Three-dimensional structure of human serum albumin.

The three-dimensional structure of human serum albumin has been solved at 6.0 angstrom (A) resolution by the method of multiple isomorphous replacement. Crystals were grown from solutions of polyethylene glycol in the infrequently observed space group P42(1)2 (unit cell constants a = b = 186.5 +/- 0.5 A and c = 81.0 +/- 0.5 A) and diffracted x-rays to lattice d-spacings of less than 2.9 A. The electron density maps are of high quality and revealed the structure as a predominantly alpha-helical globin protein in which the course of the polypeptide can be traced. The binding loci of several organic compounds have been determined.

Gene expression profiling of tolerant barley in response to Diuraphis noxia (Hemiptera: Aphididae) feeding.

Aphids are, arguably, the single most damaging group of agricultural insect pests throughout the world. Plant tolerance, which is a plant response to an insect pest, is viewed as an excellent management strategy. Developing testable hypotheses based on genome-wide and more focused methods will help in understanding the molecular underpinnings of plant tolerance to aphid herbivory. As a first step in this process, we undertook transcript profiling with Affymetrix GeneChip Barley Genome arrays using RNA extracted from tissues of tolerant and susceptible genotypes collected at three hours, three days and six days after Diuraphis noxia introduction. Acquired data were compared to identify changes unique to the tolerant barley at each harvest date. Transcript abundance of 4086 genes was differentially changed over the three harvest dates in tolerant and susceptible barley in response to D. noxia feeding. Across the three harvest dates, the greatest number of genes was differentially expressed in both barleys at three days after aphid introduction. A total of 909 genes showed significant levels of change in the tolerant barley in response to D. noxia feeding as compared to susceptible plants infested with aphids. Many of these genes could be assigned to specific metabolic categories, including several associated with plant defense and scavenging of reactive oxygen species (ROS). Interestingly, two peroxidase genes, designated HvPRXA1 and HvPRXA2, were up-regulated to a greater degree in response to D. noxia feeding on tolerant barley plants, indicating that specific peroxidases could be important for the tolerance process. These findings suggest that the ability to elevate and sustain levels of ROS-scavenging enzymes could play an important role in the tolerant response.

The mechanics of landing when stepping down in unilateral lower-limb amputees.

BACKGROUND: The ability to successfully negotiate stairs and steps is an important factor for functional independence. While work has been undertaken to understand the biomechanics of gait in lower-limb amputees, little is known about how amputees negotiate stairs and steps. This study aimed to determine the mechanics of landing in unilateral lower-limb amputees when stepping down to a new level. A secondary aim was to assess the effects of using a shank-mounted shock-absorbing device (Tele-Torsion Pylon) on the mechanics of landing. METHODS: Ten unilateral amputees (five transfemoral and five transtibial) and eight able-bodied controls performed single steps down to a new level (73 and 219 mm). Trials were repeated in amputees with the Tele-Torsion Pylon active and inactive. The mechanics of landing were evaluated by analysing peak limb longitudinal force, maximal limb shortening, lower extremity stiffness, and knee joint angular displacement during the initial contact period, and limb and ankle angle at the instant of ground-contact. Data were collected using a Vicon 3D motion analysis system and two force platforms. FINDINGS: Amputees landed on a straightened and near vertical limb. This limb position was maintained in transfemoral amputees, whereas in transtibial amputees knee flexion occurred. As a result lower extremity stiffness was significantly greater in transfemoral amputees compared to transtibial amputees and able-bodied controls (P<0.001). The Tele-Torsion Pylon had little effect on the mechanics of landing in transtibial amputees, but brought about a reduction in lower extremity stiffness in transfemoral amputees (P<0.05). INTERPRETATION: Amputees used a stepping strategy that ensured the direction of the ground reaction force vector was kept anterior of the knee joint centre. Using a Tele-Torsion Pylon may improve the mechanics of landing during downward stepping in transfemoral amputees.

The role of the surface amorphous layer of articular cartilage in joint lubrication.

Articular cartilage is a complex soft tissue that performs multiple functions in the joint. In particular, the amorphous layer that covers the surface of articular cartilage is thought to play some role in lubrication. This study aimed to characterize the surface amorphous layer (SAL) using a variety of techniques, including environmental scanning electron microscopy, transmission electron microscopy, white light interferometry, and biochemical analysis of its composition. Friction tests were conducted to investigate the role of the SAL in lubrication. A protocol to remove successfully the SAL without damaging the underlying cartilage was developed and the material removed from healthy cartilage was found to contain approximately equal quantities of glycosaminoglycan (GAG), protein, and lipid. Cartilage-on-cartilage friction tests were conducted on fresh, healthy cartilage with and without the SAL, under both dynamic and static operating conditions. Removal of the SAL was not found to change the friction coefficient. However, subsequent staining of specimens indicated that the SAL had replenished during the test following loading. The replenished SAL was characterized and found to contain lipids and sulphated GAGs with undetectable protein. This study revealed experimental evidence of surface layer replenishment in articular cartilage. It was postulated that the surface layer regeneration mechanism was purely mechanical and associated with movement of GAGs and lipids through the cartilage matrix during deformation, since the experimental set-up did not contain any means of biochemical activation.

