Pericellular heparan sulfate proteoglycans: Role in regulating the biosynthetic response of nucleus pulposus cells to osmotic loading.

Background: Daily physiologic loading causes fluctuations in hydration of the intervertebral disc (IVD); thus, the embedded cells experience cyclic alterations to their osmotic environment. These osmotic fluctuations have been described as a mechanism linking mechanics and biology, and have previously been shown to promote biosynthesis in chondrocytes. However, this phenomenon has yet to be fully interrogated in the IVD.

Template for robust soft-body crawling with reflex-triggered gripping.

Caterpillars show a remarkable ability to get around in complex environments (e.g. tree branches).

Characterization of the Staphylococcus aureus rRNA methyltransferase encoded by orfX, the gene containing the staphylococcal chromosome Cassette mec (SCCmec) insertion site.

The gene orfX is conserved among all staphylococci, and its complete sequence is maintained upon insertion of the staphylococcal chromosome cassette mec (SCCmec) genomic island, containing the gene encoding resistance to β-lactam antibiotics (mecA), into its C terminus. The function of OrfX has not been determined. We show that OrfX was constitutively produced during growth, that orfX could be inactivated without altering bacterial growth, and that insertion of SCCmec did not alter gene expression.

Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis.

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis.

Control of substrate specificity by a single active site residue of the KsgA methyltransferase.

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine.

The aminoglycoside resistance methyltransferases from the ArmA/Rmt family operate late in the 30S ribosomal biogenesis pathway.

Bacterial resistance to 4,6-type aminoglycoside antibiotics, which target the ribosome, has been traced to the ArmA/RmtA family of rRNA methyltransferases. These plasmid-encoded enzymes transfer a methyl group from S-adenosyl-L-methionine to N7 of the buried G1405 in the aminoglycoside binding site of 16S rRNA of the 30S ribosomal subunit. ArmA methylates mature 30S subunits but not 16S rRNA, 50S, or 70S ribosomal subunits or isolated Helix 44 of the 30S subunit.

Modeling locomotion of a soft-bodied arthropod using inverse dynamics.

Most bio-inspired robots have been based on animals with jointed, stiff skeletons. There is now an increasing interest in mimicking the robust performance of animals in natural environments by incorporating compliant materials into the locomotory system. However, the mechanics of moving, highly conformable structures are particularly difficult to predict.

Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly.

KsgA is an rRNA methyltransferase important to the process of small subunit biogenesis in bacteria. It is ubiquitously found in all life including archaea and eukarya, where the enzyme is referred to as Dim1. Despite the emergence of considerable data addressing KsgA function over the last several years, details pertaining to RNA recognition are limited, in part because the most accessible substrate for in vitro studies of KsgA is the 900000 Da 30S ribosomal subunit.

Binding of adenosine-based ligands to the MjDim1 rRNA methyltransferase: implications for reaction mechanism and drug design.

The KsgA/Dim1 family of proteins is intimately involved in ribosome biogenesis in all organisms. These enzymes share the common function of dimethylating two adenosine residues near the 3'-OH end of the small subunit rRNA; orthologs in the three kingdoms, along with eukaryotic organelles, have evolved additional functions in rRNA processing, ribosome assembly, and, surprisingly, transcription in mitochondria. The methyltransferase reaction is intriguingly elaborate.

Evolution of a magnetic-based biomolecular detection system.

Amongst the plethora of affinity biosensor systems based on biomolecular recognition and labeling assays, magnetic labeling and detection has emerged as a promising approach. Magnetic labels can be detected by a wide range of non-invasive methods, are physically and chemically stable, relatively inexpensive to produce, and can be easily made biocompatible. Over a decade ago, the U.

Particle tracking single protein-functionalized quantum dot diffusion and binding at silica surfaces.

We evaluate commercial QD585 and QD605 streptavidin-functionalized quantum dots (QDs) for single-particle tracking microscopy at surfaces using total internal reflectance fluorescence and measure single QD diffusion and nonspecific binding at silica surfaces in static and flow conditions. The QD diffusion coefficient on smooth, near-ideal, highly hydroxylated silica surfaces is near bulk-solution diffusivity, as expected for repulsive surfaces, but many QD trajectories on rougher, less-than-ideal surfaces or regions display transient adsorptions. We attribute the binding to defect sites or adsorbates, possibly in conjunction with protein conformation changes, and estimate binding energies from the transient adsorption lifetimes.

Structural and functional divergence within the Dim1/KsgA family of rRNA methyltransferases.

The enzymes of the KsgA/Dim1 family are universally distributed throughout all phylogeny; however, structural and functional differences are known to exist. The well-characterized function of these enzymes is to dimethylate two adjacent adenosines of the small ribosomal subunit in the normal course of ribosome maturation, and the structures of KsgA from Escherichia coli and Dim1 from Homo sapiens and Plasmodium falciparum have been determined. To this point, no examples of archaeal structures have been reported.

Scintillation proximity assay for measurement of RNA methylation.

