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At least 1,639 records · Page 91Linked to original sources
PubMed1999

Biotransformation of steroids by the fission yeast Schizosaccharomyces pombe.

The fungal biotransformation of steroids is of applied interest due to the economic importance of such stereo- and regiospecific reactions and also in the context of ergosterol pathway engineering to produce vitamin D and steroidal products. In Schizosaccharomyces pombe no steroid hydroxylation as is found in filamentous fungi was observed, but a cytosolic NAD(H)/NADP(H)-dependent hydroxysteroid dehydrogenase activity was identified. Progesterone was reduced at the delta 4 double bond (in vivo only) as well as at the C-3 and C-20 keto groups. Testosterone and 4-androstene-3,17-dione were interconverted and 5 alpha-pregnane-3,20-dione and 5 beta-pregnane-3,20-dione were reduced to 3-hydroxy products. The reactions were sometimes reversible and showed regio- and stereo specificity. In S. pombe more than one steroid dehydrogenase homologue is likely to occur, as has been observed in Saccharomyces cerevisiae. Our findings indicate that genes encoding soluble proteins should be examined as candidates for actual steroid dehydrogenase activity.

Biotransformation↗
PubMed2026

Distinct YY dinucleotide periodicity in adeno-associated virus DNA.

Dinucleotide periodicity is a hallmark of genome organization, yet its role in single-stranded (ss)DNA viruses remains poorly understood. Here, we systematically analyzed dinucleotide spacing patterns in adeno-associated virus (AAV) genomes and other viruses. Across 13 primate AAV serotypes, we identified a pronounced and highly conserved ∼15-bp periodicity specific to pyrimidine-pyrimidine (YY) dinucleotides and their reverse complements (RR). Comparative analyses across >25,000 viral sequences demonstrate that this 15-bp YY/RR periodicity is unique to the genus Dependoparvovirus and absent from other ssDNA viruses, satellite viruses, and helper viruses, which predominantly exhibit canonical ∼10- to 11-bp periodicities. Upon disruption of the YY/RR pattern using DNA family shuffling of AAV capsid genes, and subsequent iterative selection for viral production or cell entry, we found that the pattern is under positive selection. Selected sequences display increased periodicity alongside reduced sequence diversity, supporting a functional role for this genomic feature. Finally, engineered recombinant AAV genomes containing YY periodic motifs exhibit enhanced production and, for some designs, improved transduction efficiency, demonstrating that YY periodicity can modulate viral replication and infectivity. Our findings uncover a unique DNA-encoded signal in dependoparvoviruses that contributes to AAV fitness, expands our knowledge of virus biology, and has implications for vector engineering.

Dependovirus↗
PubMed1993

Cerebral transplantation for Parkinson's disease: current progress and future prospects.

Cerebral transplantation has received considerable attention from both the medical community and lay press as a potential treatment for Parkinson's disease. Animal models have demonstrated feasibility, although the experience in subhuman primates was very limited when the first human trials were initiated in the mid-1980s. The dramatic success reported for adrenal-to-brain transplantation in some initial trials could not be consistently replicated by other centers. Occasionally, however, patients benefited. Failure of the adrenal medullary graft to survive may have been a major factor in the poor outcomes. Recently, several US and European centers reported substantial clinical improvement after fetal dopaminergic mesencephalon was grafted into the striatum of patients with Parkinson's disease. Although many outcomes were impressive, in some cases the improvement was marginal; in no case was the condition completely reversed, and all but one patient still required levodopa therapy. Before this technique can be considered for routine use, further refinement is necessary, and many technical issues must be addressed. Certain animal studies have suggested that transplantation-related improvement may be derived from graft neurotrophic factors rather than from secretion of dopamine into the dopamine-depleted brain of patients with Parkinson's disease. Preliminary investigations in animals indicate that several other tissues, besides fetal mesencephalon, may also prove appropriate for grafting. Ultimately, advances in molecular biology may allow either transplantation of genetically engineered cells or direct modification of existing brain cells by transfection with viral vectors. The favorable preliminary experience with cerebral transplantation in patients with Parkinson's disease has resulted in the consideration of this strategy for other neurologic disorders.

