Real-time polymerase chain reaction and laser capture microdissection: an efficient combination tool for Chlamydophila pneumoniae DNA quantification and localization of infection in atherosclerotic lesions.

Int J Immunopathol Pharmacol. 2008 Apr-Jun;21(2):421-8

Authors: Ciervo A, Mancini F, Sale P, Russo A, Cassone A

Chlamydophila pneumoniae has been implicated in atherosclerosis, but the role of this obligate intracellular pathogen in the development of the above pathology is still unclear. In particular, its presence and quantitative distribution within lesional areas has not yet been defined. We studied 18 carotid biopsies obtained from patients undergoing endoartherectomy. By laser microdissection (LCM), two different sites (intra-plaque and plaque-adjacent areas) were taken from each lesion, and the presence and quantity of the pathogen DNA were determined by real-time polymerase chain reaction (Real-time PCR). A total of 8 plaques, exclusively from patients with unstable angina, were positive in real-time PCR. The bacterial DNA was detected in both lesional areas of 3 plaques which contained the highest number of DNA copies (1,900 to 2,200 copy numbers), while C. pneumoniae DNA was detected only in the intra-plaque area of the other 5 positive (500 to 1,600 copy numbers). No C. pneumoniae DNA was found in the other 10 plaques of which 6 were from patients with unstable angina and 4 from stable angina patients. No DNA from Helicobacter pylori or Cytomegalovirus was found in any plaque. This is the first report where both the target lesion and an adjacent reference site were evaluated for the presence of C. pneumoniae DNA by the combination of LCM and Real-time PCR assays. The integration of these two methodologies offer an excellent tool for in situ studies and may help to elucidate the putative role of C. pneumoniae in atherosclerosis. PMID: 18547488 [PubMed - in process]

Structural Basis for the Regulation Mechanism of the Tyrosine Kinase CapB from Staphylococcus aureus.

PLoS Biol. 2008 Jun 10;6(6):e143

Authors: Olivares-Illana V, Meyer P, Bechet E, Gueguen-Chaignon V, Soulat D, Lazereg-Riquier S, Mijakovic I, Deutscher J, Cozzone AJ, Laprévote O, Morera S, Grangeasse C, Nessler S

Bacteria were thought to be devoid of tyrosine-phosphorylating enzymes. However, several tyrosine kinases without similarity to their eukaryotic counterparts have recently been identified in bacteria. They are involved in many physiological processes, but their accurate functions remain poorly understood due to slow progress in their structural characterization. They have been best characterized as copolymerases involved in the synthesis and export of extracellular polysaccharides. These compounds play critical roles in the virulence of pathogenic bacteria, and bacterial tyrosine kinases can thus be considered as potential therapeutic targets. Here, we present the crystal structures of the phosphorylated and unphosphorylated states of the tyrosine kinase CapB from the human pathogen Staphylococcus aureus together with the activator domain of its cognate transmembrane modulator CapA. This first high-resolution structure of a bacterial tyrosine kinase reveals a 230-kDa ring-shaped octamer that dissociates upon intermolecular autophosphorylation. These observations provide a molecular basis for the regulation mechanism of the bacterial tyrosine kinases and give insights into their copolymerase function. PMID: 18547145 [PubMed - as supplied by publisher]

Necrotizing enterocolitis in newborns : pathogenesis, prevention and management.

Drugs. 2008;68(9):1227-38

Authors: Thompson AM, Bizzarro MJ

Necrotizing enterocolitis (NEC) is primarily a disease process of the gastrointestinal (GI) tract of premature neonates that results in inflammation and bacterial invasion of the bowel wall. Despite advances in the care of premature infants, NEC remains one of the leading causes of morbidity and mortality in this population. It occurs in 1-5% of all neonatal intensive care admissions and 5-10% of all very low birthweight (<1500 g) infants. Although research has presented an interesting array of potential contributing factors, the precise aetiology of this multifactorial disease process remains elusive. Historically, it was believed that NEC arose predominantly from ischaemic injury to the immature GI tract, yet alternate plausible hypotheses indicate that many factors are likely to be involved. These may include issues related to the introduction and advancement of enteric feeding, alterations in the normal bacterial colonization of the GI tract, bacterial translocation and activation of the cytokine cascade, decreased epidermal growth factor, increased platelet activating factor, and mucosal damage from free radical production.Clinical manifestations of NEC may be vague, including increased episodes of apnoea, desaturations, bradycardia, lethargy and temperature instability. There may also be GI-specific symptoms such as feeding intolerance, emesis, bloody stools, abdominal distention and tenderness, and abdominal wall discolouration. Laboratory values may be indicative of infection, coagulation abnormalities and fluid retention. Radiographic signs may include ileus, dilated or fixed intestinal loops, air in the intestinal wall or free air in the abdomen. Medical treatment typically consists of bowel rest and decompression, antibacterial therapy, and management of other haematological or electrolyte imbalances. Increased respiratory and cardiovascular support is sometimes needed. In neonates who do not respond adequately to medical management, or if pneumoperitoneum is present, surgical intervention may occur with either use of a peritoneal drain or laparotomy.Advances in antenatal and neonatal care have resulted in increased survival of extremely preterm neonates. As this at-risk population continues to increase, an effective preventative strategy for NEC is needed. One preventative strategy is the use of antenatal corticosteroids to enhance maturation of the fetus if preterm delivery is likely. Recommendation of use of breast milk, early initiation of trophic feeds and judicoius advancement of enteric feeds are current postnatal strategies. Other preventative strategies that have been investigated include the use of oral antibacterials, antioxidants, supplementation of arginine and epidermal growth factor, none of which have changed clinical practice. Recent promising data indicate that prophylactic use of probiotics may play a role in preventing the onset of NEC. However, more large-scale, definitive studies are needed. PMID: 18547133 [PubMed - in process]

Affinity-Capture Tandem Mass Spectrometric Characterization of Polyprenyl-Linked Oligosaccharides: Tool to Study Protein N-Glycosylation Pathways.

Anal Chem. 2008 Jun 12;

Authors: Reid CW, Stupak J, Chen MM, Imperiali B, Li J, Szymanski CM

N-Glycosylation of proteins is recognized as one of the most common post-translational modifications. Until recently it was believed that N-glycosylation occurred exclusively in eukaryotes before the discovery of the general protein glycosylation pathway (Pgl) in Campylobacter jejuni. To date, most techniques to analyze lipid-linked oligosaccharides (LLOs) of these pathways involve the use of radiolabels and chromatographic separation. Technologies capable of characterizing eukaryotic and the newly described bacterial N-glycosylation systems from biologically relevant samples in a quick, accurate, and cost-effective manner are needed. In this paper a new glycomics strategy based on lectin-affinity capture was devised and validated on the C. jejuni N-glycan pathway and the engineered Escherichia coli strains expressing the functional C. jejuni pathway. The lipid-linked oligosaccharide intermediates of the Pgl pathway were then enriched using SBA-agarose affinity-capture and examined by capillary electrophoresis-mass spectrometry (CE-MS). We demonstrate that this method is capable of detecting low levels of LLOs, the sugars are indeed assembled on undecaprenylpyrophosphate, and structural information for expected and unexpected LLOs can be obtained without further sample manipulation. Furthermore, CE-MS analyses of C. jejuni and the E. coli "glyco-factories" showed striking differences in the assembly and control of N-glycan biosynthesis. PMID: 18547063 [PubMed - as supplied by publisher]