Location and restoration of function after cerebellar tumor removal-a longitudinal study of children and adolescents.

Sequelae in children following cerebellar tumor removal surgery are well defined, and predictors for poor recovery include lesions of the cerebellar nuclei and the inferior vermis. Dynamic reorganization is thought to promote functional recovery in particular within the first year after surgery. Yet, the time course and mechanisms of recovery within this critical time frame are elusive and longitudinal studies are missing. Thus, a group of children and adolescents (n = 12, range 6-17 years) were followed longitudinally after cerebellar surgery and compared to age- and gender-matched controls (n = 11). Patients were examined (1) within the first days, (2) 3 months, and (3) 1 year after surgery. Each time behavioral tests of balance and upper limb motor function, ataxia rating, and a MRI scan were performed. Data were used for subsequent lesion-symptom mapping of cerebellar function. Behavioral improvements continued beyond 3 months, but were not complete in all patients after 1 year. At that time, remaining deficits were mild. Within the first 3 months, cerebellar lesion volumes were notably reduced by vanishing edema. Reduction in edema affecting the deep cerebellar nuclei but not reduction of total cerebellar lesion volume was a major predictor of early functional recovery. Persistent impairment in balance and upper limb function was linked to permanent lesions of the inferior vermis and the deep cerebellar nuclei.

Process analysis and economics of drinking water production from coastal aquifers containing chromophoric dissolved organic matter and bromide using nanofiltration and ozonation.

In regions characterized by water scarcity, such as coastal Southern California, groundwater containing chromophoric dissolved organic matter is a viable source of water supply. In the coastal aquifer of Orange County in California, seawater intrusion driven by coastal groundwater pumping increased the concentration of bromide in extracted groundwater from 0.4 mg l⁻¹ in 2000 to over 0.8 mg l⁻¹ in 2004. Bromide, a precursor to bromate formation is regulated by USEPA and the California Department of Health as a potential carcinogen and therefore must be reduced to a level below 10 µg l⁻¹. This paper compares two processes for treatment of highly coloured groundwater: nanofiltration and ozone injection coupled with biologically activated carbon. The requirement for bromate removal decreased the water production in the ozonation process to compensate for increased maintenance requirements, and required the adoption of catalytic carbon with associated increase in capital and operating costs per unit volume. However, due to the absence of oxidant addition in nanofiltration processes, this process is not affected by bromide. We performed a process analysis and a comparative economic analysis of capital and operating costs for both technologies. Our results show that for the case studied in coastal Southern California, nanofiltration has higher throughput and lower specific capital and operating cost, when compared to ozone injection with biologically activate carbon. Ozone injection with biologically activated carbon, compared to nanofiltration, has 14% higher capital cost and 12% higher operating costs per unit water produced while operating at the initial throughput. Due to reduced ozone concentration required to accommodate for bromate reduction, the ozonation process throughput is reduced and the actual cost increase (per unit water produced) is 68% higher for capital cost and 30% higher for operations.

Oncogene-blocking therapies: new insights from conditional mouse tumor models.

Identification of oncogene dependent signaling pathways controlling aggressive tumor growth has led to the emergence of a new era of oncogene-blocking therapies, including Herceptin and Gleevec. In the recent years conditional mouse tumor models have been established that allow switching-off the expression of specific oncogenes controlling tumor growth. The results may have two important implications for oncogene-blocking therapies: (i) downregulation of oncogenes, for instance HER2, MYC, RAS, RAF, BCR-ABL or WNT1, usually leads to a rapid tumor remission. However, it was observed that the initial remission was followed by recurrent tumor growth in most studies. Interestingly, different oncogenes controlled tumor growth in the recurrent than in the primary tumors. This could explain the astonishing clinical observation that inhibitors of a broader spectrum of protein kinases (so-called: "dirty inhibitors") may be superior over highly specific substances. Due to their additional "unspecific" inhibition of a broader spectrum of kinases, they may hamper the escape mechanisms by antagonizing also the pathways controlling recurrent tumor growth. (ii) Experiments with cell systems that allow switching-on oncogene expression point to a so far possibly underestimated cancer drug target: the dormant tumor cell. Oncogene expression (for instance: NeuT or RAS) led to a phenomenon named oncogene-induced senescence or dormancy. Dormant cells are unresponsive to mitogenic stimuli. Importantly, such cells are not at all ready to die, but can remain viable for extended periods of time. Recently, dormant tumor cells have been shown to be more resistant to stresses such as hypoxia or exposure to cytostatic drugs. It still is a matter of debate if and under which conditions dormant tumor cells can be "kissed to life". If these cells contribute to carcinogenesis, it will be important to identify substances specifically killing senescent cells. This review will focus on the possible relevance of senescence both as a pre-oncogenic condition and also for therapy.

