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Biomedical subjects

S Lavi

Publications and source records attributed to S Lavi.

At least 37 records · Page 2Linked to original sources
PubMed1996

Abnormal melatonin secretion in male patients with hypogonadism.

Recently we have demonstrated that melatonin secretion is increased in untreated male patients with GnRH deficiency. Testosterone administration to these patients decreased melatonin secretion to normal levels. These data, however, did not exclude a gonadotropic effect on melatonin secretion. To further elucidate whether gonadal steroids and/or gonadotropins modulate melatonin secretion in humans we compared untreated young males with hypogonadotropic hypogonadism (IGD, n = 6), and hypergonadotropic hypogonadism caused by KlinEfelter's syndrome (KS, n = 11) to normal pubertal male controls (n = 7). KS patients were subdivided into two groups: KS-1, with low testosterone; and KS-2, with normal testosterone levels. Serum samples for melatonin concentrations were obtained every 15 min from 7 PM to 7 AM in a controlled light-dark environment with simultaneous sleep recordings. All KS patients had elevated gonadotropin levels and decreased melatonin levels. Mean (+/- SD) dark-time nocturnal melatonin levels in KS-1 were 92 +/- 21 pmol/L and were 146 +/- 46 pmol/L in KS-2 compared with 178 +/- 64 pmol/L in controls. Integrated nocturnal melatonin secretion values (AUC) were 64 +/- 14 pmol/min x L x 10(3) in KS-1 and 96 +/- 29 pmol/min x L x 10(3) in KS-2 compared with 116 +/- 42 pmol/min x L x 10(3) in controls. All IGD patients had low gonadotropin and testosterone levels. Their dark-time melatonin levels (286 +/- 26 pmol/L) and the AUC values (184 +/- 15 pmol/min/L x 10(3)) were increased. These data indicate that melatonin secretion is increased in male patients with GnRH deficiency and decreased in low testosterone hypergonadotropic hypogonadal patients. Taken together, our results suggest that both gonadotropins and gonadal steroids modulate melatonin secretion in humans.

Adolescent↗
PubMed1996

Decreased nocturnal melatonin secretion in patients with Klinefelter's syndrome.

OBJECTIVE: We have recently demonstrated that GnRH deficient male patients have increased nocturnal melatonin secretion which decreases to normal levels during testosterone treatment. The results suggested that sex steroids, rather than LH, modulate pineal melatonin in an inverse fashion. The purpose of this study was to characterize circulating melatonin levels in untreated males with hypergonadotrophic hypogonadism due to Klinefelter's syndrome (KS). DESIGNS: Prospective, controlled. SUBJECTS: Eleven patients with Klinefelter's syndrome and seven controls. Patients were subdivided into two groups: (1) with low testosterone, and (2) with normal testosterone levels. MEASUREMENTS: Serum samples for melatonin concentrations were obtained every 15 minutes from 1900 to 0700 h in a controlled light-dark environment. RESULTS: All patients had elevated FSH, LH and oestradiol (E2) levels. Mean (+/-SD) dark time nocturnal melatonin levels were significantly lower in low testosterone KS (92 +/- 19 pmol/l) compared with 146 +/- 42 pmol/l in normal testosterone KS and 179 +/- 59 pmol/l in controls (P < 0.02). A similar pattern was observed for the mean (+/-SD) peak melatonin levels (165 +/- 41, 236 +/- 59 and 293 +/- 89 pmol/l) in low testosterone KS, normal testosterone KS and controls, respectively (P < 0.01). Integrated nocturnal melatonin secretion values (AUC) were also lower in low testosterone KS (64 +/- 13) compared with 96 +/- 26 in normal testosterone KS and 116 +/- 39 pmol/min 1 x 10(3) in controls (P < 0.02). The time of melatonin peak and the time of the nocturnal melatonin rise as well as the light-time mean (+/-SD) serum melatonin levels were similar in all three groups. No correlations were found between melatonin and LH, FSH, or E2 levels. CONCLUSIONS: Melatonin secretion is decreased in male patients with low testosterone hypergonadotrophic hypogonadism whereas in normal testosterone Klinefelter's syndrome patients, melatonin secretory profiles are normal. The results suggest that the suppression of melatonin secretion in these patients is mediated by GnRH (either directly or indirectly) and/or oestradiol.

