Calf care personnel on dairy farms and their educational opportunities.

As dairy herd sizes become larger and the organization of the business more complex, targeting communication and education to enhance animal care becomes more difficult. The purpose of this study was to describe selected demographics of calf care employees on large (>500 animals) and small (<501 animals) dairy farms that raise their own calves. Two to 8 individuals per farm involved with calf care, including owners, veterinarians, and calf managers, feeders, and treaters, were interviewed in either English or Spanish. Interviews were conducted in person on 53 dairy farms located in Arizona, Idaho, New York, Oregon, and Washington State. The number of preweaned calves on the farm ranged from 9 to 1,500 (median = 93). A total of 224 individuals were interviewed across 8 job titles. As farm size increased, personnel structure became more complex. Farms with >100 preweaned calves were 15 times more likely to have a calf manager title compared with farms with ≤100 preweaned calves. Eight farms designated the same person as calf manager, treater, and feeder, all with ≤100 preweaned calves. Thirty-two (60%) of the farms had at least 1 full-time calf feeder. Almost 30% of owners and over 40% of veterinarians interviewed were over 50 yr of age, whereas over 40% of the calf managers, feeders, and treaters were under 30 yr of age. Seventy-three percent of feeders and 72% of treaters spoke Spanish at home. For languages in which interviewees were comfortable speaking, more than 30% of owners and 33% of veterinarians were comfortable communicating in Spanish. For calf care employees, 60% of calf managers, 42% of feeders, and 38% of treaters were bilingual (English and Spanish), but most (72%) preferred to be interviewed in Spanish. The level of education varied by job title for those interviewed, but most of the calf care team had high school or less education. However, some diversity was observed in educational background within job title with almost 38% of the calf managers having at least some college education. The majority of feeders (88%) and treaters (83%) reported being trained by another employee and 66 and 58%, respectively, had not received any continuing education in the previous year. With the amount of diversity seen on these farms, understanding employees' educational backgrounds, language, and generational differences may be valuable when developing training for new procedures for animal health or other aspects of animal care.

Effect of heifer-raising practices on E. coli antimicrobial resistance and Salmonella prevalence in heifer raisers.

Although cattle movement and commingling play an important role in the inter-herd transmission of pathogens, little is known about the effect of commingling of heifers at raising operations. The objective of this study was to compare the resistance of E. coli and prevalence of Salmonella from pooled faecal pats of heifers raised off-farm at multi-source raisers (MULTI) that raised heifers from at least two farms compared with on-farm raisers (HOME), with heifers from only that farm. MULTI faecal pat samples were collected from pens with animals that had arrived at the farm within the previous 2 months (AP) and from animals that would be departing the heifer raiser in 2-3 months (DP). Corresponding age sampling was conducted at HOME raisers. Odds of ampicillin resistance were 3·0 times greater in E. coli collected from MULTI compared to HOME raisers. E. coli from AP pens had significantly (P < 0·05) higher odds of resistance to ampicillin, neomycin, streptomycin, and tetracycline compared to DP pens. Salmonella recovery was not significantly different between heifer-raising systems (P = 0·3). Heifer-raising system did not have a major overall impact on selection of resistant E. coli, which was strongly affected by the age of the animals sampled.

Cancer patient-reported outcomes assessment using wireless touch screen tablet computers.

PURPOSE: To assess the feasibility of collecting patient-reported outcomes data with wireless touch screen tablet computers in the adult oncology palliative care setting. METHODS: Patients were provided with tablet computers during scheduled clinic visits and answered online queries about their experience over the past week in the health domains of anxiety, depression, fatigue, pain interference, physical function, instrumental social support, sleep impairment, diarrhea, constipation, nausea, vomiting, anorexia, dyspnea, neuropathy, and spiritual values. RESULTS: Content analysis of patient interviews indicates that wireless touch screen tablet computers are a feasible approach for collecting patient-reported outcome measures by palliative care cancer patients presenting in clinic. Most patients indicated that the questionnaire was easy to answer. However, all but one patient requested some form of assistance, and many reported difficulties attributable to a lack of familiarity with the device, interpretation of certain questions, and wireless connectivity-related issues. CONCLUSIONS: This feasibility study demonstrates that tablet computers have the potential to efficiently and reliably collect patient-reported health status measures among palliative care cancer patients presenting in clinics. The use of these devices may lead to substantial improvements by making patient-reported outcomes available for clinical decision-making.

