Effects of high-dose soy isoflavones and equol on reproductive tissues in female cynomolgus monkeys

Soy isoflavonoids have well-established estrogenic properties in cell culture and rodent models, raising concerns that high isoflavonoid intake may promote development of uterine and breast cancers. To address this concern we evaluated the effects of high-dose isoflavonoid supplements on reproductive tissues in a postmenopausal primate model. Thirty adult female ovariectomized monkeys (Macaca fascicularis) were randomized to receive a control diet 1) alone, 2) with 509 mg/day of the soy isoflavones genistein and daidzein (IF), or 3) with 1020 mg/day of racemic equol (EQ), an isoflavan, for approximately 1 mo. Doses are expressed in aglycone units as calorically scaled human equivalents. Total serum isoflavonoid levels 4 h postfeeding were <20 nmol/L, 2570.7 nmol/L, and 6944.8 nmol/L for control, IF, and EQ groups, respectively. Equol was the predominant serum isoflavonoid in both IF (72.5%) and EQ (99.7%) groups. Aglycones represented 0.9% (IF) and 0.5% (EQ) of total serum isoflavonoids. Histologically, uteri and mammary glands were diffusely atrophic in all groups. Uterine weight, endometrial thickness, glandular area, and epithelial proliferation in the uterus were not significantly different among treatment groups (ANOVA P > 0.1 for all). Endometrial progesterone receptor gene expression was significantly increased in the IF group (P = 0.02), while protein expression was not altered (ANOVA P > 0.1). Within the mammary gland, proliferation and indicators of estrogen exposure did not differ among treatment groups (ANOVA P > 0.1 for all). These findings indicate that high doses of dietary soy isoflavonoids have minimal uterotrophic or mammotrophic effects in an established primate model.     

Influence of lifelong soy isoflavones consumption on bone mass in the rat

Soy isoflavones (IFs) have shown a bone-sparing effect through epidemiological studies in the Asian population. However, there is no evidence as to whether such protection would result from a lifelong exposure. We investigated the impact of an early exposure to IFs on bone status. Sixty female Wistar rats were fed either a standard diet (n=30) or the same food enriched with IFs (0.87 mg/g of diet) (n=30). After 1 month, they were allowed to mate, and were kept on the same regimen during the whole gestation and lactation periods. At weaning, female pups were each assigned to one of four nutritional groups; within each experimental group, animals were split into two groups, fed either the standard or the IF-rich diet. At 2, 3, 6, 12, 18, and 24 months after birth, 10 animals in each group were sacrificed. Femurs were collected for mechanical testing and bone mineral density (BMD) measurement. The rats perinatally or lifelong exposed to the IF-rich diet exhibited higher body weight and fat mass at 24 months of age. Peak bone mass was achieved between 6 and 12 months and did not differ between groups. In animals perinatally exposed to IF, BMD continued to increase. Thus, at 24 months, femoral total BMD (P<0.05), metaphyseal BMD (P<0.01), and failure load (P<0.05) were higher in the offspring born from mothers provided IF during pregnancy. Postnatal exposure alone did not improve bone parameters. This experiment provides evidence that perinatal exposure to phytoestrogens leads to a higher BMD later in life. It is suggested that these changes may have occurred as a consequence of programming effects, as has been shown for the endocrine and immune systems.     

White adipose tissue genome wide-expression profiling and adipocyte metabolic functions after soy protein consumption in rats

Obesity is associated with an increase in adipose tissue mass due to an imbalance between high dietary energy intake and low physical activity; however, the type of dietary protein may contribute to its development. The aim of the present work was to study the effect of soy protein versus casein on white adipose tissue genome profiling, and the metabolic functions of adipocytes in rats with diet-induced obesity. The results showed that rats fed a Soy Protein High-Fat (Soy HF) diet gained less weight and had lower serum leptin concentration than rats fed a Casein High-Fat (Cas HF) diet, despite similar energy intake. Histological studies indicated that rats fed the Soy HF diet had significantly smaller adipocytes than those fed the Cas HF diet, and this was associated with a lower triglyceride/DNA content. Fatty acid synthesis in isolated adipocytes was reduced by the amount of fat consumed but not by the type of protein ingested. Expression of genes of fatty acid oxidation increased in adipose tissue of rats fed Soy diets; microarray analysis revealed that Soy protein consumption modified the expression of 90 genes involved in metabolic functions and inflammatory response in adipose tissue. Network analysis showed that the expression of leptin was regulated by the type of dietary protein and it was identified as a central regulator of the expression of lipid metabolism genes in adipose tissue. Thus, soy maintains the size and metabolic functions of adipose tissue through biochemical adaptations, adipokine secretion, and global changes in gene expression.     

