Unique gangliosides synthesized in vitro by sialyltransferases from marine bacteria and their characterization: ganglioside synthesis by bacterial sialyltransferases

On the basis of the results outlined in our previous report, bacterial sialyltransferases (ST) from marine sources were further characterized using glycosphingolipids (GSL), especially ganglio-series GSLs, based on the enzymatic characteristics and kinetic parameters obtained by Line weaver-Burk plots. Among them, GA1 and GA2 were found to be good substrates for these unique STs. Thus, new gangliosides synthesized by α2-3 and α2-6STs were structurally characterized by several analytical procedures. The ganglioside generated by the catalytic activity of α2-3ST was identified as GM1b. On the other hand, when enzyme reactions by α2-6STs were performed using substrates GA2 and GA1, very unique gangliosides were generated. The structures were identified as NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer and NeuAcα2-6Galβ1-3GalNAcβ1-4Galβ1-4Glcβ-Cer, respectively. The synthesized ganglioside NeuAcα2-6GalNAcβ1-4Galβ1-4Glcβ-Cer showed binding activity to the influenza A virus {A/Panama/2007/99 (H3N2)} at a similar level to purified sialyl(α2-3)paragloboside (S2-3PG) and sialyl(α2-6)paragloboside (S2-6PG) from mammalian sources. The evidence suggests that these STs have unique features, including substrate specificities restricted not only to lacto-series but also to ganglio-series GSLs, as well as catalytic potentials for ganglioside synthesis. This evidence demonstrates that effective in vitro ganglioside synthesis could be a valuable tool for selectively synthesizing sialic acid (Sia) modifications, thereby preparing large-scale gangliosides and permitting the exploration of unknown functions.     

Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.     

α6 Integrin Is Regulated with Lens Cell Differentiation by Linkage to the Cytoskeleton and Isoform Switching

The developing chicken embryo lens provides a unique model for examining the relationship between α6 integrin expression and cell differentiation, since multiple stages of differentiation are expressed concurrently at one stage of development. We demonstrate that α6 integrin is likely to mediate the inductive effects of laminin on lens differentiation as well as to function in a matrix-independent manner along the cell–cell interfaces of the differentiating cortical lens fiber cells. Both α6 isoform expression and its linkage to the cytoskeleton were regulated in a differentiation-specific manner. The association of α6 integrin with the Triton-insoluble cytoskeleton increased as the lens cells differentiated, reaching its highest levels in the cortical fiber region where the lens fiber cells are formed. In this region of the lens α6 integrin was uniquely localized along the cell–cell borders of the differentiating fiber cells, similar to β1. α6β4, the primary transmembrane protein of hemidesmosomes, is also expressed in the lens, but in the absence of hemidesmosomes. Differential expression of α6A and α6B isoforms with lens cell differentiation was seen at both the mRNA and the protein levels. RT-PCR studies demonstrated that α6B was the predominant isoform expressed both early in development, embryonic day 4, and in the epithelial regions of the day 10 embryonic lens. Isoform switching, with α6A now the predominant isoform, occurred in the fiber cell zones. Immunoprecipitation studies showed that α6B, which is characteristic of undifferentiated cells, was expressed by the lens epithelial cells but was dramatically reduced in the lens fiber zones. Expression of α6B began to drop as the cells initiated their differentiation and then dropped precipitously in the cortical fiber zone. In contrast, expression of the α6A isoform remained high until the cells became terminally differentiated. α6A was the predominant isoform expressed in the cortical fiber region. The down-regulation of α6B relative to α6A provides a developmental switch in the process of lens fiber cell differentiation.     

The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation

Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols (trans- and cis-resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans- and cis-resveratrol 3-O-glucuronidation. Although the K(m) value for trans-resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the K(m) values for cis-resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of K(m) in trans-resveratrol, and V(max) and K(si) in cis-form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation.     

Age-related changes in GM1, GD1a, GT1b components of gangliosides in Wistar albino rats

In this study, age-related changes of GM1, GD1a, GT1b fractions of gangliosides were investigated in whole brain of male Wistar albino rats. Insignificant increases were detected in GM1 values from the third to the 24th month, whereas GD1a and GT1b concentrations of ganglioside in 24-month-old rats decreased significantly as compared to 6-month-old rats. Although there were no significant differences in the GD1a/GT1b ratio of any groups, GM1/GD1a and GM1/GT1b ratios were significantly increased as compared to 6-month-old rats. The increase in the ratios of gangliosides are not due to an increase of GM1 fractions; they result from a decrease of GD1a and GT1b fractions of gangliosides. In conclusion, the concentration of ganglioside decreased with ageing.     

