Ng Mg-1 Protein Research Articles

Neuroprotective effects of eugenol against aluminiuminduced toxicity in the rat brain.

Aluminium (Al) is a neurotoxic metal that contributes to the progression of several neurodegenerative diseases. The aim of the present study was to evaluate the protective effect of dietary eugenol supplementation against aluminium (Al)- induced cerebral damage in rats. Male Wistar rats were divided into four groups: normal controls, rats fed a diet containing 6,000 μg g-1 eugenol, rats intoxicated daily with aluminium chloride (84 mg kg-1 body weight) p. o. and fed either a basal diet or a eugenol-containing diet. Daily oral administration of Al for four consecutive weeks to rats significantly reduced brain total antioxidant status (TAS) (11.42±0.31 μmol g-1 tissue, p<0.001) with a subsequent significant enhancement of lipid peroxidation (MDA) (32.55±1.68 nmol g-1 tissue, p<0.002). In addition, Al enhanced brain acetylcholinesterase activity (AChE) (46.22±4.90 U mg-1 protein, p<0.001), tumour necrosis factor alpha (TNF-α) (118.72±11.32 pg mg-1 protein, p<0.001), and caspase 3 (Casp-3) (8.77±1.26 ng mg-1 protein, p<0.001) levels, and in contrast significantly suppressed brain-derived neurotrophic factor (BDNF) (82.74±14.53 pg mg-1 protein, p<0.002) and serotonin (5-HT) (1.54±0.12 ng mg-1 tissue, p<0.01) levels. Furthermore, decreased glial fibrillary acidic protein (GFAP) immunostaining was noticed in the striatum of Al-intoxicated rats, compared with untreated controls. On the other hand, co-administration of dietary eugenol with Al intoxication restored brain BDNF (108.76±2.64 pg mg-1 protein) and 5-HT (2.13±0.27 ng mg-1 tissue) to normal levels, enhanced brain TAS (13.43±0.24 μmol g-1 tissue, p<0.05), with a concomitant significant reduction in TNF-α (69.98±4.74 pg mg-1 protein) and Casp-3 (3.80±0.37 ng mg-1 protein) levels (p<0.001), as well as AChE activity (24.50±3.25 U mg-1 protein, p<0.001), and increased striatal GFAP immunoreactivity, compared with Al-treated rats. Histological findings of brain tissues verified biochemical data. In conclusion, eugenol holds potential as a neuroprotective agent through its hydrophobic, antioxidant, and anti-apoptotic properties, as well as its neurotrophic ability against Al-induced brain toxicity in rats.

Myocardial ferritin content is closely related to the degree of ischaemia.

Ferritin acts as an iron scavenger and thereby may reduce iron catalysed oxygen radical production during reperfusion injury. We tested the hypothesis that the myocardial ferritin concentration is enhanced during ischaemia in proportion to the blood flow reduction. In 10 anaesthetized, open chest Beagle dogs (six controls and four with 60 min coronary occlusion) regional myocardial blood flow (RMBF) was measured with the tracer microsphere technique and ferritin was determined in samples with an average mass of 125 mg (124-256 samples per heart). Under physiological conditions heart rate was 88 +/- 12 bpm, mean aortic pressure 98 +/- 8 mmHg, and RMBF 0.99 +/- 0.33 mL min-1 g-1. Data did not differ between experimental groups, P > 0.05. In the control group regional myocardial ferritin concentration averaged 11.76 +/- 3.54 ng mg-1 protein and exhibited a significant blood flow independent heterogeneity (CV(biol) = 0.27). However, between low and high flow areas (relative flow <0.5 and >1.5 times the average RMBF, respectively) no significant difference in ferritin was found, P > 0.05. In four experiments, in which regional blood flow was reduced by 40% to 0.60 +/- 0.23 mL min-1 g-1, regional ferritin content was significantly higher as compared with the control group 27.95 +/- 6.16 vs. 11.76 +/- 3.54 ng mg-1 protein, respectively. An inverse relationship was observed between ferritin and RMBF, r = -0.61, P < 0.001. Thus, a reduction of RMBF of >80% was associated with a 2.75-fold increase of the average ferritin content. Between subepicardium and subendocardium no significant difference in ferritin content was observed, neither in the control group nor in the group with induced ischaemia. Regions with control low and high flow responded similarly to the coronary constriction with regard to the local ferritin concentration: 27.88 +/- 15.22 vs. 30.10 +/- 14.91 ng mg-1, P > 0.05, respectively. A data analysis using Baye's theorem indicated that sensitivities were 0.28 and 0.94 for average flow reductions of 5 and 93%. In additional in vitro measurements (ischaemic incubation at 37 degrees C) myocardial ferritin content increased almost linearly within the first 60 min of incubation and thereafter remained unchanged. (1). Local physiological ferritin content in myocardium is heterogeneous and unrelated to control myocardial blood flow. (2). Ischaemia results in an enhanced ferritin content in relation to the degree of ischaemia. (3). The increase of myocardial ferritin requires a severe degree of ischaemia.

