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.     

Inhibition of an outwardly rectifying anion channel by HEPES and related buffers

Summary The effect of pH buffers and related compounds on the conductance of an outwardly rectifying anion channel has been studies using the patch-clamp technique. Single-channel current-voltage relationships were determined in solutions buffered by trace amounts of bicarbonate and in solutions containing N-substituted taurines (HEPES, MES, BES, TES) and glycines (glycylglycine, bicine and tricine), Tris andbis-Tris at millimolar concentrations. HEPES (pK_a=7.55) reduced the conductance of the channel when present on either side of the membrane. Significant inhibition was observed with 0.6mm HEPES on the cytoplasmic side (HEPES _i ) and this effect increased with [HEPES _i ] so that conductance at the reversal potential was diminished 25% with 10mm HEPES _i )and 70% at very high [HEPES _i ]. HEPES _i block was relieved by applying positive voltage but positive currents were not consistent with a Woodhulltype blocking scheme in that calculated dissociation constants and electrical distances depended on HEPES concentration. Results obtained by varying total HEPES _i concentration at constant [HEPES^–] and vice versa suggest both the anionic and zwitterionic (protonated) forms of HEPES inhibit. Structure-activity studies with related compounds indicate the sulfonate group and heterocyclic aliphatic groups are both required for inhibition from the cytoplasmic side. TES (pK_a=7.54), substituted glycine buffers (pK_a=8.1–8.4) andbis-Tris (pK_a=6.46) had no measurable effect on conductance and appear suitable for use with this channel.     

Epidemiologic features of Lyme disease in New York

During 1982, surveillance identified 207 cases of Lyme disease in New York State. Cases were clustered in two geographic areas, eastern Long Island and northern Westchester counties. Symptoms and signs of Lyme disease in cases were consistent with previous reports, with erythema chronicum migrans (ECM) being the most frequently (77 percent) reported sign of disease. Facial palsy was reported in a surprisingly high 18 percent of cases. Of 160 cases whose sera were submitted for Lyme spirochete specific IgG antibody testing, 112 (70 percent) had titers greater than or equal to 64, while 88 (55 percent) had titers greater than or equal to 128. Positive titers were not associated with any single sign or symptom of disease, but were significantly associated with symptom onset or tick bite occurring during the three-month period of June, July, and August. We conclude that the incidence of Lyme disease in New York is much higher than previously recognized. In addition, our data suggest that a serologic test for Lyme-spirochete IgG antibody lacks sensitivity, but can be useful in confirming the diagnosis of Lyme disease when antibody titers are high.     

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.     

The diameter and mean sarcomere length of individual muscle fibres.

Studies of the coefficients of variation and the repeatibility of the measurements indicate that a sample of 25 fibres is sufficient to provide an accurate estimate of the mean fibre diameter and sarcomere length of a muscle. There is a significant negative correlation (−0.55) between the diameter and mean sarcomere length of an individual muscle fibre. Because they affect sarcomere length postmortem mechanical influences must be strictly standardised if fibre diameter is to be a reliable parameter of muscle growth.     

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     

Investigation of Prolific Sheep from UK and Ireland for Evidence on Origin of the Mutations in BMP15 (FecXG,FecXB) and GDF9 (FecGH) in Belclare and Cambridge Sheep

This paper concerns the likely origin of three mutations with large effects on ovulation rate identified in the Belclare and Cambridge sheep breeds; two in the BMP15 gene (FecX^G and FecX^B) and the third (FecG^H) in GDF9. All three mutations segregate in Belclare sheep while one, FecX^B, has not been found in the Cambridge. Both Belclare and Cambridge breeds are relatively recently developed composites that have common ancestry through the use of genetic material from the Finnish Landrace and Lleyn breeds. The development of both composites also involved major contributions from exceptionally prolific ewes screened from flocks in Ireland (Belclare) and Britain (Cambridge) during the 1960s. The objective of the current study was to establish the likely origin of the mutations (FecX^G, FecX^B and FecG^H) through analysis of DNA from Finnish Landrace and Lleyn sheep, and Galway and Texel breeds which contributed to the development of the Belclare breed. Ewes with exceptionally high prolificacy (hyper-prolific ewes) in current flocks on Irish farms were identified to simulate the screening of ewes from Irish flocks in the 1960s. DNA was obtained from: prolific ewes in extant flocks of Lleyn sheep (n = 44) on the Lleyn peninsula in Wales; hyper-prolific ewes (n = 41); prolific Galway (n = 41) ewes; Finnish Landrace (n = 124) and Texel (n = 19) ewes. The FecX^G mutation was identified in Lleyn but not in Finnish Landrace, Galway or Texel sheep; FecX^B was only found among the hyper-prolific ewes. The FecG^H mutation was identified in the sample of Lleyn sheep. It was concluded from these findings that the Lleyn breed was the most likely source of the FecX^G and FecG^H mutations in Belclare and Cambridge sheep and that the FecX^B mutation came from the High Fertility line that was developed using prolific ewes selected from commercial flocks in Ireland in the 1960′s and subsequently used in the genesis of the Belclare.     

Potency of GABA at human recombinant GABAA receptors expressed in Xenopus oocytes: a mini review

GABA_A receptors are members of the ligand-gated ion channel superfamily that mediate inhibitory neurotransmission in the central nervous system. They are thought to be composed of 2 alpha (α), 2 beta (β) subunits and one other such as a gamma (γ) or delta (δ) subunit. The potency of GABA is influenced by the subunit composition. However, there are no reported systematic studies that evaluate GABA potency on a comprehensive number of subunit combinations expressed in Xenopus oocytes, despite the wide use of this heterologous expression system in structure–function studies and drug discovery. Thus, the aim of this study was to conduct a systematic characterization of the potency of GABA at 43 human recombinant GABA_A receptor combinations expressed in Xenopus oocytes using the two-electrode voltage clamp technique. The results show that the α-subunits and to a lesser extent, the β-subunits influence GABA potency. Of the binary and ternary combinations with and without the γ2L subunit, the α6/γ2L-containing receptors were the most sensitive to GABA, while the β2- or β3-subunit conferred higher sensitivity to GABA than receptors containing the β1-subunit with the exception of the α2β1γ2L and α6β1γ2L subtypes. Of the δ-subunit containing GABA_A receptors, α4/δ-containing GABA_A receptors displayed highest GABA sensitivity, with mid-nanomolar concentrations activating α4β1δ and α4β3δ receptors. At α4β2δ, GABA had low micromolar activity.     

Inhibition of Vancomycin-Resistant <Emphasis Type="Italic">Enterococcus</Emphasis> by Continuous-Flow Cultures of Human Stool Microflora With and Without Anaerobic Gas Supplementation

A continuous-flow competitive exclusion (CFCE) culture model of human stool microflora was used to examine whether supplemental anaerobic gas is necessary for maintenance of anaerobes and inhibition of vancomycin-resistant Enterococcus (VRE). CFCE cultures of human stool microflora were maintained with supplemental nitrogen, without supplemental nitrogen, or with percolated room air. Cultures with or without supplemental nitrogen maintained >9 log₁₀ CFU mL^–1 of obligate anaerobes and eliminated 10⁶ CFU mL^–1 of VRE. When room air was percolated into the culture, anaerobes were detected at 2 log₁₀ CFU mL^–1, and the same VRE inoculum was not eliminated (P < 0.001). These data demonstrate that human stool CFCE cultures maintain high levels of obligate anaerobes and inhibit VRE without the addition of supplemental anaerobic gas.