Assays to measure nanomolar levels of the renin inhibitor CGP 38 560 in plasma

A radioinhibitor binding assay and an enzyme inhibition assay have been developed to measure plasma levels of CGP 38 560, a potent human renin inhibitor. The detection limit of the assays was between 0.5 and 1 pmol/ml. There was a good correlation (r = 0.989) between the two assays for the measurement of human plasma spiked with CGP 38 560 in concentrations from 1.9 nM to 12 microM. Intra-assay variability was 6.1-17.3% and 4.4-27.2% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Interassay variability was 6.0-28.2% and 3.8-28.4% for the radioinhibitor binding assay and the enzyme inhibition assay, respectively. Blood samples were collected during a pharmacological study performed in normotensive human volunteers on an unrestricted diet who were infused during a 30-minute period with CGP 38 560 A (50 micrograms/kg). Similar values for the concentrations of renin inhibitor in plasma were obtained with the radioinhibitor binding assay and the enzyme inhibitor assay, and there was a significant correlation between values obtained with the two different methodologies (r = 0.94). The plasma levels of renin inhibitor reached a maximum at the end of infusion and then decreased rapidly, indicating a short plasma half-life. The changes in biochemical parameters, plasma renin activity, and plasma concentration of active renin could be related to the concentrations of CGP 38 560 measured in the plasma.     

The menstrual cycle does not affect human immunodeficiency virus type 1 levels in vaginal secretions.

To determine whether the menstrual cycle affects human immunodeficiency virus (HIV) type 1 levels in vaginal secretions, vaginal lavage samples were collected at 7, 14, and 21 days after initiation of menses, to compare virus levels during the follicular, ovulatory, and luteal phases. During 33 menstrual cycles in 25 women, HIV-1 RNA levels in vaginal secretions ranged from <1000 to 5.3x10(7) copies per lavage, and weekly changes ranged from <0.5 to 2.5 log(10) copies per lavage. HIV-1 RNA levels in vaginal lavage samples from days 7, 14, and 21 were not significantly different. No discernible pattern was found in changes of vaginal virus loads (VVLs) during the menstrual cycle. VVLs were not correlated with plasma estradiol or progesterone levels (P>.05). These results suggest that hormonal changes during the menstrual cycle do not have a significant effect on HIV-1 RNA levels in vaginal secretions.     

Most asthmatics have gastroesophageal reflux with or without bronchodilator therapy

The relationship between gastroesophageal reflux and asthma has not been clearly defined. We measured the lower esophageal sphincter pressures and studied gastroesophageal reflux patterns over 24 hours using an ambulatory Gastroreflux Recorder (Del Mar Avionics, Irvine, CA) in 44 controls and 104 consecutive adult asthmatics. The presence or absence of reflux symptoms was not used as a selection criterion for asthmatics. All asthmatics had discrete episodes of diffuse wheezing and documented reversible airway obstruction of at least 20%. Patients underwent reflux testing while receiving, if any, their usual asthmatic medications: 71.2% required chronic bronchodilators and 28.8% required no bronchodilators. Compared with controls, asthmatics had significantly decreased lower esophageal sphincter pressures, greater esophageal acid exposure times, more frequent reflux episodes, and longer clearance times in both the upright and supine positions (P less than 0.0001 for all parameters tested). There were no differences in any of the measured reflux parameters between asthmatics who required bronchodilators and those who did not. Thus, the decreased lower esophageal sphincter pressures and increased levels of acid reflux in asthmatics were not entirely caused by the effects of bronchodilator therapy. Receiver-operating characteristic analysis generated reflux values that discriminated asthmatics from controls. More than 80% of adult asthmatics have abnormal gastroesophageal reflux. We conclude that most adult asthmatics, regardless of the use of bronchodilator therapy, have abnormal gastroesophageal reflux manifested by increased reflux frequency, delayed acid clearance during the day and night, and diminished lower esophageal sphincter pressures.     

Hospital outcome of acute myocardial infarction in patients with and without diabetes mellitus.

AIMS: To assess hospital mortality and morbidity in diabetic and non-diabetic patients with acute myocardial infarction and to compare the results between the two groups. METHODS: All patients admitted in 1999 to the intensive care unit of the Schwabing City Hospital with diagnosis of acute myocardial infarction were assessed for hospital mortality and co-morbidity. RESULTS: Three hundred and thirty patients with acute myocardial infarction were admitted. Of those, 126 (38%) were diabetic and 204 (62%) were non-diabetic patients. Mortality within 24 h after admission was 13.5% in diabetic patients and 5.4% in non-diabetic patients (P<0.01). Mortality during entire hospitalization was higher in diabetic than in non-diabetic patients (29.4% vs. 16.2%; P=0.004). Diabetic patients were resuscitated more frequently than non-diabetic patients (24% vs. 11%, P<0.01). In diabetic patients, heart rate at admission was increased (91 +/- 27 vs. 82 +/- 23/min; P<0.01) and presence of angina pectoris was reported less frequently (59% (n=72) vs. 82% (n=167); P<0.001). Preceding myocardial infarction, microalbuminuria, peripheral artery disease and arterial hypertension were more frequent in diabetic than in non-diabetic patients. Diabetic patients demonstrated higher C-reactive protein (CRP) levels than non-diabetic patients (91.4 +/- 78.2 mg/l vs. 45.2 +/- 62.4 mg/l; P<0.001). CONCLUSIONS: In diabetic patients with acute myocardial infarction, early hospital mortality is increased and signs of cardiac autonomic dysfunction and microangiopathy are detected more frequently than in non-diabetic patients. The need for advanced treatment strategies early in the course of diabetic patients with myocardial infarction is emphasized.     

