Identification and characterization of two isoenzymes of methionine gamma-lyase from Entamoeba histolytica: a key enzyme of sulfur-amino acid degradation in an anaerobic parasitic protist that lacks forward and reverse trans-sulfuration pathways

To better understand the metabolism of sulfur-containing amino acids, which likely plays a key role in a variety of cell functions, in Entamoeba histolytica, we searched the genome data base for genes encoding putative orthologs of enzymes known to be involved in the metabolism. The search revealed that E. histolytica possesses only incomplete cysteine-methionine conversion pathways in both directions. Instead, this parasite possesses genes encoding two isoenzymes of methionine gamma-lyase (EC 4.4.1.11, EhMGL1/2), which has been implicated in the degradation of sulfur-containing amino acids. The two amebic MGL isoenzymes, showing 69% identity to each other, encode 389- and 392-amino acid polypeptides with predicted molecular masses of 42.3 and 42.7 kDa and pIs of 6.01 and 6.63, respectively. Amino acid comparison and phylogenetic analysis suggested that these amebic MGLs are likely to have been horizontally transferred from the Archaea, whereas an MGL from another anaerobic protist Trichomonas vaginalis has MGL isotypes that share a common ancestor with bacteria. Enzymological and immunoblot analyses of the partially purified native amebic MGL confirmed that both of the MGL isotypes are expressed in a comparable amount predominantly in the cytosol and form a homotetramer. Recombinant EhMGL1 and 2 proteins catalyzed degradation of L-methionine, DL-homocysteine, L-cysteine, and O-acetyl-L-serine to form alpha-keto acid, ammonia, and hydrogen sulfide or methanethiol, whereas activity toward cystathionine was negligible. These two isoenzymes showed notable differences in substrate specificity and pH optimum. In addition, we showed that EhMGL is an ideal target for the development of new chemotherapeutic agents against amebiasis by demonstrating an amebicidal effect of the methionine analog trifluoromethionine on trophozoites in culture (IC50 18 mum) and that this effect of trifluoromethionine was completely abolished by the addition of the MGL-specific inhibitor DL-propargylglycine.     

Inverted papilloma of the nasal cavity presenting with massive amounts of squamous metaplastic cells in sputum. A case report

BACKGROUND: Squamous metaplasic cells are rarely seen in sputum of female nonsmokers. CASE: A 47-year-old female nonsmoker presented with massive amounts of squamous metaplasic cells in sputum and an elevated level of squamous cell carcinoma (SCC) antigen in serum present for months, while no causative lesion was detected either by lung computed tomography or bronchoscopy. The patient was eventually diagnosed as having inverted papilloma in the right nasal cavity. Resection of the tumor brought about disappearance of squamous metaplastic cells in sputum and return of serum SCC antigen to the normal range. CONCLUSION: This case clearly demonstrates that squamous metaplastic cells in sputum can originate in lesions in the nasal cavity, although they are rare. It should be kept in mind that the nasal cavity is a potential site producing squamous metaplastic cells in sputum.     

Compartmentation of aluminium in leaves of an Al-accumulator,Fagopyrum esculentum Moench

Buckwheat (Fagopyrum esculentum Moench.) is an Al-accumulating plant, but the internal mechanism(s) of detoxification of Al is not fully understood. We investigated the subcellular localization of Al in the leaves of this plant (cv. Jianxi) by directly isolating protoplasts and vacuoles. Pure protoplasts and vacuoles from the leaves of buckwheat, grown hydroponically in Al solution, were obtained based on light-microscopic observation and the activities of marker enzymes of cytosol and vacuoles. More than 80% of total Al in the leaves was present in the protoplasts, and was identified as an Al-oxalate complex (1:3 ratio) by (27)Al-nuclear magnetic resonance. Oxalate and Al in the protoplasts was localized in the vacuoles. These results suggest that internal detoxification of Al in the buckwheat leaves is achieved by both complexation with oxalate and sequestration into vacuoles.     

