Use of lablab (Lablab purpureus (L.) Sweet) for bio-control by native arthropods and its effect on yield of pumpkins.

Silverleaf whitefly (SLW, Bemisia tabaci MEAM1) and aphids are sap-sucking insects, which pose a serious threat to Australian cucurbit crops and the horticulture industry. Traditional chemical control for these insect pests is becoming less effective, and there is a need to search for alternative or supplementary methods. This study aimed to manipulate the habitat of pumpkin crops in a tropical setting (Queensland, Australia), by growing pumpkins (var. Japanese pumpkin) alone and between lablab (Lablab purpureus L. Sweet). It was hypothesized that the presence of lablab will increase the populations of natural enemies, and through their control of insect pests such as SLW and aphids, will affect pumpkin yield. The population of arthropods (natural enemies and pests of pumpkin), with a focus on SLW and aphids, were sampled weekly on both lablab and pumpkin crop for a total of 21 weeks. Results showed that lablab hosted more enemies of SLW per plant than pumpkin in either treatment. In addition, adult SLW numbers were significantly higher in the pumpkin-only crop compared with the pumpkin grown between lablab, while pumpkin in the mixed plantings had significantly more ladybirds and lacewing larvae (P < 0.05). While there was no significant difference in the average fruit weight between treatments, the total weight (kg) and number of marketable pumpkins per hectare was greater (P < 0.05) for the pumpkin/lablab treatment than the pumpkin-only treatment. This study shows that growing lablab alongside a pumpkin crop may enhance natural enemies of SLW and could significantly increase the yield.

Correlation of echocardiographic and angiographic measurements of the pulmonary valve annulus in pulmonary stenosis.

OBJECTIVES: The pulmonary valve (PV) annulus is routinely measured angiographically in PV stenosis prior to balloon dilation. We sought to establish whether this radiation exposure is justified, or whether echocardiographic measurements prior to the procedure are sufficient to guide balloon selection. BACKGROUND: Previous studies have found a strong correlation between echocardiographic and angiographic measurements of the PV annulus. However, error of measurement and its implication for procedural practice has not been explored. METHODS: A total of 90 procedures in 84 patients were analyzed, at a median age 7.6 months (range 1 day to 14.2 years). The contemporaneous echocardiographic and angiographic measurements were recorded, and the original echocardiograms were re-measured in the 72 available cases by two independent reviewers. RESULTS: There was a good correlation between the two measurement methods (R(2) = 0.87). However, the echocardiographic PV measurements were smaller on average, with a significant variation in that discrepancy (mean ratio 0.941 (±0.16)). There was no significant reduction in error if extreme measurements (PV annulus z-score <-3) were excluded (P = 0.09), or if the reviewed echocardiographic measurements were used (P = 0.58). CONCLUSIONS: There is an unacceptable discrepancy between the measurement techniques: 95% of patients are predicted to have an echocardiographic measurement error between -37% and +26%. Therefore, there is no correction factor that could be employed to allow safe selection of balloon size, and balloon pulmonary valvoplasty without angiographic PV measurement cannot be advocated.

Chronic treatment with a glucagon receptor antagonist lowers glucose and moderately raises circulating glucagon and glucagon-like peptide 1 without severe alpha cell hypertrophy in diet-induced obese mice.

AIMS/HYPOTHESIS: Antagonism of the glucagon receptor (GCGR) represents a potential approach for treating diabetes. Cpd-A, a potent and selective GCGR antagonist (GRA) was studied in preclinical models to assess its effects on alpha cells. METHODS: Studies were conducted with Cpd-A to examine the effects on glucose-lowering efficacy, its effects in combination with a dipeptidyl peptidase-4 (DPP-4) inhibitor, and the extent and reversibility of alpha cell hypertrophy associated with GCGR antagonism in mouse models. RESULTS: Chronic treatment with Cpd-A resulted in effective and sustained glucose lowering in mouse models in which endogenous murine Gcgr was replaced with human GCGR (hGCGR). Treatment with Cpd-A also led to stable, moderate elevations in both glucagon and glucagon-like peptide 1 (GLP-1) levels, which were completely reversible and not associated with a hyperglycaemic overshoot following termination of treatment. When combined with a DPP-4 inhibitor, Cpd-A led to additional improvement of glycaemic control correlated with elevated active GLP-1 levels after glucose challenge. In contrast to Gcgr-knockout mice in which alpha cell hypertrophy was detected, chronic treatment with Cpd-A in obese hGCGR mice did not result in gross morphological changes in pancreatic tissue. CONCLUSIONS/INTERPRETATION: A GRA lowered glucose effectively in diabetic models without significant alpha cell hypertrophy during or following chronic treatment. Treatment with a GRA may represent an effective approach for glycaemic control in patients with type 2 diabetes, which could be further enhanced when combined with DPP-4 inhibitors.

