The response of the atrium to direct mechanical wounding in the adult heart of the newt,Notophthalmus viridescens

Summary Wound repair and proliferation were examined in the injured newt atrium with light- and electron-microscopic techniques including autoradiography. Hearts were injured by removing a piece approximately 0.5 mm² of the atrial wall. The five-day wound was an endothelial and mesothelial-lined blood clot bordered by a 150-m necrotic zone. Repair progressed from the periphery inward with areas of macrophage activity replaced by fibroblasts and connective tissue. The wound at 25 days consisted of a scar with few myocytes. There was no difference in the proliferative behavior between the right and left atria. Proliferative cells were localized to a 500-m reactive zone surrounding the wound. The maximum mesothelial cell thymidine-labeling index of 20.5% and mitotic index of 1.4% were seen 5 days after injury. The peak connective tissue cell thymidine-labeling index of 10.2% and mitotic index of 0.4% were seen 10 days after wounding. The peak thymidine-labeling index of 9.8% for myocardial cells was recorded 10 days after injury with a mitotic index of 0.2%. Proliferation returned to control levels by 25 days post-injury. Electron microscopy demonstrated that myocytes engaged in DNA synthesis were indistinguishable from control myocytes. Z-band material was not observed in mitotic myocytes, but myofilaments and junctions were present.     

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.     

Abnormal dorsal light response in teleost fish after intraocular injection of 6-hydroxydopamine

Following unilateral intraocular injection with 6-hydroxydopamine to the right eye, Midas cichlids displayed an acute abnormal dorsal light response, in favour of the right eye. This condition was reversed immediately by dopamine or dopamine receptor agonists delivered by intraocular injection but this recovery was not sustained, Retinal dopamine production reappeared gradually from day 30 post-6-hydroxydopamine treatment, and coincided with the fish beginning to adopt a normal dorsal light reaction. Dopamine production recovered completely at day 45 post-6-hydroxydopamine injection and coincided with complete recovery of the normal dorsal light reaction. These observations suggest a role for intraocular dopamine in maintenance of the visual part of the normal dorsal light reaction.     

Multi-year movements of adult and subadult bull sharks (Carcharhinus leucas): philopatry,connectivity, and environmental influences

Aquatic Ecology - Understanding the movement ecology of marine species is important for conservation management and monitoring their responses to environmental change. In this study, adult and...     

Investigations on Aberrant Glycosylation of Glycosphingolipids in Colorectal Cancer Tissues Using Liquid Chromatography and Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS)