Adrenal Medulla↗
PubMedDate not supplied

Thermoprecipitation of streptavidin via oligonucleotide-mediated self-assembly with poly(N-isopropylacrylamide).

A versatile strategy has been developed for selectively and sequentially isolating targets in a liquid-phase affinity separation environment. The strategy uses a recently developed approach for joining together molecules in linkages that are defined by the complementary pairing of oligonucleotides conjugated to the different molecules [Niemeyer, C. M., Sano, T., Smith, C. L., and Cantor, C. R. (1994) Nucleic Acids Res. 22, 5530-9]. In the work presented here, streptavidin was noncovalently coupled with the temperature-responsive poly(N-isopropylacrylamide) [poly(NIPAAM)] through the sequence-specific hybridization of oligonucleotides conjugated to the protein and polymer. A 20-mer oligonucleotide was covalently linked through a heterobifunctional linker to a genetically engineered streptavidin variant that contained a unique cysteine residue at the solvent-accessible site Glu 116. The complementary DNA sequence was conjugated to the end of a linear ester-activated poly(NIPAAM). The two conjugates were allowed to self-assemble in solution via hybridization of their complementary DNA sequences. The streptavidin-poly(NIPAAM) complex could be used to affinity-precipitate radiolabeled biotin or biotinylated alkaline phosphatase above 32 degrees C through the thermally induced phase separation activity of the poly(NIPAAM). The streptavidin-oligo species could then be reversibly separated from the precipitated polymer-oligo conjugate and recycled by lowering the salt concentration, which results in denaturation of the short double-stranded DNA connection. The use of oligonucleotides to couple polymer to streptavidin allows for selective precipitation of different polymers and streptavidin complexes based on the sequence-specific hybridization of their oligonucleotide appendages.

Acrylamides↗
PubMed2003

Ectopic B-Raf expression enhances extracellular signal-regulated kinase (ERK) signaling in T cells and prevents antigen-presenting cell-induced anergy.

T cells that receive stimulation through the T cell receptor (TCR) in the absence of costimulation become anergic and are refractory to subsequent costimulation. This unresponsiveness is associated with the constitutive activation of the small G protein, Rap1, and the lack of Ras-dependent activation of ERK. Recent studies suggest that Rap1 can activate the MAP kinase kinase kinase B-Raf that is either endogenously or ectopically expressed. Peripheral T cells generally do not express B-Raf; therefore, to test the hypothesis that ectopic expression of B-Raf could permit Rap1 to activate ERK signaling, we generated transgenic mice expressing B-Raf within peripheral T cells. This converted Rap1 into an activator of ERK, to enhance ERK activation and proliferation following TCR engagement in the absence of costimulation. When T cells were incubated with engineered APCs presenting antigen on I-Ek and expressing low levels of B7, they became anergic, displayed constitutive activation of Rap1, and were deficient in Ras and ERK activation. However, when incubated with the same APCs, T cells expressing the B-Raf transgene proliferated upon restimulation and displayed elevated ERK activation. Thus B-Raf expression and enhanced ERK activation is sufficient to prevent anergy in a model of APC-induced T cell anergy. However, studies using anti-TCR antibody-induced anergy showed that the ability of ERKs to reverse T cell anergy is dependent on the anergic model utilized.

Animals↗
PubMed2006

FGF-2 inhibits osteogenesis in mouse adipose tissue-derived stromal cells and sustains their proliferative and osteogenic potential state.

In addition to adult bone marrow-derived mesenchymal stem cells, more recently, a second large stromal compartment found in adipose tissue has received attention and is believed to contain multipotent cells. In vitro, adipose tissue-derived stromal cells (ADS) can differentiate down osteogenic, chondrogenic, myogenic, adipose, and even neuronal pathways. In this article, we explore the effect of fibroblast growth factors 2 (FGF-2) on ADS cells. Our results demonstrated that FGF-2 inhibits osteogenesis in ADS cells. The osteogenic inhibitory effects is dose-dependent and reversible, thus suggesting that the lack of osteogenesis observed in ADS cells exposed to FGF-2 is not due to a negative selection triggered by this factor on a subpopulation of osteoblast progenitors. Furthermore, either overexpression of FGF-2, or continuous FGF-2 treatment sustain the proliferative and osteogenic potential state of ADS cells. Therefore, FGF-2 appears to be a positive regulator of osteoprogenitor cells and a negative regulator of osteoblast differentiation in ADS cells. These FGF-2 functional characteristics may assist with cell selection and enrichment for the purpose of bone tissue engineering.

