Effects of 5-bromo-2'deoxyuridine (BrdU) implants on hepatic cytochrome P-450 content and beta-oxidation activity in rats and mice.

Standard regulatory toxicity tests are frequently supplemented with additional compound specific analysis. Analysis of hepatic cytochrome P-450 content, hepatic beta-oxidation activity (biochemical analysis), and cell proliferation rates are examples of these analyses that are included when past experience or similarity to other compounds, suggest that a presently tested compound may have an effect. Until now, separate subsets of animals have been designated for cell proliferation analysis and biochemical analysis, because it was unknown if implantation of 5-bromo-2'deoxyuridine (BrdU) filled osmotic pumps (BrdU implants) would effect the rate of hepatic-beta or hepatic cytochrome P-450 content. The purpose of the current study was to determine if BrdU implants had an effect on hepatic cytochrome P-450 content, beta-oxidation activity, or the measurement of these enzymes in rats and mice. The BrdU was administered through subcutaneous osmotic pump implants. The rate of hepatic peroxisomal beta-oxidation was not altered in male or female rats or mice with the BrdU implants when compared to those of the control groups. The total hepatic cytochrome P-450 content was also not altered in male or female rats or mice with the BrdU implants when compared to those of the control groups. BrdU implants do not appear to have an effect on the rate of hepatic peroxisomal beta-oxidation or the total hepatic cytochrome P-450 content in male or female rats and mice. It can be concluded that in future studies, rats or mice which are designated for cell proliferation analysis using BrdU implants are also suitable for use in evaluating chemically induced effects on hepatic peroxisomal beta-oxidation activity and/or total hepatic cytochrome P-450 content.     

An Infinitely Expandable Cloning Strategy plus Repeat-Proof PCR for Working with Multiple shRNA

Vector construction with restriction enzymes (REs) typically involves the ligation of a digested donor fragment (insert) to a reciprocally digested recipient fragment (vector backbone). Creating a suitable cloning plan becomes increasingly difficult for complex strategies requiring repeated insertions such as constructing multiple short hairpin RNA (shRNA) expression vectors for RNA interference (RNAi) studies. The problem lies in the reduced availability of suitable RE recognition sites with an increasing number of cloning events and or vector size. This report details a technically simple, directional cloning solution using REs with compatible cohesive ends that are repeatedly destroyed and simultaneously re-introduced with each round of cloning. Donor fragments can be made by PCR or sub-cloned from pre-existing vectors and inserted ad infinitum in any combination. The design incorporates several cloning cores in order to be compatible with as many donor sequences as possible. We show that joining sub-combinations made in parallel is more time-efficient than sequential construction (of one cassette at a time) for any combination of 4 or more insertions. Screening for the successful construction of combinations using Taq polymerase based PCR became increasingly difficult with increasing number of repeated sequence elements. A Pfu polymerase based PCR was developed and successfully used to amplify combinations of up to eleven consecutive hairpin expression cassettes. The identified PCR conditions can be beneficial to others working with multiple shRNA or other repeated sequences, and the infinitely expandable cloning strategy serves as a general solution applicable to many cloning scenarios.     

Treatment of ostial lesions using the Szabo technique: A case series

We present a series of 13 cases of stent treatment of a variety of ostial lesions including aortoostial, subclavian‐internal mammary artery, and coronary artery bifurcation disease using the double‐wire Szabo technique. We discuss relevant technical issues using this technique as well as potential advantages and disadvantages. © 2008 Wiley‐Liss, Inc.     

Physician management of patients with adverse outcomes

The incidence of iatrogenic complications of medical or surgical care is increasing. Such adverse events may severely strain the patient/physician relationship. The nature of the underlying patient/physician relationship in terms of the style of interaction and the handling of uncertainty and decision making may influence the impact of adverse events on the relationship. Physicians may manage these situations poorly because of perceived threats to their self-image or sense of control, or because of fear of malpractice suits. Patients may consciously or subconsciously blame the physician, fall to express their concerns, or exaggerate their complaints. If possible, continuity of the patient/physician relationship should be maintained through full disclosure of all medical facts, frank and open communication, and a renewed commitment to the therapeutic relationship.     

Use of assessment instruments in clinical settings

In recent years, increased emphasis has been placed in the field of geriatric medicine on the need for multidimensional assessment of elderly patients. There is an increasing tendency to use standardized structured assessment instruments or questionnaires in this process. Frequently, the exact question or set of questions to be answered by the instrument has not been carefully analyzed. Although comprehensive structured multidimensional instruments allow the collection of large amounts of data in multiple domains (physical, social, psychologic, economic), these instruments may require excess data collection and may not be reliable in clinical settings. Clinicians considering using a variety of assessment instruments should carefully consider their goals for data collection and carefully review the validity, reliability, and population sampled for any assessment instruments under consideration. Also, the clinical setting in which the instrument is to be used can have a negative impact on either instrument validity or reliability.     

