Subtraction of time-resolved magnetic resonance angiography images improves visualization of the pulmonary veins and left atrium in adults with congenital heart disease: a novel post-processing technique.

To describe a novel time-resolved magnetic resonance angiography (TR-MRA) postprocessing technique using the time-resolved angiography with interleaved stochastic trajectories (TWIST) method to evaluate the pulmonary veins and left atrium in adults with congenital heart disease undergoing cardiac MRI. Institutional ethics committee approved the study. 21 consecutive adult patients (14 female, 7 male patients, mean age 28 years) with known congenital heart disease who underwent a cardiac MRI were included. Post-processing of the TR-MRA sequences created novel "subtracted" datasets. Two independent observers reviewed the conventional TWIST and novel subtracted TWIST data sets in source and maximum intensity projection (MIP) coronal reformats to assess visualization of the pulmonary veins and left atrium based on a 5-point scale. Quantitative signal to noise (SNR) comparison was performed. TR-MRA yielded diagnostic image data in 20/21 patients (95.2%). The novel "subtracted" TR-MRA technique improved visualization of the pulmonary veins and left atrium compared to the source TR-MRA sequence in 16/20 patients (mean scores 3.34 ± 0.69 vs. 2.92 ± 0.69, p < 0.008). Further improved visualization of the pulmonary veins and left atrium was observed in the subtracted MIP TWIST sequences compared to the MIP TWIST images (mean scores 4.43 ± 0.80 vs. 3.02 ± 0.87 vs., p < 0.001). No significant SNR difference between the source and novel subtracted group was observed (85.4 vs. 70.4, p = 0.57). Compared to source TR-MRA images, subtraction of TR-MRA images is a novel postprocessing technique that improves visualization of the pulmonary veins and left atrium in a substantial number of patients.

The DIURNAL project: DIURNAL and circadian expression profiling, model-based pattern matching, and promoter analysis.

The DIURNAL project ( http://diurnal.cgrb.oregonstate.edu/ ) provides a graphical interface for mining and viewing diurnal and circadian microarray data for Arabidopsis thaliana, poplar, and rice. The database is searchable and provides access to several user-friendly Web-based data-mining tools with easy-to-understand output. The associated tools include HAYSTACK ( http://haystack.cgrb.oregonstate.edu/ ) and ELEMENT ( http://element.cgrb.oregonstate.edu/ ). HAYSTACK is a model-based pattern-matching algorithm for identifying genes that are coexpressed and potentially coregulated. HAYSTACK can be used to analyze virtually any large-scale microarray data set and provides an alternative method for clustering microarray data from any experimental system by grouping together genes whose expression patterns match the same or similar user-defined patterns. ELEMENT is a Web-based program for identifying potential cis-regulatory elements in the promoters of coregulated genes in Arabidopsis, poplar, and rice. Together, DIURNAL, HAYSTACK, and ELEMENT can be used to facilitate cross-species comparisons among the plant species supported and to accelerate functional genomics efforts in the laboratory.

Respirator proposal loses breath when applied to health care.

The debate continues. As Materials Management in Health Care went to press, the Occupational Safety and Health Administration was getting an earful about proposed revisions to its respiratory protection standard. The standard sets requirements for respirator use in the workplace and will be a basis for part of OSHA's proposed tuberculosis standard, which should be released later this year. Here are the American Hospital Association's comments on the proposed revisions.

Regulation of mitochondrial cAMP-dependent protein kinase activity in yeast.

We have shown that transcription of the yeast (S. cerevisiae) mitochondrial (mt) genome is cAMP-sensitive, via a mt cAMP-dependent protein kinase (cAPK). In relation to that work, we examined whether the BCY 1 gene product functions as regulatory subunit for mt cAPK, as it does for the cytoplasmic enzyme. We demonstrate that mt protein extracts from a bcy 1 strain show no cAPK activity, whereas similar extracts from an otherwise isochromosomal BCY 1 strain show high levels of such activity. Partial purification of mt cAPK from each strain confirms this difference. Photoaffinity labeling with 8-N3[32P]cAMP and highly-purified mt protein extracts from the BCY 1 strain identifies one cAMP-binding protein (M(r) approximately 47000), while similar mt extracts from the bcy 1 strain lack all cAMP-binding proteins. These data suggest that BCY 1 regulates yeast mt cAPK, and that inactivation of BCY 1 removes that mt activity from cAMP control.