Adolescent↗
PubMed1996

Nocturnal melatonin and luteinizing hormone rhythms in women with hyperprolactinemic amenorrhea.

To examine the role of melatonin in pathological hyperprolactinemia we compared untreated young females (N = 5) with hyperprolactinemic amenorrhea owing to pituitary microadenoma to healthy female controls (N = 6). Serum samples for melatonin, prolactin, and luteinizing hormone (LH) concentrations were obtained every 15 min from 1900 hr to 0700 hr in a controlled light-dark environment with simultaneous sleep recordings. The mean (+/- SD) light-time period, dark-time period, and the integrated nocturnal melatonin secretion values (area under the curve, or AUC) in patients (51 +/- 11 pmol/L, 157 +/- 33 pmol/L, and 102 +/- 19 pmol/min.L x 10(3), respectively) were similar to the values obtained in controls (79 +/- 39, 165 +/- 44, 111 +/- 31, respectively). The onset of the nocturnal melatonin rise, peak level, and peak time were similar in the two groups. A significant nocturnal prolactin rise was observed in patients (112 +/- 9 vs. 65 +/- 11 micrograms/L, P < 0.006) and controls (19 +/- 2 vs. 10 +/- 3 micrograms/L, P < 0.006). The time of prolactin peak was similar in patients and controls (0424 +/- 3:36 vs. 0350 +/- 2:21) and paralleled that of melatonin (0354 +/- 1:46 vs. 0337 +/- 1:30). The mean +/- SD light-time period, dark-time period, and the AUC values of LH were similar in patients and controls. The number of LH pulses in patients (7.2 +/- 1.9 per 12 hr) were not different from those in controls (7.7 +/- 2.1). The LH pulse interval was 100 +/- 22 min in patients compared with 94 +/- 23 min in controls. The mean (+/- SD) nocturnal estradiol (E2) levels were significantly lower in patients (84 +/- 15 pmol/L) than in controls (224 +/- 77) (P < 0.005). Analysis of LH and melatonin secretory profiles revealed significant pulses for both hormones. No significant relationship was observed between the LH and melatonin pulses. However, a negative correlation between LH pulse amplitude and the number of melatonin pulses (P < 0.04) and a positive correlation between LH amplitude and duration of melatonin pulses (P < 0.04) were observed. Taken together, these data suggest that the suppression of normal ovarian cycles in women with hyperprolactinemic amenorrhea owing to pituitary microadenoma may be mediated by blocking of gonadotropin action by prolactin at the ovarian level; yet it remains possible that chronically elevated prolactin might prevent the LH surge and thus lead to amenorrhea. Pulsatile melatonin secretion is unaltered in these patients, and frequent occurrence of amenorrhea in this population is not mediated by melatonin.

Adenoma↗
PubMed1996

Nocturnal secretory patterns of melatonin, luteinizing hormone, prolactin and cortisol in male patients with gonadotropin-releasing hormone deficiency.

To clarify whether disorders of gonadotropin releasing hormone (GnRH) deficiency are associated with altered melatonin and pituitary hormones secretory patterns, we studied male patients with hypogonadotropic hypogonadism (IGD; n = 6), delayed puberty (DP; n = 7) and age-matched pubertal controls (n = 7). Serum samples for the determination of melatonin, luteinizing hormone (LH), prolactin and cortisol levels were obtained at 15 min intervals from 1900 to 0700 in a controlled light-dark environment, complete bed-rest and fasting with simultaneous sleep recordings. Mean (+/- SD) dark-time melatonin levels were significantly higher in IGD (286 +/- 26 pmol/L) and DP (205 +/- 44 pmol/L) compared with 178 +/- 64 pmol/L in controls (P < 0.003). So were the mean (+/- SD) peak melatonin levels (453 +/- 63, 346 +/- 106 and 292 +/- 96 pmol/L) in IGD, DP and controls, respectively (P < 0.03). Integrated nocturnal melatonin (AUC) values were also higher in IGD and DP (184 +/- 15 and 134 +/- 28 pmol/min/L x 10(3)) compared with 116 +/- 42 pmol/min/L x 10(3) in controls (P < 0.003). The time of onset of the nocturnal melatonin rise was observed earlier in IGD and DP patients as compared to controls. No correlations were found between melatonin and LH levels, between melatonin and prolactin levels, or between melatonin and cortisol levels. These data indicate that melatonin secretion is enhanced in male patients with GnRH deficiency. The lack of correlation between melatonin and LH suggest that circulating gonadal steroids, rather than LH, modulate melatonin secretion in a reverse fashion.

