Coupling of retinal, protein, and water dynamics in squid rhodopsin

The light-induced isomerization of the retinal from 11-cis to all-trans triggers changes in the conformation of visual rhodopsins that lead to the formation of the activated state, which is ready to interact with the G protein. To begin to understand how changes in the structure and dynamics of the retinal are transmitted to the protein, we performed molecular dynamics simulations of squid rhodopsin with 11-cis and all-trans retinal, and with two different force fields for describing the retinal molecule. The results indicate that structural rearrangements in the binding pocket, albeit small, propagate toward the cytoplasmic side of the protein, and affect the dynamics of internal water molecules. The sensitivity of the active-site interactions on the retinal force-field parameters highlights the coupling between the retinal molecule and its immediate protein environment.     

A double blind,randomized, placebo controlled study of the efficacy and safety of 5-Loxin<Superscript>®</Superscript>for treatment of osteoarthritis of the knee

Introduction

5-Loxin^® is a novel Boswellia serrata extract enriched with 30% 3-O-acetyl-11-keto-beta-boswellic acid (AKBA), which exhibits potential anti-inflammatory properties by inhibiting the 5-lipoxygenase enzyme. A 90-day, double-blind, randomized, placebo-controlled study was conducted to evaluate the efficacy and safety of 5-Loxin^® in the treatment of osteoarthritis (OA) of the knee.

Methods

Seventy-five OA patients were included in the study. The patients received either 100 mg (n = 25) or 250 mg (n = 25) of 5-Loxin^® daily or a placebo (n = 25) for 90 days. Each patient was evaluated for pain and physical functions by using the standard tools (visual analog scale, Lequesne''s Functional Index, and Western Ontario and McMaster Universities Osteoarthritis Index) at the baseline (day 0), and at days 7, 30, 60 and 90. Additionally, the cartilage degrading enzyme matrix metalloproteinase-3 was also evaluated in synovial fluid from OA patients. Measurement of a battery of biochemical parameters in serum and haematological parameters, and urine analysis were performed to evaluate the safety of 5-Loxin^® in OA patients.

Results

Seventy patients completed the study. At the end of the study, both doses of 5-Loxin^® conferred clinically and statistically significant improvements in pain scores and physical function scores in OA patients. Interestingly, significant improvements in pain score and functional ability were recorded in the treatment group supplemented with 250 mg 5-Loxin^® as early as 7 days after the start of treatment. Corroborating the improvements in pain scores in treatment groups, we also noted significant reduction in synovial fluid matrix metalloproteinase-3. In comparison with placebo, the safety parameters were almost unchanged in the treatment groups.

Conclusion

5-Loxin^® reduces pain and improves physical functioning significantly in OA patients; and it is safe for human consumption. 5-Loxin^® may exert its beneficial effects by controlling inflammatory responses through reducing proinflammatory modulators, and it may improve joint health by reducing the enzymatic degradation of cartilage in OA patients.

Trail Registration

(Clinical trial registration number: ISRCTN05212803.)     

The alpha1.alpha2 network of collagen IV. Reinforced stabilization of the noncollagenous domain-1 by noncovalent forces and the absence of Met-Lys cross-links

Collagen IV networks are present in all metazoa and underlie epithelia as a component of basement membranes. The networks are essential for tissue function and are defective in disease. They are assembled by the oligomerization of triple-helical protomers that are linked end-to-end. At the C terminus, two protomers are linked head-to-head by interactions of their trimeric noncollagenous domains, forming a hexamer structure. This linkage in the alpha1.alpha2 network is stabilized by a putative covalent Met-Lys cross-link between the trimer-trimer interface (Than, M. E., Henrich, S., Huber, R., Ries, A., Mann, K., Kuhn, K., Timpl, R., Bourenkov, G. P., Bartunik, H. D., and Bode, W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 6607-6612) forming a nonreducible dimer that connects the hexamer. In the present study, this cross-link was further investigated by: (a) comparing the 1.5-A resolution crystal structures of the alpha1.alpha2 hexamers from bovine placenta and lens capsule basement membranes, (b) mass spectrometric analysis of monomer and nonreducible dimer subunits of placenta basement membrane hexamers, and (c) hexamer dissociation/re-association studies. The findings rule out the novel Met-Lys cross-link, as well as other covalent cross-links, but establish that the nonreducible dimer is an inherent structural feature of a subpopulation of hexamers. The dimers reflect the reinforced stabilization, by noncovalent forces, of the connection between two adjoining protomers of a network. The reinforcement extends to other types of collagen IV networks, and it underlies the cryptic nature of a B-cell epitope of the alpha3.alpha4.alpha5 hexamer, implicating the stabilization event in the etiology and pathogenesis of Goodpasture autoimmune disease.     

