Time-resolved photointermediate changes in rhodopsin glutamic acid 181 mutants

The role of glutamic acid 181 in the bovine rhodopsin retinylidene chromophore pocket was studied by expressing E181 mutants in COS cells and measuring, as a function of time, the absorbance changes produced after excitation of lauryl maltoside pigment suspensions with 7 ns laser pulses. All mutants studied except E181D showed accelerated decay of bathorhodopsin compared to wild type. Even for E181D, an anomalously large blue shift was observed in the absorption spectrum of the bathorhodopsin decay product, BSI. These observations support the idea that E181 plays a significant role in the earliest stages of receptor activation. E181 mutations have a pronounced effect on the decay of the lumirhodopsin photointermediate, primarily affecting the size of the red shift that occurs in the lumirhodopsin I to lumirhodopsin II transition that takes place on the 10 micros time scale after wild-type photoexcitation. While the spectral change that occurs in the lumirhodopsin I to lumirhodopsin II transition in wild-type rhodopsin is very small ( approximately 2 nm), making it difficult to detect, it is larger in E181D ( approximately 6 nm), making it evident even in the lower signal-to-noise ratio measurements possible with rhodopsin mutants. The change seen is even larger for the E181F mutant where significant amounts of a deprotonated Schiff base intermediate are produced with the 10 micros time constant of lumirhodopsin II formation. The E181Q mutant shows lumirhodopsin decay more similar to wild-type behavior, and no lumirhodopsin I to lumirhodopsin II transition can be resolved. The addition of chloride ion to E181Q increases the lumirhodopsin I-lumirhodopsin II spectral shift and slows the deprotonation of the Schiff base. The latter result is consistent with the idea that a negative charge at position 181 contributes to protonated Schiff base stability in the later intermediates.     

Prevalent structural disorder in E. coli and S. cerevisiae proteomes

Intrinsically unstructured proteins, which exist without a well-defined 3D structure, carry out essential functions and occur with high frequency, as predicted for genomes. The generality of this phenomenon, however, is questioned by the uncertainty of what fraction of genomes actually encodes for expressed proteins. Here, we used two independent bioinformatic predictors, PONDR VSL1, and IUPred, to demonstrate that disorder prevails in the recently characterized proteomes and essential proteins of E. coli and S. cerevisiae, at levels exceeding that estimated from the genomes. The S. cerevisiae proteome contains three times as much disorder as that of E. coli, with 50-60% of proteins containing at least one long (>30 residues) disordered segment. This evolutionary advance can be explained by the observation that disorder is much higher in Gene Ontology categories related to regulatory, as opposed to metabolic, functions, and also in categories unique to yeast. Thus, protein disorder is a widespread and functionally important phenomenon, which needs to be characterized in full detail for understanding complex organisms at the molecular level.     

Identification and validation of mannose 6-phosphate glycoproteins in human plasma reveal a wide range of lysosomal and non-lysosomal proteins

Acid hydrolase activities are normally confined within the cell to the lysosome, a membrane-delimited cytoplasmic organelle primarily responsible for the degradation of macromolecules. However, lysosomal proteins are also present in human plasma, and a proportion of these retain mannose 6-phosphate (Man-6-P), a modification on N-linked glycans that is recognized by Man-6-P receptors (MPRs) that normally direct the targeting of these proteins to the lysosome. In this study, we purified the Man-6-P glycoforms of proteins from human plasma by affinity chromatography on immobilized MPRs and characterized this subproteome by two-dimensional gel electrophoresis and by tandem mass spectrometry. As expected, we identified many known and potential candidate lysosomal proteins. In addition, we also identified a number of abundant classical plasma proteins that were retained even after two consecutive rounds of affinity purification. Given their abundance in plasma, we initially considered these proteins to be likely contaminants, but a mass spectrometric study of Man-6-phosphorylation sites using MPR-purified glycopeptides revealed that some proportion of these classical plasma proteins contained the Man-6-P modification. We propose that these glycoproteins are phosphorylated at low levels by the lysosomal enzyme phosphotransferase, but their high abundance results in detection of Man-6-P glycoforms in plasma. These results may provide useful insights into the molecular processes underlying Man-6-phosphorylation and highlight circumstances under which the presence of Man-6-P may not be indicative of lysosomal function. In addition, characterization of the plasma Man-6-P glycoproteome should facilitate development of mass spectrometry-based tools for the diagnosis of lysosomal storage diseases and for investigating the involvement of Man-6-P-containing glycoproteins in more widespread human diseases and their potential utility as biomarkers.     

