Reproduction mode in the allopolyploid facultatively apomictic hawkweed Hieracium rubrum (Asteraceae, H. subgen. Pilosella)

The versatility of the breeding system in the hybridogenous hexaploid, Hieracium rubrum, was demonstrated in emasculation and crossing experiments. The flow-cytometric ploidy analysis of 1095 seedlings amongst its' progeny enabled the determination and quantification of the reproductive pathway (apospory, haploid parthenogenesis, cross-fertilization of both reduced and unreduced female gametes) responsible for each progeny class. The progeny profiles were stable between two successive years. The percentage of hybrids arisen from crosses with tetraploid sexual H. pilosella was 7.9%. The rate of trihaploids generated by H. rubrum when crossed to H. pilosella was 3.95%, similar to that of pentaploid (n + n) and octoploid (2n + n) hybrids (3.95% and 3.39%). Unreduced pollen from H. pilosella contributed to hybridization much less frequently (0.56% of all progeny), than unreduced eggs of H. rubrum. The increased frequency of trihaploids formed by crossed compared to emasculated plants suggests an interaction between the presence of pollen and the autonomous development of meiotic embryo sacs. Although the environmental conditions (garden compared to unheated glasshouse) influenced the progeny following the emasculation, no corresponding response to environment was detected amongst the autonomously derived progeny following pollination with H. pilosella. However, there was an influence of pollen parent on progeny which varied under the different environmental treatments. The importance of residual sexuality in reproduction of apomicts is evident. The non-maternal progeny was generated with highly variable frequency especially in the glasshouse, with some capitula reaching up to 50%. Having an autonomous endosperm development, Hieracium subgen. Pilosella is almost unique among other aposporous genera, in which such variable reproduction mode is connected with pseudogamy.     

Fluvastatin in the therapy of acute coronary syndrome: Rationale and design of a multicenter, randomized, double-blind, placebo-controlled trial (The FACS Trial)[ISRCTN81331696]

Background

Stent length serves as a predictor of restenosis in use of bare metal stents (BMS). This has been demonstrated in a feasibility study that used a single short BMS implant (<9 mm) in a high proportion of lesions; the study observed a low rate of restenosis.

Methods

We performed a pilot prospective study to investigate in a series of consecutive patients the immediate and long-term effects of implantation of either 1) a single short BMS for all lesions with low probability of restenosis or 2) a drug-eluting stent (DES) for all other lesions.

Results

The 200 patients studied had 236 coronary artery lesions that were treated with short BMS in 168/236 patients (71.2%) and with DES in 68/236 patients (28.8%). Angiographic success was achieved in 230/236 lesions (97.5%) and procedural success in 194/200 patients (97.0%). Restenosis occurred in 15/153 lesions (9.8%) after short BMS, in 3/62 lesions (4.8%) after DES, and in 18/215 of all lesions (8.4%) angiographically controlled after six to eight months. Target vessel revascularization was performed in 16/218 lesion (7.4%).

Conclusion

Most of the coronary artery lesions in this small group of consecutive patients were treated sufficiently with a single BMS implant. This differential approach of treating suitable lesions in medium- to large-sized vessels with a single short BMS device and treating all other lesions with a DES implant resulted in a low incidence of restenosis.     

A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment

A papain-type cysteine endopeptidase with a molecular mass of 35 kDa for the mature enzyme, was purified from germinating castor bean (Ricinus communis L.) endosperm by virtue of its capacity to process the glyoxysomal malate dehydrogenase precursor protein to the mature subunit in vitro (C. Gietl et al., 1997, Plant Physiol 113: 863–871). The cDNA clones from endosperm of germinating seedlings and from developing seeds were isolated and sequence analysis revealed that a very similar or identical peptidase is synthesised in both tissues. Sequencing established a presequence for co-translational targeting into the endoplasmic reticulum, an N-terminal propeptide and a C-terminal KDEL motif for the castor bean cysteine endopeptidase precursor. The 45-kDa pro-enzyme stably present in isolated organelles was enzymatically active. Immunocytochemistry with antibodies raised against the purified cysteine endopeptidase revealed highly specific labelling of ricinosomes, organelles which co-purify with glyoxysomes from germinating Ricinus endosperm. The cysteine endopeptidase from castor bean endosperm, which represents a senescing tissue, is homologous to cysteine endopeptidases from other senescing tissues such as the cotyledons of germinating mung bean (Vigna mungo) and vetch (Vicia sativa), the seed pods of maturing French bean (Phaseolus vulgaris) and the flowers of daylily (Hemerocallis sp.). Received: 20 December 1997 / Accepted: 18 March 1998     

Numerical investigation of inspiratory airflow in a realistic model of the human tracheobronchial airways and a comparison with experimental results

