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The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of the unfolding force on the pulling speed, indicating that the unfolding forces measured in an AFM experiment are those required for the unfolding of the intermediate and not the native state. A mutant of TI I27 with the A-strand deleted (TI I27-A) is studied by NMR and standard biophysical techniques, combined with protein engineering. Molecular dynamics simulations show TI I27-A to be a good model for the intermediate. It has a structure very similar to the native state, and is surprisingly stable. Comparison with a Phi-value analysis of the unfolding pathway clearly shows that the protein unfolds by a different pathway under an applied force than on addition of denaturant.

The synthesis of Oxaldie-3, a synthetic 31-residue peptide with oxaloacetate decarboxylase activity, is described. Biophysical characterisation by gel filtration, CD and NMR spectroscopy indicated that the peptide adopted a folded structure in solution. Oxaldie-3 was an efficient catalyst at concentrations as low as 2 microM, 100-fold lower than the previously described Oxaldie-2, which relied on aggregating alpha-helices for activity. Oxaldie-3 speeded decarboxylation by more than three orders of magnitude relative to simple amines.

A possible pH-dependent conformational switch was investigated for cyclic ADP-ribose. NMR signals for the exchangeable protons were observed in H2O at low temperature, but there was no direct evidence for the protonation of N-3 at neutral pH that has previously been postulated. MNDO calculations indicated that pH dependent 31P chemical shift changes are attributable to protonation of the phosphate adjacent to the N-1 of adenine, and not due to trans-annular hydrogen bonding with a protonated N-3.

Objective: To estimate the prevalence of psammoma bodies in routine cervical cytology specimens and describe clinical diagnoses associated with them in pre- and postmenopausal women., Methods: We identified all reports that contained psammoma bodies from a retrospective review of 34,816 cytology reports over 4 consecutive years from the computerized pathology database at Yale New-Haven Medical Center. Slides were reviewed to confirm and qualify psammoma bodies. Medical records of women with psammoma bodies were reviewed and information on follow-up was collected., Results: We identified 18 women with psammoma bodies on their Papanicolaou smears. The median age was 49.5 years (range 17--78 years). Seven of eight postmenopausal women had gynecologic malignancies. Five presented with postmenopausal bleeding and had uterine serous or clear-cell carcinomas. One presented with a pelvic mass that was ovarian serous carcinoma. Another had a serous carcinoma of the fallopian tube with only psammoma bodies on Papanicolaou smears. Only one of the remaining 11 nonmalignant cases was a postmenopausal woman., Conclusion: The prevalence of psammoma bodies in consecutively screened Papanicolaou smears was 18 of 34,816. Psammoma bodies on Papanicolaou smears are ominous in postmenopausal women. Their presence in asymptomatic premenopausal women warrants further evaluation, but not necessarily surgical exploration.

As part of a structure activity study to examine the interaction of glucosinolates with leaf surfaces, a number of glucosinolates were synthesised bearing novel side chain functionalities. These included 7-carboxyheptyl, heptyl, and naphthyl side chains. For the carboxyheptyl glucosinolate, a novel intramolecular rearrangement reaction was observed during the final deprotection step, which generated an ester attached to the C-3 of glucose. Studies by 1H NMR spectroscopy showed that the hydrophobic side chain associated with one face of the glucose ring and it was proposed that this was the driving force for the rearrangement. Similar hydrophobic interactions were also observed between the heptyl and naphthyl side chains and the glucose.

By the observation of chemically induced dynamic nuclear polarization in (15)N NMR spectroscopy it has been shown that nitration of N-acetyltyrosine, even under acidic conditions, is largely a radical process. In the alkaline reaction of tyrosine with peroxynitrite the main products are nitrite and nitrate, both produced by a radical pathway, and tyrosine nitration is a minor reaction. It is suggested that tyrosine catalyzes the production of NO(*)(2) and HO(*) from peroxynitrite., (Copyright 2000 Academic Press.)