Regulation of stringent mitochondrial transcription in yeast following amino-acid deprivation.

In the yeast Saccharomyces cerevisiae, a phenotypically identical stringent response is induced by either nutritional downshift or starvation for a required auxotrophic amino acid (aa); in each case, the response selectively includes transcriptional curtailment for the mitochondrial (mt) genome. We have shown previously that the downshift-induced mt stringent response is governed by changing cellular cyclic AMP (cAMP) levels, via a mt cAMP-dependent protein kinase. In contrast, we demonstrate here that cAMP levels are not altered in yeast following starvation for a required aa, and we use in vitro mt transcription assays with organelles from wild-type and mutant strains to confirm that the aa starvation-induced mt stringent response is not governed by cAMP. Rather, such stringent organellar transcriptional attenuation may result from altered availability of an unidentified small molecule which is probably a product of the cytoplasmic and/or mt protein synthesis systems.

Transcription of the yeast mitochondrial genome requires cyclic AMP.

Using various mutant strains and nutritional manipulations, we investigated a potential role for cyclic AMP (cAMP) in the regulation of mitochondrial (mt) gene expression in the yeast Saccharomyces cerevisiae. In RAS mutants known to have either abnormally low or high cellular levels of this nucleotide, we show that both mt transcription rate and overall mt transcript levels vary directly with cellular cAMP levels. We further show that nutritional downshift of actively growing cells causes a severe, rapid fall in cAMP levels, and that this fall is concomitant with the stringent mt transcriptional curtailment that we and others have previously shown to follow this nutritional manipulation. In in vitro mt transcription assays using intact organelles from downshifted and actively growing cells, stringently curtailed mt gene expression can be restored to 75% of control levels by addition of cAMP to the assay mix. Consistent with these observations a RAS2vall9 mutant strain, which cannot adjust cAMP levels in response to external stimuli, shows no mt stringent response following nutritional downshift. We also demonstrate a significant but transient increase in both mt transcript levels and mt transcription rate following shift of actively respiring wild-type cells to glucose-based medium, a manipulation known to cause a short-lived pulse of cAMP in yeast; similar manipulation of the RAS2vall9 mutant strain generates no such response. Taken together all these observations indicate that cellular cAMP levels are involved in the regulation of mt transcription in yeast. Moreover, the lack of a mt stringent transcriptional response following downshift in a strain in which the BCY1 gene had been insertionally inactivated suggests that cAMP may influence mt transcription via a mt cAMP-dependent protein kinase. These results link mt gene expression with mechanisms governing growth control and nutrient adaptation in yeast, and they provide a means by which mt gene expression might be coordinated with that of related nuclear genes.

Inapparent ocular infection by Chlamydia trachomatis in experimental and human trachoma.

There is substantial indirect evidence which suggests that Chlamydia trachomatis can generate inapparent, persistent infections in human. To confirm this directly, we examined ocular chlamydial infection in both the cynomolgus monkey model of trachoma and in patient samples from a trachoma-endemic area. In monkeys, ocular infection was studied over time using direct immunofluorescence cytology (DFA) and a molecular hybridization screening system which targets chlamydial ribosomal RNA. In eleven animals infected once with B serovar, DFA and probe screening of parallel conjunctival swabs gave congruent results through day 42 post-infection. Thereafter, DFA showed clearing of chlamydia and was negative by day 70, as in previous studies. In contrast, hybridization analysis indicated a continuing presence of chlamydial RNA in all samples from all animals through the end of the experiment at day 84 post-infection. Similarly, analysis of swabs from trachoma patients showed that a number of DFA-negative samples gave clear positive signal for chlamydial RNA. Taken together these data indicate that ocular chlamydial infection persists for longer periods than previously thought, judging solely on the basis of DFA, and they support the idea that inapparent ocular chlamydial infection occurs in vivo.

Regulation of mitochondrial transcription during the stringent response in yeast.

