Comparative evaluation of proximate composition and anti-sickling potential of Annona muricata Linn seeds and leaves

Background: Annona muricata Linn is a notable, well-studied plant of therapeutic value. Based on the abundant pharmacological constituents contained in the understudied plant, it is imperative that the plant parts are investigated for nutritional value and anti-sickling potentials. Sickle cell anaemia (SCA) is an inheritable haematological disorder, caused by an amino acid substitution on the haem protein. The outcomes for SCA are poor health indices and high mortality. Therefore, the use of natural products is necessary and widely promoted in countries with poor health infrastructure. Methods: In this study, A. muricata seeds and leaves were comparatively analysed for the proximate compositions. In addition, aqueous and ethanol extracts of A. muricata seeds and leaves were respectively analysed for anti-sickling potentials with the use of spectrophotometry. Results: Proximate composition of A. muricata seeds and leaves showed that both plant parts contain ash, carbohydrate, fat, fibre, moisture and protein. However, percentage proximate composition of A. muricata seeds was not significantly different from the percentage proximate composition of A. muricata leaves (p ≤ 0.05). From anti-sickling analysis, the aqueous extracts of A. muricata seeds and leaves were observed to inhibit HbSS polymerisation, while the ethanol extracts of A. muricata seeds and leaves showed limitations to the inhibition of HbSS polymerisation. Conclusion: A. muricata seeds and leaves possess potentials as health or nutritional supplements for the management of SCA. Further studies are necessary in order to ascertain efficacy and safety in in vivo models

Pivotal role of P450–P450 interactions in CYP3A4 allostery: the case of α-naphthoflavone

We investigated the relationship between oligomerization of CYP3A4 (cytochrome P450 3A4) and its response to ANF (α-naphthoflavone), a prototypical heterotropic activator. The addition of ANF resulted in over a 2-fold increase in the rate of CYP3A4-dependent debenzylation of 7-BFC [7-benzyloxy-4-(trifluoromethyl)coumarin] in HLM (human liver microsomes), but failed to produce activation in BD Supersomes™ or Baculosomes® containing recombinant CYP3A4 and NADPH-CPR (cytochrome P450 reductase). However, incorporation of purified CYP3A4 into Supersomes™ containing only recombinant CPR reproduced the behaviour observed with HLM. The activation in this system was dependent on the surface density of the enzyme. Although no activation was detectable at an L/P (lipid/P450) ratio ≥750, it reached 225% at an L/P ratio of 140. To explore the relationship between this effect and CYP3A4 oligomerization, we probed P450–P450 interactions with a new technique that employs LRET (luminescence resonance energy transfer). The amplitude of LRET in mixed oligomers of the haem protein labelled with donor and acceptor fluorophores exhibited a sigmoidal dependence on the surface density of CYP3A4 in Supersomes™. The addition of ANF eliminated this sigmoidal character and increased the degree of oligomerization at low enzyme concentrations. Therefore the mechanisms of CYP3A4 allostery with ANF involve effector-dependent modulation of P450–P450 interactions.

The Pseudomonas aeruginosa DNR transcription factor: light and shade of nitric oxide-sensing mechanisms

In response to environmental conditions, NO (nitric oxide) induces global changes in the cellular metabolism of Pseudomonas aeruginosa, which are strictly related to pathogenesis. In particular, at low oxygen tensions and in the presence of NO the denitrification alternative respiration is activated by a key regulator: DNR (dissimilative nitrate respiration regulator). DNR belongs to the CRP (cAMP receptor protein)–FNR (fumarate and nitrate reductase regulatory protein) superfamily of bacterial transcription factors. These regulators are involved in many different pathways and distinct activation mechanism seems to be operative in several cases. Recent results indicate that DNR is a haem protein capable of discriminating between NO and CO (carbon monoxide). On the basis of the available structural data, a suggested activation mechanism is discussed.

The role of Dcytb in iron metabolism: an update

Dcytb (duodenal cytochrome b) is an iron-regulated ferric reductase highly expressed in duodenal enterocytes. Its location and strong regulation by iron has indicated it plays an important role in iron absorption. Expression of Dcytb in cells (Caco-2 and MDCK) was found to increase both ferric reductase activity and stimulate uptake of 59Fe. An additional increase in cupric reductase activity was found in MDCK (Madin–Darby canine kidney) cells expressing Dcytb. Expression and purification of Dcytb in insect cells reveals that Dcytb is a di-haem protein and that the haems are reducible by ascorbate, indicating that ascorbate is the likely intracelluar electron donor. Studies underway in Dcytb-knockout mice reveal that Dcytb is the only iron-regulated ferric reductase in the duodenal mucosa and that loss of Dcytb affects iron absorption.

Rhodobacter sphaeroides haem protein: a novel cytochrome with nitric oxide dioxygenase activity

Rhodobacter sphaeroides produces a novel cytochrome, designated as SHP (sphaeroides haem protein), that is unusual in having asparagine as a redox-labile haem ligand. The gene encoding SHP is contained within an operon that also encodes a DHC (dihaem cytochrome c) and a membrane-associated cytochrome b. DHC and SHP have been shown to have high affinity for each other at low ionic strength (Kd=0.2 μM), and DHC is able to reduce SHP very rapidly. The reduced form of the protein, SHP2+ (reduced or ferrous SHP), has high affinity for both oxygen and nitric oxide (NO). It has been shown that the oxyferrous form, SHP2+–O2 (oxygen-bound form of SHP), reacts rapidly with NO to produce nitrate, whereas SHP2+–NO (the NO-bound form of SHP) will react with superoxide with the same product formed. It is therefore possible that SHP functions physiologically as a nitric oxide dioxygenase, protecting the organism against NO poisoning, and we propose a possible mechanism for this process.

Tuning the formation of a covalent haem–protein link by selection of reductive or oxidative conditions as exemplified by ascorbate peroxidase

Previous work [Metcalfe, Ott, Patel, Singh, Mistry, Goff and Raven (2004) J. Am. Chem. Soc. 126, 16242–16248] has shown that the introduction of a methionine residue (S160M variant) close to the 2-vinyl group of the haem in ascorbate peroxidase leads to the formation of a covalent haem–methionine linkage under oxidative conditions (i.e. on reaction with H2O2). In the present study, spectroscopic, HPLC and mass spectrometric evidence is presented to show that covalent attachment of the haem to an engineered cysteine residue can also occur in the S160C variant, but, in this case, under reducing conditions analogous to those used in the formation of covalent links in cytochrome c. The data add an extra dimension to our understanding of haem to protein covalent bond formation because they show that different types of covalent attachment (one requiring an oxidative mechanism, the other a reductive pathway) are both accessible within same protein architecture.