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1.  Biochemical analysis of the stress protein response in human oesophageal epithelium 
Gut  1997;41(2):156-163.
Background—The oesophageal epithelium is exposed routinely to noxious agents in the environment, including gastric acid, thermal stress, and chemical toxins. These epithelial cells have presumably evolved effective protective mechanisms to withstand tissue damage and repair injured cells. Heat shock protein or stress protein responses play a central role in protecting distinct cell types from different types of injury. 
Aim—To determine (i) whether biochemical analysis of stress protein responses in pinch biopsy specimens from human oesophageal epithelium is feasible; (ii) whether undue stresses are imposed on cells by the act of sample collection, thus precluding analysis of stress responses; and (iii) if amenable to experimentation, the type of heat shock protein (Hsp) response that operates in the human oesophageal epithelium. 
Methods—Tissue from the human oesophagus comprised predominantly of squamous epithelium was acquired within two hours of biopsy and subjected to an in vitro heat shock. Soluble tissue cell lysates derived from untreated or heat shocked samples were examined using denaturing polyacrylamide gel electrophoresis for changes in: (i) the pattern of general protein synthesis by labelling epithelial cells with 35S-methionine and (ii) the levels of soluble Hsp70 protein and related isoforms using immunochemical protein blots. 
Results—A single pinch biopsy specimen is sufficient to extract and analyse specific sets of polypeptides in the oesophageal epithelium. After ex vivo heat shock, a classic inhibition of general protein synthesis is observed and correlates with the increased synthesis of two major proteins of molecular weight of 60 and 70 kDa. Notably, cells from unheated controls exhibit a "stressed" biochemical state 22 hours after incubation at 37°C, as shown by inhibition of general protein synthesis and increased synthesis of the 70 kDa protein. These data indicate that only freshly acquired specimens are suitable for studying stress responses ex vivo. No evidence was found that the two heat induced polypeptides are previously identified Hsp70 isoforms. In fact, heat shock results in a reduction in the steady state concentrations of Hsp70 protein in the oesophageal epithelium. 
Conclusion—Systematic and highly controlled studies on protein biochemistry are possible on epithelial biopsy specimens from the human oesophagus. These technical innovations have permitted the discovery of a novel heat shock response operating in the oesophageal epithelium. Notably, two polypeptides were synthesised after heat shock that seem to differ from Hsp70 protein. In addition, the striking reduction in steady state concentrations of Hsp70 protein after heat shock suggests that oesophageal epithelium has evolved an atypical biochemical response to thermal stress. 


Keywords: oesophagus; heat shock; Hsp70; hyperthermia; stress responses; cancer
PMCID: PMC1891457  PMID: 9301492
4.  Identification of a monooxygenase from Streptomyces coelicolor A3(2) involved in biosynthesis of actinorhodin: purification and characterization of the recombinant enzyme. 
Journal of Bacteriology  1997;179(13):4305-4310.
The oxidation of phenols to quinones is an important reaction in the oxidative tailoring of many aromatic polyketides from bacterial and fungal systems. Sequence similarity between ActVA-Orf6 protein from the actinorhodin biosynthetic cluster and the previously characterized TcmH protein that is involved in tetracenomycin biosynthesis suggested that ActVA-Orf6 might catalyze this transformation as a step in actinorhodin biosynthesis. To investigate the role of ActVA-Orf6 in this oxidation, we have expressed the actVA-Orf6 gene in Escherichia coli and purified and characterized the recombinant protein. ActVA-Orf6 was shown to catalyze the monooxygenation of the tetracenomycin intermediate TcmF1 to TcmD3, strongly suggesting that it catalyzes oxidation of a similar intermediate in actinorhodin biosynthesis. The monooxygenase obeys simple reaction kinetics and has a Km of 4.8 +/- 0.9 microM, close to the figure reported for the homologous enzyme TcmH. The enzyme contains no prosthetic groups and requires only molecular oxygen to catalyze the oxidation. Site-directed mutagenesis was used to investigate the role of histidine residues thought to be important in the reaction; mutants lacking His-52 displayed much-reduced activity, consistent with the proposed mechanistic hypothesis that this histidine acts as a general base during catalysis.
