Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

High-throughput genetic screening using matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become a powerful and widespread analytical tool in all fields of life science. Compared with other techniques, its high accuracy and sensitivity makes it a superior method, especially for the analysis of nucleic acids. Recent problems in the analysis of nucleic acids by MALDI-TOF MS can be solved using an automated MALDI-compatible sample-preparation system. Together with the reliable minisequencing assay, high-throughput genotyping of single nucleotide polymorphisms by MALDI-TOF MS is able to become a routine method in research, clinical genetics and diagnostics.     

Functional Cluster Analysis of CT Perfusion Maps: A New Tool for Diagnosis of Acute Stroke?

CT perfusion imaging constitutes an important contribution to the early diagnosis of acute stroke. Cerebral blood flow (CBF), cerebral blood volume (CBV) and time-to-peak (TTP) maps are used to estimate the severity of cerebral damage after acute ischemia. We introduce functional cluster analysis as a new tool to evaluate CT perfusion in order to identify normal brain, ischemic tissue and large vessels. CBF, CBV and TTP maps represent the basis for cluster analysis applying a partitioning (k-means) and density-based (density-based spatial clustering of applications with noise, DBSCAN) paradigm. In patients with transient ischemic attack and stroke, cluster analysis identified brain areas with distinct hemodynamic properties (gray and white matter) and segmented territorial ischemia. CBF, CBV and TTP values of each detected cluster were displayed. Our preliminary results indicate that functional cluster analysis of CT perfusion maps may become a helpful tool for the interpretation of perfusion maps and provide a rapid means for the segmentation of ischemic tissue.     

Developing resources to teach and assess the core competencies: a collaborative approach.

Graduate medical education programs face new challenges as they seek to comply with the mandate from the Accreditation Council on Graduate Medical Education to demonstrate that they are teaching and assessing residents on the six core competencies. The authors describe a project designed as a collaborative venture between the American Academy of Allergy, Asthma, and Immunology (AAAAI) and the Center for Educational Outcomes at Dartmouth College (CEdO) to provide residency programs in allergy/immunology with resources for teaching and assessing the core competencies. The goal was to create a set of learning and assessment resources that maximized the content knowledge expertise provided by the AAAAI and the learning expertise provided by CEdO. A highly interactive, iterative process was used to create a set of Web-based modules. Bilateral communication, buy-in, and active involvement in the process were seen as crucial to the development of resources and their successful implementation. Approximately 18 months after the modules were made available to training program directors, 80% of the directors surveyed were aware of and had accessed the modules. The joint creation process used in this project, designed to be generally applicable across specialties, reveals how the burden of meeting new requirements can be decreased when experts in content knowledge and experts in learning collaborate.     

The Effects of Perceived Stress on Reactions to Messages Designed to Increase Health Behaviors

This study examined the impact of perceived stress on responses to messages that encouraged the performance of health promotion and disease detection behaviors. It was hypothesized that increases in perceived stress would be associated with decreased processing of messages encouraging disease detection behaviors, and that increases in perceived stress would not effect the processing of messages encouraging health promotion behaviors. To test these hypotheses participants completed a perceived stress measure and then read a message that encouraged the performance of either a health promotion or a disease detection behavior. Then the participants were asked to indicate their agreement with the message and to attempt to recall the message. The results indicated that participants experiencing higher levels of perceived stress spent less time reading and recalled less of the messages about detection behaviors than of the messages about promotion behaviors. When participants were experiencing lower levels of perceived stress these differences disappeared.

