POSTZYGOTIC ISOLATION OVER MULTIPLE GENERATIONS OF HYBRID DESCENDENTS IN A NATURAL HYBRID ZONE: HOW WELL DO SINGLE‐GENERATION ESTIMATES REFLECT REPRODUCTIVE ISOLATION?

Understanding speciation depends on an accurate assessment of the reproductive barriers separating newly diverged populations. In several taxonomic groups, prezygotic barriers, especially preferences for conspecific mates, are thought to play the dominant role in speciation. However, the importance of postzygotic barriers (i.e., low fitness of hybrid offspring) may be widely underestimated. In this study, we examined how well the widely used proxy of postzygotic isolation (reproductive output of F₁ hybrids) reflects the long‐term fitness consequences of hybridization between two closely related species of birds. Using 40 species‐specific single nucleotide polymorphism (SNP) markers, we genotyped a mixed population of collared and pied flycatchers (Ficedula albicollis and F. hypoleuca) to identify grand‐ and great grand‐offspring from interspecific crosses to derive an accurate, multigeneration estimate of postzygotic isolation. Two independent estimates of fitness show that hybridization results in 2.4% and 2.7% of the number of descendents typical of conspecific pairing. This postzygotic isolation was considerably stronger than estimates based on F₁ hybrids. Our results demonstrate that, in nature, combined selection against hybrids and backcrossed individuals may result in almost complete postzygotic isolation between two comparatively young species. If these findings are general, postzygotic barriers separating hybridizing populations may be much stronger than previously thought.     

Liquid chromatography-mass spectrometry to study chondroitin lyase action pattern

Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.     

The degree of polymerization and sulfation patterns in heparan sulfate are critical determinants of cytomegalovirus entry into host cells

Several enveloped viruses, including herpesviruses attach to host cells by initially interacting with cell surface heparan sulfate (HS) proteoglycans followed by specific coreceptor engagement which culminates in virus-host membrane fusion and virus entry. Interfering with HS-herpesvirus interactions has long been known to result in significant reduction in virus infectivity indicating that HS play important roles in initiating virus entry. In this study, we provide a series of evidence to prove that specific sulfations as well as the degree of polymerization (dp) of HS govern human cytomegalovirus (CMV) binding and infection. First, purified CMV extracellular virions preferentially bind to sulfated longer chain HS on a glycoarray compared to a variety of unsulfated glycosaminoglycans including unsulfated shorter chain HS. Second, the fraction of glycosaminoglycans (GAG) displaying higher dp and sulfation has a larger impact on CMV titers compared to other fractions. Third, cell lines deficient in specific glucosaminyl sulfotransferases produce significantly reduced CMV titers compared to wild-type cells and virus entry is compromised in these mutant cells. Finally, purified glycoprotein B shows strong binding to heparin, and desulfated heparin analogs compete poorly with heparin for gB binding. Taken together, these results highlight the significance of HS chain length and sulfation patterns in CMV attachment and infectivity.     

Nuclear magnetic resonance quantification for monitoring heparosan K5 capsular polysaccharide production

Traditional chromatographic quantification methods for heparosan produced from the Escherichia coli K5 strain rely on extensive purification requiring laborious sample preparation. These methods are time-consuming, often resulting in sample loss during purification, and thus might not accurately reflect the amount of heparosan in the original mixture. A simple, sensitive ¹H nuclear magnetic resonance (NMR) quantification method that directly quantifies heparosan K5 polysaccharide present in E. coli fermentation supernatant is described.     

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis

Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6 h using continuous elution polyacrylamide gel electrophoresis (CE-PAGE) on commercially available equipment. The purity and structural integrity of CE-PAGE-separated oligosaccharides are confirmed by strong anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure-function relationship studies.     

