Effects of Inhibiting Dipeptidyl Peptidase-4 (DPP4) in Cows with Subclinical Ketosis

The inhibition of dipeptidyl peptidase-4 (DPP4) via specific inhibitors is known to result in improved glucose tolerance and insulin sensitivity and decreased accumulation of hepatic fat in type II diabetic human patients. The metabolic situation of dairy cows can easily be compared to the status of human diabetes and non-alcoholic fatty liver. For both, insulin sensitivity is reduced, while hepatic fat accumulation increases, characterized by high levels of non-esterified fatty acids (NEFA) and ketone bodies.Therefore, in the present study, a DPP4 inhibitor was employed (BI 14332) for the first time in cows. In a first investigation BI 14332 treatment (intravenous injection at dosages of up to 3 mg/kg body weight) was well tolerated in healthy lactating pluriparous cows (n = 6) with a significant inhibition of DPP4 in plasma and liver. Further testing included primi- and pluriparous lactating cows suffering from subclinical ketosis (β-hydroxybutyrate concentrations in serum > 1.2 mM; n = 12). The intension was to offer effects of DPP4 inhibition during comprehensive lipomobilisation and hepatosteatosis. The cows of subclinical ketosis were evenly allocated to either the treatment group (daily injections, 0.3 mg BI 14332/kg body weight, 7 days) or the control group. Under condition of subclinical ketosis, the impact of DPP4 inhibition via BI 14332 was less, as in particular β-hydroxybutyrate and the hepatic lipid content remained unaffected, but NEFA and triglyceride concentrations were decreased after treatment. Owing to lower NEFA, the revised quantitative insulin sensitivity check index (surrogate marker for insulin sensitivity) increased. Therefore, a positive influence on energy metabolism might be quite possible. Minor impacts on immune-modulating variables were limited to the lymphocyte CD4⁺/CD8⁺ ratio for which a trend to decreased values in treated versus control animals was noted. In sum, the DPP4 inhibition in cows did not affect glycaemic control like it is shown in humans, but was able to impact hyperlipemia, as NEFA and TG decreased.     

Role of tartrate-resistant acid phosphatase (TRAP) in long bone development

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.     

Ala657 and conserved active site residues promote fibroblast activation protein endopeptidase activity via distinct mechanisms of transition state stabilization

Fibroblast activation protein (FAP) and dipeptidyl peptidase-4 (DPP-4) are highly homologous serine proteases of the prolyl peptidase family and therapeutic targets for cancer and diabetes, respectively. Both proteases display dipeptidyl peptidase activity, but FAP alone has endopeptidase activity. FAP Ala657, which corresponds to DPP-4 Asp663, is important for endopeptidase activity; however, its specific role remains unclear, and it is unknown whether conserved DPP-4 substrate binding residues support FAP endopeptidase activity. Using site-directed mutagenesis and kinetic analyses, we show here that Ala657 and five conserved active site residues (Arg123, Glu203, Glu204, Tyr656, and Asn704) promote FAP endopeptidase activity via distinct mechanisms of transition state stabilization (TSS). The conserved residues provide marked TSS energy for both endopeptidase and dipeptidyl peptidase substrates, and structural modeling supports their function in binding both substrates. Ala657 also stabilizes endopeptidase substrate binding and additionally dictates FAP reactivity with transition state inhibitors, allowing tight interaction with tetrahedral intermediate analogues but not acyl-enzyme analogues. Conversely, DPP-4 Asp663 stabilizes dipeptidyl peptidase substrate binding and permits tight interaction with both transition state analogues. Structural modeling suggests that FAP Ala657 and DPP-4 Asp663 confer their contrasting effects on TSS by modulating the conformation of conserved residues FAP Glu204 and DPP-4 Glu206. FAP therefore requires the combined function of Ala657 and the conserved residues for endopeptidase activity.     

