Discrepancy between prolactin (PRL) messenger ribonucleic acid and PRL content in rat fetal pituitary cells: possible role of dopamine.

Data are controversial concerning the time when PRL-synthesizing cells are detected for the first time in the rat pituitary. Using a very sensitive immunocytochemical technique, we could visualize only a few PRL cells before day 10 after birth. At that time, pituitary PRL was still 200 times less abundant than in the adult (on a tissue weight basis) whereas PRL mRNA per mg total RNA was only 80 times lower than in the adult. However, by in situ hybridization, we could demonstrate the presence of PRL mRNA in cells from fetal day 18 on. We have also followed the expression of GH gene in rat pituitary cells during development. In contrast to results obtained with PRL cells, quantitative analysis of cDNA probe hybridization to GH mRNA correlated well with measurements of immunostained cells. We found that PRL was released in the blood from fetal day 19 onwards. Thus, at that time PRL is synthesized and secreted but not stored. We therefore measured brain dopamine levels, and the data support the idea that the rise in dopamine levels after birth contributes to PRL storage. We confirmed in vitro that newborn pituitary cells can store PRL when cultured in the presence of dopamine.

Prolactin cell subpopulations separated on discontinuous Percoll gradient: an immunocytochemical, biochemical, and physiological characterization.

A single-step procedure was devised to separate PRL cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient. Three layers were recovered. The composition of the different layers was evaluated using immunocytochemistry (with antisera to the six pituitary hormones), and in situ hybridization [with DNA complementary to PRL or to GH messenger RNA (mRNA)]. Both methods yielded identical values. PRL cells were recovered in the lower density layer (layer 1) with a good yield (that is 81% of the total PRL cells of the initial cell suspension) and in addition, markedly enriched (indeed 85% of the cells in layer 1 stained for PRL). A second layer (layer 2: intermediate density) contained most of the remaining PRL cells which were, however, heavily contaminated mainly by GH cells and cells that did not stain for any of the known pituitary hormones. A third layer (layer 3: higher density) was enriched in GH cells to 93% (representing, however, only 10% of the initial pituitary GH cells). In addition, PRL and GH were measured by RIA in culture medium and in cell lysates. Hormone biosynthesis was monitored by polyacrylamide gel electrophoresis and autoradiography after culture in the presence of [35S]methionine. These experiments confirmed that layer 1 was enriched in cells containing, and producing, PRL and depleted from GH cells. Cells in layer 2 contained and produced more GH than PRL. PRL cells from layer 1 responded to dopamine and to vasoactive intestinal polypeptide in the same way as PRL cells in the unseparated pituitary cell population. In contrast PRL cells in layer 2 had a lower basal secretion rate but a higher response to vasoactive intestinal polypeptide. Unless this represents a paracrine effect of non-PRL cells, PRL cells in layer 2 exhibit different properties and may therefore form a distinct subpopulation of PRL cells.

Gel filtration profile of immunoreactive thyrotropin and subunits of human pituitaries.

Pituitary extracts from five patients with atrophic asymptomatic thyroiditis and from five subjects without any endocrine disease, matched for sex and age, were passed through gel filtration columns and the collected fractions were analyzed in three different radioimmunoassays (hTSH-anti-hTSH; hTSHbeta-anti-pTSH; hLHAlpha-anti-LHalpha). In the alpha subunit system, every profile showed a first peak corresponding to the elution position of the intact hTSH molecule and second peak, often more important, of free alpha chain. In the beta subunit system, a high molecular weight immunoreactive protein (big hTSHBeta) was found in seven pituitary extracts; cross-reacting intact hTSH molecule was eluted as a second peak and free Beta chain was detected in all the extracts. Free alpha chain was generally found in greater amount than free Beta. In the hTSH system, every elution pattern showed a peak of intact hTSH and another in the subunit area. In six pituitary extracts (5 thyroiditis, 1 control), a high mol wt protein was eluted (big hTSH). Its predominant presence in thyroiditis extracts seems important in view of the already described immunological heterogeneity of pituitary hTSH.

Gel filtration profile of circulating immunoreactive thyrotropin and subunits of myxedematous sera.

