Adenylatcyclasen Rv2435c und Rv2212c aus Mycobacterium tuberculosis

The Mycobacterium tuberculosis genome has 15 open reading frames for class III adenylyl cyclases. Here I report on two structurally different ACs belonging to two different subclasses; the mammalian-like Rv2435c classified as class IIIa, and Rv2212c belonging to class IIIc. Rv2435c is unique in possessing an extracellular domain, two transmembrane helices as a membrane anchor and a C-terminal catalytic domain. Having four deviations from the canonical hexad in the catalytic centre raised theoretical questions concerning AC activity. Three different holoenzyme constructs were expressed and were mostly degraded. Both the catalytic domain and these unstable holoenzymes did not display enzymatic activity. Rv2212c is composed of two distinct protein modules; a C-terminal CHD and an N-terminal regulatory domain, the occurrence of which is restricted to several adenylyl cyclases present in Gram-positive bacteria including the mycobacterial Rv1264. All six amino acids annotated to be participating in catalysis are conserved. The expressed CHD was shown to function as a homodimer. Its specific activity (3.1µmol cAMP mg-1 min-1) and Vmax were high compared to other mycobacterial ACs, whereas substrate affinity was quite low. GC activity was absent. The holoenzyme had a specific activity 4.5-fold lower than the CHD, arguing against the idea of an autoinhibitory N-terminal domain. Also the function of a pH-sensing N-terminal domain was excluded as both CHD and holoenzyme demonstrated a pH-optimum at pH 6.5. Nevertheless the N-terminal domain had a role in dimerization and enzyme stabilization. Unsaturated FAs (arachidonic, oleic and linoleic) caused a significant stimulation of the enzyme by increasing its substrate affinity but without altering Vmax and conferring an increased pH-sensitivity to the protein. The effect of the FAs did not only involve the N-terminal domain because the CHD was similarly affected but to a much lesser extent. In particular, a pH-regulation in presence of FAs was not shown for the CHD, which makes the regulatory role played by the N-terminal domain in presence of FAs more relevant. Deciphering the molecular structure of this protein is required to be able to figure out how the FAs exert these effects and how exactly they interact with the enzyme.Das Genom von M. tuberculosis enthält 15 Klasse III Adenylatcyclasen. Im Rahmen der vorliegenden Arbeit wurden zwei Adenylatcyclasen mit unterschiedlichen strukturellen Eigenschaften untersucht: die Mammalia-ähnliche Rv2435c aus Klasse IIIa und Rv2212c aus Klasse IIIc. Rv2435c ist die einzige mycobakterielle AC, die aus einer extrazellulären Domäne, zwei transmembranären Helices als Membrananker und einer C-terminalen katalytischen Domäne (CHD) besteht. Sie besitzt vier Abweichungen von den sechs kanonischen Aminosäuren im katalytischen Zentrum, was theoretische Fragen über die AC Aktivität aufgeworfen hat. Drei verschiedene Holoenzym-Konstrukte wurden exprimiert. Allerdings wurden größtenteils Abbruchprodukte gebildet. Sowohl die katalytische Domäne, als auch diese instabilen Holoenzyme zeigten keine enzymatische Aktivität. Rv2212c besteht aus zwei unterschiedlichen Domänen, einer C-terminalen CHD und einer N-terminalen regulatorischen Domäne, die auch in anderen Adenylatcyclasen aus Gram-positiven Bakterien (Rv1264 inbegriffen) gefunden wurde. Alle sechs kanonischen Aminosäuren sind konserviert. Die exprimierte CHD funktioniert als Homodimer. Ihre spezifische Aktivität (3.1µmol cAMP mg-1 min-1) und Vmax waren hoch im Vergleich zu anderen mycobakteriellen Adenylatcyclasen, die Substrataffinität war allerdings gering. GC Aktivität wurde nicht gefunden. Die spezifische Aktiviät des Holoenzyms war 4,5-fach niedriger als die der CHD, was gegen eine mögliche autoinhibitorische Funktion der N-terminalen Domäne spricht. Auch eine Funktion als pH-Sensor kann ausgeschlossen werden, da CHD und Holoenzym ihr pH-Optimum bei pH 6,5 haben. Dennoch spielt die N-terminale Domäne eine Rolle bei der Dimerisierung und der Enzymstabilität. Ungesättigte Fettsäuren (Arachidonsäure, Ölsäure und Linolsäure) stimulierten das Enzym signifikant durch eine Erhöhung der Substrataffinität aber ohne eine Änderung von Vmax und verliehen dem Protein eine hohe pH-Empfindlichkeit. Diese Effekte betreffen allerdings nicht nur die N-terminale Domäne, denn auch die CHD wurde durch die Fettsäuren stimuliert in geringerem Ausmaß. Die Tatsache, dass eine pH-Regulation durch die CHD in Anwesenheit der Fettsäuren nicht beobachtet werden konnten, spricht für die Bedeutung der N-terminalen Domäne bei der Regulation in Gegenwart von Fettsäuren. Durch die Aufklärung der molekularen Struktur des Proteins könnten der Einfluss der Fettsäuren und die Interaktionen mit dem Protein genauer untersucht werden

