo disease progression

Serum thymidine kinase (TK) levels have been shown to be correlated with survival in many malignancies, including chronic lymphocytic leukaemia (CLL). This study was designed to investigate associations between TK levels and other prognostic markers, in newly and previously diagnosed Binet stage A patients. Furthermore, the use of serum TK measurement to identify subcategories of disease within those defined by IgV(H) mutational status, gene usage and chromosomal aberrations was investigated. Ninety-one CLL patients were enrolled. Serum TK levels were measured using a radioenzyme assay. IgV(H) mutational status and V(H) gene usage were determined using BIOMED-2 primers and protocol. Recurring chromosomal abnormalities were detected by interphase fluorescent in situ hybridisation (FISH). Flow cytometry and reverse transcriptase polymerase chain reaction (RT-PCR) determined CD38 and Zap-70 expression, respectively. Significantly higher serum TK levels were found in IgV(H) unmutated, compared with IgV(H) mutated, patients (P < 0.001). Elevated TK levels were also found in patients with CD38 and Zap-70 positivity (P = 0.004, P < 0.001, respectively), short lymphocyte doubling time (LDT) (P = 0.044) and poor or intermediate prognosis chromosomal aberrations (P < 0.001). A TK level of >8.5 U/L best identified patients with progressive disease. Elevated TK levels could identify patients categorised, at diagnosis, into good prognosis subgroups by the various biological markers (mutated IgV(H), good prognosis chromosomal aberrations, Zap-70(-) and CD38(-)) who subsequently showed disease progression. Additionally, patients with V(H)3-21 gene usage showed high TK levels, irrespective of mutational status, and serum TK measurement retained predictive power as disease progressed in all subcategories studied.

Multifactorial prospective study of tumoral factors related to disease-free survival (DFS) in colorectal cancer patients

3604 Background: There is still a need to identify faithful biological prognostic factors in colorectal cancer, particularly in stage 2, so as to decide upon adjuvant treatment. We thus conducted a prospective multicentric multifactorial study to this end. Methods: Primary colorectal tumors (30 stage 1, 119 stage 2, 107 stage 3) were prospectively collected in 256 patients undergoing total tumor resection (152 men, 104 women ; mean age 69, extremes 29–90). Adjuvant 5FU-based chemotherapy was administered in 92 patients. Median follow-up was 54 months (53 patients developed metastasis or recurrence). Tumors were analyzed for thymidylate synthase (TS), thymidine phosphorylase and dihydropyrimidine dehydrogenase expression (RT-PCR), TS activity (radioenzymatic assay), EGFR level (ligand-binding assay), VEGF (Elisa), DNA content and cell cycle (flow cytometry), p53 mutations (exon 4 to 8), microsatellite instability (bat 25, bat 26), EGFR genotype (CA repeats in intron 1 and -216G&gt;T), TS genotype in 3’ (6 bp deletion) and 5’ (28 bp repeats including the G&gt;C mutation), and methylenetetrahydrofolate reductase genotype (677C&gt;T and 1298A&gt;C). Results: With the exception of tumor TS expression (p = 0.034, the higher the expression, the better the DFS), none of the analyzed parameters were linked to DFS. In multivariate Cox analysis, tumor staging (p = 0.001) and TS expression (p = 0.062) were the sole factors associated to DFS. Focus on tumoral EGFR revealed large inter-patient variability (from 1 to 510 fmol/mg prot) with a significant influence of tumor localisation (p = 0.009, higher in proximal colon) and differentiation status (p = 0.01, higher in poorly differentiated tumors). EGFR within the tumors was 30 % lower than in adjacent normal mucosa (p&lt;0.001). The longer the intron 1 CA repeats, the higher the tumor EGFR (p = 0.044). EGFR -216G&gt;T polymorphism was linked to intron 1 polymorphism, although -216G&gt;T did not influence EGFR levels. Conclusions: The present results underline the major impact of TS expression as a prognostic factor and provide new insights in the knowledge of EGFR in colorectal cancer. No significant financial relationships to disclose.

