Maternal isodisomy for chromosome 7 was observed in a 4-year-old cystic fibrosis patient with very short stature. In an examination of 11 DNA polymorphisms spanning the entire length of chromosome 7, no paternal contribution could be shown in seven informative loci. Paternity was examined with probes for five polymorphic loci on the Y chromosome, for the pseudo beta-globin locus on chromosome 11 and by Jeffreys's hypervariable probes. The results with the latter gave a probability of 3.7 x 10(-9) for nonpaternity. Chromosomal examination revealed a centromeric heteromorphism of chromosome 7 in the mother, for which the proband was homozygous. Isodisomy of the patient was thus shown for the entire length of a maternal chromosome 7. The mechanisms leading to this isodisomy involve at least two events of abnormal cell division, events that may be meiotic, postzygotic, or both. This proband is the second reported maternal isodisomy; both were detected through homozygosity for CF. Both patients had short stature, which could have been caused by parental imprinting, since similar results have been observed in isodisomic mice. Homozygosity due to uniparental descent in man should be kept in mind as a mechanism for recessive disorders, especially for chromosome 7.

Recombinant, replication-deficient adenovirus (Ad) vectors have been successfully used to transfer and express the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA in vivo in the respiratory epithelium of experimental animals and humans with cystic fibrosis (CF). Since Ad-directed gene expression wanes over time, repeat administration is necessary to achieve an effective treatment for CF. A major hurdle to such a strategy is the possibility that anti-Ad humoral immunity may prevent gene expression in individuals with pre-existing anti-Ad immunity or following repeat administration. One strategy to circumvent such a problem would be alternating the use of Ad vectors belonging to different subgroups. Neutralizing antibodies developed with the administration of one Ad serotype do not cross-react with an Ad belonging to a second serotype in a manner that blocks infection and gene expression. To test this hypothesis, an immunizing dose of wild-type Ad5 (subgroup C), Ad4 (subgroup E), or Ad30 (subgroup D) was administered intratracheally to experimental animals, followed by an intratracheal administration of a replication-deficient subgroup C-derived vector coding for marker genes (chloramphenicol acetyl transferase or beta-galactosidase) or for the normal human CFTR cDNA. As expected, studies with vectors coding for marker genes or for CFTR cDNA demonstrated that airway administration of a vector does not yield efficient gene transfer, if there has been prior recent airway administration of the same Ad subgroup. In contrast, effective expression from the second administration can be achieved with an adenovirus vector belonging to a subgroup different from the first adenovirus administered. These data support the paradigm of alternating Ad vectors derived from different subgroups as strategy to circumvent anti-Ad humoral immunity, thus permitting the use of Ad vectors as a means to treat the respiratory manifestations of CF.

1) Messenger RNA obtained from spinach cotyledons directs the synthesis of all five CF1 subunits in vitro in a rabbit reticulocyte translation system. The alpha, beta and epsilon subunit polypeptides were found as translation products from ptRNA and whole-cell poly A(-)-RNA. The gamma and delta subunits were synthesized from whole-cell poly A(+)-RNA as precursors of substantially greater molecular weight indicating that they originate in the nucleus and are imported into the chloroplast. High resolution electrophoresis, immunoprecipitation with antibodies against individual CF1 subunits (Nelson et al. 1980), and proteolytic peptide mapping were employed to identify the products. 2) The genes for alpha, beta and epsilon subunits of CF1 were located by hybrid-selected translation with matrix-immobilized ptDNA fragments of known map position. The genes for all three CF1 subunit polypeptides are located in the large single-copy segment (cf. Herrmann et al. 1980b) of the circular ptDNA and each gene appears to be present once on the chromosome. The genes for the beta and epsilon subunits lie near each other in immediate vicinity to the structural gene for the large subunit of ribulose bisphosphate carboxylase/oxygenase. The gene for the alpha subunit is separated by approximately 40 kbp from this gene cluster, and located near the gene for the 32 kd photosystem II polypeptide (Driesel et al. 1980). 3) Restriction fragments of spinach ptDNA with CF1 subunit genes were cloned into pBR 322 and used to construct detailed maps.

