Comparative analyses of paediatric dental measurements using plaster and three-dimensional digital models.

AIM: Recent advances in three-dimensional imaging have led to an increased interest in the application of computer-models in paediatric dentistry. However, in evidence-based paediatric dentistry the accuracy of new methods must be validated before they are introduced to clinical practice. We aimed to compare the accuracy of measurements of digital models obtained using a non-contact 3D measuring system, with direct measurements made on plaster models (gold standard) from children. MATERIALS AND METHODS: Twelve pairs of plaster models were obtained from children with deciduous dentition; tooth size, arch width, and arch length were examined. The same parts on each cast were measured twice with at least a 2-week interval between measurements with each method by four examiners. Linear mixed-effects model analyses were performed for comparison of values from the 2 different measurement methods. RESULTS: The average difference between the 2 methods in measured values, derived from the final model, was <0.2 mm. Random effect of examiners was always the smallest component of variance, and frequently negligible. STATISTICS: Intraclass correlation coefficients were typically >90%. CONCLUSION: These results suggest that primary dentition analysis of digital models has a high accuracy level, comparable to that of direct measurement of plaster models by digital calipers.

Circular polarization in the optical afterglow of GRB 121024A.

Gamma-ray bursts (GRBs) are most probably powered by collimated relativistic outflows (jets) from accreting black holes at cosmological distances. Bright afterglows are produced when the outflow collides with the ambient medium. Afterglow polarization directly probes the magnetic properties of the jet when measured minutes after the burst, and it probes the geometric properties of the jet and the ambient medium when measured hours to days after the burst. High values of optical polarization detected minutes after the burst of GRB 120308A indicate the presence of large-scale ordered magnetic fields originating from the central engine (the power source of the GRB). Theoretical models predict low degrees of linear polarization and no circular polarization at late times, when the energy in the original ejecta is quickly transferred to the ambient medium and propagates farther into the medium as a blast wave. Here we report the detection of circularly polarized light in the afterglow of GRB 121024A, measured 0.15 days after the burst. We show that the circular polarization is intrinsic to the afterglow and unlikely to be produced by dust scattering or plasma propagation effects. A possible explanation is to invoke anisotropic (rather than the commonly assumed isotropic) electron pitch-angle distributions, and we suggest that new models are required to produce the complex microphysics of realistic shocks in relativistic jets.

Ageing, but not yet senescent, rats exhibit reduced muscle quality and sarcoplasmic reticulum function.

AIM: Reduced muscle force greater than expected from loss of muscle mass has been reported in ageing muscles. Impaired sarcoplasmic reticulum (SR) Ca(2+) release has been implicated as a possible mechanism, and attributed to several factors, including loss of ryanodine receptor (RYR) expression and protein binding. The aim of this study was to evaluate muscle quality and SR Ca(2+) release in ageing rats that were not so old that major atrophy had occurred. METHODS: We collected in situ force data from the plantarflexor muscle group and muscle mass from the constituent muscles to determine muscle quality (force/mass) in adult (6-8 months) and ageing (24 months) rats (n=8/group). We evaluated SR Ca(2+) uptake and release, and determined expression of key proteins associated with Ca(2+) release [RYR and FK506 binding protein (FKBP)] and uptake (SERCA, parvalbumin, calsequestrin). RESULTS: Plantarflexor force and muscle quality were reduced with ageing (approx. 28 and 34%, respectively), but atrophy was limited, and significant only in the medial gastrocnemius (approx. 15%). The fast phase of SR Ca(2+) release was reduced with ageing in both gastrocnemii, as was FKBP expression and FKBP-RYR binding, but RYR expression was not affected. Similar, but non-significant changes were present in the plantaris, but the soleus muscle generally showed no ageing-related changes. CONCLUSION: These data suggest a possible role for impaired SR Ca(2+) release in ageing-related loss of muscle quality, although not through loss of RYR expression.

Calcium enhanced delivery of tetraarginine-PEG-lipid-coated DNA/protamine complexes.