Effects of stress on catecholamine stores in central and peripheral tissues of long-term socially isolated rats

Both the peripheral sympatho-adrenomedullary and central catecholaminergic systems are activated by various psycho-social and physical stressors. Catecholamine stores in the hypothalamus, hippocampus, adrenal glands, and heart auricles of long-term socially isolated (21 days) and control 3-month-old male Wistar rats, as well as their response to immobilization of all 4 limbs and head fixed for 2 h and cold stress (4 degrees C, 2 h), were studied. A simultaneous single isotope radioenzymatic assay based on the conversion of catecholamines to the corresponding O-methylated derivatives by catechol-O-methyl-transferase in the presence of S-adenosyl-l-(3H-methyl)-methionine was used. The O-methylated derivatives were oxidized to 3H-vanilline and the radioactivity measured. Social isolation produced depletion of hypothalamic norepinephrine (about 18%) and hippocampal dopamine (about 20%) stores and no changes in peripheral tissues. Immobilization decreased catecholamine stores (approximately 39%) in central and peripheral tissues of control animals. However, in socially isolated rats, these reductions were observed only in the hippocampus and peripheral tissues. Cold did not affect hypothalamic catecholamine stores but reduced hippocampal dopamine (about 20%) as well as norepinephrine stores in peripheral tissues both in control and socially isolated rats, while epinephrine levels were unchanged. Thus, immobilization was more efficient in reducing catecholamine stores in control and chronically isolated rats compared to cold stress. The differences in rearing conditions appear to influence the response of adult animals to additional stress. In addition, the influence of previous exposure to a stressor on catecholaminergic activity in the brainstem depends on both the particular catecholaminergic area studied and the properties of additional acute stress. Therefore, the sensitivity of the catecholaminergic system to habituation appears to be tissue-specific.

Phosphatidylethanolamine N‐methyltransferase (PEMT) activity in pregnant and postpartum mice fed a control or choline‐deficient diet

Choline is an important nutrient during pregnancy and lactation when choline transport from mother to fetus supports fetal brain development. Choline is derived from the diet and from an endogenous pathway where phosphatidylethanolamine N-methyltransferase (PEMT) methylates phosphatidylethanolamine using S-adenosyl-L-methionine (SAM) as the methyl donor. Previous research shows that PEMT activity is increased by estrogen and by a choline-deficient diet. Pregnancy increases maternal serum estrogen levels and depletes maternal liver of choline. We investigated PEMT activity in pregnant or postpartum C57BL/6J mice fed either a control or choline-deficient diet. Hepatic PEMT activity was measured at embryonic days 15 and 18 (E15, E18) and 1, 15, and 30 days postpartum (P1, P15, P30) using a radioenzymatic assay. PEMT activity across these time points did not statistically change in dam liver from either the control or choline-deficient group. Also, changes in Pemt gene expression were measured with real-time PCR, using β-actin as an endogenous control gene. In the control diet group, Pemt gene expression at both E18 and P1 was significantly higher than at P15 and P30 (P< 0.05). In the choline-deficient group, Pemt expression at both E18 and P1 was significantly higher than expression at P15 (P<0.05). These results demonstrate that pregnancy has differential effects on Pemt gene expression in dam liver. This research was supported by grants from Mead Johnson Nutritionals and NIH (DK55865 and AG09525).

Temporal changes in biochemical indices of sulphur amino acid (SAA) metabolism in the vitamin B‐6 deficient piglet

The impact of vitamin B-6 deficiency on temporal changes in markers of SAA metabolism was determined in weanling pigs. Pigs (5.3 kg; n=6 per group) were fed a semi-purified diet (20.5% casein; 3474 kcal ME/kg) containing either 0 or 3 mg pyridoxine HCl/kg diet, using a pair-feeding design. Feed intake was measured continuously, and growth data and blood samples were collected weekly for 6 weeks. Animals were killed at the end of 6 weeks, and tissue samples harvested. Plasma pyridoxal-5-phosphate (PLP) was quantified by radioenzymatic assay. Total homocysteine (Hcy) and cysteine (Cys) were measured by reverse-phase HPLC. From the 4th week until the end of trial, vitamin B-6 deficiency decreased average daily gain and feed efficiency (p<0.05), despite the pair-feeding. In B-6 deficient pigs, plasma PLP decreased substantially after the first week (37 vs.14 nM). At 6 weeks, plasma PLP was significantly higher in control vs. B-6 deficient pigs (36 vs 5 nM; p<0.0001). Significant decreases in plasma Cys and increases in plasma Hcy, due to B-6 deficiency, were detected from the 3rd week (p<0.0001). From the 3rd to 6th week, plasma Hcy in controls was 17.70, 13.51, 11.48, and 13.28 μ M, respectively. Plasma Hcy in deficient pigs, however, was 332.48, 403.19, 337.94, and 479.91 μM, respectively. Similarly, vitamin B-6 deficiency decreased hepatic Cys (164.03 vs. 86.31 nmol/g liver, p=0.04) but increased hepatic Hcy (16.06 vs. 307.41 nmol/g liver, p=0.001). These results provide justification for a 4 week period to establish a vitamin B-6 deficiency in piglets. This data is critical to the implementation of studies designed to quantify the impact of vitamin B-6 status on rates of transsulphuration in pigs. Supported by NSERC

