Correlation of co-ordinated amino acid changes at the two-domain interface of cysteine proteases with protein stability.

The engineering of a protein containing an alternative local residue packing for a set of side-chains has proven to be a major challenge because compositional, volumetric and steric constraints must be respected. Homologous proteins should provide examples of alternative groups of residues leading to a similar functional result. The functional significance of a pair of co-ordinated changes that are observed in the cysteine proteases family has been investigated by comparing the effect of individual or double changes on secretion, stability and activity of papain. The two changes are not independent. Detrimental effects of single mutations at one of the two positions can be partly suppressed by the co-ordinated mutation that reproduces naturally occurring contacts, indicating that these changes are concerted. Single mutations at the other position produce milder effects, suggesting a pathway for evolution.     

Use of synthetic oligonucleotides in gene isolation and manipulation

Great progress has occurred in the techniques of synthesis of DNA molecules of defined sequences in terms of speed, length of the obtained oligonucleotides, and automation of the processes. Corresponding progress also occurred in the ways of using synthetic DNA in molecular biology and recombinant DNA research. Screening of cloned DNA sequence banks with long, unique oligonucleotides, provided a new approach to isolate the genes for proteins which are present in very small quantity. This technique can present considerable advantages over the more classical use of mixtures of oligonucleotides, in reducing the number of potentially positive clones on a primary screen, and enabling cloning with a minimum of amino acid sequence data. Synthetic oligonucleotides also provide the basis of a set of techniques for site-directed mutagenesis of DNA sequences. This allows the possibility of engineering the structure of particular proteins, and the properties of new variants can be tested by expressing the protein in a heterologous host. An example of this approach is the production of variants of human alpha 1-antitrypsin. A variant where valine replaces the methionine at the active site is equally active as an antielastase, but no longer susceptible to oxidative inactivation. A second variant, where arginine replaces the methionine, now functions as an antithrombin, but no longer inhibits elastase. Total gene synthesis is now feasible for larger and larger genes, and some of the recent strategies of whole gene synthesis are presented.     

Modeling human arthritic diseases in nonhuman primates

Models of rheumatoid arthritis (RA) in laboratory animals are important tools for research into pathogenic mechanisms and the development of effective, safe therapies. Rodent models (rats and mice) have provided important information about the pathogenic mechanisms. However, the evolutionary distance between rodents and humans hampers the translation of scientific principles into effective therapies. The impact of the genetic distance between the species is especially seen with treatments based on biological molecules, which are usually species-specific. The outbred nature and the closer anatomical, genetic, microbiological, physiological, and immunological similarity of nonhuman primates to humans may help to bridge the wide gap between inbred rodent strain models and the heterogeneous RA patient population. Here we review clinical, immunological and pathological aspects of the rhesus monkey model of collagen-induced arthritis, which has emerged as a reproducible model of human RA in nonhuman primates.     

Spatial heterogeneity of daphniid parasitism within lakes

Spatially explicit models show that local interactions of hosts and parasites can strongly influence invasion and persistence of parasites and can create lasting spatial patchiness of parasite distributions. These predictions have been supported by experiments conducted in two-dimensional landscapes. Yet, three-dimensional systems, such as lakes, ponds, and oceans, have received comparatively little attention from epidemiologists. Freshwater zooplankton hosts often aggregate horizontally and vertically in lakes, potentially leading to local host–parasite interactions in one-, two-, or three-dimensions. To evaluate the potential spatial component of daphniid parasitism driven by these local interactions (patchiness), we surveyed vertical and horizontal heterogeneity of pelagic Daphnia infected with multiple microparasites in several north temperate lakes. These surveys uncovered little evidence for persistent vertical patchiness of parasitism, since the prevalence of two parasites showed little consistent trend with depth in four lakes (but more heterogeneity during day than at night). On a horizontal scale of tens of meters, we found little systematic evidence of strong aggregation and spatial patterning of daphniid hosts and parasites. Yet, we observed broad-scale, basin-wide patterns of parasite prevalence. These patterns suggest that nearshore offshore gradients, rather than local-scale interactions, could play a role in governing epidemiology of this open water host–parasite system. Electronic Supplementary Material Supplementary material is available for this article at     

Transmembrane domain V of the endothelin-A receptor is a binding domain of ETA-selective TTA-386-derived photoprobes

