Bayesian evidence and epidemiological implications of environmental contamination from acute respiratory infection in long-term care facilities.

Skilled nursing home facilities (SNFs) house a vulnerable population frequently exposed to respiratory pathogens. Our study aims to gain a better understanding of the transmission of nursing home-acquired viral respiratory infections in non-epidemic settings. Symptomatic surveillance was performed in three SNFs for residents exhibiting acute respiratory symptoms. Environmental surveillance of five high-touch areas was performed to assess possible transmission. All resident and environmental samples were screened using a commercial multiplex polymerase chain reaction platform. Bayesian methods were used to evaluate environmental contamination. Among nursing home residents with respiratory symptoms, 19% had a detectable viral pathogen (parainfluenza-3, rhinovirus/enterovirus, RSV, or influenza B). Environmental contamination was found in 20% of total room surface swabs of symptomatic residents. Environmental and resident results were all concordant. Target period prevalence among symptomatic residents ranged from 5.5 to 13.3% depending on target. Bayesian analysis quantifies the probability of environmental shedding due to parainfluenza-3 as 92.4% (95% CI: 86.8-95.8%) and due to rhinovirus/enterovirus as 65.6% (95% CI: 57.9-72.5%). Our findings confirm that non-epidemic viral infections are common among SNF residents exhibiting acute respiratory symptoms and that environmental contamination may facilitate further spread with considerable epidemiological implications. Findings further emphasise the importance of environmental infection control for viral respiratory pathogens in long-term care facilities.

The management of acute hip pain in the military: femoral neck stress fractures and tears of the acetabular labrum.

Acute hip pain is a common presenting complaint amongst the military population. It can present in a variety of ways, with a broad range of differential diagnoses to consider. Most cases of acute hip pain in military patients tend to be traumatic in origin. Pathology within the hip can be a diagnostic challenge, as symptoms often overlap between differential diagnoses and examination findings are not always sensitive or specific. Any hip injury will potentially downgrade a military patient and can also be a significant cause of long-term morbidity. Being able to manage the patient with acute hip pain effectively will ensure that patients spend less time in the diagnostic chain and reach the definitive treatment they require to continue to carry out their primary role. This paper describes how best to manage military patients who present with acute hip pain. It covers the diagnostic challenges faced by clinicians, the differential diagnoses of acute hip pain and describes the management of some common injuries of the hip: tears of the acetabular labrum and femoral neck stress fractures.

Phospholamban domain IB forms an interaction site with the loop between transmembrane helices M6 and M7 of sarco(endo)plasmic reticulum Ca2+ ATPases.

Transmembrane helix M6 of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) has been shown to form a site of interaction with phospholamban (PLN). Site-directed mutagenesis was carried out in the cytoplasmic loop (L67) between M6 and M7 in SERCA1a to detect other SERCA-PLN binding sites. Mutants N810A, D813A, and R822A had diminished ability to interact functionally with PLN, but only D813A and R822A had reduced physical interaction with PLN. PLN mutants R25A, Q26A, N27A, L28A, Q29A, and N30A had enhanced physical interaction with wild-type (wt) SERCA1a, but physical interaction of these PLN mutants with SERCA1a mutants D813A and R822A was reduced about 2.5 fold (range 1.44-2.82). Exceptions were the interactions of PLN N27A and N30A with SERCA1a D813A, which were reduced by 7.3- and 5.8-fold, respectively. A superinhibitory PLN deletion mutant, PLNDelta21-29, had strong physical interactions with SERCA1a and with SERCA1a mutant D813A. Physical interactions with SERCA1a and mutant D813A were sharply diminished, however, for the PLN deletion mutant, PLNDelta21-30, lacking PLN N30. Physical interactions between SERCA1a and a PLN-cytochrome b(5) chimera containing PLN residues 1-29 were much stronger than those between a PLN-cytochrome b(5) chimera containing PLN residues 1-21 and lacking N27. These results suggest that a SERCA1-PLN interaction site occurs between L67 of SERCA1a and domain IB of PLN, which involves SERCA1a D813 and PLN N27 and N30.

Multilocus analysis of extracellular putative virulence proteins made by group A Streptococcus: population genetics, human serologic response, and gene transcription.

