Genetic immunization against cervical carcinoma: induction of cytotoxic T lymphocyte activity with a recombinant alphavirus vector expressing human papillomavirus type 16 E6 and E7.

Infection of genital epithelial cells with human papillomavirus (HPV) types 16 and 18 is closely associated with the development of cervical carcinoma. The transforming potential of these high-risk HPVs depends on the expression of the E6 and E7 early viral gene products. Since the expression of E6 and E7 is selectively maintained in premalignant and malignant cervical lesions these proteins are attractive candidates for immunotherapeutic and prophylactic strategies. This report describes the construction, characterization and the in vivo immunotherapeutic potential of recombinant Semliki Forest virus (SFV) expressing the HPV16 E6 and E7 proteins (SFV-E6E7). Western blot analysis and immunofluorescence staining demonstrated expression of E6 and E7 in BHK cells infected with SFV-E6E7. Immunization of mice with SFV-E6E7 resulted in an efficient in vivo priming of HPV-specific CTL activity. The induced CTL lysed murine tumor cells transformed with the HPV16 genome and EL4 cells loaded with an immunodominant class I-binding HPV E7 peptide. CTLs could reproducibly be induced by immunization with three injections of as few as 10(5) infectious units of SFV-E6E7. Protection from tumor challenge was studied using the tumor cell line TC-1. Immunization with 5 x 10(6) SFV-E6E7 particles protected 40% of the mice from tumor challenge. These results indicate that E6E7 expression by the efficient and safe recombinant SFV system represents a promising strategy for immunotherapy or immunoprophylaxis of cervical carcinoma.

Vectors based on Semliki Forest virus for rapid and efficient gene transfer into non-endothelial cardiovascular cells: comparison to adenovirus.

OBJECTIVE: Replication-deficient, recombinant adenovirus is used as a carrier for gene transfer, but it is unspecific and the onset of transgene expression is relatively late. Here, we evaluated the efficiency and selectivity of gene transfer mediated by recombinant Semliki Forest virus (SFV). METHODS: We compared the efficiency of a SFV-based vector with an adenoviral vector, using LacZ as a reporter gene. Firstly, the affinity for vascular smooth muscle cells, endothelial cells and cardiac myocytes was assessed. Secondly, we compared the time course of LacZ expression and cytotoxicity in vascular smooth muscle cells. RESULTS: The SFV-based vector infects vascular smooth muscle cells and cardiomyocytes as efficiently as adenovirus. In contrast to adenovirus, SFV hardly transfers LacZ to endothelial cells (2.6% or less). SFV-mediated expression was visible after 1 h, reaching a maximum after 6 h. In contrast, adenovirus-mediated expression became visible after 6 h, and reached a maximum after 48-72 h. Both vectors were cytotoxic. CONCLUSIONS: We demonstrate that SFV efficiently transfers LacZ to vascular smooth muscle cells and cardiomyocytes, but not to endothelial cells. In contrast, adenovirus causes efficient transgene expression in all cell types tested. Furthermore, SFV-mediated expression is faster than adenovirus-mediated expression. Therefore, SFV-mediated gene transfer may be a suitable alternative to adenovirus, providing a fast expression in non-endothelial cardiovascular cell types.

Genetic adaptation of bacteria to halogenated aliphatic compounds.

The bacterial degradation and detoxification of chlorinated xenobiotic compounds requires the production of enzymes that are capable of recognizing and converting compounds which do not occur at significant concentrations in nature. We have studied the catabolic route of 1,2-dichloroethane as an example of a pathway for the conversion of such a synthetic compound. In strains of Xanthobacter and Ancylobacter that have been isolated on 1,2-dichloroethane, the first catabolic step is catalyzed by a hydrolytic haloalkane dehalogenase. The enzyme converts 1,2-dichloroethane to 2-chloroethanol but is also active with many other environmentally important haloalkanes such as methylchloride, methylbromide, 1,2-dibromoethane, epichlorohydrin, and 1,3-dichloropropene. Further degradation of 2-chloroethanol proceeds by oxidation to the carboxylic acid and dehalogenation to glycolate. The aldehyde dehydrogenase prevents toxicity of the reactive chloroacetaldehyde that is formed as an intermediate and is necessary for establishing a functional 2-chloroethanol degradative pathway in a strain that is not capable of growth on this compound.

