A novel fibrillar structure in cultured cells detected by a monoclonal antibody

Using a monoclonal antibody, we have detected an antigen present in a unique fibrillar structure in the cytoplasm of cultured cells by immunofluorescence. These structures have been identified by transmission electron microscopy and ultrastructural immunocytochemistry as large single paracrystalline arrays of individual filaments morphologically similar to intermediate filaments. The antibody detects these structures in fibroblastic and epithelioid cultured cell lines of mouse, rat, bovine, and human origin but not of avian origin. Only a small percentage of the cells in a culture contains these structures; each cell usually contains only one, although two or more have been observed in a single cell. The structures are elongated vermiform arrays of filaments in the cytoplasm (approximately 0.5 X 3 microns) which have a thread-like or toroidal appearance. Because of this shape, we have named the putative antigen recognized by this antibody "nematin." Double-label experiments showed that these structures had no relationship to tubulin or vimentin. Immunocytochemical localization in human tissues revealed a high concentration of a reactive antigen in the stratum granulosum of skin and in what probably are neuroglial cells in the central nervous system. This monoclonal antibody may detect a novel intermediate filament protein and/or a shared determinant of different intermediate filament proteins.     

Cost-Minimization Analysis Favours Intravenous Ferric Carboxymaltose over Ferric Sucrose for the Ambulatory Treatment of Severe Iron Deficiency

Objective

Intravenous iron is widely used to treat iron deficiency in day-care units. Ferric carboxymaltose (FCM) allows administration of larger iron doses than iron sucrose (IS) in each infusion (1000 mg vs. 200 mg). As FCM reduces the number of infusions required but is more expensive, we performed a cost-minimization analysis to compare the cost impact of the two drugs.

Materials and Methods

The number of infusions and the iron dose of 111 consecutive patients who received intravenous iron at a gastrointestinal diseases day-care unit from 8/2007 to 7/2008 were retrospectively obtained. Costs of intravenous iron drugs were obtained from the Spanish regulatory agencies. The accounting department of the Hospital determined hospital direct and indirect costs for outpatient iron infusion. Non-hospital direct costs were calculated on the basis of patient interviews. In the pharmacoeconomic model, base case mean costs per patient were calculated for administering 1000 mg of iron per infusion using FCM or 200 mg using IS. Sensitivity analysis and Monte Carlo simulation were performed.

Results

Under baseline assumptions, the estimated cost of iron infusion per patient and year was €304 for IS and €274 for FCM, a difference of €30 in favour of FCM. Adding non-hospital direct costs to the model increased the difference to €67 (€354 for IS vs. €287 for FCM). A Monte Carlo simulation taking into account non-hospital direct costs favoured the use of FCM in 97% of simulations.

Conclusion

In this pharmacoeconomic analysis, FCM infusion reduced the costs of iron infusion at a gastrointestinal day-care unit.     

Protein expression profiles in the epidermis of cyclooxygenase-2 transgenic mice by 2-dimensional gel electrophoresis and mass spectrometry

Exposure of murine skin to tumor-promoting agents such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) causes up-regulation of cyclooxygenase-2 (COX-2) and increased prostaglandin (PG) synthesis. Pharmacological inhibition of COX-2 significantly reduces skin tumor development. However, we previously demonstrated that K14.COX-2 transgenic (TG) mice that overexpressed COX-2 in the epidermis were unexpectedly resistant to tumor development under the classical 7,12-dimethylbenz[a]anthracene-TPA protocol. In the present study, we employed a proteomic approach of 2-dimensional gel electrophoresis (2-DE) and mass spectrometry to profile differentially expressed proteins in the epidermis of K14.COX-2 TG and wild-type control mice. Various 2-DE approaches were used to identify the maximum number of differentially expressed proteins: 20 for untreated samples, 3 for acetone-treated samples, and 22 for TPA-treated samples. These proteins include 14-3-3 sigma, numerous actin fragments, actin filament related proteins cofilin-1 and destrin, galectin-3, galectin-7, prohibitin, S100A6, S100A9, and many others. The differential expression of galectin-3, galectin-7, S100A9 was validated by Western blot analysis and/or immunohistochemical analysis. The current data suggest that some of the differentially expressed proteins might increase apoptosis and cell cycle arrest, which, in turn, may provide insight into the role of COX-2 in skin tumorigenesis.     

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.     