In cystic fibrosis (CF), inflammatory mediator production by airway epithelial cells is a critical determinant of chronic airway inflammation. To determine whether altered signal transduction through the nuclear factor (NF)-kappaB pathway occurs in CF epithelial cells and results in excessive generation of inflammatory cytokines, we evaluated tumor necrosis factor (TNF)-alpha-induced production of the NF-kappaB-dependent cytokine interleukin (IL)-8 and activation of NF-kappaB in three different human bronchial epithelial cell lines: (1) BEAS cells that express wild-type CF transmembrane conductance regulator (CFTR), (2) IB3 cells with mutant CFTR, and (3) C38 cells, which are "corrected" IB3 cells complemented with wild-type CFTR. Treatment of cells with TNF-alpha (30 ng/ml) resulted in markedly elevated NF-kappaB activation and production of IL-8 by IB3 cells compared with BEAS and C38 cells. Despite the differences in NF- kappaB activation, no differences in basal levels of IkappaB-alpha or TNF-alpha- induced IkappaB-alpha processing and degradation were detected among the cell lines. In contrast, the basal level of IkappaB-beta was increased in the IB3 cells. Treatment with TNF-alpha resulted in increased formation of hypophosphorylated IkappaB-beta and increased nuclear localization of IkappaB-beta in IB3 cells compared with the other cell types. These findings provide additional evidence of a dysregulated inflammatory response in CF.

Chronic colonization and infection of the lung with Pseudomonas aeruginosa is the major cause of morbidity and mortality in cystic fibrosis (CF) patients. We found that polarized CF bronchial and pancreatic epithelia bound P. aeruginosa in a reversible and dose-dependent manner. There was significantly greater binding to CF bronchial and pancreatic cells than to their matched pairs rescued with the wild-type CF transmembrane conductance regulator. Bound P. aeruginosa were easily displaced by unlabeled P. aeruginosa but not by Escherichia coli, an organism that does not cause significant pulmonary disease in CF. In contrast, Staphylococcus aureus, a frequent pathogen in CF, could effectively displace bound P. aeruginosa from its receptor. We found undersialylation of apical proteins and a higher concentration of asialoganglioside 1 (aGM1) in apical membranes of CF compared with rescued epithelia. Incubation of P. aeruginosa with aGM1 reduced its binding, as did treatment of the epithelia with the tetrasaccharide moiety of this ganglioside (Gal beta 1-3GalNAc beta 1-4Gal beta 1-4Glc). Finally, an antibody to aGM1 effectively displaced P. aeruginosa from its binding site and blocked binding of S. aureus to CF cells but not to rescued cells. These results show that the tetrasaccharide of aGM1 is a receptor for P. aeruginosa and S. aureus and that its increased abundance in the apical membrane of CF epithelia makes it a likely contributor to the pathogenesis of bacterial infections in the CF lung.

Auditory stream segregation refers to the perceptual grouping of sounds, to form coherent representations of objects in the acoustic scene, and is a fundamental aspect of hearing and speech perception. The perceptual segregation of simple interleaved tone sequences has been studied in humans and European starlings (Sturnus vulgaris) using sequences of 2 alternating tones differing in frequency (ABA-ABA-ABA-...). The segregation of A and B tones into separate auditory streams is believed to be promoted by preattentive auditory processes that increase the separation of excitation patterns along a tonotopic gradient. We tested the hypothesis that frequency selectivity and forward masking operate as 2 preattentive processes in sequential stream segregation by recording neural responses in the auditory forebrain of awake starlings to repeated ABA- sequences in which we varied the frequency separation (DeltaF) between the A and B tones and the tone repetition time (TRT). The A tones were presented at the neurons' characteristic frequency (CF), and B tones differed from the CF over a one-octave range. Larger DeltaF values and shorter TRTs promote the perceptual segregation of alternating tone sequences in humans and also resulted in larger differences in neural responses to alternating CF (A) and non-CF (B) tones. Our results are consistent with the hypothesis that preattentive auditory processes, such as frequency selectivity and forward masking, contribute to the perceptual segregation of sequential acoustic events having different frequencies into separate auditory streams, but also suggest that additional processes may be required to account for all known perceptual effects related to sequential auditory stream segregation.

We sought to evaluate the ability of an E1(-), E3(-) adenovirus (Ad) vector (Ad(GV)CFTR.10) to transfer the normal human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to the airway epithelium of individuals with cystic fibrosis (CF). We administered Ad(GV)CFTR.10 at doses of 3 x 10(6) to 2 x 10(9) plaque-forming units over 9 months by endobronchial spray to 7 pairs of individuals with CF. Each 3-month cycle, we measured vector-derived versus endogenous CFTR mRNA in airway epithelial cells prior to therapy, as well as 3 and 30 days after therapy. The data demonstrate that (a) this strategy appears to be safe; (b) after the first administration, vector-derived CFTR cDNA expression in the CF airway epithelium is dose-dependent, with greater than 5% endogenous CFTR mRNA levels at the higher vector doses; (c) expression is transient, lasting less than 30 days; (d) expression can be achieved with a second administration, but only at intermediate doses, and no expression is observed with the third administration; and (e) the progressive lack of expression with repetitive administration does not closely correlate with induction of systemic anti-Ad neutralizing antibodies. The major advantage of an Ad vector is that it can deliver sufficient levels of CFTR cDNA to the airway epithelium so that CFTR expression protects the lungs from the respiratory manifestations of CF. However, this impressive level of expression is linked to the challenging fact that expression is limited in time. Although this can be initially overcome by repetitive administration, unknown mechanisms eventually limit this strategy, and further repetitive administration does not lead to repetitive expression.

Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.

BACKGROUND

Bio-products from stem/progenitor cells, such as extracellular vesicles, are likely a new promising approach for reprogramming resident cells in both acute and chronic kidney disease. Forty CKD patients stage III and IV (eGFR 15-60 mg/ml) have been divided into two groups; twenty patients as treatment group "A" and twenty patients as a matching placebo group "B". Two doses of MSC-derived extracellular vesicles had been administered to patients of group "A". Blood urea, serum creatinine, urinary albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) have been used to assess kidney functions and TNF-α, TGF-β1 and IL-10 have been used to assess the amelioration of the inflammatory immune activity.

RESULTS

Participants in group A exhibited significant improvement of eGFR, serum creatinine level, blood urea and UACR. Patients of the treatment group "A" also exhibited significant increase in plasma levels of TGF-β1, and IL-10 and significant decrease in plasma levels of TNF-α. Participants of the control group B did not show significant improvement in any of the previously mentioned parameters at any time point of the study period.

CONCLUSIONS

Administration of cell-free cord-blood mesenchymal stem cells derived extracellular vesicles (CF-CB-MSCs-EVs) is safe and can ameliorate the inflammatory immune reaction and improve the overall kidney function in grade III-IV CKD patients.

Successful speech perception requires that listeners map the acoustic signal to linguistic categories. These mappings are not only probabilistic, but change depending on the situation. For example, one talker's /p/ might be physically indistinguishable from another talker's /b/ (cf. lack of invariance). We characterize the computational problem posed by such a subjectively nonstationary world and propose that the speech perception system overcomes this challenge by (a) recognizing previously encountered situations, (b) generalizing to other situations based on previous similar experience, and (c) adapting to novel situations. We formalize this proposal in the ideal adapter framework: (a) to (c) can be understood as inference under uncertainty about the appropriate generative model for the current talker, thereby facilitating robust speech perception despite the lack of invariance. We focus on 2 critical aspects of the ideal adapter. First, in situations that clearly deviate from previous experience, listeners need to adapt. We develop a distributional (belief-updating) learning model of incremental adaptation. The model provides a good fit against known and novel phonetic adaptation data, including perceptual recalibration and selective adaptation. Second, robust speech recognition requires that listeners learn to represent the structured component of cross-situation variability in the speech signal. We discuss how these 2 aspects of the ideal adapter provide a unifying explanation for adaptation, talker-specificity, and generalization across talkers and groups of talkers (e.g., accents and dialects). The ideal adapter provides a guiding framework for future investigations into speech perception and adaptation, and more broadly language comprehension.

We recently reported the inflammation of the cystic fibrosis (CF) mouse small intestine, and we hypothesized bacterial overgrowth as a possible cause. Quantitative PCR of bacterial 16S genomic DNA in the CF mouse small intestine revealed an increase of greater than 40-fold compared to controls. Sequencing of 16S PCR products and Gram staining showed that the majority of bacteria in the CF mouse intestine were gram negative. Bacteria were observed to colonize the mucus that accumulates in the intestinal lumen of mice with CF. Impaired Paneth cell defenses were suggested by observation of partially dispersed Paneth granules in the mucus plugs of CF mouse intestinal crypts, and this mucus was strongly immunoreactive for Paneth cell bactericidal products. The role of bacterial overgrowth in intestinal inflammation in CF was tested by treating mice with oral antibiotics (ciprofloxacin and metronidazole) for 3 weeks, which reduced bacterial load in the CF mouse small intestine over 400-fold. Antibiotic treatment decreased the expression of the inflammation-related genes mast cell protease 2, leucine-rich alpha2 glycoprotein/leucine-rich high endothelial venule glycoprotein, suppressor of cytokine signaling 3, hematopoietic cell transcript 1, and resistin-like molecule beta/found in inflammatory zone 2, all of which were no longer expressed at levels significantly different from control levels. The reduction of intestinal bacteria also significantly improved the growth of CF mice but had no effect on the growth of wild-type mice. These data suggest that bacterial overgrowth in the CF mouse small intestine has a role in inflammation and contributes to the failure to thrive in this mouse model of CF.