PMCID: PMC179254  PMID: 9209048
5.  Relationships between fatty acid and polyketide synthases from Streptomyces coelicolor A3(2): characterization of the fatty acid synthase acyl carrier protein. 
Journal of Bacteriology  1996;178(19):5660-5667.
We have characterized an acyl carrier protein (ACP) presumed to be involved in the synthesis of fatty acids in Streptomyces coelicolor A3(2). This is the third ACP to have been identified in S. coelicolor; the two previously characterized ACPs are involved in the synthesis of two aromatic polyketides: the blue-pigmented antibiotic actinorhodin and a grey pigment associated with the spore walls. The three ACPs are clearly related. The presumed fatty acid synthase (FAS) ACP was partially purified, and the N-terminal amino acid sequence was obtained. The corresponding gene (acpP) was cloned and sequenced and found to lie within 1 kb of a previously characterized gene (fabD) encoding another subunit of the S. coelicolor FAS, malonyl coenzyme A:ACP acyl-transferase. Expression of S. coelicolor acpP in Escherichia coli yielded several different forms, whose masses corresponded to the active (holo) form of the protein carrying various acyl substituents. To test the mechanisms that normally prevent the FAS ACP from substituting for the actinorhodin ACP, acpP was cloned in place of actI-open reading frame 3 (encoding the actinorhodin ACP) to allow coexpression of acpP with the act polyketide synthase (PKS) genes. Pigmented polyketide production was observed, but only at a small fraction of its former level. This suggests that the FAS and PKS ACPs may be biochemically incompatible and that this could prevent functional complementation between the FAS and PKSs that potentially coexist within the same cells.
PMCID: PMC178404  PMID: 8824610
7.  Production of actinorhodin-related "blue pigments" by Streptomyces coelicolor A3(2). 
Journal of Bacteriology  1996;178(8):2238-2244.
The genetically well-known strain Streptomyces coelicolor A3(2) produces the pH indicator (red/blue) antibiotic actinorhodin, but not all the "blue pigment" produced by this strain is actinorhodin. When the organism was subjected to various nutrient limitations (ammonium, nitrate, phosphate, or trace elements), and also during growth cessation caused by a relatively low medium pH, blue pigment production was initiated but the pigment and its location varied. At pH 4.5 to 5.5, significant formation of actinorhodin occurred and was located exclusively intracellularly. At pH 6.0 to 7.5 a different blue pigment was produced intracellularly as well as extracellularly. It was purified and identified as gamma-actinorhodin (the lactone form of actinorhodin). Analysis of act mutants of S. coelicolor A3(2) confirmed that both pigments are derived from the act biosynthetic pathway. Mutants with lesions in actII-ORF2, actII-ORF3, or actVA-ORF1, previously implicated or suggested to be involved in actinorhodin export, were impaired in production of gamma-actinorhodin, suggesting that synthesis of gamma-actinorhodin from actinorhodin is coupled to its export from the cell. However, effects on the level of actinorhodin production were also found in some mutants.
PMCID: PMC177931  PMID: 8636024
8.  Endocytosis of fluorescent microspheres by human oesophageal epithelial cells: comparison between normal and inflamed tissue. 
Gut  1995;37(5):598-602.
This paper examines the presence and characteristics of endocytosis by oesophageal epithelial cells. Biopsy specimens from normal and inflamed oesophagus were incubated in organ culture with fluorescent microspheres (0.1 and 0.01 microns diameter). These markers were taken into early endosomes and the lysosomes of both the smaller differentiating prickle cells and the larger mature squamous cells. Confocal and electron microscopy showed that markers passed to the early endosomes and the lysosomes by endocytosis. The process was energy dependent. Larger, 1 micron microspheres adhered to the epithelial cells but were not phagocytosed. Disaggregated cells were analysed by flow cytometry. Microspheres were endocytosed in proportion to the concentration in the culture medium in a dose dependent manner. Cells from inflamed oesophagus were significantly smaller (p = 0.013) and took up significantly more microspheres than cells from normal biopsy specimens (p = 0.015). In conclusion, endocytosis occurs in oesophageal epithelial cells and is increased in inflammation.
