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PubMed2005

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures.

Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor-beta and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Adult↗
PubMed2002

Identification and sequencing analysis of intact proteins via collision-induced dissociation and quadrupole time-of-flight mass spectrometry.

Identifying unknown proteins has become a central focal point for proteomic and biopharmaceutical development laboratories. Our laboratory investigated using quadrupole time-of-flight mass spectrometry (Qq/TOFMS) for the analysis of intact proteins for the purpose of identifying unknowns while limiting the number of sample-handling steps between protein extraction and identification. Eight standard proteins, both unmodified and disulfide-bonded and ranging in mass from 5 to 66 kDa, were analyzed using nanoelectrospray and collision-induced dissociation to generate peptide sequence tags. An MS analysis, followed by MS/MS analyses on two to five individual protein charge states, were obtained to make an identification. Peptide sequence tags were extracted from the MS/MS data and used, in conjunction with molecular mass and source origin, to obtain protein identifications using the web-based search engine ProteinInfo (www.proteometrics.com). All of the proteins were unambiguously identified from the input data, after which, all of the major product ions were identified for structural information. In most cases, N- and/or C-terminal ions, and also stretches of consecutive product ions from the protein interior, were observed. This method was applied to the analysis and identification of an unknown detected via reversed-phase high-performance liquid chromatography.

Amino Acid Sequence↗
PubMed2003

A tool for understanding homologous recombination in plants.

Attempts for establishing an efficient gene targeting (GT) system in seed plants have hitherto not been successful. In contrast, GT based on homologous recombination is highly efficient in Physcomitrella, making this moss a novel tool in reverse genetics. However, why homologous and illegitimate recombination are differently regulated between Physcomitrella and seed plants is still enigmatic. Here we update the state of the art of GT in Physcomitrella and discuss approaches to unravel this enigma. Identification of molecular factors significantly enhancing GT and their subsequent transfer to crop plants will have a great impact on plant biotechnology by enabling precise genetic engineering. Physcomitrella appears to be the most useful model system in this context.

Bryopsida↗
PubMed1992

A simple and rapid method of preparing large fragments of dengue virus cDNA from replicative-form RNA using reverse transcriptase and PCR.

A method is described for cloning large fragments (1.5 kb to 2 kb) of dengue virus cDNA from replicative-form viral RNA. Aedes albopictus cells (C6/36 clone) infected with dengue virus contain double-stranded, replicative-form RNA molecules which were used as a template for an initial reverse transcription using a primer containing sequence homologous to regions of the genome at or near the 3' end of the gene being studied. The product was then used as a template for polymerase chain reaction (PCR) amplification using the same 3' primer and a second which hybridized to a region at the 5' end of the sequence to be cloned. Both primers were engineered to contain specific restriction enzyme cutting sites which enabled the PCR product to be cut and cloned directly into plasmids for sequencing and expression studies. We have used this method to construct clones of the envelope glycoprotein gene (E) and the non-structural genes 1 and 2a (NS1/2a) and 3 (NS3) of dengue type 2, Tonga 1974 strain, and E and NS1/2a from dengue type 3, H-87 strain, either as discrete genes or as constructs with long and short leader sequences, with or without anchor sequences. The method could be applied to the cloning of any gene from any flavivirus, directly from infected cell extracts, without the necessity for tedious virus purification steps.

Animals↗
PubMed2003

Inverting enantioselectivity of Burkholderia cepacia KWI-56 lipase by combinatorial mutation and high-throughput screening using single-molecule PCR and in vitro expression.

The enantioselectivity of lipase from Burkhorderia cepacia KWI-56 has been inverted using a novel in vitro technique for construction and screening of a protein library by single-molecule DNA amplification by PCR followed by in vitro coupled transcription/translation system termed single-molecule-PCR-linked in vitro expression (SIMPLEX). Four amino acid residues (L17, F119, L167, and L266) in the hydrophobic substrate-binding pocket of the lipase were selected for mutation based on a structural model of a substrate-enzyme complex, and a combinatorial mutation library was constructed by SIMPLEX and screened for (R) and (S)-configurations of p-nitrophenyl 3-phenylbutyrate. Some combinations of amino acid substitutions in the four positions of the lipase were found as effective for changing the enantiopreference from the (S)-form substrate to the (R)-form. Two variants were expressed in the original host cells and purified to homogeneity, showing completely reversed enantioselectivity for the (R)-form of ethyl 3-phenylbutyrate (selectivity factor E(R)=38 or 33), whereas the wild-type lipase was (S)-selective (selectivity factor E(S)=33). Thus the semi-rational and semi-random combinatorial design of a mutant library followed by a high-throughput screening based on their enzymatic activity should be a powerful tool to engineer the enantioselectivity of enzymes.