Human betacellulin structure modeled from other members of EGF family

We have modeled betacellulin (BTC) to gain insight into the structural elements that can explain its properties. The epidermal growth factor (EGF) signal transduction pathway, a significant mediator of several cell functions, is based on four closely related tyrosine kinase receptors. The ErbB receptors are transmembrane glycoproteins and signal transduction is initiated by ligand binding that induces receptor homo- or heterodimerization to form a complex containing two molecules of ligand and two molecules of receptor. The EGF family of ligands can be divided into three groups based on their ability to bind and activate distinct ErbB receptor homo- and heterodimers. Each member of the group formed by BTC, heparin binding EGF (HB-EGF) and epiregulin (EP) can interact with both the EGF receptor (EGFR) and heregulin receptors (ErbB-3 and ErbB-4), and are hence called bispecific ligands. BTC and EP also present the distinctive feature that they activate all possible heterodimeric ErbB receptors. BTC has been modeled with the program MODELLER, using human EGF, human transforming growth factor alpha (hTGF), human HB-EGF and human heregulin one alpha (hHRG-1) as templates. The structure of the model as well as that of the templates were optimized and a simulation of 100 ps was run for all. The main structural properties of the model and the templates were compared and in conclusion the hBTC conformation was closely similar to that of hTGF. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00894-002-0072-2.Electronic Supplementary Material available.     

Nanoparticles for the delivery of genes and drugs to human hepatocytes

Hepatitis B virus envelope L particles form hollow nanoparticles displaying a peptide that is indispensable for liver-specific infection by hepatitis B virus in humans. Here we demonstrate the use of L particles for the efficient and specific transfer of a gene or drug into human hepatocytes both in culture and in a mouse xenograft model. In this model, intravenous injection of L particles carrying the gene for green fluorescent protein (GFP) or a fluorescent dye resulted in observable fluorescence only in human hepatocellular carcinomas but not in other human carcinomas or in mouse tissues. When the gene encoding human clotting factor IX was transferred into the xenograft model using L particles, factor IX was produced at levels relevant to the treatment of hemophilia B. The yeast-derived L particle is free of viral genomes, highly specific to human liver cells and able to accommodate drugs as well as genes. These advantages should facilitate targeted delivery of genes and drugs to the human liver.     

Angiogenic network formation in the developing vertebrate trunk

We have used time-lapse multiphoton microscopy of living Tg(fli1:EGFP)y1 zebrafish embryos to examine how a patterned, functional network of angiogenic blood vessels is generated in the early vertebrate trunk. Angiogenic vascular sprouts emerge from the longitudinal trunk axial vessels (the dorsal aorta and posterior cardinal vein) in two spatially and temporally distinct steps. Dorsal aorta-derived sprouts form an initial primary network of vascular segments, followed by emergence of vein-derived secondary vascular sprouts that interact and interconnect dynamically with the primary network to initiate vascular flow. Using transgenic silent heart mutant embryos, we show that the gross anatomical patterning of this network of vessels does not require blood circulation. However, our results suggest that circulatory flow dynamics play an important role in helping to determine the pattern of interconnections between the primary network and secondary sprouts, and thus the final arterial or venous identity of the vessels in the functional network. We discuss a model to explain our results combining genetic programming of overall vascular architecture with hemodynamic determination of circulatory flow patterns.     

Design and synthesis of orally bioavailable inhibitors of inducible nitric oxide synthase. synthesis and biological evaluation of dihydropyridin-2(1H)-imines and 1,5,6,7-tetrahydro-2H-azepin-2-imines

The process of discovery and biological evaluation of alpha,beta-unsaturated cyclic amidines, as selective inhibitors of inducible nitric oxide synthase (iNOS), is reported. Dihydropyridin-2(1H)-imines and 1,5,6,7-tetrahydro-2H-azepin-2-imines were synthesized and biologically evaluated both in vitro and in vivo using a nitric oxide synthase inhibition assay. Compounds 1, 5, 6, 8-12 and 16 exhibited potent inhibition of iNOS. Among these, compounds 6, 7, 10, 11 and 16 showed 5- to 19-fold isoform selectivity. Compounds 1, 6, 10, 11 and 16 also showed potent inhibitory activity in the NOx accumulation assay in mice. Compounds 1 and 6 showed excellent bioavailability (BA) in rats when administered orally. Full details are presented here, including the structure-activity relationship (SAR) studies, the chemistry of these compounds, and the pharmacokinetic data and the computer-aided docking study of 10 with hiNOS.     

