Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.     

Cryptococcus neoformans arthritis in a renal transplant recipient

We present a Cryptococcus neoformans knee joint infection in a diabetic renal transplant patient treated with steroids, cyclosporin, and mycofenolate mofetil. We discuss reported cases of cryptococcal arthritis.     

Porphyromonas gingivalis lipopolysaccharide interferes with salivary mucin synthesis through inducible nitric oxide synthase activation by ERK and p38 kinase

Porphyromonas gingivalis is a Gram-negative periodontopathic bacterium colonizing the oral cavity and its lipopolysaccharide (LPS) is a key factor in the development of periodontitis. We investigated the effect of P. gingivalis LPS on the cellular responses associated with mucin synthesis in sublingual salivary gland acinar cells. Exposure of the acinar cells to the LPS led to a dose-dependent decrease in mucin synthesis and was accompanied by a massive induction in inducible nitric oxide synthase (NOS-2) activity and the increase in NO production, caspase-3 activity and apoptosis. Inhibition of extracellular signal-regulated kinase (ERK) with PD98059 accelerated the LPS-induced decrease in the glycoprotein synthesis and caused further increase in apoptosis and NOS-2 activity, while the blockade of p38 mitogen-activated kinase (MAPK) with SB203580 countered the LPS-induced reduction in the glycoprotein synthesis and obviated the induced increases in NOS-2 and apoptosis. Introduction of NOS-2 inhibitor, L-NAME, not only countered the LPS-induced increase in NO generation, caspase-3 activity and apoptosis, but caused the impedance of the LPS inhibition on mucin synthesis. The findings point to the upregulation in NOS-2 expression by P. gingivalis LPS as a key detrimental culprit affecting salivary mucin synthesis.     

Early diagnosis of invasive aspergillosis in neutropenic patients with bi-weekly serial screening of circulating galactomannan by Platelia Aspergillus

The diagnosis of invasive aspergillosis in neutropenic individuals is difficult and lengthy since non-invasive diagnostic tests lack sensitivity and specificity. The diagnosis of invasive aspergillosis in 154 prolonged neutropenic patients was prospectively bi-weekly validated by screening circulating galactomannan. The global sensitivity was 73% and specificity was 96%. The positive and negative predictive values were 73% and 98% respectively. False positive reactions occurred at a rate of 2%. Antigenemia was detected before clinical suspicion of invasive aspergillosis (median, 6 days before) in 30% of patients and anticipated the onset of radiologic signs 9 days in 60% of patients. CONCLUSION: the prospective screening of galactomannan is a sensitive and non-invasive tool for early diagnosis of invasive aspergillosis in high-risk adult hematology patients.     

Invasive aspergillosis

Invasive aspergillosis is one of the most frequent fungal infections in neutropenic patients, in whom it is associated with a high mortality. Its diagnosis is difficult by the traditionally used laboratory tests. In the last years, an ELISA (Platelia Aspergillus, Bio-Rad, France) to detect galactomannan in neutropenic and cancer patients with high risk of suffering invasive aspergillosis has been developed. The experience accumulated in Spain in the diagnosis of invasive aspergillosis by Platelia Aspergillus is presented in this monograph.     

Desmoglein 4 in hair follicle differentiation and epidermal adhesion: evidence from inherited hypotrichosis and acquired pemphigus vulgaris

Cell adhesion and communication are interdependent aspects of cell behavior that are critical for morphogenesis and tissue architecture. In the skin, epidermal adhesion is mediated in part by specialized cell-cell junctions known as desmosomes, which are characterized by the presence of desmosomal cadherins, known as desmogleins and desmocollins. We identified a cadherin family member, desmoglein 4, which is expressed in the suprabasal epidermis and hair follicle. The essential role of desmoglein 4 in skin was established by identifying mutations in families with inherited hypotrichosis, as well as in the lanceolate hair mouse. We also show that DSG4 is an autoantigen in pemphigus vulgaris. Characterization of the phenotype of naturally occurring mutant mice revealed disruption of desmosomal adhesion and perturbations in keratinocyte behavior. We provide evidence that desmoglein 4 is a key mediator of keratinocyte cell adhesion in the hair follicle, where it coordinates the transition from proliferation to differentiation.     

