Northern hybridization analysis of VH gene expression in murine monoclonal antibodies directed to cancer-associated ganglioside antigens having various sialic acid linkages

The expression of the VH genes in 46 murine hybridoma cells that secrete mAb directed to the cancer-associated carbohydrate Ag, especially acidic glycolipids such as gangliosides and sulfated glycoplipids, was analyzed by Northern hybridization of poly(A)+ RNA of hybridoma with cDNA probes for nine VH gene families. Different hybridomas tended to express VH genes of the same family when the cognate Ag had the same or similar carbohydrate structures; i.e., the VH genes of the J558 family (group 1) were preferentially expressed in the mAb directed to various gangliosides that have NeuAc alpha (or NeuGc alpha) 2-3 and/or 2-8 linkage (71%), the most common linkage of sialic acid residues in the gangliosides of higher animals, and the hybridomas directed to sulfated glycolipids also expressed mainly the VH genes of the J558 family (80%). In contrast, the five mAb directed to various gangliosides with NeuAc alpha 2-6 linkage were exclusively encoded by the VH genes of Q52 family (group 2, 100%), and three antibodies directed to gangliosides with a NeuAc alpha 2-9 linkage all expressed genes of J606 family (group 6, 100%). The VH family usage was largely correlated with the linkage of sialic acid residues in the cognate carbohydrate Ag, but was not correlated at all with the difference in the fine specificities toward the core neutral carbohydrate chain, to which the sialic acid residues were attached. These findings suggest that the VH gene family in these anticarbohydrate antibodies is selected, depending primarily on the linkage of the sialic acid residues in carbohydrate Ag; these residues form the immunodominant sugar residue in the respective antigenic determinant.     

Phylogeography of Fischer’s blue,Tongeia fischeri,in Japan: Evidence for introgressive hybridization

The widespread lycaenid butterfly Tongeia fischeri is distributed from eastern Europe to northeastern Asia and represented by three geographically isolated populations in Japan. In order to clarify the phylogeographic history of the species, we used sequences of three mitochondrial (COI, Cyt b and ND5) and two nuclear (Rpl5 and Ldh) genes of 207 individuals collected from 55 sites throughout Japan and five sites on the Asian continent. Phylogenetic trees and the median-joining network revealed six evolutionary mitochondrial haplotype clades, which corresponded to the geographic distribution of the species. Common ancestors of Japanese T. fischeri might have come to Japan during the mid-Pleistocene by multiple dispersals of continental populations, probably via a land bridge or narrow channel between western Japan and the Korean Peninsula. The geographical patterns of variation of mitochondrial and nuclear markers are discordant in northeastern Kyushu, possibly as a result of introgressive hybridization during the ancient contact between the Kyushu and Shikoku populations in the last glacial maximum. The phylogeographic pattern of T. fischeri in Japan are probably related to the geological history, Pleistocene climatic oscillations and distribution of the host plant.     

Effect of insulin on exocrine pancreatic secretion in healthy and diabetic anaesthetised rats

This investigation characterised the effects of exogenous insulin on exocrine pancreatic secretion in anaesthetised healthy and diabetic rats. Animals were rendered diabetic by a single injection of streptozotocin (STZ, 60 mg kg(-1) I.P.). Age-matched controls were injected citrate buffer. Rats were tested for hyperglycaemia 4 days after STZ injection and 7-8 weeks later when they were used for the experiments. Following anaesthesia (1 g kg(-1) urethane I.P.), laparotomy was performed and the pancreatic duct cannulated for collection of pure pancreatic juice. Basal pancreatic juice flow rate in diabetic rats was significantly (p < 0.001) increased whereas protein and amylase outputs were significantly (p < 0.001) decreased compared to control rats. Insulin (1 IU, I.P.) produced in healthy rats significant increases in pancreatic flow rate, amylase secretion and protein output compared to basal (p < 0.05). Insulin action also included a reduction in blood glucose (152.7 +/- 16.9 mg dl(-1), n = 6, prior to insulin and 42.0 +/- 8.4 mg dl(-1), n = 4, 100 min later). In fact, flow rate and glycaemia showed a strong negative correlation (p < 0.01, Pearson). Pretreatment with atropine (0.2 mg kg(-1), I.V.) abolished the effects of insulin on secretory parameters despite a similar reduction in glycaemia; in this series of experiments the correlation between flow rate and blood glucose was lost. In diabetic rats, insulin (4 IU, I.P.) did not modify exocrine pancreatic secretion. There was a fall in blood glucose (467.6 +/- 14.0 mg dl(-1), n = 10, prior to insulin and 386.6 +/- 43.6 mg dl(-1), n = 7, 120 min later). Rats, however, did not become hypoglycaemic. Similar results were observed in diabetic atropinized rats. The results of this study indicate that the effects of insulin on exocrine pancreatic secretion in anaesthetised healthy rats are mediated by hypoglycaemia-evoked vagal cholinergic activation.     

Dynamic neuroplasticity after human prefrontal cortex damage

Memory and attention deficits are common after prefrontal cortex (PFC) damage, yet people generally recover some function over time. Recovery is thought to be dependent upon undamaged brain regions, but the temporal dynamics underlying cognitive recovery are poorly understood. Here, we provide evidence that the intact PFC compensates for damage in the lesioned PFC on a trial-by-trial basis dependent on cognitive load. The extent of this rapid functional compensation is indexed by transient increases in electrophysiological measures of attention and memory in the intact PFC, detectable within a second after stimulus presentation and only when the lesioned hemisphere is challenged. These observations provide evidence supporting a dynamic and flexible model of compensatory neural plasticity.     

Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan

Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.     

Flow-enhanced adhesion regulated by a selectin interdomain hinge

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.     

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLβ2

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)β(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)β(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)β(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)β(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal β(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the β(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)β(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)β(2)-mediated arrest.     

A phylogenomic tree inferred with an inexpensive PCR-generated probe kit resolves higher-level relationships among Neptis butterflies (Nymphalidae: Limenitidinae)

Recent advances in obtaining reduced representation libraries for next-generation sequencing permit phylogenomic analysis of species-rich, recently diverged taxa. In this study, we performed sequence capture with homemade PCR-generated probes to study diversification among closely related species in a large insect genus to examine the utility of this method. We reconstructed the phylogeny of Neptis Fabricius, a large and poorly studied nymphalid butterfly genus distributed throughout the Old World. We inferred relationships among 108 Neptis samples using 89 loci totaling up to 84 519 bp per specimen. Our taxon sample focused on Palearctic, Oriental and Australasian species, but included 8 African species and outgroups from 5 related genera. Maximum likelihood and Bayesian analyses yielded identical trees with full support for almost all nodes. We confirmed that Neptis is not monophyletic because Lasippa heliodore (Fabricius) and Phaedyma amphion (Linnaeus) are nested within the genus, and we redefine species groups for Neptis found outside of Africa. The statistical support of our results demonstrates that the probe set we employed is useful for inferring phylogenetic relationships among Neptis species and likely has great value for intrageneric phylogenetic reconstruction of Lepidoptera. Based on our results, we revise the following two taxa: Neptis heliodore comb. rev. and Neptis amphion comb. rev.