Dedicated portable ultrasound devices generally offer a rapid, noninvasive, largely operator-independent means of assessing post-void residual urine (PVR) volume. In most published series, PVR measured by portable ultrasound correlates well with catheterized urine volume. We report 3 cases in which follow-up of falsely elevated PVR measurements on ultrasound resulted in comparatively low catheterized volumes. In all 3 cases, the elevated readings were due to cystic ovarian pathology, which was diagnosed by formal radiologic evaluation and ultimately confirmed operatively in 2 cases. Cystic pathology of the pelvis or lower abdomen may present as an elevated PVR on ultrasound and low urine volume on subsequent catheterization and should prompt further evaluation.

Background: Several documented cases of endometrial and cervical carcinoma arising in unicornuate uteri have been described; however, ovarian malignancy occurring in conjunction with this müllerian anomaly has not been reported., Case: An 18-year-old woman had a unicornuate uterus, noncommunicating rudimentary horn and homogeneous, solid, right ovarian mass found to be a dysgerminoma at surgery., Conclusion: Müllerian anomalies are unlikely to predispose women to ovarian malignancies. However, it is essential to keep in mind that women with such anomalies, though presenting at a young age, could still have cervical, uterine or even ovarian malignancies.

Objective: The aim of this study was to review the management and outcome of patients with adenocarcinoma in situ of the cervix and to evaluate the significance of endocervical cone margin status in these patients., Methods: A retrospective review of records between January 1988 and December 1996 identified 40 patients with adenocarcinoma in situ on cone biopsy for whom complete information was available. The median follow-up was 38 months., Results: The mean age was 37 years, and the mean parity was 1.3. Fifty-three percent of the patients had prior abnormal cervical cytology. The initial Pap smear that led to the patient's referral was abnormal in 39 (98%). Initial cervical biopsies showed adenocarcinoma in situ and/or glandular dysplasia in 28 (70%), squamous dysplasia in 2 (5%), chronic inflammation in 2 (5%), and no pathologic changes in 2 (5%) patients. Initially no biopsies were performed in 3 (7.5%) patients and the results of 3 (7.5%) biopsies were unknown. Subsequently, all patients had cone biopsies. The endocervical margins were positive for glandular abnormalities in 24% of cold knife cones (CKC), 75% of LEEPs, and 57% of laser cones. The ectocervical margins were positive for squamous and/or glandular abnormalities in 8% of CKCs, 13% of LEEPs, and 57% of laser cones. ECCs above the cone were obtained in 28 patients, and only 1 (3%) was positive. The definitive treatment was hysterectomy in 27, repeat cone in 5, and no additional therapy in 8 patients. The pathology showed residual disease in 44% of treated patients. From 16 cone biopsies with negative margins who had subsequent treatment, there was residual disease in 5 (31%) specimens (1 adenocarcinoma in situ, 1 mild glandular dysplasia, 3 glandular atypia). From 16 cones with positive margins who had subsequent treatment, there was residual disease in 9 (56%) specimens. The patients with negative ECCs above the cone regardless of margin status had residual disease in 58% of treated specimens., Conclusion: Women with adenocarcinoma in situ of the uterine cervix had residual disease in 31% of cases with negative margins in cone biopsies and/or with negative ECCs and in 56% of cases with positive endocervical margins. LEEP cones had higher rate of positive endocervical margins (75%) compared to CKC (24%) and laser cone (57%). If maintaining reproductive capacity is desired, we would recommend CKC; however, this does not guarantee absence of the disease., (Copyright 1999 Academic Press.)

Uterine fibroids are common benign tumors of the uterus and a major public health problem. Between 20 and 25% of women over 35 years of age are estimated to have fibroids. Three subtypes of fibroids are recognized depending on their relationship to the myometrium, namely, submucosal, subserosal and intramural. Fibroids are frequently asymptomatic, but may be associated with menorrhagia, dysmenorrhea, pregnancy loss or infertility. They are composed predominantly of smooth muscle, with a variable amount of connective tissue, and they have a characteristic smooth white whorled appearance on cross sectional examination.