Adipose Tissue↗
PubMed1996

Revertant analysis of J-K mutations in the encephalomyocarditis virus internal ribosomal entry site detects an altered leader protein.

The internal ribosomal entry site (IRES) of picornaviruses consists of various sequence and structural elements that collectively impart translational function to the genome. By engineering substitution and deletion mutations into the J-K elements of the encephalomyocarditis virus IRES, translationally defective viruses with small-plaque phenotypes were generated. From these, 60 larger-plaque revertant viruses were isolated and characterized, and their sequences were compared with a structural model of the IRES. The data provide confirming evidence for the existence of helix J3 within stem J but suggest that helix J1 is 3 bp longer than previously estimated. They also suggest that previously modeled stems L and M should be replaced by an alternative structure. One reversion mutation was mapped to the leader protein coding region. This change of leader amino acid 20 from Pro to Ser increased the viral plaque size dramatically but did not alter the cell-free translational activity of the mutated, parental IRES.

Animals↗
PubMed1995

Interventional telemedicine for noninvasive neuroradiosurgery: remote-site high-performance computing, mathematical optimization, and virtual scenario simulation.

Interventional Telemedicine may have potential utility in providing connectivity and access to specialized high performance computing and advanced software resources in support of clinical procedures in the field of Minimally-Invasive Surgery and Stereotactic Linear Accelerator (LINAC) Radiosurgery. Such interventions may benefit from application of nonlinear quadratic inverse solution methods designed to provide the capability to reverse optimize a 'best case' treatment plan. The formidable decision-making challenges posed by increasingly complex optimized data and progressively versatile LINAC delivery systems require volume visualization of projected treatment data and imaging anatomy via photorealistic rendering and virtual scenario simulation techniques. Both these new directions are heavily dependent on access to specialized high performance computing platforms solely accessible via broad-bandwidth network connectivity. This pilot project presents resimulation of retrospective radiosurgical case data using inverse solution optimization models running on workstation clusters and then volume rendered and simulated on the Princeton Graphics Engine Supercomputer. Evidence for effective utilization of such optimization and virtual simulation methods running on remotely accessed, distant high performance computing resources is discussed in view of the potential for long-term clinical investigation and eventual development of Interventional Telemedicine as a clinically practical approach for providing support to remote or non-urban radiosurgery centers in the industrialized and developing world.

Algorithms↗
PubMed2002

Artificial human telomeres from DNA nanocircle templates.

Human telomerase is a reverse-transcriptase enzyme that synthesizes the multikilobase repeating hexamer telomere sequence (TTAGGG)n at the ends of chromosomes. Here we describe a designed approach to mimicry of telomerase, in which synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes. Results show that the combination of a nanocircle and a DNA polymerase gives a positive telomere-repeat amplification protocol assay result for telomerase activity, and similar to the natural enzyme, it is inhibited by a known telomerase inhibitor. We show that artificial telomeres can be engineered on human chromosomes by this approach. This strategy allows for the preparation of synthetic telomeres for biological and structural study of telomeres and proteins that interact with them, and it raises the possibility of telomere engineering in cells without expression of telomerase itself. Finally, the results provide direct physical support for a recently proposed rolling-circle mechanism for telomerase-independent telomere elongation.

Animals↗
PubMed2000

Inhibition of Fas-mediated apoptosis in mouse insulinoma betaTC-3 cells via an anti-Fas ribozyme.