A cytotoxic C-glycosylated derivative of apigenin from the leaves of Ocimum basilicum var. thyrsiflorum

The standardized 80% ethanolic extract of the leaves of Ocimum basilicum var. thyrsiflorum (L.) Benth., Lamiaceae, growing in KSA, exhibited a significant antioxidant activity compared to the ethyl acetate and butanol extracts, which was correlated to its higher phenolic and flavonoid contents. Chromatographic separation of the 80% ethanol extract resulted in the isolation of ten known compounds; cinnamic acid, gallic acid, methylgallate, ellagic acid, methyl ellagic acid, apigenin, luteolin, vitexin, isovitexin, and 3″-O-acetylvitexin. Compound 3″-O-acetylvitexin, a C-glycosylated derivative of apigenin, was isolated for the first time from genus Ocimum. The 80% ethanolic extract and 3″-O-acetylvitexin showed significant cytotoxic activities against the HCT116 human colon cancer cell line [IC50 values 22.3 ± 1.1 and 16.8 ± 2.0 μg/ml (35.4 μM), respectively]. Keywords: Ocimum basilicum, HCT116, Cytotoxic, Antioxidant, C-glycosylation, Apigenin derivativ

Sulforaphane composition, cytotoxic and antioxidant activity of crucifer vegetables

Sulphur compounds in sulphur rich food have been shown to significantly reduce the risk of cancer development. One such compound is sulforaphane (SF), a cancer chemopreventive agent identified in broccoli (F. cruciferae). In this study, SF content was assessed in extracts of several crucifer vegetables including broccoli, brussels sprout, green cabbage, red cabbage, Chinese kale and turnip, in parallel with anticancer and antioxidant activity. Among tested crucifers, cabbage demonstrated a pronounced anticancer effect against A-549 lung cancer cells, with an IC50 value of 38 μg mL−1, and correlated with high SF levels at 540 μg g−1. Except for red cabbage and kale, crucifer extracts displayed moderate to weak activity in scavenging 2,2-diphenyl-1-picrylhydrazyl (DDPH) free radicals relative to vitamin E standard

In vitro and in vivo antidiabetic potential of extracts and a furostanol saponin from Balanites aegyptiaca