Purpose: The aim of this study was to compare the progression-free and overall survivals of women with advanced ovarian cancer treated with neoadjuvant chemotherapy followed by surgery with those treated conventionally with cytoreductive surgery followed by cytotoxic chemotherapy., Materials and Methods: Fifty-nine consecutive women with advanced malignancies compatible with ovarian cancer based on (1) physical examinations, (2) computerized tomography scans, and (3) cytologic or histologic specimens and treated with platinum-based combination chemotherapy, i.e., neoadjuvant chemotherapy, were retrospectively reviewed. Forty-one subsequently underwent cytoreductive surgery. Their overall and progression-free survivals were compared to those of 206 consecutive women with Stage IIIC and IV epithelial ovarian cancers treated with conventional cytoreductive surgery followed by platinum-based combination chemotherapy during the same era., Results: No statistical difference was observed in overall survival (P = 0.1578) or in progression-free survival between the group treated with neoadjuvant chemotherapy and the conventionally treated group (P = 0.5327) despite the neoadjuvant chemotherapy patients being statistically older (median age 67 years [range 44 to 85 years] vs a median age of 60 years [range 19 to 79 years] for conventionally treated patients; P < 0. 001) and having a statistically poorer performance status (P < 0. 001) than the conventionally treated group. Women undergoing cytoreductive surgery following neoadjuvant chemotherapy had a statistically improved overall survival (P < 0.0001) compared to those who did not undergo surgery., Conclusions: Neoadjuvant chemotherapy does not compromise the survival of women treated for advanced ovarian cancer. Prospective randomized trials comparing neoadjuvant chemotherapy to conventional therapy to determine quality of life experiences and cost/benefit outcomes are now appropriate for women presenting with advanced ovarian cancer., (Copyright 1999 Academic Press.)

Conservative surgical options for uterine myomata traditionally were abdominal myomectomy, laparoscopic myomectomy, and, more recently, myolysis. Each of these procedures has distinct advantages, but also apparent disadvantages. We attempted to introduce an additional option for conservative surgical treatment of fibroids by freezing the structures, a procedure termed cryomyolysis. In this pilot study, 14 women were pretreated with a gonadotropin-releasing hormone (GnRH) agonist for a minimum of 2 months preoperatively to minimize uterine and myoma size. Cryomyolysis was performed and the GnRH agonist was discontinued. Magnetic resonance imaging scans were performed in 10 of the 14 women after GnRH agonist treatment but before surgery, and 4 months postoperatively. Total uterine volume ranged from 41.3 to 1134.8 ml preoperatively, and 49.5 to 1320 ml postoperatively (mean increase 22% after discontinuation of GnRH agonist). Normal uterine volume ranged from 35.6 to 548.7 ml preoperatively and 45.1 to 729.6 ml postoperatively (mean increase 40%); however, myoma volume showed a mean decrease of 6% (range -87-28%). Analysis of only frozen myomata revealed a mean volume decrease of 10%. Cryomyolysis maintains at or slightly reduces these lesions to post-GnRH agonist size, and all other uterine tissue returns to pretreatment size. We believe cryomyolysis may be an effective conservative surgical approach to uterine fibroids.

Study Objective: To evaluate the effectiveness of endometrial cyroablation for abnormal uterine bleeding., Design: Prospective study with 22 months follow-up (Canadian Task Force classification II-2)., Setting: University Medical Center., Patients: Fifteen consecutive patients treated for metrorrhagia or menorrhagia refractory to medical or surgical therapy, and who were either not operative candidates or did not desire hysterectomy., Intervention: Cyroablation of the endometrium., Measurements and Main Results: Fifteen patients underwent 16 procedures for dysfunctional uterine bleeding. Uterine sounding depth was 6 to 15 cm. One patient had spinal anesthesia, seven had general anesthesia, and seven had intravenous conscious sedation with a cervical block. Eight patients underwent cryosurgery while fully anticoagulated. Posttreatment endometrial biopsies were performed on three patients and showed only granulation tissue. Life table calculations give amenorrhea rates of 75.5% at 6 months and 50.3% at 22 months. One woman underwent a repeat procedure, resulting in hypomenorrhea at 7-month follow-up., Conclusion: This pilot study suggests that endometrial cryoablation may be performed simply and effectively. Future studies should be designed to optimize the technical aspects of the procedure, determine its relative efficacy, and investigate the indications.

