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At least 901 records · Page 50Linked to original sources
PubMed2002

Physiological and pharmacological concentrations of melatonin protect against cisplatin-induced acute renal injury.

Cisplatin [cis-diaminedichloroplatinum(II), CDDP] is a widely used antineoplastic drug. However, it has major side-effects such as acute tubular necrosis (ATN). There are a number of studies concerning the role of reactive oxygen radical species in the pathophysiology of CDDP-dependent ATN. Several antioxidant agents have been reported to prevent this side-effect but there is no study regarding the protective action of either physiological or pharmacological concentrations of melatonin. Melatonin, the chief secretory product of the pineal gland, is a direct free radical scavenger and indirect antioxidant. We investigated the effects of melatonin on CDDP-induced changes of renal malondialdehyde (MDA), a lipid peroxidation product, and blood urea nitrogen (BUN) and serum creatine (Cr). The morphological changes in kidney were also examined using light microscopy. The rats were divided into two groups: pinealectomized (Px) and sham-operated (non-Px). Both CDDP and melatonin were administered to all groups. MDA levels were found to be higher in Px than non-Px animals. CDDP administration to Px or non-Px rats increased renal MDA levels and melatonin administration either before or after CDDP injection caused significant decreases in MDA in kidney compared with those in rats treated with CDDP alone. Serum levels of BUN and Cr did not change as a result of any treatment. Morphological tubule damage because of CDDP was more severe in the renal cortex than in the medulla. The damage to the kidney induced by CDDP was reversed by melatonin. The results show that pharmacological and physiological concentrations of melatonin reduce CDDP-induced renal injury.

Animals↗
PubMed2002

Bacterial proteomics and vaccine development.

Until recently, the development of vaccines for use in humans relied on the response to attenuated or whole-cell preparations, or empirically selected antigens. The post-genomic era holds the possibility of rational design of novel vaccines for important human pathogens. The discovery and development of these new vaccines is likely to be accomplished through integrated proteomic strategies. Although most proteomic studies are based on two-dimensional gel electrophoresis (2D-PAGE) as a separation technique, new methods have been developed within the past two years that provide complementary information concerning microbial protein expression. The 2D-PAGE technique in combination with Western blotting has been successfully applied in the discovery of antigens from Helicobacter pylori, Chlamydia trachomatis and Borrelia garinii. Two-dimensional semi-preparative electrophoresis has provided complementary information regarding membrane protein expression in a strain of H. pylori. Through two-dimensional liquid chromatography-tandem mass spectrometry, the most comprehensive information to date regarding protein expression in yeast was obtained. This technique may shortly become an important tool in vaccinology. This review of the current state of bacterial proteomics as applied in vaccinology presents analytical techniques for protein separation, proteomics without gels, reverse vaccinology, and functional approaches to the identification of virulence proteins in microbes.

Bacterial Proteins↗
PubMedDate not supplied

[Genetic engineering of peptide hormones. III. Cloning of the swine growth hormone cDNA and construction of the gene for expression of the hormone in bacteria].

The clones containing cDNA of porcine growth hormone were obtained using poly(A)-RNA from porcine pituitary as a template for reverse transcriptase. The analysis of their nucleotide sequences revealed that these cDNAs have differences not only on the nucleotide level but also on the amino acid level, i. e. the polymorphism of mRNA and protein occurs in the case of porcine growth hormone. To create the construction for expression of porcine growth hormone in E. coli, the 5'-part of cDNA, coding the first 15 amino acids of the mature hormone, was substituted by the artificial sequence.

Amino Acid Sequence↗
PubMed2002

Self-assembly of block copolymers derived from elastin-mimetic polypeptide sequences.