Altered metabolism of bile acids in cholestasis: Determination of 1β- and 6α-hydroxylated metabolites

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography—mass spectrometry. Five 1β-hydroxylated bile acids, viz. 1β,3α,12α-trihydroxy-, 1β,3α,7β-trihydroxy-1, 1β,3α,7α,12α-tetrahydroxy-5β-cholanoic acids and an epimer of the first compound, and two 6α-hydroxylated bile acids, viz. 3α,6α,7α-trihydroxy-, 3α,6α,7α,12α-tetrahydroxy-5β-cholanoic acids, were completely or partially identified. Large amounts of 1β-hydroxylated and 6α-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1β- or 6α-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.     

The Integrin α6β4 Functions in Carcinoma Cell Migration on Laminin-1 by Mediating the Formation and Stabilization of Actin-containing Motility Structures

Functional studies on the α6β4 integrin have focused primarily on its role in the organization of hemidesmosomes, stable adhesive structures that associate with the intermediate filament cytoskeleton. In this study, we examined the function of the α6β4 integrin in clone A cells, a colon carcinoma cell line that expresses α6β4 but no α6β1 integrin and exhibits dynamic adhesion and motility on laminin-1. Time-lapse videomicroscopy of clone A cells on laminin-1 revealed that their migration is characterized by filopodial extension and stabilization followed by lamellae that extend in the direction of stabilized filopodia. A function-blocking mAb specific for the α6β4 integrin inhibited clone A migration on laminin-1. This mAb also inhibited filopodial formation and stabilization and lamella formation. Indirect immunofluorescence microscopy revealed that the α6β4 integrin is localized as discrete clusters in filopodia, lamellae, and retraction fibers. Although β1 integrins were also localized in the same structures, a spatial separation of these two integrin populations was evident. In filopodia and lamellae, a striking colocalization of the α6β4 integrin and F-actin was seen. An association between α6β4 and F-actin is supported by the fact that α6β4 integrin and actin were released from clone A cells by treatment with the F-actin– severing protein gelsolin and that α6β4 immunostaining at the marginal edges of clone A cells on laminin-1 was resistant to solubilization with Triton X-100. Cytokeratins were not observed in filopodia and lamellipodia. Moreover, α6β4 was extracted from these marginal edges with a Tween-40/deoxycholate buffer that solubilizes the actin cytoskeleton but not cytokeratins. Three other carcinoma cell lines (MIP-101, CCL-228, and MDA-MB-231) exhibited α6β4 colocalized with actin in filopodia and lamellae. Formation of lamellae in these cells was inhibited with an α6-specific antibody. Together, these results indicate that the α6β4 integrin functions in carcinoma migration on laminin-1 through its ability to promote the formation and stabilization of actin-containing motility structures.     

Synthesis and some reactions of 6-bromoandrogens: potential affinity ligand and inactivator of estrogen synthetase.

The synthesis of epimeric 6-bromo-4-androstene-3,17-dione (1a and 1b), 6-bromotestosterone (2a and 2b) and its acetate (3a and 3b), and 6-bromo-16 alpha-acetoxy-4-androstene-3,17-dione (5a and 5b), and 6 beta-bromo-16 alpha-hydroxy-4-androstene-3,17-dione (4) is described. The interconversions among compounds 1, 2, and 3 are also studied. The 6 beta-isomer (1b, 2b, and 3b) was epimerized to the 6 alpha-isomer (1a, 2a and 3a) in carbon tetrachloride or chloroform-methanol (9:1) and the 6 alpha-isomer was isolated by fractional crystallization from the epimeric mixture. 6 alpha-Bromo isomer 1a was also epimerized back to 6 beta-bromo isomer 1b in chloroform-methanol (9:1). Two polymorphic forms of 6 beta-bromotestosterone acetate (3b) were isolated (mp. 114--117 degrees and 138--141 degrees). The 6 beta-bromo isomers were found to be unstable in methanol and decomposed to give 5 alpha-androstane-3,6-dione derivative (6). The results of irreversible inactivation of human placental androgen aromatase with some of these 6-bromoandrogens are discussed.     

