Triamterene-β-cyclodextrin systems: Preparation,characterization and in vivo evaluation

The purpose of this research was to improve the solubility and therefore dissolution and bioavailability of triamterene, a poorly water soluble diuretic, by complexation with β-cyclodextrin. Triamterene has been reported to show low bioavailability after oral administration, with wide intersubject variation. This study presents the formulation of solid dispersions of triamterene with β-cyclodextrin—by cogrinding, kneading, and coevaporation, using low pH conditions—and their characterizations, evaluation of improvement in dissolution profiles, and in vivo advantage. Phase solubility studies indicated complex with possible stoichiometry of 1∶1 and a stability constant of 167.67M⁻¹. The solid dispersions were characterized by Fourier transform infrared spectroscopy, nuclear magnetic resonance, x-ray diffraction, and differential scanning calorimetry studies. The characterization studies confirmed inclusion of the phenyl ring of triamterene within the nonpolar cavity of β-cyclodextrin in the coevaporate. Remarkable improvement in in vitro drug release profiles in 0.1 N HCl and pH 6.8 phosphate buffer was observed with all dispersions, especially the coevaporate. The coevaporate, when administered orally in rats, also exhibited improved in vivo activity, as measured by net sodium ion excretion, as compared with triamterene powder. Thus, coevaporation of the drug and β-cyclodextrin from acidified alcohol provide the optimum condition for inclusion complexation to give a binary system with remarkable improvement in in vitro drug release profile and in vivo performance.     

Myotonic dystrophy type 2: human founder haplotype and evolutionary conservation of the repeat tract

Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, can be caused by a mutation on either chromosome 19 (DM1) or 3 (DM2). In 2001, we demonstrated that DM2 is caused by a CCTG expansion in intron 1 of the zinc finger protein 9 (ZNF9) gene. To investigate the ancestral origins of the DM2 expansion, we compared haplotypes for 71 families with genetically confirmed DM2, using 19 short tandem repeat markers that we developed that flank the repeat tract. All of the families are white, with the majority of Northern European/German descent and a single family from Afghanistan. Several conserved haplotypes spanning >700 kb appear to converge into a single haplotype near the repeat tract. The common interval that is shared by all families with DM2 immediately flanks the repeat, extending up to 216 kb telomeric and 119 kb centromeric of the CCTG expansion. The DM2 repeat tract contains the complex repeat motif (TG)(n)(TCTG)(n)(CCTG)(n). The CCTG portion of the repeat tract is interrupted on normal alleles, but, as in other expansion disorders, these interruptions are lost on affected alleles. We examined haplotypes of 228 control chromosomes and identified a potential premutation allele with an uninterrupted (CCTG)(20) on a haplotype that was identical to the most common affected haplotype. Our data suggest that the predominant Northern European ancestry of families with DM2 resulted from a common founder and that the loss of interruptions within the CCTG portion of the repeat tract may predispose alleles to further expansion. To gain insight into possible function of the repeat tract, we looked for evolutionary conservation. The complex repeat motif and flanking sequences within intron 1 are conserved among human, chimpanzee, gorilla, mouse, and rat, suggesting a conserved biological function.     

Fasciola hepatica cathepsin L-like proteases: biology,function, and potential in the development of first generation liver fluke vaccines

Fasciola hepatica secretes cathepsin L proteases that facilitate the penetration of the parasite through the tissues of its host, and also participate in functions such as feeding and immune evasion. The major proteases, cathepsin L1 (FheCL1) and cathepsin L2 (FheCL2) are members of a lineage that gave rise to the human cathepsin Ls, Ks and Ss, but while they exhibit similarities in their substrate specificities to these enzymes they differ in having a wider pH range for activity and an enhanced stability at neutral pH. There are presently 13 Fasciola cathepsin L cDNAs deposited in the public databases representing a gene family of at least seven distinct members, although the temporal and spatial expression of each of these members in the developmental stage of F. hepatica remains unclear. Immunolocalisation and in situ hybridisation studies, using antibody and DNA probes, respectively, show that the vast majority of cathepsin L gene expression is carried out in the epithelial cells lining the parasite gut. Within these cells the enzyme is packaged into secretory vesicles that release their contents into the gut lumen for the purpose of degrading ingested host tissue and blood. Liver flukes also express a novel multi-domain cystatin that may be involved in the regulation of cathepsin L activity. Vaccine trials in both sheep and cattle with purified native FheCL1 and FheCL2 have shown that these enzymes can induce protection, ranging from 33 to 79%, to experimental challenge with metacercariae of F. hepatica, and very potent anti-embryonation/hatch rate effects that would block parasite transmission. In this article we review the vaccine trials carried out over the past 8 years, the role of antibody and T cell responses in mediating protection and discuss the prospects of the cathepsin Ls in the development of first generation recombinant liver fluke vaccines.     