Methylation of RNA by methyltransferases is a phylogenetically ubiquitous post-transcriptional modification that occurs most extensively in transfer RNA (tRNA) and ribosomal RNA (rRNA). Biochemical characterization of RNA methyltransferase enzymes and their methylated product RNA or RNA-protein complexes is usually done by measuring the incorporation of radiolabeled methyl groups into the product over time. This has traditionally required the separation of radiolabeled product from radiolabeled methyl donor through a filter binding assay.

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA.

While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits [small ribosomal subunits (SSUs)] and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking.

Sequence and structural evolution of the KsgA/Dim1 methyltransferase family.

Background: One of the 60 or so genes conserved in all domains of life is the ksgA/dim1 orthologous group. Enzymes from this family perform the same post-transcriptional nucleotide modification in ribosome biogenesis, irrespective of organism. Despite this common function, divergence has enabled some family members to adopt new and sometimes radically different functions.

A conserved rRNA methyltransferase regulates ribosome biogenesis.

In contrast to the diversity of most ribosomal RNA modification patterns and systems, the KsgA methyltransferase family seems to be nearly universally conserved along with the modifications it catalyzes. Our data reveal that KsgA interacts with small ribosomal subunits near functional sites, including Initiation factor 3 and 50S subunit binding sites. These findings suggest a checkpoint role for this modification system and offer a functional rationale for the unprecedented level of conservation.

Magnetic labeling, detection, and system integration.

Among the plethora of affinity biosensor systems based on biomolecular recognition and labeling assays, magnetic labeling and detection is emerging as a promising new approach. Magnetic labels can be non-invasively detected by a wide range of methods, are physically and chemically stable, relatively inexpensive to produce, and can be easily made biocompatible. Here we provide an overview of the various approaches developed for magnetic labeling and detection as applied to biosensing.

Investigation of the properties of an ion-etched plane laminar holographic grating.

We have measured the efficiency over the range 125-225 A of a bare ion-etched plane laminar holographic grating made of fused silica and with 1000 grooves/mm. The measured efficiency of each order oscillates with wavelength because of constructive and destructive interference between radiation diffracted from the lands and the grooves. We measured the grating groove profile with an atomic force microscope, and the resulting groove depth of 434 ? 6 A agrees well with the values determined independently from the oscillatory behavior of the efficiency measurements.

Efficiency calibration of the first multilayer-coated holographic ion-etched flight grating for a sounding rocket high-resolution spectrometer.

We have fabricated the four flight gratings for a sounding rocket high-resolution spectrometer using a holographic ion-etching technique. The gratings are spherical (4000-mm radius of curvature), large (160 mm x 90 mm), and have a laminar groove profile of high density (3600 grooves/mm). They have been coated with a high-reflectance multilayer of Mo/Si.

Normal-incidence efficiencies of 4800-grooves/mm-ruled replica gratings with multilayer and gold coatings in the 125-325-A wavelength region.

The normal-incidence efficiencies of two 4800-grooves/mm ruled replica gratings, one with a dual-bandpass molybdenum/silicon multilayer coating and the other with a gold coating, were measured by use of synchrotron radiation in the 125-325-A wavelength region. The peak reflectance of the multilayer coating was 22% in the first Bragg order near 235 A and 28% in the second Bragg order near 126 A. The peak efficiency of the multilayer grating was 2.

Thin-film interference effects on the efficiency of a normal-incidence grating in the 100-350-A wavelength region.

Thin-film interference effects were observed in the normal-incidence efficiency of a 2400-groove/mm replica grating. The efficiency was measured in the 100-350-A wavelength range and had an oscillatory behavior that resulted from the presence of a thin SiO(2) coating. The thicknesses of the SiO(2) and the underlying oxidized aluminum layers were inferred from computer modeling of the zero-order efficiency.

Comparison of the calculated and the measured efficiencies of a normal-incidence grating in the 125-225- A wavelength range.

The efficiency of a diffraction grating was measured near normal incidence in the 125-225-A wavelength range with synchrotron radiation. The grating pattern had 2400 grooves/mm and was recorded on a concave fused-silica blank by a holographic technique. The grooves were shaped by ion-beam etching to provide a facet with a blaze angle of 2.

Multilayer-coated laminar grating with 16% normal-incidence efficiency in the 150-A wavelength region.

We characterized a laminar grating with a Mo/Si multilayer coating by using synchrotron radiation and atomic force microscopy. The grating substrate had 2400 grooves/mm, 40-A groove depth, and 2080-A groove width. The microroughness of the grating substrate was 5 A rms.

Grazing-incidence efficiencies in the 28-42-A wavelength region of replicas of the Skylab 3600-line/mm concave grating with multilayer and gold coatings.

The efficiencies of replicas of the Skylab 3600-line/mm concave grating with multilayer and gold coatings were measured by using synchrotron radiation at an angle of incidence of 79 degrees and in the 28-42-A wavelength range. The blaze angle of the grating facets that faced the incident radiation was 3.1 degrees , and the average angle of the opposite facets was 6 degrees .