Emr, an Escherichia coli locus for multidrug resistance.

An Escherichia coli chromosomal DNA fragment cloned on a multicopy plasmid conferred resistance to carbonylcyanide m-chlorophenylhydrazone, nalidixic acid, and a number of other toxic compounds. The sequence of the cloned emr locus located at minute 57.5 of the chromosome revealed two open reading frames, emrA and emrB. emrB encodes a highly hydrophobic 56.2-kDa peptide, with 14 potential alpha-helices to span the inner membrane. The peptide is homologous to QacA, a multidrug-resistant pump from Staphylococcus aureus, and belongs to a gene family that includes tetracycline-resistant pumps of Gram-positive bacteria and the galactose/H+ symporter of E. coli. emrA encodes a putative 42.7-kDa peptide containing a single hydrophobic domain and a large C-terminal hydrophilic domain. An active pho-fusion to the C domain suggested that EmrA is a membrane protein. Disruption of emrB significantly increased sensitivity of cells to uncouplers. The cellular content of uncoupler increased in the order: overexpressed emrB cells greater than wild type greater than emrB-.     

Effects of 6-mercaptopurine treatment on sperm production and reproductive performance: a study in male mice

OBJECTIVE: Azathioprine and 6-mercaptopurine interact in purine metabolism and DNA synthesis, thus their potential mutagenic effects have been of concern in the management of inflammatory bowel disease (IBD), especially in patients of childbearing age. Although several clinical studies have indicated their safety in both reproduction and pregnancy, in a recent large epidemiological study concerns were raised about their adverse effects in pregnant patients with IBD, and experimental or basic data on this subject are limited. The aim of this study was to investigate sperm production, sperm quality, and reproductive outcome following prolonged 6-MP administration to male mice. MATERIAL AND METHODS: Highly inbred Balb/c adult male mice were used. 6-MP at doses of 2, 5, and 8 mg/kg (n = 9 for each group) was given daily for 51 days and the treatment group was compared with controls. After 45 days of treatment, the mice were mated with females. Following 13 days of pregnancy, the products of conception were evaluated and live fetuses were examined for gross malformations. Sperm production and morphology were examined after 51 days of 6-MP administration. RESULTS: Treatment with 6-MP at all doses did not affect sperm morphology and sperm production in the testicular tubules, as compared with controls (70% normal sperm). However, pregnancy rates were inversely related to escalating doses of 6-MP: 55%, 41%, 28%, and 16% for control, 2, 5, and 8 mg/kg groups, respectively. Resorption rates (abortions) were 21% in the control group as compared with 45-50% in all the treatment groups, but the incidence of major congenital malformations was not increased. CONCLUSIONS: Long-term 6-MP treatment in male mice did not impair sperm production and sperm morphology. However, a significantly high rate of embryonic resorption indicated occult sperm damage. Thus, normal sperm analysis does not necessarily imply that sperm damage at genetic level did not occur. It is difficult to extrapolate from these results to the clinical use of 6-MP/azathioprine in IBD patients; however, further basic genetic testing for DNA damage and clinical follow-up are warranted.     

Chronic shoulder pain in the community: a syndrome of disability or distress?

Objectives: To investigate two questions in a community based population of people with chronic shoulder pain. Firstly, does chronic pain lead to impaired psychological health over time? Secondly, how does restriction of daily activity influence pain perception and psychological health? Methods: Two postal surveys, two years apart, were carried out to identify a group of subjects with chronic shoulder pain. The first survey was sent to a random sample of adults (n=40026) registered with a primary care practice, and included a pain manikin, demographic information, and the Hospital Anxiety and Depression scale (HAD). The second survey was sent to those subjects who reported unilateral shoulder region pain in the first survey and it included a shoulder-specific disability scale, pain severity score, and the HAD. Results: 2606 (65.1%) people responded to the initial survey. Of these, 304 (11.7%) reported unilateral shoulder region pain at baseline. In the subsequent survey, there were 234 responders (83.3% adjusted response): 142 of these reported shoulder pain and formed our study group of "subjects with chronic shoulder pain". Within this group there was no significant change in psychological distress scores between baseline and follow up. Both the disability score and psychological distress scores correlated significantly with pain severity (disability v pain r=0.536, p<0.001; psychological distress v pain r=0.269, p=0.002). When the correlation between disability and pain severity was corrected for possible confounders, it remained significant (r=0.490, p<0.001). This was not the case for the correlation between psychological distress and pain (p>0.05). Disability was significantly correlated with psychological distress on univariate (r=0.445, p<0.001) and multivariate analysis (r=0.341, p=0.002). Conclusion: In those with chronic shoulder pain the relation between pain and psychological health seems to be linked to disability. Psychological distress was not explained by persistent pain itself.     

Restricted production of interleukin 4 by activated human T cells.

Interleukin 4 (IL-4) is secreted by activated T cells and pleiotropically modulates both B- and T-lymphocyte function. In murine helper (CD4+) T-cell clones IL-4 production appears to be regulated independently of interferon gamma and interleukin 2. To determine whether production of these lymphokines is also differentially regulated in uncloned human T cells, we studied lymphokine production by normal human peripheral T cells and T-cell subsets after in vitro polyclonal activation. After maximal induction of lymphokine expression, IL-4 mRNA was detectable in less than 5% of CD4+ and 1-2% of unfractionated T cells, whereas approximately 33% and 60% of CD4+ cells expressed detectable mRNA for interferon gamma and interleukin 2, respectively. This finding correlated with dramatically lower production of IL-4 mRNA and protein than of interferon gamma and interleukin 2 by peripheral blood and tonsillar T cells. The helper-inducer (CD4+ CD45R-) T-cell subset, which significantly enhances in vitro immunoglobulin production, accounted for the preponderance of IL-4 mRNA accumulation and protein production by CD4+ T cells; nevertheless, cells with detectable IL-4 mRNA constituted less than 10% of the CD4+ CD45R- subset. Limitation of IL-4 production to a comparatively small population of normal human T cells could selectively regulate the effects of this lymphokine in T-cell-mediated immune responses; such selective regulation may be a fundamental mechanism for restricting the potentially pleiotropic effects of certain lymphokines to appropriate responder cells.     