Context: Balanites aegyptiaca Del. (Zygophyllaceae) fruits are well-known antidiabetic drug in Egyptian folk medicine. Nevertheless, its mechanism of action is still unclear. Objectives: Searching for the possible mechanisms of action of the plant and identification of its bioactive compounds. Materials and methods: A bio-guided protocol based on the evaluation of α‐glucosidase (AG) and aldose reductase (AR) inhibitory activities was adopted to isolate the biologically active compounds from the methanol extract (MeEx). An in vivo antidiabetic study was conducted for the active extract, fraction and compound using streptozotocin-induced diabetic male albino Wistar rats at two dose levels (100 and 200 mg/kg.b/wt) for 2 weeks. Results: Three compounds were isolated and identified: a sterol, (1) stigmasterol-3-O-β-d-glucopyranoside; a pregnane glucoside, (2) pregn-5-ene-3β,16β,20(R)-trio1-3-O-β-d-glucopyranoside; a furostanol saponin, (3) 26-(O-β-d-glucopyranosyl)-22-O-methylfurost-5-ene-3β,26-diol-3-O-β-d-glucopyranosyl-(1 → 4)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside. Only compound 3 possessed significant AG and AR inhibitory activities (IC50 = 3.12 ± 0.17 and 1.04 ± 0.02 μg/mL, respectively), while compounds 1 and 2 were inactive. The in vivo antidiabetic study revealed that MeEx and furostanol saponin 3 possessed significant activities at a dose of 200 mg/kg through reducing the fasting plasma glucose level by 46.14% and 51.39%, respectively, as well as reducing the total cholesterol by 24.44% and 31.90%, respectively. Compound 3 also caused increment in insulin and C-peptide levels by 63.56% and 65%, respectively. Discussion and conclusions: We presented a scientific base for using Balanites aegyptiaca, and shed the light on one of its saponins, as an antidiabetic agent in fasting and postprandial hyperglycaemia along with the improvement of diabetic complications

Fatty acid regulation of adenylyl cyclase Rv2212 from Mycobacterium tuberculosis H37Rv

Adenylyl cyclase Rv2212 from Mycobacterium tuberculosis has a domain composition identical to the pH-sensing isoform Rv1264, an N-terminal regulatory domain and a C-terminal catalytic domain. The maximal velocity of Rv2212 was the highest of all 10 mycobacterial cyclases investigated to date (3.9 micromol cAMP.mg(-1).min(-1)), whereas ATP substrate affinity was low (SC(50) = 2.1 mm ATP). Guanylyl cyclase side activity was absent. The activities and kinetics of the holoenzyme and of the catalytic domain alone were similar, i.e. in distinct contrast to the Rv1264 adenylyl cyclase, in which the N-terminal domain is autoinhibitory. Unsaturated fatty acids strongly stimulated Rv2212 activity by increasing substrate affinity. In addition, fatty acids greatly enhanced the pH sensitivity of the holoenzyme, thus converting Rv2212 to a pH sensor adenylyl cyclase. Fatty acid binding to Rv2212 was modelled by homology to a recent structure of the N-terminal domain of Rv1264, in which a fatty acid-binding pocket is defined. Rv2212 appears to integrate three cellular parameters: ATP concentration, presence of unsaturated fatty acids, and pH. These regulatory properties open the possibility that novel modes of cAMP-mediated signal transduction exist in the pathogen

<i>In vitro</i> and <i>in vivo</i> antidiabetic potential of extracts and a furostanol saponin from <i>Balanites aegyptiaca</i>

