Iron limitation induces motility in uropathogenic E. coli CFT073 partially through action of LpdA.

More than half of women will experience a urinary tract infection (UTI) with most cases caused by uropathogenic Escherichia coli (UPEC). Bacterial swimming motility enhances UPEC pathogenicity, resulting in more severe disease outcomes including kidney infection. Surprisingly, the connection between motility and iron limitation is mostly unexplored despite the lack of free iron available in the host. We sought to investigate a potential connection between iron restriction and regulation of motility in UPEC. We cultured E. coli CFT073, a prototypical UPEC strain, under iron limitation and observed that CFT073 had elevated fliC (flagella) promoter activity, and this iron-specific response was repressed by the addition of exogenous iron. We confirmed increased flagellar expression in CFT073 by measuring fliC transcript, FliC protein, and surface-expressed flagella under iron-limited conditions. Interestingly, known motility regulator flhDC did not have altered transcription under these conditions. To define the regulatory mechanism of this response, we constructed single knockouts of eight master regulators and found the iron-regulated response was lost in crp, arcA, and fis mutants. Thus, we focused on the five genes regulated by all three regulators. Of the five genes knocked out, the iron-regulated motility response was most strongly dysregulated in the lpdA mutant, which also resulted in significantly lowered fitness in the murine model of ascending UTI, both against the WT and a non-motile fliC mutant. Collectively, we demonstrated that iron-mediated motility in CFT073 is partially regulated by lpdA, which contributes to the understanding of how uropathogens differentially regulate motility mechanisms in the iron-restricted host. IMPORTANCE: Urinary tract infections (UTIs) are ubiquitous and responsible for over five billion dollars in associated health care costs annually. Both iron acquisition and motility are highly studied virulence factors associated with uropathogenic Escherichia coli (UPEC), the main causative agent of uncomplicated UTI. This work is innovative by providing mechanistic insight into the synergistic relationship between these two critical virulence properties. Here, we demonstrate that iron limitation has pleiotropic effects with consequences that extend beyond metabolism and impact other virulence mechanisms. Indeed, targeting iron acquisition as a therapy may lead to an undesirable enhancement of UPEC pathogenesis through increased motility. It is vital to understand the full breadth of UPEC pathogenesis to adequately respond to this common infection, especially with the increase of antibiotic-resistant pathogens.

Altered motility in response to iron-limitation is regulated by lpdA in uropathogenic E. coli CFT073.

More than half of all women will experience a urinary tract infection (UTI) in their lifetime with most cases caused by uropathogenic Escherichia coli (UPEC). Bacterial motility enhances UPEC pathogenicity, resulting in more severe disease outcomes including kidney infection. Surprisingly, the connection between motility and iron limitation is mostly unexplored, despite the lack of free iron available in the host. Therefore, we sought to explore the potential connection between iron restriction and regulation of motility in UPEC. We cultured E. coli CFT073, a prototypical UPEC strain, in media containing an iron chelator. Under iron limitation, CFT073 had elevated fliC (flagella) promoter activity, driving motility on the leading edge of the colony. Furthermore, this iron-specific response was repressed by the addition of exogenous iron. We confirmed increased flagella expression in CFT073 by measuring fliC transcript, FliC protein, and surface-expressed flagella under iron-limited conditions. To define the regulatory mechanism, we constructed single knockouts of eight master regulators. The iron-regulated response was lost in crp, arcA, and fis mutants. Thus, we focused on the five genes regulated by all three transcription factors. Of the five genes knocked out, the iron-regulated motility response was most strongly dysregulated in an lpdA mutant, which also resulted in significantly lowered fitness in the murine model of ascending UTI. Collectively, we demonstrated that iron-mediated motility in CFT073 is regulated by lpdA , which contributes to the understanding of how uropathogens differentially regulate motility mechanisms in the iron-restricted host. Importance: Urinary tract infections (UTIs) are ubiquitous and responsible for over five billion dollars in associated health care costs annually. Both iron acquisition and motility are highly studied virulence factors associated with uropathogenic E. coli (UPEC), the main causative agent of uncomplicated UTI. This work is innovative by providing mechanistic insight into the synergistic relationship between these two critical virulence properties. Here, we demonstrate that iron limitation has pleiotropic effects with consequences that extend beyond metabolism, and impact other virulence mechanisms. Indeed, targeting iron acquisition as a therapy may lead to an undesirable enhancement of UPEC pathogenesis through increased motility. It is vital to understand the full breadth of UPEC pathogenesis to adequately respond to this common infection, especially with the increase of antibiotic resistant pathogens.

