Structure of the oligogalacturonate-specific KdgM porin.

The phytopathogenic Gram-negative bacterium Dickeya dadantii (Erwinia chrysanthemi) feeds on plant cell walls by secreting pectinases and utilizing the oligogalacturanate products. An outer membrane porin, KdgM, is indispensable for the uptake of these acidic oligosaccharides. Here, the crystal structure of KdgM determined to 1.9 Šresolution is presented. KdgM is folded into a regular 12-stranded antiparallel ß-barrel with a circular cross-section defining a transmembrane pore with a minimal radius of 3.1 Å. Most of the loops that would face the cell exterior in vivo are disordered, but nevertheless mediate contact between densely packed membrane-like layers in the crystal. The channel is lined by two tracks of arginine residues facing each other across the pore, a feature that is conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.

[Unilateral pigmented paravenous retinochoroidal atrophy]. / Unilaterale pigmentierte paravenöse retinochorioidale Atrophie.

We report the case of an asymptomatic unilateral pigmented paravenous retinochoroidal atrophy (PPRCA) in a 43-year-old patient. The right eye showed chorioretinal atrophy with bone-spicule-like pigmentations along the retinal veins. Visual acuity was 20 / 20 and perimetry revealed scotomas correlating to the chorioretinal atrophy. Electrophysiological examination showed decreased signals in ERG and EOG. Fundus autofluorescence and angiography findings are presented. Pathogenetically, a classification as hereditary retinal dystrophy (as in retinitis pigmentosa) as well as a post-inflammatory residuum are discussed.

Short-term reaction of choroidal neovascularization and choriocapillaris to photodynamic therapy in age-related macular degeneration.

PURPOSE: To identify the number of primary angiographic nonresponders to photodynamic therapy (PDT) with verteporfin, to determine the rate and speed of reperfusion of choroidal neovascularization (CNV) within a short observation period of only 5 weeks, and to examine the reaction of the underlying choroidal vessels. METHODS: PDT according to the TAP regimen was carried out in 36 eyes with subfoveal classic CNV secondary to age-related macular degeneration. The response to PDT was examined 1 (T1) and 5 (T2) weeks following treatment. At all visits distant visual acuity was measured and both fluorescein and indocyanine green angiography was carried out. RESULTS: One week after treatment (T1), complete closure of classic CNV had not been achieved in 17% of eyes (primary angiographic nonresponders). At T2, 91% of eyes showed reperfusion of the CNV. In 83% of the primary angiographic nonresponders the CNV size was larger than before treatment. Choroidal shadowing was present in 82% at T1 and in 48% at T2. CONCLUSIONS: Primary angiographic PDT nonresponders are relatively rare; however, in contrast to former reports, they exist and can be identified by follow-up examination 1 week after PDT. Recurrence of leakage occurred earlier than expected and may require closer follow-up and earlier retreatment than recommended by the TAP trial.

DNA-microarrays as tools for the identification of tumor specific gene expression profiles: applications in tumor biology, diagnosis and therapy.

DNA-microarrays allow the analysis of almost the complete gene expression program of tumor samples and normal control samples in a single experiment. This allows the processing of a large number of samples in a reasonable short time. Tumor specific gene expression profiles can be used for molecular tumor classification and as a new diagnostic tool. In addition, the identification of tumor specific genes can help to understand the biology of tumor cells and identified genes can be used for the development of new therapeutic strategies. However, the huge amount of data generated by DNA-microarrays creates new challenges for data analysis. In addition, accuracy and reproducibility of the available techniques require complementary methods for verification of DNA-microarray data.

Signature for vibrational to rotational evolution along the yrast line.

We present a simple method for discerning the evolution from vibrational to rotational structure in nuclei as a function of spin. The prescription is applied to the yrast cascades in the A approximately 110 region and a clear transition from vibrational to rotational motion is found.

[Interval reduction of photodynamic therapy (PDT) in age-related macular degeneration (AMD) is not advantageous. A pilot project]. / Intervallverkürzungen der photodynamischen Therapie (PDT) bei altersbedingter Makuladegeneration (AMD) bringen keine Vorteile. Pilotstudie.

