Apilarnil reduces fear and advances sexual development in male broilers but has no effect on growth.

1. An experiment was conducted to determine the possibility of stimulating sexual development at an early age in male and female broiler chickens by administration of apilarnil, a natural bee product, in the pre-pubertal period. 2. From 28 to 55 d of age, birds were given apilarnil orally. The effects of low (2.5 g/bird) and high (7.5 g/bird) doses of apilarnil on growth performance, testicular weight, secondary sexual characteristics, blood lipids, testosterone and fearful behaviour were evaluated. 3. Apilarnil administration did not cause a positive effect on growth performance of male and female broilers suggesting that apilarnil did not have an anabolic effect. 4. Apilarnil administration suppressed blood glucose and cholesterol. 5. Birds receiving apilarnil remained immobile for a shorter period in a tonic imobiliy test and showed less home-cage avoidance responses suggesting a lower level of fearfulness. 6. Increases in testicular weight, testosterone concentration and comb growth in males receiving apilarnil implied that it stimulates the sexual maturation at an early age. However, a similar stimulation of secondary sexual characteristics was not observed in females.

Zoledronic acid overcomes chemoresistance by sensitizing cancer stem cells to apoptosis.

Unlike low tumorigenic bulk tumor cells (non-CSCs), cancer stem cells (CSCs) are a subset of tumor cells that can self-renew and differentiate into different cancer subtypes. CSCs are considered responsible for tumor recurrence, distant metastasis, angiogenesis, and drug or radiation resistance. CSCs also are resistant to apoptosis. Zoledronic acid (ZA) is a third generation bisphosphonate that reduces cell proliferation and exhibits anti-tumor effects by inducing cell death in some malignancies; however, the effects of ZA on CSCs are unclear. We investigated the anti-cancer effects of ZA on two epithelial cancer cell lines, prostate DU-145 and breast MCF7, focusing primarily on induction and activation of apoptosis. Cluster of differentiation (CD) 133+/CD44+ prostate CSCs and CD 44+/CD24 breast CSCs were isolated from the DU-145 human prostate cancer and MCF-7 human breast cancer cell lines, respectively, using FACSAria flow cytometry cell sorting. CSCs and non-CSCs were exposed to increasing concentrations of ZA for 24, 48 and 72 h to determine the IC50 dose. Annexin-V assay for detecting cell death and cell cycle was performed using the Muse™ Cell Analyzer. Prostate CSCs and non-CSCs were assayed by quantitative reverse transcription PCR (qRT-PCR) array for detecting 84 key apoptosis related genes. Gene regulation at the protein level was investigated by immunofluorescence. ZA caused a dose- and time-dependent decrease in cell viability. Treatment with ZA resulted in a concomitant increase in apoptosis and cell cycle arrest at S-phase in CSCs. Significant over/under-expressions were detected in seven of the genes of ZA-treated DU-145 CSCs cells. Expressions of CASP9, CASP4, BAX and BAD genes increased, while the expressions of BIRC3, BIRC2 and BCL2 genes decreased. In the DU-145 non-CSCs, five genes exhibited changes in gene expression after ZA treatment, two exhibited increased expression (CASP7 and BAD) and three exhibited decreased expression (BIRC3, BIRC2 and BCL2). ZA caused cell death of drug resistant breast MCF-7 and prostate DU-145 cancer stem cells by activating apoptosis. ZA can facilitate the intrinsic pathway of apoptosis in human prostate CSCs by down-regulating anti-apoptotic genes and up-regulating pro-apoptotic genes. ZA may be an effective therapeutic agent for targeting chemoresistance in CSCs.

Immunoexpressions of embryonic and nonembryonic stem cell markers (Nanog, Thy-1, c-kit) and cellular connections (connexin 43 and occludin) on testicular tissue in thyrotoxicosis rat model.

In this study, possible thyrotoxicosis-related histological changes in testicular tissues of rats with experimentally induced thyrotoxicosis model were evaluated on cellular connections and stem cell markers. Two experimental groups, thyrotoxicosis and control, each consisting of eight animals were used. Rats in the thyrotoxicosis group were injected intraperitoneally with 3,3',5-triiodo-l-thyronine (50 µg/100 g body weight/day) for 10 days. At the end of the study, animals in both groups were anesthetized, and blood samples were collected for biochemical analyses. Their testes were dissected out and histological procedure was conducted to perform further histochemical, immunohistochemical analyses and tissue expression analysis by real-time polymerase chain reaction. Expression of the stem cell markers such as c-kit and Thy-1 significantly decreased in the testes of the thyrotoxicosis group compared with the control group; however, Nanog expression was not detected in any of the groups. Similarly, connexin 43 and occludin expressions were also found to be significantly lower in the thyrotoxicosis group. These results on cellular connections are supported with the tissue expression analysis. Our findings are indicative of supporting microenvironmental tissue decay rather than parenchyma damage, which has been actually ignored in the literature. In conclusion, experimental thyrotoxicosis model may have adverse effects on the cell junctional complexes, cell-cell interactions, and pluripotency capacity.