Adolescent↗
PubMed1996

A hierarchical network of interreceptor interactions determines signal transduction by Neu differentiation factor/neuregulin and epidermal growth factor.

The ErbB family includes four homologous transmembrane tyrosine kinases. Whereas ErbB-1 binds to the epidermal growth factor (EGF), both ErbB-3 and ErbB-4 bind to the Neu differentiation factors (NDFs, or neuregulins), and ErbB-2, the most oncogenic family member, is an orphan receptor whose function is still unknown. Because previous lines of evidence indicated the existence of interreceptor interactions, we used ectopic expression of individual ErbB proteins and their combinations to analyze the details of receptor cross talks. We show that 8 of 10 possible homo-and heterodimeric complexes of ErbB proteins can be hierarchically induced by ligand binding. Although ErbB-2 binds neither ligand, even in a heterodimeric receptor complex, it is the preferred heterodimer partner of the three other members, and it favors interaction with ErbB-3. Selective receptor overexpression in human tumor cells appears to bias the hierarchical relationships. The ordered network is reflected in receptor transphosphorylation, ErbB-2-mediated enhancement of ligand affinities, and remarkable potentiation of mitogenesis by a coexpressed ErbB-2. The observed superior ability of ErbB-2 to form heterodimers, in conjunction with its uniquely high basal tyrosine kinase activity, may explain why ErbB-2 overexpression is associated with poor prognosis.

Animals↗
PubMed1996

Induction of circles of heterogeneous sizes in carcinogen-treated cells: two-dimensional gel analysis of circular DNA molecules.

Extrachromosomal circular DNA molecules are associated with genomic instability, and circles containing inverted repeats were suggested to be the early amplification products. Here we present for the first time the use of neutral-neutral two-dimensional (2D) gel electrophoresis as a technique for the identification, isolation, and characterization of heterogeneous populations of circular molecules. Using this technique, we demonstrated that in N-methyl-N'-nitro-N-nitrosoguanidine-treated simian virus 40-transformed Chinese hamster cells (CO60 cells), the viral sequences are amplified as circular molecules of various sizes. The supercoiled circular fraction was isolated and was shown to contain molecules with inverted repeats. 2D gel analysis of extrachromosomal DNA from CHO cells revealed circular molecules containing highly repetitive DNA which are similar in size to the simian virus 40-amplified molecules. Moreover, enhancement of the amount of circular DNA was observed upon N-methyl-N'-nitro-N-nitrosoguanidine treatment of CHO cells. The implications of these findings regarding the processes of gene amplification and genomic instability and the possible use of the 2D gel technique to study these phenomena are discussed.

Animals↗
PubMed1996

Testosterone treatment alters melatonin concentrations in male patients with gonadotropin-releasing hormone deficiency.

