Effects of medium-chain fatty acids and tributyrin supplementation in milk replacers on growth performance, blood metabolites, and hormone concentrations in Holstein dairy calves.

This study aimed to evaluate the effects of triglycerides containing medium-chain fatty acids (MCT) and tributyrin (TB) supplementation in a milk replacer (MR) on growth performance, plasma metabolites, and hormone concentrations in dairy calves. Sixty-three Holstein heifer calves (body weight at 8 d of age, 41.1 ± 2.91 kg; mean ± SD) were randomly assigned to 1 of 4 experimental MR (28% crude protein and 18% fat): (1) containing 3.2% C8:0 and 2.8% C10:0 (in fat basis) without TB supplementation (CONT; n = 15), (2) containing 6.7% C8:0 and 6.4% C10:0 without TB supplementation (MCT; n = 16), (3) containing 3.2% C8:0 and 2.8% C10:0 with 0.6% (dry matter basis) TB supplementation (CONT+TB; n = 16), (4) containing 6.7% C8:0 and 6.4% C10:0 with 0.6% TB supplementation (MCT+TB; n = 16). The MR were offered at 600 g/d (powder basis) from 8 to 14 d, up to 1,300 g/d from 15 to 21 d, 1,400 g/d from 22 to 49 d, down to 700 g/d from 50 to 56 d, 600 g/d from 57 to 63 d, and weaned at 64 d of age. All calves were fed calf starter, chopped hay, and water ad libitum. The data were analyzed using a 2-way ANOVA via the fit model procedure of JMP Pro 16 (SAS Institute Inc.). Medium-chain fatty acid supplementation did not affect the total dry matter intake. However, calves that were fed MCT had greater feed efficiency (gain/feed) before weaning (0.74 ± 0.098 vs. 0.71 ± 0.010 kg/kg) compared with non-MCT calves. The MCT calves also had a lower incidence of diarrhea compared with non-MCT calves during 23 to 49 d of age and the weaning period (50 to 63 d of age; 9.2% vs. 18.5% and 10.5% vs. 17.2%, respectively). Calves fed with TB had a greater total dry matter intake during postweaning (3,465 vs. 3,232 g/d). Calves fed TB also had greater body weight during the weaning (90.7 ± 0.97 vs. 87.9 ± 1.01 kg) and postweaning period (116.5 ± 1.47 vs. 112.1 ± 1.50 kg) compared with that of non-TB calves. The plasma metabolites and hormone concentrations were not affected by MCT or TB. These results suggest that MCT and TB supplementation in the MR may improve the growth performance and gut health of dairy calves.

Novel biallelic mutations in the PNPT1 gene encoding a mitochondrial-RNA-import protein PNPase cause delayed myelination.

Recent studies suggest that impaired transcription or mitochondrial translation of small RNAs can cause abnormal myelination. A polynucleotide phosphorylase (PNPase) encoded by PNPT1 facilitates the import of small RNAs into mitochondria. PNPT1 mutations have been reported in patients with neurodevelopmental diseases with mitochondrial dysfunction. We report here 2 siblings with PNPT1 mutations who presented delayed myelination as well as mitochondrial dysfunction. We identified compound heterozygous mutations (c.227G>A; p.Gly76Asp and c.574C>T; p.Arg192*) in PNPT1 by quartet whole-exome sequencing. Analyses of skin fibroblasts from the patient showed that PNPase expression was markedly decreased and that import of the small RNA RNaseP into mitochondria was impaired. Exogenous expression of wild-type PNPT1, but not mutants, rescued ATP production in patient skin fibroblasts, suggesting the pathogenicity of the identified mutations. Our cases expand the phenotypic spectrum of PNPT1 mutations that can cause delayed myelination.

Diagnosis and molecular basis of mitochondrial respiratory chain disorders: exome sequencing for disease gene identification.

Mitochondrial disorders have the highest incidence among congenital metabolic diseases, and are thought to occur at a rate of 1 in 5000 births. About 25% of the diseases diagnosed as mitochondrial disorders in the field of pediatrics have mitochondrial DNA abnormalities, while the rest occur due to defects in genes encoded in the nucleus. The most important function of the mitochondria is biosynthesis of ATP. Mitochondrial disorders are nearly synonymous with mitochondrial respiratory chain disorder, as respiratory chain complexes serve a central role in ATP biosynthesis. By next-generation sequencing of the exome, we analyzed 104 patients with mitochondrial respiratory chain disorders. The results of analysis to date were 18 patients with novel variants in genes previously reported to be disease-causing, and 27 patients with mutations in genes suggested to be associated in some way with mitochondria, and it is likely that they are new disease-causing genes in mitochondrial disorders. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.

