[Transpupillary thermotherapy (TTT) for uveal melanomas. Long term results of a single TTT with an adapter for a conventional infrared laser]. / Transpupillare Thermotherapie (TTT) von Aderhautmelanomen. Langzeitergebnisse einer alleinigen TTT mit einem Adapter für einen konventionellen Infrarotlaser.

BACKGROUND: An increasing number of orbital recurrences after TTT have been reported; the aim of our paper was to present our long-term results after a maximum follow-up of 8 years and 2 months. PATIENTS AND METHOD: Among 18 eyes, 10 tumors were classified as small, and 8 as medium sized (with a maximum prominence of 5.6 mm): 5 melanomas had a juxtapapillary location, 6 a macular (or juxtamacular) location, and 7 were located in the midperiphery of the fundus. RESULTS: After a median follow-up of 7 years in seven tumors a complete regression (scar formation) could be achieved, and in six a partial regression (with a maximum residual prominence of 2.9 mm) could be seen. In three patients a recurrence was treated either by another TTT or a Ruthenium-106 plaque; in another two recurrences, enucleation had to be performed. In none of the cases has an orbital recurrence occurred so far. CONCLUSION: To prevent recurrences, adequate technique and appropriate selection of patients are mandatory in our opinion (no tumors higher than 3 mm). The higher the tumor prominence, the greater the chance of recurrences. Amelanotic melanomas and macular melanomas seem to respond poorly to thermotherapy.

[Dose-effect relation in revision surgery for consecutive strabismus divergens in adults]. / Dosis-Wirkungs-Beziehung bei Revisionsoperation des konsekutiven Strabismus divergens im Erwachsenenalter.

BACKGROUND: The aim of this study was to establish a dose-effect relationship between the reduction of the angle of deviation and the millimeters of the reoperation performed in patients with consecutive exotropia. PATIENTS AND METHODS: A total of 46 patients who had undergone reoperation for consecutive exotropia (group 1: advancement of the former recessed medial rectus muscle and group 2: additional rececession of the lateral rectus muscle) were reexamined. RESULTS: The dose-effect relationship was 2 degrees /mm in group 1 and1.6 degrees /mm far distance and 1.8 degrees /mm near in group 2. Within the first 4 months after the reoperation we found an exodrift of up to 6 degrees , which remained stable; 98% of our patients reported that their quality of life had improved. CONCLUSION: In cases of consecutive exotropia we consider the postoperative exdodrift in the planing of our reoperation. During the ensuing years patients can expect stability of the results achieved by the reoperation.

[Dose-volume histogram regression analysis of uveal melanomas after single fraction gamma knife radiosurgery]. / Dosis-Volumen-Histogramm-Regressionsanalyse von Aderhautmelanomen nach einzeitiger Leksell-Gamma-Knife-Radiochirurgie.

BACKGROUND: To evaluate tumor regression and sequelae in 30 uveal melanomas treated with Gamma Knife radiosurgery between 1992 and 1995 at the Gamma Knife center in Graz. PATIENTS: A total of 30 uveal and ciliary body melanomas were treated with a single fraction of the Gamma Knife. The mean marginal dose was 52.1 Gy, (median 50 Gy). Of the tumors, 15 were large (higher than 8 mm), 14 were medium sized (3-8 mm) and one was a small melanoma (<3 mm). Among them were 6 juxtapapillary, 10 (juxta-) maculary melanomas, 5 tumors located in the midperiphery and 9 ciliary body melanomas. METHOD: Dose-volume histogram analysis. RESULTS: After a maximum follow up of 9 years and 7 months, 8 tumors regressed into a scar, 17 tumors into a residual prominence between 50% and 80% of the initial tumor height, 3 tumors showed no change and in 2 patients recurrence was obvious. Sequelae were classified as mild (in 3 patients), moderate (3), severe (5) and very severe (19). CONCLUSIONS: Significant factors for the development of severe sequelae were the tumor volume and the marginal dose, the mean dose proved to be more important than the marginal dose and the irradiated volume more important than the selective critical dose.

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

Pseudomonas kilonensis sp. nov., a bacterium isolated from agricultural soil.

