Alginate as matrix support for yeast artificial chromosome cloning.
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Alginates↗
Biomedical subjects
E Lai
Publications and source records attributed to E Lai.
Lack of myocardial lipid peroxidation during acute reperfusion injury in perfused guinea pig hearts.
OBJECTIVE: The aim was to test the hypothesis that free radical induced lipid peroxidation contributes to the arrhythmias and contractile changes associated with reperfusion. Evidence of oxidative stress in the membrane lipids was sought using four different approaches: (1) determination of lipid peroxidation metabolites; (2) direct detection of free radical formation using a lipophilic spin trap; (3) assessment of lipid peroxidation substrate; and (4) evaluation of antioxidant levels in the heart. METHODS: Electrical and contractile functions were monitored in isolated perfused guinea pig hearts subjected to ischaemia for 20 or 60 min followed by reperfusion for 1 or 20 min. The reperfused hearts were flushed with cold phosphate buffer with EDTA and the ventricles weighed and homogenised. Malonaldehyde content was determined using both a thiobarbituric acid spectrophotometric assay and a p-aminobenzoate spectrofluorescence assay. A lipophilic spin trap, PBN, was injected into guinea pigs 2 h before they were killed. Hearts containing spin traps were perfused for 5 min, then subjected to ischaemia followed by 1 min reperfusion. Lipid extracts containing PBN were examined using electron paramagnetic resonance spectroscopy. The content of polyunsaturated fatty acids and alpha tocopherol were determined by GLC and HPLC, respectively. RESULTS: Malonaldehyde content of the heart tissues did not increase during any combination of ischaemic and reperfusion episodes. The assays were effective at detecting raised malonaldehyde in hearts perfused with either 0.5 mM malonaldehyde standard or 0.5 mM cumene hydroperoxide. No spin adducts were found in either the extracted membrane lipids or the aqueous layer. Spin adducts were detected in myocardial lipids from animals exposed to non-lethal doses of gamma irradiation, confirming the presence of the spin trap in the heart tissue and the ability to detect spin adducts when formed. There were no differences in the percentage of total fatty acids that were polyunsaturated between reperfused hearts and continously perfused controls. Furthermore, alpha tocopherol content of the hearts exposed to ischaemia and reperfusion was not decreased relative to hearts perfused without an ischaemic episode. CONCLUSIONS: The results suggest that the early functional disturbances during reperfusion are not related to peroxidation of membrane lipids.
Acute Disease↗
In vivo accumulation of sodium pump inhibitor by normal and uremic erythrocytes.
A uremic toxin fraction (fraction 2-3-1) in the middle molecular mass range (300-2000 Da) containing a sodium potassium inhibitor pump was studied. As fraction 2-3-1 was known to be present in minute quantities in urine and plasma, erythrocyte lysates were used as an alternative source. Results show that fraction 2-3-1 is more abundant in erythrocytes than in normal urine. In addition, the fraction is present in a greater quantity in uremic erythrocytes than in normal blood cells. It is concluded that fraction 2-3-1 is concentrated at the erythrocyte level in uremic patients.
Chromatography, Gel↗
Induced reciprocal translocation in transgenic mice near sites of transgene integration.
Transgenic mice (JCP0 #18), heterozygous for an insertion of approximately 50 copies of the rat peripheral myelin (P0) protein cDNA, displayed a pattern of reduced litter size that suggested a chromosome rearrangement. Chromosome banding studies of fetal cells disclosed the presence of an apparently balanced translocation between a Chromosome (Chr) 1 and 14 with breakpoints at bands 1H3 and 14C3. In situ hybridization of biotin-labeled P0 rat cDNA probe to chromosome spreads and detection of specific signal with fluorescein isothiocyanate-conjugated avidin revealed a strong signal on the 1(14) translocation chromosome at the site of the breakpoint. A weaker signal was present near the breakpoint on the 14(1) derivative chromosome. These results suggest an etiologic relationship between the insertion of the transgene and the origin of the translocation. To further elucidate possible mechanisms, we first mapped the endogenous P0 gene (gene symbol Mpp). As previously reported (You et al., Genomics 9: 751, 1991), we found that Mpp is located on Chr 1 in the region of the translocation breakpoint in JCP0 mice. Subsequently, we have carried out pulsed-field gel and standard Southern analyses with P0 gene probes, but found no evidence for a direct involvement of the endogenous P0 gene in the process that generated the balanced reciprocal translocation. Thus, we favor the hypothesis that, during repair of DNA strand breakage--possibly induced by the microinjection procedure--the transgene copies were ligated to broken ends of Chrs 1 and 14. According to convention, this translocation is designated T(1;14)1Po. Homozygotes are phenotypically normal and breed well; they will be useful for genetic and physical mapping of Chrs 1 and 14.
