Template-directed arrest of mammalian mitochondrial DNA synthesis.

Mammalian mitochondrial DNA often contains a short DNA displacement loop at the heavy-strand origin of replication. This short nascent DNA molecule has been used to study site-specific termination of mitochondrial DNA synthesis in human and mouse cells. We examined D-loop strand termination in two distantly related artiodactyls, the pig and the cow. Porcine mitochondrial DNA was unique among mammals in that it contained only a single species of D-loop single-stranded DNA. Its 3' end mapped to a site 187 nucleotides from the 5' end of the proline tRNA gene. This site was 21 and 47 nucleotides 5' to two very similar sequences (5' ACATATPyATTAT 3') which are closely related to the human and mouse termination-associated sequences noted by Doda et al. (J. N. Doda, D. T. Wright, and D. A. Clayton, Proc. Nat. Acad. Sci. USA 78:616-6120, 1981). Bovine mitochondrial DNA contained three major D-loop DNA species whose 3' ends mapped to three different sites. These sites were not found in the porcine sequence. However, the bovine termination sites were located 60 to 64 base pairs 5' from sequences which were also very similar to the termination-associated sequences present in pigs and other mammals. These results firmly establish the concept that arrest of heavy-strand DNA synthesis is an event determined, at least in part, by template sequence. They also suggest that arrest is determined by sequences which are a considerable physical distance away from the actual termination site.

Detection of transforming growth factor-alpha messenger RNA and protein in human corneal epithelial cells.

Human corneal epithelial cells are normally shed from the apical surface and replaced primarily by mitosis of basal cells. Growth factors may regulate this process, but the sources for the growth factors have not been fully established. One potential source for growth factors is tear fluid, and epidermal growth factor (EGF) has been detected in the lacrimal gland and in tears. However, the hydrophilic structure and size of growth factors such as EGF may limit penetration to basal layers of intact epithelium. It is possible that turnover of basal human corneal epithelial cells might be regulated by growth factors acting by an autocrine mechanism. To determine if human corneal epithelial cells synthesize a potential autocrine growth factor, the authors analyzed human corneal epithelial cells for transforming growth factor-alpha (TGF-alpha) messenger RNA and protein, a growth factor that is structurally related to EGF and binds to the EGF receptor. Radioimmunoassay of human corneal epithelial cell cultures detected substantial levels of immunoreactive TGF-alpha (3 ng/10(6) cells). Immunohistochemical staining of human corneas also revealed the presence of immunoreactive TGF-alpha in the corneal epithelium. Northern hybridization with a 32P-labeled complementary DNA probe for TGF-alpha generated a single intense band at 4.4 kilobases, indicating the presence of TGF-alpha messenger RNA in cultured human corneal epithelial cells. These results support the hypothesis that normal turnover of corneal epithelium is controlled by the autocrine production of growth factors, such as TGF-alpha. Growth factors present in tears may function primarily as exocrine factors to stimulate healing of epithelial injuries after the epithelial barrier has been damaged.

Application of gene therapy to acute inflammatory diseases.

The application of gene therapy to acute inflammation has not received as much research attention as has the treatment of genetically-based diseases, cancer, and viral infections. However, gene therapy as a drug delivery system offers several theoretical and practical advantages over current protein delivery systems. These include the ability to target therapies to individual tissues or cell types, to locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and to sustain new protein synthesis for periods up to several weeks after a single administration. Although retrovirus, herpes simplex, and adeno-associated virus have been proposed for gene therapy in cancer and in genetic diseases, nonviral and adenovirus approaches appear most applicable as drug delivery systems due to their rapid onset and short duration of transgene expression. The relative modest transduction efficiencies obtained at present with nonviral approaches, and the inherent inflammatory properties of first-generation adenovirus constructs, however, have limited their usefulness to date. The present review discusses the theoretical and practical benefits of specific gene therapy approaches for the treatment of acute inflammatory diseases, as well as our experiences with liposome:plasmid DNA and adenovirus-based approaches. Although a number of technical and theoretical hurdles remain before it can be evaluated in humans with acute inflammation, gene therapy offers a novel approach for the treatment of acute inflammation, and will likely enter the armamentarium of critical care physicians in the near future.