A Single-Dose Influenza A (H5N1) Vaccine Safe and Immunogenic in Adult and Elderly Patients: an Approach to Pandemic Vaccine Development

With the ongoing pandemic of influenza A (H1N1) virus infection and the threat of high fatality rates for recent human cases of infection with highly pathogenic H5N1 strains, there has been considerable interest in developing pandemic vaccines. Here we report a randomized multicenter dose-finding clinical trial of a whole-virion, inactivated, adjuvanted H5N1 vaccine in adult and elderly volunteers. Four hundred eighty patients were randomly assigned to receive one or two doses of 3.5 μg of the vaccine or one dose of 6 or 12 μg. The subjects were monitored for safety analysis, and serum samples were obtained to assess immunogenicity by hemagglutination inhibition and microneutralization tests. The subjects developed antibody responses against the influenza A (H5N1) virus. Single doses of ≥6 μg fulfilled EU and U.S. licensing criteria for interpandemic and pandemic influenza vaccines. Except for occasional injection site pain, malaise, and fever, no adverse events were observed. We found that the present vaccine is safe and immunogenic in healthy adult and elderly subjects and requires low doses and, unlike any other H5N1 vaccines, only one injection to trigger immune responses which comply with licensing criteria. A vaccine using the same methods as those described in this report, but based on a wild-type swine-origin 2009 (H1N1) influenza A virus isolate from the United States (supplied by the CDC), has been developed and is currently being tested by our group.With the ongoing pandemic of influenza A (H1N1) virus infection and the threat of high fatality rates for recent human cases of infection with highly pathogenic H5N1 strains, there has been considerable interest in developing pandemic influenza vaccines.With new cases continuing to emerge, as of June 2009, the avian influenza A (H5N1) virus subtype has caused 433 human infections in 15 countries, as confirmed by the World Health Organization (WHO), resulting in severe illness with a high fatality rate (30). Human-to-human spread has been strongly suspected and even evidenced by statistical methods (22, 33). With new human infections continuing to develop, this subtype continues to represent a potential source of an influenza pandemic (33).Mass vaccination is the most effective approach to reduce illness and death from pandemic influenza. Therefore, vaccine producers are currently developing and assessing vaccines against H5N1 viruses (2, 14, 31). The effects of split, subvirion, and whole-virion H5N1 vaccines have been tested, with various immunogenicity results (31). Three whole-virion vaccines have been tested so far, two of which required two-dose regimens (4, 14), while a one-dose regimen with the present vaccine was found to be immunogenic in 146 adult subjects (24).The objective of the present study was to determine the safety and immunogenicity of an inactivated whole-virion vaccine against influenza A/Vietnam/1194/2004, using multiple dosing and administration schedules, for adult and elderly subjects. To date, this is the only influenza pandemic prototype vaccine trial examining single-dose regimens in elderly patients.     

Malignant fibrous histiocytoma of the glans penis: a case report

BACKGROUND: Malignant fibrous histiocytoma has been regarded as the most common sarcoma of older adults. However, recent opinion regards pleomorphic malignant fibrous histiocytoma as an undifferentiated high grade pleomorphic sarcoma not otherwise classifiable utilizing current techniques available in surgical pathology. Notwithstanding controversy regarding its nomenclature, malignant fibrous histiocytoma involving the penis is exceedingly rare, with only 4 cases previously described, to our knowledge. CASE: An uncircumcised 73-year-old male presented with a painless, granular, partially necrotic lesion beneath the penile foreskin. There was no history of sexually transmitted disease, constitutional symptoms or dysuria. Examination of penile shaft, testicles, spermatic cord and inguinal lymph nodes were unremarkable. Biopsy revealed a markedly pleomorphic sarcoma. Subsequent, partial penectomy revealed the same lesion with an adjacent area of squamous cell carcinoma in situ. CONCLUSION: Malignant fibrous histiocytoma remains a diagnosis of exclusion. The investigation requires extensive tumor sampling in search of areas of differentiation and a complete battery of immunohistochemical markers. Therapeutically important entities in the differential diagnosis that must be ruled out include other poorly differentiated sarcomas, sarcomatoid squamous cell carcinoma and desmoplastic melanoma.     