Age and gender dependent alterations in the activities of glutathione related enzymes in healthy subjects.

OBJECTIVES: Oxidative stress as a result of increased free radical production is implicated in the pathogenesis of several diseases. Specific antioxidant enzymes have a crucial role in the prevention of these deleterious effects. Since the activities of these enzymes differ significantly in different populations and seem to be affected by various environmental factors, in this study we aimed to determine the reference values of glutathione related antioxidant enzyme activities in the erythrocytes of healthy subjects and to investigate the possible variations as a function of age and gender in a healthy Turkish Mediterranean population. DESIGN AND METHODS: 130 healthy subjects (12-90 yr, 82 females, 48 males) were divided into six different age groups. Erythrocyte glutathione peroxidase (GSH-PX), glutathione reductase (GR) and glutathione-s-transferase (GST) activities were measured on a Hitachi 704 autoanalyser by the modification of previously described manual UV spectrophotometric methods. RESULTS: No significant differences were observed in erythrocyte GSH-PX, GR and GST activities between different age groups. Overall, GST activities were significantly higher in females compared with males (8.08 +/- 1.39, 6.88 +/- 1.51 U/g Hb respectively, mean +/- SD, p < 0.001). A significant positive correlation between GSH-PX and GR activities was observed (r = 0.49, p < 0.001). CONCLUSION: The results of this study suggested that the activities of GSH-PX, GR and GST did not depend. GST activities overall were higher in females. The reference values that we obtained were different than the previous reports. This situation implies that each population should determine its own reference values and should investigate the influence of environmental factors and life style habits on the activities of these enzymes that constitute a major part of the antioxidant defense system in the human organism.

Association between homocysteine and neopterin in healthy subjects measured by a simple HPLC-fluorometric method.

OBJECTIVES: Neopterin and homocysteine promote vascular smooth muscle cell proliferation through the activation of nuclear factor(kappa) B. The aim of this study was to investigate the relation between these two compounds in healthy subjects by a rapid HPLC-fluorometric method which simplifies sample pretreatment for the measurement of neopterin in serum. DESIGN AND METHODS: In 40 healthy subjects (45.9 +/- 2.1 yr, mean +/- SEM, 10 males, 30 females) serum neopterin concentrations were measured by HPLC-fluorometry and enzyme-linked immunusorbant assay-ELISA and the results were compared. Urinary neopterin and plasma total homocysteine concentrations were assayed by HPLC-fluorometry. RESULTS: Serum neopterin concentrations measured by HPLC and ELISA were 7.5 +/- 0.4 and 7.4 +/- 0.3 nmol/L, respectively, r = 0.92, p < 0.01. Urinary neopterin level was 163.9 +/- 11.0 nmol/mmol creatinine and plasma total homocysteine 7.6 +/- 0.4 micromol/L. A significant positive correlation was observed between serum neopterin and plasma total homocysteine (r = 0.59, p < 0.01). CONCLUSIONS: A simple and rapid sample pretreatment for the measurement of neopterin in serum has been introduced. The significant positive correlation between neopterin and homocysteine implies that, interference with leukocyte function might be a new possible mechanism for the deleterious effects of homocysteine on vascular function.

Beta2-microglobulin and cystatin C in type 2 diabetes: assessment of diabetic nephropathy.