Amino Acid Substitution↗
PubMed2001

Sustainable systemic delivery via a single injection of lentivirus into human skin tissue.

The skin offers a tissue site accessible for delivery of gene-based therapeutics. To develop the capability for sustained systemic polypeptide delivery via cutaneous gene transfer, we generated and injected pseudotyped HIV-1 lentiviral vectors intradermally at a range of doses into human skin grafted on immune-deficient mice. Unlike Moloney murine leukemia virus (MLV)-based retrovectors, which failed to achieve detectable cutaneous gene transfer by this approach, lentivectors effectively targeted all major cell types within human skin tissue, including fibroblasts, endothelial cells, keratinocytes, and macrophages. After a single injection, lentivectors encoding human erythropoietin (EPO) produced dose-dependent increases in serum human EPO levels and hematocrit that increased rapidly within one month and remained stable subsequently. Delivered gene expression was confined locally at the injection site. Excision of engineered skin led to rapid and complete loss of human EPO in the bloodstream, confirming that systemic EPO delivery was entirely due to lentiviral targeting of cells within skin rather than via spread of the injected vector to visceral tissues. These findings indicate that the skin can sustain dosed systemic delivery of therapeutic polypeptides via direct lentivector injection and thus provide an accessible and reversible approach for gene-based delivery to the bloodstream.

Animals↗
PubMed2006

Dispersion of the RmInt1 group II intron in the Sinorhizobium meliloti genome upon acquisition by conjugative transfer.

RmInt1 is a self-splicing and mobile group II intron initially identified in the bacterium Sinorhizobium meliloti, which encodes a reverse transcriptase-maturase (Intron Encoded Protein, IEP) lacking the C-terminal DNA binding (D) and DNA endonuclease domains (En). RmInt1 invades cognate intronless homing sites (ISRm2011-2) by a mechanism known as retrohoming. This work describes how the RmInt1 intron spreads in the S.meliloti genome upon acquisition by conjugation. This process was revealed by using the wild-type intron RmInt1 and engineered intron-donor constructs based on ribozyme coding sequence (DeltaORF)-derivatives with higher homing efficiency than the wild-type intron. The data demonstrate that RmInt1 propagates into the S.meliloti genome primarily by retrohoming with a strand bias related to replication of the chromosome and symbiotic megaplasmids. Moreover, we show that when expressed in trans from a separate plasmid, the IEP is able to mobilize genomic DeltaORF ribozymes that afterward displayed wild-type levels of retrohoming. Our results contribute to get further understanding of how group II introns spread into bacterial genomes in nature.

Bacterial Proteins↗
PubMed2003

The aerodynamics of insect flight.

The flight of insects has fascinated physicists and biologists for more than a century. Yet, until recently, researchers were unable to rigorously quantify the complex wing motions of flapping insects or measure the forces and flows around their wings. However, recent developments in high-speed videography and tools for computational and mechanical modeling have allowed researchers to make rapid progress in advancing our understanding of insect flight. These mechanical and computational fluid dynamic models, combined with modern flow visualization techniques, have revealed that the fluid dynamic phenomena underlying flapping flight are different from those of non-flapping, 2-D wings on which most previous models were based. In particular, even at high angles of attack, a prominent leading edge vortex remains stably attached on the insect wing and does not shed into an unsteady wake, as would be expected from non-flapping 2-D wings. Its presence greatly enhances the forces generated by the wing, thus enabling insects to hover or maneuver. In addition, flight forces are further enhanced by other mechanisms acting during changes in angle of attack, especially at stroke reversal, the mutual interaction of the two wings at dorsal stroke reversal or wing-wake interactions following stroke reversal. This progress has enabled the development of simple analytical and empirical models that allow us to calculate the instantaneous forces on flapping insect wings more accurately than was previously possible. It also promises to foster new and exciting multi-disciplinary collaborations between physicists who seek to explain the phenomenology, biologists who seek to understand its relevance to insect physiology and evolution, and engineers who are inspired to build micro-robotic insects using these principles. This review covers the basic physical principles underlying flapping flight in insects, results of recent experiments concerning the aerodynamics of insect flight, as well as the different approaches used to model these phenomena.

