Purification of cardiac myocytes from human heart biopsies for gene expression analysis.

The collection of gene expression data from human heart biopsies is important for understanding the cellular mechanisms of arrhythmias and diseases such as cardiac hypertrophy and heart failure. Many clinical and basic research laboratories conduct gene expression analysis using RNA from whole cardiac biopsies. This allows for the analysis of global changes in gene expression in areas of the heart, while eliminating the need for more complex and technically difficult single-cell isolation procedures (such as flow cytometry, laser capture microdissection, etc.) that require expensive equipment and specialized training. The abundance of fibroblasts and other cell types in whole biopsies, however, can complicate gene expression analysis and the interpretation of results. Therefore, we have designed a technique to quickly and easily purify cardiac myocytes from whole cardiac biopsies for RNA extraction. Human heart tissue samples were collected, and our purification method was compared with the standard nonpurification method. Cell imaging using acridine orange staining of the purified sample demonstrated that >98% of total RNA was contained within identifiable cardiac myocytes. Real-time RT-PCR was performed comparing nonpurified and purified samples for the expression of troponin T (myocyte marker), vimentin (fibroblast marker), and alpha-smooth muscle actin (smooth muscle marker). Troponin T expression was significantly increased, and vimentin and alpha-smooth muscle actin were significantly decreased in the purified sample (n = 8; P < 0.05). Extracted RNA was analyzed during each step of the purification, and no significant degradation occurred. These results demonstrate that this isolation method yields a more purified cardiac myocyte RNA sample suitable for downstream applications, such as real-time RT-PCR, and allows for more accurate gene expression changes in cardiac myocytes from heart biopsies.

Identification of predicted human outer dynein arm genes: candidates for primary ciliary dyskinesia genes.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a severe inherited disorder characterised by chronic respiratory disease, male infertility, and, in approximately 50% of affected individuals, a left-right asymmetry defect called situs inversus. PCD is caused by defects in substructures of the ciliary and flagellar axoneme, most commonly loss of the outer dynein arms. Although PCD is believed to involve mutations in many genes, only three have been identified. METHODS: To facilitate discovery of new PCD genes, we have used database searching and analysis to systematically identify the human homologues of proteins associated with the Chlamydomonas reinhardtii outer dynein arm, the best characterised outer arm of any species. RESULTS: We find that 12 out of 14 known Chlamydomonas outer arm subunits have one or more likely orthologues in humans. The results predict a total of 24 human genes likely to encode outer dynein arm subunits and associated proteins possibly necessary for outer arm assembly, plus 12 additional closely related human genes likely to encode inner dynein arm subunits. CONCLUSION: These genes, which have been located on the human chromosomes for easy comparison with known or suspected PCD loci, are excellent candidates for screening for disease-causing mutations in PCD patients with outer and/or inner dynein arm defects.

Iron metabolism in mammalian cells.

Most living things require iron to exist. Iron has many functions within cells but is rarely found unbound because of its propensity to catalyze the formation of toxic free radicals. Thus the regulation of iron requirements by cells and the acquisition and uptake of iron into tissues in multicellular organisms is tightly regulated. In humans, understanding iron transport and utility has recently been advanced by a "great conjunction" of molecular genetics in simple organisms, identifying genes involved in genetic diseases of metal metabolism and by the application of traditional cell physiology approaches. We are now able to approach a rudimentary understanding of the "iron cycle" within mammals. In the future, this information will be applied toward modulating the outcome of therapies designed to overcome diseases involving metals.

Physical and neuromotor development of progeny of pyridoxine-restricted rats cross-fostered with control or isonutritional dams.