Femtosecond mode-locked Nd(3+)-doped Ba(Zr,Mg,Ta)O(3) ceramic laser.

We have demonstrated continuous wave (CW) laser operation and the first, to the best of our knowledge, sub-200 fs mode-locked laser operation of Nd(3+)-doped Ba(Zr,Mg,Ta)O(3) ceramic. Its disordered crystalline nature exhibits a broad gain bandwidth of 30 nm with a high-emission cross section. It also has higher thermal and mechanical properties than Nd:glass. In CW operation, a maximum output power of 1.5 W under 6.2 W of absorbed pump power was obtained. In mode-locked operation, a pulse duration of 196 fs with an average power of 60 mW was successfully achieved. The laser spectrum straddled both fluorescence peaks of A-site and B-site Nd(3+) ions.

Living donor liver transplantation from an asymptomatic mother who was a carrier for ornithine transcarbamylase deficiency.

Liver transplantation (LT) has been adopted as a radical treatment for ornithine transcarbamylase deficiency (OTCD), yielding favorable outcomes. Despite the fact that it is an inheritable disease, a blood relative who is heterozygous for the disorder must sometimes be used as a liver donor for living donor LT. There is ongoing discussion regarding the use of heterozygous donors, however, to our knowledge, no cases where donation was determined based on the Ornithine transcarbamylase (OTC) activity before LT have been reported. Between May 2001 and April 2011, 17 patients were indicated for living donor LT because of OTCD at our facility. There were three cases with heterozygous donor candidate (17.6%). All heterozygous candidates underwent a liver biopsy to measure their OTC activity before LT and made efforts to secure the safety of the both donor and recipient. Two of 3 candidates had headaches sometimes, and their activity was less than 40%, and thus they were not employed as the donor. One candidate with 104.4% activity was employed, yielding favorable outcomes. Our current experience supported the effectiveness of our donation criteria, however it is necessary to collect sufficient data on a large number of patients to confirm the safety of the procedure.

Living donor liver transplantation for ornithine transcarbamylase deficiency.

Ornithine transcarbamylase deficiency, the most common urea cycle disorder, causes hyperammonemic encephalopathy and has a poor prognosis. Recently, LT was introduced as a radical OTCD treatment, yielding favorable outcomes. We retrospectively analyzed LT results for OTCD at our facility. Twelve children with OTCD (six boys and six girls) accounted for 7.1% of the 170 children who underwent LDLT at our department between May 2001 and April 2010. Ages at LT ranged from nine months to 11 yr seven months. Post-operative follow-up period was 3-97 months. The post-operative survival rate was 91.7%. One patient died. Two patients who had neurological impairment preoperatively showed no alleviation after LT. All patients other than those who died or failed to show recovery from impairment achieved satisfactory quality-of-life improvement after LT. The outcomes of LDLT as a radical OTCD treatment have been satisfactory. However, neurological impairment associated with hyperammonemia is unlikely to subside even after LT. It is desirable henceforth that more objective and concrete guidelines for OTCD management be established to facilitate LDLT with optimal timing while avoiding the risk of hyperammonemic episodes.

Visualization of Lenticulostriate Arteries on CT Angiography Using Ultra-High-Resolution CT Compared with Conventional-Detector CT.