Intrathecal anaesthesia alters intracellular Ca2+/Mg2+ homeostasis in the spinal cord neurones of experimental rats.

The effect of anaesthesia induced by intrathecal injection of 6.3% MgSO4 or 4% lidocaine on intracellular electrolyte homeostasis in spinal cord neurones of a rat model was investigated. Intracellular Ca2+, Mg2+, Na+ and K+ concentrations were determined at different times after intrathecal administration of NaCl (saline, a control group), MgSO4 or lidocaine. In both thoracic and lumbar spinal cord segments, Ca2+ concentrations rose significantly 30 min and 2 h after 6.3% MgSO4 injection, and after 24 h were still significantly increased compared with the values obtained from the control group which were subjected to sham 'anaesthesia' by saline injection (172, 121 and 108 ng mg-1 protein vs. control 23 ng mg-1 protein, respectively, in the thoracic segment and 222, 229 and 176 ng mg-1 protein vs. control 43 ng mg-1 protein, respectively, in the lumbar segment). Lidocaine injection also produced a significant increase in intracellular Ca2+ in the thoracic and lumbar spinal cord segments (69, 64 and 53 ng mg-1 protein vs. control 33.4 ng mg-1 protein and 26, 94 and 46 ng mg-1 protein vs. 23 ng mg-1 protein respectively). Only a modest rise in intracellular Mg2+ was observed after intrathecal MgSO4 or lidocaine injection (27 ng mg-1 protein vs. 23 ng mg-1 protein). Na+ and K+ concentrations decreased 24 h after MgSO4 and 1 h after lidocaine injection. Intrathecal anaesthesia by MgSO4 or lidocaine alters intracellular electrolyte homeostasis in spinal cord neurones of experimental rats. A possible common mechanism of action via Ca2+ ion channels is discussed.

Characterization of the atrial natriuretic factor system in lungs of the toad Bufo paracnemis.

Blood pressure in the amphibian pulmonary circulation is relatively high because a single ventricle serves both the systemic and pulmonary circulation, creating a high degree of plasma filtration from pulmonary capillaries. Previous studies have shown that lung atrial natriuretic factor (ANF) may have an important physiological function in preventing edema in mammals. In this study, we report the presence of the complete ANF system in the lungs of the toad Bufo paracnemis. Radioimmunoassay of tissue homogenates revealed that toad lung ANF concentration was approximately twice as high (928.5 +/- 83.0 pg mg-1 protein) as that of lung tissue in mammals of a similar size. The amount of ANF was significantly higher in the left than in the right atrium (15.0 +/- 1.2 versus 1.9 +/- 0.8 ng mg-1 protein; N = 4, P < 0.001), while the ventricle contained 488.3 +/- 41.8 pg mg-1 protein. In extracts of both lungs and atria, high-performance liquid chromatography revealed two forms of the peptide; prohormone and a carboxy-terminal peptide of low molecular mass, which is the biologically active form of peptide. The presence of the prohormone suggests that ANF is synthesized in toad lungs and atria. Characterization of toad lung receptors by a competitive binding assay demonstrated three different subtypes of ANF receptors: the guanylyl cyclase (GC) receptors, GC-A and GC-B, as well as clearance (C) receptors. We conclude that the toad Bufo paracnemis has a well-developed complete ANF system in the lung, suggesting that it has a role in toad lung physiology.