Five sera from hypothyroid patients were passed through gel-filtration columns and the collected fractions were analyzed in three different radioimmunoassays (hTSH-anti-hTSH; hTSHbeta-anti-pTSH; hLH-alpha-anti hLHalpha). For each immunological system, cross reactions were carefully studied. In the hTSH system one main peak was observed at the elution position of the entire molecule, with variable contributions in the subunits area. In the alpha subunit system, every profile showed an asymmetric peak, corresponding roughly to the position of the entire molecule; only two sera exhibited small amounts of free alpha chain. In the beta subunit system, a high molecular weight (100,000 or more) immunoreactive protein was present in every serum. Moreover, cross-reacting intact molecule was eluted as a second peak and in four sera free beta chain was detected. The significance of the big beta subunit, detectable only in the beta assay, is not elucidated.

Grades of subclinical hypothyroidism in asymptomatic autoimmune thyroiditis revealed by the thyrotropin-releasing hormone test.

Serum levels of T4 T3, and TSH and peak TSH after TRH administration were determined in 60 female subjects affected with asymptomatic autoimmune thyroiditis (AAT), in 27 normal subjects, and in 8 myxoedematous patients used as controls. The AAT subjects were divided into 3 groups on the basis of their basal and peak TSH values. In the first group (grade I AAT), these parameters were similar to those of the normal controls; in the second (grade II AAT), basal TSH remained normal but peak TSH was significantly increased; and in the third (grade III AAT), both parameters were significantly increased. Serum T4 levels decreased progressively from group 1 to group 3, but all T4 values were within the normal range. T3 levels were similar in all groups. Peak and basal TSH values were highly correlated, except in grade II AAT. There seems to exist a graded process of subclinical hypothyroidism in AAT; a progressive pituitary TSH reserve is modulated by a progressive decrease in T4 levels still within the normal range.

Pituitary TSH in normal subjects and in patients with asymptomatic atrophic thyroiditis: Evidence for its immunological heterogeneity.

Biological and radioimmunological measurements of pituitary TSH concentration were performed in 22 cases of asymptomatic atrophic thyroiditis and in 18 controls. Whilst bioassay revealed the presence of a greater pituitary TSH concentration in thyroiditis cases, radioimmunoassay failed to confirm such a difference. The reason therefore seems to lie in the presence in thyroiditis pituitatries of a TSH which reacted in the bioassay but showed only a weak affinity for the anti-hTSH antiserum. The slopes of radioimmunological dilution curves of the pituitary extracts were indeed significantly lower with thyroiditis pituitaries than with controls. When the whole population sample was considered, a negative correlation existed between the ratio of biological and radioimmunological TSH determinations (B/I) and the slope of the corresponding dilution curves. Since in radioimmunoassay a low displacement slope is indicative of a weak immunological affinity of the antigen for the antiserum, this demonstrated negative correlation suggests together with a high B/I ratio, even in normal people.

Atrophic autoimmune thyroiditis: relationship between the clinical state and intrathyroidal iodine as measured in vivo in man.

The intrathyroidal iodine (ITI) was measured by means of the x-ray fluorescence method in 58 patients suffering from atrophic autoimmune thyroiditis (AAT). They were divided into 4 groups according to their basal T4, basal TSH, and the peak TSH response after TRH administration. Forty-eight normal subjects served as controls. A progressive fall of ITI was found, with less ITI in the first grade AAT patients as compared to the controls. A negative correlation between basal TSH and ITI could be shown. These data support the concept of a graded evolution of AAT, linking the clinical state to the ITI. The results also suggest that, with the hemagglutination techniques used for the determination of antithyroglobulin antibodies and antithyroid microsomal antibodies, positively at a dilution of 1:25 and 1:100, respectively, should be regraded as significant.

Effects of glucocorticoids on pituitary hormonal responses to hypoglycemia. Inhibition of prolactin release.