Effect of cholecystokinin octapeptide on diacylglycerol-PKC signaling pathway in rat pulmonary interstitial macrophages stimulated by lipopolysaccharide1

To investigate the effect of cholecystokinin octapeptide (CCK-8) on the diacylglycerol-protein kinase C (DAG-PKC) signaling pathway in rat pulmonary interstitial macrophages (PIM) stimulated by lipopolysaccaride (LPS). The PIM from rat lung tissues were isolated using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. DAG content and PKC activity were measured by radioenzymatic assay. The translocation of PKCzeta was determined by semi-quantitative immunoblot analysis. CCK-8, at high concentrations (1 x 10(-6) - 1 x 10(-5) mol/L), decreased DAG content and inhibited PKC activity and PKCzeta translocation compared with that in rat resting PIM of a control group (P< 0.01). LPS increased DAG content, and promoted PKC activity and PKCzeta translocation (P< 0.01). CCK-8 decreased LPS-induced DAG content and inhibited LPS-induced PKC activity and PKCzeta translocation significantly at 1 x 10(-8) - 1 x 10(-5) mol/L (P< 0.01). This inhibitory effect of CCK-8 could be abrogated partly by proglumide (non-selective CCK receptor antagonist), CR-1409 (selective CCK-A receptor antagonist), and CR-2945 (selective CCK-B receptor antagonist) in a concentration-dependent manner (P< 0.01). CCK-8 was a negative modulator of the DAG-PKC signaling pathway in rat resting PIM, which is very important for maintaining body homeostasis. It significantly inhibited LPS-induced DAG content, PKC activity and PKCzeta translocation in a concentration-dependent manner. The CCK receptor, especially the CCK-A receptor, might play a major role in this process.

Novel stressors affected catecholamine stores in socially isolated normotensive and spontaneously hypertensive rats

Catecholamines in some central (hypothalamus and hippocampus) and peripheral tissues (adrenal glands and heart auricles) of long-term socially isolated normotensive and spontaneously hypertensive rats exposed to novel immobilization stress were determined by a simultaneous single isotope radioenzymatic assay. Long-term isolation (21 days) produced depletion of hypothalamic norepinephrine (NE) stores and hippocampal dopamine (DA) stores in both normotensive and spontaneously hypertensive rats. Acute immobilization stress (2 h) significantly decreased NE and DA stores in hypothalamus and hippocampus of naive normotensive and spontaneously hypertensive rats controls. However, novel immobilization stress applied to normotensive rats previously subjected to long-term isolation produced no changes in catecholamine levels in hypothalamus, while resulting in somewhat higher depletion of NE stores in hypothalamus of spontaneously hypertensive rats treated in the same way. Novel immobilization stress decreased NE and DA stores in hippocampus of normotensive but was without effect on NE and DA stores of spontaneously hypertensive rats. Social isolation did not affect catecholamine stores in peripheral tissues but novel immobilization stress produced a significant decrease in catecholamine content. The results suggest that some central and peripherals tissues of spontaneously hypertensive rats and normotensive rats differ with regard to catecholamine content and that there are certain differences in their responsiveness to stress.