In this study we have designed and constructed an anti-Fas ribozyme and show that it can specifically cleave the Fas mRNA in vitro. Moreover, to test its efficacy ex vivo, we transfected the anti-Fas ribozyme into betaTC-3 insulinoma cells, using a RNA polymerase III promoter to drive its expression. Like pancreatic beta cells, betaTC-3 cells do not constitutively express Fas, but Fas expression can be induced with IL-1 and IFN-gamma. Transfected cells expressed an average of 5000 copies of anti-Fas ribozyme transcript per cell as assessed by reverse transcriptase-real-time PCR. After IL-1/IFN-gamma treatment, betaTC-3 cells transfected with the anti-Fas ribozyme expressed 80% less Fas compared with mock-transfected cells. In addition, the anti-Fas ribozyme also inhibited Fas expression in NIT-1 insulinoma cells and in primary cultures of dispersed pancreatic islet cells. Inhibition of de novo Fas expression in betaTC-3 cells expressing the anti-Fas ribozyme correlated with resistance to Fas-mediated apoptosis as determined by the number of cells exhibiting caspase 3 proteolytic activity. Hence, we have engineered a ribozyme capable of preventing Fas expression in the betaTC-3 pancreatic insulinoma cell line and conferring resistance to Fas-mediated apoptosis. We suggest that ribozymes may be potentially useful to engineer resistance to apoptosis in transplantable beta cells, a feature that may significantly improve the survival of islet cell grafts.

Animals↗
PubMed2002

Efficient transduction of pancreatic islets by feline immunodeficiency virus vectors1.

BACKGROUND: Pancreatic islets transplanted into immunocompetent diabetic subjects are rapidly lost to apoptotic or lytic death or both. Genetic engineering of islets before transplantation with protective genes may enhance their posttransplantation survival. Accomplishing this goal requires the development of a safe, efficient vector for islet gene delivery. METHODS: The ability of feline immunodeficiency virus (FIV) vectors to transfer a green fluorescent protein (GFP) gene to NIT-1 cells and primary islets was measured and compared with murine leukemia virus (MLV) and human immunodeficiency virus (HIV) vectors. Islets were examined using confocal microscopy to determine the extent and pattern of infection. Toxicity of the procedure was assessed via measurement of glucose stimulation indices and by reversion of diabetic mice using either FIV-infected or control islet transplants. RESULTS: FIV effectively transduces islets with no untoward effect on the insulin secretion capacity of the beta cells. When FIV, HIV, and MLV GFP vectors were standardized to the same 293 cell titer and used to infect NIT-1 cells or whole islets, the FIV transduced equal or greater numbers of cells relative to the HIV vector and significantly more than the MLV vector. Islets transduced with FIV GFP were transplanted in a murine model for diabetes and were shown to revert diabetes and express GFP 4 weeks after transduction and 3 weeks after transplantation. CONCLUSIONS: FIV transduction is a nontoxic and efficient method to genetically modify pancreatic islets and may prove promising for delivering genes to augment islet survival after transplantation.

Animals↗
PubMed2006

Differential gene expression between juvenile and adult dura mater: a window into what genes play a role in the regeneration of membranous bone.

BACKGROUND: Although reossification of large calvarial defects is possible in children, adults lack this tissue engineering capacity. In this study, the authors compared the differences in gene expression between juvenile and adult dura mater using a mouse cDNA microarray with 42,000 unique elements. METHODS: Non-suture-associated parietal bone was harvested from 6-day-old and 60-day-old mice. The dura mater was carefully dissected from the calvarial disk and snap-frozen. RNA was extracted from pooled dura mater for microarray analysis. The 25 most differentially expressed genes were listed, as were selected bone-related genes. In addition, quantitative real-time reverse-transcriptase polymerase chain reaction confirmation of selected genes-BMP-2, BMP-4, and BMP-7; and osteopontin (OP), osteocalcin (OC), and FGFR-1-was performed. RESULTS: Juvenile dura mater expressed significantly greater amounts of BMP-2 and OP. Minimal difference in OC expression was observed between juvenile and adult dura mater. Extracellular matrix proteins (Col3a1, 5a1, 6a1, and fibronectin 1), osteoblast differentiation markers (Runx2/Cbfa1, Itm2a, and FGFR-1), and the growth factor Ptn were among other genes with greater expression in juvenile dura mater. Markers of osteoclasts (Acp5, MMP9, Ctsk) and the multiple candidate gene Ntrk2 were also expressed at higher levels in the juvenile dura mater. CONCLUSIONS: These findings suggest a more differentiated osteoprogenitor population to exist along with a greater presence of osteoclasts in the juvenile dura mater relative to adults. In addition to establishing a baseline difference in gene expression between juvenile and adult dura mater, new genes potentially critical to the regenerative potential of juvenile calvaria were identified.

