Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

The structure of the hepatic insulin receptor and insulin binding.

Hepatocytes or hepatic plasma membranes were photoaffinity-labelled with radioiodinated N epsilon B29-monoazidobenzoyl-insulin. Analysis of the samples by SDS/polyacrylamide-gel electrophoresis and autoradiography revealed the insulin receptor as a predominant band of 450 kDa. When hepatic plasma membranes were first treated with clostridial collagenase and then photolabelled, the insulin receptor appeared as a predominant band of 360 kDa. This effect of collagenase treatment on the insulin receptor was due to Ca2+-dependent heat-labile proteinases contaminating the preparation of collagenase, and it could be mimicked by elastase. The decrease in size of the insulin receptor to 360 kDa resulted from the loss of a receptor component that was inaccessible to photolabelling. In contrast, the size of the insulin receptor of intact cells was not affected by collagenase treatment. This suggests that the site sensitive to proteolysis was located on the cytoplasmic side of the plasma membrane. In hepatic plasma membranes that were treated with collagenase or elastase, and contained the 360 kDa form of the insulin receptor, the binding affinity for insulin was increased by up to 2-fold. These findings support the concept that a component which is either a part of, or closely associated with, the insulin receptor may regulate its affinity for insulin.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

In-depth qualitative and quantitative analysis of composite glycosylation profiles and other micro-heterogeneity on intact monoclonal antibodies by high-resolution native mass spectrometry using a modified Orbitrap

Here, we describe a fast, easy-to-use, and sensitive method to profile in-depth structural micro-heterogeneity, including intricate N-glycosylation profiles, of monoclonal antibodies at the native intact protein level by means of mass spectrometry using a recently introduced modified Orbitrap Exactive Plus mass spectrometer. We demonstrate the versatility of our method to probe structural micro-heterogeneity by describing the analysis of three types of molecules: (1) a non-covalently bound IgG4 hinge deleted full-antibody in equilibrium with its half-antibody, (2) IgG4 mutants exhibiting highly complex glycosylation profiles, and (3) antibody-drug conjugates. Using the modified instrument, we obtain baseline separation and accurate mass determination of all different proteoforms that may be induced, for example, by glycosylation, drug loading and partial peptide backbone-truncation. We show that our method can handle highly complex glycosylation profiles, identifying more than 20 different glycoforms per monoclonal antibody preparation and more than 30 proteoforms on a single highly purified antibody. In analyzing antibody-drug conjugates, our method also easily identifies and quantifies more than 15 structurally different proteoforms that may result from the collective differences in drug loading and glycosylation. The method presented here will aid in the comprehensive analytical and functional characterization of protein micro-heterogeneity, which is crucial for successful development and manufacturing of therapeutic antibodies     

Characterization of histatin 5 with respect to amphipathicity, hydrophobicity, and effects on cell and mitochondrial membrane integrity excludes a candidacidal mechanism of pore formation

Histatin 5 is a 24-residue peptide from human saliva with antifungal properties. We recently demonstrated that histatin 5 translocates across the yeast membrane and targets to the mitochondria, suggesting an unusual antifungal mechanism (Helmerhorst, E. J., Breeuwer, P., van't Hof, W., Walgreen-Weterings, E., Oomen, L. C. J. M., Veerman, E. C. I., Nieuw Amerongen, A. V., and Abee, T. (1999) J. Biol. Chem. 274, 7286-7291). The present study used specifically designed synthetic analogs of histatin 5 to elucidate the role of peptide amphipathicity, hydrophobicity, and the propensity to adopt alpha-helical structures in relation to membrane permeabilization and fungicidal activity. Studies included circular dichroism measurements, evaluation of the effects on the cytoplasmic transmembrane potential and on the respiration of isolated mitochondria, and analysis of the peptide hydrophobicity/amphipathicity relationship (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623). The 14-residue synthetic peptides used were dh-5, comprising the functional domain of histatin 5, and dhvar1 and dhvar4, both designed to maximize amphipathic characteristics. The results obtained show that the amphipathic analogs exhibited a high fungicidal activity, a high propensity to form an alpha-helix, dissipated the cytoplasmic transmembrane potential, and uncoupled the respiration of isolated mitochondria, similar to the pore-forming peptide PGLa (Peptide with N-terminal Glycine and C-terminal Leucine-amide). In contrast, histatin 5 and dh-5 showed fewer or none of these features. The difference in these functional characteristics between histatin 5 and dh-5 on the one hand and dhvar1, dhvar4, and PGLa on the other hand correlated well with their predicted affinity for membranes based on hydrophobicity/amphipathicity analysis. These data indicate that the salivary protein histatin 5 exerts its antifungal function through a mechanism other than pore formation.     