Protein polymers derived from elastin-mimetic peptide sequences can be synthesized with near-absolute control of macromolecular architecture using genetic engineering techniques. Elastin-mimetic diblock and triblock copolymers have been prepared using this approach in which the individual elastin blocks display different phase behavior in aqueous solution. The selective collapse of the more hydrophobic blocks above the lower critical solution temperature was employed to drive the thermo-reversible self-assembly of elastin-mimetic diblock and triblock copolymer into protein-based nanoparticles and nano-textured hydrogels, respectively. These materials display considerable promise as biomaterials for applications in drug delivery and soft tissue augmentation.

Amino Acid Sequence↗
PubMed1990

Inhibition of adenovirus replication by the E1A antisense transcript initiated from hsp70 and VA-1 promoters.

The E1A region of the adenoviral genome, important for initiation of virus infection and activation of other viral genes, was chosen as a target for engineering antisense RNA (asRNA) to inhibit adenovirus 5 (Ad5) replication in COS-1 cell culture in vitro. The hsp70 promoter, taken from the appropriate heat-shock-protein gene of Drosophila melanogaster, and the VA-1 RNA promoter, derived from the Ad5 gene coding for low-molecular-mass VA-1 RNA and recognized by RNA polymerase III were used as regulatory elements of transcription. The two types of recombinant constructs contained E1A fragments of 710 bp (hsp70 constructs) or 380 or 740 bp (VA-1 RNA constructs) in reverse orientation relative to the promoter position, as well as a transcription termination signal, the SV40 ori, and the gene controlling Geneticin (antibiotic G418) resistance (G418R). After selection of transfected COS-1 cells in the presence of G418, a number of stable G418R cell lines were raised which expressed engineered asRNAs. Plating of Ad5 suspensions of known titre on monolayers of transfected COS-1 cells clearly showed strong inhibition of adenovirus replication by asRNAs: 75% with the hsp70 promoter and 90% with the VA-1 RNA promoter.

Adenoviridae↗
PubMed2006

Targeted and reversible disruption of the blood-testis barrier by an FSH mutant-occludin peptide conjugate.

The blood-testis barrier (BTB) is one of the tightest blood-tissue barriers in mammals. As such, it poses a challenge to deliver any drugs to the seminiferous epithelium of the testis, such as a nonhormonal male contraceptive. To circumvent this problem, a genetically engineered follicle-stimulating hormone (FSH) mutant protein was produced in Spodoptera furgiperda (Sf)-9 insect cells to serve as a testis-specific carrier. Subsequently, a 22-amino acid peptide corresponding to the second extracellular loop of occludin, which was known to disrupt BTB integrity in vivo, was inserted to the FSH mutant by polymerase chain reaction (PCR), as well as chemical cross-linking. This molecule was found to have negligible hormonal activity but was still capable of binding to FSH receptors, which are restricted to Sertoli cells in mammals. When this FSH mutant-occludin peptide conjugate was administered to adult rats at 40 microg/adult rat (approximately 300 gm b.w.) via intraperitoneally (i.p.) injection, it induced transient and reversible disruption of the BTB, while at 150 microg/rat, it induced partial germ cell loss from the testis, particularly elongating/elongate spermatids. Most importantly, this effect was limited to the BTB without compromising the TJ-barrier integrity or cell adhesion in epithelia of other organs, such as kidney, liver, and small intestine. In summary, the use of an FSH mutant-occludin peptide conjugate is a feasible nanodevice to transiently compromise the BTB.

Animals↗
PubMed1996

HIV-1 RT enhances the activity of a tethered dimer of HIV-1 proteinase.

HIV-1 reverse transcriptase (RT) can specifically enhance HIV-1 proteinase activity in vitro and in eukaryotic cells (1). To determine if the effect of RT on proteinase activity was due to changes in the equilibrium dimerization constant of the proteinase or the stability of the proteinase dimer, we studied the effect of RT on a genetically engineered covalent dimer (tethered dimer) of the proteinase. RT was found to increase the activity of the tethered dimer independent of pH and ionic strength. The effect of RT on the kinetic constants (Km and kcat) of the wild type HIV-1 proteinase and its tethered dimer were also determined. These results show that RT can increase the enzymatic catalytic efficiency and substrate affinity of the proteinase, by a mechanism independent of promoting dimer formation.