Phenotypic Assessment of Clinical Escherichia coli Isolates as an Indicator for Uropathogenic Potential.

For women in the United States, urinary tract infections (UTIs) are the most frequent diagnosis in emergency departments, comprising 21.3% of total visits. Uropathogenic Escherichia coli (UPEC) causes ~80% of uncomplicated UTIs. To combat this public health issue, it is vital to characterize UPEC strains as well as to differentiate them from commensal strains to reduce the overuse of antibiotics. It has been challenging to determine a consistent genetic signature that clearly distinguishes UPEC from other E. coli strains. Therefore, we examined whether phenotypic data could be predictive of uropathogenic potential. We screened 13 clinical strains of UPEC, isolated from cases of uncomplicated UTI in young otherwise healthy women, in a series of microbiological phenotypic assays using UPEC prototype strain CFT073 and nonpathogenic E. coli strain MG1655 K-12 as controls. Phenotypes included adherence, iron acquisition, biofilm formation, human serum resistance, motility, and stress resistance. By use of a well-established experimental mouse model of UTI, these data were able to predict the severity of the bacterial burden in both the urine and bladders. Multiple linear regression using three different phenotypic assays, i.e., growth in minimal medium, siderophore production, and type 1 fimbrial expression, was predictive of bladder colonization (adjusted R2 = 0.6411). Growth in ex vivo human urine, hemagglutination of red blood cells, and motility modeled urine colonization (adjusted R2 = 0.4821). These results showcase the utility of phenotypic characterization to predict the severity of infection that these strains may cause. We predict that these methods will also be applicable to other complex, genetically redundant, pathogens. IMPORTANCE Urinary tract infections are the second leading infectious disease worldwide, occurring in over half of the female population during their lifetime. Most infections are caused by uropathogenic Escherichia coli (UPEC) strains. These strains can establish a reservoir in the gut, in which they do not cause disease but, upon introduction to the urinary tract, can infect the host and elicit pathogenesis. Clinically, it would be beneficial to screen patient E. coli strains to understand their pathogenic potential, which may lead to the administration of prophylactic antibiotic treatment for those with increased risk. Others have proposed the use of PCR-based genetic screening methods to detect UPEC strains and differentiate them from other E. coli pathotypes; however, this method has not yielded a consistent uropathogenic genetic signature. Here, we used phenotypic characteristics such as growth rate, siderophore production, and expression of fimbriae to better predict uropathogenic potential.

CYP56 (Dit2p) in Candida albicans: characterization and investigation of its role in growth and antifungal drug susceptibility.

The complete DNA sequence of Candida albicans DIT2, encoding cytochrome P450 family 56 (CYP56), was obtained, and heterologous expression was achieved in Escherichia coli, where CYP56 was targeted to the membrane fraction. In reconstituted assays with the purified enzyme, CYP56 was shown to catalyze the conversion of N-formyl tyrosine into N,N'-bisformyl dityrosine, a reaction that was dependent on cytochrome P450 reductase, NADPH, and oxygen, yielding a turnover of 21.6 min(-1) and a k(s) of 26 microM. The Hill number was calculated as 1.6, indicating that two molecules of the substrate could bind to the protein. Azole antifungals could bind to the heme of CYP56 as a sixth ligand with high affinity. Both chromosomal alleles of CYP56 were disrupted using the SAT1 flipper technique, and CYP56 was found to be nonessential for cell viability under the culture conditions investigated. Susceptibility to azole drugs that bind to cytochromes P450 was tested, and the mutant showed unaltered susceptibility. However, the mutant showed increased susceptibility to the echinocandin drug caspofungin, suggesting an alteration in 1,3-glucan synthase and/or cell wall structure mediated by the presence of dityrosine. Phenotypically, the wild-type and mutant strains were morphologically similar when cultured in rich yeast extract-peptone-dextrose medium. However in minimal medium, the cyp56Delta mutant strain exhibited hyphal growth, in contrast to the wild-type strain, which grew solely in the yeast form. Furthermore, CYP56 was essential for chlamydospore formation.

