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Biomedical subjects

A A Szalay

Publications and source records attributed to A A Szalay.

At least 19 recordsLinked to original sources
PubMed2002

A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo.

In this study, we used a steroid-induced promoter activation system as a molecular switch to study the exogenous activation of transgene expression. This promoter activation system consists of three components: (1) a steroidal inducer drug, mifepristone (RU486), which binds to (2) a chimeric transcription factor complex, consisting of the mutant human progesterone receptor fused to the yeast GAL4 DNA-binding domain and the activation domain of the herpes simplex virus protein VP16, and (3) a synthetic promoter, consisting of a series of GAL4 recognition sequences upstream of the adenovirus major late E1B TATA box, linked to a gene construct (ruc-gfp) encoding a Renilla luciferase- Aequorea green fluorescent protein (GFP) fusion protein. Transcription of the promoter-marker gene cassette is activated by the drug (mifepristone)-bound chimeric transcription factor complex. Monitoring of induced gene expression was carried out using a low-light video camera and a UV microscope to detect luciferase and GFP, respectively. Using this activation system, we observed a 10- to 25-fold activation, depending on the inducer dose, of both luciferase and GFP expression in transiently transfected cells in comparison to cells that were not exposed to mifepristone. We further demonstrated activation of gene expression from the promoter activation system in live animals. The plasmids PAP CMV-GL914VPc'SV, carrying the chimeric transcription factor cassette, and plasmid p17x4-TATA-ruc-gfp, carrying the ruc-gfp reporter gene construct, were co-injected into limb muscles of nude mice. Following DNA injection, mifepristone (50 micro g/kg) was delivered by intraperitoneal injection. Thirty-six hours after DNA and mifepristone injection, significant Renilla luciferase activity was detectable in the limb muscles. The promoter activation system was also demonstrated in limb muscles and livers of nude mice that had received transplants of ex vivo-modified cells, which were transiently transformed with both the chimeric activator plasmid and the ruc-gfp reporter plasmid prior to implantation. Significant Renilla activity and GFP fluorescence were detected externally in limb muscles and in the livers of anesthetized animals that had received an intraperitoneal injection of inducer. This external monitoring method for observing inducible gene expression in live animals will facilitate experimental studies of fundamental questions of biological and therapeutic relevance. It will be especially valuable for the analysis of gene function at specific stages of animal development. The method should also be of general use in gene therapy, since it permits simultaneous monitoring of the expression levels of light-emitting proteins and therapeutic proteins originating from the activation of identical promoters.

Animals↗
PubMed2002

Renilla luciferase- Aequorea GFP (Ruc-GFP) fusion protein, a novel dual reporter for real-time imaging of gene expression in cell cultures and in live animals.

Light-emitting reporter proteins play an increasing role in the study of gene expression in vitro and in vivo. Here we present a ruc-gfp fusion gene construct generated by fusing a cDNA for Renilla luciferase (ruc) in-frame with a cDNA encoding the "humanized" GFP (gfp) from Aequorea. A plasmid containing the fusion gene construct was successfully transformed into, and expressed in, mammalian cells. The transformed cells exhibited both Renilla luciferase activity in the presence of coelenterazine and GFP fluorescence upon excitation with UV light. Spectrofluorometry of cells containing the Ruc-GFP fusion protein, in the absence of wavelengths capable of exciting GFP fluorescence but in the presence of the luciferase substrate, coelenterazine, showed an emission spectrum with two peaks at 475 nm and 508 nm. These two peaks correspond to the emission maximum of Renilla luciferase at 475 nm and that of GFP at 508 nm. The peak at 508 nm generated in the presence of coelenterazine alone (without UV excitation) is the result of intramolecular energy transfer from Renilla luciferase to Aequorea GFP. Southern analysis of genomic DNA purified from transformed Chinese hamster ovary (CHO) cells and fluorescence in situ hybridization (FISH) to metaphase chromosomes confirmed the integration of the ruc-gfp fusion gene on a single chromosome. The bifunctional Ruc-GFP fusion protein allows the detection of gene expression at the single-cell level based on green fluorescence, and in a group of cells based on luminescence emission. Furthermore, animal experiments revealed that light emission from the Ruc-GFP fusion protein can be detected externally in the organs or tissues of live animals bearing the gene construct.