Cytoskeletons of retinal pigment epithelial cells: Interspecies differences of expression patterns indicate independence of cell function from the specific complement of cytoskeletal proteins

Summary In vertebrate tissue development a given cell differentiation pathway is usually associated with a pattern of expression of a specific set of cytoskeletal proteins, including different intermediate filament (IF) and junctional proteins, which is identical in diverse species. The retinal pigment epithelium (RPE) is a layer of polar cells that have very similar morphological features and practically identical functions in different vertebrate species. However, in biochemical and immunolocalization studies of the cytoskeletal proteins of these cells we have noted remarkable interspecies differences. While chicken RPE cells contain only IFs of the vimentin type and do not possess desmosomes and desmosomal proteins RPE cells of diverse amphibian (Rana ridibunda, Xenopus laevis) and mammalian (rat, guinea pig, rabbit, cow, human) species express cytokeratins 8 and 18 either as their sole IF proteins, or together with vimentin IFs as in guinea pig and a certain subpopulation of bovine RPE cells. Plakoglobin, a plaque protein common to desmosomes and the zonula adhaerens exists in RPE cells of all species, whereas desmoplakin and desmoglein have been identified only in RPE desmosomes of frogs and cows, including bovine RPE cell cultures in which cytokeratins have disappeared and vimentin IFs are the only IFs present. These challenging findings show that neither cytokeratin IFs nor desmosomes are necessary for the establishment and function of a polar epithelial cell layer and that the same basic cellular architecture can be achieved by different programs of expression of cytoskeletal proteins. The differences in the composition of the RPE cytoskeleton further indicate that, at least in this tissue, a specific program of expression of IF and desmosomal proteins is not related to the functions of the RPE cell, which are very similar in the various species.     

Norisoboldine,an Anti-Arthritis Alkaloid Isolated from Radix Linderae,Attenuates Osteoclast Differentiation and Inflammatory Bone Erosion in an Aryl Hydrocarbon Receptor-Dependent Manner

Norisoboldine (NOR), the primary isoquinoline alkaloid constituent of the root of Lindera aggregata, has previously been demonstrated to attenuate osteoclast (OC) differentiation. Accumulative evidence has shown that aryl hydrocarbon receptor (AhR) plays an important role in regulating the differentiation of various cells, and multiple isoquinoline alkaloids can modulate AhR. In the present study, we explored the role of NOR in the AhR signaling pathway. These data showed that the combination of AhR antagonist resveratrol (Res) or α-naphthoflavone (α-NF) nearly reversed the inhibition of OC differentiation through NOR. NOR could stably bind to AhR, up-regulate the nuclear translocation of AhR, and enhance the accumulation of the AhR-ARNT complex, AhR-mediated reporter gene activity and CYP1A1 expression in RAW 264.7 cells, suggesting that NOR might be an agonist of AhR. Moreover, NOR inhibited the nuclear translocation of NF-κB-p65, resulting in the evident accumulation of the AhR-NF-κB-p65 complex, which could be markedly inhibited through either Res or α-NF. Although NOR only slightly affected the expression of HIF-1α, NOR markedly reduced VEGF mRNA expression and ARNT-HIF-1α complex accumulation. In vivo studies indicated that NOR decreased the number of OCs and ameliorated the bone erosion in the joints of rats with collagen-induced arthritis, accompanied by the up-regulation of CYP1A1 and the down-regulation of VEGF mRNA expression in the synovium of rats. A combination of α-NF nearly completely reversed the effects of NOR. In conclusion, NOR attenuated OC differentiation and bone erosion through the activation of AhR and the subsequent inhibition of both NF-κB and HIF pathways.     