Characterization of Sclerotinia and mycoparasite Coniothyrium minitans interaction by microscale co-culture.

AIMS: To characterize the interaction of Sclerotinia sclerotiorum and S. minor with strains of the mycoparasite and commercial biocontrol agent Coniothyrium minitans using novel perfusion chamber gasket co-culture. METHODS AND RESULTS: Sclerotinia were cultured in perfusion chamber gaskets and then flooded with Coniothyrium conidia. After germination, Coniothyrium failed to show any form of directed growth, making contact with Sclerotinia hyphae in a random manner. In turn, some Coniothyrium hyphae coiled round Sclerotinia counterparts and although no intracellular growth was observed, Coniothyrium proliferated, while the hyphae of Sclerotinia became vacuolated and lost the cytoplasm. When co-cultures of Sclerotinia with Coniothyrium were flooded with FITC-lectins, small difference in fluorescence between the fungi was found with FITC-Con A suggesting that cell walls of both the species exposed mannose. In contrast, Coniothyrium fluoresced poorly in comparison with Sclerotinia when FITC-wheat germ agglutinin was used, indicating a marked paucity of N-acetylglucosamine exposure by cell walls of Coniothyrium, hence reduced exposure to chitinolytic enzyme action. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The approach employed supported direct sequential microscopic observation of Coniothyrium and Sclerotinia as well as the utilization of representative fluorescent moieties to characterize relative carbohydrate cell wall exposure.

Biotinylation and characterization of Cryptococcus neoformans cell surface proteins.

AIMS: To develop a novel procedure for isolating and characterizing cryptococcal cell-surface proteins using biotinylation, fluorescein isothiocyanate (FITC)-streptavidin, flow cytometry and associated ligand-receptor analysis, confocal microscopy and electrophoretic separation. METHODS AND RESULTS: Cell proteins of both acapsulate and encapsulated Cryptococcus neoformans cells were labelled using sulfo-NHS-biotin which, in turn, was complexed with FITC-streptavidin. Resulting cell population fluorescence supported visualization of cell-surface protein distribution by confocal microscopy, as well as evaluation of protein exposure by flow cytometry and the calculation of the ligand-binding determinants EC(50), F(max) and H(n). Biotinylation of cell-surface proteins also supported their isolation by affinity chromatography and characterization by SDS/PAGE. Ligand-binding determinants, such as EC(50) values, indicated that acapsulate and stationary phase cells have greatest affinity for biotin. F(max) values demonstrated greatest protein exposure among stationary phase cells; in turn, encapsulated cells expose more protein than acapsulate counterparts. H(n) values of below unity potentially confirm the complex multi-receptor nature of biotin binding to cryptococcal cell surfaces under investigation. Fluorescence visualization showed marked but localized fluorescence indicative of protein exposure around sites of cell division. In turn, biotinylation of cell-surface proteins and their release under reducing conditions demonstrated at least two noncovalently linked proteinaceous entities, of 43 and 57 kDa, exposed on acapsulate cryptococcal cell walls. CONCLUSIONS: A novel method for identifying, in situ, cell-surface proteins exposed by C. neoformans was established. This novel technique was successfully implemented using both acapsulate and encapsulated C. neoformans cells, both were found to have dynamic and markedly localized protein distribution around sites of cell division and associated cell wall trauma. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel procedure, employing a versatile combination of flow cytometry, ligand-receptor analysis, confocal microscopy and biotinylation, supported the characterization and isolation of cryptococcal cell-surface proteins.

Sendai virus-mediated CFTR gene transfer to the airway epithelium.

The potential for gene therapy to be an effective treatment for cystic fibrosis has been hampered by the limited gene transfer efficiency of current vectors. We have shown that recombinant Sendai virus (SeV) is highly efficient in mediating gene transfer to differentiated airway epithelial cells, because of its capacity to overcome the intra- and extracellular barriers known to limit gene delivery. Here, we have identified a novel method to allow the cystic fibrosis transmembrane conductance regulator (CFTR) cDNA sequence to be inserted within SeV (SeV-CFTR). Following in vitro transduction with SeV-CFTR, a chloride-selective current was observed using whole-cell and single-channel patch-clamp techniques. SeV-CFTR administration to the nasal epithelium of cystic fibrosis (CF) mice (Cftr(G551D) and Cftr(tm1Unc)TgN(FABPCFTR)#Jaw mice) led to partial correction of the CF chloride transport defect. In addition, when compared to a SeV control vector, a higher degree of inflammation and epithelial damage was found in the nasal epithelium of mice treated with SeV-CFTR. Second-generation transmission-incompetent F-deleted SeV-CFTR led to similar correction of the CF chloride transport defect in vivo as first-generation transmission-competent vectors. Further modifications to the vector or the host may make it easier to translate these studies into clinical trials of cystic fibrosis.

Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation.

AIMS: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. METHODS AND RESULTS: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K(d) and relative B(max) values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, K(d) and relative B(max), which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. CONCLUSIONS: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. SIGNIFICANCE AND IMPACT OF THE STUDY: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Status epilepticus after the initial diagnosis of epilepsy in children.

OBJECTIVES: To determine the risk and predictors of status epilepticus in children after they have been diagnosed with epilepsy. METHODS: In a prospective community-based cohort study of 613 children, the occurrence of status epilepticus after the initial diagnosis of epilepsy was ascertained. Parents were called every 3 months, and interval medical records were reviewed every 6 months. Predictors of primary interest included a history of status before the diagnosis of epilepsy, age at onset, underlying etiology, and epilepsy syndrome. Data were analyzed with chi2 tests, Kaplan-Meier analyses, and Cox proportional hazards models. RESULTS: Of 613 children followed a median of 8.0 years, 58 (9.5%) had > or =1 episode of status epilepticus during follow-up evaluation. The first episode occurred a median of 2.5 years after initial diagnosis (range, <1 month to 8.8 years). A history of previous status epilepticus was strongly associated with subsequent status epilepticus (18/56 [32.1%] vs 40/557 [7.2%]; p < 0.0001). Younger age at onset and symptomatic etiology contributed independently to the risk of status epilepticus. Mortality was higher in children with status epilepticus before diagnosis, largely secondary to underlying cause. CONCLUSIONS: Status epilepticus occurs in approximately 10% of children after initial diagnosis of epilepsy. Status epilepticus before initial diagnosis, young age at onset, and symptomatic etiology independently influence the risk of status epilepticus. In those without status epilepticus before diagnosis, the risk is modest and is realized over a prolonged period. For children at highest risk, maintaining abortive therapy in the home may be a reasonable precaution.

Approaches to study of three Chinese national groups.

BACKGROUND: Although numerous studies have examined the learning approaches of Chinese students, very few comparative studies have been carried out with Chinese students from different nations. AIMS: The present research was designed to identify differences in study approach between Chinese university students drawn from Malaysia, Singapore, and Hong Kong. SAMPLES: The sample consisted of 192 Chinese students with 89 students from Malaysia (43 males and 46 females), 65 students from Hong Kong (41 males and 24 females), and 38 students from Singapore (12 males and 26 females). METHOD: All students completed Entwistle and Ramsden's (1983) Approaches to Studying Inventory by rating on a 4-point Likert scale how well each of the 64 items described their own learning behaviour. RESULTS: The hypotheses that, in comparison with their respective counterparts, Malaysian Chinese students would identify themselves as being more dependent in their learning, Singaporean students as being more adept in presenting ideas/concepts in a clear and systematic fashion, and Hong Kong students as being more anxious in their learning approach, were all supported. However, the hypothesis that Hong Kong students would be more strategic in their learning approach than their national counterparts was not supported. CONCLUSIONS: Based on the significant differences in learning approaches noted among the different Chinese subgroups, caution must therefore be taken against forming fixed conceptualisations of cultural characteristics and considerable care be given in sample definition and selection in cross-cultural research.

Indoloquinoxaline compounds that selectively antagonize P-glycoprotein.