Animals↗
PubMed2001

Microinjection and growth of bacteria in the cytosol of mammalian host cells.

Most facultative intracellular bacteria replicate in specialized phagosomes after being taken up by mammalian cells. Relatively few intracellular bacteria escape the phagosomal compartment with the help of cytolytic (pore-forming) proteins and replicate in the host cell cytosol. Without such toxins, intracellular bacteria cannot reach this cellular compartment. To circumvent the requirement of an "escape" step, we developed a procedure allowing the efficient direct injection of bacteria into the cytosol of mammalian cells. With this technique, we show that most bacteria, including extracellular bacteria and intracellular pathogens that normally reside in a vacuole, are unable to replicate in the cytosol of the mammalian cells. In contrast, microorganisms that replicate in the cytosol, such as Listeria monocytogenes, Shigella flexneri, and, to some extent, enteroinvasive Escherichia coli, are able to multiply in this cellular compartment after microinjection. Further L. monocytogenes with deletion in its PrfA-regulated hpt gene was found to be impaired in replication when injected into the cytosol. Complementation of the hpt mutation with a plasmid carrying the wild-type hpt gene restored the replication ability in the cytosol. These data indicate that cytosolic intracellular pathogens have evolved specific mechanisms to grow in this compartment of mammalian cells.

Animals↗
PubMed2001

Detection of GDNF secretion in glial cell culture and from transformed cell implants in the brains of live animals.

Current ex vivo gene therapy for Parkinson's disease using glial cell line-derived neurotrophic factor (GDNF) is limited by the lack of a monitoring mechanism to determine the expression of GDNF once the cells or other vehicles are transferred into animal models. The purpose of this study was to test whether a Renilla luciferase (RUC)-GDNF fusion protein secreted by the genetically engineered glial cell line RG-1 could be measured photometrically in cerebrospinal fluid (CSF). RG-1 was constructed by permanent transformation with a plasmid DNA construct that contains a GDNF cDNA (gdnf) fused to a RUC cDNA (ruc). The fusion protein secreted by RG-1 was shown to retain both GDNF and RUC activity. The concentration of GDNF determined by enzyme-linked immunoadsorbent assay (ELISA) was correlated with the light emission detected by assaying for RUC bioluminescence in RG-1 culture medium, indicating that RUC can be used as a reporter for GDNF in vitro. The cells were then implanted into rat brain (n=20), and the cisternal CSF was analyzed. Bioluminescence was successfully detected in the CSF samples, and was quantified over a period of 25 days, while Western blotting and ELISA failed to detect GDNF in CSF, presumably because the concentration of the RUC-GDNF fusion was too low. This study demonstrates that the transformed glial cell line RG-1 offers a sensitive self-reporting assay for GDNF expression.

Animals↗
PubMed2001

The p14ARF tumor suppressor protein facilitates nucleolar sequestration of hypoxia-inducible factor-1alpha (HIF-1alpha ) and inhibits HIF-1-mediated transcription.

Oncogenic alterations can influence tumor cell survival partly by affecting the activity of the hypoxia-inducible factor-1 (HIF-1) transcription factor. The alpha subunit of HIF-1 was found to be frequently overexpressed in advanced tumors, which was proposed to help the adaptation of tumor cells to hypoxia. Here we show that an important tumor suppressor protein, p14ARF (alternative reading frame product of the INK4A locus) can directly inhibit the transcriptional activity of HIF-1 by sequestering its alpha subunit into the nucleolus. The interaction requires neither p53 nor HDM2. This is one of the first reports that describe the interaction of p14ARF with a protein besides HDM2, which may define a p53-independent tumor suppressor activity for p14ARF.

Active Transport, Cell Nucleus↗
PubMed2001

A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP.

