Condensation of vector DNA by the chromosomal protein HMG1 results in efficient transfection.

The aim of this study was the search for a method of vector packaging using natural chromatin constituents. The interaction of the chromosomal non-histone protein HMG1 with a vector plasmid (pLTEneo) was studied by sedimentation analysis and electron microscopy at physiological salt concentration. At high protein input the complexes exist in a condensed, monodisperse form sedimenting with 80 S irrespective of the supercoiled or relaxed conformation of DNA. Saturation binding is already observed at much lower input ratios. Dilution of 80 S complexes results in decondensation of the complexes. In the decondensed complex form, HMG1 binds in a bead-like manner to specific DNA regions. Condensation by HMG1 is sufficient to introduce the vector into mammalian cells without the need for unphysiological additives. The transfection rates were similar to or even higher than those obtained by the calcium phosphate coprecipitation technique.

Effect of a mammary-derived growth inhibitor on the expression of the oncogenes c-fos, c-myc and c-ras.

A mammary-derived growth inhibitor (MDGI) inhibits the resumption of growth of stationary Ehrlich ascites carcinoma (EAC) cells in vitro. The present study shows that the resumption of growth is accompanied by a rapid increase of the steady state mRNA level of the proto-oncogenes c-fos, c-myc and c-ras, which is reduced by MDGI. EAC cells from the exponential growth phase insensitive to MDGI did not show a reduced RNA expression. The effect of MDGI represents a novel activity at the level of gene expression and suggests a link to exist between growth inhibition and the reduction of c-fos, c-myc and c-ras expression.

Properties of two epithelial cell lines derived from HPV-associated cervical and vulvar lesions.

Two epithelial cell lines were established from human papilloma virus (HPV) 18 or 16 associated tumours, characterised as poorly and well differentiated squamous cell carcinomas of the cervix uteri (EC) and the vulva (GC), respectively. The cell lines are described by their morphology, biological parameters, and immunological markers. Both cell lines have undergone approximately 35 passages in vitro. HPV16 and 18 DNA are maintained integrated into the host cell DNA. Expression of epithelial cell markers--cytokeratins K1, K10, K13, K14 and involucrin, proliferation-specific proteins, proliferating cell nuclear antigen (PCNA) and Ki67 as well as the epidermal growth factor (EGF) receptor were monitored by indirect immunofluorescence studies. The cytoplasmic and membrane-associated locations of EGF receptor molecules in EC and GC cells, respectively, suggest a differently regulated expression. Studies of the HPV18 oncogene transcription revealed marked differences of amplimers between HeLa and EC cells, such as an additional fragment, probably corresponding to a E6**--E7 splice product, and a radical shift in transcription pattern observed in various sections of the tumour tissue. Injected subcutaneously into nu/nu mice both cell lines were non-tumorigenic.

Characterization of human papillomavirus type 16 activity in separate biopsies from a carcinoma of the cervix uteri.

Human papillomavirus (HPV) 16-specific nucleic acid sequences were analysed in separate biopsies taken from a patient with a poorly differentiated squamous cell carcinoma of the uterine cervix. Biopsies were obtained from histopathologically normal epithelium adjacent to the carcinomatous epithelium, the primary carcinoma and a metastatic lymph node. Signals characterizing viral DNA and oncogene transcription were obviously differentiation dependent as shown by in situ hybridization of viral nucleic acids and immunofluorescence of epithelial differentiation specific proteins. In histologically normal parts of the epithelium viral DNA was amplified at the transition from basal to maturing cells, whereas E6/E7 genes were actively transcribed mainly in maturing epithelial cells following the basal cell layer. Some of the cells in the primary carcinoma and in the metastatic lymph node expressed involucrin at increased levels. Signals for viral DNA and HPV 16-specific E6/E7 transcripts decreased in intensity during differentiation in an inverse relationship to the observed involucrin increase in those cells. The absence of Ki67 in cells expressing large amounts of involucrin as revealed by immunostaining, support the inverse correlation between differentiation of cancer cells, HPV 16 replication and E6/E7 transcription. The changes in cytokine expression may indicate an HPV 16 associated disruption of normal cytokine expression pattern in the carcinoma.

The course of enzymatically induced lipid peroxidation in homogenized porcine kidney tissue.

