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Both pulsed- and square-wave, low-intensity ultrasound (US) signals have been reported to impact chondrocyte function and biosynthetic activity. In this study, a low-intensity diffuse ultrasound (LIDUS) signal at 5.0 MHz (0.14 mW/cm(2)) was employed to stimulate bovine chondrocytes seeded in three-dimensional (3D) chitosan-based matrices. While the duration of application was constant at 51 s, US was applied once, twice, four times and eight times/day, and the impacts of US on the biosynthetic activity of chondrocytes and the expression of chondrocyte-specific genes were evaluated. When stimulated with continuous US for predetermined time intervals, chondrocytes had higher levels of type II collagen, aggrecan, L-Sox5 and Sox9 mRNA expression when compared to controls; however, under the same conditions, the expression of MMP-3 was downregulated. Interestingly, both Sox5 and Sox9 genes coordinately responded to changes in US stimulation and generally mirrored the response of collagen type II transcript to changes in US stimulation. RT-PCR analysis revealed that US stimulation increased the gene expression of cell-surface integrins α5 and β1. The expression of integrins α2 was downregulated by US treatment, suggesting that multiple integrin subunits may be involved in the regulation of chondrocytic function in response to US stimuli. The enhancement in the abundance of the mRNA transcripts upon US stimulation was observed to correlate with the protein expression of collagen type I, collagen type II, and integrins α5 and β1. In conclusion, the US stimulation regimen employed was shown to modulate the proliferative capacity, biosynthetic activity and integrin mRNA expression of articular chondrocytes maintained in 3D matrices., (Copyright © 2011 John Wiley & Sons, Ltd.)

In an effort to control the surface-mediated activation of thrombin and clot formation, proteins and molecules which mimic the anticoagulant properties of the vascular endothelial lining were immobilized on material surfaces. When immobilized on biomaterial surfaces, thrombomodulin (TM), an endothelial glycoprotein that binds thrombin and activates protein C (PC), was shown to generate activated PC (APC) and delay clot formation. However, TM-mediated activation of PC on biomaterial surfaces was shown to be limited by the transport of PC to the surface, with maximum activation obtained at a surface density of ∼40 fmole TM cm(-2). This work investigates surface immobilized with TM and endothelial protein C receptor (EPCR), a natural cofactor to TM which increases the rate of activation of PC on the native endothelium. A sequential and ordered immobilization of TM and EPCR on polyurethane at an enzymatically relevant distance (<10 nm) resulted in higher amounts of APC compared with surfaces with immobilized TM or with TM and EPCR immobilized randomly and at TM surface densities (1400 fmole cm(-2)) which were previously shown to be transport limited. Ordered TM and EPCR samples also showed increased time to clot formation in experiments with platelet-poor plasma, as measured by thromboelastography. Surfaces immobilized with TM and its natural cofactor EPCR at an enzymatically relevant distance are able to overcome transport limitations, increasing anticoagulant activation and time to clot formation., (Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Polymerase chain assembly (PCA) is a powerful tool for basic biological research and biotechnology applications. During the last several years, major advances have been made in de novo gene synthesis. However, there is still a need for fast and reproducible methods to automatically purify the synthesized genes. Upon completion of PCA, the subsequent PCR-amplified product mixture still contains undesired shorter DNA fragments that hinder cloning efforts. To avoid tedious gel purification, an automated two-column purification has been developed and used in conjunction with rapid PCA. The system enables fast synthesis and isolation of the full-length DNA of interest, important for facile cloning of desired DNA fragments. During the PCR amplification step, forward and reverse primers tagged with iminobiotin and bromodeoxyuridine labels, respectively, were used. The automated purification was then performed on the PCR mixture using two affinity/immunocapture columns in series to isolate only the desired full-length product. The procedure has been applied to the pUC19 beta-lactamase gene (929 bp). Follow-up PCR of the purified product, cloning, and sequencing demonstrated the technique's effectiveness in obtaining the pure full-length gene. The purification has also been performed on other synthesized genes, indicating its utility as a general approach.

Polymerase chain assembly (PCA) is a technique used to synthesize genes ranging from a few hundred base pairs to many kilobase pairs in length. In traditional PCA, equimolar concentrations of single stranded DNA oligonucleotides are repeatedly hybridized and extended by a polymerase enzyme into longer dsDNA constructs, with relatively few full-length sequences being assembled. Thus, traditional PCA is followed by a second primer-mediated PCR reaction to amplify the desired full-length sequence to useful, detectable quantities. Integration of assembly and primer-mediated amplification steps into a single reaction using a high-speed thermocycler is shown to produce similar results. For the integrated technique, the effects of oligo concentration, primer concentration, and number of oligonucleotides are explored. The technique is successfully demonstrated for the synthesis of two genes encoding EPCR-1 (653bp) and pUC19 beta-lactamase (929bp) in under 20min. However, rapid integrated PCA-PCR was found to be problematic when attempted with the TM-1 gene (1509bp). Partial oligonucleotide sets of TM-1 could be assembled and amplified simultaneously, indicating that the technique may be limited to a maximum number of oligonucleotides due to competitive annealing and competition for primers.

