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Abstract: This study examined the effect of PTH and alendronate alone and in combination on the bone architecture, mineralization, and estimated mechanics in the OVX rat. Female Wistar rats aged 7–9months were assigned to one of five groups: (1) sham+vehicle, (2) OVX+vehicle, (3) OVX+PTH, (4) OVX+alendronate, and (5) OVX+PTH and alendronate. Surgery was performed at baseline (week 0), and biweekly treatment (15μg/kg of alendronate and/or daily (5days/week) 40μg/kg hPTH(1–34)) was administered from week 6 to week 14. Micro-CT scans of the right proximal tibial metaphysis were made in vivo at weeks 0, 6, 8, 10, 12 and 14 and measurements of bone microarchitecture and estimated mechanical parameters (finite element analysis) were made from the images. Synchrotron radiation micro-CT scans of the proximal tibia and fourth lumbar vertebrae were conducted ex vivo at the study endpoint to determine the degree and spatial distribution of the bone mineralization. Alendronate preserved the microarchitecture after OVX, and increased cortical (9%, p<0.05) and trabecular thickness (5%, p<0.05). PTH mono- and combined therapy induced increases in cortical (25–35%, p<0.05) and trabecular thicknesses (46–48%, p<0.05), resulting in a full restoration of bone volume in the PTH group, and an increase beyond baseline in the combined group. Improvements in estimated mechanical outcomes were observed in all treatment groups by the end of the study, with the combined group experiencing the greatest increase in predicted stiffness (63%, p<0.05). Alendronate treatment increased the peak mineral content above the other treatment groups at the trabecular (tibia: 6% above PTH, 6% above combined, L4: 4% above PTH, 4% above combined) and endocortical (tibia: 4% above PTH, 3% above combined, L4: 1% above PTH, 2% above combined) surfaces, while no differences in mineralization between the PTH and combined groups were observed. Combined treatment resulted in more pronounced improvements of the bone architecture than PTH monotherapy, while maintaining the state of mineralization observed with PTH treatment. [Copyright &y& Elsevier]

Abstract: The growing number of product variants, smaller lot sizes, reduced time to market and shorter lifecycles of products have lead to increasing demands on automation equipment and concepts. As a solution, hybrid human integrated approaches are proposed. The idea is to combine human flexibility, intelligence and skills with the advantages of sophisticated technical systems. Intelligent assist systems (IAS) represent a novel class of assembly systems capable of working with human operators in two modes: workplace sharing and time sharing. This paper presents a novel collaborative robot system (“Cobot”) capable of sharing the workspace with the human co-worker and collaborating with him through direct physical contact. [Copyright &y& Elsevier]

Sulfonated steroids are increasingly recognized as a circulating reservoir of precursors for the local production of active steroids in certain target tissues. As an alternative to sulfonation of unconjugated steroids by cytosolic sulfotransferases, their direct formation from sulfonated precursors has been described. However, productivity and physiological relevance of this sulfate pathway of steroidogenesis are still widely unclear. Applying the porcine testis as a model, conversion of pregnenolone sulfate (P5S, sulfate pathway) by CYP17A1 was assessed in comparison to the parallel conversions of pregnenolone (P5, Δ5-pathway) and progesterone (P4, Δ4-pathway). To characterize conversions in the virtual absence of competing enzyme activities, in a first series of experiments porcine recombinant CYP17A1 was incubated with the respective substrate in the presence of bovine recombinant cytochrome P450 oxidoreductase (CPR) and cytochrome b5 (b5). Moreover, porcine testicular microsomal fractions were used as a source of homologous CYP17A1, CPR and b5. Invariably 17α-hydroxylation of P5S was, if at all, only minimal and no formation of dehydroepiandrosterone sulfate from P5S was detectable. Consistent with earlier studies porcine CYP17A1 efficiently metabolized P4 and P5 in both assay systems. Metabolism of P4 and P5 by testicular microsomal protein varied substantially between the five animals tested. In conclusion, a physiologically relevant sulfate pathway for the production of C19-steroids from P5S via CYP17A1 is very unlikely in the porcine testis. [ABSTRACT FROM AUTHOR]

