Molecular cloning, expression, and characterization of myo-inositol oxygenase from mouse, rat, and human kidney

myo-Inositol oxygenase (MIOX) is a non-heme iron enzyme, which catalyzes the conversion of myo-inositol to d-glucuronic acid, the first committed step in myo-inositol catabolism. Full-length cDNAs of 858bp each coding for 33kDa protein were cloned from kidney cDNA libraries of mouse, rat, and human. The individual clones were expressed in Escherichia coli and recombinant MIOX proteins were purified to electrophoretic homogeneity. A hydrophobic interaction chromatography step yielded multiple conformers, with mouse and human MIOX showing three peaks and rat enzyme revealing two peaks. Individual MIOX peaks exhibited distinct V(max) and K(m) values. Interestingly, upon storage, the 33kDa protein was degraded to a approximately 30kDa truncated protein in each species, and formed small amounts of dimers of identical subunits. While MIOX is a highly conserved enzyme in all mammalian species, the labile nature and tendency to degrade in solution may be the source of significant differences in size previously reported in the literature. Regardless of the source, our results strongly dispel previous conflicting literature reports on the size of the protein and confirm that MIOX is a 33kDa protein.     

cDNA cloning and gene expression analysis of human myo-inositol 1-phosphate synthase

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) (IPS) is a key enzyme in myo-inositol biosynthesis pathway. This study describes the molecular cloning of the full length human myo-inositol 1-phosphate synthase (hIPS) cDNA, tissue distribution of its mRNA and characterizes its gene expression in cultured HepG2 cells. Human testis, ovary, heart, placenta, and pancreas express relatively high level of hIPS mRNA, while blood leukocyte, thymus, skeletal muscle, and colon express low or marginal amount of the mRNA. In the presence of glucose, hIPS mRNA level increases 2- to 4-fold in HepG2 cells. hIPS mRNA is also up-regulated 2- to 3-fold by 2.5 microM lovastain. This up-regulation is prevented by mevalonic acid, farnesol, and geranylgeraniol, suggesting a G-protein mediated signal transduction mechanism in the regulation of hIPS gene expression. hIPS mRNA expression is 50% suppressed by 10mM lithium ion in these cells. Neither 5mM myo-inositol nor the three hormones: estrogen, thyroid hormone, and insulin altered hIPS mRNA expression in these cells.     

Synthesis of glucopyranoside-based ligands for D-myo-inositol 1,4,5-trisphosphate receptors

Adenophostins A and B are naturally occurring glyconucleotides that interact potently with receptors for D-myo-inositol 1,4,5-trisphosphate, an important second messenger molecule in most cell types. Here we describe the design and synthesis of glucopyranoside-based analogues of adenophostin A lacking the adenine component. The key synthetic strategy involves glycosylation of selectively protected alcohols, derived from methyl beta-D-ribofuranoside or 1,4-anhydroerythritol, using glycosyl donors synthesised from 2,6-di-O-benzyl-D-glucopyranose derivatives. Further elaboration and deprotection of the coupled products gave two trisphosphate analogues; methyl 3-O-alpha-D-glucopyranosyl-beta-D-ribofuranoside 2,3',4'-trisphosphate ("ribophostin") and (3'S,4'R)-3'-hydroxytetrahydrofuran-4'-yl alpha-D-glucopyranoside 3,4,3'-trisphosphosphate ("furanophostin"). The route to furanophostin was further modified to give (3'S,4'R)-3'-hydroxytetrahydrofuran-4'-yl alpha-D-glucopyranoside 3'-phosphate 3,4-bisphosphorothioate, the first phosphorothioate-containing adenophostin analogue.     

Crystal structure of 1L-1,2:4,5-di-O-isopropylidene-allo-inositol: a comparison of its conformation in solid and solution states

The X-ray crystal structure of 1l-1,2:4,5-di-O-isopropylidene-allo-inositol, is described. The inositol ring deviate slightly from the ideal chair conformation to a flattened chair. A comparison of its conformation in solution with that in solid was made by the use of 1H NMR. This conformational analysis revealed that the title compound adopts similar conformations in solid state and in solution in low polar solvents like benzene and CHCl3 while in high polar solvents such as Me2SO, the solid state conformation is not retained.     

Solid and solution state conformation of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol

The X-ray crystal structure of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol is described. The inositol ring deviates considerably from the ideal chair conformation to a flattened chair. A comparison of its conformation in solution with that in solid was made by the use of 1H NMR. This conformational analysis revealed that the title compound adopts similar conformations in solid state and in solution states irrespective of solvent polarity.     

