beta Gal(1-->S-4)beta GlcNac--OR: a galactosidase-stable substrate for alpha(1-->3)fucosyltransferase

1'-4 Thio-N-acetyllactosamine was chemically synthesized as a galactosidase-stable substrate for alpha(1-->3)fucosyltransferase. The product of enzymatic fucose addition was confirmed to be the thio-Le X analog.     

Mitogenic activity of particulate yeast beta-(1-->3)-D-glucan and its water-soluble derivatives

Particulate beta-D-glucan was isolated from baker's yeast using autolysis and delipidization of the cells, followed by alkaline and acid treatment. The residual water-insoluble glucan termed cerevan has a beta-(1-->3)-linked backbone with beta-(1-->6)-linked short side chains. In order to achieve water solubility of the glucan, various derivatives were prepared (carboxymethyl-,carboxyethyl-,hydroxyethyl-,sulfoethyl-), and the beta-glucan was oxidized to glucuronoglucan. Their solubility, degree of substitution (DS), and molecular weight distribution (Mw) were compared. The immunomodulatory activity of these preparations was investigated in mitogenic and co-mitogenic tests on rat thymocytes. Cerevan showed higher stimulation indices compared with the known immunomodulator zymosan. Of the water-soluble derivatives, sulfoethylglucan was found to be the most active. Of the carboxymethyl derivatives of various DS, the preparation with DS = 0.75 exhibited the highest activity. Water-soluble carboxymethyl preparations with DS > 1.0 and low-molecular-weight glucuronoglucan were inactive.     

UDP-GlcNAc:Galbeta1-->3GalNAc (GlcNAc to GalNAc) beta1-->6N-acetylglucosaminyltransferase holds a key role on the control of CD15s expression in human pre-B lymphoid cell lines

Distraction osteogenesis has become an important technique to treat craniofacial skeletal dysplasia. In this study, the technique of maxillary distraction with a rigid external distraction device is presented. Cephalometric results in the first 14 consecutive patients are analyzed. The study sample consisted of 14 patients with various cleft types and maxillary hypoplasia treated with the rigid external distraction technique. Analysis of the predistraction and postdistraction cephalometric radiographs revealed significant skeletal maxillary advancement. All patients had correction of the maxillary hypoplasia with positive skeletal convexity and dental overjet after maxillary distraction. The morbidity for the procedure was minimal. Surgical and orthodontic procedures are thoroughly described.     

Measurement of beta(1-->3)-glucans in occupational and home environments with an inhibition enzyme immunoassay

beta (1-->3)-Glucans are known for their potent ability to induce nonspecific inflammatory reactions and are believed to play a role in bioaerosol-induced respiratory symptoms. An inhibition enzyme immunoassay (EIA) was developed for the quantitation of beta (1-->3)-glucans in dust samples from occupational and residential environments. Immunospecific rabbit antibodies were produced by immunization with bovine serum albumin-conjugated laminarin [beta (1-->3)-glucan] and affinity chromatography on epoxy-Sepharose-coupled beta (1-->3)-glucans. The laminarin-based calibration curve in the inhibition EIA ranged from approximately 40 to 3,000 ng/ml (15 to 85% inhibition). Another beta (1-->3)-glucan (curdlan) showed a similar inhibition curve but was three to five times less reactive on a weight basis. Pustulan, presumed to be a beta (1-->6)-glucan, showed a parallel dose-response curve at concentrations 10 times higher than that of laminarin. Control experiments with NaIO4 and beta (1-->3)-glucanase treatment to destroy beta (1-->6)- and beta (1-->3)-glucan structures, respectively, indicate that the immunoreactivity of pustulan in the assay was due to beta (1-->3)-glucan and not to beta (1-->6)-glucan structures. Other polysaccharides, such as mannan and alpha (1-->6)-glucan, did not react in the inhibition EIA. Beta (1-->3)-Glucan extraction of dust samples in water (with mild detergent) was performed by heat treatment (120 degrees C) because aqueous extracts obtained at room temperature did not contain detectable beta (1-->3)-glucan levels. The assay was shown to detect heat-extractable beta (1-->3)-glucan in dust samples collected in a variety of occupational and environmental settings. On the basis of duplicate analyses of dust samples, a coefficient of variation of approximately 25% was calculated. It was concluded that the new inhibition EIA offers a useful method for indoor beta (1-->3)-glucan exposure assessment.     