4-Epidoxycycline: an alternative to doxycycline to control gene expression in conditional mouse models.

Since the pioneering work by Gossen and Bujard in 1992 demonstrating the usefulness of the Escherichia coli derived tet resistance operon for regulating gene expression a large collection of doxycycline-controlled transgenic mice has been established. Gene switching in eukaryotic tissue culture cells or mice requires administration of tetracycline, anhydrotetracycline or doxycycline to efficiently inactivate the transactivator protein tTA (TET-OFF system) or alternatively to activate the reverse transactivator protein rtTA (TET-ON system). However, the antibiotic activity of doxycycline can create an imbalance of the intestinal flora, resulting in diarrhoea and in a smaller number of animals in colitis. Previous studies reported that 4-epidoxycycline (4-ED), a hepatic metabolite of doxycycline, does not function as an antibiotic in mice. This gave us the idea that 4-ED might be useful for controlling gene expression in mice without the unwanted antibiotic side effect. To study the applicability of 4-ED for control of gene expression we used cell lines expressing the oncogene HER2 under control of tTA (TET-OFF) as well as rtTA (TET-ON). 4-ED and doxycycline were similarly efficient in switching on or -off HER2 expression. In vivo we used a conditional mouse model that allows switching off HER2 in tumor tissue. We show that (i) doxycycline, 7.5mg/ml in drinking water (used as a positive control), (ii) 4-ED, 7.5mg/ml in drinking water, (iii) 4-ED, 10mg/kg body weight, s.c., and (iv) anhydrotetracycline, 10mg/kg, s.c. (used as a second positive control), were similarly efficient. Using mice with tumor volumes of 1.6cm(3) all four schedules led to a tumor remission of more than 95% within 7 days. In conclusion, 4-ED is similarly efficient as doxycycline to control gene expression in vitro and in mice. Since 4-ED lacks the antibiotic activity of doxycycline it may help to avoid adverse side effects and selection of resistant bacteria.

RNAi knock-down mice: an emerging technology for post-genomic functional genetics.

RNA interference (RNAi) has been extensively used for sequence-specific silencing of gene function in mammalian cells. The latest major breakthrough in the application of RNAi technology came from experiments demonstrating RNAi-mediated gene repression in mice and rats. After more than two decades of functional mouse research aimed at developing and continuously improving transgenic and knock-out technology, the advent of RNAi knock-down mice represents a valuable new alternative for studying gene function in vivo. In this review we provide some basic insight as to how RNAi can induce gene silencing to then focus on recent findings concerning the applicability of RNAi for regulating gene function in the mouse. Reviewed topics will include delivery methods for RNAi-mediating molecules, a comparison between traditional knock-out and innovative transgenic RNAi technology and the generation of graded RNAi knock-down phenotypes. Apart from the exciting possibilities RNAi provides for studying gene function in mice, we discuss several caveats and limitations to be considered. Finally, we present prospective strategies as to how RNAi technology might be applied for generating conditional and tissue-restricted knock-down mice.

Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression.

Alterations within human chromosomal region 11p15.5 are associated with the Beckwith-Wiedemann syndrome (BWS) and predisposition to a variety of neoplasias, including Wilms' tumors (WTs), rhabdoid tumors and rhabdomyosarcomas. To identify candidate genes for 11p15. 5-related diseases we compared human genomic sequence with expressed sequence tag and protein databases from different organisms to discover evolutionarily conserved sequences. Herein we describe the identification and characterization of a novel human transcript related to a putative Caenorhabditis elegans protein and the trp (transient receptor potential) gene. The highest homologies are observed with the human TRPC7 and with melastatin 1 ( MLSN1 ), whose transcript is downregulated in metastatic melanomas. Other genes related to and interacting with the trp family include the Grc gene, which codes for a growth factor-regulated channel protein, and PKD1/PKD2, involved in polycystic kidney disease. The novel gene presented here (named MTR1 for MLSN1 - and TRP -related gene 1) resides between TSSC4 and KvLQT1. MTR1 is expressed as a 4.5 kb transcript in a variety of fetal and adult tissues. The putative open reading frame is encoded in 24 exons, one of which is alternatively spliced leading to two possible proteins of 872 or 1165 amino acids with several predicted membrane-spanning domains in both versions. MTR1 transcripts are present in a large proportion of WTs and rhabdomyosarcomas. RT-PCR analysis of somatic cell hybrids harboring a single human chromosome 11 demonstrated exclusive expression of MTR1 in cell lines carrying a paternal chromosome 11, indicating allele-specific inactivation of the maternal copy by genomic imprinting.

Hepatitis B virus core promoter mutations in children with multiple anti-HBe/HBeAg reactivations result in enhanced promoter activity.