Solid and solution state conformations of (+/-)-3-O-acetyl-1,2:4,5-di-O-isopropylidene-allo-inositol and (+/-)-3-O-acetyl-1,2:4,5-di-O-isopropylidene-6-O-methyl-allo-inositol

The synthesis and conformational studies of (+/-)-3-O-acetyl-1,2:4,5-di-O-isopropylidene-allo-inositol and (+/-)-3-O-acetyl-1,2:4,5-di-O-isopropylidene-6-O-methyl-allo-inositol are described. Solid state conformations of the title compounds have been studied by solving their X-ray crystal structures. The inositol ring in both the compounds deviate considerably from the ideal chair conformation to flattened chair conformation in the solid state. Their conformations in solution were studied by the use of 1H NMR spectroscopy. These conformational analyses revealed that the title compounds adopt similar conformations in solid and solution states irrespective of the solvent polarity.     

Synthesis of galactosaminyl D-chiro-inositols

All six isomeric D-galactosaminopyranosyl-D-chiro-inositols have been prepared by glycosylation of appropriate penta-O-benzyl-D-chiro-inositols. The three requisite protected D-chiro-inositols were prepared by SmI2-promoted pinacol coupling of dialdehydes derived ultimately from L-arabinose.     

The Route of Leishmania tropica Infection Determines Disease Outcome and Protection against Leishmania major in BALB/c Mice

Leishmania tropica is one of the causative agents of leishmaniasis in humans. Routes of infection have been reported to be an important variable for some species of Leishmania parasites. The role of this variable is not clear for L. tropica infection. The aim of this study was to explore the effects of route of L. tropica infection on the disease outcome and immunologic parameters in BALB/c mice. Two routes were used; subcutaneous in the footpad and intradermal in the ear. Mice were challenged by Leishmani major, after establishment of the L. tropica infection, to evaluate the level of protective immunity. Immune responses were assayed at week 1 and week 4 after challenge. The subcutaneous route in the footpad in comparison to the intradermal route in the ear induced significantly more protective immunity against L. major challenge, including higher delayed-type hypersensitivity responses, more rapid lesion resolution, lower parasite loads, and lower levels of IL-10. Our data showed that the route of infection in BALB/c model of L. tropica infection is an important variable and should be considered in developing an appropriate experimental model for L. tropica infections.     

The first structural study on a cyclic tricoordinate phosphorochloridite and a pentacoordinate phosphorane based on 1,2,3,5-protected myo-inositol--a new conformation of 1,3,2-dioxaphosphorinane ring

Treatment of the phosphoramidite [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(O(2)P-NH-i-Pr)] with o-chloranil affords the first example of inositol-based pentacoordinate phosphorane [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(O(2)P-NH-i-Pr)(1,2-O(2)C(6)Cl(4))] (9) (X-ray structure) with a trigonal bipyramidal geometry at phosphorus. The six-membered 1,3,2-dioxaphosphorinane ring with the inositol residue has an unusual boat conformation in 9 which is quite different from that found in unrestrained rings investigated before, but is similar to that of its P(III) chloro precursor [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(O(2)PCl)] (X-ray structure). Also, a convenient and chromatography-free procedure for the protected myo-inositol derivative [myo-C(6)H(6)-2-[OC(O)Ph]-1,3,5-(O(3)CH)-4,6-(OH)(2)] is reported.     

Susceptibility of juvenile Crassostrea gigas and resistance of Panope abrupta to Mikrocytos mackini

Samples from the field and laboratory exposure to Mikrocytos mackini (a tiny protistan parasite of unknown taxonomic affiliation) confirmed that juvenile Pacific oysters (Crassostrea gigas) are susceptible to infection and the resulting disease. In the laboratory bath exposure experiment, a prevalence of infection approaching 100% and mortalities were observed in the small oysters (about 18 mm in shell length). However, in the same laboratory exposure experiment, similar aged geoduck clams (Panope abrupta, about 8mm in shell length) were resistant to infection. The main route of infection in the oysters appeared to be via the digestive tract and possibly the gills where the parasite multiplied within host cells. Other tissues such as the adductor muscle and vesicular connective tissue were subsequently colonized. Although the infection resulted in the mortality of some oysters, others appeared to overcome the disease.     