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PMCID: PMC1382860  PMID: 8549931
9.  Expression of a functional fungal polyketide synthase in the bacterium Streptomyces coelicolor A3(2). 
Journal of Bacteriology  1995;177(15):4544-4548.
The multifunctional 6-methylsalicylic acid synthase gene from Penicillium patulum was engineered for regulated expression in Streptomyces coelicolor. Production of significant amounts of 6-methylsalicylic acid by the recombinant strain was proven by nuclear magnetic resonance spectroscopy. These results suggest that it is possible to harness the molecular diversity of eukaryotic polyketide pathways by heterologous expression of biosynthetic genes in an easily manipulated model bacterial host in which prokaryotic aromatic and modular polyketide synthase genes are already expressed and recombined.
PMCID: PMC177212  PMID: 7635840
10.  Purification of a malonyltransferase from Streptomyces coelicolor A3(2) and analysis of its genetic determinant. 
Journal of Bacteriology  1995;177(14):3946-3952.
Streptomyces coelicolor A3(2) synthesizes each half molecule of the dimeric polyketide antibiotic actinorhodin (Act) from one acetyl and seven malonyl building units, catalyzed by the Act polyketide synthase (PKS). The synthesis is analogous to fatty acid biosynthesis, and there is evident structural similarity between PKSs of Streptomyces spp. and fatty acid synthases (FASs). Each system should depend on a malonyl coenzyme A:acyl carrier protein malonyltransferase, which charges the FAS or PKS with the malonyl units for carbon chain extension. We have purified the Act acyl carrier protein-dependent malonyltransferase from stationary-phase, Act-producing cultures and have determined the N-terminal amino acid sequence and cloned the structural gene. The deduced amino acid sequence resembles those of known malonyltransferases of FASs and PKSs. The gene lies some 2.8 Mb from the rest of the act cluster, adjacent to an open reading frame whose gene product resembles ketoacylsynthase III of Escherichia coli FAS. The malonyltransferase was expressed equally as well during vegetative growth (when other components of the act PKS were not expressed) as in the stationary phase, suggesting that the malonyltransferase may be shared between the FAS and PKS of S. coelicolor. Disruption of the operon containing the malonyltransferase gene proved to be impossible, supporting the idea that the malonyltransferase plays an essential role in fatty acid biosynthesis.
PMCID: PMC177122  PMID: 7608065
11.  Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2). 
Journal of Bacteriology  1995;177(2):473-476.
The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.
PMCID: PMC176614  PMID: 7814340
12.  Cell adhesion molecules in oesophageal epithelium. 
Gut  1994;35(10):1343-1347.
The distribution of a range of integrins, E-cadherin, and carcino-embryonic antigen (CEA) like molecules in normal human oesophageal epithelium was investigated immunohistochemically on frozen sections of endoscopic biopsy specimens. The integrin subunits alpha 2, alpha 3, alpha 6, alpha v, beta 1, and beta 5 were expressed throughout the epithelium. There was strong expression of alpha 2, alpha 3, and beta 1 subunits in the basal cell layer and for all the subunits studied the intensity of the staining decreased as cells moved towards the lumen. The heterodimer alpha v beta 3 was expressed weakly in the basal aspect of the basal cell layer only. The CEA molecules were not present in the basal cells layer but there was weak expression in the prickle cell layer and strong positivity in the mature functional layer. E-cadherin was found throughout the epithelium but was weakly expressed at the basal aspect of the basal cells layer and showed strong positivity in the prickle cell and squamous cell layers. These results indicate that cell-cell (E-cadherin, CEA) and cell-matrix (integrins) adhesion molecules show a well defined spatial pattern of immunoreactivity in the oesophageal mucosa and may play a part in the maintenance of normal tissue architecture and physiological homeostasis.
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PMCID: PMC1375000  PMID: 7959182
13.  tRNA genes of Streptomyces lividans: new sequences and comparison of structure and organization with those of other bacteria. 
Journal of Bacteriology  1994;176(17):5550-5553.