The gait initiation process in unilateral lower-limb amputees when stepping up and stepping down to a new level.

BACKGROUND: Unilateral lower-limb amputees lead with their intact limb when stepping up and with their prosthesis when stepping down; the gait initiation process for the different stepping directions has not previously been investigated. METHODS: Ten unilateral amputees (5 transfemoral and 5 transtibial) and 8 able-bodied controls performed single steps up and single steps down to a new level (73 and 219 mm). Duration, a-p and m-l centre of mass and centre of pressure peak displacements and centre of mass peak velocity of the anticipatory postural adjustment and step execution phase were evaluated for each stepping direction by analysing data collected using a Vicon 3D motion analysis system. FINDINGS: There were significant differences (in the phase duration, peak a-p and m-l centre of pressure displacement and peak a-p and m-l centre of mass velocity at heel-off and at foot-contact) between both amputee sub-groups and controls (P<0.05), but not between amputee sub-groups. These group differences were mainly a result of amputees adopting a different gait initiation strategy for each stepping direction. INTERPRETATION: Findings indicate the gait initiation process utilised by lower-limb amputees was dependent on the direction of stepping and more particularly by which limb the amputee led with; this suggests that the balance and postural control of gait initiation is not governed by a fixed motor program, and thus that becoming an amputee will require time and training to develop alternative neuromuscular control and coordination strategies. These findings should be considered when developing training/rehabilitation programs.

A nodule-specific gene family from Alnus glutinosa encodes glycine- and histidine-rich proteins expressed in the early stages of actinorhizal nodule development.

Two cDNAs representing different members (agNt84 and ag164) of a gene family encoding glycine- and histidine-rich proteins have been isolated from cDNA libraries from Alnus glutinosa root nodules. Expression of the corresponding genes could only be detected in nodules. With in situ hybridization, the expression in nodules was found to occur in young, infected cells of the prefixation zone (zone 2). The encoded proteins contain putative signal peptides for targeting to the endomembrane system, sharing sequence similarity with signal peptides from plant glycine-rich proteins, among them nodulin 24, a nodule-specific protein from soybean. This similarity suggests that, analogous to nodulin-24, proteins encoded by agNt84/ag164 may be located at the interface between the host plant membrane and the matrix surrounding the endosymbiont. The 3' untranslated regions of the cDNAs contain unusual poly(AT)n stretches that may play a role in the regulation of RNA stability. The protein encoded by agNt84 cDNA was expressed in Escherichia coli as a fusion with maltose-binding protein, and was shown to have the ability to bind to a nickel-chelating resin, indicating that it may function as a metal-binding protein.

Protein crystal growth aboard the U.S. space shuttle flights STS-31 and STS-32.

NASA: The first microgravity protein crystal growth experiments were performed on Spacelab I by Littke and John. These experiments indicated that the space grown crystals, which were obtained using a liquid-liquid diffusion system, were larger than crystals obtained by the same experimental system on earth. Subsequent experiments were performed by other investigators on a series of space shuttle missions from 1985 through 1990. The results from two of these shuttle flights (STS-26 and STS-29) have been described previously. The results from these missions indicated that the microgravity grown crystals for a number of different proteins were larger, displayed more uniform morphologies, and yielded diffraction data to significantly higher resolutions than the best crystals of these proteins grown on earth. This paper presents the results obtained from shuttle flight STS-32 (flown in January, 1990) and preliminary results from the most recent shuttle flight, STS-31 (flown in April, 1990).^ieng

Static and dynamic nanomechanical properties of human skin tissue using atomic force microscopy: effect of scarring in the upper dermis.

Following traumatic injury, skin has the capacity to repair itself through a complex cascade of biochemical change. The dermis, which contains a load-bearing collagenous network structure, is remodelled over a long period of time, affecting its mechanical behaviour. This study examines the nanomechanical and viscoelastic properties of the upper dermis from human skin that includes both healthy intact and scarred tissue. Extensive nanoindentation analysis shows that the dermal scar tissue exhibits stiffer behaviour than the healthy intact skin. The scar skin also shows weaker viscoelastic creep and capability to dissipate energy at physiologically relevant frequencies than the adjacent intact skin. These results are discussed in conjunction with a visual change in the orientation of collagenous fibrils in the scarred dermis compared with normal dermis, as shown by atomic force microscopy imaging.

Disordered to ordered folding in the regulation of diphtheria toxin repressor activity.