Female rats were fed diets containing graded levels of pyridoxine throughout gestation. At parturition, the pups were cross-fostered with a dam which had been fed the same diet as the biological mother throughout gestation (an isonutritional foster mother), or with one which had received a control diet containing 400% of the National Research Council recommendations for B6 throughout gestation (a control foster mother), or were left to suckle the biological mother. Physical, neuromotor, and reflexological development of the pups was assessed throughout the 3 week lactation period. There were no significant differences between pups nursed by their biological mothers and by isonutritional foster mothers. Growth, reflex acquisition, and neuromotor activities were impaired in pups subjected to pyridoxine restriction during gestation. Acquisition of a number of reflexes and time spent in neuromotor activities were responsive to cross-fostering with a control foster mother but the deficits were not completely eliminated. Growth was only slightly affected by cross-fostering and acquisition of audicular startle and negative geotaxis was not affected by the nature of the foster mother. The latter reflexes are apparently determined directly by the gestational nutrition of the fetus whereas other reflexes and neuromotor activities are influenced by long-term effects of pyridoxine restriction on the postnatal performance of the dam as a mother, in addition to being directly influenced by maternal diet during gestation.

The physical and neuromotor development of progeny of female rats fed graded levels of pyridoxine during lactation.

Female rats were maintained throughout gestation on a control diet containing 400% of the National Research Council (NRC) recommendation for pyridoxine and transferred to purified diets containing 400, 100, 75, 50, 25, or 0% of the NRC recommendations for B6 during lactation. Physical development of the offspring was assessed by measuring body weight and the occurrence of physical features, for example: eye opening, eruption of incisors, and growth of the hair to obscure the genitalia. Development of reflex reactions, neuromotor skills and coordination were also observed. Grooming was assessed as a measure of spontaneous activity. Retardation of growth, delay in the onset of reflexes such as palmar grasp, vibrissa placing, visual placing, and audicular startle, and a delay in the onset of advanced neuromotor coordination such as standing and grooming were observed in the pups of pyridoxine-restricted dams. Even progeny of dams, receiving 100% of the NRC recommendations of B6 during lactation exhibited inferior performance of skills requiring advanced neuromotor coordination such as standing, either supported or alone. These animals had weight gains comparable to neonates of maternal animals receiving 400% of the NRC recommendations for pyridoxine. The importance of factors other than growth in the assessment of nutritional requirements is demonstrated.

Maternal diet and brain fatty acids in young rats.

In order to determine to what extent maternal diet influenced the brain lipids of young rats, female rats were maintained on diets differing in fatty acid composition. Fatty acid determinations on the total brain lipids of the young from these dams indicated that the maternal dietary lipids influence the polyunsaturated fatty acid composition of these animals. A maternal diet with a high linoleic-linolenic acid ratio (corn oil) resulted in lower levels of 22ratio6omega3 and higher levels of 22ratio5omega6 than one with a low linoleic-linolenic acid ratio (grain). Transfer of young rats at birth to a foster mother, which was fed a diet differing from that of the natural dam, resulted in brain polyunsaturated fatty acid patterns at weaning similar to those of the natural young, and suckling, of the foster mother, thus indicating that the maternal diet in the immediate postnatal period can modify the brain lipids of young rats prior to weaning. The brain lipids of young rats from dams which were fed corn oil exhibited a marked tendency to incorporate 22ratio6omega3 in the immediate postnatal period in spite of a relatively high linoleic-linolenic acid ratio in the milk.

Cholesteryl ester hydrolase activity in adrenal homogenates from normal and essential fatty acid-deficient female rats.

Cholesteryl ester hydrolase was assayed in adrenal homogenates from mature female rats fed a control (corn oil-containing) or essential fatty acid (EFA)-deficient diet. Cholesteryl ester of 16:0, 18:0, 18:1, 18:2(n-6), 20:4(n-6) and 22:4(n-6) were used as substrates. In control rats, the unsaturated esters were hydrolyzed more rapidly than the saturated esters and cholesteryl arachidonate was the preferred substrate of the six investigated; cholesteryl oleate elicited the highest activity in the deficient group. Polyunsaturated esters were hydrolyzed at a significantly lower rate by homogenates from EFA-deficient rats than by those from control animals. The esters of 18:1, 18:2(n-6) and 20:4(n-6) were hydrolyzed more extensively in relation to their concentrations in adrenal tissue than were cholesteryl esters of 16:0, 18:0 and 22:4(n-6). This difference was more pronounced in control than in EFA-deficient rats. No simple relationship of adrenal cholesteryl ester hydrolase activity to ester fatty acid structure or to nutritional essentiality was evident.