Near-infrared indocyanine green videoangiography (ICGVA) and intraoperative computed tomography (iCT): are they complementary or competitive imaging techniques in aneurysm surgery?

Background

In this pilot study we compared advantages and drawbacks of near-infrared indocyanine green videoangiography (ICGVA) and intraoperative computed tomography (iCT) to investigate if these are complementary or competitive methods to acquire immediate information about blood vessels and potential critical impairment of brain perfusion during vascular neurosurgery.

Methods

A small subset of patients (n?=?10) were prospectively enrolled in this feasibility study and received ICGVA immediately after placement of the aneurysm clips. An intraoperative cranial CT angiography (iCTA) was followed by dynamic perfusion CT scan (iCTP) using a 40-slice, sliding-gantry, CT scanner. The vascular patency of major (aneurysm bearing) arteries, visualisation of arising perforating arteries and brain perfusion after clip application were analysed with both techniques.

Results

The ICGVA was able to visualise blood flow and vascular patency of all major vessels and perforating arteries within the visual field of the microscope, but failed to display vessels located within deeper areas of the surgical field. Even small coverage with brain parenchyma impaired detection of vessels. With iCTA high image quality could be obtained in 7/10 cases of clipped aneurysms. Intraoperative CTA was not sufficiently evaluable in one PICA aneurysm and one case of a previously coiled recurrent aneurysm, due to extensive coil artefacts. Small, perforating arteries could not be detected with iCTA. Intraoperative CTP allowed the assessment of global blood flow and brain perfusion in sufficient quality in 5/10 cases, and enabled adequate intraoperative decision making.

Conclusion

A combination of ICGVA and iCT is feasible, with very good diagnostic imaging quality associated with short acquisition time and little interference with the surgical workflow. Both techniques are complementary rather than competing analysing tools and help to assess information about local (ICGVA/iCTA) as well as regional (iCTA/iCTP) blood flow and cerebral perfusion immediately after clipping of intracranial aneurysms.     

An essential role for chloroplast heat shock protein 90 (Hsp90C) in protein import into chloroplasts