BACKGROUND AND PURPOSE: The newly developed ultra-high-resolution CT is equipped with a 0.25-mm detector, which has one-half the conventional section thickness, one-half the in-plane detector element width, and one-half the reconstructed pixel width compared with conventional-detector CT. Thus, the ultra-high-resolution CT scanner should provide better image quality for microvasculature than the conventional-detector CT scanners. This study aimed to determine whether ultra-high-resolution CT produces superior-quality images of the lenticulostriate arteries compared with conventional-detector CT. MATERIALS AND METHODS: From February 2017 to June 2017, thirteen patients with aneurysms (4 men, 9 women; mean age, 61.2 years) who underwent head CTA with both ultra-high-resolution CT and conventional-detector CT were enrolled. Two board-certified radiologists determined the number of all lenticulostriate arteries on the CTA coronal images of the MCA M1 segment reconstructed from 512 matrixes on conventional-detector CT and 1024 matrixes on ultra-high-resolution CT. RESULTS: There were statistically more lenticulostriate arteries identified on ultra-high-resolution CT (average, 2.85 ± 0.83; 95% CI, 2.509-3.183) than on conventional-detector CT (average, 2.17 ± 0.76; 95% CI, 1.866-2.480) (P = .009) in 16 of the total 26 MCA M1 segments. CONCLUSIONS: Improvements in lenticulostriate artery visualization were the result of the combined package of the ultra-high-resolution CT scanner plus the ultra-high-resolution scanning protocol, which includes higher radiation doses with lower than the national diagnostic reference levels and stronger adaptive iterative dose-reduction processing. This package for ultra-high-resolution CT is a simple, noninvasive, and easily accessible method to evaluate microvasculature such as the lenticulostriate arteries.

Expression of reticulon 3 in Alzheimer's disease brain.

AIMS: Reticulon 3 (RTN3), a member of the reticulon family of proteins, interacts with the beta-secretase, beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), and inhibits its activity to produce beta-amyloid protein. The aim of the present study was to clarify the biological role of RTN3 in the brain and its potential involvement in the neuropathology of Alzheimer's disease (AD). METHODS: We performed immunohistochemical and biochemical analyses using a specific antibody against RTN3 to investigate the expression and subcellular localization of RTN3 in control and AD brain tissue samples. RESULTS: Western blot analysis revealed no significant differences in the RTN3 levels between control and AD brains. Immunohistochemical staining showed that RTN3 immunoreactivity was predominantly localized in pyramidal neurones of the cerebral cortex. The patterns of RTN3 immunostaining were similar in control and AD cerebral cortices, and senile plaques were generally negative for RTN3. Biochemical subcellular fractionation disclosed that RTN3 colocalized with BACE1 in various fractions, including the endoplasmic reticulum and the Golgi apparatus. Double-immunofluorescence staining additionally indicated that RTN3 was localized in both endoplasmic reticulum and Golgi compartments in neurones. CONCLUSIONS: These results show that RTN3 is primarily expressed in pyramidal neurones of the human cerebral cortex and that no clear difference of RTN3 immunoreactivity is observable between control and AD brains. Our data also suggest that there is considerable colocalization of RTN3 with BACE1 at a subcellular level.

Mite serine protease activates protease-activated receptor-2 and induces cytokine release in human keratinocytes.

BACKGROUND: House dust mites produce serine and cysteine proteases. Mite-derived proteases have been suggested to be involved in the pathogenesis of allergies; however, whether mite-derived serine protease activity can stimulate keratinocytes remains unknown. METHODS: We examined the activation of primary human keratinocytes by serine protease-rich extract of whole mite culture and compared with that by recombinant group 1 allergens (rDer f 1 and rDer p 1), which exclusively exhibit cysteine protease activity. RESULTS: Protease activity of whole mite culture extract (WCE), rDer f 1 and rDer p 1 induced the release of IL-8 and granulocyte-macrophage colony-stimulating factor. Protease activity of WCEs induced a significant upregulation of their mRNA expression but rDer f 1 had much less effect. Protease activity of the WCE stimulated intracellular Ca(2+) mobilization but rDer f 1 and rDer p 1 did not. The mobilization induced by agonists for the human protease-activated receptor (PAR)-2, an agonist peptide or trypsin, was diminished by pre-incubation of keratinocytes with WCE. rDer f 1 inefficiently cleaved a synthetic N-terminal peptide of PAR-2 at different sites from trypsin, but the resultant peptides did not stimulate the release of interleukin-8. CONCLUSIONS: The results suggest that mite-derived serine protease activity may contribute to the pathogenesis of atopic dermatitis by activating keratinocytes via PAR-2 activation but cysteine protease activity of Der f 1 and Der p 1 acts via another mechanism.

Color-Mapping of 4D-CTA for the Detection of Cranial Arteriovenous Shunts.