RNAi-mediated germline knockdown of FABP4 increases body weight but does not improve the deranged nutrient metabolism of diet-induced obese mice.

OBJECTIVE: To investigate the impact of reduced adipocyte fatty acid-binding protein 4 (FABP4) in control of body weight, glucose and lipid homeostasis in diet-induced obese (DIO) mice. METHODS: We applied RNA interference (RNAi) technology to generate FABP4 germline knockdown mice to investigate their metabolic phenotype. RESULTS: RNAi-mediated knockdown reduced FABP4 mRNA expression and protein levels by almost 90% in adipocytes of standard chow-fed mice. In adipocytes of DIO mice, RNAi reduced FABP4 expression and protein levels by 70 and 80%, respectively. There was no increase in adipocyte FABP5 expression in FABP4 knockdown mice. The knockdown of FABP4 significantly increased body weight and fat mass in DIO mice. However, FABP4 knockdown did not affect plasma glucose and lipid homeostasis in DIO mice; nor did it improve their insulin sensitivity. CONCLUSION: Our data indicate that robust knockdown of FABP4 increases body weight and fat mass without improving glucose and lipid homeostasis in DIO mice.

Identification of immunogenic regions within the alternative reading frame protein of hepatitis C virus (genotype 3).

Hepatitis C virus (HCV) encodes ten classic proteins as well as a newly discovered alternative reading frame protein (ARFP) whose synthesis originates from the core region by a +1 frameshift. ARFP is produced by all HCV genotypes, but its function remains unknown. Although the immunogenicity of genotype 1- and 2-derived ARFP in infected hosts has been reported, no information is available for genotype 3-encoded ARFP. HCV genotype 3 core/ARFP region was PCR amplified, cloned, and sequenced. Recombinant ARFP and peptides were employed in ELISAs with patient serum samples. The effect of peptides on peripheral blood mononucleocytes (PBMCs) was also studied. DNA cloning and sequencing of HCV genotype 3 strain (PKHCV3) revealed it to encode 160 aa ARFP, which harbors a C-terminal extension of 36 aa. Serum from 74 of 88 patients (84%) contained rARFP-reactive antibodies. Peptide ELISAs showed that all regions of rARFP were immunogenic, with peptide F7 (DSLSPRRAGAKAGPGLSPGT) being the most immunodominant. When incubated with PBMCs from HCV-infected individuals, F7 stimulated the production of TNFα and IL10. PKHCV3-derived ARFP encodes a 160 aa protein and antibodies against its entire length are found in 84% of all genotype 3-infected subjects. Peptide ELISAs revealed F7 to be highly immunogenic and capable of eliciting impressive T-cell responses.

Diagnosis of Alagille syndrome-25 years of experience at King's College Hospital.