Tumor cells often develop drug resistance through overexpression of membrane transport proteins that effectively efflux anticancer agents. The pharmacologies of the two best-studied transporters, P-glycoprotein (Pgp) and MRP1, are partially overlapping but distinct. To improve the therapeutic potential of drug resistance reversing agents, we have developed a program to identify compounds with selectivity for Pgp or MRP1. Screening of a commercial library of compounds identified indoloquinoxaline compounds with transporter selectivity, and certain examples were synthesized and further evaluated. 1,4-Dibutoxy-6H-indolo[2,3-b]quinoxaline and 4,7-dibutoxy-2,3-dihydrobenzimidazole-2-spiro-3-indolin-2-one were synthesized by condensation of 3,6-dibutoxy-1,4-diaminobenzene and isatin. Neither compound was cytotoxic to MCF-7 cells, nor did either one affect the sensitivity of MCF-7/VP or HL-60/ADR cells at doses up to at least 20 microM, indicating that they do not antagonize MRP1. In contrast, each compound, at doses as low as 0.25 microM, sensitized NCI/ADR cells to vinblastine, actinomycin D, Taxol, and doxorubicin, indicating that they effectively reverse Pgp-mediated multidrug resistance (MDR). Furthermore, the compounds sensitized two additional cell lines that overexpress Pgp to this panel of anticancer drugs. However, these compounds did not affect the sensitivities of MCF-7 or T24 cells to these cytotoxic drugs, and did not alter the sensitivities of any of the tested cell lines to cisplatin or 5-fluorouracil. Both compounds enhanced the intracellular accumulation of [3H]vinblastine by NCI/ADR cells, but did not inhibit photoaffinity labeling of Pgp by [3H]azidopine at concentrations up to at least 100 microM. Therefore, these novel nontoxic indoloquinoxalines selectively sensitize Pgp-overexpressing cells to drugs that are subject to transport by this protein, without modulating the sensitivities of MRP1-overexpressing or non-Pgp cells to cytotoxic drugs. Because of this transporter selectivity, we predict that these compounds will be effective MDR modulators in vivo.

Cationic lipid-mediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a double-blind placebo-controlled trial.

BACKGROUND: We and others have previously reported significant changes in chloride transport after cationic-lipid-mediated transfer of the cystic fibrosis transmembrane conductance regulator (CFTR) gene to the nasal epithelium of patients with cystic fibrosis. We studied the safety and efficacy of this gene transfer to the lungs and nose of patients with cystic fibrosis in a double-blind placebo-controlled trial. METHODS: Eight patients with cystic fibrosis were randomly assigned DNA-lipid complex (active) by nebulisation into the lungs followed 1 week later by administration to the nose. Eight control patients followed the same protocol but with the lipid alone (placebo). Safety was assessed clinically, by radiography, by pulmonary function, by induced sputum, and by histological analysis. Efficacy was assessed by analysis of vector-specific CFTR DNA and mRNA, in-vivo potential difference, epifluorescence assay of chloride efflux, and bacterial adherence. FINDINGS: Seven of the eight patients receiving the active complex reported mild influenza-like symptoms that resolved within 36 h. Six of eight patients in both the active and placebo groups reported mild airway symptoms over a period of 12 h following pulmonary administration. No specific treatment was required for either event. Pulmonary administration resulted in a significant (p<0.05) degree of correction of the chloride abnormality in the patients receiving active treatment but not in those on placebo when assessed by in-vivo potential difference and chloride efflux. Bacterial adherence was also reduced. We detected no alterations in the sodium transport abnormality. A similar pattern occurred following nasal administration. INTERPRETATION: Cationic-lipid-mediated CFTR gene transfer can significantly influence the underlying chloride defect in the lungs of patients with cystic fibrosis.

The in vivo effects of milrinone on the airways of cystic fibrosis mice and human subjects.