Certain therapeutic microbes, including Bifidobacteria infantis (B. infantis) 35624 exert beneficial immunoregulatory effects by mimicking commensal-immune interactions; however, the value of these effects in patients with non-gastrointestinal inflammatory conditions remains unclear. In this study, we assessed the impact of oral administration of B. infantis 35624, for 6‒8 weeks on inflammatory biomarker and plasma cytokine levels in patients with ulcerative colitis (UC) (n = 22), chronic fatigue syndrome (CFS) (n = 48) and psoriasis (n = 26) in three separate randomized, double-blind, placebo-controlled interventions. Additionally, the effect of B. infantis 35624 on immunological biomarkers in healthy subjects (n = 22) was assessed. At baseline, both gastrointestinal (UC) and non-gastrointestinal (CFS and psoriasis) patients had significantly increased plasma levels of C-reactive protein (CRP) and the pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) compared with healthy volunteers. B. infantis 35624 feeding resulted in reduced plasma CRP levels in all three inflammatory disorders compared with placebo. Interestingly, plasma TNF-α was reduced in CFS and psoriasis while IL-6 was reduced in UC and CFS. Furthermore, in healthy subjects, LPS-stimulated TNF-α and IL-6 secretion by peripheral blood mononuclear cells (PBMCs) was significantly reduced in the B. infantis 35624-treated groups compared with placebo following eight weeks of feeding. These results demonstrate the ability of this microbe to reduce systemic pro-inflammatory biomarkers in both gastrointestinal and non-gastrointestinal conditions. In conclusion, these data show that the immunomodulatory effects of the microbiota in humans are not limited to the mucosal immune system but extend to the systemic immune system.

Burkholderia cepacia, which is an important pathogen in cystic fibrosis (CF) owing to the potential severity of the infections and the high transmissibility of some clones, has been recently shown to be a complex of five genomic groups, i.e., genomovars I, II (B. multivorans), III, and IV and B. vietnamiensis. B. gladioli is also involved, though rarely, in CF. Since standard laboratory procedures fail to provide an accurate identification of these organisms, we assessed the ability of restriction fragment length polymorphism (RFLP) analysis of amplified 16S ribosomal DNA (rDNA), with the combination of the patterns obtained with six endonucleases, to differentiate Burkholderia species. This method was applied to 16 type and reference strains of the genus Burkholderia and to 51 presumed B. cepacia clinical isolates, each representative of one clone previously determined by PCR ribotyping. The 12 Burkholderia type strains tested were differentiated, including B. cepacia, B. multivorans, B. vietnamiensis, and B. gladioli, but neither the genomovar I and III reference strains nor the genomovar IV reference strain and B. pyrrociniaT were distinguishable. CF clinical isolates were mainly distributed in RFLP group 2 (which includes B. multivoransT) and RFLP group 1 (which includes B. cepacia genomovar I and III reference strains, as well as nosocomial clinical isolates). Two of the five highly transmissible clones in French CF centers belonged to RFLP group 2, and three belonged to RFLP group 1. The remaining isolates either clustered with other Burkholderia species (B. cepacia genomovar IV or B. pyrrocinia, B. vietnamiensis, and B. gladioli) or harbored unique combinations of patterns. Thus, if further validated by hybridization studies, PCR-RFLP of 16S rDNA could be an interesting identification tool and contribute to a better evaluation of the respective clinical risks associated with each Burkholderia species or genomovar in patients with CF.

In recent hypotheses on muscle contraction, myosin cross-bridges cycle between two types of actin-bound configuration. These two configurations differ greatly in the stability of their actin-myosin complexes ("weak-binding" vs. "strong-binding"), and force generation or movement is the result of structural changes associated with the transition from the weak-binding (preforce generating) configuration to strong-binding (force producing) configuration [cf. Eisenberg, E. & Hill, T. L. (1985) Science 227, 999-1006]. Specifically, in this concept, the main force-generating states are only accessible after initial cross-bridge attachment in a weak-binding configuration. It has been shown that strong and weak cross-bridge attachment can occur in muscle fibers [Brenner, B., Schoenberg, M., Chalovich, J. M., Greene, L. E. & Eisenberg, E. (1982) Proc. Natl. Acad. Sci. USA 79, 7288-7291]. However, there has been no evidence that attachment in the weak-binding states represents an essential step leading to force generation. It is shown here that caldesmon can be used to selectively inhibit attachment of weak-binding cross-bridges in skeletal muscle. Such inhibition causes a parallel decrease in active force, while the kinetics of cross-bridge turnover are unchanged by this procedure. This suggests that (i) cross-bridge attachment in the weak-binding states is specific and (ii) force production can only occur after cross-bridges have first attached to actin in a weakly bound, nonforce-generating configuration.