A non-radiometric method for measuring serum thymidine kinase activity in malignant lymphoma in dogs

The aim of this study was to evaluate an enzyme-linked immunosorbent assay (ELISA), for determination of serum thymidine kinase 1 (sTK1) activity in dogs with malignant lymphoma (ML) and compare it with a thymidine kinase (TK) radioenzymatic assay (TK-REA). The TK-REA has recently been shown to be useful in determining the clinical stage and prognosis in canine ML. In addition, serum lactate dehydrogenase (LDH) was measured. Forty-five dogs were included in the study. Sixty serum samples from these dogs, stored in a tumour serum sample bank (stored at −20 °C), were analysed. Apart from 37 dogs with ML, four normal dogs as well as two dogs with mammary carcinomas, one dog with bladder carcinoma, and one dog with malignant fibrous histiocytoma were included. Staging of ML was based on the modified World Health Organization (WHO) staging system for canine ML. The diagnosis of all tumours was verified by histopathology. The TK activity (units per litre [U/L]) ranged from 1.0 to 607.9 in the TK-REA analysis and from 1.1 to 510 in the TK-ELISA (normal reference value <7 U/L). The range for LDH was between 12 and 1194 U/L (normal reference value <228 U/L). There was a significant correlation between the TK-REA and the TK-ELISA. The correlation coefficient (CC) was 0.97 and the standard error of the estimate (SEE) was 3.7 U/L. There was no correlation between LDH and either the TK-REA or the TK-ELISA (CC = 0.53 for both assays; SEE = 26.7 and 12.7 U/L, respectively). Most of the variation in LDH was still within the normal reference range. The mean LDH in dogs with high-stage (stage IV + V) disease was 201.9 U/L. The corresponding values for the TK-REA and TK-ELISA were 109 and 109.9 U/L, respectively. The significant relation between the TK-REA and the TK-ELISA was confirmed by Bland–Altman analysis. The TK-ELISA assay, because of its relative simplicity, will permit measurement of TK in cases of ML in dogs to become a routine procedure.

Production of Lysophosphatidic Acid in Blister Fluid: Involvement of a Lysophospholipase D Activity

Lysophosphatidic acid (LPA) is present in abundance in serum resulting from platelet activation and is also found in other biological fluids. LPA controls numerous cellular responses and plays a role in specific functions such as wound healing, especially in the skin. Nevertheless, its presence in the skin has never been investigated. Since re-epithelialization occurs after blister rupture, we tested the presence of endogenous LPA in blister fluid and investigated a possible mechanism for its biosynthesis and biological functions. Using a radioenzymatic assay, LPA was detected in 33 blister fluids originating from 24 bullous dermatoses, and at higher concentrations than in plasma. In parallel, blister fluids contained a lysophospholipase D (LPLD) activity but no detectable phospholipase A2 activity. The expressions of the LPLD autotaxin (ATX) and of LPA1-receptor (LPA1-R) were greatly increased in blister skin when compared with normal skin. Finally, LPA was found to have a positive effect on the migration of cultured keratinocytes. These results show that LPA is present in blister fluid synthesized by the LPLD ATX. Due to its ability to enhance keratinocyte migration, LPA in blister fluid could, via the LPA1-R, play an important role in re-epithelialization occurring after blister rupture.

Angiotensin II Exerts Positive Feedback on the Intrarenal Renin-Angiotensin System by an Angiotensin Converting Enzyme-Dependent Mechanism 1

Plasma angiotensin II (ANG II) is not increased significantly in renovascular hypertension (RVH), but tissue ANG II levels are elevated in both kidneys of renovascular rats. Because the contralateral, non-ischemic kidney is critical for maintenance of hypertension in RVH, this study sought to understand the mechanism by which intrarenal ANG II levels are augmented in the non-ischemic kidney. This study tested the hypothesis that an incremental increase in plasma ANG II induces the intrarenal renin-angiotensin system (RAS) in the non-ischemic kidney by an angiotensin converting enzyme (ACE) dependent mechanism. To simulate the incremental increase in plasma ANG II induced by the ischemic kidney in RVH, an ANG II infusion model was used. This model used a chronic infusion of ANG II (40 ng/min) or vehicle by osmotic minipump into uninephrectomized rats. Parallel groups were treated with the ACE inhibitor Enalaprilat (200 mg/kg/day). Intrarenal ACE activity was measured by radioenzymatic assay. ANG II levels were quantified by radioimmunoassay. Hypertension was evident in ANG II-infused rats, compared to control rats (155 +/- 4 versus 112 +/- 1 mmHg; P < 0.001). Concurrent treatment with Enalaprilat reversed the hypertension induced by ANG II infusion (98 +/- 3 versus 155 +/- 4 mmHg; P < 0.001). ANG II up-regulated intrarenal ACE activity in the non-ischemic kidney (59.2 +/- 11.9 versus 25.2 +/- 6.8 units/mg protein; P < 0.01). Enalaprilat significantly decreased renal ACE activity in ANG II-treated rats, compared to ANG II alone (11.4 +/- 1.0 versus 59.2 +/- 11.9 units/mg protein; P < 0.001). Intrarenal ANG II was increased in ANG II-infused rats, compared to control animals (52.9 +/- 7.1 versus 23.0 +/- 3.2 fmol/mg tissue; P < 0.001), and Enalaprilat prevented ANG II-induced increases in intrarenal ANG II (29.9 +/- 2.6 versus 52.9 +/- 7.1 fmol/mg tissue; P < 0.05). Incremental changes in plasma ANG II induce de novo production of ANG II in the non-ischemic kidney to augment intrarenal ANG II content. ACE inhibition blocks this positive feedback loop, suggesting that ANG II activates the intrarenal RAS by an ACE-dependent mechanism. The impact of ACE inhibition on blood pressure suggests that this feedback loop may be an important mechanism for maintenance of hypertension in RVH.