In this article, the results of numerical simulations using computational fluid dynamics (CFD) and a comparison with experiments performed with phase Doppler anemometry are presented. The simulations and experiments were conducted in a realistic model of the human airways, which comprised the throat, trachea and tracheobronchial tree up to the fourth generation. A full inspiration/expiration breathing cycle was used with tidal volumes 0.5 and 1 L, which correspond to a sedentary regime and deep breath, respectively. The length of the entire breathing cycle was 4 s, with inspiration and expiration each lasting 2 s. As a boundary condition for the CFD simulations, experimentally obtained flow rate distribution in 10 terminal airways was used with zero pressure resistance at the throat inlet. CCM+ CFD code (Adapco) was used with an SST k-\(\upomega \) low-Reynolds Number RANS model. The total number of polyhedral control volumes was 2.6 million with a time step of 0.001 s. Comparisons were made at several points in eight cross sections selected according to experiments in the trachea and the left and right bronchi. The results agree well with experiments involving the oscillation (temporal relocation) of flow structures in the majority of the cross sections and individual local positions. Velocity field simulation in several cross sections shows a very unstable flow field, which originates in the tracheal laryngeal jet and propagates far downstream with the formation of separation zones in both left and right airways. The RANS simulation agrees with the experiments in almost all the cross sections and shows unstable local flow structures and a quantitatively acceptable solution for the time-averaged flow field.     

Vaccination of Chickens with SPI1-lon and SPI1-lon-fliC Mutant of Salmonella enterica Serovar Enteritidis

The prevalence of Salmonella enterica serovar Enteritidis is gradually decreasing in poultry flocks in the EU, which may result in the demand for a vaccine that allows for the differentiation of vaccinated flocks from those infected by wild-type S. Enteritidis. In this study, we therefore constructed a (Salmonella Pathogenicity Island 1) SPI1-lon mutant with or without fliC encoding for S. Enteritidis flagellin. The combination of SPI1-lon mutations resulted in attenuated but immunogenic mutant suitable for oral vaccination of poultry. In addition, the vaccination of chickens with the SPI1-lon-fliC mutant enabled the serological differentiation of vaccinated and infected chickens. The absence of fliC therefore did not affect the immunogenicity of the vaccine strain and allowed for serological differentiation of the vaccinated chickens. The SPI1-lon-fliC mutant is therefore a suitable marker vaccine strain for oral vaccination of poultry.     

Phytoplankton growth in relation to network topology: time‐averaged catchment‐scale modelling in a large lowland river

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Identification and Characterization of Broadly Neutralizing Human Monoclonal Antibodies Directed against the E2 Envelope Glycoprotein of Hepatitis C Virus