Animals↗
PubMed2003

The "demise" of diagnostic and research ocular pathology: temporary or forever?

PURPOSE: Several authorities have documented a significant decrease in support for modern eye pathology/pathology research laboratories. Indeed, many laboratories have closed or suffered marked cutbacks. The purpose of this report is to ask why this is so and to seek a possible means for reversing this trend. METHODS: Observations from the senior author's experience and a case from author's facility are analyzed. RESULTS: There are several reasons for ocular pathologists' difficulties, such as financial problems, lack of vision, personality conflicts, and problems with the departmental administration. Until recently, most research and development in several subspecialty fields of ophthalmology, including biodevices research, has been done primarily by engineers and in-house workers in industry. This precludes proper independent, nonbiased control and guidance from academia. Most ocular pathologists have not participated in this relatively new and wide-open field. CONCLUSIONS: We suggest a new realm of activity for today's newly trained ocular pathologists. Based on personal experience of two decades of fruitful collaboration with industry, we believe that ocular pathologists are uniquely trained to apply their expertise to various new fields of research that most pathologists today have not utilized. An important example is research on clinicopathological aspects of implantable biodevices. In addition, support and oversight should be provided by the major ophthalmic societies, such as the American Academy of Ophthalmology, in order to retain (even regain) control over this field. This is mandatory in order to control the safety and efficacy of new drugs and devices being introduced almost daily. Only then can clear differentiation between profit and patient welfare be achieved as potentially dangerous devices and techniques are let loose on the market. The field of "routine, descriptive" eye pathology is severely wounded and will return only in an attenuated fashion. In general, full-time support for ocular pathologists will not be possible unless they seek extra support from the private sector, engage in a concurrent clinical practice, or are supported by an endowed chair (a wonderful alternative).

Financing, Government↗
PubMed2000

Chimeric melatonin mt1 and melatonin-related receptors. Identification of domains and residues participating in ligand binding and receptor activation of the melatonin mt1 receptor.

Melatonin receptors bind and become activated by melatonin. The melatonin-related receptor, despite sharing considerable amino acid sequence identity with melatonin receptors, does not bind melatonin and is currently an orphan G protein-coupled receptor. To investigate the structure and function of both receptors, we engineered a series of 14 chimeric receptor constructs, allowing us to determine the relative contribution of each transmembrane domain to ligand binding and receptor function. Results identified that when sequences encoding transmembrane domains 1, 2, 3, 5, or 7 of the melatonin mt(1) receptor were replaced by the corresponding domains of the melatonin-related receptor, the resultant chimeric receptors all displayed specific 2-[(125)I]iodomelatonin binding. Replacement of sequences incorporating transmembrane domains 4 or 6, however, resulted in chimeric receptors that displayed no detectable 2-[(125)I]iodomelatonin binding. The subsequent testing of a "reverse" chimeric receptor in which sequences encoding transmembrane domains 4 and 6 of the melatonin-related receptor were replaced by the corresponding melatonin mt(1) receptor sequences identified specific 2-[(125)I]iodomelatonin binding and melatonin-mediated modulation of cyclic AMP levels. To further investigate these findings, site-directed mutagenesis was performed on residues within transmembrane domain 6 of the melatonin mt(1) receptor. This identified Gly(258) (Gly(6.55)) as a critical residue required for high affinity ligand binding and receptor function.

Animals↗
PubMed1976

Characterization of 2-mum DNA of Saccharomyces cerevisiae by restriction fragment analysis and integration in an Escherichia coli plasmid.

Electrophoretic analysis of EcoRI and HindIII restriction fragments of 2-mum supercoiled DNA of Saccharomyces cerevisiae indicated that this class of DNA is heterogeneous and probably consists of two types of molecules. Integration of the 2-mum yeast DNA in E. coli plasmid pCR1 directly showed that existence of two types of molecules as each of these could be individually inserted into separate bacterial plasmids. The difference between the two types of 2-mum circles is due to an inversion of about 1.6 X 10(6) daltons. The inversion is flanked by a reversed duplicated sequence of 0.45 X 10(6) daltons. Possible implications of this structure are dicussed.

Chromosome Mapping↗
PubMed2004

Initial observations on the effect of medium composition on the differentiation of murine embryonic stem cells to alveolar type II cells.