As we have previously reported the delivery of plasmid DNA (DNA) complexed with oligoarginine-PEG artificial lipids (oligoarginine/DNA complexes), we focused on tetra- and decaarginine (Arg4, Arg10) to improve transfection efficiency by both the formation of oligoarginine-coated DNA complexed with protamine (PD), and the addition of Ca(2+) after formation of complexes. The efficiency of DNA condensation was determined by gel electrophoresis. Cellular uptake and transfection efficiency were evaluated in human cervical carcinoma HeLa cells using flow cytometry and luciferase assay. Oligoarginine-coated PD enhanced transfection efficiency significantly more than complexes where Arg10 in both vectors exhibited higher transfection efficiency than Arg4. As assessed by gel retardation assay, high gene expression by Arg10 may be explained by Arg4 binding DNA more strongly than Arg10. The addition of Ca(2+) to incubation medium increased transfection efficiency of Arg4-coated PD 70-fold, similar to that of Arg10-coated PD alone without an increase of cellular uptake, suggesting that Ca(2+) induced the release of DNA from complexes in endosomes. Only Arg4 with low cytotoxicity could gain an advantage from Ca(2+) in transfection, but Arg10 with relatively high cytotoxicity could not. The present results demonstrate that Arg4-coated PD with Ca(2+) has great potential as an efficient non-viral vector with low toxicity.

Subregions of human MT complex revealed by comparative MEG and direct electrocorticographic recordings.

OBJECTIVE: To locate the visual motion complex (MT+) and study its response properties in an epilepsy surgery patient. METHODS: A 17-year-old epilepsy patient underwent invasive monitoring with subdural electrodes in the right temporo-parieto-occipital area. MT+ was investigated by cortical electric stimulation and by epicortical visual evoked potentials time-locked to motion onset of sinusoidal gratings (motion VEP). Motion-related visual evoked magnetic field (motion VEF) was also recorded before the electrode implantation to complement the invasive recording. RESULTS: Motion VEPs revealed two subregions within MT+, generating early and late potentials respectively. The early activity with a peak around 130 ms was localized at a single electrode situated immediately caudal to the initial portion of the ascending limb of the superior temporal sulcus (AL-STS). The late activity, peaking at 242-274 ms, was located ventro-rostrally over three electrodes. Among the four electrodes with motion VEPs, cortical stimulation at the most caudal pair elicited motion-in-depth perception involving the whole visual field. In addition to two subregions revealed on the gyral crown, magnetoencephalography (MEG) demonstrated another subregion with a late motion VEF in AL-STS immediately rostral to the electrode with the early motion VEP. CONCLUSIONS: In combination with MEG recording, the present invasive exploration demonstrated human MT+ in a focal area of the temporo-parieto-occipital junction and delineated possible three subregions as indicated by the different latencies and distributions of the motion VEP/VEFs. SIGNIFICANCE: Comparative MEG and direct electrocorticographic recordings delineated possible subregions within the human MT complex.

Abnormal cortical mechanisms of voluntary muscle relaxation in patients with writer's cramp: an fMRI study.

Although it is hypothesized that there is abnormal motor inhibition in patients with dystonia, the question remains as to whether the mechanism related to motor inhibition is specifically impaired. The objective of the present study was to clarify the possible abnormalities of the mechanisms underlying voluntary muscle relaxation during motor preparation and execution in patients with writer's cramp, using event-related functional MRI. Eight patients with writer's cramp and 12 age-matched control subjects participated in the study. Two motor tasks were employed as an experimental paradigm. In the relaxation task, subjects were asked to hold their right wrist in the horizontal plane by maintaining moderate contraction of wrist extensor muscles in the premotor phase; they relaxed those muscles voluntarily just once during each fMRI scanning session. In the contraction task, subjects extended the right wrist voluntarily from the same premotor state as for the relaxation task. Five axial images covering the primary sensorimotor cortex (SMC) and supplementary motor area (SMA) were obtained once every second. Activated volumes in the left SMC and the SMA were significantly reduced in patients for both muscle relaxation and contraction tasks. These data suggest that there is impaired activation in both SMC and SMA in voluntary muscle relaxation and contraction in patients with writer's cramp. This implies that abnormalities of both inhibitory and excitatory mechanisms in motor cortices might play a role in the pathophysiology of focal dystonia.