The circadian cycles of plasma corticosterone and adrenal cholesteryl esters in the normal and EFA-deficient female rat.

UNLABELLED: In female rats subjected to a 12 hr light-12 hr darkness schedule and fed a semipurified diet containing 10% corn oil, plasma corticosterone concentration showed a monophasic circadian cycle with minimum and maximum concentrations at the start of the light and dark periods, respectively. Adrenal total cholesteryl ester concentration was inversely related to plasma corticosterone, as were those of several of the individual esters; changes in cholesteryl ester concentration appeared to follow rather than precede changes in plasma corticosterone. There was preferential depletion of the cholesteryl esters of 18:1, 18:2omega6, and 20:4omega6 during glucocorticoid secretion. [ ABBREVIATIONS: EFA, essential fatty acid (s);X:YomegaZ, fatty acid with X carbon atoms and Y olefinic bonds with the terminal double bond Z carbon atoms from the methyl group.] In female rats fed hydrogenated coconut oil (EFA-deficient), a monophasic cycle for plasma corticosterone was also observed, but the peak was much broader than that recorded for rats fed corn oil, although minima and maxima occurred at similar times for the two groups. No significant cycle of adrenal total cholesteryl esters was evident in the deficient rats, but the 20:3omega9 and 22:3omega9 esters did decrease significantly during the period of high plasma corticosterone concentration. Preferential net decreases in adrenal cholesteryl esters during corticosteroidogenesis were more apparent in normal than in EFA-deficient rats.

Effect of dietary vitamin C on adrenal cholesteryl ester content in the guinea pig.

Male guinea pigs fed a vitamin C-deficient diet for 3 weeks had lower concentrations of cholesteryl esters in their adrenals than did control animals fed the recommended intake of the vitamin. Not all esters were affected to the same degree, and the fatty acid profiles of the esters from control and deficient guinea pigs differed; there was proportionately more palmitic and linoleic acids and less docosatetraenoic acid [22:4 (n-6)] in the deficient guinea pig adrenal esters. [Fatty acids are designated as X:Y (n-Z), where X and Y are the numbers of carbon atoms and olefinic bonds in the acid and Z is the number of carbon atoms after the terminal olefinic bond.] A 100-fold excess of vitamin C in the diet also resulted in lower concentrations of adrenal cholesteryl esters than did the control diet, but they were not as low as in the deficient animals. Fatty acid profiles were similar for esters from control and excessively supplemented guinea pigs. Vitamin C deficiency apparently imposes a long term stress which results in a depletion of adrenal cholesteryl esters, possibly specific esters, to meet the requirements for glucocorticoid synthesis.

Cardiopathogenicity of rapeseed oils and oil blends differing in erucic, linoleic, and linolenic acid content.

Male Wistar rats were fed semipurifed diets containing 20% fat for 25 weeks. Ten different oils or oil blends were employed, including rapessed oils, simulated rapeseed-type oils, and modified rapeseed-type oils. Safflower, soybean, and hydrogenated coconut oils served as control oils. Histopathological examination of the cardiac tissue was conducted at the end of the study and an incidenceseverity rating assigned to the lesions induced by each fat. Oils containing high levels of erucic acid (26-30%) induced the most severe cardiac necrosis, irrespective of the source of erucic acid (rapeseed oil or nasturtium oil). Increasing the linoleic: :linolenic acid ratio of the high erucic oils to that of soybean oil failed to reduce necrosis, but the absence of linolenic acid from a high erucic acid oil blend resulted in a markedly reduced lesion incidenceseverity rating, comparable to those obtained for low erucic acid rapessed oil and soybean oil which were similar. Lowest lesion incidence was obtained with safflower oil and hydrogenated coconut oil. We have postulated that linolenic acid plays a role in the etiology of cardiac necrosis observed when rats are fed diets containing low erucic acid rapeseed oils.

