A Preliminary Investigation on the Chemical Composition of the Cell Surface of Five Enteropathogenic Escherichia coli Serotypes

The cell surfaces of five enteropathogenic Escherichia coli serotypes (O111:H2; O111:H12; O125:H9; O119:H6; O26:H11) were assayed by chemical methods, lectin agglutination tests and spectroscopy associated to transmission electron microscopy. Results of lectin agglutination assays showed that all strains reacted with mannosebinding lectins. Strains belonging to serotype O125:H9 also agglutinated with lectins which recognize galactose and Nacetylgalactosamine residues. The bacterial cells were treated with 0.01M phosphate buffered saline (pH 7.0) at 100oC for 2 hr and the extracts were submitted to precipitation and fractionated by Cetavlon. Phosphate, total sugar and protein contents were determined. Gas liquid chomatography-mass spectrometry analysis of alditol acetates showed the presence of galactose, mannose, fucose, glucose and traces of ribose. Spectroscopic analysis of intact cells showed the presence of a capsule-like structure which was not totally preserved after extraction. Some cells were still surrounded by an amorphous capsular-like material after polysaccharide extraction.

Studies on Cercariae from Kuwait Bay. XI. Description and surface topography of Cercaria kuwaitae XI sp.n. (Digenea: Echinostomatidae)

A new echinostome cercaria, Cercaria kuwaitae XI sp.n., from the prosobranch gastropod Cerithidea cingulata (Gmelin) from Kuwait Bay is described. The new cercaria is characterized by 23 collar spines and primary excretory tubules with distinct diverticula. The cercaria encysts in the snail host and is similar to those of Acanthoparyphium sp. The surface topography of the redia, cercaria and metacercarial cyst wall is studied by scanning electron microscopy. This is the first echinostome cercaria to be recorded in a gastropod from the Arabian Gulf region.

Rabies in tamarins (Callithrix jacchus) in the State of Ceará, Brazil, a distinct viral variant?

Presently, the State of Ceará reports the largest percentage of human rabies cases originated from wild animals in Brazil, transmitted by the principal simian species, the tamarin (Callithrix jacchus), found in various locations throughout the State, but concentrated along the coast. Epidemiological studies indicated that possibly the same virus caused the deaths in humans and non-human primates. This rabies virus seem to be different from all other identified so far.

Determinació de la regió del genoma o gens implicats en la síntesi del polièster extracel•lular present en la variant llisa de Mycobacterium vaccae mitjançant transformació

Estudi realitzat a partir d’una estada a la Universidad de Zaragoza, Espanya, entre novembre del 2007 i abril del 2008. Mycobacterium vaccae és un micobacteri ambiental de creixement ràpid molt estudiat pel seu interès com a possible ús immunoterapéutic en el tractament de la tuberculosis i altres malalties. M.vaccae a l’igual que altres micobacteris presenta dues morfologies colonials: llisa i rugosa. M.vaccae ATCC15483T té originàriament una morfologia llisa. Quant aquest es cultiva en medi sòlid a 30ºC apareixen espontàniament variants rugoses estables que no reverteixen a llises. El motiu pel qual aquest procés té lloc no es coneix, encara que s’ha descrit en Mycobacterium smegmatis i en Mycobacterium avium que els lípids de la paret cel•lular es troben involucrats en aquest canvi de morfologia colonial. L’anàlisi dels contingut en lípids i glicolípids de la paret cel•lular de les dos variants morfològiques de M.vaccae, ens ha indicat que les soques llises presenten un compost extracel•lular que no es troba en les rugoses i que mitjançant l’anàlisi estructural d’aquest compost ha sigut identificat com un polièster extracel•lular de cadena llarga. El present estudi s’ha centrat en determinar els gens implicats en la síntesis d’aquest compost. Per a realitzar aquest anàlisi genètic s’ha construit una llibreria de mutants per transposició de la soca llisa de M. vaccae mitjançant un plàsmid ts/sac i un transposó. S’han obtingut colònies de morfologia rugosa on el plàsmid s’ha insertat en la zona del genoma que codifica per aquest compost extracel•lular. Aquests nous mutants s’han analitzat mitjançant tècniques moleculars (PCR, Southern y seqüenciació). A mès, s’ha construit una llibreria genòmica amb DNA de la soca llisa en plàsmids replicatius de micobacteris derivats de pAL5000 i s’ha transformat la soca rugosa seleccionant per a un fenotip llis estudiant els gens que complementen.