PURPOSE: To find out if carrying out PDT again 6 weeks after initial PDT will improve the results at 3 months. METHODS: A total of 22 eyes were treated according to the dose regimen proposed by the TAP study. RESULTS: After the first PDT, the CNV remained permanently closed in 14% of the eyes. Repeating the treatment 6 weeks later did not lead to permanent closure of more CNVs or a diminished size at 3 months. Short-term re-treatment had no substantial effect on the underlying choriocapillaries. Visual acuity did not profit or suffer from early re-treatment. CONCLUSION: Shortening of the treatment interval has no serious side-effects, but does not seem to improve the results reported in the TAP study.

Dermal fibroblast-derived growth factors restore the ability of beta(1) integrin-deficient embryonal stem cells to differentiate into keratinocytes.

Embryonal stem (ES) cells that are homozygous null for the beta(1) integrin subunit fail to differentiate into keratinocytes in vitro but do differentiate in teratomas and wild-type/beta(1)-null chimeric mice. The failure of beta(1)-null ES cells to differentiate in culture might be the result of defective extracellular matrix assembly or reduced sensitivity to soluble inducing factors. By culturing embryoid bodies on dead, deepidermized human dermis (DED) we showed that epidermal basement membrane did not induce beta(1)-null ES cells to undergo keratinocyte differentiation and did not stimulate the differentiation of wild-type ES cells. Coculture with epidermal keratinocytes also had no effect. However, when human dermal fibroblasts were incorporated into DED, the number of epidermal cysts formed by wild-type ES cells increased dramatically, and small groups of keratin 14-positive cells differentiated from beta(1)-null ES cells. Fibroblast-conditioned medium stimulated differentiation of K14-positive cells in wild-type and beta(1)-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFalpha all stimulated differentiation of keratin 14-positive beta(1)-null cells, and KGF and FGF10 were shown to be produced by the fibroblasts used in coculture experiments. The effects of the growth factors on wild-type ES cells were much less pronounced, suggesting that the concentrations of inducing factors already present in the medium were not limiting for wild-type cells. We conclude that the lack of beta(1) integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation.

Dysbaric osteonecrosis: a reassessment and hypothesis.

Dysbaric osteonecrosis is associated with exposure to large ambient pressure changes, and comprises necrotic lesions in the fatty marrow-containing shafts of the long bones, and the ball and socket joints (hips and shoulders). The fundamental causes are still in question and the illness remains a significant health hazard. Radiological and pathological features of both dysbaric and non-dysbaric osteonecrosis are indistinguishable and both are characterized by intramedullary venous stasis, ischemia and necrosis of bone. It has been generally accepted that gas bubbles (probably by initiating intramedullary venous stasis) are the prime cause of dysbaric osteonecrosis, as well as being responsible for Type 1 Decompression Sickness or 'the bends'. Importantly, however, not all series have found a correlation between dysbaric osteonecrosis and 'the bends'. Thus even though it is likely that gas bubbles remain the prime cause of dysbaric osteonecrosis, workers have proposed that in some cases there is another etiological factor which may exaggerate the pathologic effects of gas bubbles, making the bone more susceptible to necrosis. It is proposed that rapid compression by impeding venous drainage from bone initiates intramedullary venous stasis. In the presence of intramedullary gas bubbles, this may progress to thrombosis, ischemia and bone necrosis. The review offers an explanation for total sparing of the knee joint in dysbaric osteonecrosis, and sole involvement of the hip and shoulder (in terms of sub-articular lesions and subsequent joint collapse). In addition to continued observance of proper decompression procedures, a slower rate of compression may further reduce the incidence of dysbaric osteonecrosis. Bone death or osteonecrosis is a concept which Hippocrates put forward in antiquity (1), but it was not until 1794 that James Russell of Edinburgh wrote the first modern-day descriptions. In these cases infection was the predominant etiology (1,2). In 1888 Konig described necrosis of the adult femoral head without infection (3) (aseptic necrosis of bone) and in the same year Twynam reported a case of osteonecrosis in a caisson worker (4) in which there was still a significant infective component. In 1911 Bornstein and Plate, followed later and independently by Bassoe in 1913, presented radiological confirmation of aseptic necrosis of bone in compressed air workers (5). The first report of aseptic necrosis in an underwater diver subsequently appeared in 1936 (6). The condition of aseptic necrosis of bone in association with exposure to raised ambient pressure (previously referred to as caisson disease, pressure-induced osteoarthropathy (7), 'bone rot' (8) and other synonyms (6)) is now generally known as dysbaric osteonecrosis (6). Despite detailed examination of this problem by many authorities, dysbaric osteonecrosis still remains a significant occupational hazard with serious medico-legal consequences (5-13). This suggests that preventative measures are being based upon an incomplete understanding of the pathophysiology of the disease, and that other etiological factors are perhaps being overlooked.