Identification of early microbial colonizers in human dental biofilm

AIMS: To elucidate the first colonizers within in vivo dental biofilm and to establish potential population shifts that occur during the early phases of biofilm formation. METHODS AND RESULTS: A 'checkerboard' DNA-DNA hybridization assay was employed to identify 40 different bacterial strains. Dental biofilm samples were collected from 15 healthy subjects, 0, 2, 4 and 6 h after tooth cleaning and the composition of these samples was compared with that of whole saliva collected from the same individuals. The bacterial distribution in biofilm samples was distinct from that in saliva, confirming the selectivity of the adhesion process. In the very early stages, the predominant tooth colonizers were found to be Actinomyces species. The relative proportion of streptococci, in particular Streptococcus mitis and S. oralis, increased at the expense of Actinomyces species between 2 and 6 h while the absolute level of Actinomyces remained unaltered. Periodontal pathogens such as Tannerella forsythensis(Bacteroides forsythus), Porphyromonas gingivalis and Treponema denticola as well as Actinobacillus actinomycetemcomitans were present in extremely low levels at all the examined time intervals in this healthy group of subjects. CONCLUSION: The data provide a detailed insight into the bacterial population shifts occurring within the first few hours of biofilm formation and show that the early colonizers of the tooth surface predominantly consist of beneficial micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The early colonizers of dental plaque are of great importance in the succession stages of biofilm formation and its overall effect on the oral health of the host.     

Increase in daily LH secretion in response to short-term calorie restriction in obese women with PCOS

We hypothesized that short-term calorie restriction would blunt luteinizing hormone (LH) hypersecretion in obese women with polycystic ovary syndrome (PCOS) and thereby ameliorate the anovulatory endocrine milieu. To test this hypothesis, 15 obese patients with PCOS and nine age- and body mass index-matched healthy women underwent 24-h blood sampling to quantitate plasma LH, leptin, and insulin levels. PCOS subjects were prescribed a very low caloric liquid diet (4.2 MJ/day) for 7 days and were then resampled. Basal and pulsatile LH secretion was threefold higher in PCOS subjects, but plasma insulin and leptin levels were not different in the calorie-replete state. Contrary to expectation, calorie restriction enhanced basal and pulsatile LH secretion even further. As expected, plasma glucose, insulin, and leptin concentrations decreased by 18, 75, and 50%, respectively. Serum total testosterone concentration fell by 23%, whereas serum estrone, estradiol, sex hormone-binding globulin (SHBG), and androstenedione concentrations remained unchanged. Enhanced LH secretion in the presence of normal metabolic and hormonal adaptations to calorie restriction points to anomalous feedback control of pituitary LH release in PCOS.     

The diageotropica mutation alters auxin induction of a subset of the Aux/IAA gene family in tomato

The diageotropica (dgt) mutation has been proposed to affect either auxin perception or responsiveness in tomato plants. It has previously been demonstrated that the expression of one member of the Aux/IAA family of auxin-regulated genes is reduced in dgt plants. Here, we report the cloning of ten new members of the tomato Aux/IAA family by PCR amplification based on conserved protein domains. All of the gene family members except one (LeIAA7) are expressed in etiolated tomato seedlings, although they demonstrate tissue specificity (e.g. increased expression in hypocotyls vs. roots) within the seedling. The wild-type auxin-response characteristics of the expression of these tomato LeIAA genes are similar to those previously described for Aux/IAA family members in Arabidopsis. In dgt seedlings, auxin stimulation of gene expression was reduced in only a subset of LeIAA genes (LeIAA5, 8, 10, and 11), with the greatest reduction associated with those genes with the strongest wild-type response to auxin. The remaining LeIAA genes tested exhibited essentially the same induction levels in response to the hormone in both dgt and wild-type hypocotyls. These results confirm that dgt plants can perceive auxin and suggest that a specific step in early auxin signal transduction is disrupted by the dgt mutation.