In yeast (S. cerevisiae) the stringent response is known to include rapid, selective, and severe transcriptional curtailment for genes specifying cytoplasmic rRNAs and r-proteins. We have shown that transcription of the mitochondrial 21S rRNA gene is also congruently and selectively curtailed during the yeast stringent response. Using an in vitro transcription assay with intact organelles from both rho+ and rho- strains, we show here that the mitochondrial stringent response includes not only transcription of the 21S and 16S rRNA genes, but also that of organellar genes specifying non-mitoribosome-related products. Stringent organellar transcriptional curtailment is identical when cells are starved for a required (marker) amino acid or when they are subjected to nutritional downshift, and the relative level of that transcriptional curtailment following either perturbation is the same in cells growing on fermentative (repressing) or purely respiratory carbon sources. These results confirm that the mechanism governing mitochondrial gene expression during a stringent response is specified outside the organelle, and they show that this transcriptional control mechanism is not immediately subject to glucose repression. In all strains examined, stringent organellar gene expression requires a mitochondrial promoter, suggesting that the regulatory mechanism which functions during the stringent response operates primarily at transcriptional initiation.

Mitochondrial rRNA-containing petite strains of yeast (Saccharomyces cerevisiae) show a normal nuclear-mitochondrial stringent response.

The nuclear-mitochondrial stringent response was examined in isonuclear rho+, 21S rRNA-containing rho-, and rho o strains of S. cerevisiae. By 30 min after nutritional downshift, nuclear rDNA transcription falls to 15% of control levels congruently in all strains, as assayed via whole-cell RNA or by hybrid selection of specific double-labeled transcripts. Both in vivo and in vitro, the mitochondrial stringent response is identical between the rho- strain and its parental rho+ strain, and in both, the kinetics and magnitude of the organellar response mirror those of the nuclear response. The data show that mitochondrial transcription and protein synthesis are not required for stringent regulation of either nuclear or mitochondrial rDNA transcription.

Preparation of RNA from unspheroplasted yeast cells (Saccharomyces cerevisiae).

High-quality RNA can be prepared from up to 100-ml culture volumes of unspheroplasted yeast cells (Saccharomyces cerevisiae) via homogenization in high-temperature phenol:chloroform mixtures. The yield of RNA from this preparative method is equivalent to those of other methods requiring preliminary spheroplasting of cells. Quality and quantity of recovered RNA are independent of yeast strain and cell growth medium used, and the method works equally well on cells in either log phase growth or in stationary phase. Mitochondrial RNAs recovered as part of whole cell RNA mixtures may be slightly degraded. Analyses of individual transcripts in the recovered RNA mixtures suggest that there is no selection for or against any specific single transcript or any group of transcripts when RNA is prepared by this method.

An audit of surgery in 100 patients over 75 years in a private hospital.

OBJECTIVE: To determine what operations are being done on patients over 75 years in a private hospital, to observe performance in terms of justification, morbidity and mortality. METHOD: Retrospective audit of 100 patients over 75 years having surgery at an Adelaide private hospital. RESULTS: Cystoscopy, inguinal herniorrhaphy, cataract and colon surgery were the most common procedures. One patient died--probably from a leaking aortic abdominal aneurysm. Four patients had significant morbidity in the form of burst abdomen, ruptured femoral-popliteal bypass graft, biliary collection requiring reoperation and cardiac arrhythmia requiring a pacemaker. Twenty three other patients had minor morbidity. No unnecessary operations were detected.

Tissue homogenization with sterile reinforced polyethylene bags for quantitative culture of Candida albicans.

A simple method of tissue homogenization with sterile reinforced polyethylene bags for quantitative fungal cultures was evaluated with mice infected with Candida albicans. This new method correlated well with standard methods (P less than or equal to 0.01) for quantifying viable fungus in homogenates of brain, kidney, spleen, liver, and lungs and may be applicable in clinical and experimental mycology laboratories.

The management of patients with acute myocardial infarction after successful reperfusion with streptokinase.

Intracoronary streptokinase administration has been an effective procedure for establishing reperfusion of an evolving myocardial infarction by lysing the thrombus that is usually responsible for the infarction. After reperfusion is accomplished, appropriate management of the patient must be planned to provide the best chance for assuring continued vessel patency, and appropriate management of the patient's residual coronary artery disease also must be considered. In selected patients, percutaneous transluminal coronary angioplasty of the residual coronary lesion has been performed successfully immediately following reperfusion with streptokinase. Early coronary artery bypass graft surgery has been performed with good results in other patients. The appropriate management of the patient with acute myocardial infarction is still evolving, and only with additional study and experience will the "best" approach in the management of these patients be defined.