Rapid effects of aromatase inhibition on male reproductive behaviors in Japanese quail

Non-genomic effects of steroid hormones on cell physiology have been reported in the brain. However, relatively little is known about the behavioral significance of these actions. Male sexual behavior is activated by testosterone partly through its conversion to estradiol via the enzyme aromatase in the preoptic area (POA). Brain aromatase activity (AA) changes rapidly which might in turn be important for the rapid regulation of behavior. Here, acute effects of Vorozole™, an aromatase inhibitor, injected IP at different doses and times before testing (between 15 and 60 min), were assessed on male sexual behavior in quail. To limit the risk of committing both types of statistical errors (I and II), data of all experiments were entered into a meta-analysis. Vorozole™ significantly inhibited mount attempts ( P < 0.05, size effect [ g] = 0.527) and increased the latency to first copulation ( P < 0.05, g = 0.251). The treatment had no effect on the other measures of copulatory behavior. Vorozole™ also inhibited appetitive sexual behavior measured by the social proximity response ( P < 0.05, g = 0.534) or rhythmic cloacal sphincter movements ( P < 0.001, g = 0.408). Behavioral inhibitions always reached a maximum at 30 min. Another aromatase inhibitor, androstatrienedione, induced a similar rapid inhibition of sphincter movements. Radioenzyme assays demonstrated that within 30 min Vorozole™ had reached the POA and completely blocked AA measured in homogenates. When added to the extracellular milieu, Vorozole™ also blocked within 5 min the AA in POA explants maintained in vitro. Together, these data demonstrate that aromatase inhibition rapidly decreases both consummatory and appetitive aspects of male sexual behavior.

Characterization of 11β-Hydroxysteroid Dehydrogenase Activity in Testicular Tissue of Control and GnRH-Immunized Boars as a Possible Regulator of Spermatogenesis

Glucocorticoids are involved in the regulation of spermatogenesis in the boar testis by initiating apoptosis in early stages of germ cell development. Because cortisol activity is modulated by the 11beta-hydroxysteroid dehydrogenase system (11beta-HSD), the present study determined both 11beta-activating (reductive) and inactivating (oxidative) enzyme activities in testicular tissue preparations of control boars (n = 5), GnRH-immunized boars (n = 5), and immunized, estradiol-infused boars (n = 6) by radioenzyme assay based on the conversion of tritiated cortisol to cortisone in the presence of NAD+ (inactivation) or tritiated cortisone to cortisol in the presence of NADPH (activation). The presence of both isoforms, 11beta-HSD 1 and 11beta-HSD 2, was confirmed by RTPCR in testicular tissue of control boars. Additionally, cortisol, testosterone, estradiol, and LH were determined in blood plasma sampled twice before killing. Immunization led to a drop of LH from 4.3 +/- 0.3 pmol/L to 1.0 +/- 0.3 pmol/L (testosterone: 11.63 +/- 0.83 nmol/L vs. 0.28 +/- 0.07 nmol/L; 17beta-estradiol: 512.61 +/- 47.60 pmol/L vs. 77.05 +/- 14.00 pmol/L). Low 11beta-HSD reductive activity was found in boars (1 pmol steroid x min (-1) x mg (-1)). It decreased to trace amounts in immunized boars (0.07 pmol steroid x min (-1) x mg (-1)). Oxidative activity was found in boars with 10.19 +/- 2.28 pmol steroid x min (-1) x mg (-1) protein. Immunization led to a sharp decrease (0.08 +/- 0.03 pmol x min (-1) x mg (-1)). Infusion of 17beta-estradiol significantly elevated peripheral estradiol concentrations to 752.07 +/- 24.19 pmol/L which still is a physiological concentration in this species. The infusion led to a minimal reductive activity (0.04 pmol steroid x min (-1) x mg (-1)), but led to a 6-fold rise of the 11beta-HSD oxidative activity to 0.47 +/- 0.14 pmol x min (-1) x mg (-1) compared to immunized boars. It is concluded that the 11beta-HSD system is involved in the regulation of cortisol activity in the testis and thus in the regulation of spermatogenesis.