Sera of two children were examined to determine whether specific hepatitis B virus (HBV) mutants may contribute to anti-hepatitis B e/hepatitis B e antigen (anti-HBe/HBeAg) reactivations during the course of chronic hepatitis B. The full-length HBV genome isolated from sera of patient 1 and the basic core promoter (BCP) from patient 2 were amplified and sequenced before and after several reactivations. The functional significance of the mutant BCP from patient 1 was studied using the luciferase assay. In both patients, rare mutations were found in the BCP at nucleotides 1764(G-->T)/1766(C-->G) and 1766(C-->T)/1768(T-->A) in case 1 and 2, respectively. In the BCP from patient 1, a putative new binding site for the transcription factor hepatocyte nuclear factor 3 (HNF3) was generated. The functional analyses of the mutant showed a 2.8-fold increase of core promoter activity, whereas the BCP variant of patient 2 was also identified to result in enhanced promoter activity. The alignment of full-length genomes from child 1 to the reference sequence showed 61 nucleotide substitutions. Furthermore, the time of reactivations from child 1 was always accompanied by selection of a precore mutation at nucleotide position 1899. In liver tissue of patient 1 before development of hepatocellular carcinoma only free viral sequences were found, whereas a single site integration of HBV was detected in hepatocytes after activation of carcinogenesis. Specific mutations in the HBV BCP of the two patients that are rarely present in chronic carriers were identified to increase the core promoter activity possibly by altering transcription factor binding, suggesting that these variants may be involved in the pathogenesis of frequent HBV reactivations.

The mouse mammary tumour virus promoter positioned on a tetramer of histones H3 and H4 binds nuclear factor 1 and OTF1.

Modulation of eukaryotic gene expression is influenced by the organization of regulatory DNA-elements in chromatin. The mouse mammary tumor virus (MMTV) promoter exhibits regularly positioned nucleosomes that reduce the accessibility of the binding sites for sequence-specific transcription factors, in particular nuclear factor (NF1). Hormonal induction of the MMTV promoter is accompanied by remodeling of the nucleosomal structure, but the biochemical nature of these structural changes is unknown. Using recombinant histones, we have now assembled the MMTV promoter in particles containing either an octamer of the histones H3, H4, H2A and H2B or a tetramer of histones H3 and H4, and have compared the two particles in terms of structure, positioning, and exclusion of transcription factors. Using site-directed hydroxy radicals to map histone locations, two main nucleosome positions are found with dyads at position -107 and at -127. The same two main positions are found for particles containing only the H3/H4 tetramer, showing that the absence of H2A/H2B dimers does not alter positioning. The rotational orientation of the DNA double helix in both types of particles is essentially identical. However, the ends of the nucleosomal DNA as well as its central region are more accessible to cleavage reagents in the tetramer particle than in the octamer particle. In agreement with these structural features, the transcription factors NF1 and OTF1 were able to bind to their cognate sites on the tetramer particle, while they could not gain access to the same sites on the surface of the octamer particle. The DNase I digestion pattern of octamers treated with partially purified SWI/SNF complex from HeLa cells in the presence of ATP is indistinguishable from that of tetramer particles, suggesting that the SWI/SNF complex promotes ATP-dependent remodeling of the octamer particle but not of tetramer particles. These results are compatible with a hormone-induced removal of histone H2A/H2B during MMTV induction.

Structural and functional implications of sequence diversity of Pseudomonas aeruginosa genes oriC, ampC and fliC.

Sequence analysis of three representative gene loci, oriC, ampC and fliC, in 19 Pseudomonas aeruginosa strains revealed a low sequence diversity that does not correlate with the extensive diversity of P. aeruginosa habitats. Single point mutations lead to a mean sequence diversity of 0.40%, 0.38% and 0.59% for oriC, ampC and a-type fliC, respectively, but of only 0.05% for b-type flagellin genes. The analyzed genes encode highly conserved functions that are subject to strong selective pressure. The detected nucleotide substitutions of oriC, accumulating in a central 95 bp region, affect neither the putative DnaA binding sites nor the 13 bp direct repeats that presumably provide the sites to open oriC duplex DNA. Even in P. aeruginosa strain DSM 1128, which exhibits an unusually high sequence variability in several analyzed genes, the 9 bp and 13 bp motifs are conserved, reflecting their essential functional role in replication initiation. The two flagellin types, differing by 37-38% in their primary structure, exhibit pronounced structural and functional homology, as shown by alignment of flagellin variants by hydrophobicity index, probability of surface exposure, chain flexibility and antigenicity, and by cross-reactivity between both proteins using specific antisera. Five nonsynonymous nucleotide substitutions of ampC lead to beta-lactamase variants that differ in recognition and turnover of substrate, as deduced from the three-dimensional structure of the highly homologous Enterobacter cloacae beta-lactamase and confirmed by inhibition kinetics. The identified point mutations in the three genes are classified as selectively equivalent sequence variants indicating neutral genetic drift as a mechanism of molecular evolution in P. aeruginosa, rather than positive selection.