A theoretical analysis is presented with experimental confirmation to conclusively demonstrate the critical role that annealing plays in efficient PCR amplification of GC-rich templates. The analysis is focused on the annealing of primers at alternative binding sites (competitive annealing) and the main result is a quantitative expression of the efficiency (eta) of annealing as a function of temperature (T(A)), annealing period (t(A)), and template composition. The optimal efficiency lies in a narrow region of T(A) and t(A) for GC-rich templates and a much broader region for normal GC templates. To confirm the theoretical findings, the following genes have been PCR amplified from human cDNA template: ARX and HBB (with 78.72% and 52.99% GC, respectively). Theoretical results are in excellent agreement with the experimental findings. Optimum annealing times for GC-rich genes lie in the range of 3-6s and depend on annealing temperature. Annealing times greater than 10s yield smeared PCR amplified products. The non-GC-rich gene did not exhibit this sensitivity to annealing times. Theory and experimental results show that shorter annealing times are not only sufficient but can actually aid in more efficient PCR amplification of GC-rich templates.

The gene encoding the small heat shock protein (sHSP), LeHSP21.5, has been previously cloned from tomato (GenBank accession no. AB026983). The deduced amino acid sequence of this tomato sHSP was most similar to that of other endoplasmic reticulum (ER)-localized sHSPs (ER-sHSP) and can be predicted to target the ER. We examined whether the gene product of LeHSP21.5 (probable ER-sHSP) can act as molecular chaperone. For functional analysis, LeHSP21.5 protein was expressed in Escherichia coli as His(6)-tagged protein in the C-terminal and purified. We confirmed that ER-sHSP could provide thermal protection of soluble proteins in vitro. We compared the thermal stability of E. coli strain BL21 (DE3) transformed with pET-ER-sHSP with the control E. coli strain BL21(DE3) transformed with only the pET vector under heat shock and IPTG-induced conditions. Most of the protein extracts from E. coli cells expressing ER-sHSP were protected from heat-induced denaturation, whereas extracts from cells not expressing ER-sHSP were very heat-sensitive under these conditions. A similar protective effect was observed when purified ER-sHSP was added to an E. coli cell extract. ER-sHSP prevented the thermal aggregation and inactivation of citrate synthase. These collective findings indicate that ER-sHSP can function as a molecular chaperone in vitro.

Our recent molecular studies revealed two divergent PEP-carboxylase (PEPC [Ppc]) encoding genes in the green microalga Chlamydomonas reinhardtii, CrPpc1 and CrPpc2, which are coordinately responsive to changes in inorganic-N and -C supply at the transcript level [Mamedov, T.G., Moellering, E.R. and Chollet, R. (2005) Identification and expression analysis of two inorganic C- and N-responsive genes encoding novel and distinct molecular forms of eukaryotic phosphoenolpyruvate carboxylase in the green microalga C. reinhardtii, Plant J. 42, 832-843]. Here, we report the distribution of these two encoded catalytic subunits in the minor Class-1 and predominant Class-2 PEPC enzyme-forms, the latter of which is a novel high-molecular-mass, hetero-oligomeric complex containing both CrPpc1 (p109) and CrPpc2 (p131) polypeptides. The Class-1 enzyme, however, is a typical PEPC homotetramer comprised solely of p109. We also document that the amount of both CrPpc1/2 catalytic subunits is up-/down-regulated by varying levels of NH(4)(+) supplied to the culture medium.