Understanding how metal binding regulates the activity of the diphtheria toxin repressor protein (DtxR) requires information about the structure in solution. We have prepared a DtxR mutant construct with three additional N-terminal residues, Gly-Ser-His-DtxR(Cys-102 --> Asp), that retains metal-binding capabilities, but remains monomeric in solution and does not bind DNA under conditions that effect dimerization and DNA binding in the functional DtxR(Cys-102 --> Asp) construct. Although the interaction properties of this inactive mutant in solution are very different from that of active repressors, crystallization imposes the same dimeric structure as observed in all crystal forms of the active repressor with and without bound metal. Our solution NMR analyses of active and inactive metal-free diphtheria toxin repressors demonstrate that whereas the C-terminal one-third of the protein is well ordered, the N-terminal two-thirds exhibits conformational flexibility and exists as an ensemble of structural substates with undefined tertiary structure. Fluorescence binding assays with 1-anilino naphthalene-8-sulfonic acid (ANS) confirm that the highly alpha-helical N-terminal two-thirds of the apoprotein is molten globule-like in solution. Binding of divalent metal cations induces a substantial conformational reorganization to a more ordered state, as evidenced by changes in the NMR spectra and ANS binding. The evident disorder to order transition upon binding of metal in solution is in contrast to the minor conformational changes seen comparing apo- and holo-DtxR crystal structures. Disordered to ordered folding appears to be a general mechanism for regulating specific recognition in protein action and this mechanism provides a plausible explanation for how metal binding controls the DtxR repressor activity.

Improved diffraction of antithrombin crystals grown in microgravity.

Crystals of antithrombin were grown both on earth and in microgravity aboard US Space Shuttle Flight STS-67. The quality of crystals grown in both environments was highly variable and many could not be indexed. The microgravity crystals, however, generally diffracted better, as demonstrated by a novel procedure that estimates the resolution of the Bragg scatter from single diffraction images, without requiring knowledge of the cell dimensions of the crystal. Whereas the best earth-grown crystals never diffracted beyond 3 angstroms resolution, the best microgravity crystal diffracted to 2.6 angstroms. The improvement, demonstrated here by a comparison of 23 microgravity and 12 earth-grown crystals, is attributed to better ordered crystal growth in microgravity, although other factors may have contributed also.

X-ray and primary structure of horse serum albumin (Equus caballus) at 0.27-nm resolution.

The amino-acid sequence and three-dimensional structure of equine serum albumin have been determined. The amino-acid sequence was deduced from cDNA isolated from equine liver. Comparisons of the primary structure of equine serum albumin with human serum albumin and bovine serum albumin reveal 76.1% and 73.9% sequence identity, respectively. The three-dimensional structure was determined crystallographically by the molecular-replacement method using molecular coordinates from the previously determined structure of human serum albumin, to a resolution of 0.27 nm. In accordance with the primary structure, the three-dimensional structures are highly conserved. There is a root-mean-square difference between alpha-carbons of the two structures of 0.201 nm. The association constants (Ka) for the binding of 2,3,5-triiodobenzoic acid were determined by ultrafiltration methods for equine and human serum albumins to be approximately 10(4) M-1 and 10(5) M-1, respectively. Crystallographic studies of equine serum albumin reveal two binding sites for 2,3,5-triiodobenzoic acid identical with those previously reported for human serum albumin which are located within subdomains in IIA and IIIA. Details and comparisons of the binding chemistry are discussed.

Solution structure and peptide binding studies of the C-terminal src homology 3-like domain of the diphtheria toxin repressor protein.

The diphtheria toxin repressor (DtxR) is the best-characterized member of a family of homologous proteins that regulate iron uptake and virulence gene expression in the Gram-positive bacteria. DtxR contains two domains that are separated by a short, unstructured linker. The N-terminal domain is structurally well-defined and is responsible for Fe2+ binding, dimerization, and DNA binding. The C-terminal domain adopts a fold similar to eukaryotic Src homology 3 domains, but the functional role of the C-terminal domain in repressor activity is unknown. The solution structure of the C-terminal domain, consisting of residues N130-L226 plus a 13-residue N-terminal extension, has been determined by using NMR spectroscopy. Residues before A147 are highly mobile and adopt a random coil conformation, but residues A147-L226 form a single structured domain consisting of five beta-strands and three helices arranged into a partially orthogonal, two-sheet beta-barrel, similar to the structure observed in the crystalline Co2+ complex of full-length DtxR. Chemical shift perturbation studies demonstrate that a proline-rich peptide corresponding to residues R125-G139 of intact DtxR binds to the C-terminal domain in a pocket formed by residues in beta-strands 2, 3, and 5, and helix 3. Binding of the proline-rich peptide by the C-terminal domain of DtxR presents an example of peptide binding by a prokaryotic Src homology 3-like protein. The results of this study, combined with previous x-ray studies of intact DtxR, provide insights into a possible biological function of the C-terminal domain in regulating repressor activity.