Invasive evaluation of left ventricular diastolic performance

Diastole can be divided into four phases: 1. isovolumic relaxation; 2. early filling; 3. diastasis; and 4. atrial systole. The amount of left ventricular (LV) filling that occurs during each of these phases depends on: 1. myocardial relaxation; 2. the passive characteristics of the LV; 3. the characteristics of the left atrium, pulmonary veins and mitral valve; and 4. the heart rate. When diastolic function is normal, the net effect of these factors results in LV filling sufficient to produce an adequate cardiac output, while mean pulmonary venous pressure is maintained below 12 mm Hg. Diastolic dysfunction is normally manifest as pulmonary congestion. In the absence of systolic dysfunction, abnormal diastolic performance is usually due to abnormal relaxation and/or changes in the passive LV characteristics, external compression or disease of the mitral valve and left atrium. Invasive studies can quantify the rate of myocardial relaxation from the time course of the fall of LV pressure during isovolumic relaxation and the passive LV properties from the LV diastolic pressure-volume relation. In addition, frame-by-frame analysis of contrast ventriculography and conductance determination of LV volume can quantify the pattern of LV diastolic filling. Normally, at rest, most LV filling occurs early in diastole. Conditions that produce diastolic dysfunction, such as LV hypertrophy and ischemia, are associated with reduced early diastolic filling and an augmented importance of atrial systole. It is important to recognize that such patterns can occur in patients without clinically apparent diastolic dysfunction and in normals if left atrial pressure is sufficiently elevated. Furthermore, a normal pattern can occur in patients with severe diastolic dysfunction. Reduced early diastolic filling in the absence of pulmonary congestion indicates the loss of diastolic reserve, since the left atrium is being used as a booster-pump. This pattern of diastolic filling in a patient with symptoms of pulmonary congestion suggests diastolic dysfunction, even if systolic LV performance is normal.     

Therapeutic benefits of ACE inhibitors and other antihypertensive drugs in patients with type 2 diabetes

OBJECTIVE: To assess whether ACE inhibitors are superior to alternative agents for the prevention of cardiovascular events in patients with hypertension and type 2 diabetes. RESEARCH DESIGN AND METHODS: This study is a review and meta-analysis of randomized controlled trials that included patients with type 2 diabetes and hypertension who were randomized to an ACE inhibitor or an alternative drug, were followed for > or =2 years, and had adjudicated cardiovascular events. RESULTS: A total of 4 trials were eligible. The Appropriate Blood Pressure Control in Diabetes (ABCD) trial (n = 470) compared enalapril with nisoldipine, the Captopril Prevention Project (CAPPP) (n = 572) compared captopril with diuretics or beta-blockers, the Fosinopril Versus Amlodipine Cardiovascular Events Trial (FACET) (n = 380) compared fosinopril with amlodipine, and the U.K. Prospective Diabetes Study (UKPDS) (n = 758) compared captopril with atenolol. The cumulative results of the first 3 trials showed a significant benefit of ACE inhibitors compared with alternative treatments on the outcomes of acute myocardial infarction (63% reduction, P < 0.001), cardiovascular events (51% reduction, P < 0.001), and all-cause mortality (62% reduction, P = 0.010). These findings were not observed in the UKPDS. The ACE inhibitors did not appear to be superior to other agents for the outcome of stroke in any of the trials. None of the findings were explained by differences in blood pressure control. CONCLUSIONS: Compared with the alternative agents tested, ACE inhibitors may provide a special advantage in addition to blood pressure control. The question of whether atenolol is equivalent to captopril remains open. Conclusive evidence on the comparative effects of antihypertensive treatments will come from large prospective randomized trials.     

Hypopituitarism following external radiotherapy for pituitary tumours in adults

The development of anterior pituitary hormone deficiencies has been studied in a group of 165 patients who underwent external radiotherapy for tumours of the pituitary or closely related anatomical sites, and who have been observed for up to 10 years. One hundred and forty had undergone pituitary surgery before radiotherapy. All patients received external radiotherapy by a three-field technique, giving 3750-4250 cGy in 15 or 16 fractions over 20-22 days. A combined test of anterior pituitary function using insulin hypoglycaemia or glucagon stimulation in conjunction with thyrotrophin and gonadotrophin releasing hormone tests and basal estimations of prolactin, thyroid hormones and testosterone or oestradiol was performed before radiotherapy. This was repeated six and 12 months later and subsequently annually. Before radiotherapy, 18 per cent of patients had normal growth hormone secretion, 21 per cent had normal gonadotrophin secretion, 57 per cent had normal corticotrophin reserve and 80 per cent had normal thyrotrophin secretion. Life table analysis demonstrated increasing incidences of all anterior pituitary hormone deficiencies with time: by five years all patients were growth hormone deficient, 91 per cent were gonadotrophin deficient, 77 per cent were corticotrophin deficient and 42 per cent were thyrotrophin deficient. At eight years, respective incidences of deficiencies were 100, 96, 84 and 49 per cent. Radiation-induced hyperprolactinaemia was seen in 73 patients; mean serum prolactin concentration rose from 227 +/- 11 mU/l to a peak of 369 +/- 60 mU/l at two years and subsequently declined towards the basal value. The primary diagnosis, patient age, sex, irradiated tissue volume and previous surgery were examined as variables that might influence the rate of development of anterior pituitary hormone deficiencies, but none of these factors had a significant effect. The radiation induced hyperprolactinaemia was however more marked in female patients. Although anterior pituitary hormone deficiencies most commonly developed in the order growth hormone, gonadotrophin, corticotrophin, thyrotrophin (61 per cent of patients), other sequences were evident. Most notably corticotrophin deficiency occurred before gonadotrophin deficiency. There is a high incidence of anterior pituitary hormone deficiencies in patients treated surgically for pituitary tumours and the incidence increases after external radiotherapy. Deficiencies may occur in an unpredictable sequence and endocrine testing is recommended on an annual basis.