OBJECTIVE: The aim of the study was to study the clinical and histological features of Alagille syndrome (AGS) at presentation comparing the value of the various modalities before the implementation of genetic diagnosis. PATIENTS AND METHODS: We performed a retrospective analysis of the records of 117 children diagnosed as having AGS after referral to King's College Hospital between 1980 and 2005. RESULTS: Cholestasis was seen in 104 of 117 (89%), characteristic facies in 91 of 117 (77%), posterior embryotoxon in 72 of 117 (61%), butterfly vertebrae in 44 of 117 (39%), heart disease (most often peripheral pulmonary stenosis) in 107 of 117 (91%), and renal disease in 27 of 117 (23%). Serum cholesterol levels of >5 mmol/L were seen in 52 of 86 (60.4%). Liver biopsy showed characteristic features of paucity of interlobular bile ducts in 59 of 77 (76.6%) children younger than 16 weeks of age, in 10 of 14 (71.4%) between 16 weeks and 1 year of age, and in 8 of 12 (66.66%) older than 1 year of age. Other biopsy findings were those of nonspecific hepatitis and biliary features. Iminodiacetic acid scans showed no excretion of isotope into the bowel after 24 hours in 21 of 35 (60%), and small/no gallbladder on ultrasound was seen in 29 of 104 (27.8%). Eleven of 117 (9.4%) had a diagnostic laparotomy and operative cholangiography, 2 proceeding to Kasai portoenterostomy before referral to our unit. CONCLUSIONS: Clinical features of AGS are not as consistently informative as suggested in the literature. Hypercholesterolaemia is nonspecific but may be a helpful pointer. Histology is not characteristic in 25%; hepatobiliary iminodiacetic acid scan and ultrasound may suggest a false diagnosis of biliary atresia in 60% and 28%, respectively, supporting the concept that infants with liver disease warrant early referral to a specialist centre. The advent of genetic diagnosis will redefine the syndrome with likely effects on the prognosis of the defined group.

A comparison of alternative plant mixes for conservation bio-control by native beneficial arthropods in vegetable cropping systems in Queensland Australia.

Cucurbit crops host a range of serious sap-sucking insect pests, including silverleaf whitefly (SLW) and aphids, which potentially represent considerable risk to the Australian horticulture industry. These pests are extremely polyphagous with a wide host range. Chemical control is made difficult due to resistance and pollution, and other side-effects are associated with insecticide use. Consequently, there is much interest in maximising the role of biological control in the management of these sap-sucking insect pests. This study aimed to evaluate companion cropping alongside cucurbit crops in a tropical setting as a means to increase the populations of beneficial insects and spiders so as to control the major sap-sucking insect pests. The population of beneficial and harmful insects, with a focus on SLW and aphids, and other invertebrates were sampled weekly on four different crops which could be used for habitat manipulation: Goodbug Mix (GBM; a proprietary seed mixture including self-sowing annual and perennial herbaceous flower species); lablab (Lablab purpureus L. Sweet); lucerne (Medicago sativa L.); and niger (Guizotia abyssinica (L.f.) Cass.). Lablab hosted the highest numbers of beneficial insects (larvae and adults of lacewing (Mallada signata (Schneider)), ladybird beetles (Coccinella transversalis Fabricius) and spiders) while GBM hosted the highest numbers of European bees (Apis mellifera Linnaeus) and spiders. Lucerne and niger showed little promise in hosting beneficial insects, but lucerne hosted significantly more spiders (double the numbers) than niger. Lucerne hosted sig-nificantly more of the harmful insect species of aphids (Aphis gossypii (Glover)) and Myzus persicae (Sulzer)) and heliothis (Heliothis armigera Hübner). Niger hosted significantly more vegetable weevils (Listroderes difficillis (Germar)) than the other three species. Therefore, lablab and GBM appear to be viable options to grow within cucurbits or as field boundary crops to attract and increase beneficial insects and spiders for the control of sap-sucking insect pests. Use of these bio-control strategies affords the opportunity to minimise pesticide usage and the risks associated with pollution.

Immunochemical identity of peroxisomal enoyl-CoA hydratase with the peroxisome-proliferation-associated 80,000 mol wt polypeptide in rat liver.