Coherence between cortical and muscular activities after subcortical stroke.

BACKGROUND AND PURPOSE: Functional connection between the motor cortex and muscle can be measured by electroencephalogram-electromyogram (EEG-EMG) coherence. To evaluate the functional connection to muscle between contralateral and ipsilateral motor cortices after pyramidal tract lesions, we investigated 6 patients with chronic subcortical stroke. METHODS: High-resolution EEG and EMG of the hand, forearm, and biceps muscles were recorded during 3 tonic contraction tasks: (1) elbow flexion, (2) wrist extension, and (3) power grip. To evaluate the cortical control of EMG, EEG-EMG coherence was computed. RESULTS: EEG-EMG coherence was localized over the contralateral sensorimotor area in all circumstances, and there was no significant coherence at the ipsilateral side. EEG-EMG coherence was significantly smaller on the affected side for the hand and forearm muscles but not for the biceps muscle. CONCLUSIONS: All direct functional connections to muscle after recovered subcortical stroke come from the contralateral motor cortex. The different effects of the lesion on the proximal and distal muscles appear to be associated with the strength of the corticospinal pathway.

Site-dependent effect of O-glycosylation on the conformation and biological activity of calcitonin.

We synthesized seven O-glycosylated calcitonin derivatives, each with a single GalNAc residue attached to either Ser or Thr, and studied their three-dimensional structure and biological activity to examine site-dependent effects of O-glycosylation. The CD spectra in an aqueous trifluoroethanol solution showed that the GalNAc attachment at Thr6 or Thr21 reduced the helical content of calcitonin, indicating that the O-glycosylated residue functions as a stronger helix breaker than the original amino acid residue. Only the GalNAc attachment at Ser2 or Thr21 retained the hypocalcemic activity of calcitonin. This result corresponded well to that of the calcitonin-receptor binding assay. The GalNAc attachment other than Ser2 or Thr21 perturbed the interaction with the receptor, resulting in the loss of the hypocalcemic activity. The biodistribution did not change much among the seven derivatives, but some site dependency could also be observed. Thus, we can conclude that the O-glycosylation affects both the conformation and biological activity in a site-dependent manner.

Effect of carbohydrate structure on biological activity of artificially N-glycosylated eel calcitonin.

To reveal the function of the carbohydrate portion of glycopeptides and glycoproteins, we chemo-enzymatically synthesized artificially N-glycosylated derivatives of eel calcitonin and studied their three-dimensional structure and biological activity. The CD and NMR spectra in trifluoroethanol-H(2)O solution showed that the glycosylation did not change the three-dimensional structure. All the derivatives retained the strong in vivo hypocalcemic activity of calcitonin. However, the relative activity was dependent on the structure of the attached carbohydrate. The single GlcNAc attachment best enhanced the activity, while larger carbohydrates decreased the activity. This relative activity order of compounds could be partly explained by their calcitonin-receptor binding affinity, though the affinity of the GlcNAc derivative did not exceed that of calcitonin. The enhanced hypocalcemic activity of the GlcNAc derivative was explained by its altered biodistribution. The GlcNAc attachment caused calcitonin to escape from the trap at the liver during the early circulation. Thus, the glycosylation was shown to modulate the biological activity of calcitonin depending on the carbohydrate structure without a change in the peptide backbone conformation.

Skeletal muscle adaptations in elastic resistance-trained young men and women.