Homogenization of mammalian tissue--exemplified by porcine kidney-- causes enzymatically induced lipid peroxidation (LPO) processes proven by measuring the amounts of the typical lipid peroxidation products 9- and 13-hydroxy-octadecadienoic acid (HODE) either after homogenization in aqueous (activation of enzymes) or an organic (inactivated enzymes) solvent. A kinetic study revealed that the level of the 9- and 13-isomer reached maximum values 6 hours after tissue injury. Within one day the amount of these primary oxidation products was reduced fast, indicating that they undergo degradation in their biological environment. In contrast, the level of 10-hydroxy-octadecanoic acid--obviously derived from LPO of oleic acid--increased continuously even after one day. These observations reflect that the generation and degradation of hydroperoxides occurs at different rates which might be of interest in pathological processes connected with tissue injury, e.g. myocardial infarction.

Loss of bovine papillomavirus DNA replication control in growth-arrested transformed cells.

The bovine papillomavirus type 1 (BPV-1) genome replicates as a plasmid within the nuclei of BPV-1-transformed murine C127 cells at a constant multiple copy number, and spontaneous amplification of the viral DNA is rarely observed. We report here that a mutant BPV-1 plasmid within a contact-inhibited C127 cell line replicated as a stable multicopy plasmid in exponentially growing cells but amplified to a high level in confluent cell culture. In situ hybridization analysis revealed that most of the mutant viral DNA amplification occurred in a minor subpopulation of cells within the culture. These consisted of giant nondividing cells with greatly enlarged nuclei, a cell form which was specifically induced in stationary-phase cultures. These observations indicated that expression of a viral DNA replication factor was cell growth stage specific. Consistent with this hypothesis, considerable amplification of wild-type BPV-1 DNA associated with characteristic giant cell formation was observed in typical wild-type virus-transformed C127 cultures following a period of growth arrest achieved by serum deprivation. Further observations indicated that induction of the giant-cell phenotype was dependent on BPV-1 gene expression and implicated a viral E1 replication factor in this process. Moreover, heterogeneity in virus genome copy numbers within the giant-cell population suggested a complex regulation of induction of DNA synthesis in these cells. It appears that this process represents a mechanism employed by the virus to ensure maximal viral DNA synthesis within a growth-arrested cell. Fundamental questions concerning the integration of the virus-cell control circuitry in proliferating and resting cells are discussed.

Plasmids expressing the Herpes simplex virus thymidine kinase gene in mammalian and bacterial cells.

Two plasmids containing either the complete thymidine kinase gene of Herpes simplex virus type I (pSK2) or the gene without the remote control sequence (pSK1) just behind the lac promoter and the first codons of the lacZ gene were constructed. Both plasmids efficiently transform mouse Ltk- cells as well as E. coli tk- cells to the Tk+ phenotype and are well suited for plasmid rescue from transformed mouse cells by direct functional selection for tk expression using a tk- mutant of E. coli C600.

A well transformable E. coli tdk-strain--suitable for direct rescue of tk gene plasmids from mammalian cells.

An E. coli C600 tdk strain which is suitable for the direct rescue of the eukaryotic HSV-1 tk gene by functional selection in E. coli is described. This E. coli tdk mutant failed to grow on fluorouracil selection plates and showed values for thymidine kinase activity smaller than in the tdk negative KY 895 strain. It is genetically well transformable, i.e. the transformation efficiency with plasmid DNA from pSK1 amounts to 5 X 10(6) or 3 X 10(8) depending on the transformation procedure applied.

Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells?

The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tcr region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed.

High level gene expression in mammalian cells by a nuclear T7-phase RNA polymerase.

Here we describe a novel expression system for mammalian cells which is based on transcription of hybrid genes containing T7 phage promoters by a T7 phage RNA polymerase targeted to the nucleus of the host cells. The RNA polymerase gene of T7 phage has been modified by substituting a sequence encoding the nuclear location signal of SV40 large T antigen for the N-terminal part of the polymerase gene. Expression of the modified gene is driven by the mouse metallothionein promoter in transfected mouse Ltk- cells resulting in high concentration of the polymerase in the nucleus. Nuclear T7 RNA polymerase directs efficient transcription of the cat gene under control of a T7 promoter. T7 constructs are expressed at a level at least 6 fold higher than the prototype pRSVcat. The unique properties of this heterologeous expression system are discussed.