Histidine 289 is essential for hydrolysis of the alkyl-enzyme intermediate of haloalkane dehalogenase.

Haloalkane dehalogenase (DhlA) from Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. Previous work has shown that Asp124, which is located close to the internal substrate-binding cavity, carries out a nucleophilic attack on the C-alpha of the alkylhalide, displacing the halogen. The resulting alkyl-enzyme intermediate is subsequently hydrolyzed. In order to study the role of His289 in the hydrolysis of the intermediate, a His289-->Gln mutant was constructed by site-directed mutagenesis. The purified mutant enzyme was not catalytically active with haloalkanes, but a halide burst stoichiometric to the amount of enzyme was observed with 1,2-dibromoethane. Using ion spray mass spectrometry, accumulation of the covalent alkyl-enzyme and binding of the alkyl moiety of the substrate to an Asp124-containing tryptic peptide were shown. Fluorescence-quenching experiments indicated that halide ions are strongly bound by the alkyl-enzyme but not by the substrate-free enzyme. The results show that His289 is the base catalyst for the dealkylation of the covalent intermediate, but that it is not essential for the initial nucleophilic attack of Asp124 on the C-1 atom of the haloalkane. Furthermore, the halide ion that is released in the first step probably leaves the active site only after hydrolysis of the alkyl-enzyme.

Replacement of tryptophan residues in haloalkane dehalogenase reduces halide binding and catalytic activity.

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in short-chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide-binding site in the active-site cavity, and were proposed to play a role in catalysis. The function of these residues was studied by replacing Trp125 with phenylalanine, glutamine or arginine and Trp175 by glutamine using site-directed mutagenesis. All mutants except Trp125-->Phe showed a more than 10-fold reduced kcat and much higher Km values with 1,2-dichloroethane and 1,2-dibromoethane than the wild-type enzyme. Fluorescence quenching experiments showed a decrease in the affinity of the mutant enzymes for halide ions. The 2H kinetic isotope effect observed with the wild-type enzyme in deuterium oxide was lost in the active mutants, except the Trp125-->Phe enzyme. The results indicate that both tryptophans are involved in stabilizing the transition state during the nucleophilic substitution reaction that causes carbon-halogen bond cleavage.

Activation of an Asp-124-->Asn mutant of haloalkane dehalogenase by hydrolytic deamidation of asparagine.

Haloalkane dehalogenase hydrolyses various 1-halo-n-alkanes to the corresponding alcohols by covalent catalysis with formation of an alkyl-enzyme intermediate. The carboxylate function of the nucleophilic aspartate (Asp-124) that displaces the halogen during formation of the intermediate was changed to an amide by site-directed mutagenesis (Asp-124-->Asn). Activity measurements and analysis of peptides containing the nucleophilic residue showed that the mutant enzyme was inactive, but that the activity increased by rapid deamidation of the asparagine residue, yielding wild type enzyme. There was no indication for isoaspartate formation during this process. The results suggest that a water molecule that is located close to the carboxyl function of Asp-124 in the X-ray structure is highly reactive and is responsible for the observed deamidation.

Genetics and biochemistry of 1,2-dichloroethane degradation.

Dichloroethane (1,2-DCE) is a synthetic compound that is not known to be formed naturally. Nevertheless, several pure microbial cultures are able to use it as a sole carbon source for growth. Degradation of 1,2-DCE proceeds via 2-chloroethanol, chloroacetaldehyde and chloroacetate to glycolate. The genes encoding the enzymes responsible for the conversion of 1,2-DCE to glycolic acid have been isolated. The haloalkane dehalogenase and an aldehyde dehydrogenase are plasmid encoded. Two other enzymes, the alcohol dehydrogenase and the haloacid dehalogenase, are chromosomally encoded. Sequence analysis indicates that the haloacid dehalogenase belongs to the L-specific 2-chloroproprionic acid dehalogenases. From the three-dimensional structure and sequence similarities, the haloalkane dehalogenase appears to be a member of the alpha/beta hydrolase fold hydrolytic enzymes, of which several are involved in the degradation of aromatic and aliphatic xenobiotic compounds.

Degradation of halogenated aliphatic compounds: the role of adaptation.

