Anchoring of surface proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo peptidoglycan substrate for sortase-catalyzed surface protein anchoring

Surface proteins of Staphylococcus aureus are anchored to the cell wall peptidoglycan by a mechanism requiring a C-terminal sorting signal with an LPXTG motif. Surface proteins are first synthesized in the bacterial cytoplasm and then transported across the cytoplasmic membrane. Cleavage of the N-terminal signal peptide of the cytoplasmic surface protein P1 precursor generates the extracellular P2 species, which is the substrate for the cell wall anchoring reaction. Sortase, a membrane-anchored transpeptidase, cleaves P2 between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between the carboxyl group of threonine and the amino group of cell wall cross-bridges. We have used metabolic labeling of staphylococcal cultures with [(32)P]phosphoric acid to reveal a P3 intermediate. The (32)P-label of immunoprecipitated surface protein is removed by treatment with lysostaphin, a glycyl-glycine endopeptidase that separates the cell wall anchor structure. Furthermore, the appearance of P3 is prevented in the absence of sortase or by the inhibition of cell wall synthesis. (32)P-Labeled cell wall anchor species bind to nisin, an antibiotic that is known to form a complex with lipid II. Thus, it appears that the P3 intermediate represents surface protein linked to the lipid II peptidoglycan precursor. The data support a model whereby lipid II-linked polypeptides are incorporated into the growing peptidoglycan via the transpeptidation and transglycosylation reactions of cell wall synthesis, generating mature cell wall-linked surface protein.     

Direct and quantitative single-cell analysis of human immunodeficiency virus type 1 reactivation from latency

The ability of human immunodeficiency virus type 1 (HIV-1) to establish latent infections in cells has received renewed attention owing to the failure of highly active antiretroviral therapy to eradicate HIV-1 in vivo. Despite much study, the molecular bases of HIV-1 latency and reactivation are incompletely understood. Research on HIV-1 latency would benefit from a model system that is amenable to rapid and efficient analysis and through which compounds capable of regulating HIV-1 reactivation may be conveniently screened. We describe a novel reporter system that has several advantages over existing in vitro systems, which require elaborate, expensive, and time-consuming techniques to measure virus production. Two HIV-1 molecular clones (NL4-3 and 89.6) were engineered to express enhanced green fluorescent protein (EGFP) under the control of the viral long terminal repeat without removing any viral sequences. By using these replication-competent viruses, latently infected T-cell (Jurkat) and monocyte/macrophage (THP-1) lines in which EGFP fluorescence and virus expression are tightly coupled were generated. Following reactivation with agents such as tumor necrosis factor alpha, virus expression and EGFP fluorescence peaked after 4 days and over the next 3 weeks each declined in a synchronized manner, recapitulating the establishment of latency. Using fluorescence microscopy, flow cytometry, or plate-based fluorometry, this system allows immediate, direct, and quantitative real-time analysis of these processes within single cells or in bulk populations of cells. Exploiting the single-cell analysis abilities of this system, we demonstrate that cellular activation and virus reactivation following stimulation with proinflammatory cytokines can be uncoupled.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sj?gren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunoglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunoglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (V lambda 2E) and variable kappa chain genes (V kappa A27). Moreover, clonally related V(L) chain rearrangements were identified; namely, V kappa A27-J kappa 5 and V kappa A19-J kappa 2 in the parotid gland, and V lambda 1C-J lambda 3 in the parotid gland and the peripheral blood. V kappa and V lambda rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

The fission yeast ubiquitin-conjugating enzymes UbcP3, Ubc15, and Rhp6 affect transcriptional silencing of the mating-type region