Skeletal muscle adaptations (fiber-type composition, cross-sectional area, myosin heavy chain (MHC) content, and capillarity) were assessed in the vastus lateralis muscle of young men and women after 8 weeks of training with the Sportcord, an elastic resistance device. Ten men [mean (SD) age 20 (1.1) years] and 13 women [20 (1.2) years] performed two sets each to failure of single leg squats and leg extensions at approximately 50 repetitions x min(-1). Biopsy samples were taken from the right vastus lateralis muscle before and after training. Six fiber types (I, IC, IIC, IIA, IIAB, and IIB) were classified using myofibrillar ATPase histochemistry. Training with the Sportcord caused a small, but significant, increase in one-repetition maximum using free weights and a large increase in repetitions to failure. In addition, elastic resistance training caused an increase in the percentage of fibers classified as type IIAB for both men and women, and a decrease in the percentage of type IIB fibers in the men. MHC analysis supported these findings (a significant increase in the percentage of MHCIIa for the men). The cross-sectional areas ofboth the type I and IIAB + IIB fibers increased after training for the men, whereas no area changes were found for the women. The capillary:fiber ratio and capillary contacts per fiber type increased significantly for the men, and similar trends were noted for the women. Capillary density did not change in either the men or the women. These data suggest minor changes in fiber type composition (IIB-->IIAB), fiber size, and capillarization following short-term training with elastic resistance. Although muscular changes did occur using the Sportcord, the extent of these changes was less than those reported previously for short-term resistance-training programs using free weights.

Desynchronization and synchronization of central 20-Hz rhythms associated with voluntary muscle relaxation: a magnetoencephalographic study.

To clarify the cortical mechanisms involved in motor inhibition, modulation of cortical rhythms around 20 Hz during voluntary muscle relaxation was compared to that during muscle contraction in ten normal volunteers, using a whole head type neuromagnetometer. Each subject relaxed or contracted the right forearm muscles, from which electromyograms were recorded. After bandpass filtering magnetoencephalographic signals into frequency bands of 6-10, 10-14, 14-18, 18-22, 22-26, and 26-30 Hz, the signals of each frequency band were rectified and averaged with respect to the onset of motor trial. The relaxation task showed movement-related 20-Hz desynchronization over bilateral central areas beginning a few seconds before the termination of muscle contraction. Twenty-hertz desynchronization was present also for the contraction task at the same location within each subject. The two tasks had a positive correlation among subjects in both the percent decrease (r2=0.76, P<0.01) and the peak time (r2=0.61, P<0.05) for the contralateral 20-Hz desynchronization. After the muscle relaxation, desynchronization was followed by conspicuous movement-related synchronization of the 20-Hz passband in the contralateral central areas, which was significantly larger than that after the contraction (P<0.001). The results suggest that the voluntary muscle relaxation involves the modulation of central rhythms starting a few seconds before the actual event, and the 20-Hz desynchronization has a similar temporal property in the muscle relaxation and contraction. The 20-Hz synchronization in the contralateral central area after the muscle relaxation may be associated with the temporally arrayed termination of the ongoing muscle contraction.

Effects of high-intensity resistance training on untrained older men. I. Strength, cardiovascular, and metabolic responses.

Most resistance training studies of older subjects have emphasized low-intensity, short-term training programs that have concentrated on strength measurements. The purpose of this study was, in addition to the determination of strength, to assess intramuscular and transport factors that may be associated with strength increments. Eighteen untrained men ages 60-75 years volunteered for the study; 9 were randomly placed in the resistance-training group (RT), and the other half served as untrained (UT) or control subjects. RT subjects performed a 16-week high-intensity (85-90% 1 repetition maximum (RT]) resistance training program (2 x/wk) consisting of 3 sets each to failure (6-8 repetitions based on 1 RM of 3 exercises): leg press (LP), half squat (HS), and leg extension (LE) with 1-2 minutes rest between sets. Pre- and post- training strength was measured for the 3 training exercises using a 1 RM protocol. Body fat was calculated using a 3-site skinfold method. Biopsies from the vastus lateralis m. were obtained for fiber type composition, cross-sectional area, and capillarization measurements. Exercise metabolism, electrocardiography, and arterial blood pressure were observed continuously during a progressive treadmill test, and resting echocardiographic data were recorded for all subjects. Pre- and post-training venous blood samples were analyzed for serum lipids. Resistance training caused significant changes in the following comparisons: % fat decreased in the RT group by almost 3%, strength improved for all exercises: LE = + 50.4%, LP = + 72.3%, HS = + 83.5%; type IIB fibers decreased and IIA fibers increased; cross-sectional areas of all fiber types (I, IIA, IIB) increased significantly, and capillary to fiber ratio increased but not significantly. No differences were noted for ECG and echocardiographic data. The RT group significantly improved treadmill performance and VO2max. Pre- and post-training serum lipids improved but not significantly. No significant changes occurred in any pre- to post-tests for the UT group. The results show that skeletal muscle in older, untrained men will respond with significant strength gains accompanied by considerable increases in fiber size and capillary density. Maximal working capacity, VO2max, and serum lipid profiles also benefited from high-intensity resistance training, but no changes were observed for HR max, or maximal responses of arterial blood pressure. Older men may not only tolerate very high intensity work loads but will exhibit intramuscular, cardiovascular, and metabolic changes similar to younger subjects.