Effect of preweaned dairy calf housing system on antimicrobial resistance in commensal Escherichia coli.

Group housing of preweaned dairy calves is a growing practice in the United States. The objective of this practice is to increase the average daily gain of calves in a healthy and humane environment while reducing labor requirements. However, feeding protocols, commingling of calves, and occurrence of disease in different calf-housing systems may affect the prevalence of antimicrobial drug-resistant bacteria. This study evaluated the effect of a group pen-housing system and individual pen-housing system on antimicrobial resistance trends in fecal Escherichia coli of preweaned dairy calves and on the prevalence of environmental Salmonella. Twelve farms from central New York participated in the study: 6 farms using an individual pen-housing system (IP), and 6 farms using a group pen-housing system (GP). A maximum of 3 fecal E. coli isolates per calf was tested for susceptibility to 12 antimicrobial drugs using a Kirby-Bauer disk diffusion assay. Calves in GP had a significantly higher proportion of E. coli resistant to ciprofloxacin and nalidixic acid, whereas calves in IP had a significantly higher proportion of E. coli resistant to ampicillin, ceftiofur, gentamycin, streptomycin, and tetracycline. Calf-housing system had an effect on resistance to individual antimicrobial drugs in E. coli, but no clear-cut advantage to either system was noted with regard to overall resistance frequency. No outstanding difference in the richness and diversity of resistant phenotypes was observed between the 2 calf-housing systems.

Effect of on-farm use of antimicrobial drugs on resistance in fecal Escherichia coli of preweaned dairy calves.

Respiratory disease and diarrhea are the 2 most common diseases that result in the use of antimicrobial drugs in preweaned calves. Because the use of drugs in food animals, including dairy calves, has the potential for generating cross-resistance to drugs used in human medicine, it is vital to propose farm practices that foster the judicious use of antimicrobials while assuring animal health and productivity. The objective of this study was to use dairy farm calf treatment records to identify antimicrobial drug treatments in calves and to evaluate their effects on the prevalence of antimicrobial-resistant Escherichia coli from rectal swabs of preweaned dairy calves. Eight farms from central New York participated in the study, 3 farms using individual pen housing management and 5 farms using group pen housing management. Eligible study farms could not add antimicrobial drugs to the milk fed to preweaned calves and were required to have farm records documenting antimicrobial drug treatment of calves from birth to weaning. Three fecal E. coli isolates per calf were tested for susceptibility to 12 antimicrobial drugs using a Kirby-Bauer disk diffusion assay. A total of 473 calves were sampled, from which 1,423 commensal E. coli isolates were tested. Of the 9 antimicrobial drugs used on study farms, only enrofloxacin was significantly associated with reduced antimicrobial susceptibility of E. coli isolates, although treatment with ceftiofur was associated with reduced susceptibility to ceftriaxone. The median numbers of days from treatment with ceftiofur and enrofloxacin to rectal swab sampling of calves were 16 d (range: 1-39) and 12 d (range: 6-44), respectively. At the isolate level, treatment with enrofloxacin resulted in odds ratios of 2 [95% confidence interval (CI): 1-4] and 3 (95% CI: 2-6), respectively, for isolation of nonsusceptible E. coli to nalidixic acid and ciprofloxacin compared with calves not treated with enrofloxacin. Treatment with ceftiofur resulted in an odds ratio of 3 (95% CI: 0.9-12) for isolation of nonsusceptible E. coli to ceftriaxone compared with calves not treated with ceftiofur. Treatment with enrofloxacin resulted in selection of isolates that presented phenotypic resistance to both ciprofloxacin and ceftriaxone. Treatment with ceftiofur resulted in a higher prevalence of isolates resistant to ≥3 antimicrobial drugs (97%) compared with no treatment with ceftiofur (73%). These findings reinforce the necessity for continued implementation of practices at the dairy farm that support the sustainable and judicious use of antimicrobial drugs in dairy calves.