Conformational properties of a cycle oligosaccharide: cyclotrikis-(1-->6)-[alphaD-glucopyranosyl-(1-->4)-beta-D-glucopyranosy l]

Within a larger environmental health screening program neurobehavioral measures were taken in 384 6-year-old children (mean age 74 months) in the cities of Leipzig, Gardelegen, and Duisburg. Lead concentrations in venous blood samples (PbB) and urinary mercury excretion in 24-h samples (HgU) were measured as markers of environmental exposure by electrothermal AAS. Dependent variables included two subtests from the WISC [vocabulary (V) and block design (BD)] as well as five tests from the NES2 [pattern comparison, pattern memory, tapping, simple reaction time, and the continuous performance test (CPT; child version)]. In addition, visual functions [visual acuity (TITMUS-test) and contrast sensitivity (FACT)] were tested as covariates. The overall average PbB (geometric mean) was 42.5 microg/l (upper 95% value = 89 microg/l). The overall average mercury excretion (HgU) was 0.16 microg/24 h. Whereas no significant or borderline associations between HgU and any of the target variables was found, significant negative associations were observed between PbB and verbal intelligence (WISC vocabulary but not WISC Block Design) and false-positive responses (false alarms), as well as false-negative responses (miss) in the CPT. Whereas parental education was the most important confounder for WISC performance, visual contrast sensitivity and computer familiarity also proved predictive for performance in several computer-based NES subtests. It is concluded that non-IQ measures, namely measures of sustained attention, are negatively affected in children with 95% of blood-lead levels below 90 microg/l, even after adjustment for intelligence and contrast sensitivity, whereas the causative role of lead in altering IQ functions remains somewhat equivocal, because important covariates could not be controlled for.     

Identification of an alpha3-fucosyltransferase and a novel alpha2-fucosyltransferase activity in cercariae of the schistosome Trichobilharzia ocellata: biosynthesis of the Fucalpha1-->2Fucalpha1-->3[Gal(NAc)beta1-->4]GlcNAc sequence

Knowledge of the genealogical relationships of cells during development can allow one to gain insight into when and where developmental decisions are being made. Genealogical relationships can be revealed by a variety of methods, all of which involve marking a progenitor cell and/or a group of cells and then following the progeny. The use of replication-incompetent retroviral vectors for the analysis of lineal relationships in developing vertebrate tissues is described. An overview of the relevant aspects of the retroviral life cycle is given, and the strategies and current methods in use in our laboratory are described.     

One-pot enzymatic synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc sequence with in situ UDP-Gal regeneration

The trisaccharide Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was enzymatically synthesized, with in situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4'-epimerase, UDP-Gal:GlcNAc beta 4-galactosyltransferase and UDP-Gal:Gal beta 1-->4GlcNAc alpha 3-galactosyltransferase, Gal alpha 1-->3Gal beta 1-->4GlcNAc beta 1-->O-(CH2)8COOCH3 was formed in a 2.2 mumol ml-1 yield starting from the acceptor GlcNAc beta 1-->O-(CH2)8COOCH3. This is an efficient and convenient method for the synthesis of the Gal alpha 1-->3Gal beta 1-->4GlcNAc epitope which pays an important role in various biological and immunological processes.     

Structure-activity relationships of saponins and cardiac glycosides. III. Beta-L-xylopyranosyl-(1-->6)-alpha- and -beta-D-glucopyranosides

Comparisons of the biological activities of diosgenyl, methyl glycyrrhetinate or digitoxigenyl 3-O-beta-L-xylopyranosyl-(1-->6)-beta-D-glucopyranoside with those of other previously tested glycosides confirmed our assumption that both the hemolytic and antifungal activities of steroid saponins are generally parallel to each other, while almost all hemolytic triterpenoid saponins and nonhemolytic ones have no antifungal activity, and that cardiac diglycosides having a (1-->4) sugar linkage have stronger activities than those with a (1-->6) or a (1-->2) linkage. On the other hand, the case of the diosgenyl 3-O-beta-L-xylopyranosyl-(1-->6)-alpha-D-glucopyranoside didn't conform to the above assumption, but those of methyl glycyrrhetinate and digitoxigenyl did.     

Synthesis of Hexp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O) (CH2)7 CH3 probes for exploration of the substrate specificity of glycosyltransferases: Part II, Hex = 3-O-methyl-beta-D-Gal, 3-deoxy-beta-D-Gal, 3-deoxy-3-fluoro-beta-D-Gal, 3-amino-3-deoxy-beta-D-Gal, beta-D-Gul, alpha-L-Alt, or beta-L-Gal