BACKGROUND AND PURPOSE: 4D CT angiography is increasingly used in clinical practice for the assessment of different neurovascular disorders. Optimized processing of 4D-CTA is crucial for diagnostic interpretation because of the large amount of data that is generated. A color-mapping method for 4D-CTA is presented for improved and enhanced visualization of the cerebral vasculature hemodynamics. This method was applied to detect cranial AVFs. MATERIALS AND METHODS: All patients who underwent both 4D-CTA and DSA in our hospital from 2011 to 2018 for the clinical suspicion of a cranial AVF or carotid cavernous fistula were retrospectively collected. Temporal information in the cerebral vasculature was visualized using a patient-specific color scale. All color-maps were evaluated by 3 observers for the presence or absence of an AVF or carotid cavernous fistula. The presence or absence of cortical venous reflux was evaluated as a secondary outcome measure. RESULTS: In total, 31 patients were included, 21 patients with and 10 without an AVF. Arterialization of venous structures in AVFs was accurately visualized using color-mapping. There was high sensitivity (86%-100%) and moderate-to-high specificity (70%-100%) for the detection of AVFs on color-mapping 4D-CTA, even without the availability of dynamic subtraction rendering. The diagnostic performance of the 3 observers in the detection of cortical venous reflux was variable (sensitivity, 43%-88%; specificity, 60%-80%). CONCLUSIONS: Arterialization of venous structures can be visualized using color-mapping of 4D-CTA and proves to be accurate for the detection of cranial AVFs. This finding makes color-mapping a promising visualization technique for assessing temporal hemodynamics in 4D-CTA.

Akt activation induces epidermal hyperplasia and proliferation of epidermal progenitors.

Various common signaling pathways maintain tissue stem cells, including Notch and Wnt/beta-catenin signals. Phosphoinositide-3 kinase (PI3K)/Akt signaling regulates the 'stemness' of several stem cells in culture, specifically in maintaining embryonic stem and neural stem cells, and in deriving embryonic germ cells from primordial germ cells. We examined the effect of Akt signaling in epidermal cells in transgenic mice expressing an Akt-Mer fusion protein whose kinase activity was conditionally activated by treatment with 4-hydroxytamoxifen (4OHT). The topical application of 4OHT to adult skin of the transgenic mice induced new hair growth in resting phase follicles. In addition, the mice showed hyperplasia in interfollicular epidermis (IFE) and hair follicles, which was presumably caused by the extensive proliferation of keratinocytes in basal layer of IFE and outer root sheath of hair follicles, respectively. The progenitor cell population increased consistently in 4OHT-treated transgenic mice. Our results show that PI3K/Akt signaling induces epidermal hyperplasia and proliferation of epidermal progenitors.

Change in optical properties of stratum corneum induced by protein carbonylation in vitro.

The skin is the frontier against the external environment and continuously exposed to the environmental oxidative stress such as ultraviolet (UV) irradiation. Protein carbonyls are the major oxidative products of protein and may be introduced by reaction with aldehydes derived from lipid peroxide. Acrolein is one of the most reactive aldehydes generated during degradation of lipid peroxides and protein-acrolein adducts have been found in the oxidatively damaged lesion including UV-damaged skin. Recent studies revealed that protein carbonyls are also detected in thin outermost layer of the skin, the stratum corneum (SC). However, the effect of protein carbonylation on the function of SC was still unclear. In this study, we treated the SC sheets of reconstructed human epidermis and porcine epidermis with acrolein in the experimental conditions to explore the influence of protein carbonylation on the SC. Human and porcine SC sheets treated with acrolein showed less transmission at visible light than untreated SC sheets. Attenuated total reflection-infrared spectroscopy with curve fitting analysis of amide I region showed that acrolein induced alterations in protein secondary structure of the porcine SC sheets, which were accompanied by diminished fibrous keratin structure observed by transmission electron microscopy. These results show the possibility that carbonylation of the SC caused by environmental factors is one of factors altering the fibrous structure of keratin and decreasing the light transmission of SC, which changes the quality of the skin appearance.

Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells.

