Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs.

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

Using egg yolk in a TRIS-Equex STM paste extender for freezing of dog semen is superior to egg yolk plasma, also after addition of lecithin and catalase.

We compared the results of using egg yolk plasma (EYP) instead of egg yolk (EY) in a TRIS-based Equex STM Paste freezing extender system for dog semen [25]. We also tested whether the addition of lecithin and catalase to the EYP extenders would improve results. Fractionated semen collection was done in 17 stud dogs and the sperm rich fraction diluted with different extenders in 2 steps: (I) TRIS-fructose-citric acid extender (TRIS) containing 20% egg yolk (EY) and 3% glycerol [25], (II) TRIS containing 20% egg yolk plasma (EYP) and 3% glycerol, and (III) TRIS containing 20% EYP and 0.8% lecithin (EYP-L) and 3% glycerol. After equilibration the second dilution step was done: samples with (I) were diluted with TRIS-EY with 7% glycerol and 1% Equex STM paste [25]; samples with (II) and (III) were divided in 2 aliquots each, and one part diluted with TRIS-EYP or TRIS-EYP-L, both containing 7% glycerol and 1% Equex STM paste, and the other one part with the same extenders containing additionally 300 I.U./mL catalase. After freezing and thawing, samples were analyzed by CASA and sperm chromatin structure assay (SCSA); reactive oxygen species (ROS), degree of apoptosis and zona binding ability were determined. Semen samples with TRIS-EY with a final concentration of 5% glycerol and 0.5% Equex STM paste [25] showed best post thaw progressive motility (P), most intact cells, lowest percentage of ROS, acrosome damages, dead and apoptotic cells. Curvilinear velocity (VCL), DNA fragmentation, morphological abnormalities and zona binding ability did not differ between groups. Replacement of egg yolk by EYP increased the ROS and late apoptotic cells. Addition of lecithin and catalase to EYP containing extenders decreased motility and increased complete apoptosis. We conclude that egg yolk is superior to EYP in the here investigated extenders. The TRIS-based extender [25] with EYP could not be improved by addition of lecithin and catalase; however, in-vivo fertilization capacity of the here examined extenders remains to be investigated.

Performance of the Michigan Hand Outcomes Questionnaire in hand osteoarthritis.

OBJECTIVE: To investigate the performance of the Michigan Hand Outcomes Questionnaire (MHQ) in hand osteoarthritis (OA) by evaluating truth, discrimination and feasibility. DESIGN: Symptomatic hand OA patients from the Hand Osteoarthritis in Secondary Care (HOSTAS) cohort completed questionnaires (demographics, MHQ, Australian/Canadian Hand Osteoarthritis Index [AUSCAN], Functional Index for Hand Osteoarthritis [FIHOA] and visual analogue scale [VAS] pain) at baseline (n = 383), 1- and 2-year follow-up (n = 312, n = 293). Anchor questions at follow-up assessed whether pain/function levels were (un)acceptable and had changed compared to baseline. Correlations between MHQ and other pain/function questionnaires were calculated. Validity of unique MHQ domains (work performance, aesthetics, satisfaction), discrimination across disease stages, and responsiveness were assessed by categorizing patients by external anchors (employment, joint deformities, erosions, and anchor questions). Between-group differences were assessed with linear regression, probability plots and comparison of medians. RESULTS: MHQ pain and function subscales correlated moderately-to-good with other instruments (rs 0.63-0.81). Work performance scores were worse in patients with reduced working capacity than in employed patients. Aesthetics scores were worse in patients with more deformities. Patients with unacceptable complaints had worse satisfaction scores. All pain/function instruments discriminated between patients with acceptable vs unacceptable pain/function, while only MHQ activities of daily living (ADL), FIHOA, and MHQ aesthetics could discriminate between erosive and non-erosive disease. MHQ and AUSCAN were most responsive. CONCLUSIONS: MHQ has several unique aspects and advantages justifying its use in hand OA, including the unique assessment of work performance, aesthetics, and satisfaction. However, MHQ, AUSCAN and FIHOA appear to measure different aspects of pain and function.

MiR-17-92 and miR-221/222 cluster members target KIT and ETV1 in human gastrointestinal stromal tumours.