Seven analogues of the trisaccharide beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3 have been synthesized as potential substrates for glycosyltransferases involved in the chain-termination of N-acetyllactosamine-type N-glycans. These compounds include: 3-O-methyl-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp -(1-->O) (CH2)7CH3, 3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1 -->O) (CH2)7CH3, 3-deoxy-3-fluoro-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-M anp- (1-->O)(CH2)7Ch3, 3-amino-3-deoxy-beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Ma np- (1-->O)(CH2)7CH3, beta-D-Gulp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-- >O)(CH2)7CH3, beta-L-Galp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1-->O)(CH 2)7CH3, and alpha-L-Altp-(1-->4)-beta-D-GlcpNAc-(1-->2)-alpha-D-Manp-(1- ->O) (CH2)7CH3. All trisaccharides were obtained by condensation of suitably modified glycosyl donors based on imidates or thioglycosides with the same disaccharide acceptor, octyl 3,4,6-tri-O-benzyl-2-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D- glucopyranosyl)-alpha-D-mannopyranoside, followed by deprotection.     

MP1 encodes an abundant and highly antigenic cell wall mannoprotein in the pathogenic fungus Penicillium marneffei

We characterized the inhibitory activity of several acetylenic and olefinic compounds on cytochrome P450 (CYP)-derived arachidonic acid omega-hydroxylation and epoxidation using rat renal cortical microsomes and recombinant CYP proteins. Among the acetylenic compounds, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) and N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide were found to be potent and selective inhibitors of microsomal epoxidation with IC50 values of 9 and 13 microM, respectively. On the other hand, 17-octadecynoic acid inhibited both omega-hydroxylation and epoxidation of arachidonic acid with IC50 values of 7 and 5 microM, respectively. The olefinic compounds N-methylsulfonyl-12, 12-dibromododec-11-enamide (DDMS) and 12, 12-dibromododec-11-enoic acid (DBDD) exhibited a high degree of selectivity inhibiting microsomal omega-hydroxylation with an IC50 value of 2 microM, whereas the IC50 values for epoxidation were 60 and 51 microM for DDMS and DBDD, respectively. Studies using recombinant rat CYP4A isoforms showed that PPOH caused a concentration-dependent inhibition of omega-hydroxylation and 11, 12-epoxidation by CYP4A3 or CYP4A2 but had no effect on CYP4A1-catalyzed omega-hydroxylase activity. On the other hand, DDMS inhibited both CYP4A1- and CYP4A3- or CYP4A2-catalyzed arachidonic acid oxidations. Inhibition of microsomal activity by PPOH, but not DDMS, was time- and NADPH-dependent, a result characteristic of a mechanism-based irreversible inhibitor. These studies provide information useful for evaluating the role of the CYP-derived arachidonic acid metabolites in the regulation of renal function and blood pressure.     

Crystal structure of deoxy-human hemoglobin beta6 Glu --> Trp. Implications for the structure and formation of the sickle cell fiber

An atomic-level understanding of the interactions between hemoglobin molecules that contribute to the formation of pathological fibers in sickle cell disease remains elusive. By exploring crystal structures of mutant hemoglobins with altered polymerization properties, insight can be gained into sickle cell hemoglobin (HbS) polymerization. We present here the 2.0-A resolution deoxy crystal structure of human hemoglobin mutated to tryptophan at the beta6 position, the site of the glutamate --> valine mutation in HbS. Unlike leucine and isoleucine, which promote polymerization relative to HbS, tryptophan inhibits polymerization. Our results provide explanations for the altered polymerization properties and reveal a fundamentally different double strand that may provide a model for interactions within a fiber and/or interactions leading to heterogeneous nucleation.     

Synthesis of the spacer-containing beta-D-GalpNAc-(1-->4)-beta-D- GlcpNAc-(1-->3)-alpha-D-Galp moiety, representing the non-fucosylated backbone trisaccharide of the glycocalyx glycan of the parasite Schistosoma mansoni

We have conjugated the murine monoclonal anti-CD19 antibody B43 to the tyrosine kinase inhibitor genistein to construct an effective immunoconjugate against CD19 antigen positive hematologic malignancies. The scaled-up production and purification of B43 antibody, genistein, and B43-Genistein immunoconjugate permitted the manufacturing of a highly purified clinical-grade B43-Genistein preparation. In clonogenic assays, B43-Genistein elicited selective and potent cytotoxicity against CD19 antigen positive human leukemia cells. To our knowledge, this work represents the first effort of producing a clinical-grade genistein immunoconjugate for treatment of B-lineage leukemia and lymphoma.     

The Candida albicans KRE9 gene is required for cell wall beta-1, 6-glucan synthesis and is essential for growth on glucose

We have isolated CaKRE9, a gene from Candida albicans, that is a functional homologue of the Saccharomyces cerevisiae KRE9 gene involved in beta-1,6-glucan synthesis. Disruption of the CaKRE9 gene in C. albicans shows that CaKre9p is required for the synthesis or assembly of this fungal polymer. Homozygous null disruptants of CaKRE9 grow poorly on galactose and fail to form hyphae in serum, and, in growth medium containing glucose, the gene is essential. Thus, the CaKRE9 gene product is a potentially useful candidate as a target for fungal-specific drugs.     