Embryonic stem (ES) cells can self-renew indefinitely without losing their differentiation ability to any cell types. Phosphoinositide-3 kinase (PI3K)/Akt signaling plays a pivotal role in various stem cell systems, including the formation of embryonic germ (EG) cells from primordial germ cells and self-renewal of neural stem cells. Here, we show that myristoylated, active form of Akt (myr-Akt) maintained the undifferentiated phenotypes in mouse ES cells without the addition of leukemia inhibitory factor (LIF). The effects of myr-Akt were reversible, because LIF dependence and pluripotent differentiation activity were restored by the deletion of myr-Akt. In addition, myr-Akt-Mer fusion protein, whose enzymatic activity is controlled by 4-hydroxy-tamoxifen, also maintained the pluripotency of not only mouse but also cynomolgus monkey ES cells. These results clearly demonstrate that Akt signaling sufficiently maintains pluripotency in mouse and primate ES cells, and support the notion that PI3K/Akt signaling axis regulates 'stemness' in a broad spectrum of stem cell systems.

Routine laparoscopic ultrasound can significantly reduce the need for selective intraoperative cholangiography during cholecystectomy.

BACKGROUND: The use of intraoperative cholangiography (IOC), routinely rather than selectively, during laparoscopic cholecystectomy (LC) is controversial. Recent findings have shown laparoscopic ultrasound (LUS) to be safe, quick, and effective not only for screening of the bile duct for stones, but also for evaluating the biliary anatomy. This study aimed to evaluate, on the basis of the LC outcome and the cost of LUS and IOC, whether and how much the routine use of LUS would be able to reduce the need for IOC. METHODS: During LC, LUS was used routinely to screen the bile duct for stones and to evaluate the biliary anatomy, whereas IOC was used selectively only when LUS was unsatisfactory or unsuccessful. RESULTS: For 193 (96.5%) of 200 patients, LUS was completed successfully, whereas IOC was needed for 7 patients (3.5%). Bile duct stones were identified in 20 patients (10%). For the detection of bile duct stones, LUS yielded 19 true-positive, 175 true-negative, 0 false-positive, and 1 false-negative results. It had a sensitivity of 95%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 99.4%. The postoperative complications included bile leaks from the liver bed in two patients and a retained bile duct stone in one patient. If IOC had been used selectively in a traditional manner on the basis of preoperative risk factors, IOC would have been needed for 77 patients (38.5%). The total cost of LUS plus IOC for the current 200 patients was 26,256 dollars. The total estimated cost of selective IOC, if it had been performed for the 77 patients, would have been 31,416 dollars. CONCLUSIONS: Routine LUS accurately diagnosed bile duct stones and significantly reduced the need for selective IOC from a potential 38.5% to an actual 3.5% without adversely affecting the outcome of the LC or increasing the overall cost. The routine use of LUS during LC is accurate and cost effective.

Liver Transplantation for Mitochondrial Respiratory Chain Disorder: A Single-Center Experience and Excellent Marker of Differential Diagnosis.

Mitochondrial respiratory chain disorder (MRCD) can cause liver failure requiring liver transplantation (LT), although it is often difficult to diagnose before LT. From 2005 to 2016, 9 MRCD patients with the median age at LT of 6 months underwent LT in our institute. Their clinical courses were retrospectively reviewed and the laboratory parameters were compared between the MRCD patients and 10 patients with acute liver failure unrelated to MRCD (non-MRCD). Five patients had extrahepatic manifestations, including developmental disorders in 3 and failure to thrive in 3, before LT. Only 3 patients (33.3%) were diagnosed before LT. Between MRCD and non-MRCD, lactate was significantly high and lactate-to-pyruvate ratio (L/P ratio) tended to be higher in MRCD. From the receiver operating characteristic curve, the optimal cutoff value of lactate was 50.0 mg/dL and that of L/P ratio was 23.2. Patient survival rate of MRCD was 77.8%, although 2 patients with mitochondrial depletion syndrome suffered from de novo pulmonary hypertension after LT. Our experiences showed the difficulty of preoperative diagnosis, and preoperative extrahepatic manifestations did not always mean poor outcome. Our study showed that lactate value and L/P ratio can be excellent predictors of MRCD.

Significant correlations between the flow volume of patent ductus venosus and early neonatal liver function: possible involvement of patent ductus venosus in postnatal liver function.

