Dihydrofurocoumarin glucosides from Angelica archangelica and Angelica silvestris

A water-soluble root extract of Angelica archangelica subsp. litoralis afforded, in addition to adenosine, coniferin and the two known dihydrofurocoumarin glycosides, apterin and 1′-O-β-d-glycopyranosyl-(S)-marmesin (marmesinin), two new dihydrofuranocoumarin glycosides, 1′-O-β-d-glucopyranosyl-(2S, 3R)-3-hydroxymarmesin, and 2′-β-d-glucopyranosyloxymarmesin. For the latter a 2S-configuration was demonstrated, the stereochemistry at position 1′ remaining undefined. Roots of A. silvestris similarly afforded 1′-O-β-d-glucopyranosyl-(2S, 3R)-3-hydroxymarmesin. By correlation with the aglycone 2S,3R)-3-hydroxymarmesin obtained in this work, the absolute configurations (2S,3R) were established for the known dihydrofurocoumarin diesters smirniorin and smirnioridin.     

IgG subclass antibodies to human and bacterial HSP60 are not associated with disease activity and progression over time in axial spondyloarthritis

Introduction

Spondyloarthritis (SpA), an interrelated group of rheumatic diseases, has been suggested to be triggered by bacterial infections prior to the development of an autoimmune response that causes inflammation of the spinal and peripheral joints. Because human heat shock protein 60 (HSP60), recently renamed HSPD1, and bacterial HSP60 are highly homologous, immunological cross-reactivity has been proposed as a mechanism of disease initiation. However, previous investigations of the humoral immune response to HSP60 in SpA patients have lacked determination of immunoglobulin G (IgG) subclasses and patient follow-up. In this study, we have focused on these parameters in a cohort of axial SpA patients with a well-established set of clinical characteristics, including MRI changes and human leukocyte antigen B27.

Methods

IgG subclass antibodies (IgG1, IgG2, IgG3 and IgG4) against recombinant HSP60 of three reactive arthritis-related bacteria; human HSP60; and the microorganisms Chlamydia trachomatis and C. pneumoniae were determined by ELISA. Serum samples collected from 2004 to 2006 and in 2010 and 2011 from 39 axial SpA patients were analyzed and compared with samples from 39 healthy controls. The Mann-Whitney U test and Wilcoxon matched pairs test were used to compare the antibody levels in different and paired groups, respectively. P < 0.01 was considered significant. The Spearman nonparametric correlation was used to determine correlation between antibody levels and between antibody levels and the disease parameters.

Results

Elevated levels of IgG1 and IgG3 to human HSP60 and IgG1 to HSP60 of Salmonella enterica Enteritidis were observed in SpA patients compared with healthy controls at both time points. The antibody levels were almost constant over time for IgG1, whereas high levels of IgG3 to human HSP60 tended to decrease over time. The antibody response to human HSP60 was predominantly of the IgG3 subclass, and patients with high levels of IgG3 to this antigen had low levels of IgG1, indicating an inverse association. Different IgG subclasses were produced against bacterial and human HSP60 in the same serum sample, IgG1 and IgG3, respectively, indicating that there was no cross-reaction.

Conclusions

A significant association was observed between axial SpA and the presence of IgG1/IgG3 antibodies to human HSP60 and of IgG1 to S. enterica Enteritidis and C. trachomatis. Generation of antibodies to human HSP60 was independent of the presence of antibodies to bacterial HSP60. No association was observed between clinical and MRI changes with antibodies over time. Altogether, such antibodies do not reflect the disease activity in these patients.This study has been approved by the Regional Research Ethics Committee of Central Jutland, Denmark. Trial registration numbers: 20050046 and 20100083     

Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage.     