A total of 131 bacterial isolates related to Pseudomonas corrugata were obtained from an agricultural soil from northern Germany. Based on 16S rRNA gene sequences, PCR-based genome fingerprinting and multilocus enzyme electrophoresis, they formed two groups, A (119 strains) and B (12 strains). As members of each group were highly similar, a single strain of each group was subsequently characterized by a polyphasic taxonomic approach. The selected member of group A was identified as a strain of Pseudomonas brassicacearum, whereas the selected member of group B was distinct from other species of the genus Pseudomonas. Although DNA-DNA hybridization suggested a close affiliation of the group B strain with P. brassicacearum and Pseudomonas thivervalensis and ribotyping suggested a close affiliation with P. brassicacearum, RAPD data, 16S rRNA gene sequencing and phenotypic characterization indicated the presence of a distinct taxonomic entity. This strain differed from the type strains of P. thivervalensis and P. brassicacearum in 10 and 12 metabolic properties, respectively, whereas the two organisms differ from one another by only two properties. Strains of group B are therefore considered to be members of a new species, for which the name Pseudomonas kilonensis sp. nov. is proposed. The type strain is strain 520-20T (= DSM 13647T = CFBP 5372T).

Requirement of novel competence genes pilT and pilU of Pseudomonas stutzeri for natural transformation and suppression of pilT deficiency by a hexahistidine tag on the type IV pilus protein PilAI.

The ubiquitous species Pseudomonas stutzeri has type IV pili, and these are essential for the natural transformation of the cells. An absolute transformation-deficient mutant obtained after transposon mutagenesis had an insertion in a gene which was termed pilT. The deduced amino acid sequence has identity with PilT of Pseudomonas aeruginosa (94%), Neisseria gonorrhoeae (67%), and other gram-negative species and it contains a nucleotide-binding motif. The mutant was hyperpiliated but defective for further pilus-associated properties, such as twitching motility and plating of pilus-specific phage PO4. [(3)H]thymidine-labeled DNA was bound by the mutant but not taken up. Downstream of pilT a gene, termed pilU, coding for a putative protein with 88% amino acid identity with PilU of P. aeruginosa was identified. Insertional inactivation did not affect piliation, twitching motility, or PO4 infection but reduced transformation to about 10%. The defect was fully complemented by PilU of nontransformable P. aeruginosa. When the pilAI gene (coding for the type IV pilus prepilin) was manipulated to code for a protein in which the six C-terminal amino acids were replaced by six histidine residues and then expressed from a plasmid, it gave a nonpiliated and twitching motility-defective phenotype in pilAI::Gm(r) cells but allowed transformability. Moreover, the mutant allele suppressed the absolute transformation deficiency caused by the pilT mutation. Considering the hypothesized role of pilT(+) in pilus retraction and the presumed requirement of retraction for DNA uptake, it is proposed that the pilT-independent transformation is promoted by PilA mutant protein either as single molecules or as minimal pilin assembly structures in the periplasm which may resemble depolymerized pili and that these cause the outer membrane pores to open for DNA entry.

Identification and characterization of novel competence genes comA and exbB involved in natural genetic transformation of Pseudomonas stutzeri.

After transposon mutagenesis we identified a novel gene (comA) of Pseudomonas stutzeri which is essential for natural genetic transformation. The putative amino acid sequence is similar to ComA orthologs of other transformable bacteria including Neisseria gonorrhoeae (ComA), Haemophilus influenzae (Rec-2), Bacillus subtilis (ComEC) and Streptococcus pneumoniae (CelB). Downstream of comA two partially overlapping open reading frames termed exbB and exbD were found coding for putative proteins similar to proteins required for macromolecule uptake in Escherichia coli and present in other Gram-negative bacteria. Insertional inactivation of exbB decreased the transformability to 20% of that of the wild type. The binding of 3H-labeled DNA and its uptake into a DNase-resistant state in the comA and exbB strains were similar to the wild type, suggesting that these proteins are involved in a late step of transformation, presumably in the translocation of DNA from the periplasm into the cytosol. The question of whether the translocation process occurs separately from the step of single-strand formation is discussed.

The structure of a local population of phytopathogenic Pseudomonas brassicacearum from agricultural soil indicates development under purifying selection pressure.

Among the isolates of a bacterial community from a soil sample taken from an agricultural plot in northern Germany, a population consisting of 119 strains was obtained that was identified by 16S rDNA sequencing and genomic fingerprinting as belonging to the recently described species Pseudomonas brassicacearum. Analysis of the population structure by allozyme electrophoresis (11 loci) and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR; four primers) showed higher resolution with the latter method. Both methods indicated the presence of three lineages, one of which dominated strongly. Stochastic tests derived from the neutral theory of evolution (including Slatkin's exact test, Watterson's homozygosity test and the Tajima test) indicated that the population had developed under strong purifying selection pressure. The presence of strains clearly divergent from the majority of the population can be explained by in situ evolution or by influx of strains as a result of migration or both. Phytopathogenicity of a P. brassicacearum strain determined with tomato plants reached the level obtained with the type strain of the known pathogen Pseudomonas corrugata. The results show that a selective sweep was identified in a local population. Previously, a local selective sweep had not been seen in several populations of different bacterial species from a variety of environmental habitats.