The Current Impact of Incidental Findings Found during Neuroimaging on Neurologists’ Workloads

ObjectiveNeuroimaging is an important diagnostic tool in the assessment of neurological disease, but often unmasks Incidental Findings (IFs). The negative impacts of IFs, such as ‘patient’ anxiety, present neurologists with management dilemmas, largely due to the limited knowledge base surrounding the medical significance of these IFs. In particular, the lack of evidence-based clinical trials investigating the efficacy of treatments for subclinical IFs makes management protocols challenging. The objective was to determine the impact IFs may have on neurologists’ workloads and healthcare budgets and to examine neurologists’ concerns regarding the clinical management of these ‘patients’.MethodsQualitative research based on constructivist grounded theory. Data was collected through semi-structured interviews of purposively sampled neurologists, coded, and concurrent comparative analysis performed. A substantive theory of the ‘IF impacts’ was developed after concept saturation.ResultsNeurologists managed the escalating workload caused by an increased number of referrals of ‘patients’ with IFs found during neuroimaging; however it was unclear whether this was sustainable in the future. Neurologists experienced IF management dilemmas and spent more time with ‘patients’ affected by anxiety. The lack of information provided to those undergoing neuroimaging by the referring clinician regarding the possibility of discovering IFs was highlighted.ConclusionThe impact of IFs upon the neurologist, ‘patient’ and the health institution appeared considerable. Further research determining the natural history of subclinical IFs and the efficacy of intervention will help to alleviate these issues.     

The effect of Euglena viridis on immune response of rohu, Labeo rohita (Ham.)

The study evaluated the effect of dietary doses of Euglena viridis on the immune response and disease resistance of Labeo rohita fingerlings against infection with the bacterial pathogen Aeromonas hydrophila. L. rohita fingerlings were fed with diet containing 0 (Control), 0.1 g, 0.5 g, 1.0 g Euglena powder kg⁻¹ dry diet for 90 days. Biochemical (serum total protein, albumin, globulin, albumin:globulin ratio), haematological (WBC, RBC, haemoglobin content) and immunological (superoxide anion production, lysozyme, serum bactericidal activity) parameters of fish were examined after 30, 60 and 90 days of feeding. Fish were challenged with A. hydrophila 90 days post-feeding and mortalities were recorded over 10 days post-infection. The results demonstrate that fish fed with Euglena showed increased levels of superoxide anion production, lysozyme, serum bactericidal activity, serum protein and albumin (P < 0.05) compared with the control group. Following challenge with A. hydrophila less survivability was observed in the control group (56.65%) than the group fed the experimental diets. The group fed 0.5 g Euglena kg⁻¹ dry diet showed the highest percentage survival (75%). These results indicate that Euglena stimulates the immunity and makes L. rohita more resistant to A. hydrophila infection.     

"Heads and tails" of intermediate filament phosphorylation: multiple sites and functional insights

Intermediate filaments (IFs) are major components of the mammalian cytoskeleton. They are among the most abundant cellular phosphoproteins; their phosphorylation typically involves multiple sites at repeat or unique motifs, preferentially within the "head" or "tail" domains. Phosphorylation and dephosphorylation are essential for the regulation of IF dynamics by modulating the intrinsic properties of IFs: solubility, conformation and filament organization, and, in addition, for the regulation of other IF post-translational modifications. These phosphorylation-regulated properties dictate generalized and context-dependent IF functions that reflect their tissue-specific expression. Most important among IF phosphorylation-mediated functions are the regulation of IF cellular or subcellular compartmentalization, levels and turnover, binding with associated proteins, susceptibility to cell stresses (including apoptosis), tissue-specific functions and IF-associated disease pathogenesis (where IF hyperphosphorylation also serves as a tissue-injury marker).     

Roles of nuclear receptors in the up-regulation of hepatic cholesterol 7alpha-hydroxylase by cholestyramine in rats

Nuclear receptors are involved in regulating the expression of cholesterol 7alpha-hydroxylase (CYP7A1), however, their roles in the up-regulation of CYP7A1 by cholestyramine (CSR) are still unclear. In the present study, male Wistar rats were divided into four groups and fed [high sucrose + 10% lard diet] (H), [H + 3% CSR diet] (H + CSR), [H + 0.5% cholesterol + 0.25% sodium cholate diet] (C), or [C + 3% CSR diet] (C + CSR) for 2 weeks. Cholestyramine decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but had no effect on these parameters in rats fed H-based diets. Cholestyramine raised hepatic levels of CYP7A1 mRNA and activity in both groups. The gene expression of hepatic ATP-binding cassettes A1 and G5, regulated by liver X receptor (LXR), were unchanged and down-regulated by cholestyramine, respectively. The mRNA levels of the hepatic ATP-binding cassette B11 and short heterodimer partner (SHP), regulated by farnesoid X receptor (FXR), were not changed by cholestyramine. C-based diets, which contained cholesterol and cholic acid, increased SHP mRNA levels compared to H-based diets. Consequently, in rats fed the C+CSR diet, hepatic FXR was activated by dietary bile acids, but the hepatic CYP7A1 mRNA level was increased 16-fold compared to that in rats fed an H diet. These results suggest that cholestyramine up-regulates the expression of CYP7A1 independently via LXR- or FXR-mediated pathways in rats.     