The characteristics of pituitary hormonal responses to insulin-induced hypoglycemia were investigated in 16 normal men. In all subjects, levels of blood sugar fell below 35 mg/100 ml. A statistically significant increase in mean plasma levels of prolactin, ACTH, cortisol and growth hormone was observed. Prolactin levels increased in all subjects but one; individual peak values were 1.4 minus 8.4 times greater than base levels. The kinetics of prolactin, GH and ACTH responses were similar; in particular, the onset of release (25 min) of prolactin, GH and ACTH was similar. After dexamethasone administration, insulin tolerance tests wererepeated in a number of subjects using adequate amounts of insulin to achieve hypoglycemia equivalent to that obtained in the control experiments. The administration of 1 mg of dexamethasone the evening before the test suppressed basal levels of ACTH and cortisol and the ACTH-but not the cortisol-response to hypoglycemia. Both basal levels of prolactin and prolactin response to hypoglycemia were significantly lowered but growth hormone response was not modified by administration of 1 mg of dexamethasone. The administration of larger doses of dexamethasone (1 mg every 6 h for 2 days) almost completely suppressed basal levels of ACTH, cortisol and prolactin, as well as the hypoglycemia-induced release of these hormones. In contrast, the growth hormone response to hypoglycemia was only partially inhibited. These findings demonstrate that both basal secretion and hypoglycemia-induced release of prolactin, ACTH, cortisol and growth hormone are suppressible by glucocorticoids.

Ectopic adrenocorticotropin production: disappearance after removal of inflammatory tissue.

A patient presented with the clinical and laboratory features of the ectopic ACTH syndrome. No ACTH-producing tumor was found, and bilateral adrenalectomy was performed to correct the hypercortisolism. Six months later, the removal of a pseudotumor containing only fat and inflammatory tissue resulted in normalization of both basal plasma ACTH levels and the ACTH feedback response to cortisol infusion. It is suggested that the leukocytes in the inflammatory tissue were the source of the ectopic ACTH production.

Natural history of primary myxedema.

It is generally admitted that primary myxedema in adults is the outcome of autoimmune atrophic thyroiditis. The present review traces the natural history of this process from its incipient biologic and genetic anomalies up to its protracted asymptomatic course, clinical development, and eventual lethal complications. The apprehension of preclinical hypothyroidism may change a clinician's outlook on early diagnosis and therapy.

Postnatal development of growth hormone and prolactin cells in male and female rat pituitary. An immunocytochemical light and electron microscopic study.

Localization and ultrastructural maturation of prolactin (PRL) and growth hormone (GH) cells were studied in pituitaries from neonatal, immature (4-6 weeks old), and adult rats (2-3 months old) by light and electron microscopic immunocytochemistry. The distribution pattern of these cells did not change with age. Both cell types were concentrated laterodorsally, with PRL cells adjacent to the intermediate lobe and GH cells nearer the center of the pars distalis. Labeling density of the immunogold reaction was highest for both hormones in immature rats. In neonatal and immature rats, one PRL cell type with granules 200 nm in diameter was present. In adult rats, two types of PRL cells were present: one containing polymorphous granules measuring about 500 nm (prevalent in female rats), the other with spherical granules about 200 nm (prevalent in male rats). No changes were detected in GH cells during maturation.

Effect of N omega-nitro-L-arginine methyl ester, a nitric oxide synthesis inhibitor, on stress- and morphine-induced prolactin release in male rats.

1. The effect of the nitric oxide synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) was investigated on stress- and morphine-induced prolactin (PRL) secretion in vivo in male rats, by use of a stress-free blood sampling and drug administration method by means of a permanent indwelling catheter in the right jugular vein. 2. Three doses of L-NAME were tested (1, 10 and 30 mg kg-1) and were given intraperitoneally one hour before blood sampling; control rats received saline. After the first blood sample, rats received an initial intravenous injection of morphine (3, 6 or 12 mg kg-1) or were subjected to immobilization stress. In the case of a morphine administration, rats received a second dose of morphine (3, 6 or 6 mg kg-1, respectively) 90 min later, when tolerance had developed, while rats subjected to immobilization stress received 6 mg kg-1 morphine 90 min after onset of stress. 3. L-NAME had no effect on basal plasma PRL concentration. However, it potentiated acute morphine-induced PRL secretion and attenuated the subsequent tolerance in a dose-dependent way. Immobilization stress-induced PRL secretion was inhibited dose-dependently by L-NAME, as was its subsequent tolerance to morphine; however, in this case, in a reversed dose-dependent way. 4. When the highest dose of morphine (12 mg kg-1) was combined with the highest dose of L-NAME pretreatment (30 mg kg-1), all rats showed a dramatic potentiation of the morphine-induced PRL rise compared to controls. Moreover, all of these rats died within 90 min after the administration of morphine. 5. These results show that NO plays a role in the acute opioid action on PRL release during stress as well as in the development of tolerance to the opioid effect, and some possible mechanisms are discussed.