BACKGROUND: Gastrointestinal stromal tumours (GIST) are characterised by high expression of KIT and ETV1, which cooperate in GIST oncogenesis. Our aim was to identify microRNAs that are deregulated in GIST, have a role in GIST pathogenesis, and could potentially be used as therapeutic tool. METHODS: Differentially expressed microRNAs between primary GIST (n=50) and gastrointestinal leiomyosarcomas (GI-LMS, n=10) were determined using microarrays. Selected microRNA mimics were transfected into GIST-882 and GIST-T1 cell lines to study the effects of microRNA overexpression on GIST cells. Luciferase reporter assays were used to establish regulation of target genes by selected microRNAs. RESULTS: MiR-17-92 and miR-221/222 cluster members were significantly (P<0.01) lower expressed in GIST vs GI-LMS and normal gastrointestinal control tissues. MiR-17/20a/222 overexpression in GIST cell lines severely inhibited cell proliferation, affected cell cycle progression, induced apoptosis and strongly downregulated protein and--to a lesser extent--mRNA levels of their predicted target genes KIT and ETV1. Luciferase reporter assays confirmed direct regulation of KIT and ETV1 by miR-222 and miR-17/20a, respectively. CONCLUSION: MicroRNAs that may have an essential role in GIST pathogenesis were identified, in particular miR-17/20a/222 that target KIT and ETV1. Delivering these microRNAs therapeutically could hold great potential for GIST management, especially in imatinib-resistant disease.

Child abuse: a common problem in Curaçao?

OBJECTIVE: To assess the prevalence of child abuse among high school students in Curacao. METHODS: A questionnaire survey among high school students up to 17 years of age was conducted. The questionnaire was based on existing literature and validated questionnaires. The questionnaire used was analysed and adapted to the situation in Curaçao by a panel of experts on child abuse. The primary objective was to gain insight into the incidence, prevalence and various forms of child abuse among students in Curaçao. Five forms of child abuse are distinguished in the literature: physical abuse, emotional abuse, sexual abuse, neglect and exploitation. Furthermore, the degree of confidence of the students in general practitioners (GPs) as care providers in the field of child abuse was explored. RESULTS: Questionnaires from 545 of the 628 respondents were included. In total, 43% of the respondents had ever-experienced an event which could be defined as (a form of) child abuse. More than one-third of the respondents reporting child abuse ever had an experience that could be interpreted as physical abuse. More than 15% of the respondents reporting child abuse had experienced sexual abuse. Girls experienced significantly more sexual abuse than boys. Emotional abuse in the last year was experienced by 3% of the respondents. One per cent of the respondents ever-experienced neglect. According to most respondents, GPs were not seen as care providers in cases of child abuse; they believed that GPs were mainly to be consulted for illnesses or physical symptoms and not for forms of child abuse. CONCLUSION: The prevalence of ever-having-experienced a form of child abuse is estimated at 431 per 1000 students. Child abuse, particularly physical abuse, is common in Curaçao, and is probably comparable to other surrounding countries. General practitioners were not seen as care providers in identifying and reporting cases of child abuse according to most respondents.

Contraception and induced abortion in the West Indies: a review.

BACKGROUND: Most islands in the West Indies do not have liberal laws on abortion, nor laws on pregnancy prevention programmes (contraception). We present results of a literature review about the attitude of healthcare providers and women toward (emergency) contraception and induced abortion, prevalence, methods and juridical aspects of induced abortion and prevention policies. METHODS: Articles were obtained from PubMed, EMBASE, MEDLINE, PsychlNFO and Soclndex (1999 to 2010) using as keywords contraception, induced abortion, termination of pregnancy, medical abortion and West Indies. RESULTS: Thirty-seven articles met the inclusion criteria: 18 on contraception, 17 on induced abortion and two on both subjects. Main results indicated that healthcare providers' knowledge of emergency contraception was low. Studies showed a poor knowledge of contraception, but counselling increased its effective use. Exact numbers about prevalence of abortion were not found. The total annual number of abortions in the West Indies is estimated at 300 000; one in four pregnancies ends in an abortion. The use of misoprostol diminished the complications of unsafe abortions. Legislation of abortion varies widely in the different islands in the West Indies: Cuba, Puerto Rico, Martinique, Guadeloupe and St Martin have legal abortions. Barbados was the first English-speaking island with liberal legislation on abortion. All other islands have restrictive laws. CONCLUSION: Despite high estimated numbers of abortion, research on prevalence of abortion is missing. Studies showed a poor knowledge of contraception and low use among adolescents. Most West Indian islands have restrictive laws on abortion.