US Adults' Likelihood to Participate in Dementia Prevention Drug Trials: Results from the National Poll on Healthy Aging.

BACKGROUND: Recruitment to dementia prevention clinical trials is challenging, and participants are not representative of US adults at risk. A better understanding of the general public's interest in dementia prevention research participation is needed to inform future recruitment strategies. OBJECTIVE: To examine US adults' characteristics associated with self-reported likelihood to participate in dementia prevention clinical trials. DESIGN: We conducted a cross-sectional survey using the October 2018 wave of the University of Michigan National Poll on Healthy Aging. SETTING: The National Poll on Healthy Aging is a nationally representative survey of adults using KnowledgePanel (Ipsos Public Affairs LLC), a probability-based panel of the civilian, noninstitutionalized US population. PARTICIPANTS: We analyzed data from 1,028 respondents, ages 50 to 64 years, who completed a web survey module on brain health. MEASUREMENTS: We used logistic regression models to examine associations between sociodemographic and dementia-related factors (e.g., family history) and self-reported likelihood to participate in a dementia prevention clinical trial of a new medicine ("very" or "somewhat likely" vs. "not likely" survey responses). Among respondents not likely to participate, we examined frequency of reasons endorsed for this decision, stratified by age, sex, and race and ethnicity. RESULTS: Of the 1,028 respondents, half were female, 68% Non-Hispanic White, 13% Hispanic, and 12% Non-Hispanic Black. Twelve percent of respondents reported being very likely to participate in a dementia prevention trial, 32% somewhat likely, and 56% not likely. Factors associated with higher likelihood to participate were higher perceived risk of dementia [OR, 2.17 (95% CI, 1.61, 2.93)], a positive family history of dementia [OR, 1.75 (95% CI, 1.27, 2.43)], and having discussed dementia prevention with a doctor [OR, 2.20 (95% CI, 1.10, 4.42)]. There were no differences in likelihood to participate by sociodemographic characteristics. Among 570 respondents not likely to participate, 39% said they did not want to be a guinea pig, 23% thought dementia would not affect them, 22% thought there would be too high a chance for harm, 15% indicated study participation would take too much time, and 5% reported fear of learning information about oneself. There were no differences across age, sex, and racial and ethnic groups. CONCLUSIONS: In this study, perceived risk of dementia, family history, and discussion of prevention with a doctor were associated with likelihood to participate in a dementia prevention clinical trial, whereas sociodemographic factors including race and ethnicity were not. Findings suggest that recruitment interventions focused on increasing knowledge of dementia risk and prevention trials and involving healthcare providers may be effective tools to improve enrollment rates, regardless of target community.

Improved identification of epidemiologically related strains of Salmonella enterica by use of a fusion algorithm based on pulsed-field gel electrophoresis and multiple-locus variable-number tandem-repeat analysis.

Pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat analysis (MLVA) are used to assess genetic similarity between bacterial strains. There are cases, however, when neither of these methods quantifies genetic variation at a level of resolution that is well suited for studying the molecular epidemiology of bacterial pathogens. To improve estimates based on these methods, we propose a fusion algorithm that combines the information obtained from both PFGE and MLVA assays to assess epidemiological relationships. This involves generating distance matrices for PFGE data (Dice coefficients) and MLVA data (single-step stepwise-mutation model) and modifying the relative distances using the two different data types. We applied the algorithm to a set of Salmonella enterica serovar Typhimurium isolates collected from a wide range of sampling dates, locations, and host species. All three classification methods (PFGE only, MLVA only, and fusion) produced a similar pattern of clustering relative to groupings of common phage types, with the fusion results being slightly better. We then examined a group of serovar Newport isolates collected over a limited geographic and temporal scale and showed that the fusion of PFGE and MLVA data produced the best discrimination of isolates relative to a collection site (farm). Our analysis shows that the fusion of PFGE and MLVA data provides an improved ability to discriminate epidemiologically related isolates but provides only minor improvement in the discrimination of less related isolates.

Multilocus variable-number tandem-repeat analysis and plasmid profiling to study the occurrence of blaCMY-2 within a pulsed-field gel electrophoresis-defined clade of Salmonella enterica serovar Typhimurium.

Salmonella enterica serovar Typhimurium circulating in food animal populations and carrying resistance to antimicrobial agents represents a human health risk. Recently, a new clade of S. Typhimurium, WA-TYP035/187, was reported in cattle and humans in the Pacific Northwest, United States of America. The objective of this study was to describe a possible mechanism of acquisition of expanded-spectrum cephalosporin resistance in this clade. Ceftazidime resistance increased steadily among WA-TYP035/187 isolates, from 0% (0/2) in 1999 to 77.8% (28/36) in 2006 (chi2 for linear trend, P value of <0.001). Among 112 bovine-source and 18 human-source isolates, 49 (43.8%) and 12 (66.7%) were resistant to ceftazidime, respectively. Multiple-locus variable-number tandem-repeat analysis (MLVA) and plasmid profiling suggested that resistance was acquired by multiple independent genetic events within the WA-TYP035/187 clade. Given the lack of an obvious reservoir in species other than cattle and a parallel rise in ceftiofur resistance in the bovine-specific serovar Salmonella enterica serovar Dublin in the same time frame and region, selection pressure due to the use of the expanded-spectrum cephalosporin drug ceftiofur in cattle is a likely factor driving the increasing cephalosporin resistance of WA-TYP035/187.

The prevalence of multidrug resistance is higher among bovine than human Salmonella enterica serotype Newport, Typhimurium, and 4,5,12:i:- isolates in the United States but differs by serotype and geographic region.

Salmonella represents an important zoonotic pathogen worldwide, but the transmission dynamics between humans and animals as well as within animal populations are incompletely understood. We characterized Salmonella isolates from cattle and humans in two geographic regions of the United States, the Pacific Northwest and the Northeast, using three common subtyping methods (pulsed-field gel electrophoresis [PFGE], multilocus variable number of tandem repeat analysis [MLVA], and multilocus sequence typing [MLST]). In addition, we analyzed the distribution of antimicrobial resistance among human and cattle Salmonella isolates from the two study areas and characterized Salmonella persistence on individual dairy farms. For both Salmonella enterica subsp. enterica serotypes Newport and Typhimurium, we found multidrug resistance to be significantly associated with bovine origin of isolates, with the odds of multidrug resistance for Newport isolates from cattle approximately 18 times higher than for Newport isolates from humans. Isolates from the Northwest were significantly more likely to be multidrug resistant than those from the Northeast, and susceptible and resistant isolates appeared to represent distinct Salmonella subtypes. We detected evidence for strain diversification during Salmonella persistence on farms, which included changes in antimicrobial resistance as well as genetic changes manifested in PFGE and MLVA pattern shifts. While discriminatory power was serotype dependent, the combination of PFGE data with either MLVA or resistance typing data consistently allowed for improved subtype discrimination. Our results are consistent with the idea that cattle are an important reservoir of multidrug-resistant Salmonella infections in humans. In addition, the study provides evidence for the value of including antimicrobial resistance data in epidemiological investigations and highlights the benefits and potential problems of combining subtyping methods.