A matrix metalloproteinase inhibitor prevents processing of tumor necrosis factor alpha (TNF alpha) and abrogates endotoxin-induced lethality.

Excessive tumor necrosis factor alpha (TNF alpha) production in response to Gram-negative bacteremia or endotoxemia can often lead to hypotension, shock, and increased mortality. Current approaches used to block the deleterious effects of exaggerated TNF alpha production rely on monoclonal antibodies or immunoadhesins that bind TNF alpha and thus prevent the interaction with its cellular receptors. This report examines whether a previously described inhibitor of matrix metalloproteinases, GM-6001, can inhibit TNF alpha processing and release and attenuate endotoxin-induced mortality. In human peripheral blood mononuclear cells stimulated in vitro with 1 microgram/mL endotoxin, GM-6001 at concentrations > 5 micrograms/mL blocked release of TNF alpha, but did not affect the release of either IL-1 beta or IL-6. GM-6001 also inhibited the release of soluble TNF receptor (p75) from peripheral blood mononuclear cells stimulated with endotoxin and/or TNF alpha. To confirm the role of secreted TNF alpha in endotoxic shock-induced mortality, C57BL/6 mice were challenged with either endotoxin alone (500 micrograms/mouse) or endotoxin (100 ng/mouse) plus D-galactosamine (8 mg/mouse). GM-6001 pretreatment (100 mg/kg) significantly attenuated the 90-minute plasma TNF alpha response in both models and improved survival in mice treated with low-dose endotoxin plus D-galactosamine. However, plasma IL-1 beta and IL-6 concentrations at 90 min after endotoxin treatment were unaffected by GM-6001 following lethal endotoxin challenge, confirming the in vivo specificity of this matrix metalloproteinase inhibitor for TNF alpha processing. These findings demonstrate that a novel inhibitor of matrix metalloproteinases can prevent the release of TNF alpha both in vitro and in vivo, and can abrogate the harmful sequelae of endotoxemic shock.

Five-minute treatments with fluorouracil, floxuridine, and mitomycin have long-term effects on human Tenon's capsule fibroblasts.

Proliferating human Tenon's capsule fibroblasts were exposed for 5 minutes to a wide range of concentrations of fluorouracil, floxuridine, and mitomycin. High concentrations of all three agents had prolonged effects on cell proliferation and morphologic characteristics compared with untreated control cells up to 36 days. The highest concentrations of both floxuridine (15,000 micrograms/mL) and mitomycin (1000 micrograms/mL) had an apparent cidal effect, reducing cell numbers below initial cell density. In contrast, although the highest concentration of fluorouracil (25,000 micrograms/mL) inhibited cell proliferation by more than 50% relative to the untreated control cells at 36 days, the cell numbers still increased fourfold compared with the initial cell density. These results demonstrate that 5-minute treatments with high concentrations of these drugs have prolonged effects on the proliferation of human Tenon's capsule fibroblasts in vitro. Single-dose regimens using high concentrations of these drugs at the time of operation may achieve results similar to those of protocols that involve repeated applications.

Discordant tumor necrosis factor-alpha superfamily gene expression in bacterial peritonitis and endotoxemic shock.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is a member of a large family of predominantly homotrimeric type II membrane-associated proteins with both proinflammatory and apoptosis-inducing properties. Although TNF-alpha expression has been studied extensively, little is known about the expression of other members of the TNF-alpha superfamily during acute inflammatory processes. METHODS: TNF-alpha, Fas ligand (FasL), and TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) messenger RNA (mRNA) expression were examined in liver, lung, spleen, and kidney after either a cecal ligation and puncture or endotoxemic shock with use of semiquantitative reverse transcriptase-polymerase chain reaction. RESULTS: Cecal ligation and puncture increased TNF-alpha mRNA in lung and liver (both P < .05) within 3 hours, which was paralleled by increased FasL mRNA. In the spleen TNF-alpha and FasL mRNA significantly declined (both P < .05). In contrast to TNF-alpha and FasL, TRAIL mRNA levels were unchanged in all organs except lung, where it was reduced at 24 hours (P < .05). Endotoxemic shock also increased lung TNF-alpha and FasL mRNA levels (both P < .05). CONCLUSIONS: In acute inflammatory processes TNF-alpha and FasL mRNA increase concordantly in several solid organs. In contrast, TRAIL mRNA levels do not consistently change during these acute inflammatory processes, suggesting that its expression is under independent and discordant regulatory control.