Regional differences in degree of resilin cross-linking in the desert locust, Schistocerca gregaria

Various cuticular regions from the desert locust, Schistocerca gregaria, were quantitatively analyzed for two cross-linking amino acids, dityrosine and trityrosine, characteristic constituents of the rubberlike cuticular protein, resilin. These amino acids were found in all regions of cuticle investigated, but in widely varying amounts. In fully mature adult locusts the largest amounts of di- and trityrosine were obtained from the prealar arms and wing-hinges, structures possessing long-range elasticity and being involved in energy storage in the flight system. In structures where deformations tend to occur more slowly, such as the clypeo-labral springs and tracheae, di- and trityrosine are less abundant. In sclerotized cuticle from femur and tibia, as well as in cornea and in the highly stretchable intersegmental membranes of mature females, they are only found in trace amounts and are probably unrelated to elasticity. The trityrosine-to-dityrosine ratio in the various cuticular regions vary from nearly equal amounts of the two amino acids to about ten times more dityrosine than trityrosine, indicating that the regions differ in degree of cross-linking; the tracheal wall is the material with the highest trityrosine-to-dityrosine ratio. In some cuticular regions the ratio increases during maturation from newly moulted (teneral) adults to reproductively active locusts; the most pronounced increase was observed for the wing-hinges, and only a small increase was observed for the abdominal tergal plates. In most cuticular regions in fifth instar locust nymphs the contents of di- and trityrosine corresponded to the contents measured for the adult cuticular regions, but only trace amounts of the two amino acids were obtained from the region of the nymphal wing base which corresponds to the wing-hinge containing cuticular region in adult locusts.     

Identification and characterization of a novel Chlamydia trachomatis reticulate body protein

The genome of the obligate intracellular bacterium Chlamydia trachomatis comprises 894 genes predicted by computer-based analysis. As part of a large-scale proteome analysis of C. trachomatis, a small abundant protein encoded by a previously unrecognized novel 204-bp open reading frame was identified by tandem mass spectrometry. No homology of this protein was observed to proteins from other organisms. The protein was conserved in C. trachomatis but not found in Chlamydia pneumoniae. Using proteomics, we show that the expression of the protein is initiated at the middle of the developmental cycle. The protein is rapidly degraded and is only present in reticulate or intermediate bodies, suggesting a possible function in the intracellular stage of C. trachomatis development. We have termed the protein '7-kDa reticulate body protein'.     

Rev inhibition strongly affects intracellular distribution of human immunodeficiency virus type 1 RNAs

To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.     

Mammalian ykt6 is a neuronal SNARE targeted to a specialized compartment by its profilin-like amino terminal domain

SNAREs are required for specific membrane fusion throughout the endomembrane system. Here we report the characterization of rat ykt6, a prenylated SNARE selectively expressed in brain neurons. Immunofluorescence microscopy in neuronal and neuroendocrine cell lines revealed that membrane-associated ykt6 did not colocalize significantly with any conventional markers of endosomes, lysosomes, or the secretory pathway. However, ykt6-containing membranes displayed very minor overlaps with lysosomes and dense-core secretory granules and were similar to lysosomes in buoyant density. Thus, ykt6 appears to be specialized for the trafficking of a unique membrane compartment, perhaps related to lysosomes, involved in aspects of neuronal function. Targeting of this SNARE to the ykt6 compartment was mediated by its profilin-like amino-terminal domain, even in the absence of protein prenylation. Although several other R-SNAREs contain related amino-terminal domains, only the ykt6 version was able to confer the specialized localization. Rat ykt6, which contains an arginine in its SNARE motif zero-layer, was found to behave like other R-SNAREs in its SNARE assembly properties. Interestingly, cytosolic ykt6, constituting more than half of the total cellular pool, appeared to be conformationally inactive for SNARE complex assembly, perhaps indicative of a regulatory mechanism that prevents promiscuous and potentially deleterious SNARE interactions.     

Membrane proteins PmpG and PmpH are major constituents of Chlamydia trachomatis L2 outer membrane complex

The outer membrane complex of Chlamydia is involved in the initial adherence and ingestion of Chlamydia by the host cell. In order to identify novel proteins in the outer membrane of Chlamydia trachomatis L2, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. By silver staining of the protein profile, a major protein doublet of 100-110 kDa was detected. In-gel tryptic digestion and matrix-assisted laser desorption/ionization mass spectrometry identified these proteins as the putative outer membrane proteins PmpG and PmpH.     

Mosaic structure of intragenic repetitive elements in histone H1-like protein Hc2 varies within serovars of <Emphasis Type="Italic">Chlamydia trachomatis</Emphasis>

Background  

The histone-like protein Hc2 binds DNA in Chlamydia trachomatis and is known to vary in size between 165 and 237 amino acids, which is caused by different numbers of lysine-rich pentamers. A more complex structure was seen in this study when sequences from 378 specimens covering the hctB gene, which encodes Hc2, were compared.