Oligosaccharyltransferase is highly specific for the hydroxy amino acid in Asn-Xaa-Thr/Ser.

Pig liver oligosaccharyltransferase (OST), which is involved in the en bloc transfer of the Dol-PP-linked GlcNAc(2)-Man(9)-Glc(3) precursor on to asparagine residues in the Asn-Xaa-Thr/Ser sequence, is highly stereospecific for the conformation of the 3-carbon atom in the hydroxy amino acid. Moreover, substitution of the hydroxy group by either SH as in cysteine, or NH(2) as in beta,gamma-diamino-butanoic acid as reported previously [Bause, E. et al., Biochem. J. 312 (1995) 979-985], followed by the determination of the pH optimum for enzymatic activity, indicates that neither a negative nor a positive charge in the hydroxy amino acid position is tolerated by the enzyme. Binding of the threonine beta-methyl group by OST is also specific, with serine, L-threo-beta-hydroxynorvaline and L-beta-hydroxynorleucine containing tripeptides all bound much less efficiently than the threonine peptide itself. The data are interpreted in terms of a highly stereospecific hydrophobic binding pocket for the threonine CH(3)-CH(OH) group.

Do rodent and human brains have different N-glycosylation patterns?

A large number of studies on the structure of N-glycosidically linked oligosaccharides from glycoproteins of different organs and/or different species have been carried out in the past using various combinations of techniques such as monosaccharide analysis, permethylation, peracteylation, exoglycosidase sequencing, normal and reversed phase HPLC, mass spectrometry and nuclear magnetic resonance spectroscopy. Although it is widely accepted that the processing of N-glycans in the ER and Golgi of mammalian cells follows the same principal metabolic rules, analyses have revealed that the glycosylation pattern of a particular protein may differ depending on the cell type in which it is expressed. N-glycans from brain glycoproteins have been shown to include a variety of hybrid- and complex-type structures with structural features that are not so commonly found on glycoproteins from other organs and which have, therefore, been classified as 'brain-specific'. Comparison of the N-glycans of glycoproteins from homogenates of rat, mouse and human brains confirm that, in general, glycoproteins from human brain show a similar profile of brain-specific N-glycans as glycoproteins from mouse and rat brain.

Estrogen for Alzheimer's disease in women: randomized, double-blind, placebo-controlled trial.

BACKGROUND: AD, the most prevalent cause of dementia, affects twice as many women as men. Therapeutic options are limited, but results of prior studies support the hypothesis that estrogen treatment may improve symptoms of women with this disorder. METHODS: Forty-two women with mild-to-moderate dementia due to AD were enrolled into a randomized, double-blind, placebo-controlled, parallel-group trial of unopposed conjugated equine estrogens (1.25 mg/day) for 16 weeks. RESULTS: Outcome data were available for 40 women at 4 weeks and 36 women at 16 weeks. At both 4 and 16 weeks, there were no significant differences or statistical trends between treatment groups on the primary outcome measure (the cognitive subscale of the Alzheimer's Disease Assessment Scale), clinician-rated global impression of change, or caregiver-rated functional status. Exploratory analyses of mood and specific aspects of cognitive performance also failed to demonstrate substantial group differences. CONCLUSION: Although conclusions are limited by small sample size and the possibility of a type II error, results suggest that short-term estrogen therapy does not improve symptoms of most women with AD. These findings do not address possible long-term effects of estrogen in AD, possible interactions between estrogen and other treatment modalities, or putative effects of estrogen in preventing or delaying onset of this disorder.

Estrogen replacement therapy and urinary tract infections in postmenopausal women aged 45-89.

OBJECTIVE: An elevated risk of urinary tract infections (UTIs) in postmenopausal women has been attributed to an increase in the vaginal pH. Estrogen replacement therapy (ERT) helps restore the vaginal milieu and may have a beneficial effect on risk of infection. Studies examining the association between ERT and UTIs have been inconsistent. We conducted a nested case control study to clarify this relationship in women aged 45-89. DESIGN: For each case, we selected up to five control women, matched by year of birth. We used pharmacy records to classify women as new users, past users, ongoing users (past and new users), and never users of ERT. There were 254 cases and 1,268 controls. RESULTS: The risk ratio for UTI was 1.02 [95% confidence interval (CI) 0.74, 1.40] for any use versus never use, after adjustment for health care utilization. The risk ratio for the association between new use and UTI was 1.13 (95% CI 0.46, 2.77). For ongoing users the risk ratio was 1.08 (95% CI 0.76, 1.54), whereas the risk ratio for past use was 0.77 (95% CI 0.39, 1.48). CONCLUSIONS: Our results do not support a protective effect of ERT on the risk of UTIs.

Effect of bone density information on decisions about hormone replacement therapy: a randomized trial.

