Preparation, characterisation and evaluation of curcumin with piperine-loaded cubosome nanoparticles.

OBJECTIVE: In this study, curcumin was designed into the nanoformulation called cubosome with piperine in order to improve oral bioavailability and tissue distribution of curcumin. METHODS: The characteristic of the cubosome was studied by using scanning electron microscope (SEM), Infrared spectrum and small angle X-ray scattering (SAXS) techniques. Tissue distribution of cubosome was measured by liquid chromatography-mass spectrometry (LC-MS) method in mice. RESULTS: The characteristic of the cubosome was demonstrated that the curcumin and piperine were encapsulated in the interior of the cubosome and the crystal form was Pn3m space. The pharmacokinetic test revealed that the cubosome could improve the oral bioavailability significantly compared to the suspension of curcumin with piperine and be mainly absorbed by the spleen. CONCLUSION: These findings provide the reference to a preferable choice of the curcumin formulation and contribute to therapeutic application in clinical research.

Preparation and characterisation of andrographolide niosomes and its anti-hepatocellular carcinoma activity.

OBJECTIVE: In this study, a novel andrographolide (AG) preparation formulation, niosomes, was prepared to improve the bioavailability and tissue distribution of AG. METHODS: The niosomal formulation of AG was prepared by film hydration/sonication method and tissue distribution was measured by liquid chromatography-mass spectrometry (LC-MS) method in mice, and anti-hepatocellular carcinoma (anti-HCC) activity was examined by MTT method in HepG2. RESULTS: Entrapment efficiency, drug-loading ratio and average particle size of AG niosomes were 72.36%, 5.90% and 206 nm, respectively. The tissue distribution in mice demonstrated that the AG niosomes were absorbed in liver much more than the free AG. Furthermore, the anti-HCC activity in HepG2 cells showed that there was no significant difference between free AG and AG niosomes. CONCLUSION: The present results suggest that AG niosomes may have a significant potential of liver targeting, which is valuable in chemotherapy of HCC.

Rat glutathione S-transferase M4-4: an isoenzyme with unique structural features including a redox-reactive cysteine-115 residue that forms mixed disulphides with glutathione.

Although the existence of the rat glutathione S-transferase (GST) M4 (rGSTM4) gene has been known for some time, the corresponding protein has not as yet been purified from tissue. A recombinant rGSTM4-4 was thus expressed in Escherichia coli from a chemically synthesized rGSTM4 gene. The catalytic efficiency (k(cat)/K(m)) of rGSTM4-4 for the 1-chloro-2,4-dinitrobenzene (CDNB) conjugation reaction was 50-180-fold less than that of the well-characterized homologous rGSTM1-1, and the pH optimum for the same reaction was 8.5 for rGSTM4-4 as opposed to 6.5 for rGSTM1-1. Molecular-modelling studies predict that key substitutions in the helix alpha4 region of rGSTM4-4 account for this pK(a) difference. A notable structural feature of rGSTM4-4 is the Cys-115 residue in place of the Tyr-115 of other Mu-class GSTs. The thiol group of Cys-115 is redox-reactive and readily forms a mixed disulphide even with GSH; the S-glutathiolated form of the enzyme is catalytically active. A mutated rGSTM4-4 (C115Y) had 6-10-fold greater catalytic efficiency than the wild-type rGSTM4-4. Trp-45, a conserved residue among Mu-class GSTs, is essential in rGSTM4-4 for both enzyme activity and binding to glutathione affinity matrices. Antibodies directed against either the unique C-terminal undecapeptide or tridecapeptide of rGSTM4 reacted with rat and mouse liver GSTs to reveal an orthologous mouse GSTM4-4 present at low basal levels but which is inducible in mouse liver. This subclass of rodent Mu GSTs with redox-active Cys-115 residues could have specialized physiological functions in response to oxidative stress.

Autoantibodies against ADP/ATP carrier from patients with dilated cardiomyopathy increase activity of voltage-dependent Ca channels in isolated cardiac myocytes.

The effects of autoantibodies against the ADP/ATP carrier, from sera of patients with dilated cardiomyopathy, on calcium channel current (ICa) were studied in enzymatically-isolated guinea pig ventricular myocytes by using a whole cell patch-clamp method. The results showed that the autoantibodies enhanced ICa in a concentration-dependent manner. Verapamil inhibited enhancement of ICa induced by the autoantibodies. Control sera (without the autoantibodies) did not affect ICa. This study suggests that anti-ADP/ATP carrier autoantibodies from sera of patients with dilated cardiomyopathy may enhance ICa and cause calcium overload. Disturbing Ca-channel gating by autoantibodies may contribute to the pathogenesis of dilated cardiomyopathy.