Genetic analysis of candidate genes modifying the age-at-onset in Huntington’s disease

The expansion of a polymorphic CAG repeat in the HD gene encoding huntingtin has been identified as the major cause of Huntington’s disease (HD) and determines 42–73% of the variance in the age-at-onset of the disease. Polymorphisms in huntingtin interacting or associated genes are thought to modify the course of the disease. To identify genetic modifiers influencing the age at disease onset, we searched for polymorphic markers in the GRIK2, TBP, BDNF, HIP1 and ZDHHC17 genes and analysed seven of them by association studies in 980 independent European HD patients. Screening for unknown sequence variations we found besides several silent variations three polymorphisms in the ZDHHC17 gene. These and polymorphisms in the GRIK2, TBP and BDNF genes were analysed with respect to their association with the HD age-at-onset. Although some of the factors have been defined as genetic modifier factors in previous studies, none of the genes encoding GRIK2, TBP, BDNF and ZDHHC17 could be identified as a genetic modifier for HD.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Untersuchungen zur Rolle der Folsäure im Stoffwechsel autotropher Zellen

Zusammenfassung In Massenkulturen von Chlorella pyrenoidosa wurde die Rolle der Folsäure im Stoffwechsel, besonders im Hinblick auf deren mögliche Beteiligung an frühen Vorgängen des photosynthetischen Kohlenstoffeinbaues, untersucht. Als spezifische Antimetaboliten der Folsäure-Biosynthese wurden Sulfonamide (Sulfanilamid, Sulfathiazol) eingesetzt.Bereits nach 24 Std Einwirkungsdauer ist eine deutliche Abnahme der Teilungsintensität zu verzeichnen. Parallel damit läßt sich eine etwas geringere Abnahme des Pigment-und Nucleinsäure-Gehaltes (Ges.-NS, RNS, DNS) der Zellen nachweisen. Demgegenüber liegt eine deutliche Blockierung der Substanzproduktion erst nach 48 Std Hemmdauer vor. Aus diesem unterschiedlich starken Hemmeffekt auf Teilungsintensität und Substanzproduktion resultiert eine beachtliche Zunahme der Zell-größe, die nach 96 Std Versuchsdauer beinahe den doppelten Wert der Ausgangsgröße erreicht.Bei gleichzeitiger Gabe von p-Aminobenzoesäure (10^-6 Mol/l) oder Folsäure (10^-5 Mol/l) zum sulfanilamidhaltigen Kulturmedium ist keine Hemmwirkung des Antimetaboliten auf den Stoffwechsel der Algenzellen nachweisbar.Die schädigende Wirkung des Sulfonamids ist reversibel; der Rückgang der Hemmung wird anfangs durch unterschiedliche PAB-Konzentrationen in abgestufter Weise gefördert. ¹⁴C-Fixierungsexperimente lassen eine Beteiligung von Folsäure-Coenzymen an primären Vorgängen der photosynthetischen C-Einlagerung als fraglich erscheinen, da die Fixierung, bezogen auf die Substanzproduktion, nur langsam zurückgeht. Die Abnahme der Fixierung wird als sekundäre Veränderung des für die Photosynthese notwendigen Enzymapparates gedeutet.

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Investigations concerning the role of folic acid in the metabolism of autotrophic cells

Summary The role of folic acid in metabolism was studied in mass cultures of Chlorella pyrenoidosa, especially with respect to its possible participation in early stages of photosynthetic CO₂-fixation. Sulfonamides (sulfanilamide, sulfathiazole) were used as specific antimetabolites in the biosynthesis of folic acid.After 24 hours the number of cell divisions was much more diminished than the pigment and nucleic acid content of the cells. Significant inhibition of mass production did not occur before 48 hours. This difference in the sensitivity of cell division and mass production towards the antimetabolite leads to a definite increase in cell size.Antimetabolites administered together with (10^-6 mol/l) p-aminobenzoic acid or (10^-5 mol/l) folic acid cause no inhibition.The harmful effect of the sulfonamide is strictly reversible; the speed of recovery depends on the p-aminobenzoic acid concentration. ¹⁴C-Fixation experiments gave no evidence for the participation of folic acid enzymes in primary processes of photosynthetic carbon uptake. The decline of the fixation rate may be interpreted as a secondary effect on the enzyme apparatus necessary for photosynthesis.The enormous increase of the sugar content in the individual cells indicates a general disturbance of the metabolism.

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Dissertation der Mathematisch-naturwissenschaftlichen Fakultät der Universität Tübingen.     

Local and landscape responses of biodiversity in calcareous grasslands

Biodiversity and Conservation - Across Europe, calcareous grasslands become increasingly fragmented and their quality deteriorates through abandonment and land use intensification, both affecting...     

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre’s enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.