OBJECTIVE: To determine the effect of bone density information on a woman's decision about hormone replacement therapy (HRT). METHODS: One hundred forty women were assigned randomly to receive either educational information about osteoporosis and a voucher for a bone mineral density test 12 months later or the same educational information plus an immediate dual-energy x-ray absorptiometry test for bone mineral density. Women in both groups were offered prescription for HRT. RESULTS: Of the 93 women who received a bone mineral density test, 63.4% elected HRT and filled their prescription, compared with only 20.0% of the 43 women who did not have a bone mineral density test (P < .01). Women who were classified as osteopenic (between -1 and -2.5 standard deviations [SDs] of the young normal bone mineral density) or osteoporotic (more than 2.5 SDs below young normals) were more likely to choose HRT (69.4%) than were women whose bone mineral density was in the normal range (51.6%) (above -1 SD of the young normal bone mineral density value). CONCLUSIONS: A bone mineral density test, regardless of the result, had a significant effect on women's decisions to accept HRT. Within the group having the test, women with lower bone mineral density were more likely to choose HRT.

Estrogen replacement therapy and the development of osteoarthritis.

Recent studies have indicated that estrogen users have a lower than expected rate of concurrent osteoarthritis. We assessed the association between estrogen replacement therapy and incident symptomatic osteoarthritis, using a nested case-control design. We identified all incident cases of hand, hip, and knee osteoarthritis in women members of the Fallon Community Health Plan, age 20-89 years, from January 1, 1990, to December 31, 1993. For each case, we selected a control woman matched by closest date of birth. We used pharmacy records to classify women as new users, past users, ongoing users (past and new users), and never-users of estrogen replacement therapy. There were 60 informative case-control pairs. After controlling for obesity and health care utilization, we found that new use of estrogen replacement therapy was a predictor of new osteoarthritis diagnosis. Past use was inversely associated with risk of osteoarthritis [adjusted odds ratio = 0.7; 95% confidence interval (CI) = 0.3-1.9]. For ongoing use of estrogen replacement therapy and osteoarthritis, the adjusted odds ratio was 1.4 (95% CI = 0.6-3.3). The associations between osteoarthritis and both new use of estrogen replacement therapy and utilization of services suggest that frequent medical care increases the likelihood of diagnosis of osteoarthritis.

The aqueous solution structure of a lipoteichoic acid from Streptococcus pneumoniae strain R6 containing 2,4-diamino-2,4,6-trideoxy-galactose: evidence for conformational mobility of the galactopyranose ring.

The 2D-NOESY spectra for the per-N-acetylated and the native lipoteichoic acid (LTA) oligomer from Streptococcus pneumoniae strain R6 clearly indicate a difference in conformation of the 2,4,6-trideoxy-galactopyranose ring. Whereas the 2,4-N-acetylated Gal24N adopts the usual 4C1 chair conformation, the native 2-N-acetyl-4-amino Gal24N exhibits conformational mobility with comparable populations in the 4C1 chair and 5S1 skew conformations, as determined using MD simulation for the partial trisaccharide Me-beta-D-Glc6P-(1-->3)-alpha-D-Gal24N-[6-PC]-(1-->4)-alpha- D-galNAc and from the intra-ring NOE effects. 31P-NMR spectra point to a strong electrostatic or hydrogen-bonding interaction between the free 4-NH2 group on the Gal24N and the negatively charged diester phosphate group between adjacent pentasaccharide repeating-units [Ribitol-(5-->6)-beta-D-Glc6P]. Molecular modelling and MD simulation experiments confirmed that such an interaction was feasible with the Gal24N galactopyranose ring in the inverted B1.4 or skew 5S1 conformation.

Continuous spectrophotometric assay of conjugated bile acid hydrolase.

Conjugated bile acid hydrolase (CBAH) refers to a class of enzymes which catalyze the cleavage of the amino acid moieties from conjugated bile acids. These enzymes are significant because of their role in providing substrates for further microbial metabolism in the gastrointestinal tract. They also are used in research laboratories for the deconjugation of bile acids prior to structural analyses. A continuous spectrophotometric assay for CBAH activity was developed using a conjugate of cholic acid and the chromophore, 5-amino-2-nitro-benzoic acid. The free chromophore is detected by virtue of its absorbance at 410 nm. The CBAH from Clostridium perfringens displayed a Km for this substrate of 120 microM. These results demonstrate that this new compound functions as an effective substrate of the enzyme and forms the basis for a convenient and rapid method to monitor CBAH activity.

Specific permeability and selective formation of gap junction channels in connexin-transfected HeLa cells.