Purpose: Uterine papillary serous carcinoma (UPSC) is a morphologically distinct variant of endometrial carcinoma that is associated with a poor prognosis, high recurrence rate, frequent clinical understaging, and poor response to salvage treatment. We retrospectively analyzed local control, actuarial overall survival (OS), actuarial disease-free survival (DFS), salvage rate, and complications for patients with Federation International of Gynecology and Obstetrics (FIGO) (1988) Stage I UPSC., Methods and Materials: This retrospective analysis describes 38 patients with FIGO Stage I UPSC who were treated with the combinations of radiation therapy, chemotherapy, total abdominal hysterectomy, and bilateral salpingo-oophorectomy (TAH/BSO), with or without a surgical staging procedure. Twenty of 38 patients were treated with a combination of low dose-rate (LDR) uterine/vaginal brachytherapy using 226Ra or 137Cs and conventional whole-abdomen radiation therapy (WART) or whole-pelvic radiation therapy (WPRT). Of 20 patients (10%) in this treatment group, 2 received cisplatin chemotherapy. Eighteen patients were treated with high dose-rate (HDR) vaginal apex brachytherapy using 192Ir with an afterloading device and cisplatin, doxorubicin, and cyclophosphamide (CAP) chemotherapy (5 of 18 patients). Only 6 of 20 UPSC patients treated with combination LDR uterine/vaginal brachytherapy and conventional external beam radiotherapy underwent complete surgical staging, consisting of TAH/BSO, pelvic/para-aortic lymph node sampling, omentectomy, and peritoneal fluid analysis, compared to 15 of 18 patients treated with HDR vaginal apex brachytherapy., Results: The 5-year actuarial OS for patients with complete surgical staging and adjuvant radiation/chemotherapy treatment was 100% vs. 61% for patients without complete staging (p = 0.002). The 5-year actuarial OS for all Stage I UPSC patients treated with postoperative HDR vaginal apex brachytherapy and systemic chemotherapy was 94% (18 patients). The 5-year actuarial OS for Stage I UPSC patients treated with HDR vaginal apex brachytherapy and chemotherapy who underwent complete surgical staging was 100% (15 patients). The 5-year actuarial OS for the 20 Stage I UPSC patients treated with combinations of pre- and postoperative LDR brachytherapy and postop WART was 65%. None of the 6 surgically staged UPSC patients treated with LDR radiation and WART/WPRT developed recurrent disease. For patients with FIGO Stage IA, IB, and IC UPSC who underwent complete surgical staging, the 5-year actuarial DFS by depth of myometrial invasion was 100, 71, and 40%, respectively (p = 0.006). The overall salvage rate for local and distant recurrence was 0%. Complications following HDR vaginal apex brachytherapy included only Radiation Therapy Oncology Group (RTOG) grade 1 and 2 toxicity in 16% of patients. However, complications from patients treated with WART/WPRT, and/or LDR brachytherapy, included RTOG grade 3 and 4 toxicity in 15% of patients., Conclusion: Patients with UPSC should undergo complete surgical staging, and completely surgically staged FIGO Stage I UPSC patients can be effectively and safely treated with HDR vaginal apex brachytherapy and chemotherapy. Both OS and DFS of patients with UPSC are dependent on depth of myometrial invasion. The salvage rate for both local and distant UPSC recurrences is extremely poor. Complications from HDR vaginal apex brachytherapy were minimal.

A combination of synthetic methods involving mononuclear precursors of predetermined chirality {Delta- or Lambda-[Ru(pp)(2)(CO)(2)](2+); pp = 2,2'-bipyridine or 1,10-phenanthroline} and chromatographic techniques has allowed the isolation of the meso (DeltaLambda) and rac (DeltaDelta/LambdaLambda) diastereoisomers of the dinuclear complexes [{Ru(pp)(2)}(2)(&mgr;-HAT)](4+) {HAT = 1,4,5,8,9,12-hexaazatriphenylene}. The enantiomers of the rac forms have been separated, and characterization of all species has been achieved by NMR and CD studies. Additonally, the homochiral (Delta(3)/Lambda(3)) and heterochiral (Delta(2)Lambda/Lambda(2)Delta) diastereoisomers of the trinuclear complexes [{Ru(pp)(2)}(3)(&mgr;-HAT)](6+), and the enantiomers of both forms, have been isolated and identified. Emission studies of all the dinuclear species at room temperature indicate the relative luminescence quantum yields and the emission lifetimes significantly decrease for the meso compared with the rac diastereoisomers. No significant differences were detected at room temperature in the diastereoisomeric forms of the trinuclear compounds. However, in a glass at low temperature (80 K), the luminescence lifetimes of the trinuclear heterochiral diastereoisomer were slightly shorter than those of the homochiral form.