Cloning and characterization of a novel gene (TM7SF1) encoding a putative seven-pass transmembrane protein that is upregulated during kidney development.

We have used the cDNA differential display of mRNA technique to isolate genes differentially regulated during kidney development. Here we report the identification of a novel gene, TM7SF1, which is upregulated in the course of kidney development. The full-length cDNA of TM7SF1 is about 2.4 kb and contains an open reading frame of 1197 nucleotides. The predicted secondary structure of the corresponding protein displays seven putative helical transmembrane domains, a structural feature shared by all members of the G-protein-coupled receptor class of transmembrane proteins. Two minor alternatively spliced versions of approximately 2.3 and approximately 2.2 kb could be detected, one of which contains a nearly identical open reading frame with a truncated carboxy-terminus of the deduced protein. The second alternatively spliced version harbors a completely shifted open reading frame with a potential new ATG start codon. By the use of single-chromosome hybrid cells and fluorescence in situ hybridization experiments, TM7SF1 could be localized to chromosome 1q42-q43. Human multiple tissue Northern blot analysis revealed TM7SF1 transcripts in human kidney, heart, brain, and placenta tissue. Studies on Wilms tumor samples showed variable TM7SF1 expression, ranging from nearly undetectable levels to an abundant level of expression comparable to that of adult kidney tissue.

Infections with Pseudomonas aeruginosa in patients with cystic fibrosis.

The lung infection with Pseudomonas aeruginosa is regarded as one of the major causes of health decline in patients with cystic fibrosis (CF). The CF host response to the persistent bacterial antigen load in the endobronchiolar lumen is characterized by a pronounced humoral response, local production of cytokines, influx of neutrophils into the lung and a protease-protease inhibitor imbalance predominantly sustained by released neutrophil elastase. CF is an autosomal recessive disease, and we could demonstrate for our local patient population that the age-dependent risk to become chronically colonized with P. aeruginosa can be differentiated by the disease-causing CFTR mutation genotype. The age-specific colonisation rates were significantly lower in pancreas sufficient than in pancreas insufficient patients. P. aeruginosa is occasionally detected in throat swabs already in infancy or early childhood in most patients although there is a lapse of several years amenable to preventive measures such as vaccination until onset of persistent colonization. The epidemiology of the infection with P. aeruginosa was investigated by quantitative macrorestriction fragment pattern analysis. The distribution and frequency of clones found in CF patients match that found in other clinical and environmental aquatic habitats, but the over-representation of specific clones at a CF clinic indicates a significant impact of nosocomial transmission for the prevalence of P. aeruginosa-positive patients at a particular center. Most patients remain colonized with the initially acquired P. aeruginosa clone. According to direct sputum analysis the majority of patients is carrying a single clonal variant at a concentration of 10(7)-10(9) CFU. Co-colonization with other species or other clones is infrequent. Independent of the underlying genotype, the CF lung habitat triggers a uniform, genetically fixed conversion of bacterial phenotype. Most CFP, aeruginosa strains become non-motile, mucoid, LPS-, pyocin- and phage-deficient, secrete less virulence determinants and shift the production of cytokines evoked in neutrophils. On the other hand, other properties such as antimicrobial susceptibility or adherence to bronchial mucins remain highly variable reflecting the capacity of P. aeruginosa to adapt to ongoing changes in the CF lung habitat.

Genetic diversity of flagellins of Pseudomonas aeruginosa.

Physical genome analysis of the virulence-associated fliC locus in 20 Pseudomonas aeruginosa strains by mapping and sequencing revealed groups of heterologous a-type (1164 bp; 1185 bp) and highly conserved b-type (1467 bp) flagellin genes. Whereas only two synonymous nucleotide substitutions were detected in eight b-type fliC sequences, the 12 a-type sequences exhibited 57 nucleotide substitutions, of which 39 occurred within a variable central region. Although a-type and b-type flagellins differ by 35% in their primary structure, they share strong homology in their predicted features, implying that the polymorphic proteins fold into similar structures during polymerization of the flagella.

Comparison of type IV-pilin genes of Pseudomonas aeruginosa of various habitats has uncovered a novel unusual sequence.

All known pilin sequences in Pseudomonas aeruginosa were amplified by a set of consensus primers located in the 5"-conserved region of pilA and the threonine-specific t-RNA following pilA. This also enabled the discovery of a novel pilin gene in a strain pair of clonal variants, which differs from known pilin genes in its increased GC-content. The mature protein has 173 amino acids making it the longest pilin known to date in P. aeruginosa. Different inserted sequences detected between the 3"-end of the pilin gene and the t-RNA in this strain and in strains with group I pilin genes seemed to be specific for each pilin group indicating a horizontal cotransfer of sequences.