<p><b>Context:</b><i>Balanites aegyptiaca</i> Del. (Zygophyllaceae) fruits are well-known antidiabetic drug in Egyptian folk medicine. Nevertheless, its mechanism of action is still unclear.</p> <p><b>Objectives:</b> Searching for the possible mechanisms of action of the plant and identification of its bioactive compounds.</p> <p><b>Materials and methods:</b> A bio-guided protocol based on the evaluation of α‐glucosidase (AG) and aldose reductase (AR) inhibitory activities was adopted to isolate the biologically active compounds from the methanol extract (MeEx). An <i>in vivo</i> antidiabetic study was conducted for the active extract, fraction and compound using streptozotocin-induced diabetic male albino Wistar rats at two dose levels (100 and 200 mg/kg.b/wt) for 2 weeks.</p> <p><b>Results:</b> Three compounds were isolated and identified: a sterol, (<b>1</b>) stigmasterol-3-<i>O</i>-β-d-glucopyranoside; a pregnane glucoside, (<b>2</b>) pregn-5-ene-3β,16β,20(R)-trio1-3-<i>O</i>-β-d-glucopyranoside; a furostanol saponin, (<b>3</b>) 26-(<i>O</i>-β-d-glucopyranosyl)-22-<i>O</i>-methylfurost-5-ene-3β,26-diol-3-<i>O</i>-β-d-glucopyranosyl-(1 → 4)-[α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranoside. Only compound <b>3</b> possessed significant AG and AR inhibitory activities (IC₅₀ = 3.12 ± 0.17 and 1.04 ± 0.02 μg/mL, respectively), while compounds <b>1</b> and <b>2</b> were inactive. The <i>in vivo</i> antidiabetic study revealed that MeEx and furostanol saponin <b>3</b> possessed significant activities at a dose of 200 mg/kg through reducing the fasting plasma glucose level by 46.14% and 51.39%, respectively, as well as reducing the total cholesterol by 24.44% and 31.90%, respectively. Compound <b>3</b> also caused increment in insulin and C-peptide levels by 63.56% and 65%, respectively.</p> <p><b>Discussion and conclusions:</b> We presented a scientific base for using <i>Balanites aegyptiaca</i>, and shed the light on one of its saponins, as an antidiabetic agent in fasting and postprandial hyperglycaemia along with the improvement of diabetic complications.</p

Botanical and genetic characteristics of Farsetia aegyptia Turra growing in Egypt

Farsetia aegyptia Turra is a perennial woody desert shrub native to Egypt. It is used by native Bedouins as an anti-diabetic and antispasmodic. Study of the botanical features was carried out for the root, young and old stems, leaf, fruit and seed of the plant. F. aegyptia Turra was characterized by the presence of myrosin cells and non-glandular branched unicellular two-armed hairs in the stem, leaves and fruit, while the root showed sclereids with a wide or narrow lumen and lignified pitted walls. Furthermore, the DNA of the plant was extracted from leaf samples and analysed using ten random decamer primers. A total of 58 random amplified polymorphic DNA (RAPD) markers were identified. Both the botanical study and the DNA fingerprint helped in the identification of the plant

Cytotoxic activity of phenolic constituents from Echinochloa crus-galli against four human cancer cell lines

Echinochloa crus-galli (L.) P. Beauv., Poaceae, grains are used as a feed for birds and millet for humans. The sulforhodamine B assay was used to assess its cytotoxicity against four human cancer cell lines. The ethanolic extract (70%) proved to be most active against HCT-116 and HELA cell lines (IC50 = 11.2 ± 0.11 and 12.0 ± 0.11 μg/ml, respectively). On the other hand, the chloroform and ethyl acetate fractions exhibited their highest activities against HCT-116 cell lines. The chloroform and ethyl acetate fractions were subjected to several chromatographic separations to render pure phenolic compounds (1–8). Compounds 1–8 were identified as: 5,7-dihydroxy-3′,4′,5′-trimethoxy flavone, 5,7,4′-trihydroxy-3′,5′-dimethoxy flavone (tricin), quercetin, flavone, apigenin-8-C-sophoroside, 2-methoxy-4-hydroxy cinnamic acid, p-coumaric acid and quercetin-3-O-glucoside. All the isolated phenolic compounds exhibited various significant activities against the four human carcinoma where the methoxylated flavones (1 and 2) were the most active, in a way comparable to the anticancer drug Doxorubicin®. Thus, these methoxylated flavonoids may be considered as lead compounds for the treatment of cancer, which supports previous claims of E. crus-galli traditional use. This is the first report of the occurrence of these phenolic compounds in E. crus-galli. Keywords: Barnyard grass, HCT-116, HELA, HEPG-2, MCF-7, Methoxylated flavonoid