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.     

Toll-like receptor 3 is induced by and mediates antiviral activity against rhinovirus infection of human bronchial epithelial cells

Rhinoviruses (RV) are the major cause of the common cold and acute exacerbations of asthma and chronic obstructive pulmonary disease. Toll-like receptors (TLRs) are a conserved family of receptors that recognize and respond to a variety of pathogen-associated molecular patterns. TLR3 recognizes double-stranded RNA, an important intermediate of many viral life cycles (including RV). The importance of TLR3 in host responses to virus infection is not known. Using BEAS-2B (a human bronchial epithelial cell-line), we demonstrated that RV replication increased the expression of TLR3 mRNA and TLR3 protein on the cell surface. We observed that blocking TLR3 led to a decrease in interleukin-6, CXCL8, and CCL5 in response to poly(IC) but an increase following RV infection. Finally, we demonstrated that TLR3 mediated the antiviral response. This study demonstrates an important functional requirement for TLR3 in the host response against live virus infection and indicates that poly(IC) is not always a good model for studying the biology of live virus infection.     

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     

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.     

No short‐ or long‐term effects of geolocator attachment detected in Pied Flycatchers Ficedula hypoleuca

Tracking small passerines using miniaturized location tags is a rapidly expanding field of study. In a 1‐year study, we tested whether there were any short‐ or longer‐term effects of fitting geolocators weighing 3% of body mass on male Pied Flycatchers Ficedula hypoleuca. In the deployment year, we compared adult provisioning rates to nestlings, nestling growth and nest success between nesting attempts in which adult males were fitted with a geolocator, with control nests where males had the same capture history but were not tagged. We found no difference between treatments in provisioning effort by males or their associated female 2 days after geolocator fitting, in terms of nestling growth, subsequent brood reduction or nest success. Return rate, arrival date on territories, nest timing and breeding parameters were compared between tagged and untagged males in the following breeding season. We found no difference in return rate or arrival date, and no difference in nest timing, fecundity or outcome. Our study suggests that fitting lightweight tags to small passerines need not affect behaviour, breeding or apparent between‐year survival. However, tagging new species should still require assessment and comparison with well‐matched control cohorts, and it should be recognized that tag effects could vary between years and populations, mediated by environmental conditions.     

Low and annually variable migratory connectivity in a long‐distance migrant: Whinchats Saxicola rubetra may show a bet‐hedging strategy

The spatial scale of non‐breeding areas used by long‐distance migrant animals can vary from specific, relatively small non‐breeding areas for each independent breeding population (high connectivity) to a distribution over a large non‐breeding area with mixing of breeding populations (low connectivity). Measuring variation in the degree of connectivity and how it arises is crucial to predict how migratory animals can respond to global habitat and climate change because low connectivity is likely to be an adaptation to environmental uncertainty. Here, we assess whether use of non‐breeding areas in a long‐distance migrant may be stochastic by measuring the degree of connectivity, and whether it is annually variable. Twenty‐nine wintering Whinchats tagged with geolocators over 2 years within 40 km² in central Nigeria were found to be breeding over 2.55 million km² (26% of the land area of Europe), without an asymptote being approached in the relationship between area and sample size. Ranges differed in size between years by 1.51 million km² and only 15% of the total breeding range across both years overlapped (8% overlap between years when only first‐year birds were considered), well above the range size difference and below the proportion of overlap that would be predicted from two equivalent groups breeding at random locations within the observed range. Mean distance between breeding locations (i.e. migratory spread) differed significantly between years (604 ± 18 km in 2013 and 869 ± 33 km in 2014). The results showed very low and variable connectivity that was reasonably robust to the errors and assumptions inherent in the use of geolocators, but with the caveat of having only ranges of 2 years to compare, and the sensitivity of range to the breeding locations of a small number of individuals. However, if representative, the results suggest the scope for between‐year variation (cohort effects) to determine migrant distribution on a large scale. Furthermore, for species with similarly low connectivity, we would predict breeding population trends to reflect average conditions across large non‐breeding areas: thus, as large areas of Africa become subject to habitat loss, migrant populations throughout Europe will decline.