Species of pathogenic microbes are composed of an array of evolutionarily distinct chromosomal genotypes characterized by diversity in gene content and sequence (allelic variation). The occurrence of substantial genetic diversity has hindered progress in developing a comprehensive understanding of the molecular basis of virulence and new therapeutics such as vaccines. To provide new information that bears on these issues, 11 genes encoding extracellular proteins in the human bacterial pathogen group A Streptococcus identified by analysis of four genomes were studied. Eight of the 11 genes encode proteins with a LPXTG(L) motif that covalently links Gram-positive virulence factors to the bacterial cell surface. Sequence analysis of the 11 genes in 37 geographically and phylogenetically diverse group A Streptococcus strains cultured from patients with different infection types found that recent horizontal gene transfer has contributed substantially to chromosomal diversity. Regions of the inferred proteins likely to interact with the host were identified by molecular population genetic analysis, and Western immunoblot analysis with sera from infected patients confirmed that they were antigenic. Real-time reverse transcriptase-PCR (TaqMan) assays found that transcription of six of the 11 genes was substantially up-regulated in the stationary phase. In addition, transcription of many genes was influenced by the covR and mga trans-acting gene regulatory loci. Multilocus investigation of putative virulence genes by the integrated approach described herein provides an important strategy to aid microbial pathogenesis research and rapidly identify new targets for therapeutics research.

Modeling a dehalogenase fold into the 8-A density map for Ca(2+)-ATPase defines a new domain structure.

Members of the large family of P-type pumps use active transport to maintain gradients of a wide variety of cations across cellular membranes. Recent structures of two P-type pumps at 8-A resolution have revealed the arrangement of transmembrane helices but were insufficient to reveal the architecture of the cytoplasmic domains. However, recent proposals of a structural homology with a superfamily of hydrolases offer a new basis for modeling these domains. In the current work, we have extended the sequence comparison for the superfamily and delineated domains in the 8-A density map of Ca(2+)-ATPase. The homology suggests a new domain structure for Ca(2+)-ATPase and, specifically, that the phosphorylation domain adopts a Rossman fold. Accordingly, the atomic structure of L-2 haloacid dehalogenase has been fitted into the relevant domain of Ca(2+)-ATPase. The resulting model suggests the existence of two ATP sites at the interface between two domains. Based on this new model, we are able to reconcile numerous results of mutagenesis and chemical cross-linking within the catalytic domains. Furthermore, we have used the model to predict the configuration of Mg.ATP at its binding site. Based on this prediction, we propose a mechanism, involving a change in Mg(2+) liganding, for initiating the domain movements that couple sites of ion transport to ATP hydrolysis.

Structure-function relationships in the Ca(2+)-binding and translocation domain of SERCA1: physiological correlates in Brody disease.

Alanine-scanning mutagenesis of all amino acids in transmembrane helices M4, M5, M6 and M8, which contain known Ca2+ binding residues in the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum, revealed patches of mutation-sensitivity in M4, M5 and M6, but in M8. A six-residue motif, (E/D)GLPA(T/V), in M4 and M6 and its counterpart in M5 were highlighted by mutagenesis. Site-directed disulfide mapping of helices M4 and M6 demonstrated that these transmembrane helices associate as a right-handed coiled-coil. This structural information, combined with the earlier analysis of the association of each Ca2+ binding residue with either Ca2+ binding site I or site II, permitted the development of a "side-by-side" model for the two Ca2+ binding sites in the Ca(2+)-ATPase. In about half of Brody disease families, mutations create stop codons which delete all or part of the Ca2+ binding and translocation domain, resulting in loss of SERCA1 function and muscle disease.

Structure of the calcium pump from sarcoplasmic reticulum at 8-A resolution.

The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-type cation pumps. These couple ATP hydrolysis with cation transport, generating cation gradients across membranes. Ca2+-ATPase specifically maintains the low cytoplasmic calcium concentration of resting muscle by pumping calcium into the sarcoplasmic reticulum; subsequent release is used to initiate contraction. No high-resolution structure of a P-type pump has yet been determined, although a 14-A structure of Ca2+-ATPase, obtained by electron microscopy of frozen-hydrated, tubular crystals, showed a large cytoplasmic head connected to the transmembrane domain by a narrow stalk. We have now improved the resolution to 8A and can discern ten transmembrane alpha-helices, four of which continue into the stalk On the basis of constraints from transmembrane topology, site-directed mutagenesis and disulphide crosslinking, we have made tentative assignments for these alpha-helices within the amino-acid sequence. A distinct cavity leads to the putative calcium-binding site, providing a plausible path for calcium release to the lumen of the sarcoplasmic reticulum.