Pharmacokinetics, immunogenicity, and efficacy of dimeric TNFR binding proteins in healthy and bacteremic baboon.

Immunogenicity, pharmacokinetics, and therapeutic efficacy of three novel dimeric soluble tumor necrosis factor (TNF)-receptor I constructs [TNF-binding protein (bp)] were evaluated in 28 baboons, 12 of which were healthy and 16 were challenged with a lethal Escherichia coli bacteremia. The three constructs differed only in the number of extracellular domains of the TNF receptor I and were dimerized with polyethylene glycol. Although all three constructs had generally similar pharmacokinetics when administered to a naive animal, they differed quantitatively in their immunogenicity. Antibodies were detected more frequently, and titers were significantly higher (P < 0.05) in both healthy and septic baboons that received the 4.0-domain TNF-bp construct, compared with animals receiving the 2.6-domain construct. When the TNF-bp constructs were administered a second time (21 days later), the half-lives of the three constructs were significantly shorter in animals that had an antibody response after the first injection. In contrast, all three TNF-bp constructs were equally effective at improving outcome, blocking a systemic TNF-alpha response, and attenuating the cytokine responses when administered at a dose of 1.0 mg/kg body wt 1 h before a lethal E. coli infusion. The findings suggest that immunogenicity of TNF-bp constructs can be altered by changing the number of functional domains, without affecting their capacity to neutralize TNF-alpha and to abrogate TNF-mediated pathology.

Revisiting the role of tumor necrosis factor alpha and the response to surgical injury and inflammation.

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine with diverse biological actions. Studies originally identified TNF-alpha as a systemic mediator of endotoxemic shock, cachexia, and tumor regression. We now recognize that TNF-alpha is a member of a large family of proteins, including Fas ligand, whose actions are primarily paracrine in nature, and serve to regulate both cell proliferation and apoptotic death. Although clinical trials with TNF-alpha inhibitors in sepsis syndrome have been disappointing to date, and TNF-alpha administration has not proven widely successful as an antineoplastic agent, preliminary successes with TNF-alpha inhibition have been recently reported in more chronic inflammatory diseases, including rheumatoid arthritis and ulcerative colitis. The recent description of the TNF-alpha converting enzyme responsible for the processing of cell-associated to secreted TNF-alpha has opened a new therapeutic avenue to address inflammatory diseases dependent on the release of 17-kd secreted TNF-alpha. Similarly, inhibitors of nuclear factor kappa B activation can increase TNF-alpha-mediated apoptosis and have rejuvenated efforts to explore TNF-alpha's antineoplastic potential. The multiple and often conflicting TNF-alpha signaling pathways reveal a diversity to TNF-alpha's actions not fully appreciated in the past. Such investigations have opened a number of novel therapeutic interventions to either inhibit or potentiate the actions of TNF-alpha during surgical injury or acute inflammation.

Human interleukin 10 suppresses production of inflammatory mediators by LPS-stimulated equine peritoneal macrophages.