DNAs coding for seven murine connexins (Cx) (Cx26, Cx31, Cx32, Cx37, Cx40, Cx43, and Cx45) are functionally expressed in human HeLa cells that were deficient in gap junctional communication. We compare the permeabilities of gap junctions comprised of different connexins to iontophoretically injected tracer molecules. Our results show that Lucifer yellow can pass through all connexin channels analyzed. On the other hand, propidium iodide and ethidium bromide penetrate very poorly or not at all through Cx31 and Cx32 channels, respectively, but pass through channels of other connexins. 4,6 Diamidino-2-phenylindole (DAPI) dihydrochloride shows less transfer among Cx31 or Cx43 transfectants. Neurobiotin is weakly transferred among Cx31 transfectants. Total junctional conductance in Cx31 or Cx45 transfected cells is only about half as high as in other connexin transfectants analyzed and does not correlate exactly with any of the tracer permeabilities. Permeability through different connexin channels appears to be dependent on the molecular structure of each tracer, i.e. size, charge and possibly rigidity. This supports the hypothesis that different connexin channels show different permeabilities to second messenger molecules as well as metabolites and may fulfill in this way their specific role in growth control and differentiation of cell types. In addition, we have investigated the function of heterotypic gap junctions after co-cultivation of two different connexin transfectants, one of which had been prelabeled with fluorescent dextran beads. Analysis of Lucifer yellow transfer reveals that HeLa cells expressing Cx31 (beta-type connexin) do not communicate with any other connexin transfectant tested but only with themselves. Two other beta-type connexin transfectants, HeLa-Cx26 and -Cx32, do not transmit Lucifer yellow to any of the alpha-type connexins analyzed. Among alpha-type connexins, Cx40 does not communicate with Cx43. Thus, connexins differ in their ability to form functional heterotypic gap junctions among mammalian cells.

Synthesis and characterization of novel analogs of conjugated bile acids containing reversed amide bonds.

New analogs of amino acid-conjugated bile acids were synthesized in which the amide bond was reversed from its normal configuration. These structural isomers of the beta-alanyl conjugates of cholic acid and ursodeoxycholic acid were synthesized by reaction of succinic anhydride with the 24-nor-23-amine derivatives of cholic acid and ursodeoxycholic acid. The chemical and physical properties of these reverse amide conjugated bile acid analogs were compared with those of the normal glycine and beta-alanine conjugates. The reverse amide analogs comigrated with their isomeric beta-alanine conjugates during thin-layer chromatography using a variety of solvent systems. However, the isomeric pairs could be resolved by reversed-phase high performance liquid chromatography, with the reverse amides having greater retention times compared to the beta-alanine conjugates. Critical micelle concentrations, solubility of undissociated forms, and acid dissociation constants were similar for the isomeric pairs. Significant differences in melting points were observed, however, While the isomeric pairs showed no significant differences in sensitivity to base hydrolysis, the reverse amides were not hydrolyzed by the cholylglycine hydrolase from Clostridium perfringens, even after long incubation periods.

The aqueous solution structure of the tetrasaccharide-ribitol repeat-unit from the lipoteichoic acid of Streptococcus pneumoniae strain R6 determined using a combination of NMR spectroscopy and computer calculations.

High-resolution 1D- and 2D-correlation 1H NMR and 13C NMR, at 500 and 125 MHz, respectively, permitted assignment of the majority of the resonances in the per-N-acetylated, phosphorylated tetrasaccharide-ribitol repeat-unit, and in the complete polymer (n = 5 - 7) containing between five and seven repeating units attached to the deacylated lipid anchor, for the lipoteichoic acid from Streptococcus pneumoniae strain R6; the 31P resonances were also assigned. Comparison of the 31P spectra obtained for the per-N-acetylated oligosaccharide and for the oligosaccharide having the AATG 4-NH2 group still free, indicate a conformational difference brought about by interaction between the amino group and the neighboring phosphate group.

Methyl alpha-glycoside of N-thioacetyl-D-neuraminic acid: a potential inhibitor of influenza A virus. A 1H NMR study.

The binding of influenza A virus hemagglutinin to its cell surface receptor, alpha-linked 5-N-acetylneuraminic acid (sialic acid), was studied in solution. The effect of structural modifications introduced into the N-acetyl group of the sialic acid on the binding was monitored by determining the dissociation constants by proton nuclear magnetic resonance (1H NMR) spectroscopy. Methyl alpha-glycoside of N-thioacetylneuraminic acid showed high, whereas the corresponding N-methylcarbamoylneuraminic acid exhibited relatively low binding affinity towards the hemagglutinin.

The structural elucidation of the two positional isomers of a mono-glucopyranosyl mono-acyl glycerol derivative from Cystobacter fuscus (Myxobacterales).

The structures of two glycolipids produced by Cystobacter fuscus Cb 685 have been determined by 1H-NMR spectroscopy including 2D-techniques, as the positional isomers 1- and 2-isopentadecanoyl-3-beta-D-glucopyranosyl-X-glycerol. The chain length of the fatty acid residue determined by 13C-NMR spectroscopy has been confirmed by negative ion FAB-mass spectrometry.