Previous studies have indicated that milrinone, a specific type III phosphodiesterase inhibitor, may be able to induce chloride secretion in cystic fibrosis (CF) tissues. We have now assessed the effect of this agent in vivo on the nasal epithelium of CF mutant mice and also in the nose and lungs of human subjects with CF. Wild-type mice showed a small hyperpolarization of the nasal potential difference (PD) in response to milrinone (100 microM, 1.6 +/- 0.6 mV, n = 8, P < 0.05). In contrast, CF mice carrying either the most common human mutation of the gene for the CF transmembrane regulator (CFTR), DeltaF508 (protein mislocalized), or the G551D mutation (protein normally localized) failed to demonstrate this response. Milrinone perfused alone had no significant effect on the baseline nasal PD of human subjects without CF (14.7 +/- 4.0 mV preperfusion; 15.3 +/- 4.6 mV postperfusion), but significantly (P < 0.05) augmented the hyperpolarization induced by a subsequently perfused low-chloride solution (with milrinone, 36.8 +/- 3.0 mV, n = 6; without milrinone, 18.1 +/- 2.2 mV, n = 19). In contrast, in human subjects with CF (n = 6), milrinone alone significantly (P < 0. 05) altered the nasal baseline PD (52.2 +/- 3.3 mV preperfusion; 57. 4 +/- 4.2 mV, postperfusion) but not the subsequent responses to the low-chloride solution (with milrinone, 1.1 +/- 2.2 mV, n = 4; without milrinone, 0.6 +/- 0.5 mV, n = 28) or to isoproterenol (100 microM). In a separate study in subjects (n = 6) with the DeltaF508 mutation, nasal coadministration of milrinone with isoproterenol produced no effect in the presence of amiloride and a low-chloride solution (-0.8 +/- 0.5 mV). This was also the case in the nasal epithelium of CF subjects (n = 4) carrying at least one G551D allele (-0.3 +/- 0.8 mV). Similarly, milrinone did not hyperpolarize the PD of either the tracheal (n = 6) or segmental (n = 6) airways of CF subjects (DeltaF508) when applied topically in vivo in the presence of amiloride, isoproterenol, or adenosine triphosphate (all 100 microM) in a low-chloride solution. These data do not support the use of milrinone to induce chloride secretion in CF airways in vivo.

Association of Phialophora gregata with Fusarium solani f. sp. pisi in Garbanzo Beans in California.

In 1992, a vascular disease was found in two fields of cultivar UC-15 garbanzo beans (Cicer arietinum). The fields were located on the central coast of California and Phialophora gregata was consistently isolated from the aboveground tissue in the host UC-15 plants. Diseased patches in the fields mimicked the more common Fusarium wilt with its discolored vascular tissue in the stems, but in these instances P. gregata was the sole fungus that grew out of the tissue after 5 to 8 days of incubation on isolation agar. Greenhouse pathogenicity tests yielded only minor symptoms whether a dip technique or stem puncture inoculations were used. In subsequent years it was noted that this disease was observed only in garbanzo fields where a pea crop (Pisum sativum) had been previously grown. Further isolation from small foot and root lesions of diseased field plants revealed that they were infected with Fusarium solani f. sp. pisi (Fsp) in addition to P. gregata. In greenhouse experiments with a combination of soilborne Fsp and/or stem inoculations with P. gregata, both pathogens infected the host. The symptoms from both pathogens, the strongest including occasional plant death, were similar to those observed in the fields. The effect was more than additive. Neither pathogen alone caused severe damage. In these tests Fsp was reisolated from root and foot cortical tissue along with P. gregata from stem vascular tissue near the top of the plants. Peas were an important crop along the central coast long before garbanzo beans were grown there. Fsp, a pathogen of both pea and garbanzo bean (1), has been known to attack garbanzos there since 1973 (2). The presence of yet another vascular disease of significance in this coastal area, besides those caused by Fusarium oxysporum f. sp. ciceri and Verticillium dahliae, has not been previously noted. Help in identification of P. gregata came from Diane Fogle, California Department of Food and Agriculture, Sacramento. References: (1) J. M. Kraft. Plant Dis. Rep. 53:110, 1969. (2) F. V. Westerlund Jr., et al. Phytopathology 64:432, 1974.

The Distribution of Fusarium Wilt of Blackeyed Cowpeas within California Caused by Fusarium oxysporum f. sp. tracheiphilum Race 4.

