Streptococcal M5 protein prevents neutrophil phagocytosis by interfering with CD11b/CD18 receptor-mediated association and signaling

Group A streptococci (GAS) are common human pathogens that express major surface-associated virulence factors designated M proteins. In this study, we explored directly the cellular mechanisms behind their supposed ability to prevent phagocytosis. Isolated human neutrophils killed an M-negative GAS mutant (DeltaM5), but not the wild-type parent strain (M5). After 3 h, 3-4 times as many DeltaM5 as M5 bacteria were associated with the neutrophils, and more DeltaM5 than M5 bacteria were ingested. However, there was no statistically significant difference between DeltaM5 and M5 bacteria in regard to the percentage of the neutrophil-associated bacteria that were ingested, indicating that M5 protein prevents an adhesion receptor-dependent association with neutrophils and not the phagocytic machinery per se. Different Abs against CD11b/CD18 (CR3) blocked adhesion and killing of DeltaM5 bacteria, whereas the blocking of two other complement receptors, CD11c/CD18 (CR4) and CD35 (CR1), did not. The CD11b/CD18-mediated killing of DeltaM5 bacteria resulted in protein tyrosine phosphorylations and Cdc42 activation. Furthermore, inhibition of CD11b/CD18 receptor engagement or tyrosine kinase activity blocked the DeltaM5-induced activation of Cdc42 as well as the killing of these bacteria. We conclude that M5 protein interferes with the CD11b/CD18-dependent association between GAS and neutrophils, and thereby blocks subsequent ingestion of the bacteria.     

Altered Notch signaling resulting from expression of a WAMTP1-MAML2 gene fusion in mucoepidermoid carcinomas and benign Warthin's tumors

Chromosome translocations in neoplasia commonly result in fusion genes that may encode either novel fusion proteins or normal, but ectopically expressed proteins. Here we report the cloning of a novel fusion gene in a common type of salivary and bronchial gland tumor, mucoepidermoid carcinomas (MEC), as well as in benign Warthin's tumors (WATs). The fusion, which results from a t(11;19)(q21-22;p13) translocation, creates a chimeric gene in which exon 1 of a novel gene of unknown function, designated WAMTP1, is linked to exons 2-5 of the recently identified Mastermind-like Notch coactivator MAML2. In the fusion protein, the N-terminal basic domain of MAML2, which is required for binding to intracellular Notch (Notch ICD), is replaced by an unrelated N-terminal sequence from WAMTP1. Mutation analysis of the N-terminus of WAMTP1-MAML2 identified two regions of importance for nuclear localization (amino acids 11-20) and for colocalization with MAML2 and Notch1 ICD in nuclear granules (amino acids 21-42). Analyses of the Notch target genes HES5 and MASH1 in MEC tumors with and without the WAMTP1-MAML2 fusion revealed upregulation of HES5 and downregulation of MASH1 in fusion positive MECs compared to normal salivary gland tissue and MECs lacking the fusion. These findings suggest that altered Notch signaling plays an important role in the genesis of benign and malignant neoplasms of salivary and bronchial gland origin.     

Distribution, sequence homology, and homing of group I introns among T-even-like bacteriophages: evidence for recent transfer of old introns

Self-splicing group I introns are being found in an increasing number of bacteriophages. Most introns contain an open reading frame coding for a homing endo-nuclease that confers mobility to both the intron and the homing endonuclease gene (HEG). The frequent occurrence of intron/HEG has raised questions whether group I introns are spread via horizontal transfer between phage populations. We have determined complete sequences for the known group I introns among T-even-like bacteriophages together with sequences of the intron-containing genes td, nrdB, and nrdD from phages with and without introns. A previously uncharacterized phage isolate, U5, is shown to contain all three introns, the only phage besides T4 found with a "full set" of these introns. Sequence analysis of td and nrdB genes from intron-containing and intronless phages provides evidence that recent horizontal transmission of introns has occurred among the phages. The fact that several of the HEGs have suffered deletions rendering them non-functional implies that the homing endonucleases are of no selective advantage to the phage and are rapidly degenerating and probably dependent upon frequent horizontal transmissions for maintenance within the phage populations. Several of the introns can home to closely related intronless phages during mixed infections. However, the efficiency of homing varies and is dependent on homology in regions flanking the intron insertion site. The occurrence of optional genes flanking the respective intron-containing gene can strongly affect the efficiency of homing. These findings give further insight into the mechanisms of propagation and evolution of group I introns among the T-even-like bacteriophages.     

Specific interaction between human parechovirus nonstructural 2A protein and viral RNA

The functional properties of the nonstructural 2A protein are variable among different picornaviruses. The 2A protein of the human parechovirus 1 (HPEV1) has been shown to lack the proteolytic activity found in many other picornaviruses, but no particular function has been identified for HPEV1 2A. To obtain information about the role of HPEV1 2A in the viral life cycle, the protein was expressed in Escherichia coli. A polyclonal antibody was then raised against the protein and employed to investigate its subcellular localization in the infected cells by immunofluorescence microscopy. Typically, a diffuse cytoplasmic staining pattern, concentrated to the perinuclear area, was observed in the infected cells. However, at late stages of infection some infected cells also exhibited diffuse nuclear staining. Viral RNA, visualized by fluorescent in situ hybridization, partly colocalized with 2A in the perinuclear region. Three experimental approaches including Northwestern blot, UV cross-linking, and gel retardation demonstrated that 2A possesses RNA binding activity. Competition experiments with various single-stranded RNA molecules addressed the specificity of 2A binding. These studies revealed that the 2A protein bound RNA corresponding to the 3'-untranslated region (UTR) of the viral genome with highest affinity. At the N- and C-terminal ends of the protein, two regions, necessary for RNA binding, were identified by mutagenesis. In addition, we demonstrated that 2A has affinity to double-stranded RNA containing 3'UTR(+)-3'UTR(-). In conclusion, our experiments showed that HPEV1 2A binds to viral 3'UTR RNA, a feature that could be important for the function of the protein during HPEV1 replication.     