O-acetylated gangliosides in bovine buttermilk. Characterization of 7-O-acetyl, 9-O-acetyl, and 7,9-di-O-acetyl GD3.

Three O-acetylated gangliosides, G1, G2, and G3, were purified from bovine buttermilk by using chloroform/methanol extraction, Folch partitioning, chromatography on DEAE-Sephadex A-25, and Iatrobeads columns. The final yields of gangliosides G1, G2, and G3 were 2 mg, 37 mg, and 40 mg per 1.7 kg of the buttermilk powder, respectively. On the basis of immunostaining on high performance thin layer chromatography with specific monoclonal antibodies, mild alkaline treatment, gas-liquid chromatographic analysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance studies, G1 and G2 are characterized as O-acetylated GD3 and G3 as O-acetylated GT3, and the structures of these gangliosides are as follows: [formula: see text] The major fatty acids of these gangliosides were C18:0, C22:0, C23:0, and C24:0, and the long chain base was C18-sphingosine.

Structural characterization of neutral glycosphingolipids from Trypanosoma cruzi.

The major neutral glycosphingolipids from Trypanosoma cruzi ceramide mono- and dihexosides (CMH and CDH, respectively) were analysed after chromatographic purification using 1H 500 MHz nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. The ceramide monohexoside fraction (CMH) contained both glucosyl- and galactosylceramides. After peracetylation, the CMH fraction was separated into 2 subfractions, CMH-COH and CMH-Cn, containing either hydroxy fatty acids or n-fatty acids. In the CMH-COH fraction glucose and galactose were present in a ratio of 2:1, whereas this ratio was 1:1 in the CMH-Cn fraction. The CDH fraction was identified as lactosylceramide with sphingosine as the long chain base and 16:0, 18:0, and 24:0, 24:4 fatty acids as major components.

pH-dependent changes of ganglioside biosynthesis in neuronal cell culture.

Ganglioside biosynthesis was studied in primary cultured murine cerebellar cells after labeling with [14C]galactose. A shift in biosynthesis from "a"-series to "b"-series gangliosides was observed after lowering the pH of the culture medium from 7.4 to 6.2; this effect was fully reversible on changing back to pH 7.4. The observed regulatory effect of pH is in accordance with a recent model of ganglioside biosynthesis. Sialyltransferase II (ST II), the first enzyme for biosynthesis of "b"-series gangliosides, is more active at pH 6.2 than Gal-NAc-transferase, the first enzyme for synthesis of "a"-series gangliosides, which is more active than sialyltransferase II at pH 7.4.

Treatment of coccidioidal meningitis with fluconazole.

Fluconazole was administered at doses of 50-400 mg/d to 18 patients (15 men, three women) with coccidioidal meningitis. After a mean duration of treatment of 9.8 months, 10 (67%) of 15 assessable patients had responded, one (7%) of 15 had partially responded, and four (27%) of 15 had not responded to therapy. Five (63%) of eight assessable patients receiving fluconazole as sole therapy responded or partially responded. Two patients discontinued fluconazole after initially responding to therapy, and both experienced relapse. The toxicity of fluconazole remains minimal at doses to 400 mg/d. The penetration of fluconazole into cerebrospinal fluid is substantial at all doses studied. Thus fluconazole continues to show promise even as sole therapy against coccidioidal meningitis. Not all patients respond, however, and relapse may be a problem with the currently studied doses and durations of therapy.

Isolation and structural characterization of sialic-acid-containing glycopeptides of the O-glycosidic type from the urine of two patients with an hereditary deficiency in alpha-N-acetylgalactosaminidase activity.

Glycopeptides have been isolated from the urine of two patients, aged 5 and 6, with a new lysosomal storage disease characterized by a deficiency in alpha-N-acetylgalactosaminidase activity. Isolation of these glycopeptides was achieved using gel filtration and ion-exchange chromatography. Structural determination was done using one- and two-dimensional 500 MHz 1H-NMR spectroscopy and FAB mass spectrometry of native and derivatized glycopeptides. The following structures were inferred as being present: Glycopeptide A (up to 140 mg/l urine) (1)-(3) Neu5Ac alpha 2-3Gal beta 1-3 (Neu5Ac alpha 2-6)GalNAc alpha 1-R A1: R = Ser A2: R = Thr A3: R = Thr-Pro Glycopeptide B (up to 80 mg/l urine) (4)-(6) Neu5Ac alpha 2-3Gal beta 1-4GlcNAc beta 1-6 (Neu5Ac alpha 2-3-Gal beta 1-3) GalNAc alpha 1-R B1: R = Ser B2:R = Thr B3: R = Thr-Pro