Identification of a D1 dopamine receptor, not linked to adenylate cyclase, on lactotroph cells.

1. We studied the lactotroph cells of the rat by both in vivo and in vitro pharmacological techniques for the presence of D1-receptors. Both approaches revealed the presence of D2-receptor, stimulated by quinpirole (resulting in an inhibition of prolactin secretion) and blocked by domperidone. 2. Administration of fenoldopam, the most selective D1-receptor agonist currently available, resulted in a dose-dependent decrease of prolactin secretion in vivo (after pretreatment with alpha-methyl-p-tyrosine) and in vitro (cultured pituitary cells). This increase was dose-dependently blocked by the selective D1-receptor antagonist, SCH 23390, and although the effect of fenoldopam was less than that obtained by D2-receptor stimulation, these data suggest that a D1-receptor also controls prolactin secretion. 3. In order to detect the location of these dopamine receptors, autoradiographic studies were performed by use of [3H]-SCH 23390 and [3H]-spiperone as markers for D1- and D2-receptors, respectively. Specific binding sites for [3H]-SCH 23390 were demonstrated. Fenoldopam dose-dependently reduced [3H]-SCH 23390 binding, but had no effect on [3H]-spiperone binding. Immunocytochemical labelling of prolactin cells after incubation with [3H]-SCH 23390 revealed that the granulae and hence, D1 binding sites were present on the lactotroph cells. 4. Radioligand binding studies performed on membranes from anterior pituitary cells revealed the presence of the D2-receptor (54 fmol mg-1 protein) with a Kd of 0.58 nM for [3H]-spiperone, but failed to detect D1-receptors. 5. Finally, we studied the effect of dopamine and of fenoldopam on the adenosine 3':5'-cyclic monophosphate (cyclic AMP) content of anterior pituitary cells. Although cyclic AMP increased upon prostacyclin administration, indicating an intact adenylate cyclase system, fenoldopam failed to increase the cyclic AMP production. 6. It is tempting to speculate that fenoldopam reduces prolactin secretion through interaction with a non-cyclase-linked D1-receptor on the lactotroph cells.

Functional heterogeneity with respect to oestrogen treatment in prolactin cell subpopulations separated by Percoll gradient centrifugation.

The effects of oestradiol on prolactin gene expression were studied by quantitative in situ hybridization histochemistry in different prolactin pituitary cell (sub)populations, which had been obtained by separation on a discontinuous Percoll gradient. When cells were incubated in vitro in the presence of oestradiol (10(-8) M) for a period of 4, 24, 48 and 72 h, there was an increase in the amount of prolactin mRNA, from 24 h on, only in high-density prolactin cells and lactotrophs of the total cell suspension. In contrast, the amount of prolactin mRNA in lactotrophs of low density did not change upon treatment with oestradiol. Pharmacological treatment with 50 micrograms oestradiol/day (s.c.) of random cycling female rats in vivo for 14 days increased the total number of prolactin gene-expressing cells and more lactotrophs were recovered at high density after Percoll gradient centrifugation. These results suggest a preferential stimulatory effect of oestradiol on prolactin gene transcription on a subpopulation of lactotrophs. Changes observed in prolactin cell layers after oestradiol treatment in vivo may represent a preferential effect in situ on a particular mammotroph cell subpopulation.

Multiple forms of rat prolactin and growth hormone in pituitary cell subpopulations separated using a Percoll gradient system: disulphide-bridged dimers and glycosylated variants.