Activation of human lung mast cells by monomeric immunoglobulin E

The mechanism of chronic mast cell activation in asthma is unclear. Monomeric immunoglobulin (Ig)E in the absence of allergen induces mediator release from rodent mast cells, indicating a possible role for IgE in the continued activation of mast cells within the asthmatic bronchial mucosa. In this study it was investigated whether monomeric IgE induces Ca2+ influx and mediator release from human lung mast cells (HLMC). Purified HLMC were cultured for 4 weeks and then exposed to monomeric human myeloma IgE. Ratiometric Ca2+ imaging was performed on single fura-2-loaded cells. Histamine release was measured by radioenzymatic assay; leukotriene C4 (LTC4) and interleukin (IL)-8 were measured by ELISA. At concentrations experienced in vivo, monomeric IgE induced dose-dependent histamine release, LTC4 production and IL-8 synthesis. This was associated with a rise in cytosolic free Ca2+. Enhanced histamine release was still evident 1 week after initial exposure to IgE suggesting that continued exposure maintains enhanced secretion. Monomeric immunoglobulin E alone activates cultured human lung mast cells initiating Ca2+ influx, degranulation, arachidonic acid metabolism and cytokine synthesis. These findings support the hypothesis that immunoglobulin E loading of mast cells within the asthmatic airway contributes to the disordered airway physiology of this disease.

Lysophosphatidic Acid Inhibits Adipocyte Differentiation via Lysophosphatidic Acid 1 Receptor-dependent Down-regulation of Peroxisome Proliferator-activated Receptor γ2

Lysophosphatidic acid (LPA) is a bioactive phospholipid acting via specific G protein-coupled receptors that is synthesized at the extracellular face of adipocytes by a secreted lysophospholipase D (autotaxin). Preadipocytes mainly express the LPA(1) receptor subtype, and LPA increases their proliferation. In monocytes and CV1 cells LPA was recently reported to bind and activate peroxisome proliferator-activated receptor gamma (PPARgamma), a transcription factor also known to play a pivotal role in adipogenesis. Here we show that, unlike the PPARgamma agonist rosiglitazone, LPA was unable to increase transcription of PPARgamma-sensitive genes (PEPCK and ALBP) in the mouse preadipose cell line 3T3F442A. In contrast, treatment with LPA decreased PPARgamma2 expression, impaired the response of PPARgamma-sensitive genes to rosiglitazone, reduced triglyceride accumulation, and reduced the expression of adipocyte mRNA markers. The anti-adipogenic activity of LPA was also observed in the human SGBS (Simpson-Golabi-Behmel syndrome) preadipocyte cell line, as well as in primary preadipocytes isolated from wild type mice. Conversely, the anti-adipogenic activity of LPA was not observed in primary preadipocytes from LPA(1) receptor knock-out mice, which, in parallel, exhibited a higher adiposity than wild type mice. In conclusion, LPA does not behave as a potent PPARgamma agonist in adipocytes but, conversely, inhibits PPARgamma expression and adipogenesis via LPA(1) receptor activation. The local production of LPA may exert a tonic inhibitory effect on the development of adipose tissue.