Previous studies have shown that appendage pili of Burkholderia cepacia strains isolated from patients with cystic fibrosis (CF) at The Hospital for Sick Children, Toronto, Canada, mediate adherence to mucus glycoproteins and also enhance adherence to epithelial cells. The specific pilin-associated adhesin molecule is a 22-kDa protein. In the present study we purified the major subunit pilin (17 kDa) and immunolocalized it to peritrichously arranged pili. On the basis of their novel morphological appearance as giant intertwined fibers, we refer to them as cable (Cbl) pili. Using an oligonucleotide probe corresponding to regions of the N-terminal amino acid sequence of the pilin subunit, we detected the encoding cblA gene in a chromosomal DNA library. Sequencing revealed this structural gene to be 555 bp in length, encoding a leader sequence of 19 amino acids, a cleavage site between the alanine at position 19 and the valine at position 20, and a mature pilin sequence of 165 amino acids. The calculated molecular mass is 17.3 kDa. Hydrophobic plus apolar amino acids account for 60% of the total residues. The pilin exhibits some similarities in its amino acid sequence to colonization factor antigen I and CS1 fimbriae of Escherichia coli. With the cblA gene used as a probe, hybridization assays of 59 independent isolates, including those from several geographically separated CF centers, plus environmental and clinical (non-CF) strains, gave positive results with all of the 15 CF-associated B. cepacia isolates from Toronto, plus a single strain from one other CF center (Jackson, Mississippi). The cblA gene is the first pilin subunit gene of B. cepacia to be identified.

Glutathione (GSH) is a potentially important component of antioxidant defense in the epithelial lung lining fluid. Cystic fibrosis (CF) patients have chronic inflammation in which oxidative stress can be a factor. To examine the hypothesis that the transport of GSH content was defective in CF patients, intracellular and extracellular GSH were measured by HPLC. Four cell lines were used: CFT1 cells [with defective CF transmembrane conductance regulator (CFTR), DeltaF508 homozygous, two clones] and one of the CFT1 clones transfected with either normal CFTR (CFTR repleted) or beta-galactosidase. GSH content in the apical fluid was 55% lower in CFTR-deficient cultures than in CFTR-repleted cells (P < 0.001). In contrast, intracellular GSH content was similar in CFT1 cells and CFTR-repleted cells. gamma-Glutamyl transpeptidase activity, which degrades extracellular GSH, did not account for differences in apical GSH. Rather, GSH efflux of CFTR-deficient cells was lower than that of CFTR-repleted cells. These studies suggested that decreased GSH content in the apical fluid in CF resulted from abnormal GSH transport associated with a defective CFTR.

The morbidity and mortality of patients with cystic fibrosis (CF) is primarily determined by chronic and debilitating lung infections caused by a surprisingly narrow spectrum of bacterial pathogens. Pseudomonas aeruginosa is by far the most prevalent life-threatening CF pathogen. In the absence of aggressive early therapy, it infects the majority of adult patients and determines long-term survival. The epidemiology of CF pulmonary infections continues to evolve. Amongst the most recent CF pathogens to have emerged are a group of closely related bacteria, known as the Burkholderia cepacia complex. These organisms are a particular challenge due to inherent antibiotic resistance, the potential for patient-to-patient spread, and the risk of 'cepacia syndrome', a rapid fulminating pneumonia sometimes accompanied by bacteremia. Strict cross-infection control was prompted by early epidemiological experience of the B. cepacia complex and is essential in the management of all CF pathogens.