Does emotional expression make a difference in reactions to breast cancer?

PURPOSE/OBJECTIVES: To examine the feasibility of using an emotional expression intervention with patients with cancer and test the hypothesis that emotional expression improves psychosocial adjustment. DESIGN: Sequentially randomized pretest/post-test design with repeated measures. SETTING: Two radiation therapy (RT) facilities. SAMPLE: Women completing RT for stage I or II breast cancer, who spoke and read English, were independent in self-care, and provided written consent. Subjects (N = 44) were middle-aged (mean = 53.6 years), Caucasian, married, and well educated. METHODS: Following a baseline interview, subjects were sequentially randomized to an attentional control group, a single dose, or a three-dose emotional expression writing group. Interventions were administered at the time of completion of RT. Follow-up telephone interviews were completed at 1, 4-6, 16, and 28 weeks post-RT. MAIN RESEARCH VARIABLES: Positive and negative affect, intrusiveness of thoughts, use of avoidant coping, side effect severity, trait negative affectivity, content of written essay, and themes derived from content analysis. FINDINGS: A high level of acceptance and completion of emotional expression existed, but no effect of the intervention on psychosocial adjustment was evident. Process measures in the three-dose group changed as expected. No relationship existed between content changes and outcome measures. CONCLUSIONS: Emotional expression is feasible for patients with cancer, but the efficacy of the intervention in improving mood and decreasing cognitive intrusion and avoidance was not supported. Emotional expression processes were consistent with those seen in other samples and may influence outcomes that were not addressed in this study. IMPLICATIONS FOR NURSING PRACTICE: More extensive testing is needed, including additional outcome variables. Essays reveal concerns around communication, recurrence, and health behavior changes that should be considered in practice.

Concerns, affect, and cognitive disruption following completion of radiation treatment for localized breast or prostate cancer.

PURPOSE/OBJECTIVES: To describe concerns, cognitive disruption, and affect following the completion of radiation treatment (RT) for localized breast or prostate cancer. DESIGN: Cross-sectional, descriptive. SETTING: Two RT centers associated with a university hospital. SAMPLE: Patients who completed RT for prostate (n = 9) or breast cancer (n = 11). The mean age of the sample is 64 years. Ninety percent are Caucasian, 80% are married, 50% are of Latter-day Saint faith, and 50% are retired. All had early stage disease, with a mean of 20 months post-RT. METHODS: One-time interview using qualitative methods and the Life Orientation Test, Revised Impact of Events Scale, state scale of the Positive and Negative Affect Schedule, satisfaction items of the Sarason Social Support Questionnaire, and Side Effect Severity Checklist. MAIN RESEARCH VARIABLES: Frequency of intrusive thoughts or avoidant behavior, positive and negative affect, dispositional optimism, satisfaction with social support, severity of side effects, and themes extracted from semistructured interviews. FINDINGS: Most subjects had unexpected intrusive thoughts about cancer; more than one-third avoided reminders of cancer. The level of positive affect was high, as was optimism. The level of negative affect was low. Subjects' satisfaction level with social support was high. Fatigue was the most frequently reported continuing side effect. Relative optimism, acceptance, vigilance, and trust of healthcare providers were major themes. Avoidance, comparison, maintaining normalcy, and information seeking were common coping strategies. CONCLUSIONS: Subjects perceived themselves to be doing well. Fatigue was the most common long-term side effect. Subjects continued to seek information. Symptom vigilance and interpretation were important, as were having a positive outlook and maintaining normalcy. IMPLICATIONS FOR NURSING PRACTICE: Nurses must acknowledge the potential for concerns about symptoms and continuing side effects and what they mean for the patient. Clinicians need to provide accurate information about common post-treatment experiences. Clinicians also must remember that subjects have vivid recollections of both positive and negative interactions with healthcare providers.

Planning and conducting a multi-institutional project on fatigue.