Digoxin transfer across the isolated placenta is influenced by maternal and fetal albumin concentrations.

The aim of this study was to evaluate the influence of different maternal and fetal albumin concentrations on the transplacental transfer and the placental tissue accumulation of digoxin. Digoxin passage across the isolated lobules of 15 human placentae was calculated from repeated fetal and maternal perfusate samples, and placental tissue digoxin concentrations were measured at the end of the experiments. Metildigoxin (Lanitop) was added to the maternal medium at a concentration of 5.70 +/- 0.73 ng mL-1, and maternal and fetal perfusate albumin (BSA) concentrations were kept equal either at a high concentration of 21 g L-1 (Group I; n = 5) or at a low concentration of 3 g L-1 (Group III; n = 5), or differed with a materno-fetal gradient of 21:3 g L-1 (Group II; n = 5). In the experiments with low maternal albumin concentrations (Group III), digoxin concentrations in the maternal circuit decreased to 3.56 ng mL-1, whereas digoxin concentrations in the fetal circuit reached 2.59 ng mL-1 over a 3-h period. With maternal BSA concentrations of 21 g L-1 (Group I and Group II), the decrease in digoxin concentration in the maternal circuit was lower (P < 0.05), and digoxin tissue concentrations at the end of the experiments were smaller (0.45 +/- 0.07 and 0.42 +/- 0.03 v. 0.82 +/- 0.32 ng mg-1 protein, Group I and Group II v. Group III respectively; P < 0.05). Comparing only those lobules with similar high concentrations of maternal protein, fetal BSA concentrations of 21 g L-1 resulted in a greater increase in digoxin concentrations in the fetal circuit (end-feto to initial-maternal digoxin concentrations of 0.44 +/- 0.08 v. 0.37 +/- 0.04 ng mg-1 protein (Group I v. Group II respectively), although this was not significant. The data suggest that maternal and fetal serum albumin concentrations may have an influence on transplacental digoxin transfer, and this should be considered when treating fetuses with cardiac disease transplacentally with glycosides.

Relationship of PS2 with response to tamoxifen therapy in patients with recurrent breast cancer.

PS2, an oestrogen-inducible protein, was measured in the cytosol of 230 primary tumours from patients who were subjected to first-line tamoxifen therapy for advanced disease without prior adjuvant therapy with tamoxifen. PS2 correlated positively with oestrogen receptor (ER, P < 0.01) and progesterone receptor content (PgR, P < 0.001), and with the length of progression-free survival (PFS, P = 0.05). Although not statistically significant, higher levels of PS2 (> or = 10 ng mg-1 protein) were also associated with increased probability of response to tamoxifen treatment and a longer total post-relapse survival (PRS). ER, PgR, menopausal status, site of disease and prior adjuvant chemotherapy were all associated with response to tamoxifen therapy and with PFS. In multivariate analysis for PFS, low levels of ER and PgR, visceral metastasis, a disease-free interval of less than 1 year and prior adjuvant chemotherapy were all significantly associated with an increased probability of a rapid disease progression after start of tamoxifen therapy. In the subset of 83 tumours with intermediate levels of ER and PgR (both > or = 10, but not both > or = 75 fmol mg-1 protein), PS2 was positively related with the length of PFS (P < 0.01) and PRS (P < 0.05). PS2 remained the strongest factor in multivariate analysis for PFS (P < 0.01) in this ER/PgR intermediate subgroup, but was not of predictive value in univariate or multivariate analysis for both PFS and PRS in tumours classified as ER/PgR low or high (> or = 75 fmol mg-1 protein). It is concluded that PS2 status may be used as a parameter, additional to ER and PgR, for better refinement of prediction of response to tamoxifen treatment in advanced breast cancer patients especially with intermediate ER/PgR levels in their primary tumour.

Immunoradiometric detection of pS2 and total cathepsin D in primary breast cancer biopsies: their correlation with steroid receptors.