Age Factors↗
PubMed2002

A kinetic model for enzyme interfacial activity and stability: pa-hydroxynitrile lyase at the diisopropyl ether/water interface.

A kinetic framework is developed to describe enzyme activity and stability in two-phase liquid-liquid systems. In particular, the model is applied to the enzymatic production of benzaldehyde from mandelonitrile by Prunus amygdalus hydroxynitrile lyase (pa-Hnl) adsorbed at the diisopropyl ether (DIPE)/aqueous buffer interface (pH = 5.5). We quantitatively describe our previously obtained experimental kinetic results (Hickel et al., 1999; 2001), and we successfully account for the aqueous-phase enzyme concentration dependence of product formation rates and the observed reaction rates at early times. Multilayer growth explains the early time reversibility of enzyme adsorption at the DIPE/buffer interface observed by both enzyme-activity and dynamic-interfacial-tension washout experiments that replace the aqueous enzyme solution with a buffer solution. The postulated explanation for the unusual stability of pa-Hnl adsorbed at the DIPE/buffer interface is attributed to a two-layer adsorption mechanism. In the first layer, slow conformational change from the native state leads to irreversible attachment and partial loss of catalytic activity. In the second layer, pa-Hnl is reversibly adsorbed without loss in catalytic activity. The measured catalytic activity is the combined effect of the deactivation kinetics of the first layer and of the adsorption kinetics of each layer. For the specific case of pa-Hnl adsorbed at the DIPE/buffer interface, this combined effect is nearly constant for several hours resulting in no apparent loss of catalytic activity. Our proposed kinetic model can be extended to other interfacially active enzymes and other organic solvents. Finally, we indicate how interfacial-tension lag times provide a powerful tool for rational solvent selection and enzyme engineering.

Adsorption↗
PubMedDate not supplied

Amber suppressors of Erwinia chrysanthemi.

Mutations trp1 and thyA1, both of a polyauxotrophic derivative of the Erwinia chrysanthemi strain B374, were characterized as amber mutations with an Escherichia coli suppressor, supA1P2, which inserts a glutamine in response to UAG. Simultaneous reversion of both mutations allowed us to isolate amber suppressor mutants of E. chrysanthemi. These suppressors were tested with a set of amber mutants of bacteriophage Mu which had been previously characterized on E. coli. The two independently isolated suppressors behaved as supD and supE mutants, respectively, of E. coli.

Bacteriophage mu↗
PubMed2005

Cloning and expression of earthworm fibrinolytic enzyme PM(246) in Pichia pastoris.

We have cloned, expressed, and purified a novel earthworm fibrinolytic enzyme (EFE) of Lumbricus rubellus in Pichia pastoris. Its cDNA sequence revealed a 747bp region containing an intact ORF that encodes a protein of 246 amino acid residues, designated as EFE PM(246). While EFE PM(246) is distinct, its cDNA shows a high degree of sequence homologies with four other EFE cDNAs registered in GenBank. The recombinant EFE PM(246) was active, showing a fibrinolytic activity of 7.5 x 10(6)U/L in basal salts medium, a higher fibrinolytic activity than those produced in other expression systems. The recombinant EFE PM(246) expressed in basal salts medium was purified by a three-step purification procedure with a recovery rate of about 20%. This is the first report detailing the successful purification of a genetically engineered earthworm fibrinolytic enzyme. The main physiochemical features of the EFE PM(246), including temperature stability, pH resistance, and sensitivity to some protein inhibitors, were also characterized.

Amino Acid Sequence↗
PubMed1996

Immunization against exon 1 decapeptides from the lutropin/choriogonadotropin receptor or the follitropin receptor as potential male contraceptive.