The assembly of synthetic oligonucleotides into genes and genomes is an important methodology. Several methodologies for such synthesis have been developed, but they have two drawbacks: (1) the processes are slow and (2) the error frequencies are high (typically 1-3 errors/kb of DNA). Thermal damage is a major contributor to biosynthetic errors. In this paper, we elucidate the advantages of rapid gene synthesis by polymerase chain assembly (PCA) when used in combination with smart error control strategies. We used a high-speed thermocycler (PCRJet) to effectively minimize thermal damage and to perform rapid assembly of synthetic oligonucleotides to construct two different genes: endothelial protein C receptor (EPCR) and endothelial cell thrombin receptor, thrombomodulin (TM). First, the intact EPCR gene (EPCR-1, 612 bp) and a mutant EPCR-2 (576 bp) that lacked 4 N-linked glycosylation sites were constructed from 35 and 33 oligonucleotides, respectively. Next, for direct error comparison, another longer gene, the 1548 bp TM gene was constructed from 87 oligonucleotides by both rapid and conventional PCA. The fidelity and accuracy of the synthetic genes generated in this manner were confirmed by sequencing. The combined steps of PCA and DNA amplification are completed in about 10 and 22 min for EPCR-1, 2 and TM genes, respectively with comparable low errors in the DNA sequence. Furthermore, we subcloned synthetic TM, EPCR-1, EPCR-2 and native EPCR-1 (amplified from cDNA) into a Pichia pastoris expression vector to evaluate the expression ability, and to compare them with the native gene. Here, we illustrate that the synthetic genes, assembled by rapid PCA, successfully directed the expression of functional proteins. And, importantly, the synthetic and the native genes expressed proteins with the same efficiency.

Phosphoenolpyruvate carboxylase (PEPC [Ppc]) has been previously purified and characterized in biochemical and immunological terms from two green microalgae, Chlamydomonas reinhardtii and Selenastrum minutum. The findings indicate that these algae possess at least two distinct PEPC enzyme-forms, homotetrameric Class-1 and heteromeric Class-2, that differ significantly from each other and their plant and prokaryotic counterparts. Surprisingly, however, green-algal PEPC has been unexplored to date in molecular terms. This study reports the molecular cloning of the two Ppc genes in C. reinhardtii (CrPpc1, CrPpc2), each of which is transcribed in vivo and encodes a fully active, recombinant PEPC that lacks the regulatory, N-terminal seryl-phosphorylation domain that typifies the vascular-plant enzyme. These distinct catalytic subunit-types differ with respect to their (i) predicted molecular mass ( approximately 108.9 [CrPpc1] versus approximately 131.2 kDa [CrPpc2]) and critical C-terminal tetrapeptide; and (ii) immunoreactivity with antisera against the p102 and p130 polypeptides of S. minutum PEPC1/PEPC2 and PEPC2, respectively. Only the Ppc1 transcript encodes the p102 catalytic subunits common to both Class-1 and Class-2 enzyme-forms in C. reinhardtii. The steady-state transcript levels of both CrPpc1/2 are coordinately up-/down-regulated by changes in [CO2] or [NH] during growth, and generally mirror the response of cytoplasmic glutamine synthetase (Gs1) transcript abundance to changes in inorganic [N] at 5% CO2. These collective findings provide key molecular insight into the Ppc genes and corresponding PEPC catalytic subunits in the eukaryotic algae.

Phosphoglycolate phosphatase (PGPase), a key enzyme of photorespiration in photosynthetic organisms, was purified from Chlamydomonas reinhardtii. The enzyme was an approximately 65-kDa homodimer with a pI value of 5.1 composed of approximately 32-kDa subunits not connected by any S-S bridges. It was also highly specific for phosphoglycolate with a K(m) value of 140 microm and an optimal pH between 8 and 9. The activity was strongly inhibited by CaCl(2), and it recovered competitively following the addition of MgCl(2) or EGTA. A mobility shift was observed in SDS-polyacrylamide gel electrophoresis by the addition of CaCl(2), indicating that the enzyme binds to Ca(2+). The N-terminal region of amino acid sequence deduced from cDNA sequence that was not contained in the purified PGPase had similar characteristics to those of typical stroma-targeting transit peptides in C. reinhardtii. The following region of the deduced sequence containing 302 amino acid residues was similar to p-nitrophenylphosphatase-like proteins, although the purified PGPase did not hydrolyze p-nitrophenylphosphate. Genomic DNA fragments from wild type containing the sequence homologous to the cDNA for PGPase complemented the PGPase-deficient mutant pgp1. Possible regulatory mechanisms during adaptation to limiting CO(2) were discussed based on the characteristics of the purified PGPase and the deduced amino acid sequence.

The literature data on the changes in composition, structure and properties of hemoglobin under the influence of ionizing radiation in vivo are reviewed. The algorithm of calculation of damaged hemoglobin molecule percentage is proposed. Four main realizations of radiation-chemical damage are considered. By the algorithm the estimation of the damaged molecules percentage resulted from the exposure of 10 Gy is given. Hemoglobin radiation damage is considered as one of the most important mechanisms triggering radiation sickness.

Gypsum models were constructed to study the transversal pharynx width and palate defect in 57 children with congenital opened palate clefts. Age-dependent increase in the defect width was noted and attributed to muscular dystrophy in the soft palate and lateral walls of the pharynx as a result of their dysfunction and hypodynamia.