Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines

High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes (Pvu II, Hind III, Bgl II, and Bam HI), electrophoresed for 18 or 45 h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II beta-chain probe (beta 2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B haplotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.     

Utilizable water in leaves of 8 arid species as derived from pressure-volume curves and chlorophyll fluorescence

The water-storage properties of leaves from 8 co-occurring arid species, ranging from 4 to 36 in turgid weight/dry weight ratio, were studied. Total water content at saturation varied from 75 to 97%, with 65–93% of this utilizable when bound water (B) was subtracted, and 72–93% when water content at a critical level of optimal fluorescence (F_c) was subtracted. F_c represented the turning point from slight to substantial impairment of chlorophyll fluorescence, and either coincided with B (two species) or was slightly below B (6 species). An incipient level of fluorescence was also recognized, corresponding to the lowest water content before any effect on maximum variable fluorescence/maximum fluorescence could be detected. This lay closer to B than to water content at turgor loss point (TLP), rendering TLP of no significance in dictating the fraction of stored water utilizable (UWC). Under laboratory conditions, turgid branchlets of the 8 species were estimated to take from 1.5 days to 15 weeks to reach F_c. The rate of water loss was almost completely explained by variations in leaf thickness. Under field conditions in mid-spring, UWC at F_c on a harvest weight basis ranged from 51 to 87% predawn and from 45 to 86% by early afternoon (EA). The UWC of plants severed from their root systems 6 weeks earlier ranged from 0 to 63% during the EA. Overall, two groups of species could be recognized: thick-leaved species whose UWC is high and varies little during the day and which use their stored water conservatively and have limited reliance on their root system for replenishment after winter; and thinner leaved species whose UWC is moderate and fluctuates daily, and whose stored water falls rapidly unless replenished continuously from the soil.     

Characteristics of a protease of Streptococcus sanguis G9B which degrades the major salivary adhesin

An endogenous enzyme present in cell surface extracts of Streptococcus sanguis strain G9B degraded the major salivary adhesin of the organism. The enzyme showed optimal activity between 50 and 65 degrees C and was inactivated at higher temperatures. The activity at these unusually high temperatures seemed to be a consequence of release from the cell surface since intact whole G9B cells showed greater activity at 37 degrees C. The enzyme was not found in culture supernatants of G9B cells. The pH range for the enzyme was between 5 and 9. It was inhibited by iodoacetic acid, Hg2+, Cu2+, EDTA, SDS, and PMSF, but not by TLCK, TPCK, soybean trypsin inhibitor, cysteine, dithiothreitol, leupeptin, Ca2+, Mg2+ or saliva. The enzyme did not show any activity against human or rabbit IgG or human IgA. Enzyme activity was also found in S. sanguis strains Adh- (a spontaneously occurring non-adherent mutant of G9B), and M-5.     

High summer temperatures do not interact with fire to promote germination among seeds of Cistaceae: a reinterpretation of Luna (2020) with extra data on wet/dry conditions*

Plant Ecology - In a unique study, Luna (Luna, Sci Rep 10:1–10, 2020) examined the viability and germination of 12 hard-seeded Cistaceae in the Mediterranean Basin by alternating a prolonged...     

Quantitative proteomics of intracellular Porphyromonas gingivalis

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Identification of Caveolar Resident Proteins in Ventricular Myocytes Using a Quantitative Proteomic Approach: Dynamic Changes in Caveolar Composition Following Adrenoceptor Activation