The pluripotency and high proliferative index of embryonic stem (ES) cells make them a good potential source of cells for tissue engineering purposes. We have shown that ES cells can be induced to differentiate in vitro into pulmonary epithelial cells (type II pneumocytes) using a serum-free medium designed for the maintenance of mature distal lung epithelial cells in culture (SAGM). However, the resulting cell cultures were heterogeneous. Our aim in this study was to attempt to increase pneumocyte yield and differentiation state by determining which medium components enhance the differentiation of pneumocytes and modifying the medium accordingly. Quantitative RT-PCR was used to measure changes in the expression of a type II pneumocyte-specific gene, surfactant protein C (SPC), in response to alterations in the cell culture medium. Results suggested that most individual SAGM growth factors were inhibitory for type II pneumocyte differentiation, with the largest increases in SPC expression (approximately threefold) being observed upon removal of retinoic acid and triiodothryonine. However, large standard deviations occurred between replicates, illustrating the highly variable nature of ES cell differentiation. Nevertheless, these observations represent an initial step towards achieving directed differentiation of pneumocytes from stem cells that could lead to their purification for tissue engineering purposes.

Animals↗
PubMed2002

Oestrogen-dependent tracing in the rat CNS after pseudorabies virus infection.

This study examines the hypothesis that neuronal infectivity and the spreading of the pseudorabies virus (PRV) through the synapses in the central nervous system (CNS) are influenced by the oestrogen levels. The arcuate nucleus (ARC) and the subfornical organ (SFO) were chosen as models for analysis; the neurons in both structures possess oestrogen receptors and are mutually connected. A genetically engineered pseudorabies virus (Ba-DupLac) was used as a transneuronal tract tracer. This virus is taken up preferably by axon terminals, and transported very specifically through the synapses in a retrograde manner. Ba-DupLac was injected into the ARC of rats, followed by monitoring of the PRV-immunoreactivity (PRV-IR) in the SFO 72 h following inoculation. We found no PRV immunolabelling in the SFO of ovariectomized (OVX) rats, or in those OVX animals that received oestrogen shortly (4 h) before PRV infection (OVX + E 4 h). In contrast, in those OVX animals that received oestrogen 12 h before PRV infection (OVX + E 12 h), and also in intact control animals, PRV-IR was demonstrated in the SFO in all cases. Surprisingly, a reverse labelling was observed in the OVX rats; PRV-IR appeared in the pyriform cortex, whereas PRV-IR could not be detected in the control and OVX + E 12 h animals. As far as we are aware, this is the first study to demonstrate that transneuronal PRV labelling depends on the effects of oestrogen on certain CNS structures and connections.

Animals↗
PubMed1999

Nuclear receptors: coactivators, corepressors and chromatin remodeling in the control of transcription.

A contemporary view of hormone action at the transcriptional level requires knowledge of the transcription factors including the hormone receptor that may bind to promoters or enhancers, together with the chromosomal context within which these regulatory proteins function. Nuclear receptors provide the best examples of transcriptional control through the targeted recruitment of large protein complexes that modify chromosomal components and reversibly stabilize or destabilize chromatin. Ligand-dependent recruitment of transcriptional coactivators destabilizes chromatin by mechanisms including histone acetylation and contacts with the basal transcriptional machinery. In contrast, the recruitment of corepressors in the absence of ligand or in the presence of hormone antagonists serves to stabilize chromatin by the targeting of histone deacetylases. Both activation and repression require the action of other chromatin remodeling engines of the switch 2/sucrose non-fermentable 2 (SWI2/SNF2) class. Here we summarize this information and integrate hormone action into a chromatin context.

Animals↗
PubMed2006

Thermoreversible protein hydrogel as cell scaffold.

A thermoreversible fibrillar hydrogel has been formed from an aqueous lysozyme solution in the presence of dithiothreitol (DTT). Its physical properties and potential as a tissue engineering scaffold have been explored. Hydrogels were prepared by dissolving 3 mM protein in a 20 mM DTT/water mixture, heating to 85 degrees C and cooling at room temperature. No gel was observed for the equivalent sample without DTT. The elastic nature of the gel formed was confirmed by rheology, and the storage modulus of our gel was found to be of the same order of magnitude as for other cross-linked biopolymers. Micro differential scanning calorimetry (microDSC) experiments confirmed that the hydrogel was thermally reversible and that gelation and melting occurs through a solid-liquid-like first-order transition. Infrared spectroscopy of the hydrogel and transmission electron microscopy studies of very dilute samples revealed the presence of beta-sheet-rich fibrils that were approximately 4-6 nm in diameter and 1 mum in length. These fibrils are thought to self-assemble along their long axes to form larger fibers that become physically entangled to form the three-dimensional network observed in both cryo-scanning electron microscopy (cryo-SEM) and small-angle neutron scattering (SANS) studies. The hydrogel was subsequently cultured with 3T3 fibroblasts and cells spread extensively after 7 days and stretched actin filaments formed that were roughly parallel to each other, indicating the development of organized actin filaments in the form of stress fibers in cells.