PURPOSE/OBJECTIVES: To describe the process used in proposal development and study implementation for a complex multisite project on cancer treatment-related fatigue (CRF), identify strategies used to manage the project, and provide recommendations for teams planning multisite research. DATA SOURCES: Information derived from project team meeting records, correspondence, proposals, and personal recollection. DATA SYNTHESIS: The project was built on preexisting relationships among the three site investigators who then built a team including faculty, research coordinators, staff nurses, and students. Study sites had a range of organizational models, and the proposal was designed to capitalize on the organizational and resource strengths of each setting. Three team members drawn from outside oncology nursing provided expertise in measurement and experience with fatigue in other populations. Planning meetings were critical to the success of the project. Conference calls, fax technology, and electronic mail were used for communication. Flexibility was important in managing crises and shifting responsibility for specific components of the work. The team documented and evaluated the process used for multisite research, completed a major instrumentation study, and developed a cognitive-behavioral intervention for CRF. CONCLUSIONS: Accomplishments during the one-year planning grant exceeded initial expectations. The process of conducting multisite research is complex, especially when the starting point is a planning grant with specific research protocols to be developed and implemented over one year. Explicit planning for decision-making processes to be used throughout the project, acknowledging the differences among the study settings and planning the protocols to capitalize upon those differences, and recruiting a strong research team that included a member with planning grant and team-building expertise were essential elements for success. IMPLICATIONS FOR NURSING PRACTICE: Specific recommendations for others planning multisite research are related to team-building, team membership, communication, behavioral norms, role flexibility, resources, feedback, problem management, and shared recognition.

Ultrasound treatment for breast engorgement: A randomised double blind trial.

The aim of this study was to test the efficacy of thermal ultrasound therapy as a treatment for severe post partum breast engorgement. A randomised double-blind, placebo-controlled trial was conducted on 197 breasts. Two ultrasound machines of identical appearance were used. One was working normally, the other had the crystal replaced with a resistor producing surface heat only. Assessment of effectiveness was subjective, using visual analogue scales for pain and hardness, and objective, using tonometry. Results indicate that both the true and sham machines were effective in reducing subjectively perceived pain and hardness. However, the results of this study show that the effect cannot be attributed to the ultrasound component.

Treatments to promote colonic hydration: enteral fluid therapy versus intravenous fluid therapy and magnesium sulphate.

Although large intestine impactions are commonly treated with i.v. fluids combined with the osmotic laxative MgSO4, enteral fluids are less expensive and also appear to be efficacious for impactions. Therefore, this study was conducted to compare the systemic and gastrointestinal effects of enteral fluids with the changes produced by i.v. fluids combined with MgSO4. Four horses with a fistula in the right dorsal colon alternately received both treatments in 2 periods one week apart. Sixty litres of fluids were administered continuously (10 l/h) through a venous catheter or a nasogastric tube. Magnesium sulphate (1 g/kg bwt) was administered via nasogastric tube before i.v. fluid therapy. Two horses had mild abdominal discomfort at the end of enteral fluid therapy. Pollakiuria, hypostenuria, increased bodyweight, increased faecal and ingesta hydration, and decreased PCV, plasma protein and plasma magnesium were produced by both treatments. Abdominal distention and more pronounced changes in bodyweight and ingesta hydration were seen with enteral fluids. Intravenous fluids plus MgSO4 produced hypocalcaemia and more pronounced changes in plasma protein. These results indicate that enteral fluid therapy is more effective in promoting ingesta hydration and produces less pronounced systemic effects than i.v. fluid therapy plus MgSO4.

Computer analysis of qualitative data: a comparison of three packages.

This article describes and compares three computer programs designed to assist in the analysis of narrative text. Ethnograph, GATOR, and Martin were compared in the context of a qualitative study of women and their husbands following mastectomy in order to (a) describe the fit between research purpose and program capabilities and (b) explore the influence, if any, of particular program features on research methods or analysis. The comparison suggests that methodological assistance provided by the programs involves differences in approach sufficient to make prior consideration of the fit between program capabilities and research purposes, methods, and style an important research decision. Descriptions of the programs and their application provide guidelines for comparison of these and other computer programs.