Atrial natriuretic factor receptor guanylate cyclase signaling: new ATP-regulated transduction motif

ANF-RGC membrane guanylate cyclase is the receptor for the hypotensive peptide hormones, atrial natriuretic factor (ANF) and type B natriuretic peptide (BNP). It is a single transmembrane spanning protein. Binding the hormone to the extracellular domain activates its intracellular catalytic domain. This results in accelerated production of cyclic GMP, a second messenger in controlling blood pressure, cardiac vasculature, and fluid secretion. ATP is the obligatory transducer of the ANF signal. It works through its ATP regulated module, ARM, which is juxtaposed to the C-terminal side of the transmembrane domain. Upon interaction, ATP induces a cascade of temporal and spatial changes in the ARM, which, finally, result in activation of the catalytic module. Although the exact nature and the details of these changes are not known, some of these have been stereographed in the simulated three-dimensional model of the ARM and validated biochemically. Through comprehensive techniques of steady state, time-resolved tryptophan fluorescence and Forster Resonance Energy Transfer (FRET), site-directed and deletion-mutagenesis, and reconstitution, the present study validates and explains the mechanism of the model-based predicted transduction role of the ARM’s structural motif, ⁶⁶⁹WTAPELL⁶⁷⁵. This motif is critical in the ATP-dependent ANF signaling. Molecular modeling shows that ATP binding exposes the ⁶⁶⁹WTAPELL⁶⁷⁵ motif, the exposure, in turn, facilitates its interaction and activation of the catalytic module. These principles of the model have been experimentally validated. This knowledge brings us a step closer to our understanding of the mechanism by which the ATP-dependent spatial changes within the ARM cause ANF signaling of ANF-RGC.     

Structure and Energetics of Encapsidated DNA in Bacteriophage HK97 Studied by Scanning Calorimetry and Cryo-electron Microscopy

Encapsidation of duplex DNA by bacteriophages represents an extreme case of genome condensation, reaching near-crystalline concentrations of DNA. The HK97 system is well suited to study this phenomenon in view of the detailed knowledge of its capsid structure. To characterize the interactions involved, we combined calorimetry with cryo-electron microscopy and native gel electrophoresis. We found that, as in other phages, HK97 DNA is organized in coaxially wound nested shells. When DNA-filled capsids (heads) are scanned in buffer containing 1 mM Mg²⁺, DNA melting and capsid denaturation both contribute to the complex thermal profile between 82 °C and 96 °C. In other conditions (absence of Mg²⁺ and lower ionic strength), DNA melting shifts to lower temperatures and the two events are resolved. Heads release their DNA at temperatures well below the onset of DNA melting or capsid denaturation. We suggest that, on heating, the internal pressure increases, causing the DNA to exit—probably via the portal vertex-while the capsid, although largely intact, sustains local damage that leads to an earlier onset of thermal denaturation. Heads differ structurally from empty capsids in the curvature of their protein shell, a change attributable to outwards pressure exerted by the DNA. We propose that this transition is sensed by the portal that is embedded in the capsid wall, whereupon the structure of the portal and its interactions with terminase, the packaging enzyme, are altered, thus signaling that packaging is at or approaching completion.     

HLA-Associated Clinical Progression Correlates with Epitope Reversion Rates in Early Human Immunodeficiency Virus Infection