Ouabain-induced hypertension is accompanied by increases in endothelial vasodilator factors

The involvement of nitric oxide (NO), prostaglandins, and calcium-dependent potassium channel (K(Ca)) activators on the negative modulation of phenylephrine-induced contractions was evaluated on the isolated aorta and caudal (CAU) artery obtained from rats treated with ouabain for 5 wk to induce hypertension. In ouabain-treated rats, the reactivity to phenylephrine was reduced in the endothelium-intact aorta but not the CAU segments. Endothelial modulation of phenylephrine contraction, as demonstrated by endothelium removal, NO synthase (NOS) inhibition with N(omega)-nitro-L-arginine methyl ester and aminoguanidine, as well as K(Ca) inhibition with tetraethylammonium, was more pronounced in segments from ouabain-treated animals, and here greater effects were seen in the aorta than in CAU. An increased expression of endothelial NOS and neuronal NOS was seen in the aorta after ouabain treatment. In CAU, only endothelial NOS was detected and ouabain treatment did not alter its expression. These results suggest that ouabain-induced hypertension is accompanied by increased NO release derived from endothelial NOS and neuronal NOS and increased release of an endothelial hyperpolarizing factor that presumably opens K(Ca), all of which contribute to the increased negative modulation of the phenylephrine contraction.     

Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel model system for a severely compromised oxidative stress response

Glutamate-cysteine ligase (GCL) is the rate-limiting enzyme in the GSH biosynthesis pathway. In higher eukaryotes, this enzyme is a heterodimer comprising a catalytic subunit (GCLC) and a modifier subunit (GCLM), which change the catalytic characteristics of the holoenzyme. To define the cellular function of GCLM, we disrupted the mouse Gclm gene to create a null allele. Gclm(-/-) mice are viable and fertile and have no overt phenotype. In liver, lung, pancreas, erythrocytes, and plasma, however, GSH levels in Gclm(-/-) mice were 9-16% of that in Gclm(+/+) littermates. Cysteine levels in Gclm(-/-) mice were 9, 35, and 40% of that in Gclm(+/+) mice in kidney, pancreas, and plasma, respectively, but remained unchanged in the liver and erythrocytes. Comparing the hepatic GCL holoenzyme with GCLC in the genetic absence of GCLM, we found the latter had an approximately 2-fold increase in K(m) for glutamate and a dramatically enhanced sensitivity to GSH inhibition. The major decrease in GSH, combined with diminished GCL activity, rendered Gclm(-/-) fetal fibroblasts strikingly more sensitive to chemical oxidants such as H(2)O(2). We conclude that the Gclm(-/-) mouse represents a model of chronic GSH depletion that will be very useful in evaluating the role of the GCLM subunit and GSH in numerous pathophysiological conditions as well as in environmental toxicity associated with oxidant insult.     

Characterization of the MTF-1 transcription factor from zebrafish and trout cells

Two Euphausia superba Dana endo-1.4-beta-xylanases (A, and B), hydrolysing xylan in the same manner as the enzyme classified as EC 3.2.1.8, were isolated and purified. (2) The enzymes were distinguished by their molecular mass and charge, affinities towards the oat xylan (Km of 4.1 and 7.7 mg ml(-1), respectively), values of activation energy in oat xylan hydrolysis (35.5 and 42.5 kJ mol(-1), respectively), as well as the way in which they split the substrate. (3) In vitro they showed the same optimal temperature (37-40 degrees C), optimal pH (5.7-6.0), very low thermostability, and were stabilized and activated by Ca2+ and Mg2+ ions, as well as by some unidentified substances with molecular mass less than 17 kDa, present in crude extracts of krill.     