Medical abortion in primary care: pitfalls and benefits.

We describe five pitfalls of medical abortion: ectopic pregnancy not terminated after misoprostol, but without negative side-effects; long-term vaginal blood loss with suspicious retained products which disappeared spontaneously; a patient with uterus myomatatosus with severe pain and retained products in the uterus; repetition of misoprostol because of retained products in the uterus after two weeks and an allergic reaction to methotrexate. Despite these pitfalls, there are enough benefits to consider medical abortion with methotrexate and misoprostol as a safe method with a high success rate of more than 91% and a good alternative for surgical abortion. An invasive procedure is not necessary, there are no long-term complications and it can be performed at an earlier stage, which makes it more acceptable in society. In Curaçao, where abortion is legally restricted, medical abortion is performed with methotrexate and misoprostol. In countries where abortion is legal, mifepristone and misoprostol are the first choice.

[Good results of early medicinal pregnancy termination in a general practice on Curaçao]. / Goede uitkomsten van vroege medicamenteuze zwangerschapsafbreking in een huisartspraktijk op Curaçao.

OBJECTIVE: To evaluate the results of early medicinal pregnancy termination. DESIGN: Prospective, non-blinded, clinical study. METHOD: Between 2001 and 2005, 371 women presented to a medium-sized solo general practice in the centre of Willemstad, Curaçao, to request termination of an undesired pregnancy. Ofthese women, 74 first indicated a preference for abortion via curettage, and the remaining 297 chose to be treated medicinally. In the first trimester of the pregnancy, they were given methotrexate i.m. and misoprostol vaginally 3-5 days later, or only misoprostol vaginally when methotrexate was unavailable. The result was evaluated by abdominal echography two days after the treatment. RESULTS: The result of the medicinal pregnancy termination could be determined in 271 of the 297 women. In 219 (81%) of these, the pregnancy had been terminated after a single treatment, and in 29 (11%) success was achieved after a second vaginal administration of misoprostol. The total success rate of the medicinal pregnancy termination was 92% (248/271). Curettage was required in 23 women (8%) after the medicinal treatment had failed. There was no significant difference in success rate between treatment with the methotrexate-misoprostol combination and treatment with misoprostol alone. CONCLUSION: Medicinal pregnancy termination with methotrexate and misoprostol, or with misoprostol alone, was a safe method with a relatively small risk of an ongoing pregnancy. It was a good alternative for surgical abortion.

Using Digital Tags With Integrated Video and Inertial Sensors to Study Moving Morphology and Associated Function in Large Aquatic Vertebrates.

The anatomy of large cetaceans has been well documented, mostly through dissection of dead specimens. However, the difficulty of studying the world's largest animals in their natural environment means the functions of anatomical structures must be inferred. Recently, non-invasive tracking devices have been developed that measure body position and orientation, thereby enabling the detailed reconstruction of underwater trajectories. The addition of cameras to the whale-borne tags allows the sensor data to be matched with real-time observations of how whales use their morphological structures, such as flukes, flippers, feeding apparatuses, and blowholes for the physiological functions of locomotion, feeding, and breathing. Here, we describe a new tag design with integrated video and inertial sensors and how it can be used to provide insights to the function of whale anatomy. This technology has the potential to facilitate a wide range of discoveries and comparative studies, but many challenges remain to increase the resolution and applicability of the data. Anat Rec, 300:1935-1941, 2017. © 2017 Wiley Periodicals, Inc.

SKY1 is involved in cisplatin-induced cell kill in Saccharomyces cerevisiae, and inactivation of its human homologue, SRPK1, induces cisplatin resistance in a human ovarian carcinoma cell line.