We have previously reported that Escherichia coli and mammalian cells containing a fusion protein consisting of the Renilla luciferase linked to Aequorea GFP exhibited luminescence resonance energy transfer (LRET) from luciferase to GFP in the presence of coelenterazine. In this paper, we describe the construction of two gene fusions in which the cDNA for insulin-like growth factor II (IGF-II) is connected to the cDNA for a "humanized" GFP, and the cDNA for insulin-like growth factor binding protein 6 (IGFBP-6) is linked to a cDNA encoding the Renilla luciferase (RUC). The expression of the fusion gene constructs in CHO cells resulted in single polypeptides with the molecular weights expected for IGF-II-GFP and IGFBP-6-RUC, respectively, based on the use of antibodies against GFP and Renilla luciferase. The secretion of IGF-II-GFP from CHO cells was verified by fluorescence microscopy and the presence of IGFBP-6-RUC in the culture medium was confirmed by luminometry. The interaction between the two known binding partners, IGF-II and IGFBP-6, was monitored by measuring LRET from the IGFBP-6-RUC protein to IGF-II-GFP in the presence of coelenterazine, using a low-light imaging system and spectrofluorometry. Based on these data, luciferase-to-GFP LRET holds great promise for the study of protein-protein interactions in eukaryotic cells in real time.

Animals↗
PubMed2000

Mer22-related sequence elements form pericentric repetitive DNA families in primates.

We describe a novel repetitive DNA element isolated from three primate species belonging to the family Cercopithecidae. The unusually long 2.6-kb repeat unit of this DNA element is present in high copy number in the pericentromeric region of one pair of chromosomes in both baboon and macaque, forming chromosome-specific satellite-like DNA families. Besides these two very closely related species, the novel DNA element was also detected in the more distantly related African green monkey. However, the copy number of the repeat unit in this species is significantly lower than in macaque and baboon. Sequence analysis revealed that the repeat units of the new repetitive element show similarity to the human MER22 repeat and the Y chromosome-specific TTY2 element, which also exhibits retroelement-like features. Database searches indicate that tandemly arranged MER22-related DNA sequences can also be found in human, raising the possibility that these DNA elements may correspond to a novel primate-specific repetitive DNA group. Recent studies indicate that chromosome-specific pericentric repetitive elements, besides their potential involvement in centromere function, also facilitate homolog recognition during meiosis. In addition, rapid expansion of retroelements in the pericentric regions of chromosomes during interspecific hybridization has been described. In light of these data, we hypothesize that the novel repetitive element described here might have been involved in the speciation of the family Cercopithecidae.

Animals↗
PubMed1999

An alternative intronic promoter of the Bombyx A3 cytoplasmic actin gene exhibits a high level of transcriptional activity in mammalian cells.

Previous observations have indicated that the Bombyx mori gene for A3 cytoplasmic actin and vertebrate actin genes might make use of similar mechanisms for regulation of gene expression. To examine the suggested similarities, we have analyzed the expression of a LacZ reporter plasmid construct containing the 5' and 3' regulatory regions and the first intron of the A3 actin gene in a variety of vertebrate cell lines. We found that this silkworm expression cassette could drive expression of foreign genes in both mammalian and avian cell lines. Detailed analysis, however, indicated that neither the CArG box nor any of the promoter elements previously identified in the A3 actin gene were required for expression in mammalian cells. On the other hand, the first intron contained an efficient promoter, exhibiting in mouse cells a transcriptional activity comparable to that of the SV40 early promoter. The first intron of the A3 gene was also found to contain enhancer-like DNA elements that could stimulate the heterologous SV40 early promoter in mammalian cells. Promoter activity of the first intron of the A3 actin gene has not been observed previously. Recently however, we described a rare A3 actin mRNA isoform in B. mori cells, which initiates within the first intron. We suggest that the identified intronic promoter may be active not only in vertebrate cells but also in silkworm, and that it regulates the synthesis of the alternative A3 actin mRNA isoform. The characteristics of the 5' regulatory region of the A3 gene described here can also be exploited in the construction of bi-functional insect-mammalian expression vectors.