Site-directed disulfide mapping of helices M4 and M6 in the Ca2+ binding domain of SERCA1a, the Ca2+ ATPase of fast twitch skeletal muscle sarcoplasmic reticulum.

In an attempt to define the spatial relationships among SERCA1a transmembrane helices M4, M5, M6, and M8, involved in Ca2+ binding, all six cysteine residues were removed from predicted transmembrane sequences by substitution with Ser or Ala. The cysteine-depleted protein retained 44% of wild type Ca2+ transport activity. Pairs of cysteine residues were then reintroduced to determine whether their juxtaposition would result in the formation of disulfide cross-links between transmembrane helices. In initial studies designed to map the juxtaposition of Ca2+ binding residues, Cys was substituted for Glu309 or Gly310 in transmembrane sequence M4, in combination with the substitution of Cys for Glu771 in M5; for Asn796, Thr799, or Asp800 in M6; or for Glu908 in M8. These double mutants all retained the capacity to form a phosphoenzyme intermediate from Pi (but not from ATP in the presence of Ca2+), and in all but mutants E309C/N796C and G310C/N796C, phosphoenzyme formation was insensitive to 100 microM Ca2+. These results support the view that both Glu309 and Asn796 contribute to Ca2+ binding site II, which is not required for conversion of E2, the substrate for Pi phosphorylation, to E1. Cross-linking in mutants E309C/N796C and G310C/D800C established reference points for the orientation of M4 and M6 relative to each other and provided the basis for the prediction of potential additional cross-links. Strong links were formed with the pairs T317C/A804C and T317C/L807C near the cytoplasmic ends of the two helices and with A305C/L792C and A305C/L793C near the lumenal ends. These combined results support the conclusion that M4 and M6 form a right-handed coiled-coil structure that forms part of the pathway of Ca2+ translocation. In addition to providing a possible explanation for the mutation sensitivity of several pairs of residues in these helices, the proposed association of M4 and M6 supports a new model for the orientation of the two Ca2+ binding sites among transmembrane helices M4, M5, and M6.

Structure, transmembrane topology and helix packing of P-type ion pumps.

Electron microscopy has recently provided improved structures for P-type ion pumps. In the case of Ca(2+)-ATPase, the use of unstained specimens revealed the structure of the transmembrane domain. The composition of this domain has been controversial due to the variety of methods used to study the number and exact locations of transmembrane crossings within the sequence. After reviewing the results from several members of the family, we found a consensus for 10 transmembrane segments, and also that 10 helices fitted well into the structure of Ca(2+)-ATPase. Thus, we present the most detailed model for transmembrane structure so far, in the hope of stimulating more precise experimental strategies.

A method for alpha-helical integral membrane protein fold prediction.

Integral membrane proteins (of the alpha-helical class) are of central importance in a wide variety of vital cellular functions. Despite considerable effort on methods to predict the location of the helices, little attention has been directed toward developing an automatic method to pack the helices together. In principle, the prediction of membrane proteins should be easier than the prediction of globular proteins: there is only one type of secondary structure and all helices pack with a common alignment across the membrane. This allows all possible structures to be represented on a simple lattice and exhaustively enumerated. Prediction success lies not in generating many possible folds but in recognizing which corresponds to the native. Our evaluation of each fold is based on how well the exposed surface predicted from a multiple sequence alignment fits its allocated position. Just as exposure to solvent in globular proteins can be predicted from sequence variation, so exposure to lipid can be recognized by variable-hydrophobic (variphobic) positions. Application to both bacteriorhodopsin and the eukaryotic rhodopsin/opsin families revealed that the angular size of the lipid-exposed faces must be predicted accurately to allow selection of the correct fold. With the inherent uncertainties in helix prediction and parameter choice, this accuracy could not be guaranteed but the correct fold was typically found in the top six candidates. Our method provides the first completely automatic method that can proceed from a scan of the protein sequence databanks to a predicted three-dimensional structure with no intervention required from the investigator. Within the limited domain of the seven helix bundle proteins, a good chance can be given of selecting the correct structure. However, the limited number of sequences available with a corresponding known structure makes further characterization of the method difficult.

Identification of amino acid residues photolabeled with 8-azidoadenosine 5'-diphosphate in the catalytic site of sarcoplasmic reticulum Ca-ATPase.