Exercise ventilation after balloon dilatation of the mitral valve.

BACKGROUND--Exertional dyspnoea is a limiting symptom in many patients with mitral stenosis but its causes remain incompletely understood. Ventilation during exercise is abnormal in chronic heart failure of all causes and there is increased ventilatory cost of carbon dioxide production. PATIENTS--23 patients with rheumatic mitral stenosis undergoing percutaneous balloon dilatation of the mitral valve were studied to investigate exercise ventilation. METHODS--Treadmill exercise tests with respiratory gas analysis were performed before and 1 day, 7 days, and 10 weeks after balloon dilatation of the mitral valve. The relation between ventilation (VE) and production (VCO2) was analysed by linear regression. RESULTS--The VE/VCO2 slope was linear in all patients and before balloon dilatation of the mitral valve it correlated inversely with peak minute oxygen consumption (VO2) (rs = -0.47, P < 0.05), exercise duration (rs = -0.66, P < 0.01), and mitral valve area (rs = -0.5, P < 0.05). The VE/VCO2 slope declined acutely after balloon dilatation of the mitral valve (n = 10) (mean (SD) 41 (4) v 36 (2.9), P < 0.05) and did not change again thereafter. At 10 weeks (n = 23) exercise duration (460 (230) v 630 (240) s, P < 0.01) and peak VO2 (12.7 (4.3) v 14.9 (4.8) ml/kg/min, P < 0.05) increased significantly. CONCLUSIONS--Patients with rheumatic mitral stenosis have a similar increase in the VE/VCO2 slope to that of patients with heart failure from other causes. Successful balloon dilatation of the mitral valve is associated with an acute reduction in the exercise VE/VCO2 slope.     

Overproduction of the protein product of a nonselected foreign gene carried by an adenovirus vector.

We have constructed a recombinant adenovirus that carries the herpes simplex virus type I gene for thymidine kinase (EC 2.7.1.21) and expresses thymidine kinase under control of adenovirus major late promoter. A DNA fragment carrying thymidine kinase coding sequences but lacking the thymidine kinase promoter was sandwiched between a piece of adenoviral DNA and simian virus 40 early DNA on a plasmid. The aligned fragment was then inserted into the adenoviral genome, replacing internal adenoviral DNA. Hybrid viruses carrying the thymidine kinase gene were obtained by selecting for viruses that express simian virus 40 tumor antigen (T antigen) in monkey cells. The thymidine kinase gene was positioned in the third segment of the adenovirus tripartite leader downstream from the major late promoter by in vivo DNA recombination between the duplicated adenoviral sequences present in the plasmid insert and the viral vector. Levels of thymidine kinase activity in human or monkey cells infected with this hybrid virus were several times higher than in cells infected with herpes simplex virus. Infected cells produced thymidine kinase protein at very high levels, similar to those found for adenovirus late major capsid proteins. The thymidine kinase protein represented 10% of the newly synthesized protein in late infected cells and accumulated to represent 1% of total cell protein under optimal conditions. This vector system offers a procedure by which a variety of gene products that are biologically active and properly modified can be produced at high levels in mammalian cells.     

Echocardiography in the evaluation of patients in chest pain units

Using percutaneous angioplasty to induce the ischemic cascade in the cardiac catheterization laboratory, echocardiographic wall motion abnormalities have been documented to precede electrocardiographic abnormalities and angina. Therefore, detection of cardiac wall motion abnormalities is potentially more sensitive than the history, physical examination, and ECG for identification of myocardial ischemia. Echocardiography is highly reliable for assessing cardiac wall motion and, thus, it has been used for diagnosis and risk assessment in patients presenting to the emergency department (ED) with symptoms suggestive of myocardial ischemia. In patients who have acute ST-elevation myocardial infarction (MI), echocardiography is comparable to invasive left ventriculography for detecting wall motion abnormalities. However, the usefulness of echocardiography in the low-risk population that has chest pain of uncertain origin and a nondiagnostic initial presentation is less well established.     

The molecular mechanism of excitation in visual transduction and bacteriorhodopsin

An electronic theory of excitation is proposed and described in terms of a three-dimensional excited/ground-state energy surface which elucidates the photochemical and excited-state dynamics of rhodopsins. In this theory the primary action of light is to produce significant electron redistribution in the retinal, thereby generating new interactions that vibrationally excite and perturb the ground-state protein conformation. Thus, light energy causes charge redistribution in the retinal and induces transient charge-density assisted bond rearrangements (such as proton translocation) in the protein structure which is stabilized by subsequent retinal structural alteration. In this theory the isoprenoid chain of the retinal is considered a structurally pliable molecular entity that can generate charge redistributions and can be subsequently achieve intermediate conformations or various isomeric states to minimize the energy of the new protein structure generated by light. Thus, the 11-cis to all trans isomerization of the retinylidene chromophore is not considered a primary mechanism of excitation. An alternate biological role for this molecular process (which is eventually completed in all photoreceptors but not in bacterial rhodopsins) is to provide the irreversibility needed for effective quantum detection on the time scale of a neural response. Finally, it will be demonstrated that this mechanism, which readily accounts for the photophysical and photochemical data, can also be restated in terms of the Monod, Wyman, and Changeux terminology suggesting that aggregates of these pigments may function allosterically.