The chromophore-quencher complex [Ru(Me(2)bpy)(bpy-MV(2+))(bpy-PTZ)](4+), containing one donor (phenothiazine, PTZ) and one acceptor (methyl viologen, MV(2+)) functionality, has been synthesized and separated into its four geometric isomers. This was achieved through the intermediacy of [Ru(Me(2)bpy)(bpy-MV(2+))(py)(2)](4+), the two isomers of which were separated and each reacted stereoselectively with bpy-PTZ to produce two distinct isomer pairs of the target molecule. Cation-exchange chromatography allowed separation to realize the four forms. The isomers of the product and of the intermediates were characterized by NMR spectroscopy. This is the first example of the isolation of geometric isomers of a mononuclear system containing a single donor and single acceptor functionality.

We present heteronuclear three-dimensional gradient-NMR techniques for the resonanceassignment of exchangeable (-OH and -NH) protons in uniformly 13C isotopically enrichedoligosaccharides and for the measurement of 1H-1H nuclear Overhauser enhancementsinvolving these protons. These techniques are derived from conventional HOHAHA-HSQCand NOESY(ROESY)-HSQC experiments, and are illustrated in application to a sample ofuniformly 13C-enriched Galbeta1-4GlcNAc, and demonstrate that a total of 35 ROEs involvingexchangeable protons can be detected and assigned. We present a quantitative analysis ofthese ROEs that can only be accommodated in a model of the solution behaviour of theoligosaccharide that involves considerable internal motion.

The solution conformation of an agalactosyl penta-antennary "bisected" N-linked glycan from hen ovomucoid has been determined using a combination of 1H-NMR NOE measurements and restrained molecular dynamics (MD) simulations. The majority of glycosidic linkages exhibited restricted torsional fluctuations about the global minimum energy configuration, of an extent which was generally less than that observed in N-linked glycans with a smaller number of antennae. The locations of terminal galactose residues in the native glycan, which exhibit branch specificity, could not readily be rationalized in terms of relative accessibility by the relevant galactosyltransferase of the various nonreducing terminal 2-acetamido-2-deoxy-D-glucopyranose (GlcNAc) residues in the agalactosyl glycan, suggesting either that the parent protein exhibits substantial control over glycosylation or that more than one transferase is responsible for galactosylation.

A technique is described for assisting the resonance assignment process in oligosaccharide proton NMR spectra, where multiple residues of the same type generate extreme resonance overlap in the spectrum. The approach involves the modification of a conventional HOHAHA experiment with constant-time acquisition in t1, which effectively proton decouples the C-1 protons of residues whose resonances overlap, thus affording a significant increase in effective resolution in that dimension. For a sufficiently long spin-lock time, complete one-dimensional subspectra are obtained essentially free of cross talk from adjacent resonances. Further simplification of the assignment process is illustrated by incorporation of the constant-time modification into a three-dimensional HOHAHA-HOHAHA experiment.

Aqueous solution conformations of three antigenic bacterial capsular polysaccharides (CPS) from S. pneumoniae serogroup 9 were determined using a combination of NMR data (NOE build-up rates and conformation-dependent chemical shifts), simulated annealing, and molecular dynamics simulations. Each polymer adopts a flexible extended ribbon conformation in solution. Conformations of structural elements shared by each PS are indistinguishable. Differences in conformations are minor and localised at the sites of structural variations; there is no evidence of long-range stabilisation of a secondary structure. It is likely that antigenic specificity of Group 9 PS is dominated by local structural variation rather than by conformational differences.