Prolactin and GH cells from rat pituitary glands were separated into three main fractions on discontinuous Percoll gradient layers. SDS-PAGE and subsequent immunoblotting of these fractions revealed that: (1) multiple rat prolactin (rPRL) molecular variants were present in total culture, Percoll layer 1 and 2; four variants were clear-cut: Mr approximately 23,000, Mr doublet approximately 25,000-26,000, Mr approximately 40,000 and Mr approximately 42,000; (2) cell cytosol from Percoll gradient layer 1 was particularly enriched in prolactin; (3) cells from gradient layer 1 secreted into the culture medium only prolactin in detectable amounts; (4) three distinct molecular forms of rat growth hormone (rGH) were recorded in layer 3: Mr approximately 36,000, 24,000 and 20,000; the 20,000 variant was paramount; and (5) cells from layer 3 secreted both rPRL and rGH into the culture medium. Reduction experiments showed that, on the one hand, 42,000 and 40,000 rPRL variants and, on the other hand, 36,000 rGH variants are disulphide-bridged dimers. An important finding was the presence of glycosylated rPRL and rGH: indeed Concanavalin A-Sepharose 4B affinity chromatography indicated that 26,000 rPRL and 24,000 rGH display a very strong affinity for lectin. Competitive inhibition tests showed that this affinity is specific and not due to hydrophobic binding. When rPRL was submitted to deglycosylation in conditions specific for O-linked glycoproteins, the 26,000 rPRL variant disappeared. The biological role of glycosylated rPRL is as yet unknown.

Analysis of the receptor specificity of tolerance induction in stress versus opioid-related prolactin secretion in rats.

The effects of restraint stress and opiates on prolactin secretion in male rats have been measured. Both induced a short-lived increase in prolactinaemia. Experimental evidence indicates that both opioids and restraint stress bring about their actions by the activation of opioid receptors. When restraint stress was followed by administration of the specific kappa-agonist bremazocine, a second prolactin peak was observed. In contrast, morphine (predominantly a mu-agonist) lost its prolactin-stimulating capacity when given after a period of restraint stress. This indicates cross-tolerance between restraint stress and morphine. Tolerance was overcome when the dose of morphine was doubled, and an increase in prolactin secretion could again be obtained. The cross-tolerance phenomenon argues that a common opioid receptor is involved in morphine- and restraint stress-stimulated prolactin release. In another set of experiments, in which morphine administration replaced restraint stress as a means of inducing tolerance, a second rise in prolactinaemia was seen only with bremazocine and not with a further administration of morphine. This suggests a morphine (mu) receptor-specific development of tolerance. Two consecutive administrations of bremazocine also produced tolerance, in this case for the kappa-receptor. This illustrates the rapid and receptor-specific development of tolerance for the prolactin-releasing capacity of opioid compounds.

Effect of pituitary adenylate cyclase-activating polypeptide 38 on growth hormone and prolactin expression.

Time- and dose-dependent effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on prolactin (PRL) and growth hormone (GH) release were examined in static and dynamic rat pituitary cell incubations and on different pituitary cell (sub)populations separated according to their density on a discontinuous Percoll gradient. Quantitative in situ hybridization histochemistry allowed us to examine in parallel the effects of PACAP on PRL and GH gene expression. PACAP did not alter GH or PRL secretion in a dynamic superfusion system, in any cell population tested. Static incubations (30 min, 2-36 h) with PACAP 38 resulted in a significant increase in GH release and stimulated GH synthesis, as measured by the cytoplasmic accumulation of GH mRNA in the somatotrophs. These effects on synthesis and release were also observed after the enrichment of GH cells on Percoll gradients. PRL release was not altered by longer periods of incubation. Although no significant changes were observed in PRL secretion after 38 h, accumulation of cytoplasmic PRL mRNA was significantly stimulated in total pituitary cell suspension. After fractioning lactotrophs on Percoll gradients, the stimulatory effect of PACAP on PRL synthesis was lost. These results suggest that PACAP stimulates GH release and synthesis, and that it may act as a physiological regulator of this cell type. The PRL cell is not the most likely target cell type for PACAP. Effects observed on PRL synthesis in the total cell population may involve paracrine action of other hormone- or non-hormone-secreting cell types.

Differential dopamine-induced prolactin mRNA levels in various prolactin-secreting cell (sub)populations.

We have examined the effects of dopamine on prolactin gene expression using quantitative in-situ hybridization histochemistry in different pituitary cell (sub)populations separated according to their density on a discontinuous Percoll gradient. Administration of dopamine resulted in a drastic reduction in hybridization of 35S-labelled DNA probe complementary to prolactin mRNA in total pituitary cells and in lactotrophs with low density. In contrast, dopamine significantly stimulated mRNA accumulation in prolactin-secreting cells with high density compared with other cell layers. The combined use of Percoll gradient and quantitative in-situ hybridization is a valuable and sensitive method with which to examine prolactin-secreting cell response to a given stimulation. Prolactin-secreting cells with high and low density clearly show functional heterogeneity in their response to dopamine.