To investigate the ability of recombinant human interleukin 10 (rhuIL-10) to suppress the release of inflammatory mediators from lipopolysaccharide (LPS)-stimulated equine macrophages, rhuIL-10 was added to equine peritoneal macrophage monolayers at concentrations of 0, 0.1, 1, 10, or 100 ng/ml. Thirty minutes later, LPS (E. coli O55:B5) was added at final concentrations of 0, 1, 10, 100 ng/ml. Macrophages were incubated for 16 h at 37 degrees C, then supernates were harvested and assayed for tumor necrosis factor (TNF) activity (L929 cytotoxicity), interleukin-6 (IL-6) activity (B9 proliferation), prostaglandin E2 concentration (ELISA), and nitric oxide (Griess reaction for nitrite). Preincubation of LPS-stimulated peritoneal macrophages with rhuIL-10 caused significant (P<0.05) reduction in secretion of TNF, IL-6, and PGE2, in a dose-dependent manner. Of the inflammatory mediators, TNF was most sensitive to the effects of rhuIL-10. At concentrations of rhuIL-10> or =1 ng/ml, TNF activity in the supernate was inhibited significantly at all concentrations of LPS. At one or more LPS concentrations, there was significant inhibition of each mediator in the presence of 1 ng rhuIL-10/ml and, at 100 ng/ml, rhuIL-10 significantly inhibited production of each mediator at all LPS concentrations tested. When data were expressed as a percentage of control values and pooled across all LPS concentrations, both PGE2 and TNF values were significantly reduced at rhuIL-10 concentrations of > or =1 ng/ml, whereas IL-6 was inhibited significantly at concentrations of > or =10 ng rhuIL-10/ml. Tumor necrosis factor production was more completely suppressed (7.8% of control) by the highest concentration of rhuIL-10(100 ng/ml) than was PGE2 (27.2%) or IL-6 (43.8%). Nitrite was not detected in any supernate from peritoneal macrophage monolayers.

Affinity of the C9 molecule for the C5b-8 complex compared with that for the complex containing C9 molecules.

Gram-negative bacterial cells exposed to a complement source may carry membrane attack complexes containing variable numbers of C9 molecules per C5b-8 site. In order to investigate the assembly of this complex, the ability of C9 molecules to bind to C5b-8 complexes was compared with the binding characteristics of C9 for C5b-8 complexes containing variable numbers of bound C9 molecules. The apparent dissociation constant (Kd) of the C9 molecule for the C5b-8 site on a complement-sensitive strain of Escherichia coli was 1.2 (+/- 0.15) nM at 0 degree C. These conditions allow the binding of one C9 molecule per C5b-8 site. The C5b-8 site containing one C9 molecule bound a second C9 molecule at 0 degree C only after incubation at 37 degrees C. The binding of C9 to a C5b-8 site containing one C9 molecule was found to be 1.3 (+/- 0.2) nM. Therefore, the presence of a C9 molecule did not significantly alter the binding capacity of the C5b-8 site for additional C9 molecules. A similar result was obtained by using rabbit erythrocytes bearing either C5b-8 sites or C5b-8 sites containing one molecule of C9 per complex at 0 degree C. The similarity of binding characteristics for the first and second C9 molecules argues that the initial C9 molecule in the complex does not affect the binding of subsequent C9 molecules. This suggests that a unique C9 binding site that does not involve previously bound C9 molecules may exist on the forming membrane attack complex.

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites.

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.

Transcribed heteroplasmic repeated sequences in the porcine mitochondrial DNA D-loop region.

The mitochondrial D-loop region of the pig, Sus scrofa, was found to be several hundred base pairs larger than the corresponding region in cow, a related artiodactyl species, primarily because of an insertion containing the tandemly repeated sequence CGTGCGTACA. Porcine mitochondrial DNA from the tissue of a single animal exhibits a large population of length polymorphs, each member of which may have as few as 14 or as many as 29 of these repeat units. This intracellular variability may be due to the repeated and self-complementary properties of this sequence, which would favor mispairing and lead to replication slippage. The repeat domain is unusual in that symmetry properties suggest it may assume alternative conformations including cruciforms and left-handed (Z) DNA. It also appears to be the longest known, naturally occurring, alternating purine-pyrimidine sequence. In order to understand the functional significance of this heteroplasmic domain that potentially disrupts a key regulatory region in the mitochondrial genome, RNA and DNA mapping studies were conducted which located this region between the H-strand replication origin and the putative L-strand transcriptional start site. H-strand RNA analysis demonstrated that this heteroplasmic region is transcribed and, therefore, that priming for H-strand DNA replication in mitochondria is independent of the primer RNA length or secondary structure.