Nearly all livers transplanted into hepatitis C virus (HCV)-positive patients become infected with HCV, and 10 to 25% of reinfected livers develop cirrhosis within 5 years. Neutralizing monoclonal antibody could be an effective therapy for the prevention of infection in a transplant setting. To pursue this treatment modality, we developed human monoclonal antibodies (HuMAbs) directed against the HCV E2 envelope glycoprotein and assessed the capacity of these HuMAbs to neutralize a broad panel of HCV genotypes. HuMAb antibodies were generated by immunizing transgenic mice containing human antibody genes (HuMAb mice; Medarex Inc.) with soluble E2 envelope glycoprotein derived from a genotype 1a virus (H77). Two HuMAbs, HCV1 and 95-2, were selected for further study based on initial cross-reactivity with soluble E2 glycoproteins derived from genotypes 1a and 1b, as well as neutralization of lentivirus pseudotyped with HCV 1a and 1b envelope glycoproteins. Additionally, HuMAbs HCV1 and 95-2 potently neutralized pseudoviruses from all genotypes tested (1a, 1b, 2b, 3a, and 4a). Epitope mapping with mammalian and bacterially expressed proteins, as well as synthetic peptides, revealed that HuMAbs HCV1 and 95-2 recognize a highly conserved linear epitope spanning amino acids 412 to 423 of the E2 glycoprotein. The capacity to recognize and neutralize a broad range of genotypes, the highly conserved E2 epitope, and the fully human nature of the antibodies make HuMAbs HCV1 and 95-2 excellent candidates for treatment of HCV-positive individuals undergoing liver transplantation.Hepatitis C virus (HCV) is a major cause of liver failure and infects more than 170 million people worldwide. HCV is a member of the Flaviviridae family and contains a 9.6-kb positive-strand RNA genome. The genome is translated into a single polypeptide that is cleaved by viral and cellular proteases into at least nine different proteins. The major HCV surface glycoproteins, E1 and E2, form a noncovalent heterodimer on the virion surface (23) and are believed to mediate viral entry via a complex set of poorly understood interactions with cellular coreceptors, including CD81 (28), claudin-1 (8), occludin (29), scavenger receptor class B type I (30), and others (38). The E2 glycoprotein has been shown to interact directly with receptors (38); currently, no function has been assigned to E1, although it is known to be required for viral infection. These viral glycoproteins provide an obvious target for neutralizing monoclonal antibodies (MAbs).Isolation of potently neutralizing HCV-specific MAbs has been complicated by the lack of an in vitro cell culture system to study the full infection cycle of the virus. Recently, systems have been developed that allow for the generation of infectious viral particles, highlighting the importance of E1 and E2 in viral binding and entry. A novel in vitro infection system employs HCV pseudotyped viral particles (HCVpp) generated from a lentivirus that are devoid of native glycoproteins and engineered to contain HCV glycoproteins E1 and E2 (4, 15). HCVpp specifically infect cell lines derived from human liver cells and can be neutralized by polyclonal and MAbs directed against the HCV envelope glycoproteins.HCVpp have allowed the identification of antibodies that can neutralize HCV infection in cell culture. E1 has proven to be a difficult target for MAb-mediated neutralization, possibly because it appears to have low immunogenicity (32), has no identified binding proteins on the cell surface, and has an undefined role in cell entry. Despite this challenge, two groups have identified HCV neutralizing MAbs directed to E1: these MAbs are H-111, which has moderate neutralizing activity (17), and the recently isolated IGH505 and IGH526, which neutralize numerous HCV genotypes (1a, 1b, 2a, 4a, 5a, and 6a but not 2b and 3a) (22). Although they are predicted to inhibit viral binding or fusion, the mechanism by which these E1-directed MAbs neutralize HCV infection is unclear.A diverse group of mouse anti-E2 antibodies, recognizing both linear and discontinuous epitopes, has been generated. Many of these MAbs showed broad neutralization of multiple HCV genotypes, but not surprisingly, several HCV isolates were refractory to neutralization. In contrast, AP33, a mouse MAb that largely recognizes a highly conserved linear epitope in the N terminus of E2 (amino acids 412 to 423), was identified as a broadly cross-reactive antibody that neutralized strains from all genotypes tested (1a, 1b, 2a, 2b, 3a, 4, 5, and 6), with the exception of one genotype 5 virus (UKN5.14.4; GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY894682","term_id":"58220837","term_text":"AY894682"}}AY894682) (24). Recently, several cross-reactive neutralizing MAbs have been identified that are of human origin and have the capacity to neutralize a significant fraction of the genotypes tested (1, 5, 12, 13, 27, 31) or to neutralize all genotypes tested (16, 20, 25). As with the vast majority of previously described human MAbs (HuMAbs), these MAbs recognize conformation-dependent epitopes of E2. One broadly neutralizing human antibody, AR3B, was tested in a mouse model of infection and showed significant protection from viremia (20). Given the known function of the E2 envelope glycoprotein, the high level of immunogenicity, the surface vulnerability, and the abundance of data pertaining to E2 and HCV neutralization, E2 provides a promising target for the development of fully human neutralizing antibodies.Liver deterioration due to HCV infection is the leading reason for liver transplantation in the United States. Unfortunately, it is highly likely that the transplanted liver will also become infected with HCV, and 10 to 25% of these patients develop cirrhosis within 5 years of transplant (9, 40). Here we describe the characterization of HuMAbs directed against the HCV E2 envelope glycoprotein, generated using transgenic mice. Based on epitope conservation and broad neutralization capacity, HuMAbs HCV1 and 95-2 provide excellent candidates for prevention of graft reinfection of HCV-infected individuals undergoing liver transplantation.     

Backbone cyclic insulin

Backbone cyclic insulin was designed and prepared by reverse proteolysis in partial organic solvent of a single‐chain precursor expressed in yeast. The precursor contains two loops to bridge the two chains of native insulin. The cyclisation method uses Achromobacter lyticus protease and should be generally applicable to proteins with C‐terminal lysine and proximal N‐terminal. The presence of the ring‐closing bond and the native insulin disulfide patterns were documented by LC–MS peptide maps. The cyclic insulin was shown to be inert towards degradation by CPY, but was somewhat labile towards chymotrypsin. Intravenous administration of the cyclic insulin to Wistar rats showed the compounds to be equipotent to HI despite much lower insulin receptor affinity. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of Dimethyl Sulfoxide on Extracellular Enzyme Production by Pleurotus ostreatus

Dimethyl sulfoxide (DMSO) is commonly used as a co-solvent to dissolve poorly water-soluble biologically active agents to assess their biological activities such as for enzyme induction. The question addressed was whether DMSO can be assumed to be an inert co-solvent. The influence of DMSO on the production of extracellular enzymes by Pleurotus ostreatus was investigated. DMSO functioned as either an inducer or a repressor, depending on the enzyme studied. The production of laccase and endo-1,4-β-xylanase increased by 29 and 250%, respectively, in presence of DMSO. However, DMSO repressed the activities of manganese peroxidase, β-glucosidase, β-xylanase, and endo-1,4-β-glucanase by 30, 33, 99 and 16%, respectively. These results raise concerns about the interpretation of bioactivity measurements when DMSO is assumed to function as an inert co-solvent to solubilize water-insoluble molecules. Revisions requested 20 December 2005; Revisions received 6 February 2006