The Woolley and Roe case.

Albert Woolley and Cecil Roe were healthy, middle-aged men who became paraplegic after spinal anaesthesia for minor surgery at the Chesterfield Royal Hospital in 1947. The spinal anaesthetics were given by the same anaesthetist, Dr Malcolm Graham, using the same drug on the same day at the same hospital. The outcome for the patients and their families was devastating, as it was for the use of spinal anaesthesia in the UK. At the trial 6 yr later, and against the opinion of leading neurologists, the judge accepted Professor Macintosh's suggestion that phenol, in which the ampoules of local anaesthetic had been immersed, had contaminated the local anaesthetic through invisible cracks. In an interview 30 yr after the verdict, Dr Graham believed tha the tragedy was caused by contamination of the spinal needles or syringes during the sterilization process. The subsequent explanation that, on the day in question, descaling liquid in the sterilizing pan had not been replaced by water, supported his belief and finally offered a credible explanation. We review the Woolley and Roe case, the status of spinal anaesthesia before and after 1947, and the relevant medico-legal judgments in claims for negligence in the early days of the National Health Service.

Microsatellite variation in Drosophila melanogaster and Drosophila simulans: a reciprocal test of the ascertainment bias hypothesis.

Interspecific comparisons of microsatellite loci have repeatedly shown that the loci are longer and more variable in the species from which they are derived (the focal species) than are homologous loci in other (nonfocal) species. There is debate as to whether this is due to directional evolution or to an ascertainment bias during the cloning and locus selection processes. This study tests these hypotheses by performing a reciprocal study. Eighteen perfect dinucleotide microsatellite loci identified from a Drosophila simulans library screen and 18 previously identified in an identical Drosophila melanogaster library screen were used to survey natural populations of each species. No difference between focal and nonfocal species was observed for mean PCR fragment length. However, heterozygosity and number of alleles were significantly higher in the focal species than in the nonfocal species. The most common allele in the Zimbabwe population of both species was sequenced for 31 of the 36 loci. The length of the longest stretch of perfect repeat units is, on average, longer in the focal species than in the non-focal species. There is a positive correlation between the length of the longest stretch of perfect repeats and heterozygosity. The difference in heterozygosity can thus be explained by a reduction in the length of the longest stretch of perfect repeats in the nonfocal species. Furthermore, flanking-sequence length difference was noted between the two species at 58% of the loci sequenced. These data do not support the predictions of the directional-evolution hypothesis; however, consistent with the ascertainment bias hypothesis, the lower variability in nonfocal species is an artifact of the microsatellite cloning and isolation process. Our results also suggest that the magnitude of ascertainment bias for repeat unit length is a function of the microsatellite size distribution in the genomes of different species.

The mutation rates of di-, tri- and tetranucleotide repeats in Drosophila melanogaster.