Amino Acid Sequence↗
PubMed2006

Achilles tendon characterization in GDF-7 deficient mice.

Growth/differentiation factors (GDFs) play a significant role in numerous skeletal tissues and processes. Previous work using the brachypod mouse has suggested that GDF-5 affects Achilles tendon composition, ultrastructure, and material behavior, as well as tendon repair. The aim of the present study was to examine the role of a related GDF family member, GDF-7 (BMP-12), in intact tendon by studying the Achilles tendon of genetically engineered knockout mice. Achilles tendons from 16-week-old GDF-7 -/- mice contained 14% less GAG/DNA than did wild type littermates (p = 0.0481), although collagen content was comparable to controls. Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) results show that GDF-5 was upregulated two-threefold in response to the absence of GDF-7 protein. GDF-6 was also upregulated in knockouts, but to a lesser extent (twofold, p = 0.0013). On an ultrastructural level, GDF-7 deficient Achilles tendons exhibited a shift towards smaller diameter fibrils which resulted in a small but significant reduction in mean fibril diameter (-8%, p = 0.05). GDF-7 deficiency did not noticeably affect the expression of fibrillar collagens (I, III, V) or tendon proteoglycans (decorin, fibromodulin, lumican, biglycan, versican, aggrecan). Differences in tendon composition and ultrastructure were not biologically significant enough to have a noticeable effect on the structural or material behavior of the tendons. These results demonstrate that GDF-7 deficiency has a subtle effect on the composition and ultrastructure of murine Achilles tendon. The small magnitude of the observed differences may be due to overcompensation by related GDF family members.

Achilles Tendon↗
PubMed2000

Dynamics of gene expression in rat hepatocytes under stress.

The response of cells to physical or biochemical stress involves concerted changes in the expression of a large number of genes encoding various functions. We have used a quantitative kinetic RT-PCR technique to follow the dynamics of changes in transcription factor and acute-phase mRNA levels in cultured rat hepatocytes subjected to either elevated temperature (40 degrees C) or exposure to the inflammatory cytokine interleukin-6. The profiles of transcription factor gene expression displayed rapid and coordinate regulation, attainment of new steady-states, transitions in some instances from up-regulation to down-regulation (or vice versa), and, for elevated temperature, multiple spikes of up-regulation. Transcripts of acute-phase genes generally displayed relatively small changes during the first few hours followed by more significant changes over the course of tens of hours (elevated temperature) to days (IL-6 exposure). These observations are all consistent with the notion of genetic reprogramming due to a network of interacting transcription factor proteins and transcripts. We utilized a simple transcription/translation model incorporating autoregulation to describe the dynamics of transcription factor gene expression. This model successfully described key features of the transcription factor dynamics, most notably the multiple spikes observed after exposure to elevated temperature. The dynamics of gene expression are rich in information that, with considerably more study, may eventually be exploited to provide insights into the interplay of genetic networks in regulating a variety of cellular responses.

Acute-Phase Reaction↗
PubMed2006

Non-invasive intraoperative monitoring of blood pressure and arterial pCO2 during surgical anesthesia in mice.

Despite the constantly increasing use of genetically engineered mice in biomedical research, control of crucial physiological parameters such as blood pressure and arterial blood gases is difficult to achieve in temporarily anesthetized mice due to lack of techniques for reversible arterial cannulation. Here we report that arterial blood pressure and blood gases can be measured reliably in anaesthetized and artificially ventilated mice using non-invasive technology. C57Bl6 mice were anaesthetized by i.p. injection of midazolam, fentanyl, and medetomidin, intubated, and ventilated for 3h. End tidal pCO2 was monitored by micro-capnometry. Arterial blood pressure was measured non-invasively using a tail cuff. Non-invasive blood pressure (NIBP) correlated strongly with the invasive arterial blood pressure measured at the external carotid artery (r = 0.99, P < 0.001) and end tidal pCO2 values correlated very well with arterial blood pCO2 (r = 0.93, P < 0.001). The current results demonstrate that it is possible to reliably measure and control the most relevant physiological parameters in anesthetized mice. Thereby the current study may help to reduce animal numbers and perform mice experiments under more defined and controlled physiological conditions in the future.