Peroxisome proliferators, which induce proliferation of hepatic peroxisomes, have been shown previously to cause a marked increase in an 80,000 mol wt polypeptide predominantly in the light mitochondrial and microsomal fractions of liver of rodents. We now present evidence to show that this hepatic peroxisome-proliferation-associated polypeptide, referred to as polypeptide PPA-80, is immunochemically identical with the multifunctional peroxisome protein displaying heat-labile enoyl-CoA hydratase activity. This conclusion is based on the following observations: (a) the purified polypeptide PPA-80 and the heat- labile enoyl-CoA hydratase from livers of rats treated with the peroxisome proliferators Wy-14,643 {[4-chloro-6(2,3-xylidino)-2-pyrimidinylthio]acetic acid} exhibit identical minimum molecular weights of approximately 80,000 on SDS polyacrylamide gel electrophoresis; (b) these two proteins are immunochemically identical on the basis of ouchterlony double diffusion, immunotitration, rocket immunoelectrophoresis, and crossed immunoelectrophoresis analysis; and (c) the immunoprecipitates formed by antibodies to polypeptide PPA-80 when dissociated on a sephadex G-200 column yield enoyl-CoA hydratase activity. Whether the polypeptide PPA-80 exhibits the activity of other enzyme(s) of the peroxisomal beta-oxidation system such as fatty acyl-CoA oxidase activity or displays immunochemical identity with such enzymes remains to be determined. The availability of antibodies to polypeptide PPA-80 and enoyl-CoA hydratase facilitated immunofluorescent and immunocytochemical localization of the polypeptide PPA- 80 and enoyl-CoA hydratase in the rat liver. The indirect immunofluorescent studies with these antibodies provided direct visual evidence for the marked induction of polypeptide PPA-80 and enoyl-CoA hydratase in the livers of rats treated with Wy-14,643. The present studies also provide immunocytochemical evidence for the localization of polypeptide PPA- 80 and the heat-labile enoyl-CoA hydratase in the peroxisome, but not in the mitochondria, of hepatic parenchymal cells. These studies, therefore, provide morphological evidence for the existence of fatty acyl-CoA oxidizing system in peroxisomes. An increase of polypeptide PPA-80 on SDS polyacrylamide gel electrophoretic analysis of the subcellular fractions of liver of rodents treated with lipid-lowering drugs should serve as a reliable and sensitive indicator of enhanced peroxisomal beta- oxidation system.

Induction and origin of hepatocytes in rat pancreas.

2-[4(2,2- Dichlorocyclopropyl )phenoxy]2-methyl propionic acid (ciprofibrate), a peroxisome proliferator , induced hepatocytes in the pancreas of adult male F-344 rats when added to their diet at a dosage of 10 mg/kg body weight for 60-72 wk. These cells are morphologically indistinguishable from hepatic hepatocytes and were usually localized adjacent to islets of Langerhans with extensions into surrounding acinar tissue. A significant increase in the volume density of peroxisomes, together with immunochemically detectable amounts of two peroxisome-associated enzymes, was observed in pancreas with hepatocytes of rats maintained on ciprofibrate. Uricase-containing crystalloid nucleoids, specific for rat hepatocyte peroxisomes, were present in pancreatic hepatocytes. These structures facilitated the identification of cells with hybrid cytoplasmic features characteristic of pancreatic acinar and endocrine cells and hepatocytes. Such cells are presumed to represent a transitional state in which pancreas specific genes are being repressed while liver specific ones are simultaneously expressed. The presence of exocrine and/or endocrine secretory granules in transitional cells indicates that acinar/intermediate cells represent the precursor cell from which pancreatic hepatocytes are derived.

Transcription: new insights from studies on Archaea.

Molecular-genetic analyses have revealed that the Archaea (archaebacteria) are phylogenetically distinct from both eukaryotes and eubacteria. Archaea lack nuclei and resemble eubacteria in morphology and genomic organization, but their molecular design shares many features with eukaryotes. Here, we review recent work that indicates that the archaeal transcriptional machinery is strikingly similar to the RNA polymerase I, II, and III systems of eukaryotic cell nuclei. These findings provide important insights into the evolution of the transcriptional apparatus.

Cholera toxin induces expression of the immediate-early response gene JE via a cyclic AMP-independent signaling pathway.

Cholera toxin (CT) activates expression of two immediate-early response genes (JE and TIS10) in quiescent BALB/c 3T3 cells. Increases in cyclic AMP (cAMP) levels in response to CT are likely responsible for the induction of TIS10 gene expression, since treatment with 8-Br-cAMP and increasing the intracellular levels of cAMP by treatment with forskolin induce TIS10 gene expression. In contrast, neither forskolin nor 8-Br-cAMP induces JE gene expression. 3-Isobutyl-1-methylxanthine, which stabilizes intracellular cAMP, potentiates CT-induced TIS10 gene expression but has no effect on CT-induced JE gene expression. Thus, induction of JE by CT is independent of the cAMP produced in response to CT. Induction of JE by CT does not require protein kinase C (PKC), since depleting cells of PKC activity has no effect on the induction of JE by CT. CT-induced expression of JE can be distinguished from CT-induced TIS10 gene expression by using protein kinase inhibitors and inhibitors of arachidonic acid metabolism, further suggesting distinct signaling pathways for CT-induced JE and TIS10 gene expression. Thus, induction of JE gene expression by CT results from the activation of an intracellular signaling pathway that is independent of cAMP production. This pathway is independent of PKC activity and uniquely sensitive to inhibitors of protein kinases and arachidonic acid metabolism.