Complete 1H and 13C NMR assignments, determined by one- and two-dimensional homo- and hetero-nuclear experiments, are reported for the antigenic capsular polysaccharide (CPS) from Streptococcus pneumoniae serotype 9N (S9 in American nomenclature). Distance constraints derived from 1D NOE difference experiments were combined with energy minimisation (simulated annealing) and molecular dynamics (MD) calculations to determine the most favoured conformation of S9 in aqueous solution at 70 degrees C. NOE values simulated for several static conformational models using the NOEMOL program did not correlate well with experimental values, whereas time averaged interproton distances calculated from 500 ps of restrained MD (using the Tropp formalism to account for rapid internal mobility) were in close agreement with experimentally derived distance estimates.

We have examined the dynamic behavior in solution of the biantennary glycan GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)Man beta 1- 4GlcNAc beta 1-4GlcNAc, and its "bisected" analogue GlcNAc beta 1-2Man alpha 1-6(GlcNAc beta 1-2Man alpha 1-3)(GlcNAc beta 1- 4)Man beta 1-4GlcNAc beta 1-4GlcNAc, by use of both free dynamics simulations and restrained dynamics simulations using distance restraints derived from 1H NMR rotating frame Overhauser effect measurements. Data resulting from each type of simulation are compared with experimental data and are critically evaluated. Both methods suggest that most glycosidic linkages exhibit significant torsional oscillations in solution, and the dynamic behavior of certain linkages, notably Man alpha 1-6Man and Man alpha 1-3Man, were found to be restricted by the presence of the bisecting GlcNAc residue. The average structures so obtained were found to agree closely with those predicted in previous investigations where torsional oscillations about glycosidic linkages were not considered. In particular, the characteristic chemical shift perturbations induced by the bisecting GlcNAc residue could be explained in terms of the dynamic differences between the two glycans.

The range of internal motions of the sialyl Lewis-X (SLe(x)) tetrasaccharide (NeuNAc alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc) [where Fuc is L-fucopyranose, Gal is D-galactopyranose, GlcNAc is 2-acetamido-2-deoxy-D-glucopyranose and NeuNAc is D-neuraminic acid (sialic acid)] was studied by restrained simulated annealing and restrained molecular dynamics (MD) calculations. Transitions between predominantly two conformational states were observed for the NeuNAc alpha (2-->3)Gal linkage, consistent with previous observations for this linkage in sialyl-N-acetyllactosamine. The MD trajectory was simulated for 5 ns of real time, in order to observe a statistically significant number of these relatively low-frequency transitions. The Fuc alpha(1-->3)GlcNAc and Gal beta (1-->4)GlcNAc linkages, however, showed more restricted flexibility within a single energy well (RMS differences for the time-averaged glycosidic torsion angles, and , were 50% lower than for the NeuNAc alpha(2-->3)Gal linkage), and approximate to a rigid conformation. NMR parameters [relative rotating-frame Overhauser enhancement (r.O.e.) and inter-glycosidic 3JCH] back-calculated from the MD simulation were in close agreement with experimentally measured values for the free reducing oligosaccharide in D2O solution.

Sulphated blood group Lewis(a)/Lewis(x) (Le(a)/Le(x)) type sequences, with sulphate at the 3-position of galactose, have emerged as potent ligands for the endothelial adhesion molecule E-selectin and the leukocyte adhesion molecule L-selectin. As a first step in elucidating the molecular basis of the strong interactions with the selectins, we have performed conformational studies of the sulphated Le(a) in comparison with the non-sulphated analogue which is less strongly bound by E-selectin and not at all by L-selectin. Experimental NMR parameters [nuclear Overhauser effects (NOE) and interglycosidic 3JC,H] and theoretical values back-calculated from the minimum energy structures are in excellent agreement for both molecules. Molecular dynamics calculations for SuLe(a) depict only minor torsional fluctuations around the glycosidic linkages over the time course of the 500 ps simulations, leading to the conclusion that the conformation of SuLe(a) approximates to a single-rigid structure, as does the previously investigated Le(a) molecule. Comparison of experimentally and theoretically obtained parameters for SuLe(a) with those for the non-sulphated Le(a) molecule indicate that no detectable changes occur in the three-dimensional structure of the trisaccharide upon sulphation. Thus, the enhanced selectin binding to the sulphated Le(a) is most likely due to favourable electrostatic interactions between the charged sulphate group and corresponding charged groups on the selectin protein.