Animals↗
Murine chromosomal location of four hepatocyte-enriched transcription factors: HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4.
The genes for rat hepatocyte nuclear factors 3 and 4 (HNF-3 alpha, HNF-3 beta, HNF-3 gamma, and HNF-4) have been mapped in mouse by analysis of restriction fragment length polymorphisms in interspecific backcross mice. These hepatocyte-enriched transcription factors are positive-acting transcription factors with binding sites in regulatory regions of many genes expressed in hepatocytes. Both HNF-3 alpha, beta, and gamma and HNF-4 are also expressed in intestine. They have recently been implicated as potential participants in endodermal development from early gut cells because of their close homology to Drosophila genes, which themselves are expressed in the developing gut. Despite having similar functional roles and highly conserved DNA binding domains, the three loci from the Hnf-3 family of genes mapped to three different mouse chromosomes, suggesting that the Hnf-3 family has become widely dispersed during evolution and implying the necessity for independent activation of each member of the HNF-3 family.
Animals↗
Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain.
We have previously characterized a novel transcription factor family in mammals, the HNF-3 family, by the members' homology to one another and to the Drosophila homeotic gene fork head. The expression of fork head is restricted to the anterior and posterior termini of the early fly embryo. Brain factor 1 (BF-1) is a new member of this family isolated from rat brain with an expression pattern and DNA binding specificity distinct from the HNF-3 genes. Expression is highly restricted in the developing neural tube to its rostral end, which gives rise to the telencephalon. These results suggest that BF-1 plays an important role in the establishment of the regional subdivision of the developing brain and in the development of the telencephalon.
Amino Acid Sequence↗
Regulation of hepatic gene expression and development.
Tissue-specific expression in the hepatocyte appears to require the actions of multiple factors in specific combinations for each gene. The interactions between two or more positive-acting factors that raise transcription to optimal levels and those between positive and negative factors that enforce strict cell-specific transcription remain to be elucidated. However, recent progress in the identification and cloning of the genes for many of these factors will make it possible to begin to answer these important questions.
Animals↗
Hepatocyte nuclear factor 3 beta contains two transcriptional activation domains, one of which is novel and conserved with the Drosophila fork head protein.
The hepatocyte nuclear factor 3 (HNF-3) gene family is composed of three proteins (alpha, beta, and gamma) that are transcription factors involved in the coordinate expression of several liver genes. All three proteins share strong homology in their DNA binding domains (region I) and are able to recognize the same DNA sequence. They also possess two similar stretches of amino acids at the carboxyl terminus (regions II and III) and a fourth segment of homology at the amino terminus (region IV). Furthermore, the HNF-3 proteins demonstrate homology with the Drosophila homeotic gene fork head in regions I, II, and III, suggesting that HNF-3 may be its mammalian homolog. In order to define HNF-3 beta protein domains involved in transcriptional activation, we have used a reporter gene, whose transcription is dependent on HNF-3 binding, for hepatoma cell cotransfection assays with expression vectors that produced different truncated HNF-3 beta proteins. A position-independent activation domain which contained conserved regions II and III was identified at the carboxyl terminus of the HNF-3 beta protein (amino acids 361 to 458). Moreover, site-directed mutations that altered the sequences within regions II and III demonstrated their importance to transactivation. The region II-III domain does not possess amino acid sequences in common with other transcription factors and may define a novel activation motif. HNF-3 beta amino-terminal sequences defined by conserved region IV also contributed to transactivation, but region IV activity required the participation of the region II-III domain. Region IV is abundant in serine amino acids and contains two putative casein kinase I phosphorylation sites, a feature similar to protein motifs described for the transcription factors Pit-1/GHF-1 and HNF-1.
Amino Acid Sequence↗
Polymerase chain reaction analysis for specific HTLV-1 sequences from cerebrospinal fluid and peripheral blood cells in Sardinian multiple sclerosis patients.
Using polymerase chain reaction and specific primers, we found no gag and env sequences of HTLV-1 in DNA samples from peripheral blood mononuclear cells of 26 multiple sclerosis (MS) patients with relapsing-remitting, relapsing-progressive and progressive course from onset of the disease, and from 8 patients affected with other neurological diseases (OND). A Positive signal for the gag region was found in DNA samples from cerebrospinal fluid mononuclear cells (CSFMC) of 6/17 (27.3%) MS patients (either with relapsing-remitting, or relapsing-progressive and progressive course from onset of the disease), and in 2/11 (18.2%) CSFMC OND samples. Positive hybridization for the env sequence was evident in 2/11 (18.2%) CSFMC from OND and none of MS samples. The finding of positive hybridization for gag and env sequences in a few samples of CSFMC may be related to the presence in the CSF of a great number of activated cells, which could express cross-reacting sequences of endogenous retrovirus.