Recently, we demonstrated that melatonin secretion is increased in untreated male patients with GnRH deficiency. As testosterone (T) can be aromatized to estradiol (E2), and both T and E2 increase during T enanthate treatment, we were interested in determining whether T treatment (when T and E2 levels were well matched with pubertal control values) has an effect on melatonin levels in these patients. We measured nocturnal serum melatonin levels during the administration of 250 mg testosterone enantale/month for 4 months in 12 male patients with idiopathic hypogonadotropic hypogonadism (IGD; n = 6) and delayed puberty (DP; n = 6). Serum samples for melatonin and LH determinations were obtained every 15 min from 1900-0700 h in a controlled light-dark environment. The results of melatonin profiles were compared with the pretreatment values in each group and with values obtained in six normal pubertal male controls. After 4 months of testosterone treatment, all patients attained normal serum testosterone (19.5 +/- 3.7 in IGD vs. 20.8 +/- 4.1 nmol/L in DP) and E2 levels (83 +/- 12 in IGD vs. 84 +/- 9 pmol/L in DP). Serum LH levels were suppressed in all patients during T treatment (0.12 +/- 0.1 in IGD vs. 0.12 +/- 0.2 IU/L in DP). Before T treatment, patient melatonin levels were greater than those in age-matched pubertal controls. Melatonin levels were equal in patients and controls when T and E2 levels were well matched. Mean (+/- SD) dark-time melatonin levels decreased from 286 +/- 23 to 157 +/- 36 pmol/L in IGD and from 217 +/- 32 to 133 +/- 47 pmol/L in DP (vs. 183 +/- 64 pmol/L in controls). The integrated melatonin values decreased to normal (from 184 +/- 16 to 102 +/- 21 in IGD and from 142 +/- 19 to 90 +/- 26 pmol/min.L x 10(3) in DP vs. 119 +/- 61 pmol/min.L x 10(3) in controls). The intraindividual variations in melatonin levels ranged from 7.2-14.5%. These data indicate that male patients with GnRH deficiency have increased nocturnal melatonin secretion. T treatment decreased melatonin secretion to normal levels. The results suggest that in GnRH-deficient male patients, sex steroids, rather than LH, modulate pineal melatonin in a reverse fashion.

Adolescent↗
PubMed1995

ErbB-3 mediates differential mitogenic effects of NDF/heregulin isoforms on mouse keratinocytes.

The family of Neu differentiation factors (NDFs, or heregulins) includes a dozen secreted glycoproteins, whose receptor binding domain displays two variants, alpha and beta, and they bind to two receptor tyrosine kinases, ErbB-3 and ErbB-4. Certain isoforms were reported to induce growth-arrest and differentiation of mammary tumor cells, while other breast cancer cell lines responded mitogenically. The present study addressed the biologic effects of various NDF isoforms on normal EGF-dependent epithelial cells, Balb/MK keratinocytes, that can undergo either proliferation or differentiation. We found that beta isoforms of NDF induced a mitogenic effect, that was significantly smaller than the maximal response to EGF. By contrast with NDF-beta, NDF-alpha isoforms exerted almost no mitogenic effect, but they were sufficient to maintain keratinocytes in culture. Consistent with their higher mitogenic potency, NDF-beta isoforms bound to Balb/MK cells with higher affinity (Kd = 2.2 nM) than alpha isoforms, however both groups shared their receptor, that we identified as ErbB-3. No transcript of ErbB-4 was detectable in the keratinocytes, but these cells express multiple NDF mRNAs and also ErbB-2. We conclude that different isoforms of NDF induce distinct growth regulatory effects on cultured keratinocytes, through direct interaction with ErbB-3.

Animals↗
PubMed1995

Increased nocturnal melatonin secretion in male patients with hypogonadotropic hypogonadism and delayed puberty.