The lipid raft concept proposes that membrane environments enriched in cholesterol and sphingolipids cluster certain proteins and form platforms to integrate cell signaling. In cardiac muscle, caveolae concentrate signaling molecules and ion transporters, and play a vital role in adrenergic regulation of excitation–contraction coupling, and consequently cardiac contractility. Proteomic analysis of cardiac caveolae is hampered by the presence of contaminants that have sometimes, erroneously, been proposed to be resident in these domains. Here we present the first unbiased analysis of the proteome of cardiac caveolae, and investigate dynamic changes in their protein constituents following adrenoreceptor (AR) stimulation.Rat ventricular myocytes were treated with methyl-β-cyclodextrin (MβCD) to deplete cholesterol and disrupt caveolae. Buoyant caveolin-enriched microdomains (BCEMs) were prepared from MβCD-treated and control cell lysates using a standard discontinuous sucrose gradient. BCEMs were harvested, pelleted, and resolubilized, then alkylated, digested, and labeled with iTRAQ reagents, and proteins identified by LC-MS/MS on a LTQ Orbitrap Velos Pro. Proteins were defined as BCEM resident if they were consistently depleted from the BCEM fraction following MβCD treatment. Selective activation of α-, β1-, and β2-AR prior to preparation of BCEMs was achieved by application of agonist/antagonist pairs for 10 min in populations of field-stimulated myocytes.We typically identified 600–850 proteins per experiment, of which, 249 were defined as high-confidence BCEM residents. Functional annotation clustering indicates cardiac BCEMs are enriched in integrin signaling, guanine nucleotide binding, ion transport, and insulin signaling clusters. Proteins possessing a caveolin binding motif were poorly enriched in BCEMs, suggesting this is not the only mechanism that targets proteins to caveolae. With the notable exception of the cavin family, very few proteins show altered abundance in BCEMs following AR activation, suggesting signaling complexes are preformed in BCEMs to ensure a rapid and high fidelity response to adrenergic stimulation in cardiac muscle.Caveolae are specialized invaginated lipid rafts (1), around 50–100 nm in diameter, enriched in cholesterol and sphingolipids, and characterized by the presence of caveolin and cavin proteins. The lipid environment, caveolin content, and morphology of caveolae are central to their diverse functional roles, which include coordination of signal transduction, cholesterol homeostasis, and endocytosis (2). Clustering of elements of particular signal cascades within a caveola promotes efficiency and fidelity of signaling. Although caveolae and noncaveolar rafts coexist, evidence suggests that most proteins are clustered by caveolae in the cardiac cell (3). Caveolin exists as three major isoforms: caveolin 1 and caveolin 2, which are expressed in most cell types, and caveolin 3, which is the muscle-specific isoform. Caveolins 1 and 3 are the predominant forms found in the adult cardiac myocyte (4, 5). Four members of the cavin family of related proteins exist, and all have been detected in the heart (6).One of caveolae''s best-characterized roles is as a signalosome, a compartment that brings together components of signal transduction cascades (including receptors, effectors, and targets (7)). Within caveolae, the 20-residue scaffolding domain of caveolin (CSD)¹ has been proposed to interact with a complementary caveolin-binding motif (CBM) in proteins. This enables oligomeric caveolin to act as a regulatory scaffold for macromolecular signaling complex formation (8). However, the ability of this simple and commonly occurring motif to interact with caveolin (directing proteins to caveolae and regulating their activity) has recently been challenged, because it is often buried within mature proteins (9, 10). Palmitoylation of juxtamembrane cysteine residues has also been proposed to partition proteins to ordered detergent-resistant membranes such as caveolae (11).The organization of proteins in caveolae suggests that they have a key role in regulation of signaling in the heart. We adopt the convention of the field here to assign proteins as caveolar if they are present in buoyant caveolin-containing membrane fractions obtained by sucrose gradient fractionation or in morphologically identifiable caveolae by immunogold electron microscopy. For example, α1- and β2-adrenoceptors (AR) are found exclusively in caveolae-containing membrane fractions of the adult heart (12, 13), whereas β1-AR are in both caveolar and bulk sarcolemmal fractions (14). Cardiac caveolae are also sites of enrichment of G proteins (12, 15), effectors of AR (including adenylyl cyclase V/VI, protein kinase A (RII), GRK2, phospholipase Cβ, PP2A, and eNOS (13–16)), and their downstream targets. Importantly, the distribution of receptors, effectors, and their targets is key to the efficiency and fidelity of their coupling (13, 17, 18). For example, altered β1- and β2-AR responses have been observed following cholesterol depletion (which disrupts