Analysis of cell type-specific responses mediated by the type IV secretion system of Helicobacter pylori

Helicobacter pylori persistently infects the human stomach and can cause gastritis, gastric ulceration, and gastric cancer. The type IV secretion system (TFSS) of virulent H. pylori strains translocates the CagA protein, inducing the dephosphorylation of host cell proteins and leading to changes in the morphology or shape of AGS gastric epithelial cells. Furthermore, the TFSS is involved in the induction of proinflammatory cytokines. While the H. pylori genes required for TFSS function have been investigated systematically, little is known about possible host cell factors involved. We infected 19 different mammalian cell lines individually with H. pylori and analyzed CagA translocation, dephosphorylation of host cell proteins, chemokine secretion (interleukin-8 and macrophage inflammatory protein 2), and changes in cellular phenotypes. Our results demonstrate that not only bacterial but also host cell factors determine the cellular response to infection. The identification of such unknown host cell factors will add to our understanding of host-pathogen interactions and might help in the development of new therapeutic strategies.     

Filamentous phage morphogenetic signal sequence and orientation of DNA in the virion and gene-V protein complex

The circular single-stranded viral DNA (ssDNA) of filamentous phage is oriented in the virus with a hairpin forming sequence called mos at the leading end of the virion-the end that emerges from the cell first. In the experiments that originally defined mos it was also found to enhance virion production 100-fold, though in other circumstances it has no such effect. Using a new electron-microscopic method, we have ascertained the orientation of ssDNA in phage having mutations involving mos and other viral functions, and also in the intracellular precursor to the virion-a rod-shaped complex between the ssDNA and the phage-encoded gene-V protein, pV. The results show (1) that the ssDNA is oriented in the complex as in the virion, with mos at one end; (2) that orientation is maintained even if assembly is not mediated by the complex; (3) that orientation is manifested in polyphage--abnormal particles in which many unit-length ssDNA molecules are sheathed in a single, extremely long capsid; (4) that orientation is imposed by mos itself (or something very nearby) since it disappears in a mos deletion; and (5) that a 229-base segment including the minus strand of mos is also effective at imposing orientation. On the basis of our findings, we speculate that mos determines orientation in two stages, one imposing an axis on the ssDNA loop during formation of the pV/ssDNA complex, the other imposing a direction on the loop during initiation of particle assembly. The sequence requirements for the two stages may be different.     

The Topography of Non-Linear Cortical Dynamics at Rest, in Mental Calculation and Moving Shape Perception

Differential cortical activation by cognitive processing was studied using dimensional complexity, a measure derived from nonlinear dynamics that indicates the degrees of freedom (complexity) of a dynamic system. We examined the EEG of 32 healthy subjects at rest, during a visually presented calculation task, and during a moving shape perception task. As a nonlinear measure of connectivity, the mutual dimension of selected electrode pairs was used. The first Lyapunov coefficient was also calculated. Data were tested for non-linearity using a surrogate data method and compared to spectral EEG measures (power, coherence). Surrogate data testing confirmed the presence of nonlinear structure in the data. Cognitive activation led to a highly significant rise in dimensional complexity. While both tasks activated central, parietal and temporal areas, mental arithmetic showed frontal activation and an activity maximum at T3, while the moving shape task led to occipital activation and a right parietal activity maximum. Analysis of mutual dimension showed activation of a bilateral temporal-right frontal network in calculation. The Lyapunov coefficent showed clear topographic variation, but was not significantly changed by mental tasks (p<.09). While dimensional complexity was almost unrelated to power values, nonlinear (mutual dimension) and linear (coherence) measures of connectivity shared up to 37% of variance. Data are interpreted in terms of increased cortical complexity as a result of recruitment of asynchronously active, distributed neuronal assemblies in cognition. The topography of nonlinear dynamics are related to neuropsychological and neuroimaging findings on mental calculation and moving shape perception.     

The nitric oxide pathway in pre-eclampsia: pathophysiological implications

Pre-eclampsia, one of the most significant health problems inhuman pregnancy, complicates 6-7% of all gestations and is theleading cause of fetal growth retardation, infant morbidityand mortality, premature birth and maternal death. Recent researchimplicates free radicals in the pathophysiology of pre-eclampsia.This review covers the biochemistry of nitric oxide (NO) andpossible interactions with other free radicals. Studies in therat show that pregnancy is associated with enhanced productionand responsiveness to NO in both reproductive tissues and bloodvessels. Rats infused with N^G-nitro-L-arginine methyl ester(L-NAME, a NO synthase inhibitor) have been used as an animalmodel of pre-eclampsia, and the effects of steroid hormoneson blood pressure in this model have been tested. Results suggestthat pre-eclampsia may be a state of NO deficiency. However,in humans there seem to be contradictions regarding the involvementof NO in maternal adaptation to pregnancy. It is suggested thatNO may be one of several systems that act in concert to maintaina symbiotic relationship between mother and fetus. However,the input of each system may be genetically determined.     