Molecular heterogeneity and glycosylation modulation of rat pituitary prolactin isoforms synthesized and secreted in vitro in postnatal ontogeny, gestation, lactation and weaning.

The modulation of both the molecular size heterogeneity and the relative distribution of rat prolactin variants, synthesized and secreted in vitro by rat pituitary cells in the course of postnatal ontogeny and in gestation, lactation and weaning was investigated by SDS-PAGE, immunoblotting, radioimmunological techniques and O-sialoendopeptidase digestion. The outcome of the experiments is as follows: 1) from day 1 of postnatal life 20-, 23-, 26-, 40-44 kDa and oligomeric rat prolactin isoforms were stored and secreted; 2) perinatal life is characterized by a high degree of variability of prolactin size isoforms and their respective repartition in storage and release; in addition to the major variants, transient ones of M, 25-, 28-, 33- and 36 kDa were secreted and/or stored; 3) O-sialoglycoprotease digestion of pituitary cell lysate gave good evidence for 25 kDa prolactin being a glycoform; 4) at 1 month of age 16 kDa rat prolactin appeared and persisted over the whole postnatal span (1 day-->1 year) but only in stored form; 5) the physiology of gestation was essentially characterized by the M(r)-modulation of the glycoform (26 kDa-->26.3 kDa) and the virtual absence of stored 26 kDa rat prolactin at week 1 of pregnancy; 6) in lactation and weaning uncommon multiple banding was observed in secreted oligomeric prolactin; 7) in pregnancy, lactation and weaning the differential distribution of released and stored prolactin isoforms displayed a considerable intra- and intervariability; 8) in the vast array of size isoforms observed in all our experiments monomeric 23 kDa prolactin was always the dominating variant. In conclusion, the molecular size heterogeneity and the differential distribution of secreted and stored rat pituitary prolactin is considerably influenced by age and physiological stimuli. The nature of polymeric prolactin and of the transient variants is presently unclear, and the exact physiological role of molecular heterogeneity modulation is unknown, both in humans and rat, but the patterns of change we observed in definite stages of life, suggest that this phenomenon is important in the maturation of the hypothalamus-pituitary axis and in the metabolic and hormonal changes accompanying gestation.

Effect of tunicamycin, swainsonine, castanospermine, Beta-hydroxynorvaline and monensin on the post-translational processing of rat prolactin molecular forms.

Abstract Prolactin cells derived from the anterior pituitaries of female rats were cultured in the presence of tunicamycin, swainsonine, castanospermine, beta-hydroxynorvaline and monensin in order to study their effect on the post-translational processing of the M(r) 17,000, 23,000 and 26,000 prolactin molecular forms. Sodium-dodecyl-sulphate polyacrylamide electrophoresis and subsequent immunoblotting revealed that: 1) tunicamycin, swainsonine and castanospermine, compounds that are essentially known as inhibitors of the N-glycosylation processus, had no effect on M(r) 17,000, 23,000 and 26,000 rat prolactin; 2) betahydroxynorvaline, which has been assumed to inhibit processing of pre-prolactin to mature 23,000 prolactin, did not increase the synthesis of 26,000 rat prolactin. In case of inhibition of the processing of a pre-prolactin to mature prolactin, one would expect an increase of the pre-prolactin; consequently, we could not establish the 26,000 rat prolactin, we revealed in immunoblotting, as a pre-prolactin; 3) monensin affected the post-translational processing of 17,000 and 26,000 rat prolactin, but left the 23,000 mature form intact. This is an important finding for the following reasons: monensin blocks the transport of secretory and membrane proteins, and this blockade prevents the cleavage of these molecules; indeed, production of 17,000 rat prolactin, a form of cleaved prolactin, was inhibited. Monensin also affects glycosylation and 26,000 rat prolactin has been identified as a presumably O-iinked glycosylated variant. The fact that its synthesis is inhibited by monensin treatment, but not by inhibitors of the N-linked process, particularly tunicamycin, and that 26,000 rat prolactin is susceptible to mild alkali and decomposition via beta-elimination are decisive arguments in favour of the O-linked glycosidic linkage.