Hydrogenation is a core technology in chemical synthesis. High rates and selectivities are attainable only by the coordination of structurally well-designed catalysts and suitable reaction conditions. The newly devised [RuCl(2)(phosphane)(2)(1,2-diamine)] complexes are excellent precatalysts for homogeneous hydrogenation of simple ketones which lack any functionality capable of interacting with the metal center. This catalyst system allows for the preferential reduction of a C=O function over a coexisting C=C linkage in a 2-propanol solution containing an alkaline base. The hydrogenation tolerates many substituents including F, Cl, Br, I, CF(3), OCH(3), OCH(2)C(6)H(5), COOCH(CH(3))(2), NO(2), NH(2), and NRCOR as well as various electron-rich and -deficient heterocycles. Furthermore, stereoselectivity is easily controlled by the electronic and steric properties (bulkiness and chirality) of the ligands as well as the reaction conditions. Diastereoselectivities observed in the catalytic hydrogenation of cyclic and acyclic ketones with the standard triphenylphosphane/ethylenediamine combination compare well with the best conventional hydride reductions. The use of appropriate chiral diphosphanes, particularly BINAP compounds, and chiral diamines results in rapid and productive asymmetric hydrogenation of a range of aromatic and heteroaromatic ketones and gives a consistently high enantioselectivity. Certain amino and alkoxy ketones can be used as substrates. Cyclic and acyclic alpha,beta-unsaturated ketones can be converted into chiral allyl alcohols of high enantiomeric purity. Hydrogenation of configurationally labile ketones allows for the dynamic kinetic discrimination of diastereomers, epimers, and enantiomers. This new method shows promise in the practical synthesis of a wide variety of chiral alcohols from achiral and chiral ketone substrates. Its versatility is manifested by the asymmetric synthesis of some biologically significant chiral compounds. The high rate and carbonyl selectivity are based on nonclassical metal-ligand bifunctional catalysis involving an 18-electron amino ruthenium hydride complex and a 16-electron amido ruthenium species.

The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring recognition of various pathogen molecules and plant hormones. We report here a large-scale structure-function analysis of an eLRR protein. A total of 66 site-directed mutants of Cf-9 were analyzed for activity in Avr9 recognition and for protein stability and the results interpreted with the help of a homology model of the Cf-9 structure. Conserved Trp and Cys pairs in the N-terminal LRR-flanking domain appear to be important for Cf-9 activity and are probably exposed at the putative concave inner surface of the Cf-9 protein, where recognition specificity also resides. Removal of each of the 22 putative N-linked glycosylation sites (PGS) revealed that many PGSs contribute to Cf-9 activity and that the PGSs in the putative alpha-helices of the LRR modules are essential. Immunoblot analysis and mass spectrometry showed that all but one of the PGSs are N-glycosylated. Introduction of glycosylation at the putative concave beta-sheet surface blocks Cf-9 activity, in some cases probably by disturbing specific recognition, and in another case by steric hindrance with existing N-glycans. The glycosylation pattern and several other features are conserved in other eLRR proteins, where similar mutations show similar phenotypes.

OBJECTIVE

To review the effect of assisted reproductive technology (ART) on perinatal outcomes, to provide guidelines to optimize obstetrical management and counselling of Canadian women using ART, and to identify areas specific to birth outcomes and ART requiring further research.

METHODS

Perinatal outcomes of ART pregnancies in subfertile women are compared with those of spontaneously conceived pregnancies. Perinatal outcomes are compared between different types of ART.

RESULTS

This guideline discusses the adverse outcomes that have been recorded in association with ART, including obstetrical complications, adverse perinatal outcomes, multiple gestations, structural congenital abnormalities, chromosomal abnormalities, imprinting disorders, and childhood cancer.

METHODS

The Cochrane Library and MEDLINE were searched for English-language articles from 1990 to February 2005, relating to assisted reproduction and perinatal outcomes. Search terms included assisted reproduction, assisted reproductive technology, ovulation induction, intracytoplasmic sperm injection (ICSI), embryo transfer, and in vitro fertilization (IVF). Additional publications were identified from the bibliographies of these articles as well as the Science Citation Index. Studies assessing gamete intrafallopian transfer (GIFT) and zygote intrafallopian transfer (ZIFT) were excluded since they are rarely used in Canada. All study types were reviewed. Randomized controlled trials were considered evidence of the highest quality, followed by cohort studies. Key studies and supporting data for each recommendation are summarized with evaluative comments and referenced.

METHODS

The evidence collected was reviewed by the Genetics Committee and the Reproductive Endocrinology Infertility Committee of the Society of Obstetricians and Gynaecologists of Canada (SOGC) and quantified using the Evaluation of Evidence Guidelines developed by the Canadian Task Force on the Periodic Health Examination.

RESULTS

The type and magnitude of benefits, harms, and costs expected for patients from guideline implementation. This guideline has been reviewed by the Genetics Committee and the Reproductive Endocrinology and Infertility Committee, and approved by the Executive and Council of the Society of Obstetricians and Gynaecologists of Canada and the Board of the Canadian Fertility and Andrology Society.