Genes transcribed by RNA polymerase II are silenced when introduced near the mat2 or mat3 mating-type loci of the fission yeast Schizosaccharomyces pombe. Silencing is mediated by a number of gene products and cis-acting elements. We report here the finding of novel trans-acting factors identified in a screen for high-copy-number disruptors of silencing. Expression of cDNAs encoding the putative E2 ubiquitin-conjugating enzymes UbcP3, Ubc15 (ubiquitin-conjugating enzyme), or Rhp6 (Rad homolog pombe) from the strong nmt1 promoter derepressed the silent mating-type loci mat2 and mat3 and reporter genes inserted nearby. Deletion of rhp6 slightly derepressed an ade6 reporter gene placed in the mating-type region, whereas disruption of ubcP3 or ubc15 had no obvious effect on silencing. Rhp18 is the S. pombe homolog of Saccharomyces cerevisiae Rad18p, a DNA-binding protein that physically interacts with Rad6p. Rhp18 was not required for the derepression observed when UbcP3, Ubc15, or Rhp6 was overproduced. Overexpressing Rhp6 active-site mutants showed that the ubiquitin-conjugating activity of Rhp6 is essential for disruption of silencing. However, high dosage of UbcP3, Ubc15, or Rhp6 was not suppressed by a mutation in the 26S proteasome, suggesting that loss of silencing is not due to an increased degradation of silencing factors but rather to the posttranslational modification of proteins by ubiquitination. We discuss the implications of these results for the possible modes of action of UbcP3, Ubc15, and Rhp6.     

Erythropoietin therapy for acute stroke is both safe and beneficial

BACKGROUND: Erythropoietin (EPO) and its receptor play a major role in embryonic brain, are weakly expressed in normal postnatal/adult brain and up-regulated upon metabolic stress. EPO protects neurons from hypoxic/ ischemic injury. The objective of this trial is to study the safety and efficacy of recombinant human EPO (rhEPO) for treatment of ischemic stroke in man. MATERIALS AND METHODS: The trial consisted of a safety part and an efficacy part. In the safety study, 13 patients received rhEPO intravenously (3.3 X 10(4) IU/50 ml/30 min) once daily for the first 3 days after stroke. In the double-blind randomized proof-of-concept trial, 40 patients received either rhEPO or saline. Inclusion criteria were age <80 years, ischemic stroke within the middle cerebral artery territory confirmed by diffusion-weighted MRI, symptom onset <8 hr before drug administration, and deficits on stroke scales. The study endpoints were functional outcome at day 30 (Barthel Index, modified Rankin scale), NIH and Scandinavian stroke scales, evolution of infarct size (sequential MRI evaluation using diffusion-weighted [DWI] and fluid-attenuated inversion recovery sequences [FLAIR]) and the damage marker S100ss. RESULTS: No safety concerns were identified. Cerebrospinal fluid EPO increased to 60-100 times that of nontreated patients, proving that intravenously administered rhEPO reaches the brain. In the efficacy trial, patients received rhEPO within 5 hr of onset of symptoms (median, range 2:40-7:55). Admission neurologic scores and serum S100beta concentrations were strong predictors ofoutcome. Analysis of covariance controlled for these two variables indicated that rhEPO treatment was associated with an improvement in follow-up and outcome scales. A strong trend for reduction in infarct size in rhEPO patients as compared to controls was observed by MRI. CONCLUSION: Intravenous high-dose rhEPO is well tolerated in acute ischemic stroke and associated with an improvement in clinical outcome at 1 month. A larger scale clinical trial is warranted.     

A phylogenetic supertree of the bats (Mammalia: Chiroptera)

We present the first estimate of the phylogenetic relationships among all 916 extant and nine recently extinct species of bats Mammalia: Chiroptera), a group that accounts for almost one-quarter of extant mammalian diversity. This phylogeny was derived by combining 105 estimates of bat phylogenetic relationships published since 1970 using the supertree construction technique of Matrix Representation with Parsimony (MRP). Despite the explosive growth in the number of phylogenetic studies of bats since 1990, phylogenetic relationships in the order have been studied non-randomly. For example, over one-third of all bat systematic studies to date have locused on relationships within Phyllostomidae, whereas relationships within clades such as Kerivoulinae and Murinae have never been studied using cladistic methods. Resolution in the supertree similarly differs among clades: overall resolution is poor (46.4%, of a fully bifurcating solution) but reaches 100% in some groups (e.g. relationships within Mormoopidae). The supertree analysis does not support a recent proposal that Microchiroptera is paraphyletic with respect to Megachiroptera, as the majority of source topologies support microbat monophyly. Although it is not a substitute for comprehensive phylogenetic analyses of primary molecular and morphological data, the bat supertree provides a useful tool for future phylogenetic comparative and macroevolutionary studies. Additionally, it identifies clades that have been little studied, highlights groups within which relationships are controversial, and like all phylogenetic studies, provides preliminary hypotheses that can form starting points for future phylogenetic studies of bats.     