Sulfation of flavonoids and other phenolic dietary compounds by the human cytosolic sulfotransferases

The protective effects of diet, especially soya products, tea, and many fruits, against a variety of human cancers, as suggested by epidemiological studies, has focused attention on flavonoids, isoflavonoids, and other phenolic dietary compounds as chemoprotectants. Among the mechanisms suggested for their chemoprotective action, their ability to inhibit the bioactivation of carcinogens by the human cytosolic sulfotransferases (STs) and the direct effects of their sulfoconjugates are being increasingly studied. We report here a systematic study on the sulfation of representative flavonoids, isoflavonoids, anti-oxidants, and other phenolic dietary compounds by all ten known human cytosolic STs. All ten recombinant human cytosolic STs were prepared in a pure form and tested for their sulfating activities with a variety of these compounds. P-form (SULT1A1) phenol ST (PST) showed high sulfating activity with most of these compounds. M-form (SULT1A3) PST showed high activity with the flavonoids but not with the isoflavonoids. SULT1C ST #2 showed high activity with the isoflavonoids and also sulfated most of the other compounds. Possible relevance of these results to the chemoprotective effects of these dietary compounds is discussed.     

RhoA-Binding Kinase α Translocation Is Facilitated by the Collapse of the Vimentin Intermediate Filament Network

The regulation of morphological changes in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). The putative effector of RhoA, RhoA-binding kinase α (ROKα), is a serine/threonine kinase that has been implicated in the reorganization of actin filaments and in myosin contractility. Here, we show that ROKα also directly affects the structural integrity of IFs. Overexpression of active ROKα, like that of RhoA, caused the collapse of filamentous vimentin, a type III IF. A RhoA-binding-deficient, kinase-inactive ROKα inhibited the collapse of vimentin IFs induced by RhoA in HeLa cells. In vitro, ROKα bound and phosphorylated vimentin at its head-rod domain, thereby inhibiting the assembly of vimentin. ROKα colocalized predominantly with the filamentous vimentin network, which remained intact in serum-starved cells. Treatment of cells with vinblastine, a microtubule-disrupting agent, also resulted in filamentous vimentin collapse and concomitant ROKα translocation to the cell periphery. ROKα translocation did not occur when the vimentin network remained intact in vinblastine-treated cells at 4°C or in the presence of the dominant-negative RhoAN19 mutant. Transient translocation of ROKα was also observed in cells subjected to heat shock, which caused the disassembly of the vimentin network. Thus, the translocation of ROKα to the cell periphery upon overexpression of RhoAV14 or growth factor treatment is associated with disassembly of vimentin IFs. These results indicate that Rho effectors known to act on microfilaments may be involved in regulating the assembly of IFs. Vimentin when phosphorylated also exhibits reduced affinity for the inactive ROKα. The translocation of ROKα from IFs to the cell periphery upon action by activated RhoA and ROKα suggests that ROKα may initiate its own cascade of activation.     

Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

Nestin is the characteristic intermediate filament (IF) protein of rapidly proliferating progenitor cells and regenerating tissue. Nestin copolymerizes with class III IF-proteins, mostly vimentin, into heteromeric filaments. Its expression is downregulated with differentiation. Here we show that a strong nestin expression in mouse embryo tissue coincides with a strong accumulation of the glucocorticoid receptor (GR), a key regulator of growth and differentiation in embryonic development. Microscopic studies on cultured cells show an association of GR with IFs composed of vimentin and nestin. Cells lacking nestin, but expressing vimentin, or cells expressing vimentin, but lacking nestin accumulate GR in the nucleus. Completing these networks with an exogenous nestin, respectively an exogenous vimentin restores cytoplasmic anchoring of GR to the IF system. Thus, heteromeric filaments provide the basis for anchoring of GR. The reaction pattern with phospho-GR specific antibodies and the presence of the chaperone HSC70 suggest that specifically the unliganded receptor is anchored to the IF system. Ligand addition releases GR from IFs and shifts the receptor into the nucleus. Suppression of nestin by specific shRNA abolishes anchoring of GR, induces its accumulation in the nucleus and provokes an irreversible G1/S cell cycle arrest. Suppression of GR prior to that of nestin prevents entry into the arrest. The data give evidence that nestin/vimentin specific anchoring modulates growth suppression by GR. We hypothesize that expression of nestin is a major determinant in suppression of anti-proliferative activity of GR in undifferentiated tissue and facilitates activation of this growth control in a precise tissue and differentiation dependent manner.     