CONCLUSIONS

1. Spontaneous pregnancies in untreated infertile women may be at higher risk for obstetrical complications and perinatal mortality than spontaneous pregnancies in fertile women. Further research is required to clarify the contribution of infertility itself to adverse obstetrical and perinatal outcomes. (II-2A) 2. All men with severe oligozoospermia or azoospermia should be offered genetic/clinical counselling for informed consent and offered karyotyping for chromosomal abnormalities before attempting IVF-ICSI. They should be made aware of the availability of tests for Y chromosome microdeletion. Some patients may consider the option of donor insemination. (II-3B) 3. Couples exploring IVF-ICSI when the man has obstructive azoospermia should be offered genetic/clinical counselling for informed consent and offered genetic testing for alterations in genes associated with cystic fibrosis (CF) before attempting IVF-ICSI. (II-2A) 4. Pregnancies achieved by ovarian stimulation with gonadotropins and intrauterine insemination are at higher risk for perinatal complications, and close surveillance during pregnancy should be considered. It remains unclear if these increased risks are attributable to the underlying infertility, characteristics of the infertile couple, or use of assisted reproductive techniques. Multiple gestations remain a significant risk of gonadotropin treatment. (II-2A) 5. Pregnancies achieved by IVF with or without ICSI are at higher risk for obstetrical and perinatal complications than spontaneous pregnancies, and close surveillance during pregnancy should be considered. It remains unclear if these increased risks are attributable to the underlying infertility, characteristics of the infertile couple, or use of assisted reproductive techniques. (II-2A) 6. Women undergoing ART should be informed about the increased rate of obstetrical interventions such as induced labour and elective Caesarean delivery. (II-2A) 7. Couples suffering from infertility who are exploring treatment options should be made aware of the psychosocial implications of ART. Further research into the psychosocial impact of ART is needed. (II-2A) 8. Singleton pregnancies achieved by assisted reproduction are at higher risk than spontaneous pregnancies for adverse perinatal outcomes, including perinatal mortality, preterm delivery, and low birth weight, and close surveillance during pregnancy should be available as needed. (II-2A) 9. A significant risk of ART is multiple pregnancies. Infertile couples need to be informed of the increased risks of multifetal pregnancies. Although dichorionic twins are most common, the incidence of monochorionic twins is also increased. Risks of multiple pregnancies include higher rates of perinatal mortality, preterm birth, low birth weight, gestational hypertension, placental abruption, and placenta previa. Perinatal mortality in assisted conception twin pregnancies appears to be lower than in spontaneously conceived twin pregnancies. (II-2A) 10. When multifetal reduction is being considered for high-order multiple pregnancies, psychosocial counselling should be readily available. Careful surveillance for fetal growth problems should be undertaken after multifetal reduction. (II-2A) 11. To reduce the risks of multiple pregnancies associated with ART and to optimize pregnancy rates, national guidelines should be developed on the number of embryos replaced according to characteristics such as patient's age and grade of embryos. (II-2A) 12. Further epidemiologic and basic science research is needed to help determine the etiology and extent of the increased risks to childhood and long-term growth and development associated with ART. (II-2A) 13. Discussion of options for prenatal screening for congenital structural abnormalities in pregnancies achieved by ART is recommended, including appropriate use of biochemical and sonographic screening. (II-2A) 14. Further epidemiologic and basic science research is needed to help determine the etiology and extent of the increased risks of congenital abnormalities associated with ART. (II-2A) 15. Couples considering IVF-ICSI for male-factor infertility should receive information, and if necessary formal genetic counselling, about the increased risk of de novo chromosomal abnormalities (mainly sex chromosomal anomalies) associated with their condition. Prenatal diagnosis by chorionic villus sampling (CVS) or amniocentesis should be offered to these couples if they conceive. (II-2A) 16. Further epidemiologic and basic science research is needed to help determine the etiology and extent of the increased risks of chromosomal abnormalities associated with ART. (II-2A) 17. Discussion of options for prenatal screening and testing for aneuploidy in pregnancies achieved by ART, adapted for maternal age and number of fetuses, is recommended, including appropriate use of biochemical and sonographic screening. (II-2A) 18. The precise risks of imprinting and childhood cancer from ART remain unclear but cannot be ignored. Further clinical research, including long-term follow-up, is urgently required to evaluate the prevalence of imprinting disorders and cancers associated with ART. (II-2A) 19. The clinical application of preimplantation genetic diagnosis must balance the benefits of avoiding disease transmission with the medical risks and financial burden of in vitro fertilization. Further ethical discussion and clinical research is required to evaluate appropriate indications for preimplantation genetic diagnosis. (III-B).