The therapeutic potential of cisplatin, one of the most active and widely used anticancer drugs, is severely limited by the occurrence of cellular resistance. In this study, using budding yeast Saccharomyces cerevisiae as a model organism to identify novel drug resistance genes, we found that disruption of the yeast gene SKY1 (serine/arginine-rich protein-specific kinase from budding yeast) by either transposon insertion or one-step gene replacement conferred cellular resistance to cisplatin. Heterologous expression of the human SKY1 homologue SRPK1 (serine/arginine-rich protein-specific kinase) in SKY1 deletion mutant yeast cells restored cisplatin sensitivity, suggesting that SRPK1 is a cisplatin sensitivity gene, the inactivation of which could lead to cisplatin resistance. Subsequently, we investigated the role of SRPK1 in cisplatin sensitivity and resistance in human ovarian carcinoma A2780 cells using antisense oligodeoxynucleotides. Treatment of A2780 cells with antisense oligodeoxynucleotides directed against the translation initiation site of SRPK1 led to down-regulation of SRPK1 protein and conferred a 4-fold resistance to cisplatin. The human SRPK1 gene has not been associated with drug resistance before. Our new findings strongly suggest that SRPK1 is involved in cisplatin-induced cell kill and indicate that SRPK1 might potentially be of importance for studying clinical drug resistance.

Circular dichroism studies on the proline-rich glycoprotein of human parotid saliva.

Previous results on the primary structure of the human parotid saliva proline-rich glycoprotein established the concept of repetitive domains in the sequence of that glycoprotein, tryptic glycopeptides of proline-rich glycoprotein representing the basic structure. The present work is concerned with the study of the secondary structure of the proline-rich glycoprotein, and of tryptic glycopeptides with and without the glycan moiety, using circular dichroism. CD spectra exhibit the same secondary structure with about 60% of polyproline II helical structure for the proline-rich glycoprotein, tryptic glycopeptides and their deglycosylated homologues. The present results are in fair agreement with the amino acid sequence results and suggest a model for the schematic representation of the proline-rich glycoprotein.

Expression of p53, p21/WAF/CIP, Bcl-2, Bax, Bcl-x, and Bak in radiation-induced apoptosis in testicular germ cell tumor lines.

PURPOSE: Testicular germ cell tumors (TGCTs) represent one of the few tumor types that are curable by antineoplastic therapy, probably due to the high sensitivity of this neoplasm to induction of apoptosis by chemotherapeutic agents and/or ionizing radiation. Here, we tested cell susceptibility to radiation-induced apoptosis in a panel of TGCT cell lines and attempted to correlate this with the known potentially relevant molecular determinants (p53 gene status and Bcl-2 family proteins) of apoptosis. METHODS AND MATERIALS: Induction of apoptosis by gamma-radiation was morphologically recognized in NT2, NCCIT, S2, and 2102 EP using Hoechst/PI staining and additionally confirmed by Western blot analysis of PARP cleavage. The p53 gene status was estimated by sequence analysis. Expression of p21/WAF/CIP was determined by Northern blot analysis and immunoblotting was used to monitor p53, Bax, Bcl-2, Bcl-x, and Bak protein levels. In vitro colony formation was studied to establish clonogenic survival curves. RESULTS: NT2 and NCCIT appeared to be susceptible for radiation-induced apoptosis, contrasting 2102 EP and S2 which were highly resistant. Sequence analysis showed that NT2, S2, and 2102 EP are homozygous for wild-type p53 (wtp53), whereas NCCIT contains mutant p53 (mtp53). NT2 and 2102 EP cells showed radiation-induced p53 upregulation, while NCCIT (mtp53) and S2 (no p53 protein) cells did not. Consistently, gamma-radiation-induced DNA damage resulted in a p53-dependent transactivation of the p21/WAF/CIP gene in NT2 and 2102 EP, but not in mtp53-containing NCCIT cells and p53 nonexpressing S2 cells. Constitutive expression of Bax, Bcl-2, Bcl-x, and Bak was not affected by radiation and showed no correlation with cell susceptibility to radiation-induced apoptosis. A discrepancy was found between apoptosis and reproductive death. CONCLUSIONS: The present study revealed that: i) the presence of wtp53 may not be absolutely required for the hypersensitivity for radiation-induced apoptosis in TGCT cell lines, ii) the molecular mechanism underlying the unique radiosensitivity was independent of the expression of Bcl-2 family proteins, and iii) cell susceptibility to apoptosis induction is not sufficiently informative to predict intrinsic radiosensitivity as determined by clonogenic survival.