A limited number of halogenated aliphatic compounds can serve as a growth substrate for aerobic microorganisms. Such cultures have (specifically) developed a variety of enzyme systems to degrade these compounds. Dehalogenations are of critical importance. Various heavily chlorinated compounds are not easily biodegraded, although there are no obvious biochemical or thermodynamic reasons why microorganisms should not be able to grow with any halogenated compound. The very diversity of catabolic enzymes present in cultures that degrade halogenated aliphatics and the occurrence of molecular mechanisms for genetic adaptation serve as good starting points for the evolution of catabolic pathways for compounds that are currently still resistant to biodegradation.

The role of spontaneous cap domain mutations in haloalkane dehalogenase specificity and evolution.

The first step in the utilization of the xenobiotic chlorinated hydrocarbon 1,2-dichloroethane by Xanthobacter autotrophicus is catalyzed by haloalkane dehalogenase (Dh1A). The enzyme hydrolyses 1-haloalkanes to the corresponding alcohols. This allows the organism to grow also on short-chain (C2-C4) 1-chloro-n-alkanes. We have expressed Dh1A in a strain of Pseudomonas that grows on long-chain alcohols and have selected 12 independent mutants that utilize 1-chlorohexane. Six different mutant enzymes with improved Km or Vmax values with 1-chlorohexane were obtained. The sequences of the mutated dh1A genes showed that several mutants had the same 11-amino acid deletion, two mutants carried a different point mutation, and three mutants had different tandem repeats. All mutations occurred in a region encoding the N-terminal part of the cap domain of Dh1A, and it is concluded that this part of the protein is involved in the evolution of activity toward xenobiotic substrates.

Site-directed mutagenesis and oxygen isotope incorporation studies of the nucleophilic aspartate of haloalkane dehalogenase.

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon-halogen bonds in a broad range of halogenated aliphatic compounds. The X-ray structure suggests that Asp124, which is located close to an internal cavity, carries out a nucleophilic attack on the C alpha of the substrate, releasing the halogen. To study the mechanism of hydrolysis, this aspartate residue was mutated to alanine, glycine, or glutamate. The mutant enzymes showed no activity toward 1,2-dichloroethane and 1,2-dibromoethane. Incubation of purified wild-type dehalogenase with 1,2-dichloroethane in the presence of H2(18)O resulted in the incorporation of 18O in 2-chloroethanol and in the carboxylate group of Asp124. This shows that the reaction proceeds by covalent catalysis with the formation of an alkyl-enzyme intermediate that is hydrolyzed by attack of solvent water on the carbonyl carbon of Asp124. On the basis of amino acid sequence similarity between haloalkane dehalogenase and epoxide hydrolases, it is proposed that a conserved aspartate residue is also involved in covalent catalysis by the latter enzymes.

Genetics and biochemistry of dehalogenating enzymes.

Microorganisms that can utilize halogenated compounds as a growth substrate generally produce-enzymes whose function is carbon-halogen bond cleavage. Based on substrate range, reaction type and gene sequences, the dehalogenating enzymes can be classified in different groups, including hydrolytic dehalogenases, glutathione transferases, monooxygenases and hydratases. X-ray crystallographic and biochemical studies have provided detailed mechanistic insight into the action of haloalkane dehalogenase. The essential features are nucleophilic substitution of the halogen by a carboxylate group and the presence of a distinct halogen binding site, formed by tryptophan residues. This review summaries current knowledge on a variety of other dehalogenating enzymes and indicates the existence of a widespread and diverse microbial potential for dechlorination of natural and xenobiotic halogenated compounds.

Construction of an expression and site-directed mutagenesis system of haloalkane dehalogenase in Escherichia coli.

Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21 (DE3) and E. coli JM101. After introduction of restriction sites by PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (phi 10), trc, and tac promoters. This resulted in expression at 30 degrees C up to 38 and 18% of the total soluble cellular protein with the T7 and trc promoters, respectively. Dehalogenase expression under control of the tac promoter was below 1% of the soluble cell protein, however. Aggregation of haloalkane dehalogenase into inclusion bodies was found during growth at 37 degrees C but not at 30 degrees C. Aggregates were also formed from intact enzyme upon incubation at 37 degrees C of cells or crude extracts containing active mature dehalogenase. The high level of expression resulted in a short purification procedure in which 30-35 mg highly enriched haloalkane dehalogenase was obtained from an 0.51 culture. For the production of single-stranded DNA an f1(+) origin was introduced in the T7 expression system.

