The extent of polylactosamine glycosylation of MDCK LAMP-2 is determined by its Golgi residence time

The increased polylactosamine glycosylation of LAMP-2 in MDCK cells cultured for 1 day relative to cells cultured for 3 days has been correlated with its slower rate of Golgi transit (Nabi and Rodriguez- Boulan, 1993, Mol. Biol. Cell., 4, 627-635). To determine if the differential polylactosamine glycosylation of LAMP-2 is a consequence of glycosyltransferase expression levels, the activities of beta1- 6GlcNAc-TV, beta1-3GlcNAc-T(i), beta1-2GlcNAc-TI, beta1, 4Gal-T, alpha2- 6sialyl-T, and alpha2-3sialyl-T were assayed and no significant differences in the activities of these enzymes in 1 and 3 day cell extracts were detected. During MDCK epithelial polarization, the Golgi apparatus undergoes morphological changes and apiconuclear Golgi networks were more evident in 3 day cells. Treatment with nocodazole disrupted Golgi networks and generated numerous Golgi clusters in both 1 day and 3 day cells. In the presence of nocodazole the differential migration of LAMP-2 in 1 and 3 day MDCK cells was maintained and could be eliminated by treatment with endo-beta-galactosidase, indicating that gross Golgi morphology did not influence the extent of LAMP-2 polylactosamine glycosylation. Nocodazole treatment did, however, result in the faster migration of LAMP-2 which was not due to modification of core N-glycans as the precursor form of the glycoprotein migrated with an identical molecular size. Following incubation at 20 degrees C, which prevents the exit of proteins from the trans-Golgi network, the molecular size of LAMP-2 increased to a similar extent in both 1 and 3 day MDCK cells. Extending the time of incubation at 20 degrees C did not influence the size of LAMP-2, demonstrating that its glycosylation is modified not by its retention within the Golgi but rather by its equivalent slower Golgi passage at the lower temperature in both 1 and 3 day cells. An identical effect was observed in nocodazole treated cells, demonstrating that Golgi residence time determines the extent of LAMP-2 polylactosamine glycosylation, even in isolated Golgi clusters.      

Development of the hypothalamic-pituitary-axis in the ovine fetus: ontogeny of action of ovine corticotropin-releasing factor

In samples from twenty chronically cannulated ovine fetuses the plasma immunoreactive adrenocorticotrophin (ACTH) concentrations were 12.5 +/- 3.2(8), 15.2 +/- 4.1(9) and 21.2 +/- 5.6(8) pg/ml at periods, prior to parturition, of -30 to -35, -25 to -29 and -20 to -24 days respectively. Values are mean +/- SEM (number of samples). These values were not significantly different from each other but were significantly lower (P less than 0.02) than values in the next two age groups -36.0 +/- 4.9(7) pg/ml at -19 to -15 days, and 39.6 +/- 6.6(11) pg/ml at -14 to -9 days. A further significant increase (P less than 0.05) occurred in the -8 to -3 day period, ACTH being 53.9 +/- 5.4(12) pg/ml. On day of delivery two samples had values of 325 and 360 pg/ml. A single injection, intravenously of 1.0 microgram ovine corticotrophin-releasing factor (O-CRF), caused a significant increase in fetal plasma ACTH concentrations in fetuses of -6 to -23 days prior to delivery but not in fetuses -24 to -35 days prior to parturition. The maximum values of ACTH after O-CRF were significantly greater in fetuses -2 to 0 days prior to parturition than in younger fetuses (P less than 0.01). In 6 experiments in 4 fetuses (parturition -1 to -13 days) the effect of 1.0 microgram O-CRF persisted for at least 2.5 h. The results support the hypothesis that the pituitary release of ACTH changes sensitivity to hypothalamic O-CRF at least twice during the last fifth of gestation; an increasing sensitivity is seen as term approaches.     