Characterization in vitro and in vivo of hammerhead ribozymes directed against murine tumor necrosis factoralpha.

A hammerhead ribozyme directed against murine TNFalpha (mTNFalpha) mRNA has been constructed. In vitro studies showed that this ribozyme was released from the parent molecule by flanking cis-acting hammerhead and hairpin ribozymes. This same anti-mTNFalpha ribozyme specifically cleaved both synthetically derived substrate RNA and mTNFalpha mRNA within a pool of total cellular RNA. Endogenous delivery of this anti-mTNFalpha ribozyme via the self-cleaving cassette reduced mTNFalpha mRNA and protein levels in lipopolysaccharide (LPS)-stimulated, stably transfected murine macrophage RAW 264.7 cells. When complexed to liposomes and exogenously delivered to mouse peritoneal macrophages, the same ribozyme, with and without the cis-acting ribozymes, reduced mTNFalpha protein levels. However, an irrelevant ribozyme delivered in an identical fashion was also effective at reducing mTNFalpha protein levels. These data suggest that anti-mTNFalpha ribozymes can be constructed which efficiently cleave mTNFalpha mRNA, but irrelevant RNA/liposome complexes also effectively limit TNFalpha mRNA expression and can mimic functional ribozyme activity under in vitro conditions.

Liposome-mediated, nonviral gene transfer induces a systemic inflammatory response which can exacerbate pre-existing inflammation.

Cationic liposome and plasmid-mediated gene transfer has emerged as a novel technique for the targeted delivery of protein-based therapies in acute inflammatory diseases. However, concerns have arisen that cationic liposomes and plasmid DNA have inherent proinflammatory properties which could exacerbate pre-existing inflammatory processes. In healthy mice, intraperitoneal administration of cationic liposomes (200 nmol) complexed to plasmid DNA (100 microg) induced a proinflammatory response characterized by the induction of tumor necrosis factor alpha and interleukin-1beta mRNA expression. The plasma concentrations of the hepatic acute phase proteins interleukin-6, amyloid A, amyloid P, and seromucoid were also increased (P<0.05), and this response was seen in endotoxin-resistant (C3H/HeJ) mice. The inflammatory response associated with gene transfer increased the mortality and severity of experimentally induced sterile inflammation (pancreatitis). We conclude that systemic administration of cationic liposomes and plasmid DNA is associated with induction of the innate immune response which may exacerbate pre-existing inflammatory processes.

Colon cancer cells that are not growth inhibited by TGF-beta lack functional type I and type II TGF-beta receptors.