Rescue of transfected genes from mammalian cells by functional selection in Escherichia coli.

We have established procedures for reisolating a transfected gene from mammalian cells by selection in Escherichia coli for the function of the gene product using the Herpes simplex virus thymidine kinase gene as a model. Rescue of the gene is accomplished by three different methods. The tk gene is recloned into a plasmid in which it is hooked up by either the lac promoter or a lac/tk hybrid promoter, or the original plasmid is cut out of the host cell DNA. As the lac/tk hybrid gene can be expressed and selected both in the mammalian and E. coli cells, this type of gene rescue allows investigations on mutagenesis and methylation processes. Additionally, it offers a simple way of studying the integration of the transfected gene into the mammalian genome.

Transfection by DNA-nuclear protein HMG1 complexes: raising of efficiency and role of DNA topology.

We have developed a novel and efficient transfection method based on the introduction of foreign DNA into mammalian cells in form of complexes of vector DNA with the nuclear protein HMG1. In this study, it is shown that a stabilization of the complexes against dilution dissociation by addition of soluble CaCl2 or by excessive HMG1 enhances the transfection efficiency. Furthermore, there are no differences in the transfection abilities between the 3 topological DNA forms, viz., supercoiled, open relaxed and linear DNA, if delivered to cells as HMG1-DNA complexes. It is further shown that transfection-inactive complexes of the core histones with foreign DNA can be activated in transfection by the addition of HMG1.

Pharmacological modulation of IL-6 and IL-8 secretion by the H1-antagonist decarboethoxy-loratadine and dexamethasone by human mast and basophilic cell lines.

Mast cells and basophils are central effector cells of allergic reactions and are involved in inflammatory diseases. These cell types produce an array of mediators including a broad spectrum of cytokines. In order to examine whether antiallergic drugs modulate the release of these mediators, we have investigated the influence of dexamethasone and decarboethoxy-loratadine (DEL), the active metabolite of the H1-blocking agent loratadine, on the release of IL-6 and IL-8 by the human mast cell line HMC-1 and the human basophilic cell line KU812 by ELISA. Dexamethasone (10(-6)-10(-11) M) or Del (10(-5)-10(-14) M) were added to the cells either 1 h prior to or simultaneously with PMA and Ca-ionophore A23187. When preincubated with the cells, DEL dose-dependently suppressed IL-6 release by up to 40% and IL-8 release by up to 50%. Dexamethasone potently suppressed secretion of both cytokines if simultaneously added to the cells with the stimuli by up to 60% and after preincubation by up to 80%. Since both antihistamines and glucocorticoids are used for treatment of allergic diseases, the findings reported here indicate that these drugs may modulate allergic reactions via inhibition of cytokine release from mast cells and basophils.

Transfer of a human preproinsulin gene containing plasmid into non-pancreatic mammalian cells.

A recombinant plasmid containing the human preproinsulin gene fused to the mouse metallothionein-I-promoter was transferred into Vero cells from a monkey kidney cell line by protoplast fusion. The transient formation of immunoreactive insulin was demonstrated by RIA and studied in the absence and in the presence of zinc and cadmium ions. The recombinant plasmid was encapsulated into reverse-phase evaporated vesicles and the influence of the sonication time on the encapsulated DNA has been studied. It was demonstrated that, although DNA fragmentation occurs, at least a part of the encapsulated DNA retains its biological activity.

DNA transformation of a pluripotent mouse embryonal stem cell line with a dominant selective marker.

The mouse embryonal stem cell line BLC 1 growing on feeder layer was treated with a calcium phosphate/DNA precipitate prepared with DNA of plasmid pAG60 which harbors the Tn5-derived neo gene, thus encoding resistance to G418, an aminoglycoside antibiotic. Transfection performed on feeder layer resulted in the formation of G418-resistant clones T1 and T2/K26. The stable integration of the transformed neo gene was confirmed by dot hybridization in all descendant cultures of clones T1 and T2/K26 as well as in the tumors derived from them. In vivo and in vitro differentiation revealed the pluripotent status of the transformants. Tumors derived from T1 and T2/K26 contained various tissues with derivatives of all three primary germ layers.

Incidence and prevalence of juvenile chronic arthritis in East Berlin 1980-88.