Cancer is a leading cause of death and alterations of glycosylation are characteristic features of malignant cells. Colorectal cancer is one of the most common cancers and its exact causes and biology are not yet well understood. Here, we compared glycosylation profiles of colorectal tumor tissues and corresponding control tissues of 13 colorectal cancer patients to contribute to the understanding of this cancer. Using MALDI-TOF(/TOF)-MS and 2-dimensional LC-MS/MS we characterized enzymatically released and 2-aminobenzoic acid labeled glycans from glycosphingolipids. Multivariate data analysis revealed significant differences between tumor and corresponding control tissues. Main discriminators were obtained, which represent the overall alteration in glycosylation of glycosphingolipids during colorectal cancer progression, and these were found to be characterized by (1) increased fucosylation, (2) decreased acetylation, (3) decreased sulfation, (4) reduced expression of globo-type glycans, as well as (5) disialyl gangliosides. The findings of our current research confirm former reports, and in addition expand the knowledge of glycosphingolipid glycosylation in colorectal cancer by revealing new glycans with discriminative power and characteristic, cancer-associated glycosylation alterations. The obtained discriminating glycans can contribute to progress the discovery of biomarkers to improve diagnostics and patient treatment.Worldwide, cancer is a leading cause of death. With estimated 1.2 million diagnoses in 2008, colorectal cancer is the third most common cancer in the world and the fourth most common cause of death with an annual mortality of ∼600 000 (1). The exact causes of colorectal cancer are unknown, but different risk factors such as age, polyps, personal and family history, ulcerative colitis, or Crohn''s colitis have been proposed (2). Standard screening procedures include flexible sigmoidoscopy, colonoscopy, and immunological fecal occult blood testing. Each of them has its advantages and drawbacks such as invasiveness or low sensitivity and specificity (3). The method of choice for the treatment of colorectal cancer is surgery and therapeutic decisions are based on the tumor, lymph node, and metastasis staging-system as a prognostic factor (4). Current research has led to improved treatment strategies of colorectal cancer, however, the clinical outcome, the progression of the disease, and the response to the treatment remain variable among individuals. The heterogeneity of colorectal cancer at the molecular level—caused by accumulation of multiple genetic changes—may be one of the main reasons for this variability (5). Genetic factors such as instabilities, but also expression levels (6) can explain part of the cancer biology, but glycomics is gaining importance to complement the overall picture as aberrant glycosylation of proteins and lipids has been shown to be correlated with disease and malignancy (7, 8).Glycosylation is involved in many biological processes and especially its functional role in cellular interaction with respect to adhesion, cell growth, and signaling is prone to be affected in cancer progression, invasion, and metastasis (9). Several cancer-associated alterations in protein glycosylation have been reported: (1) increased branching of N-glycans, (2) higher density of O-glycans, and (3) incomplete synthesis of glycans. More particularly, an increased or induced expression of GlcNAc transferase V resulting in N-glycan structures with β1–6GlcNAc antennae (5, 10), and the expression of (sialyl) Tn-antigens (11) as aberrant O-glycosylation have been reported (10).Altered glycosphingolipid (GSL)¹ glycosylation of the cell surface membrane during malignancy can affect cell recognition, adhesion, and signal transduction (12) and is found to reflect: (1) incomplete synthesis with or without precursor accumulation, (2) neosynthesis (9), (3) increased sialylation, and (4) increased fucosylation (13). In many cancers, including colorectal cancer, an overexpression of the (sialyl) Lewis X antigen (10, 14) and the expression of (sialyl) Lewis A (15) are considered to be related to malignant transformation—reflecting incomplete synthesis of sialyl 6-sulfo Lewis X and disialyl Lewis A (16) as well as neosynthesis (17). Studies on gangliosides showed an overexpression of these sialylated GSLs in human malignant melanoma (18). Furthermore, the involvement of gangliosides in cell adhesion and motility was reported, which contributes to tumor metastasis (19). Specifically, the gangliosides GD3 (Hex2NeuAc2ceramide) and GM2 (Hex2HexNAc1NeuAc1ceramide) have been found to be associated with tumor-angiogenesis (19). The up-regulation of fucosyltransferases in cancer was shown to cause a higher degree of fucosylation in malignant tissues (20) and Moriwaki et al. proposed that the increase in the fucosylation for GSLs was an early event in cancer (21). Misonou et al. investigated glycans derived from GSLs in colorectal cancer tissues showing aberrant glycan structures based on linkage differences as well as increased sialylation and fucosylation compared with control tissue (22), which is in line with observed changes in GSL glycosylation with regard to cancer progression (9, 13).Recently, we investigated the N-glycosylation profiles of colorectal tumors and correlating control tissues for biomarker discovery. Statistical analyses revealed an increase of sulfated glycan structures as well as paucimannosidic glycans and glycans containing sialylated Lewis type epitopes in the tumor tissue, whereas structures with bisecting GlcNAc were found to be decreased in malignancy (23). To further progress the understanding of colorectal cancer biology and the improvement of diagnostic tools and patient treatment, we complemented this recent study on N-glycosylation by an investigation of the glycosphingolipid-derived glycans (named GSL-glycans in the following) from frozen tumor tissues and corresponding control tissues from the same 13 colorectal cancer patients. GSL-glycans were enzymatically released, labeled with 2-aminobenzoic acid (AA) and analyzed by hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection as well as matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Employing multivariate statistical analysis, this approach revealed an intricate GSL-glycosylation pattern of tumor tissues and specific glycosylation differences in comparison to the corresponding control tissue.     