Three closely linked Streptomyces lividans tRNA genes encoding two tRNA(Lys)s and a tRNA(Gly) were cloned and sequences. The structure of tRNA(Gly) is unusual for eubacterial tRNAs. Including those in previous reports (R. Sedlmeier and H. Schmieger, Nucleic Acids Res. 18:4027, 1990, and R. Sedlmeier, G. Linti, K. Gregor, and H. Schmieger, Gene 132:125-130, 1993), 18 S. lividans tRNA genes were physically mapped on the chromosome of the closely related strain Streptomyces coelicolor A3(2). The structure and organization of tRNA genes of S. lividans and S. coelicolor are compared with those of Escherichia coli and Bacillus subtilis.
PMCID: PMC196748  PMID: 8071238
15.  The prognostic significance of the accumulation of p53 tumour-suppressor gene protein in gastric adenocarcinoma. 
British Journal of Cancer  1994;69(5):943-946.
We have studied the expression of p53 in 206 patients with gastric adenocarcinomas. A standard immunohistochemical technique employing the CM-1 anti-p53 polyclonal antibody was applied to the routinely fixed and paraffin-embedded material from these tumours; overexpression of p53 was defined as positive nuclear staining: 46% (94/206) of gastric carcinomas expressed high levels of p53. There was no significant correlation between p53 positivity and the tumour grade, growth pattern, the Lauren type or lymph node metastases. Correlation with disease stage was only marginally significant (P = 0.05). Life table analysis revealed a highly significant association between p53 expression and survival (P = 0.0062), the odds ratio of death being 1.89 (95% confidence interval 1.33-2.69). The overall 5-year survival of patients with p53-positive tumours was 3% compared with 16% for those with p53-negative tumours (median survival time being 5.6 and 11.4 months respectively). These data suggest that overexpression of the p53 oncoprotein is an independent marker of shortened survival in gastric cancer patients.
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PMCID: PMC1968903  PMID: 8180028
16.  Cloning, sequencing, and analysis of the griseusin polyketide synthase gene cluster from Streptomyces griseus. 
Journal of Bacteriology  1994;176(9):2627-2634.
A fragment of DNA was cloned from the Streptomyces griseus K-63 genome by using genes (act) for the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor as a probe. Sequencing of a 5.4-kb segment of the cloned DNA revealed a set of five gris open reading frames (ORFs), corresponding to the act PKS genes, in the following order: ORF1 for a ketosynthase, ORF2 for a chain length-determining factor, ORF3 for an acyl carrier protein, ORF5 for a ketoreductase, and ORF4 for a cyclase-dehydrase. Replacement of the gris genes with a marker gene in the S. griseus genome by using a single-stranded suicide vector propagated in Escherichia coli resulted in loss of the ability to produce griseusins A and B, showing that the five gris genes do indeed encode the type II griseusin PKS. These genes, encoding a PKS that is programmed differently from those for other aromatic PKSs so far available, will provide further valuable material for analysis of the programming mechanism by the construction and analysis of strains carrying hybrid PKS.
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PMCID: PMC205401  PMID: 8169211
17.  Characterization of phi HAU3, a broad-host-range temperate streptomyces phage, and development of phasmids. 
Journal of Bacteriology  1994;176(7):2096-2099.
phi HAU3 is a temperate Streptomyces phage with cohesive ends and a broad host range that includes Streptomyces hygroscopicus 10-22, a producer of antifungal compounds, but it fails to grow on Streptomyces lividans 66. Two phasmid derivatives were constructed that function as lambda cosmid vectors in Escherichia coli and as phages in Streptomyces spp.
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PMCID: PMC205316  PMID: 8144476
18.  Development of a gene cloning system for Streptomyces hygroscopicus subsp. yingchengensis, a producer of three useful antifungal compounds, by elimination of three barriers to DNA transfer. 
Journal of Bacteriology  1994;176(7):2090-2095.