OBJECTIVE: The authors determined the molecular mechanisms for the failure of transforming growth factor-beta (TGF-beta) to inhibit the growth of SW1116 and SW48 colon cancer cell lines. BACKGROUND: Transforming growth factor-beta is a bifunctional regulator of cell growth that typically stimulates proliferation of mesenchymal cells, but inhibits proliferation of normal epithelial cells. In the colon, TGF-beta appears to arrest proliferation of enterocytes as they leave the intestinal crypt and move to the villus tip. Transforming growth factor-beta actions are mediated by binding to heteromeric complexes of type I and type II TGF-beta receptors. Loss of TGF-beta responsiveness may contribute to uncontrolled cell growth and cancer. METHODS: The effects of TGF-beta 1 on DNA synthesis were measured by incorporation of tritiated thymidine into DNA of cultures of moderately differentiated adenocarcinoma (SW48) and poorly differentiated adenocarcinoma (SW1116) colon cell lines and a mink lung epithelial cell line (CCL-64). The effects of TGF-beta on the expression of c-myc, TGF-alpha, and TGF-beta in SW48 cells, SW1116 cells, and CCL-64 cells (c-myc only) were measured by Northern blot analysis. Expression of TGF-beta receptors in the cell lines was measured using competitive binding assays, receptor affinity labelling techniques, and reverse transcriptase-polymerase chain reaction. RESULTS: Incubation with TGF-beta 1 (50 ng/mL) did not decrease serum-stimulated uptake of [3H]-thymidine into actively growing cultures of SW48 or SW1116 cells, but suppressed DNA synthesis of actively growing CCL-64 cells by 90%. Similarly, incubation with TGF-beta 1 (12 ng/mL) for 4 hours did not substantially alter the mRNA levels of c-myc, TGF-alpha, and TGF-beta 1 in either colon tumor cell line, although levels of c-myc mRNA in CCL-64 cells were reduced by TGF-beta 1 treatment. Competitive displacement of [125I]-TGF-beta 1 binding detected high levels (16,500 TGF-beta receptors per cell) of specific, high-affinity (200 pmol/L half-displacement) TGF-beta receptors on CCL-64 cells. In marked contrast, very low levels of TGF-beta 1 binding to SW1116 cells (250 receptors per cell) and SW48 cells (260 receptors per cell) were detected. Autoradiograms of CCL-64 cells affinity labelled with [125I]TGF-beta 1 revealed the presence of type I, type II, and type III TGF-beta receptors. No TGF-beta receptors were identified on SW1116 cells, and only very low levels of the nonsignaling type III TGF-beta receptors were detected on SW48 cells. Reverse transcriptase-polymerase chain reaction amplification detected mRNAs for type I, type II, and type III TGF-beta receptors in CCL-64 cells. SW48 cells, and SW1116 cells. CONCLUSIONS: These results suggest that the lack of growth inhibition by TGF-beta in SW48 and SW1116 colon cancer cells may be caused by a lack of expression of functional TGF-beta receptors.

LPS-induced liver injury in D-galactosamine-sensitized mice requires secreted TNF-alpha and the TNF-p55 receptor.

Lipopolysaccharide and D-galactosamine induced lethality and apoptotic liver injury is dependent on endogenously produced tumor necrosis factor (TNF)-alpha. The present study was undertaken to determine whether membrane-associated or secreted TNF-alpha signaling through the p55 or p75 receptor was responsible for survival and hepatic injury after lipopolysaccharide administration in D-galactosamine-sensitized mice. Transgenic mice expressing null forms of TNF-alpha, the p55 and p75 receptor, and mice expressing only a cell-associated form of TNF-alpha were challenged with 8 mg D-galactosamine and 100 ng lipopolysaccharide. Mortality and apoptotic liver injury were only seen in wild-type and p75 knockout mice. p75 Knockout mice had significantly higher concentrations of plasma TNF-alpha than any other experimental group (P

Enhanced collagen accumulation following direct transfection of the inducible nitric oxide synthase gene in cutaneous wounds.

Inducible nitric oxide synthase (iNOS) is expressed during cutaneous wound repair. Mounting evidence suggests that wound nitric oxide (NO) augments collagen accumulation. We hypothesized that in vivo transfection of wound cells with the iNOS gene would increase physiological wound NO levels and thus augment collagen accumulation. Polyvinyl alcohol sponges were instilled with a mammalian expression plasmid (pMP6) containing either the chloramphenicol acetyl transferase (CAT) reporter or murine iNOS gene driven by a CMV immediate-early promoter. Plasmid DNA was injected alone or in complex with cationic liposomes, and the sponges were placed subcutaneously in male Sprague-Dawley rats which had received a longitudinal dorsal midline incision. Animals were sacrificed at different time points post-wounding and the sponges assayed for CAT activity, transfected iNOS mRNA, total nitrate and nitrite concentration (NOx) (as an index of wound NO synthesis), and hydroxyproline content (as an index of sponge collagen accumulation). The results demonstrate that wound cells were more efficiently transfected by naked DNA than by liposome mediated transfection and that maximal expression of both iNOS and CAT occurred at 48 hrs with a rapid decline after this time point. After 7 days, iNOS transfected sponges had accumulated significantly more collagen than those transfected with CAT. We conclude that cutaneous wounds can be successfully transfected by direct injection of naked DNA and that increased iNOS expression precedes an increase in collagen synthesis.