HSP70 and Its Cochaperone CPIP Promote Potyvirus Infection in Nicotiana benthamiana by Regulating Viral Coat Protein Functions

This study demonstrates that heat shock protein 70 (HSP70) together with its cochaperone CPIP regulates the function of a potyviral coat protein (CP), which in turn can interfere with viral gene expression. HSP70 was copurified as a component of a membrane-associated viral ribonucleoprotein complex from Potato virus A–infected plants. Downregulation of HSP70 caused a CP-mediated defect associated with replication. When PVA CP was expressed in trans, it interfered with viral gene expression and replication-associated translation (RAT). However, CP produced in cis interfered specifically with RAT. CPIP binds to potyviral CP, and overexpression of CPIP was sufficient to restore RAT inhibited by expression of CP in trans. Restoration of RAT was dependent on the ability of CPIP to interact with HSP70 since expression of a J-domain mutant, CPIP^Δ66, had only a minor effect on RAT. CPIP-mediated delivery of CP to HSP70 promoted CP degradation by increasing its ubiquitination when assayed in the absence of virus infection. In conclusion, CPIP and HSP70 are crucial components of a distinct translation activity that is associated with potyvirus replication.     

Africanizing scientific knowledge: the Multilateral Initiative on Malaria as a model?

In November 2009, the fifth Pan African Malaria conference was held in Nairobi. Thirteen years after the founding initiative in Dakar, the first African Secretariat based in Africa (TANZANIA) organized this major event for the malaria community. Looking back, it has been a long way: changes in the research landscape, new funding opportunities came out and establishment of new partnerships between Europe, America and Africa. Goals identified in 1997 have not all been achieved because the critical mass of scientists has not been reached yet. However a new generation of African scientists have emerged through MIM/TDR funding and advocacy for more support remains on the agenda. Could it be rightly stated today that the MIM concept reflects the africanization of malaria research?     

The expression of syndecan-1, syndecan-4 and decorin in healthy human breast tissue during the menstrual cycle

Background  

In order to unravel the interactions between the epithelium and the extra cellular matrix (ECM) in breast tissue progressing to cancer, it is necessary to understand the relevant interactions in healthy tissue under normal physiologic settings. Proteoglycans in the ECM play an important role in the signaling between the different tissue compartments. The proteoglycan decorin is abundant in the breast stroma. Decreased expression in breast cancer tissue is a sign of a poor tumor prognosis. The heparane sulphate proteoglycans syndecan-1 and syndecan-4 promote the integration of cellular adhesion and proliferation. The aim of this study was to investigate the gene expression and location of decorin, syndecan-1 and syndecan-4 in the healthy breast during the menstrual cycle.     

Neutralisation of uPA with a Monoclonal Antibody Reduces Plasmin Formation and Delays Skin Wound Healing in tPA-Deficient Mice

Background

Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds.

Methodology/Principal Findings

To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficent mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficent mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure.

Conclusions/Significance

Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.     

Specific haptoglobin expression in bronchoalveolar lavage during differentiation of circulating fibroblast progenitor cells in mild asthma

Haptoglobin is an acute-phase glycoprotein considered to be involved in tissue repair and is produced by fibroblasts and inflammatory cells. By using a gel-based proteomic approach, we show for the first time a possible role for haptoglobin in the differentiation of fibroblast progenitor cells, termed fibrocytes, in patients with mild asthma. Bronchoalveolar lavage fluid (BALF) was performed to sample circulating fibrocytes from patients with mild asthma and nonasthmatic control subjects. Fibrocytes from the airway lumen were characterized by triple staining of the markers CD34/CD45R0/alpha-smooth muscle actin, and subjected to confocal microscopy. The protein expression pattern was analyzed using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF). Elevated levels of haptoglobin expression in BALF was reported in a sub-group of patients with mild asthma (p < 0.05) when compared to the other subjects. In addition, this increase in haptoglobin was accompanied by differentiation of fibrocytes into fibroblast-like cells. When further analyzing the expression pattern of haptoglobin isoforms, a heterozygous expression was detected in the patients where fibrocyte differentiation could be observed. These data raise the possibility that an acute and specific inflammatory state facilitates the differentiation of fibroblast progenitor cells into activated fibroblasts. Furthermore, this study proposes a novel role for haptoglobin in airway remodeling in patients with asthma.