Gene expression profiles in chondrosarcoma cells subjected to cyclic stretching and hydrostatic pressure. A cDNA array study

Mechanical forces have a profound effect on cartilage tissue and chondrocyte metabolism. Strenuous loading inhibits the cellular metabolism, while optimal level of loading at correct frequency raises an anabolic response in chondrocytes. In this study, we used Atlas Human Cancer cDNA array to investigate mRNA expression profiles in human chondrosarcoma cells stretched 8% for 6 hours at a frequency of 0.5 Hz. In addition, cultures were exposed to continuous and cyclic (0.5 Hz) 5 MPa hydrostatic pressure. Cyclic stretch had a more profound effect on the gene expression profiles than 5 MPa hydrostatic pressure. Several genes involved with the regulation of cell cycle were increased in stretched cells, as well as mRNAs for PDGF-B, glucose-1-phosphate uridylyltransferase, Tiam1, cdc37 homolog, Gem, integrin alpha6, and matrix metalloproteinase-3. Among down-regulated genes were plakoglobin, TGF-alpha, retinoic acid receptor-alpha and Wnt8b. A smaller number of changes was detected after pressure treatments. Plakoglobin was increased under cyclic and continuous 5 MPa hydrostatic pressure, while mitogen-activated protein kinase-9, proliferating cell nuclear antigen, Rad6, CD9 antigen, integrins alphaE and beta8, and vimentin were decreased. Cyclic and continuous pressurization induces a number of specific changes. In conclusion, a different set of genes were affected by three different types of mechanical stimuli applied on chondrosarcoma cells.     

Neuroprotection in hypothermia linked to redistribution of oxygen in brain

Hypothermia improves the outcome of acute ischemic stroke, traumatic injury, and inflammation of brain tissue. We tested the hypothesis that hypothermia reduces the energy metabolism of brain tissue to a level that is commensurate with the prevailing blood flow and hence allows adequate distribution of oxygen to the entire tissue. To determine the effect of 32 degrees C hypothermia on brain tissue, we measured the sequential changes of physiological variables by means of PET in pigs. Cerebral blood flow and oxygen consumption (cerebral metabolic rate of oxygen) declined to 50% of the baseline in 3 and 5 h, respectively, thus elevating the oxygen extraction fraction to 140% of the baseline at 3 h. The results are consistent with the claim that cooling of the brain to 32 degrees C couples both energy metabolism and blood flow to a lower rate of work of the entire tissue.     

Determination of asymmetric structure of ganglioside-DPPC mixed vesicle using SANS,SAXS, and DLS

Functions of mammalian cell membrane microdomains being rich in glycosphingolipids, so-called rafts, are now one of the current hot topics in cell biology from the intimate relation to cell adhesion and signaling. However, little is known about the role of glycosphingolipids in the formation and stability of the domains. By the use of the inverse contrast variation method in small-angle neutron scattering (SANS), combined with small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS), we have determined an asymmetric internal structure of the bilayer of the small unilamellar vesicle (SUV) of monosialoganglioside (G(M1))-dipalmitoylphosphatidylcholine (DPPC) mixture ([G(M1)]:[DPPC] = 0.1:1). A direct method using a shell-model fitting with a size distribution function describes consistently all experimental results of SANS, SAXS, and DLS. We have found that G(M1) molecules predominantly localize at SUV outer surface to form a highly hydrophilic layer which is dehydrated with the rise of temperature from 25 degrees C to 55 degrees C accompanied by the conformational change of the oligosaccharide chains. The average SUV size determined is approximately 200 A, which is comparable to the reported value 260 +/- 130 A of glycosphingolipids microdomains. The present results suggest that the preferential asymmetric distribution of gangliosides is essential to define the size and stability of the domains.