Positional Scanning Mutagenesis of α-Conotoxin PeIA Identifies Critical Residues That Confer Potency and Selectivity for α6/α3β2β3 and α3β2 Nicotinic Acetylcholine Receptors

The nicotinic acetylcholine receptor (nAChR) subtype α6β2* (the asterisk denotes the possible presence of additional subunits) has been identified as an important molecular target for the pharmacotherapy of Parkinson disease and nicotine dependence. The α6 subunit is closely related to the α3 subunit, and this presents a problem in designing ligands that discriminate between α6β2* and α3β2* nAChRs. We used positional scanning mutagenesis of α-conotoxin PeIA, which targets both α6β2* and α3β2*, in combination with mutagenesis of the α6 and α3 subunits, to gain molecular insights into the interaction of PeIA with heterologously expressed α6/α3β2β3 and α3β2 receptors. Mutagenesis of PeIA revealed that Asn¹¹ was located in an important position that interacts with the α6 and α3 subunits. Substitution of Asn¹¹ with a positively charged amino acid essentially abolished the activity of PeIA for α3β2 but not for α6/α3β2β3 receptors. These results were used to synthesize a PeIA analog that was >15,000-fold more potent on α6/α3β2β3 than α3β2 receptors. Analogs with an N11R substitution were then used to show a critical interaction between the 11th position of PeIA and Glu¹⁵² of the α6 subunit and Lys¹⁵² of the α3 subunit. The results of these studies provide molecular insights into designing ligands that selectively target α6β2* nAChRs.     

A practical route to partially protected pyrrolidines as precursors for the stereoselective synthesis of alexines

Either 3-O-benzoyl- (2a) or 3-O-benzyl-1,2-O-isopropylidene-beta-D-fructopyranose (2b) were regioselectively O-benzylated at C-4 to give 4a and 4b, respectively, which were transformed into 5-azido-3-O-benzoyl-4-O-benzyl- (6a) and 5-azido-3,4-di-O-benzyl-5-deoxy-1,2-O-isopropylidene-alpha-L-sorbopyranose (6b) by nucleophilic displacement of the corresponding 5-O-mesyl derivatives 5a and 5b by sodium azide in DMF, respectively. Compound 6b was also prepared from 4b in one step by the Mitsunobu methodology. Deacetonation of 6a and 6b gave the partially protected free azidouloses 8a and 8b, respectively, that were protected as their 1-O-TBDPS derivatives 9a and 9b. Hydrogenation of 9b over Raney nickel gave stereoselectively (2R,3R,4R,5S)-3,4-dibenzyloxy-2'-O-tert-butyldiphenylsilyl-2,5-bis(hydroxymethyl)pyrrolidine (12) which was identified by transformation into the well known (2R,3R,4R,5S)-3,4-dihydroxy-2,5-bis(hydroxymethyl)pyrrolidine (1, DGDP).     

Simultaneous determination of 6β- and 6α-hydroxycortisols and 6β-hydroxycortisone in human urine by stable isotope dilution mass spectrometry

A capillary gas chromatographic–mass spectrometric method for the simultaneous determination of 6β-hydroxycortisol (6β-OHF, 6β,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione), 6α-hydroxycortisol (6α-OHF, 6α,11β,17α,21-tetrahydroxypregn-4-ene-3,20-dione) and 6β-hydroxycortisone (6β-OHE, 6β,17α,21-trihydroxypregn-4-ene-3,11,20-trione) in human urine is described. Deuterium-labelled compounds, 6β-[1,1,19,19,19-²H₅]OHF (6β-OHF-d₅), 6α-[1,1,19,19,19-²H₅]OHF (6α-OHF-d₅) and 6β-[1,1,19,19,19-²H₅]OHE (6β-OHE-d₅) were used as internal standards. Quantitation was carried out by selected-ion monitoring of the characteristic fragment ions ([M-31]⁺) of the methoxime–trimethylsilyl (MO–TMS) derivatives of 6β-OHF, 6α-OHF and 6β-OHE. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring 6β-OHF, 6α-OHF and 6β-OHE in human urine.     