A detailed investigation has been undertaken on the extent and nature of torsional fluctuations about the glycosidic linkage of the model disaccharide Man alpha 1-3Man alpha 1-OMe. In particular, we sought to determine whether the three nuclear Overhauser effects and the two long-range heteronuclear 3JCH spin coupling constants measurable across the glycosidic linkage were consistent with a single conformation or multiple conformations about that linkage. Within experimental error, we have found that these five parameters can be interpreted in terms of a single, rigid geometry. Alternatively, the data are also consistent with a model in which the glycosidic torsional angles exhibit significant but restricted fluctuations about the global minimum energy conformation. Evidence from restrained molecular dynamics simulations both in vacuo and with explicit inclusion of solvent water and from 13C relaxation measurements upon an oligomannose glycan in covalent association with protein suggests that the latter model is the most accurate representation of the conformational behavior of oligosaccharides in solution.

A new homonuclear NMR experiment is described for the assignment of the proton NMR spectra of oligosaccharides, namely HOHAHA-HOHAHA-COSY (where HOHAHA is homonuclear Hartmann-Hahn spectroscopy and COSY is correlated spectroscopy). While this technique may formally be thought of as a four-dimensional NMR experiment, by use of selective pulses it is demonstrated that the analogous pseudo-four-dimensional experiment is a valuable improvement over conventional three-dimensional HOHAHA-COSY, in that the degree of resonance overlap is markedly reduced by the dispersion of resonances into a fourth effective dimension. The technique is demonstrated by application to the biantennary nonasaccharide Gal beta 1-4-GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man-beta 1-4GlcNAc beta 1-4GlcNAc.

The 1H-n.m.r. spectra of the Streptococcus pneumoniae type 9V (S68 in American nomenclature) capsular polysaccharide (PS) and its O-deacetylated derivative [which is structurally identical to the S. pneumoniae type 9A (S33) PS] were assigned using COSY, relayed-COSY, and 2D-NOESY experiments. The positions of the OAc groups in the alpha-GlcA, beta-ManNAc, and alpha-Glc residues of the native 9V PS were established using 2D-n.m.r. and chemical shift arguments, and the relative proportions of different O-acetylated species were estimated by integration of well-resolved 1H-n.m.r. signals. The locations of the OAc substituents differ from those previously reported. [formula: see text].

The gene for human galactosyltransferase (EC 2.4.1.22) has been localized to the short arm of chromosome 9 by in situ hybridization to human metaphase chromosomes of a 985 bp cDNA probe for the gene.

Human milk galactosyltransferase (EC 2.4.1.22) was purified to homogeneity using affinity chromatography. Edman degradation was used to determine the amino acid sequences of eight peptide fragments isolated from the purified enzyme. A 60-mer "optimal" oligonucleotide probe that corresponded to the amino acid sequence of one of the galactosyltransferase peptide fragments was constructed and used to screen a lambda gt10 cDNA library. Two hybridization-positive recombinant phages, each with a 1.7 Kbp insert, were detected among 3 X 10(6) recombinant lambda gt10 phages. Sequencing of one of the cDNA inserts revealed a 783 bp galactosyltransferase coding sequence. The remainder of the sequence corresponded to the 3'-region of the mRNA downstream from the termination codon.

Galactosyltransferase (EC 2.4.1.22), purified to homogeneity from human milk by affinity chromatography, had an apparent molecular weight of 53,000 as determined by denaturing polyacrylamide gel electrophoresis. Subtraction of the estimated contribution of the oligosaccharide portion of the molecule leaves a Mr of 47,000. An N-terminal amino acid sequence analysis of the isolated protein revealed a sequence similar to that found near the 5' end of a cDNA clone isolated by Shaper et al, which encodes a 35,000 molecular weight protein. Either the molecular weight of galactosyltransferase, has been overestimated, or a discrepancy exists between the actual molecular weight of galactosyltransferase and that predicted by the bovine cDNA clone isolated by Shaper et al.