Fusarium wilt of blackeyed cowpeas has been known in California since the 1930s, and breeding for resistance to this disease pathogen has been a continuous effort. During the 1960s and 1970s, California Blackeye 5 (CB 5) cowpea (Vigna unguiculata L. Walp.), a widely grown cultivar of the time, became increasingly diseased by Fusarium oxysporum f. sp. tracheiphilum (Fot) Race 3 (2) throughout the growing regions of California. University of California cultivars CB 46 and CB 88 (1) were developed for resistance to Fot Races 1, 2, and 3. CB 46 is currently the principal blackeye cultivar grown on the majority of the acreage in the San Joaquin Valley. In 1989, a new race we designate "Fot Race 4" was isolated from wilted plants at a single field site in Stanislaus County. In years prior to identification, Fot Race 4 had caused severe wilt of CB 46 and CB 88 in this field. Even though the new Fot Race 4 remained confined to a small area for a number of years, sources of host plant resistance to Fot Race 4 were identified, hybridized, and screened, resulting in new progeny with desirable commercial agronomic characteristics. As observed in Stanislaus County, F. oxysporum f. sp. tracheiphilum Race 4 has the potential to cause serious crop damage, depending on virulence and soil inoculum levels, which may vary from year to year. In 1997 and 1998, an entirely different area in the southern San Joaquin Valley, about 140 miles from the original site in Stanislaus County, was found to have plants infected with Fot Race 4. Diseased plants were collected from patches in three separate CB 46 or CB 88 field sites in Tulare County. About 30 cultures were isolated from the diseased plants, which showed stunting, yellowing, and vascular discoloration. In greenhouse fusarium dip tests CB 46, CB 88, CB 5, and several Fot Race 4 resistant breeding lines were inoculated with all the collected isolates and evaluated. CB 46, CB 88, and CB 5 proved to be susceptible to these isolates, showing typical Fot Race 4 symptoms. The Fot Race 4 pathogen was then reisolated from greenhouse-grown, diseased stem tissue of CB 46, CB 88, and CB 5. These findings emphasize the importance of vigilance and necessity of continual disease surveys. They serve as an early alert for the University of California breeding program, and validate local cooperation with University of California Extension Farm Advisors. As a result of this effort new cultivar candidates with resistance to Fot Race 4 are in the final phases of multi-year commercial testing. References: (1) D. M. Helms et al. Crop Sci. 31:1703, 1991. (2) K. S. Rigert and K. W. Foster. Crop Sci. 27:220, 1987.

Effect of IBMX and alkaline phosphatase inhibitors on Cl- secretion in G551D cystic fibrosis mutant mice.

Some cystic fibrosis transmembrane conductance regulator (CFTR) mutations, such as G551D, result in a correctly localized Cl- channel at the cell apical membrane, albeit with markedly reduced function. Patch-clamp studies have indicated that both phosphatase inhibitors and 3-isobutyl-1-methylxanthine (IBMX) can induce Cl- secretion through the G551D mutant protein. We have now assessed whether these agents can induce Cl- secretion in cftrG551D mutant mice. No induction of Cl- secretion was seen with the alkaline phosphatase inhibitors bromotetramisole or levamisole in either the respiratory or intestinal tracts of wild-type or cftrG551D mice. In contrast, in G551D intestinal tissues, IBMX was able to produce a small CFTR-related secretory response [means +/- SE: jejunum, 1.8 +/- 0.9 microA/cm2, n = 7; cecum, 3.7 +/- 0.8 microA/cm2, n = 7; rectum (in vivo), 1.9 +/- 0.9 mV, n = 5]. This was approximately one order of magnitude less than the wild-type response to this agent and, in the cecum, was significantly greater than that seen in null mice (cftrUNC). In the trachea, IBMX produced a transient Cl- secretory response (37.3 +/- 14.7 microA/cm2, n = 6) of a magnitude similar to that seen in wild-type mice (33.7 +/- 4.7 microA/cm2, n = 9). This response was also present in null mice and therefore is likely to be independent of CFTR. No effect of IBMX on Cl- secretion was seen in the nasal epithelium of cftrG551D mice. We conclude that IBMX is able to induce detectable levels of CFTR-related Cl- secretion in the intestinal tract but not the respiratory tract through the G551D mutant protein.

Inoculum Densities of Fusarium oxysporum f. sp. vasinfectum and Meloidogyne incognita in Relation to the Development of Fusarium Wilt and the Phenology of Cotton Plants (Gossypium hirsutum).

ABSTRACT Development of Fusarium wilt in upland cotton (Gossypium hirsutum) usually requires infections of plants by both Meloidogyne incognita and Fusarium oxysporum f. sp. vasinfectum. In this study, the soil densities of M. incognita and F. oxysporum f. sp. vasinfectum and the incidence of Fusarium wilt in three field sites were determined in 1982-1984. Multiple regression analysis of percent incidence of Fusarium wilt symptoms on population densities of M. incognita and F. oxysporum f. sp. vasinfectum yielded a significant fit (R (2) = 0.64) only on F. oxysporum f. sp. vasinfectum. Significant t-values for slope were also obtained for the interaction of M. incognita and F. oxysporum f. sp. vasinfectum, but densities of M. incognita and F. oxysporum f. sp. vasinfectum were also related on a log(10) scale. The physiological time of appearance of first foliar symptoms of Fusarium wilt, based on a degree-days threshold of 11.9 degrees C (53.5 degrees F), was used as a basis for determining disease progress curves and the phenology of cotton plant growth and development. Effects of Fusarium wilt on plant height and boll set were determined in three successive years. Increases in both of these plant characteristics decreased or stopped before foliar symptoms were apparent. Seed cotton yields of plant cohorts that developed foliar wilt symptoms early in the season (before 2,000 F degree-days) were variable but not much different in these years. This contrasted with cohorts of plants that first showed foliar symptoms late in the season (after 2,400 F degree-days) and cohorts of plants that showed no foliar symptoms of wilt. Regression analyses for 1982-1984 indicated moderate to weak correlations (r = 0.16-0.74) of the time of appearance of the first foliar symptoms and seed cotton yields.