Pseudomonas stutzeri has two closely related pilA genes (Type IV pilus structural protein) with opposite influences on natural genetic transformation.

Pseudomonas stutzeri has type IV pili for which the pilA gene (here termed pilAI) provides the structural protein and which are required for DNA uptake and natural genetic transformation. Downstream of pilAI we identified a gene, termed pilAII, coding for a deduced protein with a size similar to that of PilAI with 55% amino acid sequence identity and with a typical leader peptide including a leader peptidase cleavage site. Fusions to lacZ revealed that pilAII is expressed only about 10% compared to pilAI, although the genes are cotranscribed as shown by reverse transcription-PCR. Surprisingly, insertional inactivation of pilAII produced a hypertransformation phenotype giving about 16-fold-increased transformation frequencies. Hypertransformation also occurred in pilAI pilAII double mutants expressing heterologous pilA genes of nontransformable bacteria, like Pseudomonas aeruginosa or Dichelobacter nodosus. The overexpression of pilAII decreased transformation up to 5,000-fold compared to that of the pilAII mutant. However, neither inactivation of pilAII nor its overexpression affected the amounts of [(3)H]thymidine-labeled DNA that were competence-specifically bound and taken up by the cells. In the pilAII mutant, the transformation by purified single-stranded DNA (which depends on comA and exbB, as does transformation by duplex DNA) was also increased 17-fold. It is concluded that PilAII suppresses a step in transformation after the uptake of duplex DNA into the cell and perhaps before its translocation into the cytoplasm. The idea that the degree of the transformability of cells could be permanently adjusted by the expression level of an antagonistic protein is discussed.

The natural transformation of the soil bacteria Pseudomonas stutzeri and Acinetobacter sp. by transgenic plant DNA strictly depends on homologous sequences in the recipient cells.

The nptII(+) gene present in the genome of transgenic potato plants transforms naturally competent cells of the soil bacteria Pseudomonas stutzeri and Acinetobacter BD413 (both harboring a plasmid with an nptII gene containing a small deletion) with the same high efficiency as nptII(+) genes on plasmid DNA (3x10(-5)-1x10(-4) transformants per nptII(+)) despite the presence of a more than 10(6)-fold excess of plant DNA. However, in the absence of homologous sequences in the recipient cells the transformation by nptII(+) dropped by at least about 10(8)-fold in P. stutzeri and 10(9)-fold in Acinetobacter resulting in the latter strain in < or =1x10(-13) transformants per nptII(+). This indicated a very low probability of non-homologous DNA fragments to be integrated by illegitimate recombination events during transformation.

Safety considerations of DNA in food.

Recombinant DNA techniques are capable of introducing genetic changes into food organisms that are more predictable than those introduced through conventional breeding techniques. This review discusses whether the consumption of DNA in approved novel foods and novel food ingredients derived from genetically modified organisms (GMOs) can be regarded as being as safe as the consumption of DNA in existing foods. It concludes that DNA from GMOs is equivalent to DNA from existing food organisms that has always been consumed with human diets. Any risks associated with the consumption of DNA will remain, irrespective of its origin, because the body handles all DNA in the same way. The breakdown of DNA during food processing and passage through the gastrointestinal tract reduces the likelihood that intact genes capable of encoding foreign proteins will be transferred to gut microflora. The review does not specifically address food safety issues arising from the consumption of viable genetically modified microorganisms but it shows that the likelihood of transfer and functional integration of DNA from ingested food by gut microflora and/or human cells is minimal. Information reviewed does not indicate any safety concerns associated with the ingestion of DNA per se from GMOs resulting from the use of currently available recombinant DNA techniques in the food chain.

A broad-host-range expression vector series including a Ptac test plasmid and its application in the expression of the dod gene of Serratia marcescens (coding for ribulose-5-phosphate 3-epimerase) in Pseudomonas stutzeri.

A series of expression vectors for gram-negative bacteria was constructed which combine broad-host-range, inducible expression from the tac promoter and diverse antibiotic resistance determinants. The tac promoter activity and the repression by lacIq can be quantitated with a separate test plasmid in the strain to be studied. The dod gene of Serratia marcescens was expressed in Pseudomonas stutzeri and was shown to code for D-ribulose-5-phosphate 3-epimerase.

Increased killing of Bacillus subtilis on the hair roots of transgenic T4 lysozyme-producing potatoes.