caveolae) and severing of normal caveolin 3 interactions with a caveolin 3 CSD peptide (19, 20).A considerable number of cardiac ion transporters are resident in cardiac caveolae: voltage-gated sodium channels (21), L-type calcium channels (16), voltage gated potassium channels (22), ATP-sensitive potassium channels (23), the sodium-calcium exchanger (24) (NCX - although this has been challenged (25)), the sodium potassium ATPase (sodium pump) (26), and the plasma membrane calcium ATPase (PMCA) (27). Physical colocalization of ion transporters in the caveolar compartment may functionally link ion flow by providing a restricted diffusional space (28) and facilitates hormonal regulation of these transporters by placing them physically adjacent to signaling molecules. For example, regulation of a subpopulation of L-type calcium channels by β2-AR requires their colocalization in caveolae (16), and regulation of the cardiac sodium pump by phospholemman (PLM) relies on phosphorylation and dephosphorylation of PLM in caveolae (29, 30). The presence of ion transporters in caveolae is likely to have functional relevance beyond signal transduction because the lipid composition of the bilayer in which an ion transporter resides will influence its activity. Membrane cholesterol modulates many aspects of ion channel function: the sodium pump, for example, is regulated by the cholesterol content of the membranes within which it resides (31).Using traditional biochemical techniques (such as sucrose density gradient fractionation followed by Western blotting), the presence of certain proteins in cardiac caveolae has been shown to be dynamic on an acute timescale (minutes), however, no unbiased assessment of global changes in caveolar composition during signaling has been reported. PKC isoforms translocate into caveolae upon activation (32), and various G-protein coupled receptors translocate in and out of caveolae upon activation: muscarinic M2 (33) receptors into caveolae, and β2-AR out of caveolae (14, 34). Pivotally, this redistribution of proteins has been closely linked with functional responses. For example, the negative inotropic effect of the muscarinic agonist carbachol is ascribed to the ability of M2 receptors to move to caveolae and couple therein to eNOS, which is exclusively found in this compartment (33). Conversely, one suggestion for the limited functional response following β2-AR receptor stimulation is that translocation from the caveolar compartment (where it normally couples to its effector adenylyl cyclase) to clathrin-coated pits terminates its downstream effects by internalization of the receptor (14, 34).Hence, it has been proposed that many signaling proteins and ion transporters are located in caveolae and that the distribution between caveolar and noncaveolar membranes is dynamically regulated. Although quantitative proteomics has been used to highlight the breadth of proteins localized to caveolae in nonexcitable cells (for example (35)), to date no study has used an unbiased proteomic approach to define resident proteins of cardiac caveolae, or changes in the composition of caveolae during signaling events in any cell type.The principal challenge to overcome in using proteomics to characterize a particular membrane compartment is purifying this compartment to homogeneity. The high sensitivity of detection possible in modern proteomics means that even minor contamination of caveolar membranes with another membrane compartment leads to mis-assignment of proteins as caveolar residents. In practice, because contamination of membrane preparations cannot be reduced to zero, an alternative approach has been developed: For proteins to be considered genuinely localized to lipid rafts/caveolae, their presence must be sensitive to cholesterol depletion (36). Hence stable isotope labeling with amino acids in cell culture (SILAC) and cholesterol depletion with methyl-beta-cyclodextrin (MβCD) in noncardiac cultured cells has distinguished genuine caveolar proteins from the mitochondrial, sarco/endoplasmic reticulum, and cytosolic proteins that routinely contaminate caveolae prepared by density gradient centrifugation (35). Using such an approach, contaminating proteins appear in the caveolar fraction whether or not cholesterol is depleted with MβCD, but genuine caveolar residents are specifically depleted. This approach has refuted research that implies that mitochondrial proteins are enriched in caveolae (37, 38), including data from cardiac tissue that suggested that principal components of cardiac caveolae are mitochondrial and structural proteins (39). The current study employed a similar approach—with the modification that because cardiac myocytes cannot be maintained in culture for SILAC (because of marked phenotypic changes), we used iTRAQ-based quantitative proteomics.Here we identify 249 high-confidence caveolar proteins in the cardiac cell and show for the first time that the caveolar proteome is remarkably stable on an acute timescale following adrenoreceptor stimulation, suggesting that signaling complexes are preformed in this microdomain.     