BACKGROUND: Changes in glomerular filtration rate (GFR) provide a valuable indicator of the progression of diabetic nephropathy (DN). This study was designed to demonstrate the clinical values of serum cystatin C (Cys C) and beta2-microglobulin in the assessment of renal function in type 2 diabetics by comparing them with the GFR, estimated from the uptake phase of 99 m technetium dimetiltriamino pentaacetic acid renogram (GFR-DTPA) and creatinine clearances. MATERIALS AND METHODS: 68 type 2 diabetic patients with (urinary albumin excretions (UAE) 30 - 300 mg/24 h) (n = 39) and without (UAE < 30 mg/24 h) (n = 29) microalbuminuria and 32 controls were enrolled in the study. Serum Cys C, beta2-microglobulin, creatinine, urinary microalbumin levels, creatinine clearances and GFR-DTPA values were determined in all groups. Non-parametric ROC curves, using a cut-off GFR-DTPA of 60 mL/min/1.73 m (2), were obtained for these markers. RESULTS: Serum Cys C, beta2-microglobulin, glucose and HbA1c concentrations were significantly higher in the group with diabetes compared to controls. In the patients with microalbuminuria, serum Cys C and glucose concentrations increased significantly in comparison to patients with normoalbuminuria, while no differences were observed for beta2-microglobulin levels. Serum creatinine concentrations, GFR-DTPA values and creatinine clearances were not different between both diabetic groups and controls. Cys C was positively correlated with beta2-microglobulin and creatinine and negatively with GFR values; beta2-microglobulin was also positively correlated with serum creatinine in microalbuminurics. A significant inverse correlation was found between beta2-microglobulin and GFR values in both microalbuminurics and normoalbuminurics. CONCLUSIONS: Increased Cys C and beta2-microglobulin in diabetics may be early indicators of incipient DN. The diagnostic accuracies of Cys C and beta2-microglobulin are superior to that of serum creatinine in distinguishing between mild and moderately reduced GFR.

Glucose metabolism and catecholamine responses during physical exercise in non-insulin-dependent diabetes.

Blood glucose, lactate, insulin, C-peptide, norepinephrine and epinephrine concentrations were determined in non-insulin-dependent diabetic patients and in healthy controls before, during and after moderate exercise, to evaluate the effects of physical exercise on glucoregulation. Ten diabetic and ten healthy control females bicycled 14 minutes at 60% of their maximal heart rates. In the diabetic patients, there were no significant changes in blood glucose levels post-exercise, while in controls the 60 minute post-exercise levels were higher than those measured in mid-exercise (p < 0.05). Lactate concentrations increased with exercise in both groups in a similar manner, with highest values at the end of exercise. No significant changes in insulin and C-peptide levels were induced with exercise in either group. Norepinephrine and epinephrine concentrations increased 2.5-3 fold with exercise in both groups (p < 0.05 for all values) but in the diabetics an earlier and prolonged catecholamine response was observed. We propose that catecholamines prevent hypoglycaemia during exercise when changes in insulin and C-peptide do not occur. In diabetic patients with good metabolic control, the glucoregulatory response to exercise is not worse than in anthropometrically similar controls with similar levels of fitness.

Plasma homocysteine levels in acute coronary syndromes.

Hyperhomocysteinemia is currently regarded as an independent and modifiable risk factor for ischemic vascular diseases and thrombosis. We measured fasting plasma total homocysteine levels by HPLC with fluorescence detection in 30 patients presenting with acute coronary syndromes and 30 age and sex-matched control subjects. Demographic data, classical risk factors (systolic blood pressure, diabetes mellitus, smoking, ethanol intake, family history of ischaemic heart disease) and life-style habits were recorded. Lipid fractions including total cholesterol, triglycerides, HDL-cholesterol, total cholesterol/HDL-cholesterol ratio, serum creatinine, LDL-cholesterol and vitamins involved in the metabolism of homocysteine, folic acid and vitamin B12 were also assessed. Total fasting homocysteine concentrations were significantly higher in the patient group (12.2 +/- 1.01 micromol/l) than in the control subjects (7.05 +/- 0.36 micromol/l; p < 0.0001). Homocysteine correlated positively with age (r = 0.617; p < 0.01) and serum creatinine (r = 0.457; p < 0.01) in the patient group. Hyperhomocysteinemia was not associated with vitamin B12 or folate deficiency states. Vitamin B12 concentration was 273 +/- 16.4 ng/l in the control group and 284.3 +/- 32.2 ng/l in the patient group (p = NS). Serum folate concentration also was not significantly different between controls and patients; 7.57 +/- 0.58 microg/l and 8.05 +/- 0.72 microg/l, respectively. Since no significant difference was observed in the lipid parameters between patients and controls, the hyperhomocysteinemia in the patient group supports the view that homocysteine is an independent risk factor for cardiovascular diseases. Our results strongly suggest that elevated homocysteine levels are among the interacting factors in the complex, multifactorial pathophysiology of ischemic heart disease.