Transgenic potato plants expressing the phage T4 lysozyme gene which are resistant to the plant-pathogenic enterobacterium Erwinia carotovora subsp. carotovora have been constructed. The agricultural growth of these potatoes might have harmful effects on soil microbiota as a result of T4 lysozyme release into the rhizosphere. To assess the bactericidal effect of roots, we have developed a novel method to associate the cells of Bacillus subtilis with hair roots of plants and to quantify the survival of cells directly on the root surface by appropriate staining and fluorescence microscopy. With this technique, we found that the roots of potato plants (Désirée and transgenic control lines) without T4 lysozyme gene display measurable killing activity on root-adsorbed B. subtilis cells. Killing was largely independent of the plant age and growth of plants in greenhouse or field plots. Roots from potato lines expressing the T4 lysozyme gene always showed significantly (1.5- to 3.5-fold) higher killing. It is concluded that T4 lysozyme is released from the root epidermis cells and is active in the fluid film on the root surface. We discuss why strong negative effects of T4 lysozyme-producing potatoes on soil bacteria in field trials may not be observed. We propose that the novel method presented here to study interactions of bacteria with roots can be applied not only to bacterial killing but also to interactions leading to growth-sustaining effects of plants on bacteria.

Type IV pilus genes pilA and pilC of Pseudomonas stutzeri are required for natural genetic transformation, and pilA can be replaced by corresponding genes from nontransformable species.

Pseudomonas stutzeri lives in terrestrial and aquatic habitats and is capable of natural genetic transformation. After transposon mutagenesis, transformation-deficient mutants were isolated from a P. stutzeri JM300 strain. In one of them a gene which coded for a protein with 75% amino acid sequence identity to PilC of Pseudomonas aeruginosa, an accessory protein for type IV pilus biogenesis, was inactivated. The presence of type IV pili was demonstrated by susceptibility to the type IV pilus-dependent phage PO4, by occurrence of twitching motility, and by electron microscopy. The pilC mutant had no pili and was defective in twitching motility. Further sequencing revealed that pilC is clustered in an operon with genes homologous to pilB and pilD of P. aeruginosa, which are also involved in pilus formation. Next to these genes but transcribed in the opposite orientation a pilA gene encoding a protein with high amino acid sequence identity to pilin, the structural component of type IV pili, was identified. Insertional inactivation of pilA abolished pilus formation, PO4 plating, twitching motility, and natural transformation. The amounts of (3)H-labeled P. stutzeri DNA that were bound to competent parental cells and taken up were strongly reduced in the pilC and pilA mutants. Remarkably, the cloned pilA genes from nontransformable organisms like Dichelobacter nodosus and the PAK and PAO strains of P. aeruginosa fully restored pilus formation and transformability of the P. stutzeri pilA mutant (along with PO4 plating and twitching motility). It is concluded that the type IV pili of the soil bacterium P. stutzeri function in DNA uptake for transformation and that their role in this process is not confined to the species-specific pilin.

Characterization of a dam mutant of Serratia marcescens and nucleotide sequence of the dam region.

The DNA of Serratia marcescens has N6-adenine methylation in GATC sequences. Among 2-aminopurine-sensitive mutants isolated from S. marcescens Sr41, one was identified which lacked GATC methylation. The mutant showed up to 30-fold increased spontaneous mutability and enhanced mutability after treatment with 2-aminopurine, ethyl methanesulfonate, or UV light. The gene (dam) coding for the adenine methyltransferase (Dam enzyme) of S. marcescens was identified on a gene bank plasmid which alleviated the 2-aminopurine sensitivity and the higher mutability of a dam-13::Tn9 mutant of Escherichia coli. Nucleotide sequencing revealed that the deduced amino acid sequence of Dam (270 amino acids; molecular mass, 31.3 kDa) has 72% identity to the Dam enzyme of E. coli. The dam gene is located between flanking genes which are similar to those found to the sides of the E. coli dam gene. The results of complementation studies indicated that like Dam of E. coli and unlike Dam of Vibrio cholerae, the Dam enzyme of S. marcescens plays an important role in mutation avoidance by allowing the mismatch repair enzymes to discriminate between the parental and newly synthesized strands during correction of replication errors.

Interaction of RecBCD enzyme with DNA at double-strand breaks produced in UV-irradiated Escherichia coli: requirement for DNA end processing.

