The effects of calystegines isolated from edible fruits and vegetables on mammalian liver glycosidases

The polyhydroxylated nortropane alkaloids called calyste-ginesoccur in many plants of the Convolvulaceae, Solanaceae, andMoraceae families. Certain of these alkaloids exhibit potentinhibitory activities against glycosidases and the recentlydemonstrated occurrence of calystegines in the leaves, skins,and sprouts of potatoes (Solatium tuberosum), and in the leavesof the eggplant (S.melongena), has raised concerns regardingthe safety of these vegetables in the human diet. We have surveyedthe occurrence of calystegines in edible fruits and vegetablesof the families Convolvulaceae, Solanaceae, and Moraceae byGC-MS. Calystegines A3, B1, B2, and C1 were detected in allthe edible fruits and vegetables tested; sweet and chili peppers,potatoes, eggplants, tomatoes, Physalis fruits, sweet potatoes,and mulberries. Calystegines B1 and C1 were potent competitiveinhibitors of the bovine, human, and rat β-glucosidaseactivities, with K₁ values of 150, 10, and 1.9 µM, respectivelyfor B1 and 15,1.5, and 1 µM, respectively, for C1. CalystegineB2 was a strong competitive inhibitor of the -galactosidaseactivity in all the livers. Human β-xylosidase was inhibitedby all four nortropanes, with calystegine C1 having a K₁ of0.13 µM. Calystegines A3 and B2 selectively inhibitedthe rat liver β-glucosidase activity. The potent inhibitionof mammalian β-glucosidase and -galactosidase activitiesin vitro raises the possibility of toxicity in humans consuminglarge amounts of plants that contain these compounds. edible plants calystegines glycosidase inhibitors bovine, human, and rat liver     

Gestational Heat Stress Alters Postnatal Offspring Body Composition Indices and Metabolic Parameters in Pigs

The study objectives were to test the hypothesis that heat stress (HS) during gestational development alters postnatal growth, body composition, and biological response to HS conditions in pigs. To investigate this, 14 first parity crossbred gilts were exposed to one of four environmental treatments (TNTN, TNHS, HSTN, or HSHS) during gestation. TNTN and HSHS dams were exposed to thermal neutral (TN, cyclical 18–22°C) or HS conditions (cyclical 28–34°C) during the entire gestation, respectively. Dams assigned to HSTN and TNHS treatments were heat-stressed for the first or second half of gestation, respectively. Postnatal offspring were exposed to one of two thermal environments for an acute (24 h) or chronic (five weeks) duration in either constant TN (21°C) or HS (35°C) environment. Exposure to chronic HS during their growth phase resulted in decreased longissimus dorsi cross-sectional area (LDA) in offspring from HSHS and HSTN treated dams whereas LDA was larger in offspring from dams in TNTN and TNHS conditions. Irrespective of HS during prepubertal postnatal growth, pigs from dams that experienced HS during the first half of gestation (HSHS and HSTN) had increased (13.9%) subcutaneous fat thickness compared to pigs from dams exposed to TN conditions during the first half of gestation. This metabolic repartitioning towards increased fat deposition in pigs from dams heat-stressed during the first half of gestation was accompanied by elevated blood insulin concentrations (33%; P = 0.01). Together, these results demonstrate HS during the first half of gestation altered metabolic and body composition parameters during future development and in biological responses to a subsequent HS challenge.     

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of <Emphasis Type="Italic">Paenibacillus graminis</Emphasis>

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of α- and β-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10^T). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.     