Role of vitamins B6, B12 and folic acid on hyperhomocysteinemia in a Pakistani population of patients with acute myocardial infarction

Pakistani people belong to an ethnic group which has the highest rate of coronary artery disease (CAD). We investigated the possible correlation between deficiency of vitamins B6, B12 or folic acid and hyperhomocysteinemia in Pakistani patients with acute myocardial infarction (AMI). A case-control study was carried out involving 224 AMI patients (age 30-70 years; 55 females and 169 males) and 126 normal healthy subjects (age 31-70 years; 35 females and 91 males). Fasting venous blood was obtained from cases and controls. Serum was analyzed for folic acid and B12 using radioassays. Plasma was analyzed for pyridoxal phosphate (PLP; coenzymic form of B6) using a radioenzymatic assay and for total homocysteine using a fluorescence polarization immunoassay. Mean serum B12 concentration in AMI patients was found to be significantly lower than the mean for controls (241+/-185 pg/ml vs 608+/-341 pg/ml; p < 0.001). Mean serum folate level in patients was also found to be lower than controls (3.35+/-3.78 ng/ml vs 4.93+/-2.93 ng/ml), however, the differences were not statistically significant. Similarly, mean PLP concentration in plasma of cases (19.4+/-24.4 nmol/l) was lower than the concentration in controls (23.2+/-17.6 nmol/l), but the difference was not statistically significant. Mean plasma homocysteine level in AMI cases (18+/-8.36 micromol/l) was higher than the mean level in controls (16.4+/-4.9 micromol/l), but not to a significant extent. However, this mean homocysteine concentration in normal healthy subjects was among the highest reported in the literature and was significantly more than mean values reported in most Eastern and Western studies. Compared to controls, there was significantly greater deficiency of folate (32.5% vs 67.1%), B12 (3.2% vs 63.4%) and PLP (49.2% vs 74.1%) in AMI patients. Deficiencies of folate, B12 and PLP were defined as serum folate levels less than 3.5 ng/ml, serum levels of B12 less than 200 pg/ml and plasma PLP levels less than 20 nmol/l. Mean plasma homocysteine levels in smokers were found to be significantly higher in both cases and controls. Similarly, mean serum folate levels in smokers (compared to nonsmokers) were significantly lower in both cases and controls. Substantial nutritional deficiencies of these three vitamins along with mild hyperhomocysteinemia, perhaps through an interplay with the classical cardiovascular risk factors (highly prevalent in this population), could be further aggravating the risk of CAD in the Pakistani population.

Co-cultivation of mast cells and FceRIalpha+ dendritic-like cells from human hip bone marrow

Mast cells contribute to the pathogenesis of asthma and allergy through the release of a plethora of pro-inflammatory mediators and cytokines. Their study is hampered by the difficult access to human tissue samples. Human mast cells have been cultured from CD34+ progenitors in the bone marrow of normal volunteers following iliac crest bone marrow biopsy but this is invasive. Hip bone marrow could provide a more convenient less invasive source of mast cell progenitors. To characterize mast cells cultured from human bone marrow obtained at routine hip surgery. Mononuclear cells were isolated from the bone marrow reamings of patients undergoing routine hip replacement surgery and were cultured with recombinant stem cell factor (SCF), IL-6 and IL-10. Cell surface markers were examined using flow cytometry, protease expression monitored using immunohistochemistry, histamine measured by radioenzymic assay, Ca2+ responses analysed using ratiometric Ca2+ imaging, and ion currents recorded via the patch-clamp technique. Mast cells were absent at baseline, but accounted for 65 +/- 7% of cells after 8-12 weeks of culture, equating to a mean 0.6 +/- 0.14 x 10(6) mast cells per culture. Fifty-three percent of tryptase+ cells also contained chymase. The remaining cells comprised a population of large CD1a+ HLA-DR+ and Fc epsilon RI alpha+ cells, most likely dendritic cells. All mast cells expressed CD117 and the high-affinity IgE receptor alpha-chain (Fc epsilon RI alpha) constitutively, and developed a Ca2+ response following IgE-dependent activation. These cells exhibited 7.8 +/- 2.9% net IgE-dependent histamine release, and demonstrated a similar ion channel profile to human lung mast cells. In particular, the intermediate conductance Ca(2+)-activated K+ channel opened following IgE-dependent activation. Mast cells grown from human hip marrow provide a rich non-invasive source of functionally mature mast cells. In addition, this culture system may be useful for the generation of Fc epsilon RI alpha+ dendritic cells.