Laboratory and clinical studies in support of cystic fibrosis gene therapy using pCMV-CFTR-DOTAP.

The first phase I study of cystic fibrosis gene therapy using cationic liposomes to deliver the cystic fibrosis conductance regulator gene to the nose reported partial and transient correction of the nasal transepithelial ion transport defect, While encouraging, further improvements will be required if this form of treatment is to be of therapeutic value. We tested a new formulation, pCMV-CFTR-DOTAP. The complex is stable for 10 days and effective at correcting the electrophysiological deficit in the trachea of CF mutant mice at 8 or 9 days after intratracheal instillation. Reliable protocols for consistent detection of as few as 10 molecules of CFTR mRNA and DNA in nasal brushing samples are described, Both vector and DNA have been produced to Good Manufacturing Practice standard, Nasal potential difference measurements developed at the National Heart and Lung Institute to assess the CFTR ion channel activity in CF patients replicated well at the Scottish Adult Cystic Fibrosis Service. The SPO fluorescence assay for halide ion conductance in nasal brushings has also been tested. These establish baseline conditions in the Scottish CF cohort from which evidence for correction can be judged under clinical trial conditions. These studies formed the basis for regulatory approval of a randomised, placebo controlled double-blind phase I research study.

Local anaesthesia to the airway reduces sedation requirements in patients undergoing artificial ventilation.

Patients in the intensive care unit require large doses of sedative/analgesic drugs to tolerate the presence of a tracheal tube and other unpleasant stimuli. The ideal regimen for sedatives and analgesics has not yet been found. We have investigated the effects of topical local anaesthesia to the pharynx and airway on sedative/analgesic requirements in 30 ICU patients (25-75 yr old) with no obvious brain injury, undergoing mechanical ventilation. Oral tracheal tubes were changed to a modified tube which allowed instillation of local anaesthetic solutions onto the pharyngeal, laryngeal and tracheal mucosa. Lignocaine 1% (5 ml) or 5 ml of 0.9% saline were instilled hourly for 12 h each for a total of 24 h, in a double-blind, randomized crossover design. Baseline sedation was maintained with propofol or alfentanil infusions, or both, which were titrated to patient comfort and to maintain an optimum sedation score throughout. Twenty-five patients completed the study. Mean total propofol and alfentanil requirements were 766 (SD 524) mg and 17 (7.6) mg, respectively, during 12 h of lignocaine instillation, and 1321 (862) mg and 25 (11.4) mg, respectively, during 12 h of saline instillation. There was a significant reduction (P < 0.05) in the requirements for both agents during the period of lignocaine instillation.

A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction.

Quantifying the level of transgene expression necessary for phenotypic effect is an important consideration in designing somatic gene therapy protocols. A nonlinear relationship between phenotype and gene activity is predicted by control analysis for any autosomal recessive condition. The unaffected phenotype of heterozygotes for autosomal recessive disorders demonstrates that 50% of the normal level of gene expression is sufficient to prevent disease. By extension, an exaggerated and positive effect on the mutant phenotype is predicted to arise from only a small addition of normal transgene expression delivered by gene therapy. We tested this expectation directly by intercrossing mice carrying different Cftr alleles which modulate Cftr gene expression from 0 to 100%. We demonstrate that 5% of the normal level of Cftr gene expression results in a disproportionately large correction of the chloride ion transport defect (50% of normal) and essentially complete rescue of the intestinal disease (100% survival). It follows that even modest levels of transgene expression and only partial correction of CFTR channel activity may have a significant clinical impact.