Chronic High Fructose Intake Reduces Serum 1,25 (OH)2D3 Levels in Calcium-Sufficient Rodents

Excessive fructose consumption inhibits adaptive increases in intestinal Ca²⁺ transport in lactating and weanling rats with increased Ca²⁺ requirements by preventing the increase in serum levels of 1,25(OH)₂D₃. Here we tested the hypothesis that chronic fructose intake decreases 1,25(OH)₂D₃ levels independent of increases in Ca²⁺ requirements. Adult mice fed for five wk a high glucose-low Ca²⁺ diet displayed expected compensatory increases in intestinal and renal Ca²⁺ transporter expression and activity, in renal CYP27B1 (coding for 1α-hydroxylase) expression as well as in serum 1,25(OH)₂D₃ levels, compared with mice fed isocaloric glucose- or fructose-normal Ca²⁺ diets. Replacing glucose with fructose prevented these increases in Ca²⁺ transporter, CYP27B1, and 1,25(OH)₂D₃ levels induced by a low Ca²⁺ diet. In adult mice fed for three mo a normal Ca²⁺ diet, renal expression of CYP27B1 and of CYP24A1 (24-hydroxylase) decreased and increased, respectively, when the carbohydrate source was fructose instead of glucose or starch. Intestinal and renal Ca²⁺ transporter activity and expression did not vary with dietary carbohydrate. To determine the time course of fructose effects, a high fructose or glucose diet with normal Ca²⁺ levels was fed to adult rats for three mo. Serum levels of 1,25(OH)₂D₃ decreased and of FGF23 increased significantly over time. Renal expression of CYP27B1 and serum levels of 1,25(OH)₂D₃ still decreased in fructose- compared to those in glucose-fed rats after three mo. Serum parathyroid hormone, Ca²⁺ and phosphate levels were normal and independent of dietary sugar as well as time of feeding. Thus, chronically high fructose intakes can decrease serum levels of 1,25(OH)₂D₃ in adult rodents experiencing no Ca²⁺ stress and fed sufficient levels of dietary Ca²⁺. This finding is highly significant because fructose constitutes a substantial portion of the average diet of Americans already deficient in vitamin D.     

Arrangement of trichokeratin intermediate filaments and matrix in the cortex of Merino wool

Tomograms of transverse sections of Merino wool fibers obtained from fleeces differing in fiber curvature were reconstructed from image series collected using a 300 kV transmission electron microscope. Trichokeratin intermediate filaments (IFs) from the ortho-, para- and mesocortices were modeled from the tomograms. IFs were predominantly arranged in left-handed concentric helices with the relative angle of IFs increasing progressively from the center to the periphery of orthocortex macrofibrils. The median increase in IF angle between adjacent IFs between the center and periphery was 2.5°. The length of one turn of the helical path of an IF was calculated to be approximately 1 μm for an IF tilted at 30° and positioned 100 nm from the macrofibril center. With the exception of one paracortex macrofibril that weakly resembled an orthocortex macrofibril, all para- and mesocortex macrofibrils modeled had a parallel arrangement of the IFs, with a more ordered arrangement found in the mesocortex. Within the limited sample set, there appeared to be no significant relationship between IF angle and fiber curvature. We examined the matrix/IF ratio (in the form of proportion of matrix to one IF, calculated from IF center-to-center distance and IF diameter) for 28 macrofibrils used for modeling. The proportion of matrix was significantly different in the different cortex cell types, with paracortex having the most (0.61), orthocortex having the least (0.42), and mesocortex being intermediate (0.54). Fibers of different crimp type (high, medium or low crimp) were not significantly different from each other with respect to matrix proportion.