Functional role of BLAP75 in BLM-topoisomerase IIIalpha-dependent holliday junction processing

The BLAP75 protein combines with the BLM helicase and topoisomerase (Topo) IIIalpha to form an evolutionarily conserved complex, termed the BTB complex, that functions to regulate homologous recombination. BLAP75 binds DNA, associates with both BLM and Topo IIIalpha, and enhances the ability of the BLM-Topo IIIalpha pair to branch migrate the Holliday junction (HJ) or dissolve the double Holliday junction (dHJ) structure to yield non-crossover recombinants. Here we seek to understand the relevance of the biochemical attributes of BLAP75 in HJ processing. With the use of a series of BLAP75 protein fragments, we show that the evolutionarily conserved N-terminal third of BLAP75 mediates complex formation with BLM and Topo IIIalpha and that the DNA binding activity resides in the C-terminal third of this novel protein. Interestingly, the N-terminal third of BLAP75 is just as adept as the full-length protein in the promotion of dHJ dissolution and HJ unwinding by BLM-Topo IIIalpha. Thus, the BLAP75 DNA binding activity is dispensable for the ability of the BTB complex to process the HJ in vitro. Lastly, we show that a BLAP75 point mutant (K166A), defective in Topo IIIalpha interaction, is unable to promote dHJ dissolution and HJ unwinding by BLM-Topo IIIalpha. This result provides proof that the functional integrity of the BTB complex is contingent upon the interaction of BLAP75 with Topo IIIalpha.     

In vivo consequences of cholesterol-24S-hydroxylase (CYP46A1) inhibition by voriconazole on cholesterol homeostasis and function in the rat retina

Cholesterol 24S-hydroxylase (CYP46A1) converts cholesterol into 24S-hydroxycholesterol in neurons and participates in cholesterol homeostasis in the central nervous system, including the retina. We aimed to evaluate the consequences of CYP46A1 inhibition by voriconazole on cholesterol homeostasis and function in the retina. Rats received daily intraperitoneal injections of voriconazole (60 mg/kg), minocycline (22 mg/kg), voriconazole plus minocycline, or vehicle during five consecutive days. The rats were submitted to electroretinography to monitor retinal functionality. Cholesterol and 24S-hydroxycholesterol were measured in plasma, brain and retina by gas chromatography-mass spectrometry. The expression of CYP46A1, and GFAP as a marker for glial activation was analyzed in the retina and brain. Cytokines and chemokines were measured in plasma, vitreous, retina and brain. Voriconazole significantly impaired the functioning of the retina as exemplified by the reduced amplitude and increased latency of the b-wave of the electroretinogram, and altered oscillary potentials. Voriconazole decreased 24S-hydroxycholesterol levels in the retina. Unexpectedly, CYP46A1 and GFAP expression was increased in the retina of voriconazole-treated rats. ICAM-1 and MCP-1 showed significant increases in the retina and vitreous body. Minocycline did not reverse the effects of voriconazole. Our data highlighted the cross talk between retinal ganglion cells and glial cells in the retina, suggesting that reduced 24S-hydroxycholesterol concentration in the retina may be detected by glial cells, which were consequently activated.     

Adiposity in Early,Middle and Later Adult Life and Cardiometabolic Risk Markers in Later Life; Findings from the British Regional Heart Study

Objectives: This research investigates the associations between body mass index (BMI) at 21, 40–59, 60–79 years of age on cardiometabolic risk markers at 60–79 years. Methods: A prospective study of 3464 British men with BMI measured at 40–59 and 60–79 years, when cardiometabolic risk was assessed. BMI at 21 years was ascertained from military records, or recalled from middle-age (adjusted for reporting bias); associations between BMI at different ages and later cardiometabolic risk markers were examined using linear regression. Sensitive period, accumulation and mobility life course models were devised for high BMI (defined as BMI≥75^th centile) and compared with a saturated BMI trajectory model. Results: At ages 21, 40–59 and 60–79 years, prevalences of overweight (BMI≥25 kg/m²) were 12%, 53%, 70%, and obesity (≥30 kg/m²) 1.6%, 6.6%, and 17.6%, respectively. BMI at 21 years was positively associated with serum insulin, blood glucose, and HbA1c at 60–79 years, with increases of 1.5% (95%CI 0.8,2.3%), 0.4% (0.1,0.6%), 0.3% (0.1,0.4%) per 1 kg/m², respectively, but showed no associations with blood pressure or blood cholesterol. However, these associations were modest compared to those between BMI at 60–79 years and serum insulin, blood glucose and HbA1c at 60–79 years, with increases of 8.6% (8.0,9.2%), 0.7% (0.5,0.9%), and 0.5% (0.4,0.7%) per 1 kg/m², respectively. BMI at 60–79 years was also associated with total cholesterol and blood pressure. Associations for BMI at 40–59 years were mainly consistent with those of BMI at 60–79 years. None of the life course models fitted the data as well as the saturated model for serum insulin. A sensitive period at 50 years for glucose and HbA1c and sensitive period at 70 years for blood pressure were identified. Conclusions: In this cohort of men who were thin compared to more contemporary cohorts, BMI in later life was the dominant influence on cardiovascular and diabetes risk. BMI in early adult life may have a small long-term effect on diabetes risk.     