Alpha-inhibin gene expression occurs in the ovine adrenal cortex, and is regulated by adrenocorticotropin

Inhibin is a glycoprotein hormone composed of two nonidentical subunits. It is produced by the ovary and testis and plays a vital role in gonadal function by inhibiting the secretion of FSH. More recently, additional activities associated with inhibin peptides have been identified. Inhibin heterodimers (alpha-beta) are reported to act directly on ovarian granulosa cells and inhibit estrogen production induced by FSH. Furthermore, homodimers of beta-inhibin subunits stimulate the secretion of FSH, an activity that is directly opposite to that of inhibin. Each of these inhibin-related activities are concerned with the hypothalamic-pituitary-gonadal axis. We have investigated further the complexity of inhibin activity by determining whether inhibin genes are expressed in nongonadal tissue. RNA hybridization experiments demonstrate that the alpha-inhibin gene is expressed in the sheep adrenal cortex and hybridization histochemistry shows that this gene is expressed in each of the functional zones within the cortex. Dot blot analysis showed that the level of alpha mRNA within the adrenal is influenced by ACTH, one of the major regulators of adrenal cortex function. These observations imply that there are inhibin-related peptides not directly associated with the gonads. beta-inhibin gene expression was not clearly detected in the adrenal and we conclude that if expression occurs then it does so at extremely low levels.     

Cross-bridge number, position, and angle in target zones of cryofixed isometrically active insect flight muscle

Electron micrographic tomograms of isometrically active insect flight muscle, freeze substituted after rapid freezing, show binding of single myosin heads at varying angles that is largely restricted to actin target zones every 38.7 nm. To quantify the parameters that govern this pattern, we measured the number and position of attached myosin heads by tracing cross-bridges through the three-dimensional tomogram from their origins on 14.5-nm-spaced shelves along the thick filament to their thin filament attachments in the target zones. The relationship between the probability of cross-bridge formation and axial offset between the shelf and target zone center was well fitted by a Gaussian distribution. One head of each myosin whose origin is close to an actin target zone forms a cross-bridge most of the time. The probability of cross-bridge formation remains high for myosin heads originating within 8 nm axially of the target zone center and is low outside 12 nm. We infer that most target zone cross-bridges are nearly perpendicular to the filaments (60% within 11 degrees ). The results suggest that in isometric contraction, most cross-bridges maintain tension near the beginning of their working stroke at angles near perpendicular to the filament axis. Moreover, in the absence of filament sliding, cross-bridges cannot change tilt angle while attached nor reach other target zones while detached, so may cycle repeatedly on and off the same actin target monomer.     

Bacitracin significantly reduces degradation of peptides in plant cell cultures

Production of useful peptides using recombinant plant cells is often limited by the stability of the newly translated peptides in the plant expression system. As an initial step toward producing peptides in transformed plant cell cultures, we examined the stability of exogenously added arginine vasopressin (AVP) peptide in a suspension culture of Nicotiana plumbaginifolia cells. The peptide was lost rapidly from the plant cell culture, but the rate of loss was slowed significantly by the addition of the peptide, bacitracin, at a concentration of 150 mug/mL. At higher concentrations, bacitracin substantially inhibited the growth of the plant cells in culture. (c) 1997 John Wiley & Sons, Inc.     

Rapid semi-on-line monitoring of Fmoc solid-phase peptide synthesis by matrix-assisted laser desorption/ionization mass spectrometry

A simple yet highly effective application of matrix-assisted laser desorption/ionization massspectrometry (MALDI-MS) for the rapid monitoring of Fmoc solid-phase peptide synthesisis described. A few beads of the resin are removed at any desired step during synthesis, thefully protected peptide is cleaved from the resin and an MS spectrum of the analytes presentis produced. Some standard side-chain protecting groups may be cleaved off during samplepreparation for MS analysis; however, these cleavages are readily identified. Using thisapproach, incomplete amino acid acylations are readily detected in approximately the sametime as by traditional tests such as ninhydrin. The semi-on-line method also lends itself toready optimization of synthesis protocols and to the examination of resin-bound peptide sidereactions which may not be detectable by chemical means.