ITS 2 sequences heterogeneity in Phlebotomus sergenti and Phlebotomus similis (Diptera,Psychodidae): possible consequences in their ability to transmit Leishmania tropica

An intraspecific study on Phlebotomus sergenti, the main and only proven vector of Leishmania tropica among the members of the subgenus Paraphlebotomus was performed. The internal transcribed spacer 2 (ITS2) sequences of 12 populations from 10 countries (Cyprus, Egypt, Italy, Lebanon, Morocco, Pakistan, Portugal, Spain, Syria, and Turkey) were compared. Samples also included three species closely related to P. sergenti: Phlebotomus similis (three populations from Greece and Malta), Phlebotomus jacusieli and Phlebotomus kazeruni. Our results confirm the validity of the taxa morphologically characterised, and imply the revision of their distribution areas, which are explained through biogeographical events. At the Miocene time, a migration route, north of the Paratethys sea would have been followed by P. similis to colonise the north of the Caucasus, Crimea, Balkans including Greece and its islands, and western Turkey. Phlebotomus sergenti would have followed an Asiatic dispersion as well as a western migration route south of the Tethys sea to colonise North Africa and western Europe. This hypothesis seems to be well supported by high degree of variation observed in the present study, which is not related to colonisation or to intra-populational variation. Two groups can be individualised, one oriental and one western in connection with ecology, host preferences and distribution of L. tropica. We hypothesise that they could be correlated with differences in vectorial capacities.     

Bioassay evidence for the transmission of WSSV by the harpacticoid copepod Nitocra sp

White Spot Syndrome Virus (WSSV) is now one of the most devastating and virulent viral agents threatening the penaeid shrimp culture industry and has been responsible for serious economic losses for shrimp farms worldwide. One remarkable characteristic of WSSV is its wide reservoir range, which contributes to its wide geographical distribution. Among epizootiological surveys, there is substantial evidence for WSSV-positive copepods found in shrimp farming ponds. Therefore, copepods are suspected to be the vector of WSSV. In the present study, nested-PCR analysis showed positive results in the harpacticoid copepod Nitocra sp. exposed to WSSV by virus-phytoplankton adhesion route. Oral route and intramuscular injection were used to test the pathogenicity of WSSV isolated from the WSSV-positive Nitocra sp. For the oral route of infection, Marsupenaeus japonicus postlarvae were fed with WSSV-positive copepods. The shrimp postlarvae in the infected treatment became WSSV-positive and occurred 52.50+/-5.00% mortality which was significant higher (P <0.05) than that in the control treatment (20.00+/-0.00%) when postlarvae were fed with WSSV free copepods. In the intramuscular injection challenge, M. japonicus juveniles were injected with the copepods inoculum extracted from the WSSV-positive Nitocra sp., and showed 72.50+/-9.57% mortality which was also significant higher (P <0.05) than that in the control treatment (22.50+/-5.00%) when juveniles were received mock injection of a tissue homogenate prepared from WSSV-negative Nitocra sp. Based on these laboratory challenge studies, it was confirmed that the copepods can serve as a vector in WSSV transmission.     

Fatal experimental transplacental Babesia gibsoni infections in dogs

A Babesia gibsoni infected bitch was mated with an uninfected dog in order to determine whether this parasite could be vertically transmitted. The bitch delivered a litter of four live and one stillborn pup. The four pups died from congenital babesiosis between 14 and 39 days post-birth. Babesia gibsoni DNA was detected in tissue from all five pups. These results show that vertical transmission occurred by the uterine route and not via the transmammary route. This is the first confirmed report of transplacental Babesia infection in any animal species.     

Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells

To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [³H]d-chiro-inositol (DCI) by 159-fold. [³H]myo-Inositol uptake increased by 37-fold. In contrast, [¹⁴C]d-glucose, [¹⁴C]2-deoxy-d-glucose, or [¹⁴C]3-O-methyl-d-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The K_m of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 μM, respectively, whereas glucose competed for DCI uptake with a K_i of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 μM for 24 h) increased [³H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.     

Maculalactone M from the marine cyanobacterium Kyrtuthrix maculans

Maculalactone M, a seco-dibenzyldiphenyl-4,5,6,7-tetrahydrobenzofuranone, was isolated from the epilithic encrusting cyanobacterium Kyrtuthrix maculans. A possible biogenetic route to this compound is proposed involving cleavage of an allylic hydroperoxide.     

