Impact of clarification strategy on chromatographic separations: Pre-processing of cell homogenates

This research focused on how the extent and type of primary solid-liquid separation can affect the performance of guard filtration and chromatography, in this instance hydrophobic interaction chromatography. The system used in the study was yeast (Saccharomyces cerevisiae) with the target molecule being an intracellular protein; alcohol dehydrogenase (ADH). As expected, loading more poorly clarified suspensions (both centrates and primary filtrates) required proportionally larger guard filtration areas. In addition for feed suspensions prepared by centrifugation, increased clarification led to greater column capacity. However, where filtration was used to achieve similar clarification considerably lower column capacity was achieved. These results were attributed to centrifugation leading to the aggregation of lipids and their subsequent removal in this form before application to the column. Clarification by filtration leaves such lipids in their original "soluble" state and hence they are not removed. The importance of the need to examine such interactive effects in bioprocess studies is discussed. This observation was confirmed with further analytical work into the nature of the aggregated material formed in the supernatant under centrifugation conditions. This material was only soluble in an organic solvent, and identified as phophatidylcholine and ergosterol as among the components removed by centrifugation and guard filtration as opposed to filtration and guard filtration.     

Adaptive developmental plasticity in growing nestlings: sibling competition induces differential gape growth

Sibling competition has been shown to affect overall growth rates in birds. However, growth consists on the coordinated development of a multitude of structures, and there is ample scope for developmental plasticity and trade-offs among these structures. We would expect that the growth of structures that are used in sibling competition, such as the gape of altricial nestlings, should be prioritized under intense competition. We conducted an experiment in the spotless starling (Sturnus unicolor), cross-fostering nestlings to nests with different levels of sibling competition. We predicted that nestlings subjected to higher levels of sibling competition should develop larger gapes than control birds. We found that, halfway through the nestling period, overall size (a composite index of mass, wing, tarsus and bill) was reduced in nests with intense sibling competition, whereas gape width remained unaffected. At the end of the nestling period, experimental nestlings had wider gapes than controls. Additionally, a correlative study showed that nestling gape width increased when feeding conditions worsened and overall size decreased. These patterns could either be due to increased growth of gape flanges or to delayed reabsorption of this structure. Our results show that birds can invest differentially in the development of organs during growth, and that the growth of organs used in sibling competition is prioritized over structural growth.     

Parent-absent begging: evidence for sibling honesty and cooperation in the spotless starling (Sturnus unicolor)

Begging in avian nestlings is a highly conspicuous behaviorwith important implications for the study of parent–offspringconflict. In some species, nestlings also call for long boutsin the absence of parents, and it has been proposed that thisbehavior is used by nestlings as a means of negotiating accessto food. We studied this phenomenon in the spotless starling(Sturnus unicolor). We found that parent-absent calls were acousticallydistinct from parent-present calls. Observations showed thatthe probability of parent-absent begging increased with nestlingage and brood size, whereas it decreased with increasing bodycondition. This result was confirmed by an experiment that showedthat nestlings produced higher parent-absent begging rates whenfood deprived than when satiated. Finally, we carried out aplayback experiment to test the reaction of nestlings to parent-absentbegging by fellow nestlings. Principle components analyses yielded2 independent components of begging: 1) a general begging componentand 2) a second factor that measures the relative contributionof communicative begging over competitive begging. Nestlingsexposed to playback decreased their general begging levels andsimultaneously increased the relative contribution of communicativeover competitive begging. This behavior may favor needy nestlingsto obtain impending feedings while keeping high levels of foodsolicitation from parents and is consistent with a cooperativestrategy among nestlings. Future research should consider theactual response of parents to these signals.     

Phylogeny and systematics of the bee genus Osmia (Hymenoptera: Megachilidae) with emphasis on North American Melanosmia: subgenera,synonymies and nesting biology revisited