Role of STAT2 in the alpha interferon signaling pathway.

We have isolated U6A, a mutant cell line which lacks the STAT2 subunit of the transcription factor interferon (IFN)-stimulated gene factor 3 (ISGF3). The response of U6A cells to IFN-alpha is almost completely defective, but the response to IFN-gamma is normal. Complementation of U6A cells with a cDNA encoding STAT2 restores the IFN-alpha response, proving that STAT2 is required in this pathway. Binding of IFNs to their receptors triggers tyrosine phosphorylation and activation of the receptors, JAK family kinases, STAT1, and STAT2. In IFN-alpha-treated U6A cells, phosphorylation of the essential tyrosine kinases TYK2 and JAK1 is normal, but the phosphorylation of STAT1 is weak. A mutant STAT2 protein in which the phosphorylated tyrosine at position 690 is changed to phenylalanine does not restore normal phosphorylation of STAT1 in response to IFN-alpha. The dependence of STAT1 phosphorylation on the presence of STAT2 but not vice versa (T. Improta, C. Schindler, C. M. Horvath, I. M. Kerr, G. R. Stark, and J. E. Darnell, Jr., Proc. Natl. Acad. Sci. USA 91:4776-4780, 1994) indicates that in the formation of ISGF3, these two proteins may be phosphorylated sequentially in response to IFN-alpha and that phosphorylated STAT2 may be required to allow unphosphorylated STAT1 to bind to the activated IFN-alpha receptor.

Function of Stat2 protein in transcriptional activation by alpha interferon.

Alpha interferon (IFN-alpha)-induced transcriptional activation requires the induction of a complex of DNA-binding proteins, including tyrosine-phosphorylated Stat1 and Stat2, and of p48, a protein which is not phosphorylated on tyrosine and which comes from a separate family of DNA-binding proteins. The isolation and characterization of U6A cells, which lack Stat2, have allowed the introduction of normal and mutant forms of Stat2 so that various functions of the Stat2 protein can be examined. As reported earlier, Stat1, which is the second target of tyrosine phosphorylation in IFN-alpha-treated cells, is not phosphorylated in the absence of Stat2. We show that all mutations that block Stat2 phosphorylation also block Stat1 phosphorylation. These include not only the mutations of Y-690 and SH2 domain residues that are involved in tyrosine phosphorylation but also short deletions at the amino terminus of the protein. Two mutants of Stat2 that are not phosphorylated on tyrosine can act as dominant negative proteins in suppressing wild-type Stat2 phosphorylation, most likely by competition at the receptor-kinase interaction site(s). We also show that the COOH-terminal 50 amino acids are required for transcriptional activation in response to IFN-alpha. Mutants lacking these amino acids can be phosphorylated, form IFN-stimulated gene factor 3, and translocate to the nucleus but cannot stimulate IFN-alpha-dependent transcription. Seven acidic residues are present in the deleted COOH-terminal residues, but 24 acidic residues still remain in the 100 carboxy-terminal amino acids after deletion. Thus, transcriptional activation is unlikely to depend on acidic amino acids alone.

Response of hepatocytes transplanted into syngeneic hosts and heterotransplanted into athymic nude mice to peroxisome proliferators.

