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Biomedical subjects

P Berg

Publications and source records attributed to P Berg.

At least 163 records · Page 9Linked to original sources
PubMed1987

DNA cross-linked by cisplatin: a new probe for the DNA repair defect in xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is an inherited disease characterized by the defective repair of DNA damaged by ultraviolet radiation and a number of chemicals. In this paper, plasmid DNA carrying a marker gene is cross-linked in vitro by the antitumor drug cisplatin and successfully introduced into tissue culture cells by both calcium phosphate coprecipitation and electroporation. Transient expression of the marker gene is greatly decreased in XP cells compared to wild-type. As few as seven lesions will inactivate the marker gene in XP cells. Furthermore, the biochemical defect must include an impaired capacity for repair of cisplatin-DNA intrastrand cross-links. Since the host cell itself is not exposed to chemical modification, a cisplatin cross-linked plasmid shuttle vector can be used as a specific probe for the DNA repair capacity of cultured cells. Paradoxically, when cisplatin cross-linked plasmid carrying the selectable marker neo is introduced into cells, there is an increase in the number of stable neo+ transformants in both XP and wild-type cells. Thus, cisplatin damage appears to stimulate the integration of transfected DNA into the host chromosome by a mechanism that is independent of the defective repair pathway in XP.

Cell Line↗
PubMed1986

[IE and IgG antibodies against flavonoids following therapy with flavonoid-containing drugs].

During treatment with flavonoid drugs a number of patients developed adverse reactions such as fever, various skin eruptions and intravascular hemolysis. The appearance of flavonoid-specific IgE and IgG antibodies and its possible relation to the observed side effects was studied on a total of 168 individuals treated with flavonoid drugs: 71 patients received troxerutin (Venoruton) parenterally to improve tolerance of radiation therapy, 12 patients were treated intravenously with silymarin (Legalon) for amanita intoxication and 77 patients received various flavonoid drugs for other indications. Flavonoid treatment often induced specific IgG antibodies and less frequently IgE antibodies. After short treatment IgE antibodies were more frequently detected than after treatment of longer duration which nearly always induced IgG antibodies. Patients with hemolysis had the highest IgG titers. In cases with fever and skin eruptions no correlation with antibody titers became evident. Antibody production in patients undergoing radiation therapy appeared to be lower than in non-irradiated patients. Both the IgE and IgG antibody test show a remarkable cross-reactivity between four different flavonoids. Prospective studies will be necessary to decide whether or not this method for the detection of anti-flavonoid antibodies will be suitable for recognizing increased risk of side reactions upon reexposure to flavonoid drugs.

Catechin↗
PubMed1986

Immortalization of xeroderma pigmentosum cells by simian virus 40 DNA having a defective origin of DNA replication.

A simian virus 40 (SV40) DNA fragment, encompassing the whole early region and having a defective origin of DNA replication, has been used to transform human fibroblast cells derived from two xeroderma pigmentosum (XP) patients. Two of the SV40-transformed XP cell lines, belonging to complementation group C, had acquired the characteristic of indefinite life-span in culture. These XP cell lines synthesize T antigen as shown by immunofluorescence and retain the high sensitivity to UV irradiation. Detailed karyotype analysis shows very few chromosomal changes, while the transfecting SV40 DNA is integrated into cellular DNA sequences. These are the first immortalized XP cell lines derived from complementation group C. In view of the extreme difficulty in obtaining immortalized human fibroblasts, we suggest a possible advantage of replication defective SV40 DNA molecules for immortalizing human fibroblast cells of any source.

Cell Line↗
PubMed1986

Ultrasonography as a routine examination before treatment of retinal detachment due to macular hole.

Twelve of 25 patients were treated at the University Eye Clinic in Münster because of retinal detachment due to a macular hole. Partial fluid gas exchange (SF6), vitrectomy and SF6, or vitrectomy and silicone instillation, were used, and the patients were examined both pre- and postoperatively by ultrasonography with particular attention to the situation at the posterior pole. These echographical examinations enabled us to evaluate the following three criteria: staphyloma posticum, stiffness of the retina, and vitreous adhesions. All of these conditions mean that the prognosis will be worse if SF6 gas instillation is used alone. In these cases, a vitrectomy should also be performed or a Klöti-Klemme procedure is useful when stiffness of the retina or staphyloma posticum is present. Preoperative ultrasonography is useful when setting up an adequate therapy plan.