Hypogonadotropic hypogonadism (IGD) and constitutional delayed puberty (DP) share a common pathophysiologic process, i.e. GnRH deficiency. Both conditions are heterogenous and exhibit different grades of GnRH deficiency. To discern whether these disorders of GnRH deficiency are associated with altered melatonin secretion profiles, we compared untreated young males IGD (n = 7) and DP (n = 7) to normal pubertal male controls (n = 6). Serum samples for melatonin, LH, and prolactin concentrations were obtained every 15 min from 1900 h to 0700 h in a controlled light-dark environment with simultaneous sleep recordings. Mean (+/- SD) darktime nocturnal melatonin levels were significantly higher in IGD (259 +/- 73 pmol/L) and DP (217 +/- 29 pmol/L) compared with 182 +/- 69 pmol/L in controls (P < 0.02). So were the mean (+/- SD) peak melatonin levels (410 +/- 117, 327 +/- 97 and 298 +/- 95 pmol/L in IGD, DP, and controls, respectively (P < 0.05). Integrated nocturnal melatonin secretion values (AUC) were also higher in IGD and DP (168 +/- 45 and 134 +/- 28) compared with 119 +/- 45 pmol/min.1 x 10(3) in controls (P < 0.02). The time of melatonin peak and the time of onset of the nocturnal melatonin rise were observed earlier in IGD and DP. Light-time mean (+/- SD) serum melatonin levels were similar in all three groups. No correlations were found between melatonin and LH levels, nor between melatonin and prolactin levels. These data indicate that melatonin secretion is increased in male patients with GnRH deficiency. The lack of correlations between melatonin and LH suggest that circulating sex steroids, rather than LH, modulate melatonin secretion in a reverse fashion.

Activity Cycles↗
PubMed1994

ErbB-3 and ErbB-4 function as the respective low and high affinity receptors of all Neu differentiation factor/heregulin isoforms.

Neu differentiation factor (NDF or heregulin) elevates tyrosine phosphorylation of the ErbB-2 receptor tyrosine kinase, and it was, therefore, thought to function as a ligand of this receptor. However, several lines of evidence raised the possibility that the interaction between NDF and ErbB-2 involves another molecule, which belongs to the family of epidermal growth factor receptors. To address this question we constructed soluble chimeric proteins between alkaline phosphatase and the extracellular domains of ErbB-2 and either ErbB-3 or ErbB-4, two newly recognized members of the epidermal growth factor receptor family. Using the soluble proteins we found that beta isoforms of NDF specifically bind to the ErbB-3 and ErbB-4 receptors but not to the soluble ErbB-2 protein. When ectopically expressed in monkey fibroblasts, the full-length ErbB-3 and ErbB-4 receptors conferred specific binding to NDF. In these cells ErbB-3 displayed lower ligand binding affinity than ErbB-4, but like the latter receptor it preferred to bind the beta isoform over the alpha class of NDFs. These results indicate that both ErbB-3 and ErbB-4 function as physiological receptors of all NDF isoforms and suggest that a still unknown ligand of ErbB-2 exists.

Amino Acid Sequence↗
PubMed1994

Brain neurons and glial cells express Neu differentiation factor/heregulin: a survival factor for astrocytes.

Neu differentiation factor (NDF, also called heregulin) was isolated from mesenchymal cells on the basis of its ability to elevate phosphorylation of ErbB proteins. Earlier in situ hybridization analysis showed that NDF was transcribed predominantly in the central nervous system during embryonic development. To gain insights into the role of NDF in brain we analyzed its distribution by immunohistochemistry and in situ hybridization. Late-gestation (day 17) rat embryos displayed high NDF immunoreactivity in both motor (e.g., putamen) and limbic (e.g., septum) regions. Lower levels of the factor were exhibited by adult brain, except for the cerebellum, where NDF expression was increased postnatally. Both neurons and glial cells were identified by immunohistochemistry as NDF-producing cells (e.g., pyramidal neurons in the cerebral cortex and glial cells in the corpus callosum). By establishment of primary cultures of rat brain cells we confirmed that NDF was expressed in neurons as well as in astrocytes. In addition, by using such primary cultures we observed that NDF treatment exerted only a limited mitogenic effect, which was accompanied by significant acceleration of astrocyte maturation. Furthermore, long-term incubation with the factor specifically protected astrocytes from apoptosis, implying that NDF functions in brain as a survival and maturation factor for astrocytes.

Animals↗
PubMed1994

Hairpin structures are the primary amplification products: a novel mechanism for generation of inverted repeats during gene amplification.