Anesthesia↗
PubMed2003

Induction of specific T-helper and cytolytic responses to epitopes displayed on a virus-like protein scaffold derived from the pyruvate dehydrogenase multienzyme complex.

The icosahedral protein scaffold (1.5MDa) generated by self-assembly of the catalytic domains of the dihydrolipoyl acetyltransferase core of the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus has been engineered to display 60 copies of one or more peptide epitopes on a single molecule (E2DISP). An E2DISP scaffold displaying pep23, a 15-residue B- and T-helper epitope from the reverse transcriptase of HIV-1, was able to induce a pep23-specific T-helper response in cell lines in vitro. The same scaffold displaying both pep23 and peptide RT2, a nine-residue CTL epitope from HIV-1 reverse transcriptase, was able to prime an RT2-specific CD8(+) T-cell response in human cell lines in vitro and in HLA-A2 transgenic mice in vivo. This was accompanied by a humoral antibody response specific for E2DISP-presented epitopes. Thus, the icosahedral acetyltransferase core constitutes a simple and flexible scaffold for multiple epitope display with access to both cellular and humoral immune response pathways.

Acetyltransferases↗
PubMed2005

Phase transition between nanostructures of titanate and titanium dioxides via simple wet-chemical reactions.

Titanate nanofibers of various sizes and layered structure were prepared from inorganic titanium compounds by hydrothermal reactions. These fibers are different from "refractory" mineral substances because of their dimension, morphology, and significant large ratio of surface to volume, and, surprisingly, they are highly reactive. We found, for the first time, that phase transitions from the titanate nanostructures to TiO(2) polymorphs take place readily in simple wet-chemical processes at temperatures close to ambient temperature. In acidic aqueous dispersions, the fibers transform to anatase and rutile nanoparticles, respectively, but via different mechanisms. The titanate fibers prepared at lower hydrothermal temperatures transform to TiO(2) polymorphs at correspondingly lower temperatures because they are thinner, possess a larger surface area and more defects, and possess a less rigid crystal structure, resulting in lower stability. The transformations are reversible: in this case, the obtained TiO(2) nanocrystals reacted with concentrate NaOH solution, yielding hollow titanate nanotubes. Consequently, there are reversible transformation pathways for transitions between the titanates and the titanium dioxide polymorphs, via wet-chemical reactions at moderate temperatures. The significance of these findings arises because such transitions can be engineered to produce numerous delicate nanostructures under moderate conditions. To demonstrate the commercial application potential of these processes, we also report titanate and TiO(2) nanostructures synthesized directly from rutile minerals and industrial-grade rutiles by a new scheme of hydrometallurgical reactions.

Journal Article↗
PubMed1999

Spliceosome-mediated RNA trans-splicing as a tool for gene therapy.

We have developed RNA molecules capable of effecting spliceosome-mediated RNA trans-splicing reactions with a target messenger RNA precursor (pre-mRNA). Targeted trans-splicing was demonstrated in a HeLa nuclear extract, cultured human cells, and H1299 human lung cancer tumors in athymic mice. Trans-splicing between a cancer-associated pre-mRNA encoding the beta-subunit of human chorionic gonadotropin gene 6 and pre-trans-splicing molecule (PTM) RNA was accurate both in vitro and in vivo. Comparison of targeted versus nontargeted trans-splicing revealed a moderate level of specificity, which was improved by the addition of an internal inverted repeat encompassing the PTM splice site. Competition between cis- and trans-splicing demonstrated that cis-splicing can be inhibited by trans-splicing. RNA repair in a splicing model of a nonfunctional lacZ transcript was effected in cells by a PTM, which restored significant beta-galactosidase activity. These observations suggest that spliceosome-mediated RNA trans-splicing may represent a general approach for reprogramming the sequence of targeted transcripts, providing a novel approach to gene therapy.