Adolescent↗
Ultraviolet nicking of large DNA molecules from pulsed-field gels for southern transfer and hybridization.
Large DNA molecules separated in pulsed-field gels are not efficiently transferred from the gel for Southern hybridization. Various procedures for fragmenting the DNA prior to transfer are in use, but quantitative details that permit reproducible application have not been reported. We have determined the optimum level of energy for uv nicking of large DNA needed to promote efficient Southern transfer and detection by hybridization. To ensure consistent results we have used a uv oven equipped with a detector that measures only 200-400 nm wavelengths, and we report the total energy delivered. Using uv nicking and the transfer techniques described, we can obtain hybridization signals overnight with single-copy DNA probes on Southern blots of large DNA fragments separated by pulsed-field gel electrophoresis.
Blotting, Southern↗
Transcriptional control in hepatocytes: a window on development.
The unique phenotype of each differentiated cell in an animal (or plant) arises from selective expression of genes in a cell- or tissue-specific fashion, which is controlled primarily at the level of transcription. This review will focus on transcription factors that regulate cell-specific transcription in one intensely studied cell type, the hepatocyte or parenchymal liver cell. All of the recently isolated transcription factors that are important in hepatocyte-specific gene expression were identified in adult liver and there are strong hints that some of these may play an important role in embryogenesis.
Animals↗
Hepatocyte nuclear factor 3 alpha belongs to a gene family in mammals that is homologous to the Drosophila homeotic gene fork head.
By analysis of cDNA clones that cross-hybridized with a portion of the cDNA encoding the recently described rat protein hepatocyte nuclear factor 3 alpha (HNF-3 alpha, previously called HNF-3A), we now describe two additional members, HNF-3 beta and HNF-3 gamma, of this gene family. A 110-amino-acid region in the DNA-binding domain of this family is not only very highly conserved in rodents (HNF-3 alpha, -3 beta, and -3 gamma are identical in 93 of 110 amino acids in this region) but also in Drosophila where the homeotic gene fork head has 88 of the 93 residues that are identical in the three rat genes. The HNF-3 family in rodents is expressed in cells that derive from the lining of the primitive gut; some of the embryonic Drosophila cells in which fork head is expressed also give rise to gut and salivary glands. Thus, it appears that this gene family, the DNA-binding portion of which is unlike that of any previously recognized DNA-binding proteins, may contribute to differentiation of cells in internal organs in both vertebrates and invertebrates.
Amino Acid Sequence↗
Erythropoietin and Cooley disease.
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Adolescent↗
[Anesthesia in opioid dependent patients in orthopedic traumatic pathology: our experience].
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Adolescent↗
Differential regulation of hepatocyte-enriched transcription factors explains changes in albumin and transthyretin gene expression among hepatoma cells.
To investigate the regulation of genes whose expression is enriched in liver we studied expression of the albumin and transthyretin (TTR) genes in a series of rat hepatoma cell lines (FaO, C2, C2rev7, and H5) that express these genes at different rates. The level of expression of albumin and TTR was compared to the expression and DNA-binding activity of four transcription factors, HNF1/LFB1, C/EBP, HNF3, and HNF4, that are found at high concentrations in liver. We conclude that the levels of these factors are controlled both transcriptionally (HNF-3, HNF-4, and C/EBP) and post-transcriptionally (HNF-1/LFB1), and that the cellular concentration of these DNA-binding proteins helps explain the level of transcriptional activity observed for the genes they regulate.
Albumins↗
Rapid restriction map constructions using a modified pWE15 cosmid vector and a robotic workstation.
This paper describes a number of techniques for rapid restriction mapping of cosmid clones. First, we have replaced the cloning site of cosmid vector pWE15 with a polylinker containing 15 infrequently cleaved restriction enzyme sites that are placed asymmetrically on each side of the BamHI cloning site. DNA cloned into this vector can be fully recovered by using several pairs of restriction enzymes. Second, we have designed a simple electrical circuit device that allows the performance of asymmetric voltage gradient field inversion gel electrophoresis (AFIGE) to improve the resolution of DNA molecules in the range of 20-50 kbp. AFIGE can be obtained by simply placing the device in between a commercially available switching unit and the gel box in a standard field inversion system. Finally, the restriction digestion procedure has been automated by using a Beckman Biomek 1000 robotic workstation. Using this automated system, 96 restriction reactions, including gel loading, can be performed in less than two hours. In summary, these methods represent at least a tenfold improvement in the speed and/or mapping data that can be obtained in a single gel.
Animals↗
Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75- and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum.
Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.
Animals↗
The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels.
We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.
Base Sequence↗