Inactivation of plasmid reporter gene expression by one benzo(a)pyrene diol-epoxide DNA adduct in adult rat hepatocytes.

Polycyclic aromatic hydrocarbons such as benzo(a)pyrene diol-epoxide (BPDE-I) cause hepatocellular carcinoma. To identify short-term carcinogen effects, we studied hepatocytes transfected with nonreplicating plasmids, adducted covalently with BPDE-I, varying in promoter structure and encoded reporter gene (beta-galactosidase or luciferase). BPDE inactivated gene expression as a first-order function of BPDE concentration in adduction reactions. No evidence of cytotoxicity, diminished coprecipitation and availability, enhanced nicking of supercoiled forms and reduced cellular uptake, or instability of adducted plasmids was observed. At low BPDE:plasmid ratios, inactivation occurred with 1 adduct/plasmid within a target 23-27% of plasmid bases. Using nuclear extracts and BPDE-adducted G-free cassette-encoding plasmids, the fraction of full-length RNA polymerase II-initiated transcripts also declined as a first-order function of BPDE concentration when approximately 3 adducts were distributed among 48% of plasmid bases. These observations suggest that carcinogens such as BPDE block mRNA transcription along DNA templates by forming limited numbers of persistent adducts at coding or noncoding sites.

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes.

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.

Attenuation of gene expression by a trinucleotide repeat-rich tract from the terminal exon of the rat hepatic polymeric immunoglobulin receptor gene.

A 359 bp terminal exon fragment of the rat polymeric immunoglobulin receptor gene has been tested for biological effects. The fragment contains an S1 nuclease-sensitive microsatellite with d(GGA) and d(GAA) trinucleotide repeats that are expressed discordantly in the 3'UTRs of liver mRNAs encoded by the single copy gene. When human A293 cells are transfected with expression plasmids carrying this fragment in forward orientations, flanking or replacing poly(A) cassettes in the 3' ends of the transcription units, luciferase reporter gene expression is attenuated 47 to 59% or 98.5%, respectively. In contrast, when the fragment is tested similarly in reverse orientation, there is significantly less or no attenuation of gene expression. These observations, and computer models of partial triplet repeat DNA tertiary and RNA secondary structures, suggest that this fragment might regulate gene expression by orientation and position-dependent mechanisms at transcriptional and post-transcriptional levels.

Functional pleiotropy of an intramolecular triplex-forming fragment from the 3'-UTR of the rat Pigr gene.

A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3'-untranslated region (3'-UTR) and inserted into 3'-UTR or 3' flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5' flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5' and 3' flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3'-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.

Site-specific integration of targeted DNA into animal cell genomes.

Novel genetically engineered retroviral vectors and targeting plasmids are described that enable the site-specific targeting of exogenous DNA into the genomes of cultured animal cells. The protocol involves the transduction of competent cells by a chimeric retroviral vector containing a transcription unit composed of two linked cassettes: an upstream marker gene under the control of the viral 5' LTR; and a downstream reporter trap containing a strong promoter 5' to a 48bp yeast FRT element. When cells containing such integrated units are co-transfected with a plasmid encoding yeast FLP recombinase and a promoterless targeting plasmid containing a reporter cDNA tract 3' to an homologous FRT element, the targeting plasmid recombines at the chromosomally preconfigured FRT site, and a new hemizygous function is introduced into the downstream cassette. These reagents provide a new portable system for site-specific targeting of chemically modified genes into uniform and unique sites in genomically integrated transcription units.

Construction of a fusion protein between protein A and green fluorescent protein and its application to western blotting.

Aequorea green fluorescent protein (GFP) and protein A were fused and expressed in Escherichia coli. The fluorescent native fusion protein (PA-GFP) migrated at 47 kDa in SDS-PAGE. However, the non-fluorescent denatured PA-GFP migrated at 57 kDa which corresponds to the theoretical molecular mass. Although the reason(s) for this mobility shift between fluorescent and non-fluorescent molecules remains unclear, the small ring structure within the native molecules may affect their mobility. The cell extract, prepared from an E. coli strain producing PA-GFP, was used in Western and dot blots. The sensitivity and specificity of the PA-GFP detection were sufficient for rapid and easy screening.

Immediate reproducibility of tilt-table test results in elderly patients referred for evaluation of syncope or presyncope.

We studied the immediate reproducibility of the results of the head-up tilt-table test in elderly patients (age > or =60 years). Twenty-seven consecutive men underwent 51 tests. The overall reproducibility was 98%, including 92% of a positive test and 100% of a negative test. The finding of this study validates the use of intravenous pharmacologic intervention in the elderly population.

Proliferation of hepatocytes.

Hepatocyte proliferation may be controlled by reversible patterns of endocrine changes, monitored by the liver, involving known hormones and their receptors. A two-programme model of related interactions among nutrients, specific lipoproteins, and highly phosphorylated nucleotides is postulated. This hypothesis stems from in vitro studies of rat hepatocyte proliferation under chemically defined conditions and from in vivo studies using partially hepatectomized, hormone-infused, developing and lipotrope-deficient rats. Certain findings are discussed with regard to receptor systems which show negatively cooperative properties; to problems of proliferative specificity; and to novel approaches for defined studies of chemical hepatocarcinogenesis.

Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation.