The development of a transplantation system by which rat hepatocytes can be implanted and remain viable in the dorsal fascia of two-thirds hepatectomized syngeneic hosts provides an opportunity to examine whether such transplanted hepatocytes retain the capacity to recognize and respond to the peroxisome proliferators 2-[4-(2,2- dichlorocyclopropyl )phenoxy]-2- methylpropionic acid (ciprofibrate), a hypolipidemic drug, and di-(2-ethylhexyl)phthalate (DEHP), an industrial plasticizer. Male F344 rats with transplanted rat hepatocytes were fed a control diet or a diet containing either 0.05% ciprofibrate (w/w) or 2% DEHP (v/w). After 4 weeks, the animals were sacrificed, and transplanted hepatocytes revealed a significant increase in the numerical density of peroxisomes in both ciprofibrate- and DEHP-fed rats. The volume density of peroxisomes in transplanted hepatocytes increased 9.2- and 5.3-fold, respectively, in ciprofibrate- and DEHP-fed rats, whereas the volume density of mitochondria remained essentially unchanged. The magnitude of increase in peroxisome volume density in transplanted hepatocytes was comparable to increases in the volume density of these organelles in the liver parenchymal cells of syngeneic hosts. The present study also demonstrates that hepatocytes isolated from cat liver and heterotransplanted into partially hepatectomized athymic nude mice retain their biological integrity and respond to the peroxisome proliferative effect of ciprofibrate. This observation suggests the possibility that hepatocytes obtained from small segments of liver of humans, primates, and other species and heterotransplanted into nude mice might provide a valuable model system for toxicological evaluation of chemicals. These studies suggest that hepatocytes, irrespective of their location in the body, recognize the peroxisome proliferator or its active metabolite(s), which stimulates the expression of peroxisome-specific genes in these cells.

Ha-Ras functions downstream from protein kinase C in v-Fps-induced gene expression mediated by TPA response elements.

v-Fps activates promoters under the control of the 12-O-tetradecanoyl phorbol 13-acetate (TPA) response element (TRE). The induction of TRE-mediated transcription by v-Fps was sensitive to a dominant-negative mutant of Ha-Ras. An activated derivative of Ha-Ras, v-Ha-Ras, also activated TRE-mediated transcription. v-Fps-induced TRE-mediated gene expression was sensitive to depleting cells of protein kinase C (PKC), whereas v-Ha-Ras-induced TRE-mediated transcription was insensitive to PKC depletion, suggesting that Ha-Ras functions downstream from PKC in v-Fps-induced TRE-mediated gene expression. Consistent with this hypothesis, the induction of TRE-mediated gene expression by phorbol esters that activate PKC directly was blocked by the dominant-negative Ha-Ras mutant. Thus, v-Fps-induced activation of TRE-mediated gene expression is via an intracellular signaling mechanism that is dependent upon both PKC and Ha-Ras and Ha-Ras functions downstream from PKC.

Cloning and characterization of a thermolabile v-src gene for use in reversible transformation of mammalian cells.

The use of temperature-sensitive (ts) src mutants for studies of cell transformation and differentiation has been limited by the availability of cloned ts-src genes that are inactivated at temperatures compatible with growth of mammalian cells. In this report, we describe the cloning and characterization of the tsLA90src gene, which displays tight thermal sensitivity at 39.5 degrees C. Nucleotide sequence comparison of tsLA90 and wild-type src genes from the Schmidt-Ruppin subgroup A and D strains of Rous sarcoma virus (RSV) revealed four amino acid differences in tsLA90src. Substitution of one of these residues (Lys-280) from tsLA90src with its wild-type homolog (Glu-280) caused a reversion to a wild-type src phenotype. The cloned tsLA90 gene, designated tsUP1, was introduced into avian and mammalian retroviral vectors. Chicken embryo fibroblasts and immortalized mouse 3T3 cells infected with these viral vectors displayed a temperature-dependent transformed phenotype as assessed by cell morphology, secretion of plasminogen activator, transcriptional activation of the primary response genes, Egr-1 and TIS 10, and stimulation of tyrosine phosphorylation. In addition, chicken myoblasts (infected with RSVtsUP1) showed a temperature-dependent differentiation into myotubes. Thus, this cloned src gene should be ideally suited for inducing reversible transformation and differentiation of mammalian cells in culture.

v-Src activates both protein kinase C-dependent and independent signaling pathways in murine fibroblasts.