Detailed DEM analysis of a rockslide scar to characterize the basal sliding surface of active rockslides

The basal sliding surfaces in large rockslides are often composed of several surfaces and possess a complex geometry. The exact morphology and location in three dimensions of the sliding surface remains generally unknown, in spite of extensive field and subsurface investigations, such as those at the Åknes rockslide (western Norway). This knowledge is crucial for volume estimations, failure mechanisms, and numerical slope stability modeling. This paper focuses on the geomorphologic characterization of the basal sliding surface of a postglacial rockslide scar in the vicinity of Åknes. This scar displays a stepped basal sliding surface formed by dip slopes of the gneiss foliation linked together by steeply dipping fractures. A detailed characterization of the rockslide scar by means of high-resolution digital elevation models permits statistical parameters of dip angle, spacing, persistence, and roughness of foliation surfaces and step fractures to be obtained. The characteristics are used for stochastic simulations of stepped basal sliding surfaces at the Åknes rockslide. These findings are compared with previous models based on geophysical investigations. This study discusses the investigation of rockslide scars and rock outcrops for a better understanding of potential rockslides. This work identifies possible basal sliding surface locations, which is a valuable input for volume estimates, design and location of monitoring instrumentation, and numerical slope stability modeling.

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Atomic force and electron microscopic-based study of sarcolemmal surface of living cardiomyocytes unveils unexpected mitochondrial shift in heart failure.

Loss of T-tubules (TT), sarcolemmal invaginations of cardiomyocytes (CMs), was recently identified as a general heart failure (HF) hallmark. However, whether TT per se or the overall sarcolemma is altered during HF process is still unknown. In this study, we directly examined sarcolemmal surface topography and physical properties using Atomic Force Microscopy (AFM) in living CMs from healthy and failing mice hearts. We confirmed the presence of highly organized crests and hollows along myofilaments in isolated healthy CMs. Sarcolemma topography was tightly correlated with elasticity, with crests stiffer than hollows and related to the presence of few packed subsarcolemmal mitochondria (SSM) as evidenced by electron microscopy. Three days after myocardial infarction (MI), CMs already exhibit an overall sarcolemma disorganization with general loss of crests topography thus becoming smooth and correlating with a decreased elasticity while interfibrillar mitochondria (IFM), myofilaments alignment and TT network were unaltered. End-stage post-ischemic condition (15days post-MI) exacerbates overall sarcolemma disorganization with, in addition to general loss of crest/hollow periodicity, a significant increase of cell surface stiffness. Strikingly, electron microscopy revealed the total depletion of SSM while some IFM heaps could be visualized beneath the membrane. Accordingly, mitochondrial Ca(2+) studies showed a heterogeneous pattern between SSM and IFM in healthy CMs which disappeared in HF. In vitro, formamide-induced sarcolemmal stress on healthy CMs phenocopied post-ischemic kinetics abnormalities and revealed initial SSM death and crest/hollow disorganization followed by IFM later disarray which moved toward the cell surface and structured heaps correlating with TT loss. This study demonstrates that the loss of crest/hollow organization of CM surface in HF occurs early and precedes disruption of the TT network. It also highlights a general stiffness increased of the CM surface most likely related to atypical IFM heaps while SSM died during HF process. Overall, these results indicate that initial sarcolemmal stress leading to SSM death could underlie subsequent TT disarray and HF setting.

Benign pineal cysts in children with bilateral retinoblastoma: a new variant of trilateral retinoblastoma?