OBJECTIVE: To estimate the incidence and prevalence rates of juvenile chronic arthritis (JCA). METHODS: The study population was children under 16 years of age living in the East Berlin area (part of the former German Democratic Republic). By admission order that was effective up to 1990, all children with symptoms of a rheumatic disease living in the East Berlin area had to be referred to the 2nd Children's Hospital at Berlin-Buch. This specific condition allowed us to ascertain cases from the clinical records and to calculate population rates. Based upon this data, the results of surveys with different methods of case ascertainment are compared. RESULTS: An incidence rate of 3.5 per 100,000 and a prevalence rate of 2.0 per 10,000 children were calculated. The frequency of JCA is higher for girls, with an incidence of 4.3 per 100,000 and a prevalence of 2.3 per 10,000. The figures for boys are 2.7 per 100,000 and 1.7 per 10,000, respectively. CONCLUSION: Because of the specific prerequisites, the population rates of prevalence and incidence that were based on clinical records can be regarded as valid in this study. Deviant results of other surveys can be explained by differences in the study design or in the diagnostic procedures used.

Rescue of a tk-plasmid from transgenic mice reveals its episomal transmission.

This communication demonstrates the usefulness of the plasmid rescue procedure for recovery of plasmids from transgenic mice. We have microinjected the plasmid pSK1 harbouring the Herpes simplex virus thymidine kinase gene into fertilized mouse oocytes and succeeded in recovering plasmids from newborns by transformation of E. coli either with HindIII cut cellular DNA or with uncut DNA. The majority of the rescued plasmids were indistinguishable from pSK1 by restriction analysis. The rescued plasmids proved to be functionally active in a transient expression assay in mouse Ltk- cells. The pSK1 DNA sequences were inherited by up to 90% of the second generation progeny mice, which is not in agreement with a Mendelian transmission of heterozygous markers integrated into a single site of the chromosome. These data support the assumption that germ line transmission of non-integrated episomal plasmids can occur.

The efficiency of genetic transformation of mammalian cells by transfection and microinjection depends on the transferred gene.

The efficiency of genetic transformation of mammalian cells was analysed with respect to the kind of the transferred gene and the selective system. Plasmids pAGO and pAG60 harboring the thymidine kinase gene of Herpes simplex virus type 1 and the bacterial neomycin resistance gene, respectively, were compared concerning their ability to transform mouse Ltk-aprt- cells. Using the calcium phosphate technique the neomycin resistance gene transformed at least ten times more efficiently than the thymidine kinase gene (3 X 10(-3) versus 2 X 10(-4] whereas the difference is even more impressive following microinjection of the plasmids into the nuclei (2 X 10(-1) versus 2.5 X 10(-3]. The neomycin system also proved to be more effective in secondary gene transfer experiments and, thus, seems to be the most convenient marker for cotransfer experiments.

Prognosis of patients with juvenile chronic arthritis and juvenile spondyloarthropathy.

OBJECTIVE: Evaluation of the course and the prognosis of juvenile chronic arthritis (JCA) and juvenile spondyloarthropathy (JSpA). METHODS: The entire medical histories of 171 patients with JCA or JSpA were reviewed. The study cohort comprised 102 patients with oligoarticular, 17 with systemic, and 24 with polyarticular onset of JCA; 28 patients had a SpA; 91 patients with JCA from a population based cohort were included in that study cohort. The mean period of followup was 7.4 years. The probability of remission was estimated by survival analysis methods (Kaplan-Meier method). RESULTS: After a disease duration of 10 years the highest probability of complete remission was estimated for patients with oligoarticular or systemic onset of JCA (54% and 38%, respectively). In the oligoarthritis group with late onset of JCA, a lower probability of remission was found for the HLA-B27+ patients compared with HLA-B27- patients. Patients with polyarticular onset of JCA had the poorest prognosis, with a significantly lower probability of complete remission (15%) within 10 years, more secondary injuries, and a lower functional capacity at followup. Patients with JSpA showed a 17% probability of remission after a disease duration of 5 years and ranged between the remission rates for oligoarticular and polyarticular JCA. The estimated remission rates for the patients with JCA in the population based cohort and in the whole cohort were quite similar. CONCLUSION: Our data suggest a favorable prognosis for JCA and JSpA in general, but with differences among the subtypes. It seems that more than 50% of the patients with JCA and JSpA reach adulthood with active arthritis and need further rheumatological care.