Abstract: Sorghum flour was fermented by the traditional Sudanese method of fermentation for 24 h, taking samples every 8 h, and selected functional properties were studied. Results showed that fermentation increased the protein solubility of sorghum flour in the acidic range (pH 2–4). Fermented sorghum flour had a least gelation concentration of 6% after 16 h of fermentation, while it was 18% for unfermented sorghum. Fermentation also increased oil-binding capacity, emulsifying capacity and emulsifying stability, while it decreased the water-binding capacity. Sorghum flour, fermented or unfermented, showed no foam capacity. [Copyright &y& Elsevier]

The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(β)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased K m and decreased k cat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites. [ABSTRACT FROM AUTHOR]

We report the first direct electrochemistry of CYP109C1, CYP109C2 and CYP109D1 from the myxobacterium Sorangium cellulosum So ce56 immobilized on the screen-printed graphite electrode (SPE) modified by a didodecyldimethylammonium bromide (DDAB) film. Electrochemical response was investigated by cyclic voltammetry. Cyclic voltammograms in a deoxygenated argon saturated 0.1 M potassium phosphate buffer, pH 7.4, demonstrated a reversible redox process with E ° ’ of −0.305 ± 0.005 V, −0.313 ± 0.005 V, −0.309 ± 0.005 V ( vs. Ag/AgCl) for CYP109C1, CYP109C2 and СYP109D1, respectively. The apparent surface coverages were (2.5 ± 0.2) × 10 −11 , (2.5 ± 0.2) × 10 −11 , (3.1 ± 0.2) × 10 −11 mol cm −2 , which corresponded to 5-6% of the total amount of protein that was consumed by the immobilization process. The rate constant k s of heterogeneous electron transfer between the hemoproteins and the SPE modified by DDAB film (SPE/DDAB) were determined as 0.39 ± 0.01 s −1 , 0.35 ± 0.01 s −1 , and 0.43 ± 0.02 s −1 for CYP109C1, CYP109C2 and СYP109D1, respectively. The investigated CYPs (CYP109C1, CYP109C2 and CYP109D1) demonstrated electrocatalytic activity detected by an increase of the reduction current in the presence of dissolved oxygen. Upon addition of the substrates (myristic acid and the norisoprenoid α-ionone) in the air-saturated solution, the reduction peak current of dissolved oxygen increased, which manifested the catalytic behavior of CYP109C2 and CYP109D1 towards the substrates. CYP109D1-depended electrocatalytic hydroxylation of the myristic acid was analyzed by mass-spectrometry after electrolysis at controlled working electrode potential. [ABSTRACT FROM AUTHOR]

The biosynthesis of steroid hormones in vertebrates is initiated by the cytochrome P450 CYP11A1, which performs the side-chain cleavage of cholesterol thereby producing pregnenolone. In this study, we report a direct stimulatory effect of the estrogens estradiol and estrone onto the pregnenolone formation in a reconstituted in vitro system consisting of purified CYP11A1 and its natural redox partners. We demonstrated the formation of new products from 11-deoxycorticosterone (DOC), androstenedione, testosterone and dehydroepiandrosterone (DHEA) during the in vitro reaction catalyzed by CYP11A1. In addition, we also established an Escherichia coli -based whole-cell biocatalytic system consisting of CYP11A1 and its redox partners to obtain sufficient yields of products for NMR-characterization. Our results indicate that CYP11A1, in addition to the previously described 6β-hydroxylase activity, possesses a 2β-hydroxylase activity towards DOC and androstenedione as well as a 16β-hydroxylase activity towards DHEA. We also showed that CYP11A1 is able to perform the 6β-hydroxylation of testosterone, a reaction that has been predominantly attributed to CYP3A4. Our results are the first evidence that sex hormones positively regulate the overall production of steroid hormones suggesting the need to reassess the role of CYP11A1 in steroid hormone biosynthesis and its substrate-dependent mechanistic properties. [ABSTRACT FROM AUTHOR]

In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a K d -value of 74.8 ± 4.2 μM for the CYP17A1–PregS complex, which is 2.5-fold higher compared to the CYP17A1–pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher K m - and a lower k cat -value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b 5 ( b 5 ) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon–carbon bond occurs, when b 5 is added to the reconstituted in-vitro system, while b 5 promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids. [ABSTRACT FROM AUTHOR]