In a recent study, we reported that the combined average mutation rate of 10 di-, 6 tri-, and 8 tetranucleotide repeats in Drosophila melanogaster was 6.3 x 10(-6) mutations per locus per generation, a rate substantially below that of microsatellite repeat units in mammals studied to date (range = 10(-2)-10(-5) per locus per generation). To obtain a more precise estimate of mutation rate for dinucleotide repeat motifs alone, we assayed 39 new dinucleotide repeat microsatellite loci in the mutation accumulation lines from our earlier study. Our estimate of mutation rate for a total of 49 dinucleotide repeats is 9.3 x 10(-6) per locus per generation, only slightly higher than the estimate from our earlier study. We also estimated the relative difference in microsatellite mutation rate among di-, tri-, and tetranucleotide repeats in the genome of D. melanogaster using a method based on population variation, and we found that tri- and tetranucleotide repeats mutate at rates 6.4 and 8.4 times slower than that of dinucleotide repeats, respectively. The slower mutation rates of tri- and tetranucleotide repeats appear to be associated with a relatively short repeat unit length of these repeat motifs in the genome of D. melanogaster. A positive correlation between repeat unit length and allelic variation suggests that mutation rate increases as the repeat unit lengths of microsatellites increase.

Genetic variation and differentiation at microsatellite loci in Drosophila simulans. Evidence for founder effects in new world populations.

Drosophila simulans isofemale lines from Africa, South America, and two locations in North America were surveyed for variation at 16 microsatellite loci on the X, second, and third chromosomes, and 18 microsatellites, which are unmapped. D. simulans is thought to have colonized New World habitats only relatively recently (within the last few hundred years). Consistent with a founder effect occurring as colonizers moved into these New World habitats, we find less microsatellite variability in North and South American D. simulans populations than for an African population. Population subdivision as measured at microsatellites is moderate when averaged across all loci (FST = 0.136), but contrasts sharply with previous studies of allozyme variation, which have showed significantly less differentiation in D. simulans than in D. melanogaster. There are substantially fewer private alleles observed in New World populations of D. simulans than seen in a similar survey of D. melanogaster. In addition to possible differences in population size during their evolutionary histories, varying colonization histories or other demographic events may be necessary to explain discrepancies in the patterns of variation observed at various genetic markers between these closely related species.

Mutation and evolution of microsatellites in Drosophila melanogaster.

Levels of nucleotide polymorphism in the Drosophila melanogaster genome are correlated with rates of recombination. This relationship may be due to hitchhiking of advantageous mutations (selective sweeps) or to continual removal of deleterious mutations from the genome (background selection). One test of the relative contributions of selective sweeps and background selection to the observed levels of variation in the genome of D. melanogaster is to compare levels of nucleotide variability (with a mutation rate on the order of 10(-9) per nucleotide per generation) with more rapidly evolving DNA loci such as microsatellites. This test depends critically on details of the mutational process of microsatellites. In this paper, we summarize our studies of microsatellite characteristics and mutation rates in D. melanogaster. We find that D. melanogaster microsatellites are short and have a mutation rate (6.5 x 10(-6) per locus per generation) several orders of magnitude lower than mammals studied to date. We further show that genetic variation at 18 dinucleotide repeat microsatellites in a population of D. melanogaster from Maryland is correlated with regional rates of recombination. These and other microsatellite data suggest that both background selection and selective sweeps may contribute to the correlation between DNA sequence variation and recombination in Drosophila.

The distribution and frequency of microsatellite loci in Drosophila melanogaster.

We report the results of a comprehensive search of Drosophila melanogaster DNA sequences in GenBank for di-, tri-, and tetranucleotide repeats of more than four repeat units, and a DNA library screen for dinucleotide repeats. Dinucleotide repeats are more abundant (66%) than tri- (30%) or tetranucleotide (4%) repeats. We estimate that 1917 dinucleotide repeats with 10 or more repeat units are present in the euchromatic D. melanogaster genome and, on average, they occur once every 60 kb. Relative to many other animals, dinucleotide repeats in D. melanogaster are short. Tri- and tetranucleotide repeats have even fewer repeat units on average than dinucleotide repeats. Our WorldWide Web site (http://www.bio.cornell.edu/genetics/aquadro/+ ++aquadro.html) posts the complete list of 1298 microsatellites (> or = five repeat units) identified from the GenBank search. We also summarize assay conditions for 70 D. melanogaster microsatellites characterized in previous studies and an additional 56 newly characterized markers.