The photoreactive ADP analogue 8-N3-ADP binds in the dark to the catalytic site of the sarcoplasmic reticulum Ca-ATPase. An apparent Kd value of 30 microM has been deduced from competition with ADP in the presence of EGTA. Photoirradiation of Ca-ATPase with 8-N3-[3H]ADP in the presence of calcium results in irreversible inhibition of ATPase activity with corresponding stoichiometries of covalently and specifically photolabeled Ca-ATPase. The site of photolabeling of the Ca-ATPase in the presence of calcium has been explored. Controlled trypsin digestion of the labeled protein shows that 8-azido-ADP is incorporated in the B subfragment. Extensive trypsin digestion of the labeled protein releases a small peptide as revealed by gel filtration chromatography (Sephadex G-50). Further HPLC purification on a reverse-phase column (C8) eluted with a water/acetonitrile gradient buffered at pH 6 or at pH 2 gives a single labeled peptide. Edman degradation of that isolated peptide, as well as the amino acid composition, shows that it contains five amino acid residues (Val-530-Arg-534) with the radioactivity localized on Thr-532 and Thr-533.

Structural modelling of P-type ion pumps.

Over forty sequences of P-type ion pumps have been determined. They fall into five families showing between 20% and 50% identity in sequence. The conserved residues are concentrated in several regions which are found in all the pumps. All the defined functional sites are associated with conserved segments and provide a basis for subdivision into domains, to which tentative secondary and tertiary structures can be assigned. The domains have been assembled into a structure consisting of a conserved core with variable loops and deletions on the surface, which accommodates site mutants, affinity labels and known epitopes. This model has been correlated with the results of an electron crystallographic study of the Ca++ pump. Two types of crystal have been examined in negative stain and in amorphous ice; thin plates which diffract to 4A and long helical tubes which diffract to 15A. The plates have given a 6A projection map, some of which can be interpreted by difference from the map of the negatively stained crystal, as transmembrane helices. Three dimensional interpretation will require a tilt series. In the meantime, analysis of the tubes has given a 14A 3D map which clearly defines the cytoplasmic domains and their relation to the transmembrane region (Stokes and Toyoshima in preparation). Although it is not yet possible to assign specific functions to the cytoplasmic lobes, the structure at this resolution is consistent with the model.

A hypothetical model for the peptide binding domain of hsp70 based on the peptide binding domain of HLA.

The sequences of the peptide binding domains of 33 70 kd heat shock proteins (hsp70) have been aligned and a consensus secondary structure has been deduced. Individual members showed no significant deviation from the consensus, which showed a beta 4 alpha motif repeated twice, followed by two further helices and a terminus rich in Pro and Gly. The repeated motif could be aligned with the secondary structure of the functionally equivalent peptide binding domain of human leucocyte antigen (HLA) class I maintaining equivalent residues in structurally important positions in the two families and a model was built based on this alignment. The interaction of this domain with the ATP domain is considered. The overall model is shown to be consistent with the properties of products of chymotryptic cleavage.

Calreticulin is a candidate for a calsequestrin-like function in Ca2(+)-storage compartments (calciosomes) of liver and brain.

In a search for the non-muscle equivalent of calsequestrin (the low-affinity high-capacity Ca2(+)-binding protein responsible for Ca2+ storage within the terminal cisternae of the sarcoplasmic reticulum), acidic proteins were extracted from rat liver and brain microsomal preparations and purified by column chromatography. No calsequestrin was observed in these extracts, but the N-terminal amino acid sequence of the major Ca2(+)-binding protein of the liver microsomal fraction was determined and found to correspond to that of calreticulin. This protein was found to bind approx. 50 mol of Ca2+/mol of protein, with low affinity (average Kd approx. 1.0 mM). A monoclonal antibody, C6, raised against skeletal-muscle calsequestrin cross-reacted with calreticulin in SDS/PAGE immunoblots, but polyclonal antibodies reacted with native calreticulin only weakly, or not at all, after SDS denaturation. Immuno-gold decoration of liver ultrathin cryosections with affinity-purified antibodies against liver calreticulin revealed luminal labelling of vacuolar profiles indistinguishable from calciosomes, the subcellular structures previously identified by the use of anti-calsequestrin antibodies. We conclude that calreticulin is the Ca2(+)-binding protein segregated within the calciosome lumen, previously described as being calsequestrin-like. Because of its properties and intraluminal location, calreticulin might play a critical role in Ca2+ storage and release in non-muscle cells, similar to that played by calsequestrin in the muscle sarcoplasmic reticulum.