Fasciola hepatica: parasite-secreted proteinases degrade all human IgG subclasses: determination of the specific cleavage sites and identification of the immunoglobulin fragments produced

The study was focused on the relationship of Fasciola hepatica-secreted proteinases and human IgG subclasses. Each IgG was incubated at different pH values and lengths of time with either the adult parasite excretion-secretion products or the purified cysteinyl proteinases cathepsin L1 and cathepsin L2. The Ig fragments produced were isolated and characterized by Western blot analysis, and the specific cleavage sites were determined by amino acid sequence analysis. Parasite excretion-secretion products and both cathepsins L produced similar degradation patterns and cleaved all human IgG subclasses at the hinge region, yielding at pH 7.3 and 37 degrees C Fab and Fc fragments in the case of IgG1 and IgG3 or Fab(2) and Fc in IgG2 and IgG4. While IgG1 and IgG3 were readily degraded by E/S products either in the presence or in the absence of reducing agents, IgG2 and IgG4 were resistant to proteolysis and were only digested in the presence of 0.1 M dithiothreitol. The cathepsins L needed the presence of dithiothreitol to digest IgG1, IgG2, and IgG4 whereas IgG3 was identically cleaved under both reducing and nonreducing conditions. The main cleavage sites produced by E/S products, CL1, or CL2 were located at the positions peptide bonds: His237-Thr238, Glu237-Cys239, Gly233-Asp234, and Ser241-Cys242 for gamma1, gamma2, gamma3, or gamma4, respectively. The enzymes gave additional splitting sites on the middle hinge of IgG3 to produce shorter Fc fragments and also produce Fd degradation of the IgG4. No cleavage specificity differences were found between CL1 and CL2, but they differed in the kinetics of IgG3 degradation. By lowering the pH, only the E/S products produced concomitant destruction of the Fc while preserving the Fab portion. Under all the conditions assayed the enzymes produced an Fc'-like fragment of 14-15 kDa corresponding to the whole CH3 domain of the immunoglobulin. Contrary to the extensive degradation produced by cathepsins on digested proteins, its actions on IgG subclasses were specific and restricted; thus, all the fragments produced could be potentially involved in the mechanisms used by the parasite to evade the host immune response.     

Knowledge and Uses of African Pangolins as a Source of Traditional Medicine in Ghana

Traditional medicine has been practised in Ghana for centuries with the majority of Ghanaians still patronising the services of traditional healers. Throughout Africa a large number of people use pangolins as a source of traditional medicine, however, there is a dearth of information on the use of animals in folk medicine in Ghana, in particular the use of pangolins. The aim of this study was to determine the prevalent use of pangolins and the level of knowledge of pangolin use among traditional healers in Ghana for the treatment of human ailments. Data was gathered from 48 traditional healers using semi-structured interviews on the traditional medicinal use of pangolin body parts in the Kumasi metropolis of Ghana. The cultural importance index, relative frequency of citation, informant agreement ratio and use agreement values were calculated to ascertain the most culturally important pangolin body part as well as the level of knowledge dissemination among traditional healers with regards pangolin body parts. Our study revealed that 13 body parts of pangolins are used to treat various medicinal ailments. Pangolin scales and bones were the most prevalent prescribed body parts and indicated the highest cultural significance among traditional healing practices primarily for the treatment of spiritual protection, rheumatism, financial rituals and convulsions. Despite being classified under Schedule 1 of Ghana’s Wildlife Conservation Act of 1971 (LI 685), that prohibits anyone from hunting or being in possession of a pangolin, our results indicated that the use of pangolins for traditional medicinal purposes is widespread among traditional healers in Ghana. A study on the population status and ecology of the three species of African pangolins occurring in Ghana is urgently required in order to determine the impact this harvest for traditional medical purposes has on their respective populations as current levels appear to be unmonitored and unsustainable.