Degradation of 1,2-dichloroethane by Ancylobacter aquaticus and other facultative methylotrophs.

Cultures of the newly isolated bacterial strains AD20, AD25, and AD27, identified as strains of Ancylobacter aquaticus, were capable of growth on 1,2-dichloroethane (DCE) as the sole carbon and energy source. These strains, as well as two other new DCE utilizers, were facultative methylotrophs and were also able to grow on 2-chloroethanol, chloroacetate, and 2-chloropropionate. In all strains tested, DCE was degraded by initial hydrolytic dehalogenation to 2-chloroethanol, followed by oxidation by a phenazine methosulfate-dependent alcohol dehydrogenase and an NAD-dependent aldehyde dehydrogenase. The resulting chloroacetic acid was converted to glycolate by chloroacetate dehalogenase. The alcohol dehydrogenase was induced during growth on methanol or DCE in strain AD20, but no activity was found during growth on glucose. However, in strain AD25 the enzyme was synthesized to a higher level during growth on glucose than on methanol, and it reached levels of around 2 U/mg of protein in late-exponential-phase cultures growing on glucose. The haloalkane dehalogenase was constitutively produced in all strains tested, but strain AD25 synthesized the enzyme at a level of 30 to 40% of the total cellular protein, which is much higher than that found in other DCE degraders. The nucleotide sequences of the haloalkane dehalogenase (dhlA) genes of strains AD20 and AD25 were the same as the sequence of dhlA from Xanthobacter autotrophicus GJ10 and GJ11. Hybridization experiments showed that the dhlA genes of six different DCE utilizers were all located on an 8.3-kb EcoRI restriction fragment, indicating that the organisms may have obtained the dhlA gene by horizontal gene transmission.

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.

[Leiomyomas of the stomach--diagnostic possibilities]. / Leiomyome des Magens--diagnostische Möglichkeiten.

The article reports on three gastric leiomyomas, each belonging to a different histological sub-type. The influence of imaging methods is described taking sonography and computed tomography into account which are also used for diagnostic purposes.

[Myelolipoma of the adrenals]. / Myelolipom der Nebenniere.

A 47-year-old man reported increasing feeling of fullness in the upper abdomen and pain in both flanks, radiating to both groins. Physical examination did not reveal any abnormalities. Biochemical tests demonstrated hypertriglyceridaemia and hypercholesteremia. Urography, abdominal ultrasound, computed and magnetic resonance tomography revealed a large, fat-containing tumour of the right adrenal. At operation a soft, somewhat elastic adrenal tumour, about 10 cm in diameter, was removed. Histologically it proved to be a myelolipoma. The correct preoperative diagnosis of such a tumour is possible in about 90% of cases.

Value of image enhancement and injection of contrast medium for right ventricular volume determination by two-dimensional echocardiography in congenital heart disease.

To determine factors that influence the accuracy of echocardiographically estimated right ventricular volume and to improve the echocardiographic input information by applying image enhancement techniques, quantitative contrast echocardiography (4-chamber view) and biplane angiocardiography were performed in 23 children during routine diagnostic cardiac catheterization. Volumes calculated on the basis of unprocessed and processed echocardiographic cross sections (area-length method and sphere model) underestimated angiocardiographic volumes significantly (p less than 0.01), and more so in end-diastole (50.6%) than in end-systole (35.9%). Thus, ejection fraction was significantly (p less than 0.01) underestimated; mean values were 0.48 +/- 0.12 and 0.60 +/- 0.08, respectively. The best comparison between echocardiography and angiocardiography at end-diastole was achieved with the sphere model using image enhancement techniques and injection of contrast media, where y = 0.54x - 6.8, r = 0.97, sy.x = 7.3. Correlations, however, in which unprocessed echocardiograms were used showed only slightly less good correlations. With the 6 image-enhancement techniques, a more homogeneous structure of the image and a more distinct outline of the internal surface was achieved. The statistical error improved only slightly. The echocardiographic 4-chamber view allows right ventricular volume determination with an acceptable accuracy. Its underestimation is related to inadequate visualization of trabeculations and mainly to the models used. Application of image enhancement techniques allows easier outlining of the internal cavity surface. The advantage gained by the combination of contrast infection and image enhancement techniques does not warrant the routine central injection of available contrast material.