Pituitary gonadotropin hormones lutropin (LH) and follitropin (FSH) control steroidogenesis and gametogenesis in male and female gonads through interaction with G protein-coupled receptors, LHR and FSHR. In the male, LH acts on leydig cells and is mostly responsible for the acquisition of puberty and the production of androgens while FSH, together with androgens, regulates spermatogenesis within Sertoli cells. We have engineered filamentous phages displaying mouse LHR and human FSHR decapeptides chosen in hormone binding regions. Peptides from both receptors displayed on phages belong either to the receptor specific exon 1 (amino acids 18-27) or to the homologous exon 4 (amino acids 98-107). Vaccination of prepubertal BALB/c male mice with hybrid phages using sub-cutaneous or intraperitoneal injections induced immunity against receptors. Anti-receptor immunization produced agonist or antagonist effects depending only on the circulating levels of the antibodies. Both anti-LHR and anti-FSHR vaccines induced efficient as well as reversible male contraception, through different mechanisms: targeting LH receptors inhibited or hyperstimulated Leydig cell testosterone production while targeting FSH receptors did not affect testosterone levels.

Amino Acid Sequence↗
PubMed2006

High-performance elastomeric nanocomposites via solvent-exchange processing.

The incorporation of nanoparticles into engineering thermoplastics affords engineers an opportunity to synthesize polymer nanocomposites that potentially rival the most advanced materials in nature. Development of these materials is difficult because thermodynamic and kinetic barriers inhibit the dispersal of inorganic, often hydrophilic nanoparticles in hydrophobic polymer matrices. Using a new solvent-exchange approach, we preferentially reinforce the hard microdomains of thermoplastic elastomers with smectic clay of similar characteristic dimensions. The strong adhesion between the clay and the hard microdomains coupled with the formation of a percolative network not only stiffens and toughens, but increases the heat distortion temperature of the material and induces reversible thermotropic liquid-crystalline transitions. The discotic clay platelets induce morphological ordering over a range of length scales, which results in significant thermomechanical enhancement and expands high-temperature applications. Merging block-copolymer processing techniques with this method for preferential ordering of nanoparticle facilitates the development of new, hierarchically ordered materials.

Elastomers↗
PubMed2001

Chinese hamster ovary (CHO) cells may express six beta 4-galactosyltransferases (beta 4GalTs). Consequences of the loss of functional beta 4GalT-1, beta 4GalT-6, or both in CHO glycosylation mutants.

Six beta4-galactosyltransferase (beta4GalT) genes have been cloned from mammalian sources. We show that all six genes are expressed in the Gat(-)2 line of Chinese hamster ovary cells (Gat(-)2 CHO). Two independent mutants termed Pro(-)5Lec20 and Gat(-)2Lec20, previously selected for lectin resistance, were found to have a galactosylation defect. Radiolabeled biantennary N-glycans synthesized by Pro(-)5Lec20 were proportionately less ricin-bound than similar species from parental CHO cells, and Lec20 cell extracts had a markedly reduced ability to transfer Gal to GlcNAc-terminating acceptors. Northern blot analysis revealed a severe reduction in beta4GalT-1 transcripts in Pro(-)5Lec20 cells. The Gat(-)2Lec20 mutant expressed beta4GalT-1 transcripts of reduced size due to a 311-base pair deletion in the beta4GalT-1 gene coding region. Northern analysis with probes from the remaining five beta4GalT genes revealed that Gat(-)2 CHO and Gat(-)2Lec20 cells express all six beta4GalT genes. Unexpectedly, the beta4GalT-6 gene is not expressed in either Pro(-)5 or Pro(-)5Lec20 cells. Thus, in addition to a deficiency in beta4GalT-1, Pro(-)5Lec20 cells lack beta4GalT-6. Nevertheless, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry data of N-glycans released from cellular glycoproteins showed that both the beta4GalT-1(-) (Gat(-)2Lec20) and beta4GalT-1(-)/beta4GalT-6(-) (Pro(-)5Lec20) mutants have a similar Gal deficiency, affecting neutral and sialylated bi-, tri-, and tetraantennary N-glycans. By contrast, glycolipid synthesis was normal in both mutants. Therefore, beta4GalT-1 is a key enzyme in the galactosylation of N-glycans, but is not involved in glycolipid synthesis in CHO cells. beta4GalT-6 contributes only slightly to the galactosylation of N-glycans and is also not involved in CHO cell glycolipid synthesis. These CHO glycosylation mutants provide insight into the variety of in vivo substrates of different beta4GalTs. They may be used in glycosylation engineering and in investigating roles for beta4GalT-1 and beta4GalT-6 in generating specific glycan ligands.

Amino Acid Sequence↗