Transfection of the type II TGF-beta receptor into colon cancer cells increases receptor expression, inhibits cell growth, and reduces the malignant phenotype.

OBJECTIVE: To determine if transfection of SW48 colon cancer cells with the type II transforming growth factor-beta (TGF-beta) receptor restores growth inhibition and reverses the in vitro and in vivo malignant phenotype. SUMMARY BACKGROUND DATA: The authors have previously shown that SW48 colon cancer cells that are replication error positive in both alleles lack functional cell surface TGF-beta type I (RI) and type II (RII) receptors and are insensitive to TGF-beta1-induced growth inhibition. METHODS: SW48 cells were stably transfected with the cDNA for the normal type II TGF-beta receptor (RII). Once transfected, the cells were evaluated for in vitro phenotypic changes and in vivo changes in tumor growth. RESULTS: Denaturing sequencing gel electrophoresis of the reverse transcriptase-polymerase chain reaction product from SW48 cells revealed that the RII coding sequence contained a single base deletion mutation. When these cells were transfected with normal RII cDNA, Northern and Western blot analyses revealed increased levels of RII mRNA and protein. Affinity labeling techniques revealed that RII-transfected SW48 cells produced functional RI and RII protein. Transfection of SW48 cells also led to changes in cell phenotype, as shown by inhibition of both in vitro growth rate and incorporation of [3H]-thymidine. SW48 cells expressing normal RII also exhibited reduced cloning efficiency in semisolid medium and reduced growth as a xenograft in NOD/LtSz-scid/J mice. CONCLUSIONS: The results confirm that RII is a tumor-suppressor protein that is required for TGF-beta-induced growth inhibition in SW48 colon cancer cells.

Increased leptin expression in mice with bacterial peritonitis is partially regulated by tumor necrosis factor alpha.

Plasma leptin and ob gene mRNA levels were increased in mice following bacterial peritonitis, and blocking an endogenous tumor necrosis factor alpha (TNF-alpha) response blunted the increase. However, plasma leptin concentrations did not correlate with the associated anorexia. We conclude that leptin expression is under partial regulatory control of TNF-alpha in peritonitis, but the anorexia is not dependent on increased leptin production.

Effects of benzoyltropine and tropacocaine on several cholinergic processes in the rat brain.

Benzoyltropine and tropacocaine are two contaminants of street-cocaine reported to have parasympatholytic activity. Because the mechanism underlying this activity is obscure, we investigated the effects of these compounds on several cholinergic processes: sodium-dependent choline uptake, sodium-independent choline uptake, acetylcholine synthesis, acetylcholine release (spontaneous and veratridine-induced) and binding of [3H]quinuclidinyl benzylate to muscarinic receptors. These studies used rat cerebral cortical synaptosomes, except for the receptor-binding studies, which used whole brain plasma membranes. Benzoyltropine and tropacocaine each inhibited sodium-dependent choline uptake and acetylcholine synthesis in a dose-related manner that was competitive with extracellular choline. Benzoyltropine was 4 to 5 times more potent in both actions than tropacocaine. Sodium-independent choline uptake was not affected by either compound. Benzoyltropine (30 microM) had no effect on the sodium-dependent uptake of norepinephrine, gamma-amino-butyric acid, glutamate or serotonin; tropacocaine (30 microM) inhibited only norepinephrine uptake at this concentration. Benzoyltropine and tropacocaine each inhibited the spontaneous and veratridine-induced release of newly synthesized acetylcholine, but not via activation of presynaptic muscarinic receptors. Instead, each compound was able to attenuate the oxotremorine-induced inhibition of the release of acetylcholine, suggesting antimuscarinic activity. Binding experiments showed that benzoyltropine and tropacocaine were, respectively, about 1,000- and 10,000-fold less potent than scopolamine as receptor antagonists. Finally, we demonstrated that benzoyltropine accumulates in the rat brain after its peripheral injection (10 mg/kg i.p.) and remains there with a half-life similar to that of cocaine.(ABSTRACT TRUNCATED AT 250 WORDS)