Effects of glucagon-like peptide-1 on endothelial function in type 2 diabetes patients with stable coronary artery disease

GLP-1 stimulates insulin secretion, suppresses glucagon secretion, delays gastric emptying, and inhibits small bowel motility, all actions contributing to the anti-diabetogenic peptide effect. Endothelial dysfunction is strongly associated with insulin resistance and type 2 diabetes mellitus and may cause the angiopathy typifying this debilitating disease. Therefore, interventions affecting both endothelial dysfunction and insulin resistance may prove useful in improving survival in type 2 diabetes patients. We investigated GLP-1's effect on endothelial function and insulin sensitivity (S(I)) in two groups: 1) 12 type 2 diabetes patients with stable coronary artery disease and 2) 10 healthy subjects with normal endothelial function and S(I). Subjects underwent infusion of recombinant GLP-1 or saline in a random crossover study. Endothelial function was measured by postischemic FMD of brachial artery, using ultrasonography. S(I) [in (10(-4) dl.kg(-1).min(-1))/(muU/ml)] was measured by hyperinsulinemic isoglycemic clamp technique. In type 2 diabetic subjects, GLP-1 infusion significantly increased relative changes in brachial artery diameter from baseline FMD(%) (3.1 +/- 0.6 vs. 6.6 +/- 1.0%, P < 0.05), with no significant effects on S(I) (4.5 +/- 0.8 vs. 5.2 +/- 0.9, P = NS). In healthy subjects, GLP-1 infusion affected neither FMD(%) (11.9 +/- 0.9 vs. 10.3 +/- 1.0%, P = NS) nor S(I) (14.8 +/- 1.8 vs. 11.6 +/- 2.0, P = NS). We conclude that GLP-1 improves endothelial dysfunction but not insulin resistance in type 2 diabetic patients with coronary heart disease. This beneficial vascular effect of GLP-1 adds yet another salutary property of the peptide useful in diabetes treatment.     

Purification and characterization of a recombinant listeriolysin O expressed in Escherichia coli and possible diagnostic applications

The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol. This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed by a large number of Gram-positive bacteria. A protocol for large-scale expression and purification of recombinant LLO was previously optimized. By a simple two-step purification method, we achieved a high-level LLO synthesis (4.5 mg l(-1) of cell culture) in a hemolytically active form (1.2 x 10(6) HU mg(-1) of protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures which is a difficult task, mainly owing to the low levels of toxin released in the culture media. Here we report the characterization of toxin properties and its preliminary application in an ELISA diagnostic test for listeriosis.     

Spinal p38beta isoform mediates tissue injury-induced hyperalgesia and spinal sensitization

Antagonist studies show that spinal p38 mitogen-activated protein kinase plays a crucial role in spinal sensitization. However, there are two p38 isoforms found in spinal cord and the relative contribution of these two to hyperalgesia is not known. Here we demonstrate that the isoforms are distinctly expressed in spinal dorsal horn: p38alpha in neurons and p38beta in microglia. In lieu of isoform selective inhibitors, we examined the functional role of these two individual isoforms in nociception by using intrathecal isoform-specific antisense oligonucleotides to selectively block the expression of the respective isoform. In these rats, down-regulation of spinal p38beta, but not p38alpha, prevented nocifensive flinching evoked by intraplantar injection of formalin and hyperalgesia induced by activation of spinal neurokinin-1 receptors through intrathecal injection of substance P. Both intraplantar formalin and intrathecal substance P produced an increase in spinal p38 phosphorylation and this phosphorylation (activation) was prevented when spinal p38beta, but not p38alpha, was down-regulated. Thus, spinal p38beta, probably in microglia, plays a significant role in spinal nociceptive processing and represents a potential target for pain therapy.     

Pinoresinol-lariciresinol reductases with different stereospecificity from Linum album and Linum usitatissimum

Recently it was found that cell cultures and plants of Linum species contain lignans of various chemical structures. The stereochemistry of these compounds differ among species. Cell cultures of L. album accumulate (-)-podophyllotoxin together with pure (-)-secoisolariciresinol. The presence of both enantiomers of the precursor pinoresinol indicates that in L. album cell cultures the reactions from pinoresinol to secoisolariciresinol are the first steps determining enantiospecificity in biosynthesis of podophyllotoxin. Seeds of L. usitatissimum contain almost enantiomerically pure (+)-secoisolariciresinoldiglucosid derived from (+)-secoisolariciresinol. A cell culture of this species contains a mixture of both enantiomers of pinoresinol and pure (+)-secoisolariciresinol. In order to get more insight into the mechanism of (-)- and (+)-secoisolariciresinol biosynthesis, respectively, we isolated a cDNA encoding pinoresinol-lariciresinol reductase (PLR) from L. album. The heterologously expressed PLR-La1 converts only (+)-pinoresinol into (-)-secoisolariciresinol. In contrast, the heterologously expressed PLR from L. usitatissimum converts only (-)-pinoresinol to (+)-secoisolariciresinol confirming the results from others. Comparison of all available PLR protein sequences resulted in a few amino acids which may be responsible for the action of the PLRs with respect to the different enantioselectivity. A mutagenesis approach could not confirm this hypothesis. Aspects about the evolution of PLRs are discussed.