Identification of a gene disrupted by a microdeletion in a patient with X-linked retinitis pigmentosa (XLRP)

The gene for the most frequent from of X-linked retinitis pigmentosa (XLRP), RP3, has been assigned by genetic and physical mapping to a segment of less than 1000 kbp, which is flanked by the marker DXS1110 and the ornithine transcarbamylase (OTC) gene. In search of microdeletions, we have screened the DNA of 30 unrelated patients with XLRP by employing a representative set of YAC-derived DNA fragments that were generated by restriction enzyme digestion and PCR amplification. In one of these patients, a 6.4 kbp microdeletion was detected which was not present in the DNA of 444 male controls. A cosmid contig spanning the deletion was constructed and used to isolate cDNAs from retina-specific libraries. Exons corresponding to these expressed sequences as well as other putative exons were identified by sequencing more than 30 kbp of the critical region. So far, no point mutations in these putative exon sequences have been identified.      

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Impaired sodium excretion, decreased glomerular filtration rate and elevated blood pressure in endothelin receptor type B deficient rats

The renal endothelin (ET) system, particularly the ET type B receptor, has been implicated in the regulation of sodium excretion and glomerular filtration rate (GFR). We analyzed kidney morphology and function in a rat strain characterized by complete absence of a functional ETB receptor. Due to Hirschsprung's disease limiting lifetime in these rats, studies were performed in 23-day-old rats. Kidney size and morphology (glomerular and interstitial matrix content, glomerular size and cell density and intrarenal vascular morphology) were normal in ETB-deficient rats. There were also no evidence of altered kidney cell cycle regulation in these rats. GFR was significantly lower, by 72% (P<0.001), in homozygous ETB-deficient rats than in wild-type rats. Fractional sodium excretion was likewise markedly reduced by 84% in homozygous ETB-deficient rats (P<0.001 versus wild-type rats). Treatment with the specific epithelial sodium channel blocker amiloride led to a much higher increase in fractional sodium excretion in ETB-deficient rats (934.2+/-73% in ETB-deficient rats versus 297+/-20% in wild-type rats, expressed as percentage of corresponding placebo treated control; P<0.001). Mean arterial blood pressure was elevated by 7.9 mmHg in homozygous ETB-deficient rats (P<0.05 versus wild-type rats). Our study demonstrates that ETB-deficiency causes early onset kidney dysfunction characterized by a markedly reduced sodium excretion, decreased GFR, and slightly elevated blood pressure. The complete absence of the ETB receptor causes in the kidney--in contrast to the colon--a functional rather than a developmental, neural crest cell dependent disease, since kidney morphology was normal in ETB-deficient rats. The much higher increase in the fractional sodium excretion in ETB-deficient rats after pharmacological blockade of the epithelial sodium channel indicates that the decreased fractional sodium excretion in ETB-deficient rats is most probably due to a lack of the inhibitory property of the ETB receptor on the epithelial sodium channel activity.     

Surgical sperm retrieval and intracytoplasmic sperm injection as treatment of obstructive azoospermia

Male genital tract obstructions may result from infections, previous inguinal and scrotal surgery (vasectomy) and congenital bilateral absence of the vas deferens (CBAVD). Microsurgery can sometimes be successful in treating the obstruction. In other cases and in cases of failed surgical intervention, the patient can be treated by microsurgical or percutaneous epididymal sperm aspiration (MESA, PESA) or testicular sperm extraction (TESE) and intracytoplasmic sperm injection (ICSI). We present the results of 39 ICSI procedures for obstructive azoospermia in 24 couples. The aetiology of the obstruction was failed microsurgery in 11 patients, CBAVD in nine and genital infections in four. Sperm retrieval was accomplished via MESA in four cases, PESA in 18 cases and via TESE in 11 cases. TESE was only applied when PESA failed to produce enough spermatozoa for simultaneous ICSI. In six patients, the ICSI procedure was performed with cryopreserved spermatozoa after an initial PESA procedure. Fertilization occurred in 47% of the metaphase II oocytes; embryo transfer was performed in 92% of procedures and resulted in a clinical pregnancy in 13/39 procedures. Ongoing pregnancy was achieved in 10/39 procedures. One pregnancy was terminated early after prenatal investigation showed a cytogenetic abnormality (47,XX+18, Edwards syndrome). The other nine pregnancies resulted in the live birth of 10 children, without any congenital abnormalities. Epididymal and testicular retrieved spermatozoa were successfully used for ICSI to treat obstructive azoospermia, and resulted in an ongoing pregnancy in 10 of 24 couples (41.6%) after 39 ICSI procedures, a success rate of 25.6% per treatment cycle and of 27.7% per embryo transfer.      

Differences in host susceptibility to disease progression in the human challenge model of Haemophilus ducreyi infection

With human volunteers inoculated at two sites with Haemophilus ducreyi, outcomes for a subject were not independent. In a reinfection trial, 2 of 11 previous pustule formers and 6 of 10 previous resolvers resolved all sites of infection. There was no correlation between serum bactericidal or phagocytic activity and outcome in the trial. These data indicate that different hosts are differentially susceptible to disease progression versus resolution in the model.