Animals↗
PubMed2003

Targeted regulation of imprinted genes by synthetic zinc-finger transcription factors.

Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.

Beckwith-Wiedemann Syndrome↗
PubMed2002

Genetic engineering of mouse embryonic stem cells by Nurr1 enhances differentiation and maturation into dopaminergic neurons.

Nurr1 is a transcription factor critical for the development of midbrain dopaminergic (DA) neurons. This study modified mouse embryonic stem (ES) cells to constitutively express Nurr1 under the elongation factor-1alpha promoter. The Nurr1-expression in ES cells lead to up-regulation of all DA neuronal markers tested, resulting in about a 4- to 5-fold increase in the proportion of DA neurons. In contrast, other neuronal and glial markers were not significantly changed by Nurr1 expression. It was also observed that there was an additional 4-fold increase in the number of DA neurons in Nurr1-expressing clones following treatment with Shh, FGF8 and ascorbic acid. Several lines of evidence suggest that these neurons may represent midbrain DA neuronal phenotypes; firstly, they coexpress midbrain DA markers such as aromatic L-amino acid decarboxylase, calretinin, and dopamine transporter, in addition to tyrosine hydroxylase and secondly, they do not coexpress other neurotransmitters such as GABA or serotonin. Finally, consistent with an increased number of DA neurons, the Nurr1 transduction enhanced the ability of these neurons to produce and release DA in response to membrane depolarization. This study demonstrates an efficient genetic manipulation of ES cells that facilitates differentiation to midbrain DA neurons, and it will serve as a framework of genetic engineering of ES cells by key transcription factor to regulate their cell fate.

Animals↗
PubMed2026

Generation of spCAS9 expressing human mesenchymal stem cell line to study gene function during osteoblast differentiation.

Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.

CRISPR-Cas9↗
PubMed2006

Circadian rhythms lit up in Chlamydomonas.

Recent work on the circadian clock of the unicellular green alga Chlamydomonas reinhardtii strengthens its standing as a convenient model system for circadian study. It was shown to be amenable to molecular engineering using a luciferase-based real-time reporter for circadian rhythms. Together with the completed draft genomic sequence, the new system opens the door for genome-scale forward and reverse genetic analysis.

Animals↗
PubMed2005

[Construction of eukaryotic expression vector for human angiopoietin 1 and its expression in the marrow mesenchymal stem cells of rabbit].

OBJECTIVE: To investigate the possibility of constructing eukaryotic expression vector for human angiopoietin 1 (hAng-1), transfecting it to bone marrow mesenchymal stem cells (MSCs) so as to repair bone defect. METHODS: The eukaryotic expression vector pcDNA3-hAng-1 was constructed by recombinant DNA technique, transfected into MSCs by liposome DOTAP, and selected with G418. The hAng-1 expression of mRNA and protein was detected by reverse transcript-PCR and Western Blot. RESULTS: After the recombinant eukaryotic expression vector for hAng-1 was digested with Xho-I and BamH-I, electrophoresis revealed 1.4 kb fragment for hAng-1 gene and 5.4 kb fragment for pcDNA3 vector. In the transfected MSCs, the mRNA and protein expression of hAng-1 gene were detected with reverse transcript-PCR and Western Blot. CONCLUSION: The constructed eukaryotic expression vector hAng-1 could be expressed in the transfected MSCs, thus to provide the basis for bone repair with tissue engineering.

Angiopoietin-1↗