Serum-free media containing 10-50 ng insulin, glucagon and epidermal growth factor (EGF) ml-1 stimulate adult rat hepatocyte proliferation in 10-15 day old primary liver cell cultures. The kinetics of this response simulate hepatocellular transitions that accompnay liver regeneration after 67% hepatectomy. Amiloride, a Na+ influx inhibitor, reversibly blocks these transitions in vitro (ID50 approximately 0.02 mM) and in vivo (ID50 approximately 25 mg kg-1). Inhibition is observed with other cation flux modulators, including ouabain (ID50 approximately 0.2 mM), 0.2 microM monensin and 0.2 microM nigericin, but not with 0.3 mM furosemide or tetrodotoxin. The prereplicative interval in culture (0-12 hr) is characterized by preferential cellular responsiveness to EGF (0-3 hr) followed by insulin plus glucagon (3-12 hr). Parallel culture and animal studies show that the amiloride-sensitive and prereplicative intervals coincide. In culture, a "burst" of 22Na+ influx, stimulated by peptide-supplemented media within 1 min but decreased later at 12 hr, is retarded by amiloride. This drug also blocks delayed prereplicative events involving increased amino acid "A" transport system function at 4-8 hr, and 3H-uridine and 3H-leucine incorporation into RNA and protein, respectively, at 8-12 hr. These findings suggest that at least two time-ordered processes are necessary to initiate hepatic growth fully: first, activation of Na+ flux systems by peptides similar or identical to EGF; and second, potentiation of these and subsequent cellular events by the combined action of insulin plus glucagon. [Amiloride: N-amidino-3,5-diamino-6-chloropyrazinecarboxamide; furosemide: 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid; AIB: alpha-aminoisobutyric acid; ID50: administered dose giving 50% inhibition of a maximal response; dFBS: dialyzed fetal bovine serum; L.I.: 3H-dT nuclear labeling index.]

Giant hairpins formed by CUG repeats in myotonic dystrophy messenger RNAs might sterically block RNA export through nuclear pores.

Energy minimization calculations were used to generate secondary structures of partial and full-length myotonic dystrophy messenger RNAs (DMPK mRNAs) carrying variable numbers of CUG triplet repeats (n = 0 to 500). The results suggest that (1) unitary hairpins are the most stable structures formed; (2) long-axis distances of unfolded hairpins are directly proportional to CUG repeat numbers; and (3) hairpins composed of CUG repeats might form interstem clusters that are stabilized by hydrogen or ionic bonds. A model is proposed whereby DMPK mRNAs are sterically impeded from transport through nuclear pores, by giant hairpins or hairpin clusters formed by CUG repeats above a limit size (n > or = 44).

Primary rat hepatocytes express cyclin D1 messenger RNA during their growth cycle and during mitogenic transitions induced by transforming growth factor-alpha.

Cyclin D1 messenger RNA expression was investigated in differentiated proliferation-competent primary cultures of adult rat hepatocytes. Periodic expression was observed during the 12-day growth cycle (approximately two population doublings). Upon reaching a quiescent G(o)-state, 4-kb cyclin D1 mRNA levels were undetectable. When G(o) cultures were shifted into defined media without or with 0.2-0.8 nM TGF-alpha, conditions that reinitiate full proliferative transitions synchronously, cyclin D1 mRNA levels were elevated 1.2-4.6-fold, respectively. These findings support the hypothesis that hepatic mitogens stimulate periodic cyclin mRNA expression directly.

Profound hypotension in a tetraplegic patient following angiotensin-converting enzyme inhibitor lisinopril. Case report.

We present the case of a 60 year old C6 complete tetraplegic patient who developed profound hypotension following initiation of the angiotensin-converting enzyme inhibitor lisinopril to control blood pressure. Other causes of hypotension, such as myocardial infarction and sepsis was ruled out. Inhibition of the renin-angiotensin-aldosterone system was the probable cause of hypotension. This case demonstrates the critical importance of the renin-angiotensin-aldosterone axis in the maintenance of blood pressure in tetraplegic patients, who may lack input from the brain to sympathetic neurons, and therefore have increased reliance on the renin-angiotensin-aldosterone axis for the maintenance of blood pressure. Angiotensin-converting enzyme inhibitors should be avoided in tetraplegic patients, unless other treatment modalities are ineffective.

Transforming growth factor-alpha stimulates proto-oncogene c-jun expression and a mitogenic program in primary cultures of adult rat hepatocytes.

Human transforming growth factor-alpha (TGF-alpha, MW 5547) initiates a mitogenic program in "quiescent" 11-to 13-day-old primary cultures of adult rat hepatocytes. Using validated growth reinitiation assays and chemically defined conditions (Koch and Leffert, 1979a) that simulate proto-oncogene expression in regenerating liver (Kruijer et al., 1986), we find that 5.4 nM TGF-alpha stimulates: (i) increases in rates of amiloride-sensitive 22Na+ uptake; (ii) a transient induction in steady-state mRNA levels of proto-oncogene c-jun; (iii) specific increases in hepatocyte nuclear [3H]dT labeling indices, augmented synergistically by insulin and glucagon; and (iv) increases in rates of S-phase entry. Comparative studies indicate that TGF-alpha is a more effective hepatocyte growth promoter than mouse epidermal growth factor. These observations, and published reports linking normal and cancerous liver as biosynthetic sources of TGF-alpha, suggest an autocrine or paracrine role for TGF-alpha in the control of hepatic growth, regeneration, and gene expression.