Salmonella enterica serotype 4,5,12:i:-, an emerging Salmonella serotype that represents multiple distinct clones.

The prevalence, among human clinical cases, of Salmonella enterica serotype 4,5,12:i:-, a serotype antigenically similar to Salmonella enterica serotype Typhimurium but lacking second-phase flagellar antigens, has increased considerably over the last 10 years. To probe the evolution and ecology of this emerging serotype, we characterized 190 Salmonella isolates initially classified as Salmonella serotypes 4,5,12:i:- (n = 90) and Typhimurium (n = 100) and obtained from various sources in the United States and Spain. These isolates were characterized into six sequence types (determined by multilocus sequence typing [MLST]) and 79 pulsed-field gel electrophoresis types. The majority of Salmonella serotype 4,5,12:i:- and Typhimurium isolates (85 and 84 isolates, respectively) represented a single MLST type. Existing genome information revealed different genome deletions (which included genes responsible for phase 2 flagellum expression) in four Spanish Salmonella serotype 4,5,12:i:- isolates and one U.S. Salmonella serotype 4,5,12:i:- isolate. Fifty-nine isolates of both serotypes, representing different sources and geographical locations as well as different molecular subtypes, were thus screened for the presence of six genes and one specific region, all of which were previously found to show variable presence among Salmonella serotype 4,5,12:i:- and Typhimurium strains. All Salmonella serotype 4,5,12:i:- isolates lacked the phase 2 flagella genes fljA and fljB, which were present in all Salmonella serotype Typhimurium isolates. While all Spanish Salmonella serotype 4,5,12:i:- isolates carried the same deletion surrounding fljAB, all but two U.S. isolates showed a different genomic deletion; the two atypical U.S. isolates represented the "Spanish" deletion genotype and a unique deletion genotype. Salmonella serotype 4,5,12:i:- thus appears to represent at least two common clones, which cannot easily be differentiated with standard diagnostic procedures.

Radioactive labeling of antibody: a simple and efficient method.

A simple and efficient method of covalently coupling the strong chelator diethylenetriaminepentaacetic acid to proteins was developed for radiolabeling immunoglobulin G antibodies. After being coupled and labeled with indium-111, a monoclonal antibody to carcinoembryonic antigen retained its ability to bind to its antigen in vitro and in vivo. In nude mice with a human colorectal xenograft, 41 percent of the injected radioactivity became localized in each gram of xenograft at 24 hours compared with 9 percent for control antibody and 19 percent for radioiodinated antibody to carcinoembryonic antigen.

Introduction of new multidrug-resistant Salmonella enterica strains into commercial dairy herds.

A longitudinal observational study of 59 dairy herds was conducted in Washington State to estimate the rate of introduction of new multidrug-resistant (MDR) Salmonella enterica strains onto commercial dairy herds. Samples were collected on these herds over 7 visits separated by intervals of 2 to 4 mo over a period of 15 to 21 mo. Samples were cultured for Salmonella spp. and serogroup, serovar, and antimicrobial susceptibility patterns were identified for MDR Salmonella isolates. Fingerprinting generated by pulsed-field gel electrophoresis (PFGE) using XbaI restriction enzyme digestion generated genotyping profiles for all MDR isolates identified in the study. The rate of new MDR Salmonella strain introduction was 0.9 per herd-year (95% confidence interval: 0.6-1.4). The rates for the most commonly introduced MDR Salmonella serovars were 0.4/herd-year for Typhimurium, 1.2/herd-year for Newport, and 0.1/herd-year for Dublin. Thirty-three of 59 herds (56%) had at least one new MDR Salmonella introduction during the study period. The number of new MDR Salmonella strains acquired by dairy herds ranged from zero to 8. Thirteen of the 59 herds had a history of clinical salmonellosis. Among these 13 herds, 6 herds acquired new MDR Salmonella strains, although these strains were different than historical clinical strains. These data indicate that acquisition of new MDR Salmonella strains by dairy herds was a common event in participating herds, although the number of strains introduced varied greatly among herds.