The Impact of Increased Food Availability on Reproduction in a Long-Distance Migratory Songbird: Implications for Environmental Change?

Many populations of migratory songbirds are declining or shifting in distribution. This is likely due to environmental changes that alter factors such as food availability that may have an impact on survival and/or breeding success. We tested the impact of experimentally supplemented food on the breeding success over three years of northern wheatears (Oenanthe oenanthe), a species in decline over much of Europe. The number of offspring fledged over the season was higher for food-supplemented birds than for control birds. The mechanisms for this effect were that food supplementation advanced breeding date, which, together with increased resources, allowed further breeding attempts. While food supplementation did not increase the clutch size, hatching success or number of chicks fledged per breeding attempt, it did increase chick size in one year of the study. The increased breeding success was greater for males than females; males could attempt to rear simultaneous broods with multiple females as well as attempting second broods, whereas females could only increase their breeding effort via second broods. Multiple brooding is rare in the study population, but this study demonstrates the potential for changes in food availability to affect wheatear breeding productivity, primarily via phenotypic flexibility in the number of breeding attempts. Our results have implications for our understanding of how wheatears may respond to natural changes in food availability due to climate changes or changes in habitat management.     

RhoA as a mediator of clinically relevant androgen action in prostate cancer cells

Recently, we have identified serum response factor (SRF) as a mediator of clinically relevant androgen receptor (AR) action in prostate cancer (PCa). Genes that rely on SRF for androgen responsiveness represent a small fraction of androgen-regulated genes, but distinguish benign from malignant prostate, correlate with aggressive disease, and are associated with biochemical recurrence. Thus, understanding the mechanism(s) by which SRF conveys androgen regulation to its target genes may provide novel opportunities to target clinically relevant androgen signaling. Here, we show that the small GTPase ras homolog family member A (RhoA) mediates androgen-responsiveness of more than half of SRF target genes. Interference with expression of RhoA, activity of the RhoA effector Rho-associated coiled-coil containing protein kinase 1 (ROCK), and actin polymerization necessary for nuclear translocation of the SRF cofactor megakaryocytic acute leukemia (MAL) prevented full androgen regulation of SRF target genes. Androgen treatment induced RhoA activation, increased the nuclear content of MAL, and led to MAL recruitment to the promoter of the SRF target gene FHL2. In clinical specimens RhoA expression was higher in PCa cells than benign prostate cells, and elevated RhoA expression levels were associated with aggressive disease features and decreased disease-free survival after radical prostatectomy. Overexpression of RhoA markedly increased the androgen-responsiveness of select SRF target genes, in a manner that depends on its GTPase activity. The use of isogenic cell lines and a xenograft model that mimics the transition from androgen-stimulated to castration-recurrent PCa indicated that RhoA levels are not altered during disease progression, suggesting that RhoA expression levels in the primary tumor determine disease aggressiveness. Androgen-responsiveness of SRF target genes in castration-recurrent PCa cells continued to rely on AR, RhoA, SRF, and MAL and the presence of intact SRF binding sites. Silencing of RhoA, use of Rho-associated coiled-coil containing protein kinase 1 inhibitors, or an inhibitor of SRF-MAL interaction attenuated (androgen-regulated) cell viability and blunted PCa cell migration. Taken together, these studies demonstrate that the RhoA signaling axis mediates clinically relevant AR action in PCa.