PACAP enhances the expression of CD11b,CD66b and CD63 in human neutrophils

Pituitary adenylate cyclase-activating peptide (PACAP) is a neuropeptide with strong bronchodilator capacity, present in the human airways. There is recent evidence that PACAP decreases the release of proinflammatory cytokines. We have previously shown that PACAP inhibits neutrophil chemotaxis, but altogether little is known about the effects of PACAP on granulocytes. The present study was designed to investigate if PACAP and the closely related peptide vasoactive intestinal peptide (VIP) could affect the cell surface expression of CD11b, CD63 and CD66b in human neutrophils. Neutrophils isolated from 12 healthy blood donors were incubated with either PACAP or VIP, and the expression of neutrophil cell surface markers was assessed using flowcytometry. Neutrophils incubated with PACAP38 exhibited a marked, concentration-dependent increase in their expression of CD11b, CD63 and CD66b. In contrast, neutrophils incubated with VIP showed no increase of the investigated surface markers. This indicates a role for PACAP in granulocyte activation, mediated via a pathway not shared with VIP. Together with the previously presented data on leukocyte migration it suggests that PACAP acts as a regulator of neutrophil inflammation.     

Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands

The role of the N-terminal domains of corticotropin-releasing factor (CRF) and CRF-like peptides in receptor subtype selectivity, ligand affinity and biological potency was investigated. Therefore, human CRF(12-41), human URP(12-38) and antisauvagine-30 (aSvg) were N-terminally prolonged by consecutive addition of one or two amino acids. The peptides obtained were tested for their binding affinities to rat CRF1 and murine CRF(2beta) receptor, and their capability to stimulate cAMP-release by HEK cells producing either receptor.It was observed that human CRF N-terminally truncated by eight residues was bound with high affinity to CRF2 receptor (Ki=5.4nM), whereas affinity for CRF1 receptor was decreased (Ki=250 nM). A similar shift of affinity was found with sauvagine (Svg) analogs. Truncation of human URP analogs did not affect their preference for CRF(2beta) receptor, but reduced their affinity. Changes in affinity were positively correlated with changes in potency. These results indicated that CRF1 receptor was more stringent in its structural requirements for ligands to exhibit high affinity binding than CRF(2beta) receptor.     

Mathematical modelling in the post-genome era: understanding genome expression and regulation--a system theoretic approach

This paper introduces a mathematical framework for modelling genome expression and regulation. Starting with a philosophical foundation, causation is identified as the principle of explanation of change in the realm of matter. Causation is, therefore, a relationship, not between components, but between changes of states of a system. We subsequently view genome expression (formerly known as 'gene expression') as a dynamic process and model aspects of it as dynamic systems using methodologies developed within the areas of systems and control theory. We begin with the possibly most abstract but general formulation in the setting of category theory. The class of models realised are state-space models, input--output models, autoregressive models or automata. We find that a number of proposed 'gene network' models are, therefore, included in the framework presented here. The conceptual framework that integrates all of these models defines a dynamic system as a family of expression profiles. It becomes apparent that the concept of a 'gene' is less appropriate when considering mathematical models of genome expression and regulation. The main claim of this paper is that we should treat (model) the organisation and regulation of genetic pathways as what they are: dynamic systems. Microarray technology allows us to generate large sets of time series data and is, therefore, discussed with regard to its use in mathematical modelling of gene expression and regulation.