Functional Expression and Properties of Sec14p-Like Protein with Molecular Mass 45 kD from Rat Olfactory Epithelium

cDNA of Sec14p-like water-soluble protein with molecular mass 45 kD from rat olfactory epithelium was expressed in Escherichia coli Rosetta cells. The expression product was purified by a two-step chromatographic procedure on DEAE-Sepharose and Sephacryl S-200. The identity of structural and functional characteristics of the recombinant and native proteins was demonstrated by CD, mass spectrometry, and Western blotting. Using several lipids immobilized on nitrocellulose membranes, it was shown that phosphatidylinositol-3,4,5-triphosphate is the specific ligand for the studied protein.     

Goodpasture autoantibodies unmask cryptic epitopes by selectively dissociating autoantigen complexes lacking structural reinforcement: novel mechanisms for immune privilege and autoimmune pathogenesis

Rapidly progressive glomerulonephritis in Goodpasture disease is mediated by autoantibodies binding to the non-collagenous NC1 domain of alpha3(IV) collagen in the glomerular basement membrane. Goodpasture epitopes in the native autoantigen are cryptic (sequestered) within the NC1 hexamers of the alpha3alpha4alpha5(IV) collagen network. The biochemical mechanism for crypticity and exposure for autoantibody binding is not known. We now report that crypticity is a feature of the quaternary structure of two distinct subsets of alpha3alpha4alpha5(IV) NC1 hexamers: autoantibody-reactive M-hexamers containing only monomer subunits and autoantibody-impenetrable D-hexamers composed of both dimer and monomer subunits. Goodpasture antibodies only breach the quaternary structure of M-hexamers, unmasking the cryptic epitopes, whereas D-hexamers are resistant to autoantibodies under native conditions. The epitopes of D-hexamers are structurally sequestered by dimer reinforcement of the quaternary complex, which represents a new molecular solution for conferring immunologic privilege to a potential autoantigen. Dissociation of non-reinforced M-alpha3alpha4alpha5(IV) hexamers by Goodpasture antibodies is a novel mechanism whereby pathogenic autoantibodies gain access to cryptic B cell epitopes. These findings provide fundamental new insights into immune privilege and the molecular mechanisms underlying the pathogenesis of human autoimmune Goodpasture disease.     

Evolution of anaerobic ciliates from the gastrointestinal tract: phylogenetic analysis of the ribosomal repeat from Nyctotherus ovalis and its relatives

The 18S and 5.8S rDNA genes and the internal transcribed spacers ITS-1 and ITS-2 of ciliates living in the hindgut of frogs, millipedes, and cockroaches were analyzed in order to study the evolution of intestinal protists. All ciliates studied here belong to the genus Nycrotherus. Phylogenetic analysis revealed that these ciliates from a monophyletic group that includes the distantly related anaerobic free-living heterotrichous ciliates Metopus palaeformis and Metopus contortus. The intestinal ciliates from the different vertebrate and invertebrate hosts are clearly divergent at the level of their rDNA repeats. This argues for the antiquity of the associations and a predominantly vertical transmission. This mode of transmission seems to be controlled primarily by the behavior of the host. The different degrees of divergence between ciliates living in different strains of one and the same cockroach species most likely reflect the different geographical origins of the hosts. In addition, host switches must have occurred during the evolution of cockroaches, since identical ciliates were found only in distantly related hosts. These phenomena prevent the reconstruction of potential cospeciation events.      

Expression of lauroyl-acyl carrier protein thioesterase in brassica napus seeds induces pathways for both fatty acid oxidation and biosynthesis and implies a set point for triacylglycerol accumulation

Expression of a California bay lauroyl-acyl carrier protein thioesterase (MCTE) in developing seeds of transgenic oilseed rape alters the fatty acid composition of the mature seed, resulting in up to 60 mol% of laurate in triacylglycerols. In this study, we examined the metabolism of lauric acid and 14C-acetate in developing seeds of oilseed rape that express high levels of MCTE. Lauroyl-CoA oxidase activity but not palmitoyl-CoA oxidase activity was increased several-fold in developing seeds expressing MCTE. In addition, isocitrate lyase and malate synthase activities were six- and 30-fold higher, respectively, in high-laurate developing seeds. Control seeds incorporated 14C-acetate almost entirely into fatty acids, whereas in seeds expressing MCTE, only 50% of the label was recovered in lipids and the remainder was in a range of water-soluble components, including sucrose and malate. Together, these results indicate that the pathways for beta-oxidation and the glyoxylate cycle have been induced in seeds expressing high levels of MCTE. Although a substantial portion of the fatty acid produced in these seeds is recycled to acetyl-CoA and sucrose through the beta-oxidation and glyoxylate cycle pathways, total seed oil is not reduced. How is oil content maintained if lauric acid is inefficiently converted to triacylglycerol? The levels of acyl carrier protein and several enzymes of fatty acid synthesis were increased two- to threefold at midstage development in high-laurate seeds. These results indicate that a coordinate induction of the fatty acid synthesis pathway occurs, presumably to compensate for the lauric acid lost through beta-oxidation or for a shortage of long-chain fatty acids.