Age-associated changes in nitric oxide metabolites nitrite and nitrate.

Aging is an important determinant of vascular disease. Endothelial dysfunction accompanying vascular disease may be related to cardiovascular risk factors such as aging, hypertension, and atherosclerosis. Experimental models suggest that endothelium-derived nitric oxide is reduced with aging, and this reduction is implicated in atherogenesis. The aim of this study was to determine whether increased age resulted in altered serum nitrite and nitrate levels, end-products of nitric oxide, in healthy subjects. Sixty-nine healthy individuals were divided into five different age groups: group I (6-15 years), group II (16-30 years), group III (31-45 years), group IV (46-60 years), and group V (>61 years). In these subjects, serum nitrite was measured by the Griess reaction and nitrate by the nitrate reductase method. Statistical analysis showed that serum nitrite levels were not significantly different in any of the groups, while serum nitrate concentrations exhibited significant differences (P<0.001). These findings suggest that nitric oxide synthesis and/or secretion is reduced with age and consequently endothelium-dependent vasodilation is impaired.

Adequate immune response to tetanus toxoid and failure of vitamin A and E supplementation to enhance antibody response in healthy children.

The effects of vitamin A and vitamin E supplementation on the IgG response to tetanus toxoid after primary immunization were evaluated in a prospective, randomized controlled clinical trial involving 89 healthy infants with normal serum vitamin A and E levels at 2 months of age. Before the first dose of DPT vaccine, the infants were randomly enrolled into four different study groups [Group I (n=24): 30,000 IU vitamin A for 3 days just after each three doses of primary vaccination, Group II (n=21): 150 mg oral vitamin E for only 1 day after the injections for primary immunization, Group III (n=21): vitamins A and E together in the same order, Group IV (n=23) no vitamin after DPT vaccines]. Serum tetanus antitoxin (IgG) titres were measured three times; initially at 2 months of age before the first dose of DPT, secondly at 5 months of age 1 month after primary immunization and thirdly at 16-18 months of age before the booster dose of DPT. Before the first dose of the DPT vaccine, 1 month after the third DPT injection and at 16-18 months before the booster dose of DPT, there was no significant difference in serum tetanus antitoxin levels between these four groups. A significant increase was observed in all the groups when serum tetanus antitoxin levels before (2 months) and after (5 months) primary immunization were compared. In addition, serum antibody levels against tetanus significantly decreased in the four groups before booster vaccination. Before the beginning of primary immunization, 15 infants (16.8%) had serum tetanus antitoxins (IgG) below protective level. After three doses of DPT, all the infants had protective antitoxin levels. At 16-18 months of age before booster dose, four infants (10%) also had serum tetanus antitoxins (IgG) below the protective level. No side-effects were observed except bulging fontanelle in two infants in Group I.

Urinary hypoxanthine and xanthine levels in acute coronary syndromes.

Ischemia leads to impaired ATP metabolism, with increased production of purine degradation products, such as hypoxanthine and xanthine, which are useful markers of tissue hypoxia. These extracellular markers of ischemia have been studied extensively in many clinical conditions of oxidative stress, including perinatal asphyxia, acute respiratory distress syndrome, cerebral ischemia, and preeclampsia. The aim of this study was to explore the usefulness of urinary hypoxanthine and xanthine as ischemia markers in acute coronary syndromes. Urinary excretion of hypoxanthine and xanthine was assessed by high-performance liquid chromatography in 30 patients with acute coronary syndromes and in 30 age- and sex-matched controls. Serum and urine uric acid, creatinine, and urea concentrations were also determined. Hypoxanthine excretion was significantly elevated in patients compared with healthy controls (84.37+/-8.63 and 42.70+/-3.97 nmol/mg creatinine, mean+/-SEM, P<0.0001). Urinary xanthine levels were also increased in patients with acute coronary syndromes (100.13+/-12.14 and 34.74+/-4.07 nmol/mg creatinine patients and controls, respectively; P<0.0001). Hypoxanthine and xanthine excretion showed a strong positive correlation in both groups. Significant negative correlations between urinary hypoxanthine and uric acid and xanthine and uric acid were observed in the patients, but not in controls. In conclusion, increased levels of ATP degradation products hypoxanthine and xanthine are observed in various hypoxic clinical conditions. This study suggests that these parameters may be useful markers of ischemia in patients with acute coronary syndromes.

Hyperhomocysteinaemia in Behçet's disease.