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Rapid Report

Coexpression of KCNQ2 and KCNQ3 channels results in a 10-fold increased current amplitude compared to that of KCNQ2 alone, suggesting the formation of heteromultimeric channels. There is no interaction of either channel with KCNQ1. We evaluated the C-terminus as a potential interaction domain by construction of chimeras with interchanged C-termini of KCNQ1, KCNQ2 and KCNQ3 and functional expression in Xenopus oocytes. The chimera of KCNQ1 with a KCNQ2 C-terminus (Q1ctQ2) showed an 8-fold increase in current amplitude, and Q1ctQ3 a 3-fold increase when coexpressed with KCNQ3 and KCNQ2, respectively, indicating that the C-terminus contains an interaction domain. To characterize this interacting region, we studied further chimeras of KCNQ1 containing different parts of the KCNQ3 C-terminus for interaction with KCNQ2. We also evaluated short sequences of the KCNQ2 C-terminus for a dominant-negative effect on Q1ctQ3. According to the results of these experiments, functional interaction of KCNQ2 and KCNQ3 requires a highly conserved region of about 80 amino acids, previously called the A-domain, plus either 40 residues downstream of the A-domain (B-domain) or the proximal C-terminus between S6 and the A-domain. Furthermore, the chimeras Q1ctQ3 and Q2ctQ3 showed > 10-fold increased current amplitudes compared to KCNQ1 or KCNQ2 alone and a strong depolarizing shift of voltage-dependent activation. The proximal part of the KCNQ3 C-terminus was necessary to produce these effects. Our results indicate that specific parts of the C-terminus enable the interaction between KCNQ2 and KCNQ3 channels and that different parts of the KCNQ3 C-terminus are important for regulating current amplitude.     

Fear-Potentiated Startle in Humans: Effects of Anticipatory Anxiety on the Acoustic Blink Reflex

The effects of fear/anticipatory anxiety on the acoustic startle reflex were investigated in humans using a paradigm involving anticipation of electric shocks. The eyeblink component of the startle reflex, elicited by an abrupt auditory stimulus, was measured in 9 normal volunteers during either the anticipation of electric shocks (anticipatory anxiety) or periods in which no shocks were anticipated (safe period). The eyeblink was consistently higher in amplitude, and shorter in latency, during periods when the subjects anticipated shocks, compared to the safe periods. This effect could not be attributed solely to a reduction in habituation and was statistically significant before the subjects actually received any shock (a single 30 mA stimulation on the median nerve). These results indicate that anticipatory anxiety can be measured objectively in humans using the fear-potentiated startle reflex in a paradigm not actually requiring any shock. Because a great deal is known about the neuroanatomical and pharmacological mechanisms of fear-potentiated startle in laboratory animals, this test procedure may be especially useful in humans to investigate the neurobiological substrates of anxiety disorders and their pharmacological treatments.     

Expression of adhesion molecules in allergic lung diseases

Endothelial adherence and migration of leukocytes into tissue is mediated by different sets of adhesion molecules. The expression of these sets might not only preselect the types of leukocytes that enter the inflammatory sites, but also activate these leukocytes, induce adherence to epithelial cells, and cause the release of cytokines. Atopic asthma, extrinsic allergic alveolitis, and sarcoidosis as examples of immunologic lung diseases were investigated for the expression of adhesion molecules. Bronchial biopsies in chronic obstructive lung disease (COPD) and resected lung tissue of juvenile emphysema were chosen for controls. Immunohistochemistry was done on sections from bronchial and transbronchial biopsies and on smears from bronchoalveolar lavage cells. In all three types of immune disorders, lymphocytes expressed the integrins alpha4/beta1 (VLA4) and ICAM3, whereas lymphocytes in COPD bronchitis and in emphysema controls were unreactive. Eosinophils in atopic asthma bronchitis in contrast to COPD bronchitis also expressed both VLA4 and ICAM3. The expression of VCAM1 on endothelial cells was only seen in atopic asthma and was related to disease activity. The expression of other adhesion molecules was nonspecific. Expression of VCAM1 on endothelial cells and its ligand VLA4 on lymphocytes and eosinophils seems to be a specific event in atopic asthma. Expression of VLA4 and ICAM3 on lymphocytes, however, might be a specific event in all three immune reactions.     

Rice as a model for centromere and heterochromatin research

Rice (Oryza sativa) has become an important model plant species in numerous research projects involving genome, molecular and evolutionary biology. In this review we describe the reasons why rice provides an excellent model system for centromere and heterochromatin research. In most multicellular eukaryotes, centromeres and heterochromatic domains contain long arrays of repetitive DNA elements that are recalcitrant to DNA sequencing. In contrast, three rice centromeres and the majority of the cytologically defined heterochromatin in the rice genome have been sequenced to high quality, providing an unparalleled resource compared to other model multicellular eukaryotes. Most importantly, active genes have been discovered in the functional domains of several rice centromeres. The centromeric genes and sequence resources provide an unprecedented opportunity to study function and evolution of centromeres and centromere-associated genes.