Usefulness of (1-->3)-beta-D-glucan measurement for diagnosis of deep mycosis

The number of deep mycosis has been increasing because of increases in immunocompromised hosts and in fungal colonization associated with increasing use of broad-spectrum antibacterial antibiotics. Based on these phenomenon, a simple test method for an early diagnosis of deep mycosis is urgently desired. We therefore investigated the usefulness of assaying a fungal cell component, (1-->3)-beta-D-glucan (beta-glucan). The amount of beta-glucan was obtained from the difference between the amounts determined using Toxicolor and Endospecy, and the serum levels of more than 10 pg/ml were considered positive signs for beta-glucan. The following results were obtained: We found that beta-glucan was positive in 75% of the patients who had been definitely diagnosed to have mycosis, and in 58.3% of the patients strongly suspected of mycosis. The numbers of beta-glucan positive patients' in these 2 groups of patients were significantly different from that in those without mycosis (14.7%, P < 0.05). Thus a usefulness of beta-glucan measurement for the diagnosis of mycosis was demonstrated. However, beta-glucan was sometimes negative even in patients with fungemia at an early phase of the disease and turned positive several days later. Even in a patient with definite lung mycosis, who had a latent circumscribed lesion (afebrile and CRP-negative), beta-glucan was also negative. From these findings, one should be aware that the beta-glucan test produces false negatives even in patients with definite mycosis and that the test should be repeated during the course of the disease.     

Synthesis of a neoglycoprotein containing the Lewis X analogous trisaccharide beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc

The trisaccharide allyl glycoside 36 and related disaccharide part structures have been prepared using the 2-trichloroacetamido-2-deoxy-alpha-D-galactopyranosyl trichloroacetimidate derivative 9 as glycosyl donor under promotion with TMSOTf or Sn(OTf)2, respectively, to produce the beta-(1-->4) linkage to suitably protected glucosamine derivatives in fair yields. Fucosylation was effected employing the ethyl 1-thio glycosyl donor 20 in the presence of IDCP. Deprotection of the intermediates afforded the disaccharide allyl glycosides beta-D-GalpNAc-(1-->4)- beta-D-GlcpNAc 13, beta-D-GalpNClAc-(1-->4)-beta-D-GlcpNAc 14, alpha-L-Fucp-(1-->3)-beta-D-GlcpNAc 24, alpha-L-Fucp-(1-->4)-beta-D- GlcpNAc 31 and the branched trisaccharide allyl glycoside beta-D-GalpNAc-(1-->4)[alpha-L-Fucp-(1-->3)]-beta-D-GlcpNAc 36. The trisaccharide which corresponds to a structural motif occurring in N-glycoprotein glycans from human urokinase, human recombinant protein C, phospholipase A2 as well as O-glycans, was converted into a neoglycoprotein following introduction of a cysteamine-derived spacer group and subsequent activation with thiophosgene.     

Preparation of novel (1-->3)-beta-D-glucans having reducing glucose side chains

Novel (1-->3)-beta-D-glucans (GPBCD, GPECD, GP6CD, and GP3CD) having reducing glucose side chains were prepared from a linear (1-->3)-beta-D-glucan (curdlan: CD) with halogeno glucose isopropylidene derivatives in dimethyl sulfoxide containing dimsyl sodium, followed by treatment with 40% trifluoroacetic acid to remove protecting isopropylidene groups. The side chain glucose moiety was linked or directly or through a spacer at various positions except for its anomeric carbon.     

Expeditious synthesis of a new hexasaccharide using transglycosylation reaction catalyzed by Bacillus (1-->3),(1-->4)-Beta-D-glucan 4-glucanohydrolase

In this study, we report the structural characterization of photosystem II complexes obtained from partially solubilized photosystem II membranes. Direct observation by electron microscopy, within a few minutes after a mild disruption of the membranes with the detergent n-dodecyl-alpha,D-maltoside, revealed the presence of several large supramolecular complexes. Images of these complexes were subjected to multivariate statistical analysis and classification procedures, resolving a new complex consisting of the previously characterized dimeric supercomplex of photosystem II and light-harvesting complex II [Boekema et al., Proc. Natl. Acad. Sci. USA 92 (1995) 175-179] and two additional, symmetrically organized protein masses each containing a second type of trimeric light-harvesting II complex. We conclude that large and labile integral membrane proteins, such as photosystem II, can be quickly structurally characterized without extensive purification.