OBJECTIVE: Arterial and venous thrombosis are among the clinical features of Behçet's disease (BD), the pathogenesis of which is not completely understood. In this study, we investigated whether hyperhomocysteinaemia, being a well known risk factor for thrombosis, is also a contributive risk factor for the arterial and venous thrombosis of BD. METHODS: Eighty-four patients fulfilling the criteria of the International Study Group for Behçet's Disease (54 males, 30 females, mean age 36+/-9 yr) were enrolled. All the patients were carefully screened for a history of venous thrombosis and were separated into two groups with respect to thrombosis history. Thirty-six healthy individuals (23 males, 13 females), matched for age and sex with the BD group, were included as a negative control group. Patients were excluded if they had any condition that might affect plasma homocysteine concentration. As methotrexate (MTX) causes hyperhomocysteinaemia, we also included 29 rheumatoid arthritis patients (five males, 24 females) receiving MTX weekly. Fasting plasma homocysteine concentrations were measured by high-performance liquid chromatography. The data were analysed with the chi(2) test and Student's t-test. RESULTS: The highest homocysteine concentrations were found in the MTX group (17.5+/-5.3 micromol/l). Mean plasma homocysteine concentrations in BD patients were significantly higher than in the healthy controls (11.5+/-5.3 vs. 8.8+/-3.1 micromol/l, P<0.001). Among BD patients with a history of thrombosis, 20 of 31 (64%) had hyperhomocysteinaemia, and this was significantly higher than in those without thrombosis (9%). On the other hand, there was no significant difference between patients with non-thrombotic BD and healthy controls (P>0.05). In patients with thrombosis, we found no correlation between the duration of the post-thrombotic period and homocysteine concentration. Among all the variables investigated, only hyperhomocysteinaemia was found to be related to thrombosis. CONCLUSION: Hyperhomocysteinaemia may be assumed to be an independent risk factor for venous thrombosis in BD. Unlike the factor V Leiden mutation, hyperhomocysteinaemia is a correctable risk factor. This finding might lead to new avenues in the prophylaxis of thrombosis in BD.

Effects of defibrotide on aorta and brain malondialdehyde and antioxidants in cholesterol-induced atherosclerotic rabbits.

The effects of a high-cholesterol diet in the presence and absence of defibrotide, a single-stranded polydeoxyribonucleotide compound, on the lipid peroxidation product malondialdehyde, endogenous antioxidant enzymes catalase, glutathione peroxidase, and the antioxidant thiol compound GSH were investigated. Forty male New Zeland white rabbits were divided into four groups each consisting of 10 rabbits. Group I received a regular rabbit chow diet and group II 1% cholesterol plus regular chow, group III was given defibrotide (60 mg/kg per day p.o. in water) and was fed with regular chow, and group IV received defibrotide plus 1% cholesterol for 9 weeks. Blood cholesterol and malondialdehyde, catalase, glutathione peroxidase, and GSH were determined before starting the experimental diet regimen (basal). After 9 weeks, the same parameters were determined in blood, aorta, and brain tissues (end -experiment). Aortic tissue was examined under a light microscope for morphological alterations indicative of atherosclerosis. The increase in serum total cholesterol was greater in group II than group IV. Plasma malondialdehyde in group II was higher than in group III. Brain malondialdehyde in group II was higher than all other groups, and aortic malondialdehyde in this group was higher than group I and III. Serum catalase activity decreased in group II and increased in group III, compared with basal values. Brain catalase activity in group I was higher than group II, and aorta catalase in group IV was higher than in group I and III. Blood glutathione peroxidase activity in group III and IV was higher than basal. GSH concentrations decreased significantly in the cholesterol-fed groups (group II and IV). Histological alterations in the cholesterol-fed groups were more pronounced in group II. The increased levels of malondialdehyde in plasma, aorta, and brain tissue of group II suggest a role of oxygen free radicals in the pathogenesis of cholesterol-induced atherosclerosis. The higher malondialdehyde values in the brain tissues of animals in group II compared with group IV suggest a protective role of defibrotide in the brain against lipid peroxidation in the oxidant stress of cholesterol-induced atherosclerosis. Increased catalase activities in the blood and aortic tissues and increased glutathione peroxidase activities in the blood of rabbits receiving defibrotide suggest an induction of these antioxidant enzyme activities by defibrotide. These results imply that anti-atherosclerotic, anti-ischemic effects of this drug may be due to the beneficial effects on the oxidant-antioxidant balance of various tissues.