Helicobacter pylori-induced downregulation of the secretory leukocyte protease inhibitor (SLPI) in gastric epithelial cell lines and its functional relevance for H. pylori-mediated diseases

The secretory leukocyte protease inhibitor (SLPI) exerts antiproteolytic activity towards serine proteases, as well as anti-microbial and anti-inflammatory effects. To investigate its role in H. pylori-mediated diseases, SLPI expression was analyzed by RT-PCR, ELISA and immunohistochemistry in clinical samples and gastric tumor cell lines. Determination of the mucosal SLPI levels in 126 patients confirmed the previously reported downregulation of SLPI in H. pylori-infected patients. The lower SLPI levels in antral biopsies of H. pylori-positive subjects were associated with a 30-fold increase (p<0.01) in neutrophil elastase activity, and a significant negative correlation was demonstrated for both parameters (R=-0.63, p=0.0002). Eradication of the bacterium in a long-term study (5-7 years) led to a recovery of mucosal SLPI expression. In vitro experiments using four gastric tumor cell lines (AGS, MKN-28, MKN-45, NCI-N87) generally confirmed the clinical findings. While the co-incubation of these cell lines with H. pylori resulted in lower or unchanged SLPI protein levels, the corresponding SLPI mRNA amounts were upregulated by up to five-fold (p=0.006) in all cell lines. Taken together, these results indicate that the reduction in antral SLPI levels in H. pylori-infected subjects has a functional relevance for gastric mucosa and the H. pylori-induced decrease in SLPI is primarily regulated at the posttranslational level.     

Normal mode analysis of the horseradish peroxidase collective motions: correlation with spectroscopically observed heme distortions

Horseradish peroxidase C is a class III peroxidase whose structure is stabilized by the presence of two endogenous calcium atoms. Calcium removal has been shown to decrease the enzymatic activity of the enzyme and significantly affect the spectroscopically detectable properties of the heme, such as the spin state of the iron, heme normal modes, and distortions from planarity. In this work, we report on normal mode analysis (NMA) performed on models subjected to 2 ns of molecular dynamics simulations to describe the effect of calcium removal on protein collective motions and to investigate the correlation between active site (heme) and protein matrix fluctuations. We show that in the native peroxidase model, heme fluctuations are correlated to matrix fluctuations while they are not in the calcium-depleted model.     

Interactions of mitochondria with the actin cytoskeleton

Interactions between mitochondria and the cytoskeleton are essential for normal mitochondrial morphology, motility and distribution. While microtubules and their motors have been established as important factors for mitochondrial transport, emerging evidence indicates that mitochondria interact with the actin cytoskeleton in many cell types. In certain fungi, such as the budding yeast and Aspergillus, or in plant cells mitochondrial motility is largely actin-based. Even in systems such as neurons, where microtubules are the primary means of long-distance mitochondrial transport, the actin cytoskeleton is required for short-distance mitochondrial movements and for immobilization of the organelle at the cell cortex. The actin cytoskeleton is also involved in the immobilization of mitochondria at the cortex in cultured tobacco cells and in budding yeast. While the exact nature of these immobilizations is not known, they may be important for retaining mitochondria at sites of high ATP utilization or at other cellular locations where they are needed. Recent findings also indicate that mutations in actin or actin-binding proteins can influence mitochondrial pathways leading to cell death. Thus, mitochondria-actin interactions contribute to apoptosis.     

Common Buzzards wintering strategies as an effect of weather conditions and geographic barriers

AimMigration is a constantly changing adaptation due to the climate condition evolution. The struggle for surviving during harsh winter season is different across Europe, being more complex toward the inner parts of the continent. The current approach explores the Common Buzzard number variation during the cold season and the climatic predictors of birds of prey wintering movements in relation to the possible influences of the Carpathian Mountains, which may act as a geographical barrier providing shelter from cold air outbreak from north and northeast of the continent.LocationRomania (45°N25°E).TaxonBirds of Prey.MethodsWe applied a GLMM to investigate the relation between continental and local climatic factors with the number of Common Buzzard observations in two regions. The first region is located inside the Carpathian Arch and the other one outside, east of this large mountains chain.ResultsThe Common Buzzard numbers wintering Eastern from the Carpathian Mountains are highly influenced by AO (Z = 2.87, p < .05%), while those wintering western are influenced by NAO (Z = 2.17, p < .05%). This is the first proof of separating influences for biodiversity of AO and NAO at continental scale, outlining the influence limit placed over the Eastern Carpathian Mountains.Main conclusionsThe Carpathian Mountains act like a geographic barrier, separating the wintering Common Buzzard populations from both sides of the mountain range. While the high number of individuals in Moldova is related to their eastern and northeastern Europe origins, in Transylvania the large number of individuals observed is related to the more sheltered characteristics of the region attracting individuals from central Europe. Also, since Transylvania region is well sheltered during cold air outbreak, it represents a more favorable region for wintering. From this point of view, we can consider that the Carpathian Mountains are a geographic barrier for wintering birds of prey.