Chloroplast heat shock protein 90 (Hsp90C) represents a highly conserved subfamily of the Hsp90 family of molecular chaperones whose function has not been defined. We identified Hsp90C as a component that interacts with import intermediates of nuclear-encoded preproteins during posttranslational import into isolated chloroplasts. Hsp90C was specifically coprecipitated with a complex of protein import components, including Tic110, Tic40, Toc75, Tic22, and the stromal chaperones, Hsp93 and Hsp70. Radicicol, an inhibitor of Hsp90 ATPase activity, reversibly inhibited the import of a variety of preproteins during translocation across the inner envelope membrane, indicating that Hsp90C functions in membrane translocation into the organelle. Hsp90C is encoded by a single gene in Arabidopsis thaliana, and insertion mutations in the Hsp90C gene are embryo lethal, indicating an essential function for the chaperone in plant viability. On the basis of these results, we propose that Hsp90C functions within a chaperone complex in the chloroplast stroma to facilitate membrane translocation during protein import into the organelle.The function of chloroplasts in photosynthesis, and the metabolism of amino acids, lipids, and secondary metabolites, positions them at the central hub of plant metabolism and signaling (1). Chloroplast biogenesis and function rely on the coordinate expression of genes from both the plastid and nuclear genomes, and several thousand nuclear-encoded proteins are imported into the organelle following their synthesis on cytoplasmic ribosomes. The majority of these proteins are synthesized as preproteins with an amino-terminal cleavable targeting signal, the transit peptide, which directs their import into chloroplasts through multiprotein membrane complexes or translocons located in the outer (TOC) and inner (TIC) chloroplast envelope membranes (2, 3). During or shortly after transport through the TOC and TIC translocons, proteins fold and assemble or engage secondary targeting pathways within the organelle that mediate sorting and insertion to the inner envelope and thylakoid membrane systems (4).The targeting and translocation of preproteins at the chloroplast envelope is facilitated by a variety of molecular chaperones (5). Cytosolic molecular chaperones, Hsc70 and Hsp90, have been proposed to assist in delivery of preproteins to the TOC receptors, Toc34 and Toc159, and the intrinsic GTPase activities of the receptors initiate outer membrane translocation via the β-barrel membrane channel, Toc75 (2, 3). Preprotein translocation occurs simultaneously through the TOC and TIC complexes with the assistance of Tic22, a chaperone in the intermembrane space (6, 7), and the putative channel components, Tic20 and Tic21 (2, 3). The TIC machinery interacts with three ATP-driven molecular chaperones in the stroma (3, 5). Members of the Hsp70 family, cpHsp70, and the Hsp100 AAA+ (ATPases associated with various cellular activities) family, Hsp93, associate with the TIC translocon in the stroma to facilitate import.Two functionally overlapping genes encode both cpHsp70 (cpHsp70-1 and -2) and Hsp93 (Hsp93-III and -V) in Arabidopsis. Mutants with reduced or altered cpHsp70 or Hsp93 activity exhibit reduced levels of protein import and marked defects in chloroplast biogenesis (8–11), leading to proposals that both chaperones function as molecular motors for preprotein translocation across the envelope. The chloroplast also contains an Hsp60/GroEL chaperonin family member, cpn60, which is proposed to function in the folding of newly imported preproteins downstream of cpHsp70 and Hsp93 (12). All three chaperone systems have been shown to directly or indirectly immunoprecipitate with the major TIC component, Tic110 (10, 12–14). Tic110 also contains a docking site for preproteins at the stromal side of the inner membrane (15). At least one cochaperone, the integral inner membrane protein, Tic40, participates in import by facilitating the interaction of Hsp93, Tic110, and preproteins during the import reaction (9, 16, 17). Together, these observations have led to the proposal that the stromal domain of Tic110 functions as a scaffold to assemble the chloroplast chaperone network at the TIC translocon and mediate transfer of the preprotein from the translocon channel to the chaperones (2, 3, 5).Despite strong evidence for their participation in protein import, the precise roles of individual chaperones in membrane translocation, protein folding, or suborganellar targeting has not been defined. In this study, we attempted to obtain a more complete accounting of the factors associated with protein import intermediates as a foundation for understanding the functions of the chloroplast import-associated chaperone network. We identified chloroplast heat shock protein 90, Hsp90C (18, 19), a member of the Hsp90 family of molecular chaperones, as a component of purified complexes containing protein import intermediates. We demonstrate that Hsp90C specifically associates with the TOC–TIC apparatus and several preproteins during protein import. Hsp90C is encoded by a single essential gene in Arabidopsis thaliana, indicating a requirement of Hsp90C for chloroplast function. Radicicol, an inhibitor of Hsp90 ATPase activity, reversibly blocked the import of a variety of nuclear-encoded preproteins at early protein import intermediates. On the basis of these results, we propose that Hsp90C cooperates with TIC components and other molecular chaperones to drive transport of preproteins into chloroplasts.     

Cholinergic Innervation of Fetal Neocortical Transplants Is Increased after Neutralization of Myelin-Associated Neurite Growth Inhibitors

Fetal neocortical transplants placed into adult neocortical sensorimotor aspiration lesions are known to receive afferent input from the adult host rat brain. As this input is less dense than normal, the present study was designed to investigate whether neutralization of myelin-associated neurite growth inhibitors NI-35/250 might promote host derived cholinergic innervation of fetal neocortical transplants. Adult rats received unilateral sensorimotor cortical aspiration lesions, and block grafts from embryonic day 14–15 neocortical tissue were placed immediately into the lesion cavities. Mouse hybridoma cells secreting either the monoclonal antibody IN-1, which blocks neurite growth inhibitors NI-35/250, or a control antibody or medium without cells were applied in millipore filter capsules directly over the fetal graft tissue. The brains were processed 12 weeks later for the visualization of acetylcholinesterase-positive, presumptive cholinergic fibers. We found an enhancement in the cholinergic innervation of fetal grafts in the recipients treated with the antibody IN-1 both in terms of fibers growing into the graft and of density within the center of the grafts. These results indicate that myelin-associated neurite growth inhibitors are involved in the development of host–transplant connectivity in the adult brain.     

Segregation Analysis of Asthma and Respiratory Allergy in Population‐Based Samples of Families

Class A regressive multiple logistic segregation models with a sibling covariate were used to investigate the underlying determinants of asthma and respiratory allergy (defined from specific IgE levels to inhaled allergens) in the randomly recruited Caucasian families from the Genetic Analysis Workshop 12 asthma data sets — Perth, Busselton, and Southampton. For asthma, both a purely multifactorial model and a major gene (dominant or recessive) model with multifactorial effects fitted the data. For respiratory allergy, a dominant, dominant with multifactorial effects and a purely multifactorial model all fitted the data. However, homogeneity of the three studies was rejected for both traits indicating that the three populations are significantly different and should be analyzed separately. This finding has implications for the meta‐analysis of asthma linkage studies. © 2001 Wiley‐Liss, Inc.