5' Untranslated Regions↗
PubMed1999

Genotyping the baboon ABO histo-blood group locus by two-color fluorescence SSCP.

The use of baboons in experimental medicine, especially as organ and tissue donors, would be facilitated by the availability of ABO histo-blood group O animals, which are currently rare. To meet our need in breeding and identifying such animals, we have developed a single-stranded conformational polymorphism (SSCP)-based genotyping assay using fluorescence detection. Various buffers and labeling schemes were evaluated to optimize allele discrimination. Through the use of a nested PCR protocol, single-cell sensitivity was achieved, making the assay applicable to preimplantation diagnosis following in vitro fertilization. We discuss the comparative advantages of SSCP vs. alternative methodologies for genotyping.

ABO Blood-Group System↗
PubMed1998

Molecular characterization of a stably transformed Bombyx mori cell line: identification of alternative transcriptional initiation sites of the A3 cytoplasmic actin gene.

We have isolated a stably transformed Bormbyx mori cell line containing a novel selectable marker gene, puromycin N-acetyl transferase, under control of transcriptional regulatory signals from the A3 cytoplasmic actin gene. By using this cell line we have identified alternative transcriptional initiation sites for the A3 actin gene. One of these start sites is located approximately 35 bp upstream from the previously determined transcription initiation site. The two mRNA start sites are utilized with a similar efficiencies in the BmN cell line. In addition, we detected transcripts that initiated in the first intron of the A3 actin gene. These transcripts may be synthesised under control of an alternative promoter. The stably transformed B. mori cell line used in this study was also extensively characterized. Integration of the plasmid molecules into the host genome was demonstrated by Southern and in situ hybridization analyses. Establishment and characterization of stably transformed insect cell lines, like the one described here, represents an important step in the development of nonlytic insect expression systems.

Acetyltransferases↗
PubMed1998

Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes.

Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes. L. monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin. Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L. monocytogenes strain. After delivery by L. monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).

Animals↗
PubMed1998

Baboon/human homologies examined by spectral karyotyping (SKY): a visual comparison.

Baboon (Papio hamadryas) metaphase chromosomes were analyzed using spectral karyotyping (SKY), a technique combining fluorescence microscopy, CCD-imaging, and Fourier spectroscopy. Results from a comparison of SKY analyses using probes derived from human chromosomes on baboon metaphases were consistent with the majority of comparative gene mapping data between the two species. These data were also compatible with earlier studies comparing macaque and human chromosomes. Human (HSA) chromosome 2 was homologous to baboon (PHA) chromosomes 12 (HSA 2q) and 13 (HSA 2p), whereas three baboon chromosomes corresponded to two different human chromosomes: PHA 3 to HSA 7 and HSA 21, PHA 7 to HSA 14 and HSA 15, and PHA 10 to HSA 20 and HSA 22. These results support the retained synteny between the Hominidae and Cercopithecidae genomes.

Animals↗
PubMed1997

The effect of quick-freezing in ethylene glycol on morphological survival and in vitro development of mouse embryos frozen at different preimplantation stages.