Participation of the left posterior inferior temporal cortex in writing and mental recall of kanji orthography: A functional MRI study.

To examine the neuropsychological mechanisms involved in writing kanji (morphograms), we used functional MRI (fMRI) in 10 normal volunteers, all right-handed, native Japanese speakers. The experimental paradigms consisted of kana-to-kanji transcription, mental recall of kanji orthography and oral reading and semantic judgement of kana words. The first two tasks require manual and mental transcription of visually presented kana words into kanji, respectively, whereas the last two tasks involve language processing of the same set of stimulus words without recall of kanji. The transcription and mental recall tasks yielded lateralized activation of the left posterior inferior temporal cortex (PITC). By contrast, neither oral reading nor semantic judgement produced similar activation of the area. These results, in good accordance with lesion data, provide converging evidence that the left PITC plays an important role in writing kanji through retrieval of their visual graphic images, and suggest language-specific cerebral organization of writing. The set of fMRI experiments also provides new neuroimaging data on the cortical localization of basic language functions in people using a non-alphabetical language.

Fiber type composition of the vastus lateralis muscle of young men and women.

This study presents data collected over the past 10 years on the muscle fiber type composition of the vastus lateralis muscle of young men and women. Biopsies were taken from the vastus lateralis muscle of 55 women (21.2+/-2.2 yr) and 95 men (21.5+/-2.4 yr) who had volunteered to participate in various research projects. Six fiber types (I, IC, IIC, IIA, IIAB, and IIB) were classified using mATPase histochemistry, and cross-sectional area was measured for the major fiber types (I, IIA, and IIB). Myosin heavy chain (MHC) content was determined electrophoretically on all of the samples from the men and on 26 samples from the women. With the exception of fiber Type IC, no significant differences were found between men and women for muscle fiber type distribution. The vastus lateralis muscle of both the men and women contained approximately 41% I, 1% IC, 1% IIC, 31% IIA, 6% IIAB, and 20% IIB. However, the cross-sectional area of all three major fiber types was larger for the men compared to the women. In addition, the Type IIA fibers were the largest for the men, whereas the Type I fibers tended to be the largest for the women. Therefore, gender differences were found with regard to the area occupied by each specific fiber type: IIA>I>IIB for the men and I>IIA>IIB for the women. These data establish normative values for the mATPase-based fiber type distribution and sizes in untrained young men and women.

Humoral immunity against the proline-rich peptide epitope of the IgA1 hinge region in IgA nephropathy.