Early events of DNA amplification which occur during perturbed replication were studied by using simian virus 40 (SV40)-transformed Chinese hamster cells (CO60) as a model system. The amplification is observed shortly after carcinogen treatment, and the amplified sequences contain molecules organized as inverted repeats (IRs). SV40 amplification in vitro was studied by using extracts from carcinogen-treated CO60 cells. In the amplified DNA the SV40 origin region was rereplicated, while more distal sequences were not replicated even once. Using several experimental procedures such as sucrose gradients, "snap-back" assay, and two-dimensional gel electrophoresis, we show that the overreplicated DNA contains IRs which are synthesized de novo as hairpins or stem-loop structures which were detached from the template molecules. The fully replicated SV40 molecules synthesized by the HeLa extracts do not contain such IRs. We propose "U-turn replication" as a novel mechanism for gene amplification, accounting for the generation of extrachromosomal inverted duplications as a result of perturbed replication and template switching of the DNA polymerases.

Animals↗
PubMed1993

Replication protein A is the major single-stranded DNA binding protein detected in mammalian cell extracts by gel retardation assays and UV cross-linking of long and short single-stranded DNA molecules.

Band shift and UV cross-linking assays were used to analyze the major single-stranded DNA (ssDNA) binding activity in lysates of primate and rodent cells. The ssDNA binding activity behaved chromatographically similar to that of replication protein A (RP-A), a multisubunit protein containing three polypeptides of molecular mass 70, 34, and 14 kDa. A 70-kDa protein was found to harbor the ssDNA binding activity when UV cross-linked to long ssDNA or to oligonucleotide probes. Monoclonal antibodies against the 70- and the 34-kDa subunits produced super-gel-shift patterns, demonstrating that the reactive protein is indeed RP-A and that the retarded native binding complex included both subunits. RP-A displayed oligonucleotide-specific binding dependent on oligomer length. Increasing oligonucleotide length led to the formation of slow migrating complexes harboring multiple RP-A molecules, suggesting that an interval of about 20-30 bases is required for the binding of RP-A molecules. While similar binding activity was detected in cell extracts derived from proliferating and quiescent cells, a sharp decline in ssDNA binding activity was observed in the SV40-transformed Chinese hamster cell line 631 following UV irradiation. The nature of this decrease in activity and its possible effect on DNA replication is discussed.

Animals↗
PubMed1992

Carcinogen-induced activation of SV40 gene expression in a semi-permissive environment.

Carcinogen-induced expression of the integrated viral genome was examined on SV40-transformed Chinese hamster cells. Carcinogen treatment markedly increased the transcription rate and the steady state mRNA level of both early and late viral transcripts. Carcinogen-induced transcription was mediated by RNA polymerase II. The increase in viral gene expression was also detected at the protein level, although at a reduced amplitude. Enhanced transcription was apparent as early as 12 hr postexposure and was considerably elevated after 24-36 hr. The increased gene expression depended on the existence of a functional replication machinery, as indicated by two lines of evidence. First, a cell line that harbors origin-deleted SV40 failed to respond to carcinogen treatment by increasing transcription and expression of T antigen. Furthermore, carcinogen-induced overtranscription was inhibited by aphidicolin, an inhibitor of DNA polymerase alpha. The involvement of the replication apparatus in the enhanced expression points to mechanistic similarities between the carcinogen-induced viral gene expression in the drug-treated semipermissive cells and the SV40 lytic pathway under permissive conditions. It is therefore suggested that cellular permissivity to viral development is enhanced following exposure to carcinogens. The implications of these findings for the nature of cellular permissivity to viral infection and the synergistic effects of carcinogens and tumor viruses are discussed.

Amanitins↗
PubMed1992

Modulation of the cellular phenotype by integrated adeno-associated virus.