The role of animal movement, including off-farm rearing of heifers, in the interherd transmission of multidrug-resistant Salmonella.

Fifty-nine commercial dairy farms were sampled 7 times over 15 to 21 mo to determine the role of animal movement, including off-farm rearing of heifers, in the interherd transmission of multidrug-resistant (MDR) Salmonella spp. Farm management data were collected by on-site inspections and questionnaires on herd management practices before and after the study. Forty-four percent (26/59) of herds did not acquire any new MDR Salmonella strains. The number of newly introduced MDR Salmonella strains acquired by the remaining 56% (33/59) of herds ranged from 1 to 8. Logistic regression models indicated that off-farm heifer raising, including contract heifer raising where heifers commingle with cattle from other farms [commingled heifers, odds ratio (OR) = 8.9, 95% confidence interval (CI): 2.4, 32.80], and herd size per 100-animal increment (herd size, OR = 1.04, 95% CI, 1.01, 1.05) were significantly associated with the introduction of new MDR Salmonella strains. The negative binomial regression similarly revealed that commingled heifers [relative risk (RR) = 2.3, 95% CI: 1.1, 4.7], herd size per 100 animals (RR = 1.02, 95% CI, 1.01, 1.03), and a history of clinical salmonellosis diagnosed before the study (RR = 2.5, 95% CI, 1.3, 5.0) were significantly associated with the number of new MDR Salmonella strains that were introduced. Factors not associated with the introduction of new MDR Salmonella strains were housing of heifers and cows in the same close-up pen, a common hospital-maternity pen, and the number of purchased cattle. This study highlights the role of animal movement in the interherd transmission of MDR Salmonella spp.

Role of epidermal growth factor receptor degradation in gemcitabine-mediated cytotoxicity.

We have recently reported that treatment with gemcitabine, a potent chemotherapeutic agent and radiation sensitizer, stimulates phosphorylation of the epidermal growth factor receptor (EGFR). Because phosphorylation of EGFR is known to precede receptor degradation, we hypothesized that gemcitabine treatment may also result in EGFR degradation. In two human head and neck cancer cell lines, UMSCC-1 and UMSCC-6, we demonstrated an approximately 80% decrease in total EGFR levels at 72 h after a 2-h treatment with 1 muM gemcitabine. Neither cisplatin nor 5-fluorouracil, which are used to treat head and neck cancer, caused EGFR degradation. EGFR downregulation did not occur at the level of transcription, as assessed by reverse transcription-polymerase chain reaction (RT-PCR), but instead occurred via phosphorylation and ubiquitination of the receptor along a proteosome/lysosome-mediated pathway. Inhibition of EGFR degradation, by either pretreatment with the EGFR tyrosine kinase inhibitor gefitinib or by exposure to the proteosome/lysosome inhibitor MG132, significantly reduced gemcitabine-induced cell death. These results suggest that EGFR degradation may be a novel mechanism for gemcitabine-mediated cell death. These findings also indicate that caution should be exercised when combining gemcitabine with agents that may prevent EGFR degradation, such as EGFR tyrosine kinase inhibitors administered in a suboptimal sequence or proteosome inhibitors.

Associations between bovine, human, and raw milk, and beef isolates of non-O157 Shiga toxigenic Escherichia coli within a restricted geographic area of the United States.

A survey for Shiga toxigenic Escherichia coli in raw milk and beef was conducted within a defined geographic region of the United States. Prevalence rates based on detection of Shiga toxin gene (stx) were 36% for retail beef, 23% for beef carcasses, and 21% for raw milk samples, which were significantly higher than were Shiga toxigenic E. coli isolation rates of 7.5, 5.8, and 3.2%, respectively. Seasonal prevalence differences were significant for stx positivity among ground beef and milk samples. Distribution of stx subtypes among isolates varied according to sample type, with stx1 predominating in milk, stx2 on carcasses, and the combination of both stx1 and stx2 in beef. Ancillary virulence markers eae and ehx were evident in 23 and 15% of isolates, respectively. Pulsed-field gel electrophoresis demonstrated associations between food isolates and sympatric bovine fecal, and human clinical isolates. These data demonstrate that non-O157 Shiga toxigenic E. coli is present in the food chain in the Pacific Northwest, and its risk to health warrants critical assessment.