BACKGROUND: The human IgA1 hinge region is a unique mucin-like O-linked proline-rich glycopeptide, and its core peptide was found to be exposed aberrantly by the underglycosylation in IgA nephropathy (IgAN). We describe here the presence of humoral immunity against the IgA1 hinge peptide epitope in IgAN and evaluate the relationship between the underglycosylation of the IgA1 hinge region and humoral immunity. METHOD: The serum anti-IgA1 hinge peptide antibody (anti-alpha1HP ab) titre was measured and compared between the IgAN (n=37) and control groups (n=34) by enzyme-linked immunosorbent assay (ELISA) using a synthetic peptide corresponding to the human IgA1 hinge region, PVPSTPPTPSPSTPPTPSPS, as an antigen. Next, to evaluate the relationship between the underglycosylation of the IgA1 hinge region and the humoral immunity, the reactivity of the serum IgG from the patients with IgAN against monoclonal IgA1 which had been digested enzymatically to remove the carbohydrates from the IgA1 hinge region was measured by ELISA. RESULTS: The anti-alpha1HP ab titre was significantly higher in the IgAN group than in the control group (OD value: IgG class, 0.564+/-0.344 vs 0. 331+/-0.154, P=0.0014; IgM class, 0.272+/-0.148 vs 0.141+/-0.072, P<0.0001) and it was positive in approximately 40% of the patients with IgAN. In addition, the reactivity of the serum IgG from the IgAN patients against the monoclonal IgA1 was found to be increased as the carbohydrates were enzymatically removed from the IgA1 hinge region (when native=100; asialo, 122+/-9.5; agalacto, 167+/-11.5; naked, 188+/-3.9). CONCLUSION: These results suggested that the peptide epitope of the IgA1 hinge region which was aberrantly exposed by underglycosylation could induce the humoral immune response in IgAN.

Transient neural activity in the medial superior frontal gyrus and precuneus time locked with attention shift between object features.

To investigate the contribution of the superior frontal gyrus and precuneus to the cognitive process of attention set shift, we examined the correlation between change in neural activity in these areas and the timing of attention set shift using event-related functional magnetic resonance imaging. Seven subjects underwent a card-sorting task in which they matched a test card to one of two target cards according to color or shape. The subjects had to determine the correct category based only on feedback and shift the sorting principle when the feedback changed from "correct" to "incorrect." Transient increase of neural activity time locked with attention shift phases was detected in the medial superior frontal gyrus (the rostral part of the supplementary motor area) and precuneus. During the control task, in which the feedback and the motor responses were preserved without any attention shift, this type of change in neural activity was not observed. Our findings indicate that increase in neural activity in these brain areas may be closely related to attention set shift between object features and suggest that these areas may play a role in the shifting of cognitive sets.

Study of the relationship between sticky human serum IgA1 and its O-glycan glycoform.

The high-affinity IgA1 toward jacalin was mostly composed of aggregated IgA1 and abundantly contained the asialo disaccharide, Galbeta1,3GalNAc, in the O-linked oligosaccharide in the hinge region [Journal of Biochemistry 120, 92-97 (1996)]. Meanwhile, the removal of sialic acid from IgA1 accelerated the aggregation of the IgA1 molecule [J. Am. Soc. Nephrol. 9, 2048-2054 (1998)]. In order to examine the nature of such a sticky IgA1, affinity chromatography using asialo-IgA1 (deSIgA1)-Sepharose was carried out. Seventeen percent of normal human serum IgA1, 27% of asialo-IgA1 (IgA1-S), and 48% of asialo-, agalacto-IgA1 (IgA1-SG) were bound to the column. Removal of the N-acetylgalactosamine residue from IgA1-SG resulted in a decreasing affinity toward deSIgA1-Sepharose. Thus, the binding ability toward the column was the highest for the IgA1-SG among the deglycosylated IgA1s. On the other hand, heat treatment of IgA1 accelerated the aggregation but decreased its binding ability toward the column. Such heat denaturation probably destroys the structure of the binding site. Since the enzymatic removal of the N-glycan sugar chains did not induce the aggregation and exhibited no effect on the binding, the incomplete O-linked sugar chain on the hinge portion should be directly related to the sticky characteristics of the IgA1 molecule. The binding was non-covalent and not strong because the asialo-, agalacto-hinge glycopeptide was eluted slightly slower than the native one from the column and the bound IgA1 was dissociated in the presence of 1 M NaCl.

Abnormal cortical processing of voluntary muscle relaxation in patients with focal hand dystonia studied by movement-related potentials.