Human immunodeficiency virus type 1 (HIV-1) can evade immunity shortly after transmission to a new host but the clinical significance of this early viral adaptation in HIV infection is not clear. We present an analysis of sequence variation from a longitudinal cohort study of HIV adaptation in 189 acute seroconverters followed for up to 3 years. We measured the rates of variation within well-defined epitopes to determine associations with the HLA-linked hazard of disease progression. We found early reversion across both the gag and pol genes, with a 10-fold faster rate of escape in gag (2.2 versus 0.27 forward mutations/1,000 amino acid sites). For most epitopes (23/34), variation in the HLA-matched and HLA-unmatched controls was similar. For a minority of epitopes (8/34, and generally associated with HLA class I alleles that confer clinical benefit), new variants appeared early and consistently over the first 3 years of infection. Reversion occurred early at a rate which was HLA-dependent and correlated with the HLA class 1-associated relative hazard of disease progression and death (P = 0.0008), reinforcing the association between strong cytotoxic T-lymphocyte responses, viral fitness, and disease status. These data provide a comprehensive overview of viral adaptation in the first 3 years of infection. Our findings of HLA-dependent reversion suggest that costs are borne by some escape variants which may benefit the host, a finding contrary to a simple immune evasion paradigm. These epitopes, which are both strongly and frequently recognized, and for which escape involves a high cost to the virus, have the potential to optimize vaccine design.The dynamics of viral replication in acute and early human immunodeficiency virus (HIV) infection are not well understood as longitudinal data from large cohorts of seroconverters are hard to assemble. Recent studies have shown that new HIV infections may be the result of a single transmitted variant, that new env gene mutations can be detected within a few weeks (25), and that early immune escape can be detected at sites across the HIV genome (9). These data add to a body of work showing that cytotoxic T cells act early, contributing to the early reduction in viremia (8, 30).Whether early cytotoxic T-lymphocyte (CTL) immune responses influence longer-term clinical outcome is not clear. Antigen-specific CTLs capable of producing gamma interferon and other cytokines are detectable at all stages of HIV infection (1, 3, 24, 41). Much weight is placed on the macaque/simian immunodeficiency virus model in which nearly total peripheral blood CD8⁺ T-cell elimination using monoclonal antibodies results in rising viremia (42). The role of other forms of host immunity (e.g., neutralizing antibodies, natural killer cells, and macrophages) has, to some extent, been pursued with less intensity in light of persuasive evidence that CTLs can control retrovirus infection (46). The extent to which the simian model mirrors HIV infection has been questioned (5) and, despite exhaustive cellular assays of T-cell function—from gamma interferon enzyme-linked immunospot assays(1, 27, 38) to polyfunctional cytokine matrices (2, 6)—no CTL function correlates robustly with HIV plasma viral load or viral dynamics. Moreover, analyses of evolutionary data suggest that CTLs are inefficient at killing HIV-infected cells (4).However, statistical analysis of data from large cross-sectional studies link HLA class I alleles with specific genome-wide HIV polymorphisms, suggestive of a pervasive selection pressure enacted by CTLs (7, 10, 18, 36, 40). It is clear that associations between some HLA class I alleles and particular amino acid polymorphisms are robust although it is disputed whether immune escape influences disease progression. The viral fitness costs resulting from immune escape may even contribute to better clinical outcomes associated with the possession of HLA class I alleles such as B*27, B*57, and B*58 (18).Evolutionary studies of HIV require longitudinal data from large cohorts of patients sampled since seroconversion to detect adaptation in new hosts as it accrues. HIV is one of the few pathogens where it is possible to do this within individuals because of the high viral turnover and rapid intrahost evolution. Here, we investigate a cohort of 189 acute seroconverters—the largest cohort reported to date—followed for up to 3 years to study the rates of viral mutation in individual epitopes within internal HIV proteins and to determine the association between HLA class I alleles and rates of immune escape and reversion.     

657WTAPELL663 motif of the photoreceptor ROS-GC1: a general phototransduction switch

This study documents the identity of an intriguing transduction mechanism of the [Ca(2+)](i) signals by the photoreceptor ROS-GC1. Despite their distal residences and operational modes in phototransduction, the two GCAPs transmit and activate ROS-GC1 through a common Ca(2+) transmitter switch (Ca(2+)TS). A combination of immunoprecipitation, fluorescent spectroscopy, mutational analyses and reconstitution studies has been used to demonstrate that the structure of this switch is (657)WTAPELL(663). The two Ca(2+) signaling GCAP pathways converge in Ca(2+)TS, get transduced, activate ROS-GC1, generate the LIGHT signal second messenger cyclic GMP and yet functionally perform divergent operations of the phototransduction machinery. The findings define a new Ca(2+)-modulated photoreceptor ROS-GC transduction model; it is depicted and discussed for its application to processing the different shades of LIGHT.     

Characterization of anti-ECA antibodies in rabbit antiserum against rough <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> O:3

Enterobacterial common antigen (ECA) is a characteristic surface component in bacteria belonging to the Enterobacteriaceae family. It is generally integrated in the outer membrane via a linkage to phosphatidylglycerol (ECA_PG) and at the same time in some special cases via a linkage to lipopolysaccharide (ECA_LPS); the latter form is immunogenic. Yersinia enterocolitica O:3 expresses both ECA_PG and ECA_LPS. To study whether ECA-immunogenicity of Y. enterocolitica O:3 is temperature-regulated, rabbits were immunized with ECA-expressing Y. enterocolitica O:3 bacteria grown at 22 and 37°C. To induce minimal amount of anti-LPS antibodies, immunization was performed with YeO3-c-trs8-R, an LPS mutant missing both O-polysaccharide and the outer core hexasaccharide. However, abundant antibodies specific for LPS core were still present in the obtained antisera such that the reactivity of ECA-specific antibodies could not be detected. To obtain “monovalent” anti-ECA antisera, the sera were absorbed with ECA-negative bacteria. Absorption with live bacteria removed efficiently the anti-LPS antibodies, whereas this was not the case with boiled bacteria. Western blotting revealed that the specificity of the monovalent anti-ECA antiserum was different from that of a monoclonal anti-ECA antibody (mAb 898) as it did not react with ECAPG, and this suggested that in Y. enterocolitica O:3 ECA_LPS only one or two ECA repeat unit(s) is/are linked to LPS. Both ECA_PG and ECA_LPS expression were found to be regulated by temperature and repressed at 37°C.     