The adeno-associated virus (AAV) rep gene encodes a series of overlapping, multifunctional, nonstructural proteins (Rep proteins) which regulate the viral life cycle and which are also capable of trans-regulating nonviral gene expressions (reviewed in Berns, 1990, Microbiol. Rev. 54, 316-329). To investigate the expression of the AAV rep gene in a cellular chromosomal context, SV40-transformed Chinese hamster embryo (OD4) cells were infected with an AAV/neo hybrid virus and progeny resistant to the antibiotic G418 were selected and amplified. Chromosomal integration and RNA transcription of the AAV and neo DNA inserts were confirmed by Southern and Northern blotting procedures. One of the G418R cell lines stably expressed a protein which reacted specifically with AAV anti-Rep antiserum in Western immunoblots. The stable integration of AAV rep DNA, which did not interfere with cell proliferation under normal growth conditions, was associated with two changes in cellular phenotype: eight of nine lines were markedly more sensitive to UV light (254 nm) than were the parental OD4 cells; and seven of the nine lines had lost the capacity to promote SV40 origin DNA amplification in vitro, in contrast to the parental OD4 cells. OD4 cells transformed to G418R by AAV/neo DNA constructs with a deleted rep gene, or by a neo DNA construct lacking AAV DNA, did not display these phenotypic changes. It is suggested that stable integration of the AAV rep gene interferes with cellular processes connected with DNA repair and gene amplification.

Animals↗
PubMed1992

The mechanism of carcinogen-induced DNA amplification: in vivo and in vitro studies.

Exposure to chemical carcinogens provides a means for the enhancement of the frequency of gene amplification and for the facilitation of research into its mechanism(s). Using carcinogen-induced SV40 amplification as a model system it was shown that amplification of the viral sequences occurs via a replication-dependent mode. This process involves overactivation of the origin region and the generation of inverted repeats. Carcinogen-induced enhancement of gene amplification is triggered by cellular factors that could act in trans. An in vitro amplification system, based on extracts from carcinogen-treated cells and SV40 template sequences, was used to further characterize the amplification intermediates. The major products of overreplication in this system consist of sequences derived from the origin region. Our studies suggest that the ability to overreplicate the origin region in vitro derives from the combined action of carcinogen-induced factors that trigger overinitiation, with the inherent inability of Chinese hamster cell extracts to fully replicate large plasmid templates. The newly replicated sequences are not associated with the parental molecule and contain hairpin or stem and loop structures. Based on these findings we propose a novel replicative mechanism for DNA amplification that allows the de novo formation of hairpin structures. According to this model, an obstruction of the replication fork may cause an overturning of the DNA polymerase, followed by a template switch that leads to the use of the newly replicated strand as a template. This mode of replication results in the generation of hairpin structures which can function as precursors for the duplicated inverted repeats which are commonly observed in amplified genomes. This model is supported by our in vitro and in vivo studies. The relevance of this model for the amplification of cellular sequences is discussed.

Animals↗
PubMed1991

Minimally oxidized low-density lipoprotein induces tissue factor expression in cultured human endothelial cells.

Oxidatively modified low-density lipoprotein is present in atherosclerotic lesions and has been proposed to play an important role in atherogenesis through its biologic effects on vascular cells. This study examined the effects of minimally oxidized preparations of LDL (MM-LDL) on tissue factor (TF) expression by cultured human endothelial cells. Low-density lipoprotein purified from normal donors was modified by exposure to iron or by prolonged storage, resulting in levels of thiobarbituric acid-reacting substances of approximately 2.5 to 4 nmoles/mg cholesterol. Preparations had less than 2.5 pg of endotoxin per microgram LDL and had no intrinsic procoagulant activity. This form of modified but not native LDL induced TF expression in endothelial cells in a time- and dose-dependent manner. Peak TF coagulant activity in cells exposed to 40 micrograms/ml MM-LDL were observed at 4 to 6 hours, and ranged from 50 to 500 pg/10(5) cells, compared with less than 10 pg/10(5) cells exposed to native LDL. Northern blot analysis showed TF mRNA levels to increase approximately 30-fold with exposure to MM-LDL for 2 hours. Induction of TF activity was dependent on the concentration of MM-LDL from 1 microgram/ml to 80 micrograms/ml, a range in which cell viability and morphology were unaffected. The findings suggest that minimally oxidized LDL may be a local mediator promoting thrombosis in atherosclerotic lesions.

Blood Coagulation↗