Enzymatic cleavage of pyroglutamyl-histidyl-dopamine, a TRH-related pseudo-peptide by porcine serum and brain hoimogenate.

The Pseudo-peptide (pGlu-His-Dopamine) was subjected to enzymatic degradation by porcine serum and brain homogenate. The digests were quantitatively analyzed by HPLC to provide evidence for liberation of pyroglutamic acid and dopamine in serum and of pyroglutamic acid in brain. The yield of liberation of pGlu is about 3% in serum digests and about 0.70% in brain digests after 3 minutes incubation. The time course of the yield of liberation of both pGlu and dopamine in serum digests is determined. The "stability in vitro" toward enzymes of serum and brain homogenate of a new type of drug based on the combination of peptidic fragment of TRH-(Thyrotropin-Releasing Hormone:pGlu-His-Pro-NH2) with a non peptide moiety (dopamine) is considered and discussed.

Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro.

The cytotoxicity of cisplatin alone and in combination with topotecan (TPT) or SN-38, two novel topoisomerase I (topo I) inhibitors, was determined in a panel of eight well-characterized human solid-tumor cell lines. Interactions between cisplatin and these topo I inhibitors were investigated using three different administration schedules: (1) simultaneous incubation (C + T and C + S), (2) cisplatin followed by TPT or SN-38 (C --> T and C --> S), and (3) TPT or SN-38 followed by cisplatin (T --> C and S --> C). Median-effect analysis revealed synergistic cytotoxicity in seven of the eight cell lines used. In addition, a significant schedule-dependent synergistic cytotoxicity was found in three of the cell lines used, with C --> T (or C --> S) being the most active schedule. The formation and repair of total cisplatin-DNA adducts in the IGROV-1 ovarian cancer cell line and its cisplatin-resistant subline IGROV(CDDP) was not significantly affected by TPT on simultaneous incubation. In contrast, the number of cisplatin-DNA interstrand cross-links detected in the IGROV-1 and IGROV(CDDP) lines at certain time points was significantly lower after coincubation of the cells with TPT. Assessment of the cell-cycle distribution revealed an accumulation of cells in the G2/M phase after exposure to cisplatin. After exposure to TPT a different pattern was observed that was cell-type-specific and dependent upon the TPT concentration. Although up to 4-fold differences in topo I activity were observed in this panel of cell lines, these differences did not appear to be related to the synergy observed between cisplatin and TPT or SN-38. The observed synergy may at least partly be explained by the increased retention of cisplatin-DNA interstrand cross-links in the presence of topo I inhibitors.

Objectively measured sperm motility and sperm head morphometry in boars (Sus scrofa): relation to fertility and seminal plasma growth factors.

This study was conducted to investigate the relationships between results of computer-assisted semen analysis (spermatozoal motility and sperm head morphometry) and fertility of boars. In addition, concentrations of insulin-like growth factor (IGF)-I and IGF-II in seminal plasma were determined. The nonreturn rate (NRR) and the number of live-born piglets were compatible with the requirements of artificial insemination for all boars included in this study. Semen samples of 12 boars (Pietrain; 3 ejaculates each) were evaluated for spermatozoal motility and sperm head dimensions using computer-assisted methods. Native semen samples were centrifuged, and seminal plasma was frozen at -20 degrees C until assayed for IGF-I and IGF-II by specific radioimmunoassays. Spermatozoa of boars with a higher NRR (>86%) had a significantly slower average velocity of motile spermatozoa when compared with that of boars with an NRR below 86%. High-fertility boars (NRR > 86%) had significantly smaller sperm heads than did boars with an NRR below 86%, and their sperm heads were less elongated. Substantial concentrations of IGF-I (8.4-22.2 ng/mL) and IGF-II (12.1-19.8 ng/mL) could be measured in porcine seminal plasma; however, there was no correlation between IGF levels and semen parameters or individual fertility.