Genetic diversity of Flavobacterium psychrophilum recovered from commercially raised rainbow trout, Oncorhynchus mykiss (Walbaum), and spawning coho salmon, O. kisutch (Walbaum).

Flavobacterium psychrophilum is the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. This study examined the genetic diversity of F. psychrophilum isolates retrieved from multiple epizootics at rainbow trout, Oncorhynchus mykiss, rearing facilities and from spawning coho salmon, O. kisutch. A total of 139 isolates were confirmed as F. psychrophilum by PCR assay and were further typed using pulsed-field gel electrophoresis (PFGE). Multiple epizootics at three proximally located rainbow trout rearing facilities were numerically dominated by three PFGE profiles, which accounted for 76% of all trout isolates. In coho salmon, 19 PFGE profiles were differentiated by PFGE and four numerically dominant PFGE profiles represented 56% of all coho salmon isolates. PFGE analysis also indicated that the average similarity of macrorestriction patterns of F. psychrophilum isolates was greater in rainbow trout than in coho salmon (88% vs. 70%). Furthermore, it was not unusual to isolate multiple PFGE profiles from a single coho salmon sample whereas only two PFGE profiles were shared between two sample dates separated by 1 month. It is clear that the domestic rainbow trout aquaculture facilities studied here were primarily affected by a complex of genetically related strains whereas spawning coho salmon supported a much more genetically diverse collection of F. psychrophilum.

Regulatory genes in Aspergillus nidulans.

A major area for the study of gene regulation in lower eukaryotes has been the coordinated control of catabolic enzyme synthesis. Studies of catabolic gene regulation aim to define how interactions between input signals and regulatory proteins are transmitted to the transcription machinery to bring about changes in gene expression. In the past, mutants altered in the utilization of a wide variety of substrates have been characterized in Aspergillus nidulans. Recently, the development of a transformation system for A. nidulans has meant that molecular techniques can now be combined with the traditional genetic approach.

AnCF, the CCAAT binding complex of Aspergillus nidulans, is essential for the formation of a DNase I-hypersensitive site in the 5' region of the amdS gene.

The CCAAT sequence in the amdS promoter of Aspergillus nidulans is recognized by AnCF, a complex consisting of the three evolutionary conserved subunits HapB, HapC, and HapE. In this study we have investigated the effect of AnCF on the chromatin structure of the amdS gene. The AnCF complex and the CCAAT sequence were found to be necessary for the formation of a nucleosome-free, DNase I-hypersensitive region in the 5' region of the amdS gene. Deletion of the hapE gene results in loss of the DNase I-hypersensitive site, and the positioning of nucleosomes over the transcriptional start point is lost. Likewise, a point mutation in the CCAAT motif, as well as a 530-bp deletion which removes the CCAAT box, results in the loss of the DNase I-hypersensitive region. The DNase I-hypersensitive region and the nucleosome positioning can be restored by insertion of a 35-bp oligonucleotide carrying the CCAAT motif. A DNase I-hypersensitive region has been found in the CCAAT-containing fmdS gene and was also hapE dependent. These data indicate a critical role for the AnCF complex in establishing an open chromatin structure in A. nidulans.

AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR.

CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapC gene, encoding a protein with significant similarity to Hap3p of Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the genes hapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae, Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB, hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression of amdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on gamma-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdS and gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.

ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane.

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser(269)) in the cytosol and the analogous residue in ACAT2 (Ser(249)) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.