In order to clarify the abnormality in cortical motor preparation for voluntary muscle relaxation of the hand in patients with focal hand dystonia, Bereitschaftspotentials (BPs) preceding voluntary muscle contraction and relaxation were recorded in eight patients (three with simple writer's cramp and five with dystonic writer's cramp), and were compared with those from 10 normal subjects. Voluntary muscle relaxation: after keeping the right wrist in an extended position for > 5 s, the subject let the hand drop by voluntarily terminating muscle contraction of the wrist extensor without any associated muscle contraction. Voluntary muscle contraction: the right wrist was flexed by voluntarily contracting the wrist flexor muscle. Scalp EEGs were recorded from 11 electrodes placed over the frontal, central and parietal areas. In the control group, the BP measured at the movement onset was maximal at the left central area (C1), and distributed predominantly over the left hemisphere equally in both the contraction and relaxation tasks. In the focal hand dystonia group, BP was maximal at C1 in the contraction task, whereas, in the relaxation task, it was maximal at the midline central area (Cz) and symmetrically distributed. At the left central area, the BP amplitude in the focal hand dystonia group was diminished significantly in the relaxation task compared with the contraction task (P < 0.05). The present results demonstrate for the first time that the cortical preparatory process for voluntary muscle relaxation, or motor inhibition, is abnormal in focal hand dystonia.

Effects of glycosylation on the structure and dynamics of eel calcitonin in micelles and lipid bilayers determined by nuclear magnetic resonance spectroscopy.

The three-dimensional structures of eel calcitonin (CT) and two glycosylated CT derivatives, [Asn(GlcNAc)3]-CT (CT-GlcNAc) and [Asn(Man6-GlcNAc2)3]-CT (CT-M6), in micelles were determined by solution NMR spectroscopy. The topologies of these peptides associated with oriented lipid bilayers were determined with solid-state NMR. All of the peptides were found to have an identical conformation in micelles characterized by an amphipathic alpha-helix consisting of residues Ser5 through Leu19 followed by an unstructured region at the C-terminus. The overall conformation of the peptide moiety was not affected by the glycosylation. Nevertheless, comparison of the relative exchange rates of the Leu12 amide proton might suggest the possibility that fluctuations of the alpha-helix are reduced by glycosylation. The presence of NOEs between the carbohydrate and the peptide moieties of CT-GlcNAc and CT-M6 and the amide proton chemical shift data suggested that the carbohydrate interacted with the peptide, and this might account for the conformational stabilization of the alpha-helix. Both the unmodified CT and the glycosylated CT were found to have orientations with their helix axes parallel to the plane of the lipid bilayers by solid-state NMR spectroscopy.

Mutual separation of hinge-glycopeptide isomers bearing five N-acetylgalactosamine residues from normal human serum immunoglobulin A1 by capillary electrophoresis.

Immunoglobulin A1 (IgA1) from normal human serum is known to have O-linked sugar chains, sialylated Galbeta1,3GalNAc, in the hinge portion. In order to reduce the microheterogenity of the sugar chain, the hinge glycopeptide prepared from IgA1 was sequentially treated with neuraminidase and beta-galactosidase. The asialo-, agalacto-hinge glycopeptide (HGP-SG) composed of a 33-mer peptide (HP33) and N-acetylgalactosamine (GalNAc) residues was obtained. The HGP-SG was separated into three major peaks, A, B and C, by high-performance liquid chromatography (HPLC). Each glycopeptide fraction was further separated by capillary electrophoresis (CE). Peaks A, B and C with HPLC abundantly contained HP33 bearing five and six N-acetylgalactosamine residues (HGP33-5,6GN), HGP33-4,5GN and HGP33-3,4GN, respectively. Among these glycopeptide peaks, only the HGP33-5GN peak was partly split into two peaks based on the CE analysis - HGP33-5GN-alpha and -beta. The glycopeptide, HGP25-5GN shortened by the thermolysin digest of HGP33-SG was also well separated into the alpha and beta forms by CE analysis. No differences in their mass and peptide portion were observed between HGP25-5GN-alpha and -beta. Therefore, the obtained result might indicate that HGP25-5GN-alpha was an isomer of HGP25-5GN-beta differing in its stereospecific structure of the peptide portion and/or the attachment site of the GalNAc residue.