[Biochemical study of the glycosaminoglycan peptides obtained from osteoarthrotic and normal femoral heads (author's transl)]. / Etude biochimique comparative des glycosaminoglycannes peptides du cartilage articulaire de la tête fémorale humaine normale et arthrosique.

Glycosaminoglycan peptides prepared by papain hydrolysis of different regions were obtained from osteoarthrotic and normal human femoral heads. Data obtained in these experiments show that in osteoarthrosis a decrease in keratan sulfate and an increase in chondroitin sulfate are observed. Since keratan sulfate appeared to play an important role in proteoglycan aggregation, we suggest that the keratan sulfate decrease is one of the factors involved in the cartilage disorder observed in patients suffering from osteoarthrosis.

Gonadotropic dysfunction produced by Trypanosoma brucei brucei in the rat.

Hormonal disorders have been frequently observed in humans and animals infected with tsetse-transmitted (African) trypanosomes. We studied the pituitary gonadal axis (plasma concentrations of testosterone, luteinizing hormone (LH), follicle stimulating hormone (FSH) and the pituitary gonadotropin (LH, FSH) concentrations) in rats as an experimental model infected with an acute stock of Trypanosoma brucei brucei (AnTat 1.1A). The same investigations in vivo were carried out with rats inoculated by trypanosomal preparations: surface coat components slowly released at pH 5.5 and the parasitic cellular pellet. The releasing procedure as firstly described by Baltz et al. (1976) was performed in the presence or the absence of protease inhibitors. We noted a testicular hypogonadism produced by the acute infection with the decrease of the testosterone level and an increase of the pituitary LH concentration, although the other circulating FSH and LH hormone levels were stable. The injection of the trypanosomal pellet, obtained in the presence of antiproteases, generated a similar clinical hormonal picture: decrease of testosterone level; increase in pituitary LH, FSH content; absence of significant variation of circulating FSH and LH rates. When the trypanosomal pellet was prepared in absence of antiproteases the circulating gonadostimuline levels were significantly decreased. In the same conditions (absence of antiproteases) the trypanosomal supernatant pH 5.5 induced the decrease of the testosterone and plasma LH levels. These results suggested that component(s) from trypanosomes generated hormonal perturbations.

Structural studies of the carbohydrate component of a human, parotid-saliva, proline-rich glycoprotein.

The carbohydrate chains of the human, parotid-saliva, proline-rich glycoprotein were released as oligosaccharides by hydrazinolysis and were fractionated by high-pressure-liquid chromatography. Four oligosaccharides were characterized. On the basis of compositional analysis, sequential enzymic degradation, and methylation data, the carbohydrate moiety of the glycoprotein was found to be a complex-type oligosaccharide containing a tri-D-mannosyl-di-N-acetylchitobiose core with two and three lactosamine branches, and an L-fucosyl group linked to O-6 of the asparagine-linked 2-acetamido-2-deoxy-D-glucose residue. In addition, several L-fucosyl groups, linked at O-6 of the penultimate D-galactose residues, are present in variable proportions. The biological significance of these results is discussed.

Evidence for the presence of the trimannosyl-di-N-acetylchitobiose core in the carbohydrate chains of human-parotid, proline-rich glycoprotein.

The carbohydrate chains of the human-parotid, proline-rich glycoprotein are linked through a single type of carbohydrate-peptide linkage (asparaginyl-N-acetylglucosamine). The structure of the internal part of the carbohydrate chains, determined by chemical, enzymic, and g.l.c.--m.s. methods, includes the trimannosyl-di-N-acetylchitobiose core involved in the carbohydrate-peptide linkage. Furthermore, an L-fucose residue is linked to the 2-acetamido-2-deoxy-D-glucosyl residue linked to the L-asparaginyl residue. The sequence of the peripheral part of the chains has also been determined as alpha-L-Fuc rho leads to beta-D-Gal rho leads to beta-D-Glc rho NAc leads to alpha-D-Man rho, suggesting a double-branched, basic carbohydrate structure.