On the basis of the structure of TTA-386, a specific antagonist of the endothelin-A receptor subtype (ET(A)), photosensitive analogues were developed to investigate the binding domain of the receptor. Among those, a derivative containing, in position 6, the photoreactive amino acid D- or L-p-benzoyl-phenylalanine showed pharmacological properties very similar to those of TTA-386. Affinity of the probes were also evaluated on transfected CHO cells overexpressing the human ET(A) receptor. Data showed that binding of the radiolabeled peptides were inhibited by ET-1 and BQ-610. Therefore, these photolabile probes were used to label the ET(A) receptor found in CHO cells. Photolabeling produced a ligand-protein complex appearing on SDS-PAGE at around 66 kDa. An excess of ET-1 or BQ-610 completely abolished the formation of the complex showing the selectivity of the photoprobes. Digestions of the [125I-Tyr5, D- or L-Bpa6]TTA-386-ET(A) complex were carried out, and receptor fragments were analyzed to define the region of the receptor where the ligand interacted. Results showed that Endo Lys-C digestion gave a 4.8 kDa fragment corresponding to the Asp256-Lys299 segment, whereas migration after V8 digestion revealed a fragment of 2.9 kDa. Because the fragments of these two digestions must overlap, the latter would be the Trp257-Glu281 stretch. A cleavage with CNBr confirmed the identity of the binding domain by giving a fragment of 3.9 kDa corresponding to Glu249-Met278. Thus, the combined cleavage data strongly suggested that the binding domain of ET(A) includes a portion of the fifth transmembrane domain, between residues Trp257 and Met278.     

Leaf longevity of Oxalis acetosella (Oxalidaceae) in the Catskill Mountains, New York, USA

Leaf habit correlates with multiple physiological traits. Understanding ecophysiology is therefore dependent on knowledge of leaf habit. A variety of leaf habits exists within forest understory plant communities. Oxalis acetosella is one such understory plant and has long been considered a wintergreen, meaning that it keeps a set of leaves for one full year, replacing them with a new set during spring. To assess the leaf habit of O. acetosella and place it into a classification scheme of leaf habits, leaves of four populations of O. acetosella were repeatedly censused for two years in a northern hardwood forest of the Catskill Mountains, New York, USA. New leaves developed and old leaves senesced throughout the year, yielding a continual replacement of leaves and a summer peak in leaf number. Leaves that developed in the fall and winter had longer maximum life spans than leaves that developed during the summer. The name "seasonalgreen" is suggested to describe the continual development, senescence, and presence of leaves and annual peak in leaf number within O. acetosella. The functional significance of this leaf habit in this species and the possibility of its presence in other species deserve further study.     

The relationship between adiposopathy and glucose-insulin homeostasis is not affected by moderate-intensity aerobic training in healthy women with obesity

The contribution of adiposopathy to glucose-insulin homeostasis remains unclear. This longitudinal study examined the potential relationship between the adiponectin/leptin ratio (A/L, a marker of adiposopathy) and insulin resistance (IR: homeostasis model assessment (HOMA)), insulin sensitivity (IS: Matsuda), and insulin response to an oral glucose tolerance test before and after a 16-week walking program, in 29 physically inactive pre- and postmenopausal women with obesity (BMI, 29–35 kg/m²; age, 47–54 years). Anthropometry, body composition, VO₂max, and fasting lipid-lipoprotein and inflammatory profiles were assessed. A/L was unchanged after training (p =?0.15), despite decreased leptin levels (p <?0.05). While the Matsuda index tended to increase (p =?0.07), HOMA decreased (p <?0.05) and fasting insulin was reduced (p <?0.01) but insulin area under the curve (AUC) remained unchanged (p =?0.18) after training. Body fatness and VO₂max were improved (p <?0.05) while triacylglycerols increased and HDL-CHOL levels decreased after training (p <?0.05). At baseline, A/L was positively associated with VO₂max, HDL-CHOL levels, and Matsuda (0.37?< ρ <?0.56; p <?0.05) but negatively with body fatness, HOMA, insulin AUC, IL-6, and hs-CRP levels (??0.41?< ρ <???0.66; p <?0.05). After training, associations with fitness, HOMA, and inflammation were lost. Multiple regression analysis revealed A/L as an independent predictor of IR and IS, before training (partial R² =?0.10 and 0.22), although A/L did not predict the insulin AUC pre- or post-intervention. A significant correlation was found between training-induced changes to A/L and IS (r =?0.38; p <?0.05) but not with IR or insulin AUC. Although changes in the A/L ratio could not explain improvements to glucose-insulin homeostasis indices following training, a relationship with insulin sensitivity was revealed in healthy women with obesity.     

Identification and Crystallization of a Protease-Resistant Core of Calnexin That Retains Biological Activity

Calnexin is a molecular chaperone that facilitates folding of glycoproteins in the endoplasmic reticulum (ER). The cloned lumenal domain of canine calnexin, cnxΔTMC, retains its biological activity without the transmembrane and cytosolic region. For the purpose of structure determination we generated a crystallizable core by mild proteolysis and identified its termini by N-terminal sequencing and molecular mass determination. A truncated gene was cloned accordingly. Its product, cnxΔN25C15, was purified to apparent homogeneity and crystallized. This truncated variant remains biologically active as shown by its binding to monoglucosylated oligosaccharides and functional interaction with ERp57. A heavy atom derivative was identified.