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing: a finite element study based on sheep experiments

During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.     

The Prohead-I structure of bacteriophage HK97: implications for scaffold-mediated control of particle assembly and maturation

Virus capsid assembly requires recruiting and organizing multiple copies of protein subunits to form a closed shell for genome packaging that leads to infectivity. Many viruses encode scaffolding proteins to shift the equilibrium toward particle formation by promoting intersubunit interactions and stabilizing assembly intermediates. Bacteriophage HK97 lacks an explicit scaffolding protein, but the capsid protein (gp5) contains a scaffold-like N-terminal segment termed the delta domain. When gp5 is expressed in Escherichia coli, the delta domain guides 420 copies of the subunit into a procapsid with T = 7 laevo icosahedral symmetry named Prohead-I. Prohead-I can be disassembled and reassembled under mild conditions and it cannot mature further. When the virally encoded protease (gp4) is coexpressed with gp5, it is incorporated into the capsid and digests the delta domain followed by autoproteolysis to produce the metastable Prohead-II. Prohead-I^+P was isolated by coexpressing gp5 and an inactive mutant of gp4. Prohead-I and Prohead-I^+P were compared by biochemical methods, revealing that the inactive protease stabilized the capsid against disassembly by chemical or physical stress. The crystal structure of Prohead-I^+P was determined at 5.2 Å resolution, and distortions were observed in the subunit tertiary structures similar to those observed previously in Prohead-II. Prohead-I^+P differed from Prohead-II due to the presence of the delta domain and the resulting repositioning of the N-arms, explaining why Prohead-I can be reversibly dissociated and cannot mature. Low-resolution X-ray data enhanced the density of the relatively dynamic delta domains, revealing their quaternary arrangement and suggesting how they drive proper assembly.     

Paraphyly and budding speciation in the hairy snail (Pulmonata,Hygromiidae)

Delimitation of species is often complicated by discordance of morphological and genetic data. This may be caused by the existence of cryptic or polymorphic species. The latter case is particularly true for certain snail species showing an exceptionally high intraspecific genetic diversity. The present investigation deals with the Trochulus hispidus complex, which has a complicated taxonomy. Our analyses of the COI sequence revealed that individuals showing a T. hispidus phenotype are distributed in nine highly differentiated mitochondrial clades (showing p‐distances up to 19%). The results of a parallel morphometric investigation did not reveal any differentiation between these clades, although the overall variability is quite high. The phylogenetic analyses based on 12S, 16S and COI sequences show that the T. hispidus complex is paraphyletic with respect to several other morphologically well‐defined Trochulus species (T. clandestinus, T. villosus, T. villosulus and T. striolatus) which form well‐supported monophyletic groups. The nc marker sequence (5.8S–ITS2–28S) shows only a clear separation of T. o. oreinos and T. o. scheerpeltzi, and a weakly supported separation of T. clandestinus, whereas all other species and the clades of the T. hispidus complex appear within one homogeneous group. The paraphyly of the T. hispidus complex reflects its complicated history, which was probably driven by geographic isolation in different glacial refugia and budding speciation. At our present state of knowledge, it cannot be excluded that several cryptic species are embedded within the T. hispidus complex. However, the lack of morphological differentiation of the T. hispidus mitochondrial clades does not provide any hints in this direction. Thus, we currently do not recommend any taxonomic changes. The results of the current investigation exemplify the limitations of barcoding attempts in highly diverse species such as T. hispidus.     

Age-Related Loss of Lumbar Spinal Lordosis and Mobility – A Study of 323 Asymptomatic Volunteers

Background

The understanding of the individual shape and mobility of the lumbar spine are key factors for the prevention and treatment of low back pain. The influence of age and sex on the total lumbar lordosis and the range of motion as well as on different lumbar sub-regions (lower, middle and upper lordosis) in asymptomatic subjects still merits discussion, since it is essential for patient-specific treatment and evidence-based distinction between painful degenerative pathologies and asymptomatic aging.

Methods and Findings

A novel non-invasive measuring system was used to assess the total and local lumbar shape and its mobility of 323 asymptomatic volunteers (age: 20–75 yrs; BMI <26.0 kg/m²; males/females: 139/184). The lumbar lordosis for standing and the range of motion for maximal upper body flexion (RoF) and extension (RoE) were determined. The total lordosis was significantly reduced by approximately 20%, the RoF by 12% and the RoE by 31% in the oldest (>50 yrs) compared to the youngest age cohort (20–29 yrs). Locally, these decreases mostly occurred in the middle part of the lordosis and less towards the lumbo-sacral and thoraco-lumbar transitions. The sex only affected the RoE.

Conclusions

During aging, the lower lumbar spine retains its lordosis and mobility, whereas the middle part flattens and becomes less mobile. These findings lay the ground for a better understanding of the incidence of level- and age-dependent spinal disorders, and may have important implications for the clinical long-term success of different surgical interventions.