Proteomic Profiling of Cranial (Superior) Cervical Ganglia Reveals Beta-Amyloid and Ubiquitin Proteasome System Perturbations in an Equine Multiple System Neuropathy

Equine grass sickness (EGS) is an acute, predominantly fatal, multiple system neuropathy of grazing horses with reported incidence rates of ∼2%. An apparently identical disease occurs in multiple species, including but not limited to cats, dogs, and rabbits. Although the precise etiology remains unclear, ultrastructural findings have suggested that the primary lesion lies in the glycoprotein biosynthetic pathway of specific neuronal populations. The goal of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS. Here, we use a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. In what appears to be the proteomic application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease), we identified 2,311 proteins in CCG extracts, with 320 proteins increased and 186 decreased by greater than 20% relative to controls. Further examination of selected proteomic candidates by quantitative fluorescent Western blotting (QFWB) and subcellular expression profiling by immunohistochemistry highlighted a previously unreported dysregulation in proteins commonly associated with protein misfolding/aggregation responses seen in a myriad of human neurodegenerative conditions, including but not limited to amyloid precursor protein (APP), microtubule associated protein (Tau), and multiple components of the ubiquitin proteasome system (UPS). Differentially expressed proteins eligible for in silico pathway analysis clustered predominantly into the following biofunctions: (1) diseases and disorders, including; neurological disease and skeletal and muscular disorders and (2) molecular and cellular functions, including cellular assembly and organization, cell-to-cell signaling and interaction (including epinephrine, dopamine, and adrenergic signaling and receptor function), and small molecule biochemistry. Interestingly, while the biofunctions identified in this study may represent pathways underpinning EGS-induced neurodegeneration, this is also the first demonstration of potential molecular conservation (including previously unreported dysregulation of the UPS and APP) spanning the degenerative cascades from an apparently unrelated condition of large animals, to small animal models with altered neuronal vulnerability, and human neurological conditions. Importantly, this study highlights the feasibility and benefits of applying modern proteomic techniques to veterinary investigations of neurodegenerative processes in diseases of large animals.Equine grass sickness (EGS, or equine dysautonomia) is a predominantly fatal, rapid multiple system neuropathy of grazing horses with reported incidence rates of 2.1–2.3% (reviewed by (1, 2)). An apparently identical disease occurs in cats, dogs, hares, rabbits, llamas, and possibly sheep (3–9). EGS is associated with chromatolysis of sympathetic and parasympathetic postsynaptic neurons, particularly in the enteric nervous system, as well as autonomic presynaptic and somatic lower motor neurons in the brainstem and spinal cord (10). EGS is subdivided into acute, subacute, and chronic forms according to the severity of clinical signs that largely reflect enteric and autonomic neurodegeneration, including dysphagia, generalized ileus, sweating, salivation, ptosis, rhinitis sicca, and tachycardia. While the etiology of EGS remains unknown, some evidence supports it being a toxic infection with Clostridium botulinum type C or D (11, 12). Ultrastructural studies suggest that the lesion in EGS primarily involves the glycoprotein biosynthetic pathway of specific neurons since the rough endoplasmic reticulum and Golgi complexes are consistently affected, while other organelles, including mitochondria, appear relatively normal (13). However, while the ultrastructural and cellular appearance of affected neurons has been studied extensively, little is known about the molecular mechanisms that contribute to neurodegeneration.The overarching aim of this study was therefore to identify the molecular processes underpinning neurodegeneration in EGS using a bottom-up approach beginning with the application of modern proteomic tools to the analysis of cranial (superior) cervical ganglion (CCG, a consistently affected tissue) from EGS-affected patients and appropriate control cases postmortem. The cranial (superior) cervical ganglion (CCG), which supplies sympathetic innervation to the head and neck, was selected because chromatolysis of a high proportion of CCG neurons is a consistent feature of EGS (Fig. 1 and Supplemental Fig. 1 (14)). Here, proteomic analysis was carried out using isobaric tag for relative and absolute quantitation (iTRAQ) tools, which are now well established in small animal models of human neurodegenerative conditions but which are not routinely utilized in large animal models or large animal intrinsic conditions. This proteomic analysis was coupled with quantitative fluorescent Western blotting (QFWB), immunohistochemistry (IHC), and in silico based techniques in an attempt to identify the molecular pathways and processes that may be contributing to neurodegeneration in EGS. Here, we report widespread changes in the CCG of EGS horses, including significant disruption to a broad range of functional pathways clustering around candidates commonly associated with protein misfolding/aggregation responses in human neurodegenerative conditions.Open in a separate windowFig. 1.Equine grass sickness is a predominantly fatal, acute multiple system neuropathy of grazing horses. (A) Example photograph of a horse exhibiting typical appearance associated with chronic EGS. There is ptosis, and generalized muscle weakness as evidenced by the base narrow stance, low head and neck carriage, and leaning against a wall for support. Generalized muscle atrophy and reduced abdominal volume are also evident. (B) Example BIII-tubulin-stained sections from cranial cervical ganglia (CCG), which are known to exhibit neuronal perturbations in this disease. The visible puncta are BIII positive neurons. (C) High power micrographs stained CCG sections from B showing BIII positive neuronal profiles. (D) Quantification of BIII positive neurons demonstrates that there is still equivalent neuronal density in ganglia at terminal stages of the disease (control 6.25 ± 0.12, EGS 6.10 ± 0.20 cells per 100 μm², mean ± Standard Error (S.E) n = 4 cases per condition, n = 116 grids measured. See Materials and Methods for more information). (E) Example H and E stained sections from control and EGS-affected CCG demonstrates that while the neuronal density may be similar, many of the neurons exhibit chromatolysis. Scale bar = 0.75ft (A), 0.5 cm (B), 35 μm (C), 100 μm (E).This study therefore represents the first application of modern proteomic tools to equine neuronal tissues and/or to an inherent neurodegenerative disease of large animals (not a model of human disease). It is also the first to demonstrate correlation and conservation spanning the degenerative molecular cascades from an apparently unrelated condition of large animals to small animal models with altered neuronal vulnerability and a range of human neurological conditions from childhood neurodegenerative conditions such as spinal muscular atrophy through to diseases associated with advancing age such as Alzheimer''s. Finally, this study highlights the feasibility and benefits of applying differential proteomics techniques to the investigation of the neurodegenerative processes in diseases of large animals.