Lessons from Plectocephalus (Compositae, Cardueae-Centaureinae): ITS disorientation in annuals and Beringian dispersal as revealed by molecular analyses

Background and Aims

The geographic distribution of the genus Plectocephalus comprises a single species in Ethiopia, two in North America and possibly four more in South America, in a striking disjunction that is exceptional for genera of the tribe Cardueae. The enormity of this disjunction cast doubts on the precise taxonomic delineation of the genus, which is not unanimously recognized as a natural entity. The aims of this study were to define the generic boundaries of Plectocephalus and to formulate a hypothesis that would explain its natural range.

Methods

A combined molecular approach, using nuclear internal transcribed spacers (ITS) and external transcribed spacers (ETS), and plastid trnL-trnL-F, rpl32-trnL^UAG and ndhF markers, was chosen for phylogenetic reconstruction by maximum parsimony and Bayesian inference.

Key Results

Phylogenetic analysis shows that Plectocephalus is a natural genus that includes the African species P. varians, together with all the native South American species, currently classified as Centaurea, C. cachinalensis, C. floccosa and C. tweediei. The recognition of Centaurodendron as an independent genus, which we consider appropriate, would make Plectocephalus paraphyletic. Affinities of Plectocephalus should lie with eastern representatives of Centaureinae. Geographic disjunction is explained as a consequence of dispersal via the Bering Land Bridge during the Miocene–Pliocene. The phylogeny of the basal grade of Centaureinae differs from previous phylogenies, and artefacts resulting from differences in mutation rates of annual and perennial taxa are confirmed. Sensitivity of ITS to these differences was the highest observed for all DNA regions used in this study.

Conclusions

The natural status of the genus Plectocephalus is confirmed and several nomenclatural combinations are proposed. New evidence contributes to the debate concerning problems posed by the use of ITS in the phylogenetic reconstruction of groups that differ in terms of their life cycles. Dispersal from Caucasus and Anatolia along the Siberian route and then across the Bering Land Bridge follows a route previously proposed for other taxonomic groups.     

Synthesis of the 'primer-adaptor' trisaccharide moiety of Escherichia coli O8, O9, and O9a lipopolysaccharide

Described is the synthesis of the trisaccharide alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->3)-beta-D-GlcpNAcO(CH2)8N3, the glycan portion of which corresponds to the 'adaptor-primer' moiety linking the O-chain and core oligosaccharide in the lipopolysaccharide of several Escherichia coli and Klebsiella pneumoniae serotypes. This report represents the first synthesis of this trisaccharide motif, and in the route involved, a key step is a [2+1] coupling of a protected Manp-(1-->3)-alpha-D-Manp glycosyl donor with a GlcpNAc acceptor. The azido group was included in the target to facilitate future preparation of neoglycoconjugates.     

Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier

Using an in vitro model of the human blood-brain barrier consisting of human brain microvascular endothelial cells we recently demonstrated that Trypanosoma brucei gambiense bloodstream-forms efficiently cross these cells via a paracellular route while Trypanosoma brucei brucei crosses these cells poorly. Using a combination of techniques that include fluorescence activated cell sorting, confocal and electron microscopy, we now show that some T.b. gambiense blood stream form parasites have the capacity to enter human brain microvascular endothelial cells. The intracellular location of the trypanosomes was demonstrated in relation to the endothelial cell plasma membrane and to the actin cytoskeleton. These parasites may be a terminal stage within a lysosomal compartment or they may be viable trypanosomes that will be able to exit the brain microvascular endothelial cells. This process may provide an additional transcellular route by which the parasites cross the blood-brain barrier.     

Synthesis of a C-galactopyranosyl-linked N-substituted 1,2-ethylenediamine

A straightforward route to a C-galactopyranosyl-linked 1,2-ethylenediamine is described. The five-step synthetic procedure involves: (i) C-allenylation of d-galactopyranose pentaacetate with propargyl trimethylsilane in the presence of a Lewis acid, (ii) iodination of allenyl galactopyranosyl tetraacetate to diiodoallyl galactopyranosyl tetraacetate, (iii) displacement of the allylic iodide with N-Boc-ethylenediamine, (iv) catalytic hydrogenation of vinyl iodide to alkane, (v) deprotection of the acetyl and N-Boc-groups using acid-catalyzed hydrolysis. This method demonstrates a general method to access a new class of carbohydrate-ethylenediamine C-glycosyl chelators.