Streptomyces hygroscopicus 10-22 could not be transformed with any of the commonly used Streptomyces plasmid vectors and was resistant to plaque formation by the Streptomyces phages phi C31 and R4. Repeated selection resulted in the isolation of derivatives of S. hygroscopicus 10-22 that could be transformed with pIJ101- and pJV1-derived cloning vectors and of restriction-deficient derivatives that could accept DNA propagated in Streptomyces lividans 66. These new strains, which include three that still produce the original antibiotics, can be used as hosts for gene cloning. Insertion of nonreplicating vectors by homologous recombination and transposition of Tn4560 were demonstrated in S. hygroscopicus 10-22.
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PMCID: PMC205315  PMID: 8144475
19.  Analysis of type II polyketide beta-ketoacyl synthase specificity in Streptomyces coelicolor A3(2) by trans complementation of actinorhodin synthase mutants. 
Journal of Bacteriology  1994;176(6):1801-1804.
Complementation of defined actinorhodin beta-ketoacyl synthase (KS) mutants by various other KS genes suggested that the ORF1-encoded KS may be relatively generalized in function, whereas the ORF2-encoded KS component may provide specificity in polyketide chain construction. Evidence for differential temporal-spatial expression of the actinorhodin and spore pigment KSs in Streptomyces coelicolor was obtained.
PMCID: PMC205275  PMID: 8132481
20.  Evidence for field change in oral cancer based on cytokeratin expression. 
British Journal of Cancer  1993;67(6):1324-1330.
It was hypothesised that one may be able to visualise field changes, which are proposed to exist around tumours, as alterations in keratin intermediate filament protein expression. Standard immunohistochemical analysis using a panel of monoclonal anti-keratin antibodies was applied to fresh tissue sections to look for subtle changes in epithelial differentiation not visible in H&E sections. Such changes were observed in clinically normal epithelium from oral cancer patients, involving primarily substantial expression of keratins K8/K7 (using CAM 5.2) in the basal cells of 12 out of 34 biopsies, and also a trend towards a reduction in the complexity of keratin differentiation. Monitoring such changes may prove to be a valuable adjunct to conventional H&E staining if found to have prognostic and diagnostic significance.
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PMCID: PMC1968511  PMID: 7685618
21.  Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins. 
Journal of Bacteriology  1993;175(8):2197-2204.
The gene that encodes the acyl carrier protein (ACP) of the actinorhodin polyketide synthase (PKS) of Streptomyces coelicolor A3(2) was replaced with homologs from the granaticin, oxytetracycline, tetracenomycin, and putative frenolicin polyketide synthase gene clusters. All of the replacements led to expression of functional synthases, and the recombinants synthesized aromatic polyketides similar in chromatographic properties to actinorhodin or to shunt products produced by mutants defective in the actinorhodin pathway. Some regions within the ACP were also shown to be interchangeable and allow production of a functional hybrid ACP. Structural analysis of the most abundant polyketide product of one of the recombinants by electrospray mass spectrometry suggested that it is identical to mutactin, a previously characterized shunt product of an actVII mutant (deficient in cyclase and dehydrase activities). Quantitative differences in the product profiles of strains that express the various hybrid synthases were observed. These can be explained, at least in part, by differences in ribosome-binding sites upstream of each ACP gene, implying either that the ACP concentration in some strains is rate limiting to overall PKS activity or that the level of ACP expression also influences the expression of another enzyme(s) encoded by a downstream gene(s) in the same operon as the actinorhodin ACP gene. These results reaffirm the idea that construction of hybrid polyketide synthases will be a useful approach for dissecting the molecular basis of the specificity of PKS-catalyzed reactions. However, they also point to the need for reducing the chemical complexity of the approach by minimizing the diversity of polyketide products synthesized in strains that produce recombinant polyketide synthases.
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PMCID: PMC204504  PMID: 8468280
22.  Hepatic iron in dialysed patients given intravenous iron dextran. 
Journal of Clinical Pathology  1990;43(2):119-124.