Animals↗
PubMed2006

Characterization of human bone cells derived from the maxillary alveolar ridge.

In this study, we have characterized bone cell cultures derived from the human maxillary alveolar ridge, which could be a potential cell source for tissue engineering of the severely resorbed maxilla. From 10 individuals, an osseous core was obtained. Without the use of collagenase, 10 explant cultures were established and the morphology of the cells (human maxilla-derived cells (hMDCs)) was studied with light microscopy (LM). Explant cultures were analyzed by flow cytometry with respect to size, granularity and surface marker expression. Fluorochrom-conjugated monoclonal antibodies (CD13, CD31, CD44, CD90 or CD73) were used. hMDCs were cultured in standard medium (SCM) or osteoinductive medium (OIM) for 21 days and analyzed for the presence of alkaline phosphatase (ALP) and calcium deposits (Von Kossa). Furthermore, osteogenic gene expression (osteocalcin [OC], ALP, collagen type 1) were analyzed by reverse transcription polymerase chain reaction (RT-PCR). LM demonstrated that hMDCs had a polygonal morphology containing a central nucleus with two to three nucleoli. Size/granularity analysis revealed differences between individuals. Immunophenotypically, these cells were positive for CD13, CD44, CD90 and CD73 while negative for CD31. Cells cultured in SCM for 21 days showed moderate ALP staining and many calcium deposits. Culturing cells in OIM for 21 days significantly increased both ALP staining and the number of calcium deposits. RT-PCR demonstrated expression of osteogenic marker genes and the ability to upregulate osteocalcin and ALP in response to osteogenic inducers. To our knowledge, it is the first time that surface marker expression has been studied on bone cells originating from this site. Cells were positive for markers characteristic for immature mesenchymal stem cells and had osteogenic differentiation capability. This study indicates that cells derived from maxillary biopsies could be a potential cell source for bone tissue engineering.

5'-Nucleotidase↗
PubMed2000

Promoter strength influences polyamine metabolism and morphogenic capacity in transgenic rice tissues expressing the oat adc cDNA constitutively.

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat adc (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline biochemical parameters to elucidate the role of polyamines (PAs) during morphogenesis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedifferentiated callus. Polyamine levels were also quantified in two subsequent developmental stages--regenerating tissue and differentiated shoots. We observed significant (P < 0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spermidine (Spd) in dedifferentiated transgenic callus were lower than those in the wild type or in hpt (hygromycin resistant)-controls, whereas the amount of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P < 0.05), and lower levels of Spm (P < 0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated shoots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P < 0.05); on occasion reaching six times the level observed in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expression. mRNA measurements and ADC enzyme activity were consistently higher (P < 0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high adc expression leading to production of Put above a basal level is necessary to generate a big enough metabolic pool to trigger PA flux through the pathway leading to an increase in the concentration of Spd and Spm. This can be best accomplished by a strong constitutive promoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.

Avena↗
PubMed2006

Angiogenesis enhances factor IX delivery and persistence from retrievable human bioengineered muscle implants.

Human muscle progenitor cells transduced with lentiviral vectors secreted high levels of blood clotting factor IX (FIX). When bioengineered into postmitotic myofibers as human bioartificial muscles (HBAMs) and subcutaneously implanted into immunodeficient mice, they secreted FIX into the circulation for >3 months. The HBAM-derived FIX was biologically active, consistent with the cells' ability to conduct the necessary posttranslational modifications. These bioengineered muscle implants are retrievable, an inherent safety feature that distinguishes this "reversible" gene therapy approach from most other gene therapy strategies. When myofibers were bioengineered from human myoblasts expressing FIX and vascular endothelial growth factor, circulating FIX levels were increased and maintained long term within the therapeutic range, consistent with the generation of a vascular network around the HBAM. The present study implicates an important role for angiogenesis in the efficient delivery of therapeutic proteins using tissue engineered stem cell-based gene therapies.

Animals↗