Formation of Hyodeoxycholic Acid from Muricholic Acid and Hyocholic Acid by an Unidentified Gram-Positive Rod Termed HDCA-1 Isolated from Rat Intestinal Microflora

From the rat intestinal microflora we isolated a gram-positive rod, termed HDCA-1, that is a member of a not previously described genomic species and that is able to transform the 3α,6β,7β-trihydroxy bile acid β-muricholic acid into hyodeoxycholic acid (3α,6α-dihydroxy acid) by dehydroxylation of the 7β-hydroxy group and epimerization of the 6β-hydroxy group into a 6α-hydroxy group. Other bile acids that were also transformed into hyodeoxycholic acid were hyocholic acid (3α,6α,7α-trihydroxy acid), α-muricholic acid (3α,6β,7α-trihydroxy acid), and ω-muricholic acid (3α,6α,7β-trihydroxy acid). The strain HDCA-1 could not be grown unless a nonconjugated 7-hydroxylated bile acid and an unidentified growth factor produced by a Ruminococcus productus strain that was also isolated from the intestinal microflora were added to the culture medium. Germfree rats selectively associated with the strain HDCA-1 plus a bile acid-deconjugating strain and the growth factor-producing R. productus strain converted β-muricholic acid almost completely into hyodeoxycholic acid.     

Acetylcholinesterase/butyrylcholinesterase inhibition activity of some new carbacylamidophosphate derivatives

Eight newly synthesized carbacylamidophosphates with the general formula RC(O)NHP(O)Cl2 with R = pCl-C6H4 1a, pBr-C6H4 2a, C6H5 3a, and pMe-C6H4 4a and RC(O)NHP(O)(NC4H8O)2 R = pCl-C6H4 1b, pBr-C6H4 2b, C6H5 3b, pMe-C6H4 4b, were selected to compare the inhibition kinetic parameters, IC50, Ki, kp and KD, on human erythrocyte acetylcholinesterase (hAChE) and bovine serum butyrylcholinesterase (BuChE), Also, the in vivo inhibition potency of compound 2a, 2b and 3a, were studied. The data demonstrates that compound 2a and compound 2b are the potent sensitive as AChE and BuChE inhibitors respectively, and the inhibition of hAChE is about 10-fold greater than that of BuChE.     

Parallel DNA containing pyrazolo[3,4-D]pyrimidine analogues of isoguanine

The phosphoramidites of 8-aza-7-deaza-2'-deoxyisoguanosine (1a) and its bromo derivative 1b as well as of 6-aza-2'-deoxyisocytidine and its 5-methyl derivative (3a,b) were synthesized. Parallel-stranded duplexes containing the nucleosides 1a,b show a significantly enhanced duplex stability compared to those containing 2'-deoxyisoguanosine.     

Synthesis and β-lactamase inhibitory activity of 6-fluoropenicillanic acids

The benzyl 6-fluoro-penicillanate sulfides 4a, 6a, 7a; and sulfones 6c, 7d were synthesized. The conversion to their free acids 4b, 4b, 6d, 7b, 7e and potassium salts 7c, 7f are described. These acids and salt 7c were evaluated as β- lactamase inhibitors using β-lactamase I from Bacillus cereus. The data indicate that substitution of the 6-hydrogen by a 6- fluorine atom on 6β-bromopenicillanic acid (1), leads to loss of β-lactamase inhibitory activity. In the case of the isomers 6β- and 6-fluoropenicillanic acids the 6β-enantiomer proved to be considerably more potent. Potassium salts of 6β- fluoropenicillanate sulfide and sulfone were unstable in solid state and in water solution. The fragmentation of the sulfone in two parts in water solution is consistent with the hydrolytic behavior of the penicillanic acid sulfone (2) with 0.5 N NaOH.     

