Hygrophorones A-G: fungicidal cyclopentenones from Hygrophorus species (Basidiomycetes)

Twenty new 5-(hydroxyalkyl)-2-cyclopentenone derivatives (hygrophorones) could be isolated from Hygrophorus latitabundus, H. olivaceoalbus, H. persoonii, and H. pustulatus. Their fungicidal activity was exemplarily tested. The hygrophorones have structural similarities to the antibiotic pentenomycin. Chemically, hygrophorones are 2-cyclopentenones with hydroxy or acetoxy substituents at C-4 and/or C-5. An odd-numbered 1' oxidized alkyl chain (C(11), C(13), C(15), or C(17)) is attached at C-5. In addition, from H. persoonii the new gamma-butyrolactone derivative [5-(E)-2-hydroxytetradexylidene-5H-furan-2-one] could be isolated. Some hygrophorones are responsible for the color reaction of the stipes of these fungi upon treatment with potassium hydroxide solution. Structural elucidations are based on 1D ((1)H, (13)C) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectroscopic analyses as well as HR-FT-ICR-MS investigations.     

Effect of partial outlet obstruction on nitrotyrosine content and distribution within the rabbit bladder

Purpose: Evidence indicates that free radicals are etiological factors in obstructive bladder disease. However, it is not clear which species of reactive oxygen or nitrogen species mediate the damage. The current studies were designed to determine if partial outlet obstruction in rabbits results in the generation of nitrotyrosine (NT). Materials and methods: Sixteen rabbits were separated into four groups of four. The rabbits in groups 1 and 2 underwent sham operation while rabbits in groups 3 and 4 underwent partial outlet obstruction. The rabbits in groups 1 and 3 were evaluated after 1 week of obstruction and the rabbits in groups 2 and 4 were evaluated after 2 weeks of obstruction. A separate group of four controls were evaluated simultaneously with the sham and obstructed rabbits. Four rabbits from each group were evaluated after 1 and 2 weeks of obstruction. Four control rabbits were also evaluated. Isolated strips were evaluated for contractile responses and NT content of the mucosa and muscle were quantitated by Western blot analysis. Results: (1) The mucosa contains both 42 and 62 kD proteins exhibiting a strong nitrotyrosine signal; the muscle presents a signal only at 62 kD. (2) The sham operations had no effect on nitrotyrosine distribution or content. (3) The nitrotyrosine of both mucosal proteins and the muscle protein are increased in the 1 week obstructed bladder; whereas, only the 62 kD signal is increased in the two week obstructed bladder mucosa. (4) The contractile response to FS are reduced to a significantly greater degree than the responses to carbachol, KCl, or ATP. Conclusions: These studies clearly demonstrated that partial outlet obstruction in rabbits results in significant increases in nitrotyrosine within the bladder and may contribute to the contractile dysfunctions mediated by partial outlet obstruction. (Mol Cell Biochem 276: 143–148, 2005)     

A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC

The most sensitive screening technique for genes that predispose patients for particular cancers is direct sequencing. However, sequencing of complex genes is technically demanding, costly and time-consuming. We have tested alternate screening techniques to find a fast sensitive method for detecting alterations of DNA in the large BRCA1 gene prior to sequencing. Sequencing of this gene is particularly arduous because it lacks clearly defined mutation sites. The single-strand conformation polymorphism (SSCP) technique is one of the most frequently used pre-screening methods but its sensitivity and efficiency is not completely satisfying. We have compared the SSCP assay with a newly developed technique called denaturing high performance liquid chromatography (DHPLC) to screen the BRCA1gene. We studied 23 patients at high risk for early onset breast and ovarian cancer and four controls. In these patients, a total of 113 fragments with sequence variations in the BRCA1 gene could be identified. The DHPLC technique resolved 100% of the DNA alterations that were observed in cycle sequencing. In contrast, mutation analysis by SSCP accounted for 94% of the detected variations. In addition, DHPLC screening allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the DNA molecules. Polymorphisms that were present in our samples could be predicted by means of DHPLC testing independently of sequence analysis. We conclude that DHPLC is a highly potent screening method for genetic analyses. It is highly sensitive, efficient and economical and can be automated. Electronic Publication     

Mechanism of incomplete mitral leaflet coaptation--interaction of chordal restraint and changes in mitral leaflet coaptation geometry. Insight from in vitro validation of the premise of force equilibrium

Clinically observed incomplete mitral leaflet coaptation was reproduced in vitro by altering the balance of the chordal tethering and chordal coapting force components. Mitral leaflet coaptation geometry was distorted by changes of the spatial relations between the papillary muscles and the mitral valve as well as hemodynamics. Mitral leaflet malalignment was accentuated by a redistribution of the chordal tethering and coapting force components. For the overall assessment of systolic mitral leaflet configuration in functional mitral regurgitation it is important to consider the interaction between chordal restraint and an altered mitral leaflet coaptation geometry.     

The glycan shield of HIV is predominantly oligomannose independently of production system or viral clade

The N-linked oligomannose glycans of HIV gp120 are a target for both microbicide and vaccine design. The extent of cross-clade conservation of HIV oligomannose glycans is therefore a critical consideration for the development of HIV prophylaxes. We measured the oligomannose content of virion-associated gp120 from primary virus from PBMCs for a range of viral isolates and showed cross-clade elevation (62-79%) of these glycans relative to recombinant, monomeric gp120 (～30%). We also confirmed that pseudoviral production systems can give rise to notably elevated gp120 oligomannose levels (～98%), compared to gp120 derived from a single-plasmid viral system using the HIV(LAI) backbone (56%). This study highlights differences in glycosylation between virion-associated and recombinant gp120.     

Class II MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition.

T cell expression of class II MHC/peptide complexes may be important for maintenance of peripheral self-tolerance, but mechanisms underlying the genesis of class II MHC glycoproteins on T cells are not well resolved. T cell APC (T-APC) used herein were transformed IL-2-dependent clones that constitutively synthesized class II MHC glycoproteins. When pulsed with myelin basic protein (MBP) and injected into Lewis rats, these T-APC reduced the severity of experimental autoimmune encephalomyelitis, whereas unpulsed T-APC were without activity. Normal MBP-reactive clones cultured without APC did not express class II MHC even when activated with mitogens and exposed to IFN-gamma. However, during a 4-h culture with T-APC or macrophage APC, recognition of MBP or mitogenic activation of responder T cells elicited high levels of I-A and I-E expression on responders. Acquisition of class II MHC glycoproteins by responders was resistant to the protein synthesis inhibitor cycloheximide, coincided with transfer of a PKH26 lipophilic dye from APC to responders, and resulted in the expression of syngeneic and allogeneic MHC glycoproteins on responders. Unlike rested I-A- T cell clones, rat thymic and splenic T cells expressed readily detectable levels of class II MHC glycoproteins. When preactivated with mitogens, naive T cells acquired APC-derived MHC class II molecules and other membrane-associated proteins when cultured with xenogeneic APC in the absence of Ag. In conclusion, this study provides evidence that APC donate membrane-bound peptide/MHC complexes to Ag-specific T cell responders by a mechanism associated with the induction of tolerance.