PURPOSE: Patients with hereditary retinoblastoma (Rb) develop in 4%-8% a malignant midline tumor called trilateral Rb (TRb). We report in this study on benign pineal cysts observed in patients investigated for TRb. PATIENTS AND METHODS: Between September 1990 and December 2001, 172 patients were screened for TRb. Ninty-five had bilateral, 77 unilateral disease. The median age at diagnosis of Rb was 7 months (range 1-26). Treatment included enucleation, local treatment with cryotherapy or photocoagulation, first-line chemotherapy (CT), thermo-chemotherapy (TCT), Ruthenium plaque, and, rarely, external beam radiation (EBR). RESULTS: TRb was found in 5/95 patients (5.3%) with bilateral disease. Interestingly, five other patients (5.3%) presented a pineal cyst on magnetic resonance imaging (MRI). No cysts were recorded in the 77 patients with unilateral disease. This difference was statistically significant (P < 0.05). The median age at diagnosis of the pineal cyst was 26 months (range 16-80), much younger than reported in literature for healthy children. Four of five patients with TRb died of the disease, while all the patients with pineal cysts remained stable and asymptomatic during a median follow-up of 41 months (range 37-54). CONCLUSIONS: This report describes benign cystic lesions of the pineal gland in patients with hereditary Rb, suggesting a benign variant of TRb. Underlying possible pathogenetic mechanisms are discussed.

Relation between Hepatitis B Carrier Status and Antibody against Synthetic Plasmodium falciparum Erythrocyte Surface (pf155 - RESA) Antigen

A survey on Plasmodium infection was carried out in gold mine camps located in the Brazilian Amazon. Antibody against P. falciparum ring-infected erythrocyte surface antigen (RESA) was quantified by an enzyme-immunoassay in order to assess P. falciparum exposure. Hepatitis B, a common infection in this area, was also investigated by serologic markers. Among 520 sampled subjects, 517 (99.4%) admitted previous symptomatic malaria, 106 (20.4%) had positive thick smears for malaria, 82.9% had HBV markers, and 7.1% were HBsAg positive. Anti-RESA titers was significantly lower in HBV carriers than in people with resolved HBV infection suggesting that the anti-RESA immune response could be supressed by HBV carrier status. Moreover, immunedeficient responses to both infections may take place in some subjects causing concomitant lower anti-RESA response and incapacity to clear HBV.

Effect of the Contents and Form of Rabies Glycoprotein on the Potency of Rabies Vaccination in Cattle

One of the methods used for controlling cattle rabies in Brazil consists of vaccination. Sometimes, however, rabies occurs in cattle supposedly protected. Since rabies vaccine batches are officially controlled by tests performed on laboratory animals, it is questionable whether the minimal mandatory requirements really correspond to immunogenicity in the target species. We have analyzed the association among potencies of rabies vaccines tested by the NIH test, the contents and form (free-soluble or virus-attached) of rabies glycoprotein (G) in the vaccine batches, and the virus-neutralizing antibodies (VNA) titers elicited in cattle. No correlation was found between G contents in the vaccine batches and the NIH values, whatever the presentation of G. There was no correlation either between NIH values and VNA titers elicited in cattle. There was, however, a positive correlation (r = 0.8681; p = 0.0001) between the amounts of virion-attached G present in the vaccine batches and VNA elicited in cattle. This was not observed when the same analysis was performed with total-glycoprotein or free-soluble glycoprotein. The study demonstrated that NIH values can not predict the effect of the immunogen in cattle. On the other hand, the quantification of virus-attached rabies glycoprotein has a strong correlation with VNA elicited in cattle.

Interaction of Diocleinae Lectins with Glycoproteins Based in Surface Plasmon Resonance

Interaction of glucose/mannose-binding lectins in solution with immobilized glycoproteins was followed in real time using surface plasmon resonance technology. The lectins which share many biochemical and structural features could be clearly differentiated in terms of their specificity for complex glycoconjugates. The most prominent interaction of the lectins with PHA-E comparing with soybean agglutinin, both glycoproteins exhibiting high mannose oligosaccharides, suggests that the whole structure of the glycoproteins themselves, may interfere in affinity. These findings also support the hypothesis that minor amino acid replacements in the primary sequence of the lectins might be responsible for their divergence in fine specificity and biological activities. This is the first report using surface plasmon resonance technology that evidences differences of Diocleinae lectins in respect their fine glycan-specificity.

Surface ultrastructure of third-instar Megaselia scalaris (Diptera: Phoridae)

We describe some ultrastructure of the third-instar Megaselia scalaris (Diptera: Phoridae) using scanning electron microscopy, with the cephalic segment, anterior spiracle and posterior spiracle being emphasized. This study provides the taxonomic information of this larval species, which may be useful to differentiate from other closely-related species.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.