A molecular genetic approach for the identification of essential residues in human glutathione S-transferase function in Escherichia coli.

The common substrate for glutathione S-transferases (GSTs), 1-chloro-2,4-dinitrobenzene (CDNB), is an inhibitor of Escherichia coli growth. This growth inhibition by CDNB is enhanced when E. coli expresses a functional GST. Cells under growth inhibition have reduced intracellular GSH levels and form filaments when they resume growth. Based on this differential growth inhibition by CDNB we have developed a simple procedure to select for null-mutants of a human GST in E. coli. Null mutations in the human GST gene from hydroxylamine mutagenesis or oligonucleotide-directed mutagenesis can be selected for on agar plates containing CDNB after transformation. The molecular nature of each mutation can be identified by DNA sequence analysis of the mutant GST gene. We have identified three essential amino acid residues in an alpha class human GST at Glu31, Glu96, and Gly97. Single substitution at each of these residues, E31K, E96K, G97D, resulted in mutant GST proteins with loss of CDNB conjugation activity and failure in binding to the S-hexyl GSH affinity matrix. In contrast, a mutant GST (Y8F) resulting from substitution of the conserved tyrosine near the N terminus has much reduced CDNB conjugation activity but was still capable of binding to the S-hexyl GSH-agarose. Additional mutant GSTs with substitutions at position 96 (E96F, E96Y) and 97 (G97P, G97T, G97S) resulted in changes in both Km and kcat to different extents. The in vitro CDNB conjugation activity of the purified mutant enzymes correlate negatively with the plating efficiencies of strains encoding them in the presence of CDNB. Based on the x-ray structure model of human GST 1-1, two of these residues are involved in salt bridges (Arg19-Glu31, Arg68-Glu96) and the third Gly97 is in the middle of the helix alpha 4. Our results provide evidence in vivo that Tyr8, Gly97, and the two salt bridges are important for GST structure-function. This molecular genetic approach for the identification of essential amino acids in GSTs should be applicable to any GSTs with CDNB conjugation activity. It should also complement the x-ray crystallographic approach in understanding the structure and function of GSTs.

Thermodynamics of oxidative phosphorylation in bovine heart submitochondrial particles.

The rates of both forward and reverse electron transfer in phosphorylating submitochondrial particles from bovine heart can be controlled by the thermodynamic phosphorylation potential (deltaGp) of the adenine nucleotide system. deltaGp is the Gibbs free energy of ATP synthesis and is defined by the relationship deltaGp = -deltaG'o + RTln([ATP]/[ADP][Pi]) where deltaG'o is the standard free energy of ATP hydrolysis. Studies of the effects of deltaGp on NADH respiration and the reduction of NAD+ by succinate show that increasing values of deltaGp cause an inhibition of forward electron transfer and a stimulation of reverse electron transfer. Between deltaGp values of 7.6 and 13.0 kcal/mol the rate of NADH respiration decreased 3-fold and the rate of NAD+ reduction by succinate increased 3-fold. Indirect phosphorylation potential titration experiments as well as direct chemical measurements indicate that steady state levels of ATP, ADP, and Pi are established during NADH respiration which correspond to a deltaGp equal to 10.7 to 11.4 kcal/mol.

Relationship of nuclease activity and synthesis to senescence of corn (Zea mays L.) stalk pith, cob parenchyma and first developed leaf tissues.

An increase in total RNase activity was associated with three patterns of cell senescence in corn (Zea mays L.) (cv. WF9 X 38-11) cob parenchyma during the first two weeks following silking, stalk pith tissue after internode elongation and the first developed leaf of seedlings. Stalk pith tissue had two RNase activities, one inhibited by EDTA and one not. Both remained in approximately equal amounts in young to old pith tissue. In the first developed leaf of seedlings, the activity not inhibited by EDTA remained at a constant low level during the period studied, while the other activity varied. No inhibition by EDTA was found in cob parenchyma tissue. Incubation of sections of cob parenchyma and stalk pith tissues suggested that the total RNase activity of cob parenchyma is very stable and that of stalk pith tissue is relatively stable. An age-related increase in DNase activity was found in stalk pith tissue and in the first developed leaf of seedlings, but not in cob parenchyma tissue.