The mycoestrogen zearalenone in Portuguese flowing waters and its potential environmental impact

This study provides the first environmental risk assessment (ERA) for zearalenone (ZEN), the only known mycoestrogen, based on a broad-scale investigation on its occurrence in rivers and creeks from Portugal. Water sample filtration and immunoaffinity columns (IAC) clean-up followed by liquid chromatograph with tandem mass spectrometry (LC/MS ⁿ ) provided an analytical method with good analytical performance. ZEN levels were determined for seven Portuguese rivers and one creek, during two different seasons, in a total of 38 samples collected upstream wastewater treatment plants (WWTPs). Overall, 23.7% were contaminated with ZEN at levels ranging between 5.6 and 82.6 ng/L. The highest concentration was observed during spring, although no statistically significant differences were observed between spring and autumn sampling campaigns. The potential ecotoxicological risk from ZEN to different trophic levels of aquatic organisms was evaluated by means of risk quotients (RQs) calculation. Although all the RQs obtained were lower than 1, our results confirm that ZEN is a relatively frequent contaminant in flowing waters in Portugal and might contribute to the overall estrogenic activity in the environment.     

Interphase cytogenetics on paraffin-embedded sections of ovary for detection of genomic constitution in a patient with Turner's syndrome and chromosomal mosaicism

The difficulty of detecting sex chromosome mosaicism cytogenetically hinders the finding of an acceptable explanation for phenotypic-genotypic discrepancy amongst those patients. Fluorescence in situ hybridization (FISH) permits the genomic identification of patients with mosaic karyotypes in interphase nuclei by utilising an X chromosome-specific DNA probe (interphase cytogenetics). We evaluated the efficiency of interphase cytogenetics in the detection of the genomic constitution of the ovary from a patient with Turner's syndrome having mosaicism (46,XX/45,X0) previously established by blood lymphocyte karyotyping. We used a biotin-labelled alphoid repetitive sequence, pBAMX5, specific for the centromeric region of the human X chromosome. Although examination of ovarian sections and blood lymphocytes by FISH showed the presence of both 46,XX and 45,X0 cell lines, the genomic constitution of the germ cells/oocytes in ovarian primordial follicles was shown to be normal (46,XX). Our results (1) show the high applicability of interphase cytogenetics on paraffin sections, (2) indicate the possibility of genomic screening of different tissues that are otherwise not amenable to routine cytogenetic investigation and (3) offer a reliable methodological approach to defining accurate by the percentage of abnormal karyotypes in mosaicism of different organs and non-dividing tissues.     

PbsP,a cell wall‐anchored protein that binds plasminogen to promote hematogenous dissemination of group B Streptococcus

Streptococcus agalactiae (Group B Streptococcus or GBS) is a leading cause of invasive infections in neonates whose virulence is dependent on its ability to interact with cells and host components. We here characterized a surface protein with a critical function in GBS pathophysiology. This adhesin, designated PbsP, possesses two Streptococcal Surface Repeat domains, a methionine and lysine‐rich region, and a LPXTG cell wall‐anchoring motif. PbsP mediates plasminogen (Plg) binding both in vitro and in vivo and we showed that cell surface‐bound Plg can be activated into plasmin by tissue plasminogen activator to increase the bacterial extracellular proteolytic activity. Absence of PbsP results in a decreased bacterial transmigration across brain endothelial cells and impaired virulence in a murine model of infection. PbsP is conserved among the main GBS lineages and is a major plasminogen adhesin in non‐CC17 GBS strains. Importantly, immunization of mice with recombinant PbsP confers protective immunity. Our results indicate that GBS have evolved different strategies to recruit Plg which indicates that the ability to acquire cell surface proteolytic activity is essential for the invasiveness of this bacterium.     

Neurobehavioral Abnormalities in the HIV-1 Transgenic Rat Do Not Correspond to Neuronal Hypometabolism on 18F-FDG-PET

Motor and behavioral abnormalities are common presentations among individuals with HIV-1 associated neurocognitive disorders (HAND). We investigated whether longitudinal motor and behavioral performance in the HIV-1 transgenic rat (Tg), a commonly used neuro-HIV model, corresponded to in vivo neuronal death/dysfunction, by using rotarod and open field testing in parallel to [¹⁸F] 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). We demonstrated that age-matched non-Tg wild type (WT) rats outperformed the HIV-1 Tg rats at most time points on rotarod testing. Habituation to rotarod occurred at 8 weeks of age (fifth weekly testing session) in the WT rats but it never occurred in the Tg rats, suggesting deficits in motor learning. Similarly, in open field testing, WT rats outperformed the Tg rats at most time points, suggesting defective exploratory/motor behavior and increased emotionality in the Tg rat. Despite the neurobehavioral abnormalities, there were no concomitant deficits in ¹⁸F-FDG uptake in Tg rats on PET compared to age-matched WT rats and no significant longitudinal loss of FDG uptake in either group. The negative PET findings were confirmed using ¹⁴C- Deoxy-D-glucose autoradiography in 32 week-old Tg and WT rats. We believe that the neuropathology in the HIV-1 Tg rat is more likely a consequence of neuronal dysfunction rather than overt neurodegeneration/neuronal cell death, similar to what is seen in HIV-positive patients in the post-ART era.