A novel improved method for Aspergillus nidulans transformation

We systematically investigated the efficiency of Aspergillus nidulans transformation using protoplasts prepared from different stages of conidiospore germination and young mycelium. Using standard integrative plasmids, increased transformation yields were obtained with protoplasts isolated from a specific stage coincident with germ tube emergence. This increase ranged, on the average, from two- to eightfold depending on different plasmids used. Transformation efficiencies with a replicative plasmid were similar to those obtained using previously described methods. Although this observation suggests that elevated transformation efficiencies might be due to increased efficiency of recombination between plasmid and genomic sequences, we cannot exclude other factors associated with the particular developmental stage used. In the course of this study, we also examined the effect of other parameters that might enhance transformation yields. The method described is also significantly easier and faster than other current methods.     

Molecular phylogeny and phylogeography of free-living Bryozoa (Cupuladriidae) from both sides of the Isthmus of Panama

Genetic data were used to identify Recent species of free-living bryozoans (Cupuladriidae) from both sides of the Isthmus of Panama, and to examine their phylogenetic relationships, species richness, and population structures. An approximately 480bp fragment of the 16S mitochondrial rRNA gene was sequenced from 182 individuals from Panama, the Gulf of Mexico, and El Salvador. Ten haplotype groups (Cupuladria 4, 5, and 6; Discoporella 1, 2, 3A, 3B, 3C, 7, and 8) were identified. Genetic distances between haplotype groups (3.2-26.5%; K2P+Gamma) were 1-2 orders of magnitude greater than within groups (0.1-1.4%). Seven of the haplotype groups represent morphologically distinct species; Discoporellas 3A-C appear to be cryptic species. Phylogenetic analyses identified two pairs of transisthmian sister clades. An average divergence rate derived from other taxa suggests that Cupuladrias 4 and 5 diverged approximately 7Ma, a Discoporella 7 clade diverged from a 3A-C clade approximately 11Ma, and the 3A-C clade radiated approximately 6-4Ma; these events all predated final closure of the isthmus? 3Ma. The Caribbean side of the isthmus, with 5 species, is only marginally richer in cupuladriids than the Pacific side, with 4, but has greater phylogenetic depth. The Caribbean retains lineages stemming from a New World Miocene radiation that are not represented in the eastern Pacific; extant eastern Pacific cupuladriids share most recent common ancestry with only two of the Caribbean lineages. Species in the eastern Pacific tend to show shallow population structures, with high levels of gene flow between geographically separate populations, whereas Caribbean species tend to show deeper populations structures, with indications of restricted gene flow between Bocas del Toro/Gulf of Mosquitos and Costa Arriba/San Blas. The population structures derive from Pleistocene histories and may be of limited value in interpreting the macroevolutionary pattern, as our results provide no evidence of speciation on either side of the isthmus following closure in the late Pliocene.     

Biphasic role of platelet-activating factor in oral mucosal ulcer healing

Platelet-activating factor (PAF) is a potent phospholipid-derived messenger molecule involved in a number of pathological conditions, including mediation of inflammatory cascades associated with wound healing. We investigated prophylactic and therapeutic effects of a specific PAF antagonist, BN52020, on the course of experimentally induced oral mucosal ulcer healing. The prophylactic BN52020 administration produced an accelerated ulcer healing that was characterized by a marked induction in COX-2 enzyme protein expression and the substantial decline in apoptosis, TNF-alpha, and NOS-2 activity. A delay in ulcer healing, however, occurred with the therapeutic BN52020 administration, and this effect of the agent was reflected in a decreased expression of COX-2 protein, higher rate of apoptosis, and the elevated level of TNF-alpha and NOS-2. Our findings implicate PAF requirement in orderly progression of the events involved in oral tissue repair, and suggest that the interference with its actions during healing process results in the suppression of COX-2-derived anti-inflammatory prostaglandins that delay the mucosal repair.