This study was conducted to examine the effect of a quick-freezing protocol on morphological survival and in vitro development of mouse embryos cryopreserved in ethylene glycol (EG) at different preimplantation stages. One-cell embryos were harvested from 6-to 8-wk-old CB6F1 superovulated mice, 20 to 23 h after pairing with males of the same strain and hCG injection. The embryos were cultured in human tubal fluid (HTF) containing 4 mg/ml BSA under mineral oil at 37 degrees C in 5% CO(2) plus 95% room air at maximal humidity. Twenty-four to 96 h after collection, the embryos were removed from culture and frozen at the 2 cell, 4 to 8-cell, compact morula, early blastocyst, expanding blastocyst and expanded blastocyst stages. To perform the quick-freeze procedure, embryos were equilibrated in Dulbecco's phosphate buffered saline (DPBS) + 10 % fetal bovine serum (FBS) + 0.25 M sucrose + 3 M ethylene glycol (freeze medium) for 20 min at room temperature (22 to 26 degrees C) and loaded in a single column of freeze medium into 0.25-ml straws (4 to 5 embryos per straw). The straws were held in liquid nitrogen vapor for 2 min and immersed in liquid nitrogen. Embryos were thawed by gentle agitation in a 37 degrees C water bath for 20 sec and transferred to DPBS + 10 % FBS + 0.5 M sucrose (re-hydration medium) for 10 min at room temperature, rinsed 2 times in HTF plus 4 mg/ml BSA and then cultured for 24 to 96 h. Survival of embryos was based on their general morphological appearance after thawing and their ability to continue development upon subsequent culture in vitro. Survival of blastocysts after thawing also required expansion or reexpansion of the blastocoel after several hours in culture. Significant differences were found in the survival and development of mouse embryos at different developmental stages quick-frozen in ethylene glycol and sucrose: 2-cell embryos 43/84 (51%), 4 to 8-cell embryos 44/94 (47%), morulae and early blastocysts 56/70 (80%; P</=0.05), expanding and expanded blastocysts 10/59 (17%; P</=0.05). Our data indicate that the developmental stage in which mouse embryos are subjected to this quick-freeze protocol affects survival and development in vitro and that most (80%) morula and early blastocyst stage embryos survive the procedure.

Journal Article↗
PubMed1997

Sequence comparison of baboon ABO histo-blood group alleles: lesions found in O alleles differ between human and baboon.

Histo-blood group O has only rarely been observed in baboon. Recent discovery of such an animal has provided use the opportunity to investigate the molecular genetics of the ABO locus in baboons. The major baboon prototype O allele, observed in two homozygous and several heterozygous animals, is related to the A allele as is the case in humans. Additional apparent prototype O alleles have been observed in heterozygotes, one of which is related to the B allele. The nucleotide changes conferring the O phenotype in the two known human O alleles have not been observed in any baboon allele. This information will aid the identification of baboons useful for the development of xenotransplantation in humans.

ABO Blood-Group System↗
PubMed1996

Luciferase assembly after transport into mammalian microsomes involves molecular chaperones and peptidyl-prolyl cis/trans-isomerases.

The assembly of a heterodimeric luciferase was studied after de novo synthesis of corresponding precursor proteins in reticulocyte lysate and concomitant transport into dog pancreas microsomes. This cytosolic luciferase from a prokaryotic organism (Vibrio harveyi) was specifically used as a model protein to investigate (i) whether the eukaryotic cytosol and the microsomal lumen have similar folding capabilities and (ii) whether the requirements of a polypeptide for certain molecular chaperones and folding catalysts are determined by the polypeptide or the intracellular compartment. The two luciferase subunits were fused to the preprolactin signal peptide. Data indicate that efficient assembly of luciferase occurs in the mammalian microsomes. Furthermore, it was observed that luciferase assembly can be separated in time from synthesis and membrane transport, depends on ATP hydrolysis, is partially sensitive to cyclosporin A and FK506, and in the absence of lumenal proteins is less efficient as compared with the presence of lumenal proteins. Thus, heterodimeric luciferase depends on functionally related molecular chaperones and folding catalysts during its assembly in either the eukaryotic cytosol or the microsomal lumen.

Adenosine Triphosphate↗
PubMed1996

De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes.

Chromosomes formed de novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of approximately 30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formed de novo by an H-type amplification revealed that it is composed of a tandem array of 10-12 large (approximately 30 Mb) amplicons each marked with integrated "foreign' DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of approximately 7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are approximately 7.5-Mb blocks of satellite DNA flanked by non-satellite sequences. We suggest that the formation de novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

Animals↗
PubMed1996

Evidence for a megareplicon covering megabases of centromeric chromosome segments.

We have analysed the replication of the heterochromatic megachromosome that was formed de novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons approximately 30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.

Animals↗