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.     

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     

Hearing dysfunction in heterozygous MitfMi‐wh/+ mice,a model for Waardenburg syndrome type 2 and Tietz syndrome

The human deafness‐pigmentation syndromes, Waardenburg syndrome (WS) type 2a, and Tietz syndrome are characterized by profound deafness but only partial cutaneous pigmentary abnormalities. Both syndromes are caused by mutations in MITF. To illuminate differences between cutaneous and otic melanocytes in these syndromes, their development and survival in heterozygous Microphthalmia‐White (Mitf^Mi‐wh/+) mice were studied and hearing function of these mice characterized. Mitf^Mi‐wh/+ mice have a profound hearing deficit, characterized by elevated auditory brainstem response thresholds, reduced distortion product otoacoustic emissions, absent endocochlear potential, loss of outer hair cells, and stria vascularis abnormalities. Mitf^Mi‐wh/+ embryos have fewer melanoblasts during embryonic development than their wild‐type littermates. Although cochlear melanocytes are present at birth, they disappear from the Mitf^Mi‐wh/+ cochlea between P1 and P7. These findings may provide insight into the mechanism of melanocyte and hearing loss in human deafness‐pigmentation syndromes such as WS and Tietz syndrome and illustrate differences between otic and follicular melanocytes.     

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.     

Biochemical isolation and purification of ovulation-inducing factor (OIF) in seminal plasma of llamas

Background  

The objective of the present study was to isolate and purify the protein fraction(s) of llama seminal plasma responsible for the ovulation-inducing effect of the ejaculate.     

Test of the principle of initial value in rat genetic models of exercise capacity

An inverse relationship between initial level of physical capacity and the magnitude of response to training is termed the principle of initial value. We tested the operation of this principle under experimental conditions of minimal genetic and environmental variation. Inbred rat strains previously identified as genetic models of low [Copenhagen (COP)] and high [Dark Agouti (DA)] intrinsic (untrained) exercise capacity were trained for 8 wk on a treadmill using two disparate protocols: 1) a relative mode where each rat exercised daily according to its initial capacity, and 2) an absolute mode where both strains received the same amount of training independent of initial capacity. Response to exercise was the change in running capacity as estimated by meters run to exhaustion before and after training. When trained with the relative mode, COP rats gained 88 m (+21%; NS) whereas DA rats increased distance run by 228 m (+36%; P < 0.001). When each strain trained with the same absolute amount of training, the COP strain showed essentially no change (-6 m, -2%) and the DA strain gained 325 m (+49%; P < 0.009). Differences in response to exercise between the COP and DA could not be explained by body mass differences, oxidative enzyme activity (citrate synthase or ATP), or spontaneous behavioral activity. Our data demonstrate that genetic factors causative of high response to exercise are not uniquely associated with genetic factors for low intrinsic capacity and thus are not in accord with the principle of initial value.     

Enhanced mitochondrial sensitivity to creatine in rats bred for high aerobic capacity.

Qualitative and quantitative measures of mitochondrial function were performed in rats selectively bred 15 generations for intrinsic aerobic high running capacity (HCR; n = 8) or low running capacity (LCR; n=8). As estimated from a speed-ramped treadmill exercise test to exhaustion (15 degrees slope; initial velocity of 10 m/min, increased 1 m/min every 2 min), HCR rats ran 10 times further (2,375+/-80 m) compared with LCR rats (238+/-12 m). Fiber bundles were obtained from the soleus and chemically permeabilized. Respiration was measured 1) in the absence of ADP, 2) in the presence of a submaximally stimulating concentration of ADP (0.1 mM ADP, with and without 20 mM creatine), and 3) in the presence of a maximally stimulating concentration of ADP (2 mM). Although non-ADP-stimulated and maximally ADP-stimulated rates of respiration were 13% higher in HCR compared with LCR, the difference was not statistically significant (P>0.05). Despite a similar rate of respiration in the presence of 0.1 mM ADP, HCR rats demonstrated a higher rate of respiration in the presence of 0.1 mM ADP+20 mM creatine (HCR 33% higher vs. LCR, P<0.05). Thus mitochondria from HCR rats exhibit enhanced mitochondrial sensitivity to creatine (i.e., the ability of creatine to decrease the Km for ADP). We propose that increased respiratory sensitivity to ADP in the presence of creatine can effectively increase muscle sensitivity to ADP during exercise (when creatine is increased) and may be, in part, a contributing factor for the increased running capacity in HCR rats.