Five percutaneous biopsy and 17 necropsy liver specimens were analysed histologically and chemically for iron content in 22 patients receiving dialysis for chronic renal failure, 13 of whom were given intravenous iron-dextran. Brissot scores for assessing histological hepatic iron deposition and chemically measured liver iron concentrations correlated closely. Both variables depended on total cumulative dose of iron, and to a lesser extent, on time since the last dose. Fibrosis (seen in five patients) was minimal and non-specific. Electron microscopic examination showed that there was no generalised damage and confirmed the presence of iron in the hepatocytes in the form of ferritin. High liver iron concentrations, in excess of 1000 micrograms/100 mg dry weight, were seen in two patients. Four others given comparable cumulated amounts (18-23 g iron) did not have such high concentrations. Plasma ferritin concentrations were high in eight patients, some with and some without fibrosis. The risk of temporarily high iron deposition in the liver causing damage seemed to be minimal when weighed against the benefit of increased haemoglobin in most of the patients. Intravenous iron treatment merits further evaluation, particularly with the advent of erythropoietin treatment, which requires continuously available iron.
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PMCID: PMC502291  PMID: 2318987
23.  A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp. 
Journal of Bacteriology  1992;174(23):7585-7594.
A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQ1 or afsQ2 on the S. coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQ1 gene on pIJ922 suppressed the S. coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation. Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.
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PMCID: PMC207469  PMID: 1339426
24.  Epidermal growth factor in the oesophagus. 
Gut  1992;33(11):1448-1453.
Epidermal growth factor (EGF) has been implicated in mitogenesis and oncogenesis in the gastrointestinal tract. To determine the role of EGF in oesophageal disease, its quantity and distribution in the oesophageal mucosa of control subjects and patients with oesophageal disease were studied. Oesophageal biopsy specimens, taken 20-40 cm from the incisors in 72 patients, were graded histologically and adjacent specimens were taken for immunohistochemical analysis of the distribution of EGF. In patients with Barrett's columnar lined oesophagus, specimens were also taken from the gastric cardia for comparison. Twenty two biopsy specimens showed oesophagitis, 20 Barrett's mucosa, and 30 were histologically normal. EGF was found in the capillary endothelium of the normal oesophageal papillae and basal mucosa. Significantly more EGF positive papillae were found in the normal mucosa (81%) than in the inflamed mucosa (42%) (p < 0.001). The 20 patients with Barrett's mucosa showed abnormal expression of EGF in 25% of the isthmus and superficial epithelial cells. This study has shown that EGF is found only in the endothelial cells of the capillaries of the normal oesophageal mucosa and that the peptide is detectable significantly less frequently than normal in the inflamed oesophageal mucosa. EGF is also abnormally present, in large quantities, in the cytoplasm of the epithelial cells of Barrett's mucosa compared with gastric mucosa.
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PMCID: PMC1379525  PMID: 1452065
25.  Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway. 
Journal of Bacteriology  1992;174(19):6184-6190.
Streptomyces coelicolor A3(2) and Streptomyces violaceoruber Tü22 produce the antibiotics actinorhodin and granaticin, respectively. Both the aglycone of granaticin and the half-molecule of actinorhodin are derived from one acetyl coenzyme A starter unit and seven malonyl coenzyme A extender units via the polyketide pathway to produce benzoisochromane quinone moieties with identical structures (except for the stereochemistry at two chiral centers). In S. coelicolor and S. violaceoruber, the type II polyketide synthase (PKS) is encoded by clusters of five and six genes, respectively. We complemented a series of S. coelicolor mutants (act) defective in different components of the PKS (actI for carbon chain assembly, actIII for ketoreduction, and actVII for cyclization-dehydration) by the corresponding genes (gra) from S. violaceoruber introduced in trans on low-copy-number plasmids. This procedure showed that four of the act PKS components could be replaced by a heterologous gra protein to give a functional PKS. The analysis also served to identify which of three candidate open reading frames (ORFs) in the actI region had been altered in each of a set of 13 actI mutants. It also proved that actI-ORF2 (whose putative protein product shows overall similarity to the beta-ketoacyl synthase encoded by actI-ORF1 but whose function is unclear) is essential for PKS function. Mutations in each of the four complemented act genes (actI-ORF1, actI-ORF2, actIII, and actVII) were cloned and sequenced, revealing a nonsense or frameshift mutation in each mutant.
PMCID: PMC207686  PMID: 1400167

Results 1-25 (56)