Commercially available immunoradiometric assays were used for pS2 and total cathepsin D determination in the cytosol fraction obtained from 266 primary breast cancers. We show that pS2 and cathepsin D values were significantly associated (Spearman's rank correlation: P < 0.0001) in tumours from lymph node-positive patients (N+), while such association did not reach significance in tumours taken from patients with negative lymph nodes (N-). Moreover, cathepsin D concentrations in pS2-rich tumours (pS2 above the median value, 5 ng mg-1 protein) were significantly higher (Mann-Whitney-Wilcoxon's rank-sum test: P = 0.00001) than those obtained in the samples expressing less than 5 ng of pS2 per mg of protein. pS2 was also correlated to both the oestrogen receptor (ER) (Spearman's rank correlation: P < 0.0001) and the progesterone receptor (PR) (Spearman's rank correlation: P = 0.022). No significant differences in the expression of pS2 and cathepsin D taken from N+ and N- patients were found. Furthermore, no significant differences in pS2 and cathepsin D expression were obtained by stratifying tumours on the basis of their size (T). pS2 and cathepsin D values obtained in ER-positive/PR-positive tumours did not significantly differ from the values obtained in ER-positive/PR-negative and in ER-negative/PR-positive tumours. We conclude that pS2 could have a role in cathepsin D expression, and that it can be used in the assessment of a functioning oestrogen response machinery in those tumours that express only ER.

Aromatase activity and oestrogen concentrations in bovine cotyledons and caruncles during gestation and parturition

Two peaks of aromatase activities were detected in bovine cotyledons, 2.76 +/- 0.96 pmol min-1 mg-1 protein at month five of gestation and 3.62 +/- 0.78 pmol min-1 mg-1 protein immediately after parturition, but aromatase activity in caruncles was very low throughout gestation. Oestrone concentrations in the cotyledons and caruncles were 0.93 +/- 0.50 and 1.51 +/- 0.69 ng mg-1 protein at month five of gestation, but 4.31 +/- 1.66 and 3.36 +/- 0.98 ng mg-1 protein immediately after parturition, indicating a biphasic pattern. Our findings suggest that oestrogen synthesis in the bovine placenta increased to a maximum at least twice during the period from gestation to parturition; at month five of gestation and at parturition the changes in aromatase activity may only be partially explained by changes in oestrogen production.

Cathepsin D and epidermal growth factor in human breast cyst fluid

Cathespin D (Cath D) is a proteolytic enzyme secreted by human breast cancer cells with a growth promoting activity in vitro. In the present study, we measured Cath D and Epidermal Growth Factor/alpha Transforming Growth Factor (EGF/alpha-TGF) concentrations in the breast cyst fluid (BCF) of 43 patients with gross cystic disease of the breast. Both Cath D (median 2.45 pmoles mg-1 protein; range 0-4.84 vs 0.98 pmoles mg-1 protein; range 0-3.11) and EGF/alpha-TGF (28.71 ng mg-1 protein; range 7.05-50.63 vs 10.83 ng mg-1 protein; range 0.06-30.55) levels were higher in BCF of apocrine than flattened cysts (P less than 0.0005 and P less than 0.01, respectively). Premenopausal patients showed higher concentrations of Cath D (P less than 0.05) and EGF/alpha-TGF (P less than 0.05) than postmenopausal patients. A positive correlation was obtained between intracystic concentrations of Cath D and EGF/alpha-TGF (P less than 0.00001). The higher levels of Cath-D and EGF/alpha-TGF found in apocrine cysts could provide an explanation for the increased risk of subsequent breast cancer in women with this type of cyst.

Mechanisms of hypercholesterolaemia in glycogen storage disease type I: defective metabolism of low density lipoprotein in cultured skin fibroblasts

Hyperlipidaemia is a feature of glycogen storage disease type I (GSD-I) (Levy et al.). High levels of LDL cholesterol (200 +/- 25 mg dl-1) and apo B (387 +/- 44 mg dl-1) were found in association with hypercholesterolaemia in GSD-I. Related causative factors might be attributed to overproduction and/or delayed removal of LDL. In this study, a possible alteration in the clearance of LDL was examined. Using cultured fibroblasts for LDL receptor activity, the following observations were made: 1. GSD-I fibroblasts revealed only a slight decrease in LDL binding (65 +/- 7) when compared with controls (74 +/- 4 ng mg-1 protein), however, LDL internalization (382 +/- 24 vs. 570 +/- 52 ng mg-1 protein) and proteolytic degradation (2082 +/- 280 vs. 2916 +/- 12.5 ng mg-1 protein) were significantly affected (P less than 0.01). 2. Binding, internalization and proteolytic degradation of LDL from GSD-I were compared with that of controls, and were found to be significantly lower (P less than 0.01). 3. Substitution of control lipoprotein-deficient serum (LPDS) by GSD-I LPDS further diminished the above processes (P less than 0.05). Our results demonstrate that increased plasma cholesterol in GSD-I is due to a decreased catabolism of LDL. The data suggest that the problem may well be multifactorial, due to diminished receptor expression, abnormal LDL composition and impaired LDL receptor interaction due to a circulating inhibitory factor.