The authors present a computational model that builds a holographic lexicon representing both word meaning and word order from unsupervised experience with natural language. The model uses simple convolution and superposition mechanisms (cf. B. B. Murdock, 1982) to learn distributed holographic representations for words. The structure of the resulting lexicon can account for empirical data from classic experiments studying semantic typicality, categorization, priming, and semantic constraint in sentence completions. Furthermore, order information can be retrieved from the holographic representations, allowing the model to account for limited word transitions without the need for built-in transition rules. The model demonstrates that a broad range of psychological data can be accounted for directly from the structure of lexical representations learned in this way, without the need for complexity to be built into either the processing mechanisms or the representations. The holographic representations are an appropriate knowledge representation to be used by higher order models of language comprehension, relieving the complexity required at the higher level.

OBJECTIVE

The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes.

RESULTS

Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT-PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of beta-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes.

CONCLUSIONS

Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.

Pseudomonas aeruginosa is an important opportunistic pathogen that can cause chronic and often life-threatening infections of the respiratory tract, particularly in individuals with cystic fibrosis (CF). Because infections with P. aeruginosa remain the major cause of the high morbidity and mortality of CF, a vaccine against P. aeruginosa would be very useful for preventing this disorder. The outer membrane protein F (OprF) of P. aeruginosa is a promising vaccine candidate and various B cell epitopes within OprF have been identified. Given that adenovirus (Ad) vectors have strong immunogenic potential and can function as adjuvants for genetic vaccines, the present study evaluates the immunogenic and protective properties of a novel replication-deficient Ad vector in which the Ad hexon protein was modified to include a 14-amino acid epitope of P. aeruginosa OprF (Epi8) in loop 1 of the hypervariable region 5 of the hexon (AdZ.Epi8). Immunization of C57BL/6 mice with AdZ.Epi8 resulted in detectable serum anti-P. aeruginosa and anti-OprF humoral responses. These responses were haplotype dependent, with higher serum anti-OprF titers in CBA mice than in BALB/c or C57BL/6 mice. AdZ.Epi8 induced Epi8-specific IFN-gamma-positive CD4 and CD8 T cell responses and resulted in protection against a lethal pulmonary challenge with agar-encapsulated P. aeruginosa. Importantly, repeated administration of AdZ.Epi8 resulted in boosting of the anti-OprF humoral and anti-Epi8 cellular response, whereas no boosting effect was present in the response against the transgene beta-galactosidase. These observations suggest that Ad vectors expressing pathogen epitopes in their capsid will protect against an extracellular pathogen and will allow boosting of the epitope-specific humoral response with repeated administration, a strategy that should prove useful in developing Ad vectors as vaccines where humoral immunity will be protective.

Extracellular ATP and UTP produced a rapid accumulation of inositol phosphates in human airway epithelial cells (CF/T43). The order of agonist potencies for a series of nucleotide analogues differed markedly from that of the classically described P2x- or P2y-purinergic receptors. UTP was the most potent agonist and was fully efficacious; ATP and adenosine-5'-O-(3-thiotriphosphate) were also full agonists. In contrast, 2-methylthio-ATP, adenosine-5'-O-(2-thiodiphosphate) and alpha,beta-methylene-ATP were without effect. ADP and UDP had little or no effect at concentrations as high as 100 microM, and deoxyribose and dideoxyribose compounds were inactive. The effects of ATP and UTP were not additive, whereas bradykinin- or histamine-stimulated inositol phosphate production was additive with the effects of ATP or UTP. Preincubation of cells with either UTP or ATP resulted in a parallel loss of responsiveness to both agonists. Desensitization was specific for the response to nucleotides, because no ATP- or UTP-induced effect on the response to histamine or bradykinin was observed. Pertussis toxin treatment of CF/T43 cells produced a 30-40% decrease in the response to ATP or UTP, which correlated with the ADP-ribosylation of 41- and 43-kDa proteins. Bradykinin and histamine responses were not modified by pertussis toxin. Guanine nucleotides had little effect on the inositol phosphate response in intact CF/T43 cells at concentrations below 100 microM. However, in streptolysin-O-permeabilized cells GTP-gamma S produced a concentration-dependence activation of inositol phosphate formation. UTP or ATP had little effect in permeabilized cells in the absence of guanine nucleotides but markedly increased inositol phosphate formation in the presence of guanine nucleotides. Taken together, these results suggest that UTP and ATP activate a 5'-nucleotide receptor on CF/T43 cells that is distinct from the classically defined P2x- and P2y-purinergic receptors. Activation of phospholipase C by this receptor involves, at least in part, a guanine nucleotide-binding regulatory protein.