Eye Diseases↗
PubMed1986

[Iatrogenic eye perforation in para- and retrobulbar injections].

The authors report on 5 cases of inadvertent iatrogenic perforation of the globe in the course of retrobulbar and parabulbar injections. Perforation occurred twice by injection of the drug into the eyeball itself (subretinally or intravitreally), while in 3 cases a double perforation occurred, as a result of which the drug penetrated behind the eye. The prognosis is better in the latter case. Even though perforation of the globe during parabulbar and retrobulbar injections is extremely rare, it must always be considered if there is a sudden deterioration in vision and/or severe pain immediately after the injection; depending on the situation, retinal and/or vitreous surgery must then be performed at once.

Adult↗
PubMed1986

Formation of functional asialoglycoprotein receptor after transfection with cDNAs encoding the receptor proteins.

The rat asialoglycoprotein receptor (ASGP-R) has been expressed in cultured rat hepatoma cells (HTC cells) after transfection with cloned cDNAs. Fluorescence-activated cell sorting of transfected cells was used to identify the functional cDNA clones and to isolate cells expressing the ASGP-R. Simultaneous or sequential transfections with two cloned cDNAs that encode related but distinctive polypeptide chains were needed to obtain ASGP-R activity; transfection with either cDNA alone failed to produce detectable ASGP-R. The affinity of transduced ASGP-R for asialo orosomucoid is less than that of the native rat ASGP-R, and the number of surface receptors in clones expressing ASGP-R is about one-fifth that found on rat hepatocytes.

Animals↗
PubMed1986

Isolation and characterization of expressible cDNA clones encoding the M1 and M2 subunits of mouse ribonucleotide reductase.

Mammalian ribonucleotide reductase consists of two nonidentical subunits, proteins M1 and M2, which are differentially regulated during the cell cycle. We have isolated expressible cDNA clones of both subunits from an Okayama-Berg cDNA library made with mRNA from hydroxyurea-resistant, M2 protein-overproducing mouse TA3 cells. Expression of M2 protein could be demonstrated by electron paramagnetic resonance spectroscopy after transfection of COS-7 monkey cells with the plasmid. Electrophoresis and blot analyses of the parent and hydroxyurea-resistant TA3 mRNA revealed two M2 transcripts, a major one of 2.1 kilobases and a minor one of about 1.6 kilobases. Restriction endonuclease mapping of the corresponding cDNAs indicated that the two mRNAs differed only in the length of the 3' untranslated ends. By contrast, there was only one mRNA corresponding to the M1 protein, and its mobility corresponded to about 3.1 kilobases. The hydroxyurea-resistant TA3 cells contained a 50- to 100-fold excess of the M2 mRNAs over that of the parent cells and a 10-fold excess of the M1 mRNA. However, a Southern blot analysis of the corresponding genomic DNA sequences showed that the M2 gene was amplified fivefold but the M1 gene was still single copy. The complete nucleotide sequence of the 2,111-base-pair-long M2 cDNA revealed an open reading frame coding for 390 amino acids, which corresponds to a molecular weight of 45,100. The mouse M2 protein sequence was quite homologous to the equivalent protein in the clam Spisula solidissima, while the homology to the smaller subunits of Epstein-Barr virus, herpes simplex virus type 2, and Escherichia coli ribonucleotide reductases were less pronounced.

Amino Acid Sequence↗
PubMed1986

Effects of the position of the simian virus 40 enhancer on expression of multiple transcription units in a single plasmid.

We have examined the ability of the simian virus 40 72-base pair enhancer segment to simultaneously activate multiple transcription units with plasmids that contain one, two, or three simian virus 40-based transcription units in various arrangements. After transfection into CV1 cells, the expression of a marker gene, Ecogpt, was determined as a function of the position of that marker gene relative to the other transcription units and the position of the marker gene relative to enhancer elements on the plasmids. Two types of position effects were revealed by that analysis. The first, promoter occlusion, causes reduced transcription at a downstream promoter if transcription is initiated at a nearby upstream promoter. This effect does not involve enhancer elements directly, even though the effect is most pronounced when the downstream promoter lacks an enhancer element. The second effect stems from the ability of promoter sequences to reduce the effect of a single enhancer element on other promoters in the same plasmid. This latter effect is mediated by either promoters adjacent to the enhancer element or promoters interposed between the enhancer element and the other promoters on the plasmid.