Activating the protein-tyrosine kinase activity of v-Src rapidly induced expression of the two 'primary response' genes, TIS10 and Egr-1, in Balb/c 3T3 cells. Depleting cells of protein kinase C (PKC) by prolonged exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA), blocked v-Src-induced TIS10 expression, but had no effect on v-Src-induced Egr-1 gene expression. In addition, the induction of TIS10 and Egr-1 by v-Src could be distinguished using protein kinase inhibitors. Thus, v-Src induced gene expression in murine fibroblasts via two distinguishable signaling pathways: one dependent upon PKC and another that is independent of PKC. Consistent with the use of PKC-mediated signaling pathway by v-Src in murine fibroblasts, we found that activating the kinase activity of v-Src led to increased phosphorylation of a major PKC substrate. Thus, data presented here suggest that v-Src-induced transformation involves the activation of multiple signalling pathways, one of which requires PKC.

Evidence that activation of the Egr-1 promoter by v-Raf involves serum response elements.

The constitutively active serine/threonine kinase encoded by the v-raf oncogene, v-Raf, activates the Egr-1 promoter in transient expression assays. To characterize the v-Raf-responsive transcriptional control elements, deletion mutants of the Egr-1 promoter were used in transient expression assays. A v-Raf expression vector was co-transfected into NIH3T3 cells with reporter chloramphenicol acetyl transferase (CAT) expression vectors under the control of the Egr-1 promoter or the Egr-1 promoter containing various deletions. Responsiveness to v-Raf was restricted to a region that contained repeated CC(A/T)6GG sequences, known as CArG boxes. CArG boxes form the core of serum response elements (SREs). v-Raf-induced Egr-1 promoter activation was lost by removal of the four tandemly repeated SREs. This region, between -425 and -250, which was necessary for v-Raf responsiveness, was also found to be sufficient for maximal Egr-1 induction by v-Raf when placed upstream from a minimal heterologous promoter. Three out of four SREs from this region were able to respond to v-Raf, however the activation of the individual SREs was lower than the clustered SREs. This cluster of SREs has previously been shown to be responsive to several mitogenic stimuli and the oncogene v-src. Thus, the SREs contained in this cluster may be an important target for cell division signals.

Sustained induction of egr-1 by v-src correlates with a lack of fos-mediated repression of the egr-1 promoter.

Serum stimulation of quiescent fibroblasts leads to a transient induction of the transcription factor egr-1. However, the induction of egr-1 by v-src was found to be sustained rather than transient. The proto-oncogene fos has been reported to be co-regulated with egr-1 and to repress serum-induced egr-1 expression. We found that c-fos prevents v-src-induced gene expression regulated by the egr-1 promoter. Thus, the sustained induction of egr-1 by v-src could be explained by a lack of c-fos induction by v-src. Consistent with this hypothesis, egr-1 and c-fos were co-induced by serum, but not by v-src, in Balb/c 3T3 cells; v-src did not induce c-fos expression in these cells. We propose that sustained expression of egr-1 induced by v-src in Balb/c 3T3 cells is due to a lack of c-fos down-regulation of egr-1.

Transcatheter embolization in the treatment of coronary artery fistulas.

Seven patients with a coronary artery fistula underwent percutaneous transcatheter embolization (five were male and two female; the age range was 2 to 67 years [median 17]). Three patients were symptomatic. The left to right shunt ranged from 1.6 to 2.6:1. In six patients, the fistula was an isolated congenital anomaly; in one, it was acquired. The fistula arose from branches of the left (n = 5) and right (n = 2) coronary arteries and drained to the right ventricle (n = 2), right atrium (n = 2), coronary sinus (n = 1), pulmonary artery (n = 1) and a bronchial artery (n = 1). Different embolization techniques were used to occlude eight feeding arteries. The embolization materials included a detachable balloon (n = 3), coaxial embolization with platinum microcoils (n = 3), a combination of detachable balloon and microcoil (n = 1) and standard steel coils (n = 1). Satisfactory occlusion was achieved in six patients. In one case, the valve of the detachable balloon was damaged, resulting in early balloon deflation and a residual fistula. There were no associated complications in any patient. Follow-up investigation by Doppler ultrasound or coronary angiography 4 months to 4 years later showed that permanent occlusion was achieved in all six patients in whom embolization was initially successful. Transcatheter embolization should be considered the treatment of choice for coronary artery fistulas.