Zonal heterogeneity of prostaglandin and thromboxane release in the dog kidney

The release of prostaglandin(PG) and thromboxane(TX) was examined in the six different areas of the normal dog kidney, i.e., renal arterial and venous strips(RA and RV), superficial and deep cortical slices (SC and DC) and outer and inner medullary slices(Om and IM). These tissues were incubated in Krebs-bicarbonate buffer(pH 7.4, 37°C), and the released PGE2, PGF2α, 6-keto-PGF1α and TXB2(as stable metabolites of PG12 and TXA2, respectively) were determined by radioimmunoassay. In RA, RV, SC and DC, 6-keto-PGF1α was predominant, however, there were no quantitative differences between RA and RV, or SC and DC. The release of 6-keto-PGF1α reached a maximum in IM, similar to findings on the release of PGE2 and PGF2α. The release of TXB was uniform in OM and IM. The amount of PGE2, PGF2α, 6-keto-PGF1α and TXB2 released from IM was 2800, 400, 60 and 50 times higher, respectively, than the extent of the release from the cortical slices.These results suggest that PG12 as well as PGE2 and PGF2α, may be involved in renal PG, and that TXA2 is biosynthesized in the normal dog kidney.     

Reactivation of Herpes Simplex Virus Type 1 in the Mouse Trigeminal Ganglion: an In Vivo Study of Virus Antigen and Cytokines

Reactivation of herpes simplex virus type 1 (HSV-1) in the trigeminal ganglion (TG) was induced by UV irradiation of the corneas of latently infected mice. Immunocytochemistry was used to monitor the dynamics of cytokine (interleukin-2 [IL-2], IL-4, IL-6, IL-10, gamma interferon [IFN-γ], and tumor necrosis factor alpha [TNF-α]) and viral antigen production in the TG and the adjacent central nervous system on days 1 to 4, 6, 7, and 10 after irradiation. UV irradiation induced increased expression of IL-6 and TNF-α from satellite cells in uninfected TG. In latently infected TG, prior to reactivation, all satellite cells were TNF-α⁺ and most were also IL-6⁺. Reactivation, evidenced by HSV-1 antigens and/or infiltrating immune cells, occurred in 28 of 45 (62%) TG samples. Viral antigens were present in the TG in neurons, often disintegrating on days 2 to 6 after irradiation. Infected neurons were usually surrounded by satellite cells and the foci of immune cells producing TNF-α and/or IL-6. IL-4⁺ cells were detected as early as day 3 and were more numerous by day 10 (a very few IL-2⁺ and/or IFN-γ⁺ cells were seen at this time). No IL-10 was detected at any time. Our observations indicate that UV irradiation of the cornea may modulate cytokine production by satellite cells. We confirm that neurons are the site of reactivation and that they probably do not survive this event. The predominance of TNF-α and IL-6 following reactivation parallels primary infection in the TG and suggests a role in viral clearance. The presence of Th2-type cytokines (IL-4 and IL-6) indicates a role for antibody. Thus, several clearance mechanisms may be at work.     

Cytokine interactions in mesenchymal stem cells from cord blood

We used cytokine protein array to analyze the expression of cytokines from human cord blood-derived mesenchymal stem cells (CB-MSCs). Several cytokines, interleukins (IL), and growth factors, including ENA-78, GM-CSF, GRO, IL-1β, IL-6, IL-8, MCP-1, OSM, VEGF, FGF-4, FGF-7, FGF-9, GCP-2, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IP-10, LIF, MIF, MIP-3α, osteoprotegerin, PARC, PIGF, TGF-β2, TGF-β3, TIMP-1, as well as TIMP-2, were secreted by CB-MSCs, while IL-4, IL-5, IL-7, IL-13, TGF-β1, TNF-α, and TNF-β were not expressed under normal growth conditions. IL-6, IL-8, TIMP-1, and TIMP-2 were the most abundant interleukins expressed by CB-MSCs. A set of growth factors were selected to evaluate their stimulatory effects on the IL6 secretion for CB-MSCs. IL-1β was the most important factor inducing CB-MSC to secret IL-6. The mechanism by which IL-1β promoted IL-6 expression in CB-MSCs was studied. By using various inhibitors of signal transduction, we found that activation of p38 mitogen-activated protein kinases (MAPK) and MAPK kinase (MEK) is essential in the IL-1β stimulated signaling cascade which leads to the increase in IL-6 synthesis. Additionally, continuous supplement of IL-1β in the CB-MSCs culture will facilitate adipogenic maturation of CB-MSCs as evidenced by the presence of oil drops in the CB-MSCs and secretion of leptin, a molecule marker of adipocytes. These results strongly suggest that cytokine induction and signal transduction are important for the differentiation of CB-MSCs.