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     

The ‘Antiretrovirals,Sexual Transmission Risk and Attitudes’ (ASTRA) Study. Design,Methods and Participant Characteristics

Life expectancy for people diagnosed with HIV has improved dramatically however the number of new infections in the UK remains high. Understanding patterns of sexual behaviour among people living with diagnosed HIV, and the factors associated with having condom-less sex, is important for informing HIV prevention strategies and clinical care. In addition, in view of the current interest in a policy of early antiretroviral treatment (ART) for all people diagnosed with HIV in the UK, it is of particular importance to assess whether ART use is associated with increased levels of condom-less sex. In this context the ASTRA study was designed to investigate current sexual activity, and attitudes to HIV transmission risk, in a large unselected sample of HIV-infected patients under care in the UK. The study also gathered background information on demographic, socio-economic, lifestyle and disease-related characteristics, and physical and psychological symptoms, in order to identify other key factors impacting on HIV patients and the behaviours which underpin transmission. In this paper we describe the study rationale, design, methods, response rate and the demographic characteristics of the participants. People diagnosed with HIV infection attending 8 UK HIV out-patient clinics in 2011-2012 were invited to participate in the study. Those who agreed to participate completed a confidential, self-administered pen-and-paper questionnaire, and their latest CD4 count and viral load test results were recorded. During the study period, 5112 eligible patients were invited to take part in the study and 3258 completed questionnaires were obtained, representing a response rate of 64% of eligible patients. The study includes 2248 men who have sex with men (MSM), 373 heterosexual men and 637 women. Future results from ASTRA will be a key resource for understanding HIV transmission within the UK, targeting prevention efforts, and informing clinical care of individuals living with HIV.     

Failure modelling of trabecular bone using a non-linear combined damage and fracture voxel finite element approach

Trabecular bone tissue failure can be considered as consisting of two stages: damage and fracture; however, most failure analyses of 3D high-resolution trabecular bone samples are confined to damage mechanisms only, that is, without fracture. This study aims to develop a computational model of trabecular bone consisting of an explicit representation of complete failure, incorporating damage criteria, fracture criteria, cohesive forces, asymmetry and large deformation capabilities. Following parameter studies on a test specimen, and experimental testing of bone sample to complete failure, the asymmetric critical tissue damage and fracture strains of ovine vertebral trabecular bone were calibrated and validated to be compression damage ?1.16 %, tension damage 0.69 %, compression fracture ?2.91 % and tension fracture 1.98 %. Ultimate strength and post–ultimate strength softening were captured by the computational model, and the failure of individual struts in bending and shear was also predicted. This modelling approach incorporated a cohesive parameter that provided a facility to calibrate ductile–brittle behaviour of bone tissue in this non-linear geometric and non-linear constitutive property analyses tool. Finally, the full accumulation of tissue damage and tissue fracture has been monitored from range of small magnitude (normal daily loading) through to specimen yielding, ultimate strength and post–ultimate strength softening.     

Ca2+-dependent Structural Changes in the B-cell Receptor CD23 Increase Its Affinity for Human Immunoglobulin E

Immunoglobulin E (IgE) antibodies play a fundamental role in allergic disease and are a target for therapeutic intervention. IgE functions principally through two receptors, FcϵRI and CD23 (FcϵRII). Minute amounts of allergen trigger mast cell or basophil degranulation by cross-linking IgE-bound FcϵRI, leading to an inflammatory response. The interaction between IgE and CD23 on B-cells regulates IgE synthesis. CD23 is unique among Ig receptors in that it belongs to the C-type (calcium-dependent) lectin-like superfamily. Although the interaction of CD23 with IgE is carbohydrate-independent, calcium has been reported to increase the affinity for IgE, but the structural basis for this activity has previously been unknown. We have determined the crystal structures of the human lectin-like head domain of CD23 in its Ca²⁺-free and Ca²⁺-bound forms, as well as the crystal structure of the Ca²⁺-bound head domain of CD23 in complex with a subfragment of IgE-Fc consisting of the dimer of Cϵ3 and Cϵ4 domains (Fcϵ3-4). Together with site-directed mutagenesis, the crystal structures of four Ca²⁺ ligand mutants, isothermal titration calorimetry, surface plasmon resonance, and stopped-flow analysis, we demonstrate that Ca²⁺ binds at the principal and evolutionarily conserved binding site in CD23. Ca²⁺ binding drives Pro-250, at the base of an IgE-binding loop (loop 4), from the trans to the cis configuration with a concomitant conformational change and ordering of residues in the loop. These Ca²⁺-induced structural changes in CD23 lead to additional interactions with IgE, a more entropically favorable interaction, and a 30-fold increase in affinity of a single head domain of CD23 for IgE. Taken together, these results suggest that binding of Ca²⁺ brings an extra degree of modulation to CD23 function.