The predominantly Holarctic bee genus Osmia Panzer is species‐rich and behaviourally diverse. A robust phylogeny of this genus is important for understanding the evolution of the immense variety of morphological and behavioural traits exhibited by this group. We infer a phylogeny of Osmia using DNA sequence data obtained from three nuclear genes (elongation factor 1‐α, LW‐rhodopsin and CAD) and the mitochondrial gene COI. Our taxon sampling places special attention on North American members of the subgenus Melanosmia Schmiedeknecht; we discuss the novel placement of a number of species traditionally assigned to O. (Melanosmia) and examine the relative support for alternative classifications of this species‐rich subgenus. We use this new phylogeny to guide a reassessment of morphological and behavioural characters within Osmia. Our results provide support for the recognition of Osmia (Hapsidosmia), subgen.n ., a monotypic subgenus containing Osmia iridis Cockerell & Titus. We synonymize Osmia (Mystacosmia) Snelling under O. (Melanosmia), syn.n . We synonymize Osmia (Acanthosmioides) Ashmead under O. (Melanosmia), syn.n ., propose ‘odontogaster species group’ as a replacement for the subgeneric name Acanthosmioides, and refine the morphological characters that serve to diagnose the species group. We additionally propose ‘nigrifrons species group’ for a clade within O. (Melanosmia) containing most species formerly placed in Osmia (Centrosmia) Robertson. We demonstrate more cohesive patterns of nest substrate use in the nigrifrons and odontogaster species groups than was previously believed to occur, reconsider character polarity of aspects of the female mandible, and show that a large number of morphological characters have evolved convergently within the genus. In order to facilitate discussion of relevant taxa, we propose the following 15 new synonymies: O. bakeri Sandhouse under O. melanopleura Cockerell; O. crenulaticornis Michener under O. pinorum Cockerell; O. claremontensis Michener under O. sedula Sandhouse; O. cockerelli Sandhouse under O. dakotensis Michener; O. francisconis White under O. enixa Sandhouse; O. hurdi White under O. austromaritima Michener; O. sladeni Sandhouse under O. nifoata Cockerell; O. titusi Cockerell under O. phenax Cockerell; O. subtrevoris Cockerell, O. physariae Cockerell, and O. erecta Michener under O. giliarum Cockerell; and O. universitatis Cockerell, O. integrella Cockerell, O. amala Cockerell, and O. metitia Cockerell under O. nigrifrons Cresson, syn.n . We remove O. wyomingensis Michener from synonymy with O. nifoata Cockerell, stat.n ., and O. pinorum Cockerell from synonymy with O. physariae Cockerell, stat.n . This published work has been registered in ZooBank, http://zoobank.org/urn:lsid:zoobank.org:pub:A3E7D63B‐5C4C‐4ACF‐BF33‐48E5C5DD1B0D .     

Endogenous chloride channels of insect sf9 cells. Evidence for coordinated activity of small elementary channel units

The endogenous Cl- conductance of Spodoptera frugiperda (Sf9) cells was studied 20-35 h after plating out of either uninfected cells or cells infected by a baculovirus vector carrying the cloned beta-galactosidase gene (beta-Gal cells). With the cation Tris+ in the pipette and Na+ in the bath, the reversal potential of whole-cell currents was governed by the prevailing Cl- equilibrium potential and could be fitted by the Goldman-Hodgkin-Katz equation with similar permeabilities for uninfected and beta-Gal cells. In the frequency range 0.12 < f < 300 Hz, the power density spectrum of whole-cell Cl- currents could be fitted by three Lorentzians. Independent of membrane potential, >50% of the total variance of whole-cell current fluctuations was accounted for by the low frequency Lorentzian (fc = 0.40 +/- 0.03 Hz, n = 6). Single-Cl- channels showed complex gating kinetics with long lasting (seconds) openings interrupted by similar long closures. In the open state, channels exhibited fast burst-like closures. Since the patches normally contained more than a single channel, it was not possible to measure open and closed dwell-time distributions for comparing single-Cl- channel activity with the kinetic features of whole-cell currents. However, the power density spectrum of Cl- currents of cell-attached and excised outside-out patches contained both high and low frequency Lorentzian components, with the corner frequency of the slow component (fc = 0.40 +/- 0.02 Hz, n = 4) similar to that of whole-cell current fluctuations. Chloride channels exhibited multiple conductance states with similar Goldman-Hodgkin-Katz-type rectification. Single-channel permeabilities covered the range from approximately 0.6.10(-14) cm5/s to approximately 6.10(-14) cm3/s, corresponding to a limiting conductance (gamma 150/150) of approximately 3.5 pS and approximately 35 pS, respectively. All states reversed near the same membrane potential, and they exhibited similar halide ion selectivity, P1 > PCl approximately PBr. Accordingly, Cl- current amplitudes larger than current flow through the smallest channel unit resolved seem to result from simultaneous open/shut events of two or more channel units.     

Characterization of intraepithelial lymphocytes in human endometrium

Intraepithelial lymphocytes (IELs) were characterized and quantitated in normal non-pregnant endometrium and in early pregnancy decidua using H & E and phloxine tartrazine stains and a panel of monoclonal antibodies in an indirect immunoperoxidase technique. The relative numbers of granulated and non-granulated IELs varied according to menstrual cycle stage and in early pregnancy all IELs appeared to be granulated. There was a higher surface:gland ratio for IELs in proliferative endometrium compared with late secretory phase and early pregnancy endometrium. In proliferative endometrium most IELs were T cells, predominantly of the CD8 + subset. In first trimester decidua, higher numbers of CD56 + cells were observed, in keeping with the increased proportion of granulated IELs. IEL populations in human endometrium vary according to menstrual cycle stage and endometrial IELs appear to show phenotypic differences compared with IELs in the human gastrointestinal tract.