An investigation of the pharmacological response to vitamin K1 in the rabbit.

1. The relationship between pharmacological response and disposition of a dose of vitamin K1 (10 mgkg-1, i.v.) in normal rabbits and in rabbits treated with the coumarin anticoagulant brodifacoum, has been studied. 2. High performance liquid chromatography (h.p.l.c.) with electrochemical detection (EC) was used to determine concentrations of vitamin K1 in plasma, whole liver homogenate, and liver microsomes. 3. After intravenous administration of vitamin K1, plasma concentrations of the vitamin declined in a tri-exponential fashion. There were no differences between the two groups over the first 24 h of the experiment. However, between 24 h and the end of the study, plasma concentrations of vitamin K1 in the presence of brodifacoum were significantly (P less than or equal to 0.05) below those of vehicle-treated rabbits. 4. Seventy-two hours after administration of vitamin K1, plasma concentrations of the vitamin were not different from normal. 5. Three hours after administration of vitamin K1, the concentrations of the vitamin in whole liver were 46.6 +/- 4.3 micrograms g-1 in the presence of brodifacoum, and 32.8 +/- 6.4 micrograms g-1 in the absence of brodifacoum; and were significantly (P less than or equal to 0.05) greater than normal (127.7 +/- 44.3 ng g-1). Likewise, microsomal concentrations of vitamin K1 (4.00 +/- 2.38 micrograms mg-1 protein, and 2.65 +/- 1.01 micrograms mg-1 protein, in the presence and absence of brodifacoum, respectively) were significantly (P less than or equal to 0.01) greater than normal (16.0 +/- 3.5 ng mg-1 protein). 6 In conclusion, there appears to be no direct effect of coumarins on clearance of vitamin K1 from either plasma or liver; the need for large doses of vitamin K1 during coumarin poisoning is due to a greatly increased requirement for the vitamin.

Effects of adrenocorticotropic hormone on low density lipoprotein receptors of human fetal adrenal tissue.

In the present investigation, the mechanism(s) whereby ACTH stimulates low density lipoprotein (LDL) metabolism in human fetal adrenal was evaluated. Plasma membrane fractions were prepared from fetal adrenal tissue fragments incubated in lipoprotein-poor serum with or without ACTH, and the binding of [125I]iodo-LDL to such membrane fractions was examined. The mean specific binding capacity for [125I]iodo-LDL by membrane fractions prepared from four separate fresh human fetal adrenal glands was 1370 +/- 168 ng mg-1 protein (mean +/- SE), and the concentration of [125I]iodo-LDL producing half-maximal binding was 20.8 +/- 1.2 ng ml-1. Thus, the presence of high affinity, low capacity binding sites for LDL in human fetal adrenal tissue was confirmed. When human fetal adrenal tissue was maintained in organ culture for 2 days in medium containing lipoprotein-poor serum in the absence of ACTH, and plasma membrane fractions were subsequently prepared, the binding capacity for LDL in such membrane fractions was the same as or slightly greater than that in membrane fractions prepared from fresh tissue. When ACTH was present in the culture medium, the binding capacity for LDL was doubled compared to that in membrane fractions prepared from tissues incubated in the absence of ACTH. The rate of [125I]iodo-LDL degradation by human fetal adrenal tissue maintained in medium containing ACTH was also twice that of tissue maintained in the absence of ACTH. These results demonstrate that ACTH causes an increase in the number of LDL-binding sites in human fetal adrenal tissue in vitro. This is one mechanism whereby ACTH stimulates LDL metabolism in this tissue.