DNA Restriction Enzymes↗
PubMed1986

Termination-reinitiation occurs in the translation of mammalian cell mRNAs.

Many examples of internal translation initiation in eucaryotes have accumulated in recent years. In many cases terminators of upstream reading frames precede the internal initiation site, suggesting that translational reinitiation may be a mechanism for initiation at internal AUGs. To test this idea, a series of recombinants was constructed in the mammalian expression vector pSV2. Each contained a dicistronic transcription unit comprising the coding sequence for mouse dihydrofolate reductase (DHFR) followed by the gene for xanthine-guanine phosphoribosyl transferase (XGPRT) from Escherichia coli. Various versions of this pSV2dhfr-gpt recombinant plasmid altered the location at which the DHFR reading frame was terminated relative to the XGPRT initiation codon and demonstrated that this is a critical factor for the expression of XGPRT activity in transfected Cos-1 cells. Thus, when the DHFR frame terminated upstream or a very short distance downstream of the XGPRT initiator AUG, substantial levels of XGPRT activity were observed. When the DHFR frame terminated 50 nucleotides beyond the XGPRT initiator, activity was reduced about twofold. However, when the DHFR and XGPRT sequences were fused in-frame so that ribosomes which initiated at the DHFR AUG did not terminate until they encountered the XGPRT terminator, production of XGPRT activity was abolished. This dependence of internal translation initiation on the position of terminators of the upstream reading frame is consistent with the hypothesis that mammalian ribosomes are capable of translational reinitiation.

Animals↗
PubMed1986

Effect of upstream reading frames on translation efficiency in simian virus 40 recombinants.

In a previous report (S. Subramani, R. Mulligan, and P. Berg, Mol. Cell. Biol. 1:854-864, 1981), it was shown that mouse dihydrofolate reductase (DHFR) could be efficiently expressed from simian virus 40 recombinant viruses containing the DHFR cDNA in different locations in the viral late region. This was true even in the case of the SVGT7dhfr26 recombinant, which had the DHFR coding sequence 700 to 800 nucleotides from the 5' end of the mRNA, where it was preceded by the VP2 and VP3 initiator AUGs and a number of other noninitiator AUGs. To investigate the process of internal translation initiation in mammalian cells, we constructed a series of SVGT7dhfr recombinants in which the upstream VP2 and VP3 reading frame was terminated in various positions relative to the DHFR initiation codon. The efficient production of DHFR in infected CV1 cells depended on having the terminators of the VP2-VP3 reading frame positioned upstream or nearby downstream from the DHFR initiation codon. These results reinforce the notion that mammalian ribosomes are capable of translational reinitiation.

Base Sequence↗
PubMed1985

Transcription from a plant gene promoter in animal cells.

The promoter segment of a plant gene (maize alcohol dehydrogenase 1 (Adh 1)) has been fused to two bacterial reporter genes, Ecogpt (1) and neo (2), in pSV2-derived vectors and introduced into cultured mammalian cells by DNA transfection. The pAdh1-gpt plasmids transformed the recipient cells for resistance to mycophenolic acid plus xanthine (3) and the analogous pAdh1-neo plasmid transformed cells to G418 resistance (2). S1 analysis of transient transfections of CV1 cells with various derivatives of pAdh1-gpt confirmed that production of gpt mRNA is initiated at the Adh1 promoter at and near the same site used in transcription of the intact Adh1 gene in maize. Moreover, expression of the Adh1 promoter was increased 10-20 fold if the SV40 early region enhancer sequence was included in the same molecule.

Animals↗
PubMed1985

Rapid assay for detection of Escherichia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells.

Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1). This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-xanthosine (XR) by thin layer chromatography. The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt. During our studies, we have found that a human cell line (XP20S) converts xanthine to XMP. This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine. A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X.

Animals↗
PubMed1985

[Intraocular therapy of macular hole-induced retinal changes].

The treatment of retinal detachments due to macular holes without further central peripheral holes with intraocular tamponades (SF6, silicone oil) is preferred to episcleral techniques. After treatment of 24 cases of retinal detachments due to macular holes a therapeutic program is proposed.

Adult↗