The RecBCD enzyme has a powerful duplex DNA exonuclease activity in vivo. We found that this activity decreased strongly when cells were irradiated with UV light (135 J/m2). The activity decrease was seen by an increase in survival of phage T4 2(-) of about 200-fold (phage T4 2(-) has defective duplex DNA end-protecting gene 2 protein). The activity decrease depended on excision repair proficiency of the cells and a postirradiation incubation. During this time, chromosome fragmentation occurred as demonstrated by pulsed-field gel electrophoresis. In accord with previous observations, it was concluded that the RecBCD enzyme is silenced during interaction with duplex DNA fragments containing Chi nucleotide sequences. The silencing was suppressed by induction or permanent derepression of the SOS system or by the overproduction of single-strand DNA binding protein (from a plasmid with ssb+) which is known to inhibit degradation of chromosomal DNA by cellular DNases. Further, mutations in xonA, recJ, and sbcCD, particularly in the recJ sbcCD and xonA recJ sbcCD combinations, impeded RecBCD silencing. The findings suggest that the DNA fragments had single-stranded tails of a length which prevents loading of RecBCD. It is concluded that in wild-type cells the tails are effectively removed by single-strand-specific DNases including exonuclease I, RecJ DNase, and SbcCD DNase. By this, tailed DNA ends are processed to entry sites for RecBCD. It is proposed that end blunting functions to direct DNA ends into the RecABCD pathway. This pathway specifically activates Chi-containing regions for recombination and recombinational repair.

Detection of nptII (kanamycin resistance) genes in genomes of transgenic plants by marker-rescue transformation.

We have developed a novel system for the sensitive detection of nptII genes (kanamycin resistance determinants) including those present in transgenic plant genomes. The assay is based on the recombinational repair of an nptII gene with an internal 10-bp deletion located on a plasmid downstream of a bacterial promoter. Uptake of an nptII gene by transformation restores kanamycin resistance. In Escherichia coli, promoterless nptII genes provided by electroporation were rescued with high efficiency in a RecA-dependent recombinational process. For the rescue of nptII genes present in chromosomal plant DNA, the system was adapted to natural transformation, which favours the uptake of linear DNA. When competent Acinetobacter sp. BD413 (formerly A. calcoaceticus) cells containing the mutant nptII gene on a plasmid were transformed with DNA from various transgenic plants carrying nptII as a marker gene (Solanum tuberosum, Nicotiana tabacum, Beta vulgaris, Brassica napus, Lycopersicon esculentum), kanamycin-resistant transformants were obtained roughly in proportion to the concentration of nptII genes in the plant DNA. The rescue of nptII genes occurred in the presence of a more than 6 x 10(6)-fold excess of plant DNA. Only 18 ng of potato DNA (2.5 x 10(3) genome equivalents, each with one copy of nptII) was required to produce one kanamycin-resistant transformant. These experiments and others employing DNA isolated from soil samples demonstrate that the system allows reliable and highly sensitive monitoring of nptII genes in transgenic plant DNA and in DNA from environmental sources, such as soil, without the need for prior DNA amplification (e.g. by PCR).

Identification of multiple plasmids released from recombinant genomes of Hansenula polymorpha by transformation of Escherichia coli.

Total DNAs isolated from two Hansenula polymorpha (Pichia angusta) strains having chromosomal single or tandem multiple integrations of a pUC18-derived expression plasmid produced Escherichia coli transformants which contained plasmids of different size and/or organization than that of the expression plasmid. Evidence that plasmid-like structures are formed in H. polymorpha and that their formation is stimulated by DNA damage is presented in this study.

Interaction with the recombination hot spot chi in vivo converts the RecBCD enzyme of Escherichia coli into a chi-independent recombinase by inactivation of the RecD subunit.

The RecBCD enzyme of Escherichia coli promotes recombination preferentially at chi nucleotide sequences and has in vivo helicase and strong duplex DNA exonuclease (exoV) activities. The enzyme without the RecD subunit, as in a recD null mutant, promotes recombination efficiently but independently of chi and has no nucleolytic activity. Employing phage lambda red gam crosses, phage T4 2- survival measurements, and exoV assays, it is shown that E. coli cells in which RecBCD has extensive opportunity to interact with linear chi-containing DNA (produced by rolling circle replication of a plasmid with chi or by bleomycin-induced fragmentation of the cellular chromosome) acquire the phenotype of a recD mutant and maintain this for approximately 2 h. It is concluded that RecBCD is converted into RecBC during interaction with chi by irreversible inactivation of RecD. After conversion, the enzyme is released and initiates recombination on other DNA molecules in a chi-independent fashion. Overexpression of recD+ (from a plasmid) prevented the phenotypic change and providing RecD after the change restored chi-stimulated recombination. The observed recA+ dependence of the downregulation of exoV could explain the previously noted "reckless" DNA degradation of recA mutants. It is proposed that chi sites are regulatory elements for the RecBCD to RecBC switch and thereby function as cis- and trans-acting stimulators of RecBC-dependent recombination.