BACKGROUND: The biochemical features of portosystemic venous shunt with high flow volume are hypergalactosaemia, hyperammonaemia, prolonged blood coagulation time, and raised serum bile acid concentration. The ductus venosus remains open with shunt flow in most neonates for a certain period after birth. However, the effects of blood flow through the ductus venosus on neonatal liver function remain unclear. OBJECTIVE: To elucidate the effect of patency of the ductus venosus on liver function in early neonates. METHODS: Subjects were divided into three groups by gestational age (group I, 29-32 weeks; group II, 33-36 weeks; group III, 37-41 weeks). The shunt flow volume through the ductus venosus was examined serially using ultrasonography, and correlations between flow volume and liver function in the respective groups were calculated during the first week after birth. RESULTS: Group I had a higher flow volume and later functional closure than the other two groups. Plasma ammonia and serum total bile acid concentrations correlated with flow volume in groups I and II, and blood galactose and galactose 1-phosphate concentrations correlated significantly with flow volume in group III. Percentage hepaplastin also correlated significantly with flow volume in all groups, but plasma vitamin K concentration did not in any group. CONCLUSIONS: Patent ductus venosus has a considerable effect on crucial liver functions such as ammonia detoxification, blood coagulation, and regulation of serum total bile acid concentration in early neonates.

De novo guanylate synthesis in the commitment to replication in hepatoma 3924A cells.

This work tested the relationship of guanylate and adenylate biosynthesis during the display of the proliferative program of rat hepatoma 3924A cells. Since serine, the major source of one-carbon units, competed with the substrate [14C]formate for purine labeling, serine-free medium was used in the assays. The initial rates of purine de novo synthesis with [14C]formate or L-[3-14C]serine followed Michaelis-Menten kinetics yielding similar Vmax values with apparent Kms of 0.5 and 0.038 mM, respectively. During the transition of cancer cells from plateau phase into logarithmic proliferation the specific activity of 5-phosphoribosyl 1-pyrophosphate synthase (EC 2.7.6.1, ribose phosphate pyrophosphokinase) increased 2.2-fold, followed by a 14-fold elevation of the concentration of 5-phosphoribosyl 1-pyrophosphate with a subsequent 8-fold rise in de novo purine synthesis. The ratio of guanylate to adenylate synthesis from IMP in plateau phase cells was 0.24 to 1. After replating the resting cells there was a sharp increase in the relative labeling of guanylates with a concurrent marked decrease in that of the adenylates, reaching an 8-fold rise in the ratio of guanylate to adenylate synthesis from IMP at the maximum deviation in the late lag phase at 20 to 24 h after seeding. This striking redirection in the distribution of label from IMP utilization to the preferential synthesis of guanylates during the expression of the biochemical proliferative program of cancer cells supports the potential significance of this pathway as a target of chemotherapy.

Qualitative differences in position of sialylation and surface expression of glycolipids between murine lymphomas with low metastatic (Eb) and high metastatic (ESb) potentials and isolation of a novel disialoganglioside (GD1 alpha) from Eb cells.

Glycolipids of murine lymphoma cell lines with low metastatic (Eb) and high metastatic (ESb) potentials have been investigated. The Eb cell line was characterized by a high quantity of gangliotriaosylceramide (Gg3), gangliotetraosylceramide (Gg4), GM1b, and a new type of disialoganglioside, termed GD1 alpha. In contrast, the high metastatic ESb cell line was characterized by the absence of these glycolipids and instead by the presence of GM3, GM2, GM1a, GD1a, and GD1b gangliosides. A clear cell surface reactivity with monoclonal antibody anti-Gg3 (2D4) was observed only in Eb cells. Thus, Eb cells are distinct from ESb cells in their ability to add the GalNAc residue to LacCer, supplying Gg3 for synthesis of a series of glycolipids via an asialogangliotetraosyl pathway, while ESb cells are capable of synthesizing GM3, which initiates synthesis of ganglio-series gangliosides GM2, GM1a, GD1a, and GD1b. While disialogangliosides of ESb cells were identified as GD1a and GD1b, a disialoganglioside isolated from Eb cells was characterized as having a novel structure (referred to as GD1 alpha) as follows: (formula; see text) Thus, Eb and ESb cells are clearly different in their qualitative sialylation patterns, i.e., the position of sialic acid residues. Cell surface labeling with galactose-oxidase/NaB[3H]4 revealed a high exposure of Gg3 and Gg4 at the Eb cell surface, while both labels were absent in ESb cells. In contrast, ESb cells showed a substantial label at GM1a, which was greatly enhanced after sialidase treatment.