A small region of the beta-adrenergic receptor is selectively involved in its rapid regulation.

Plasma membrane receptors that couple to guanine nucleotide-binding regulatory proteins (G proteins) undergo a variety of rapid (minutes) and longer term (hours) regulatory processes induced by ligands. For the beta 2-adrenergic receptor (beta 2AR), the rapid processes include functional desensitization, mediated by phosphorylation of the receptor by the cAMP-dependent protein kinase and the beta-adrenergic receptor kinase, as well as a loss of hydrophilic ligand binding proposed to represent sequestration of receptors into a cellular compartment distinct from the plasma membrane. The slower processes include beta 2AR down-regulation (i.e., a decrease in the total cellular complement of receptors). It is not yet known whether beta 2AR phosphorylation and/or sequestration are prerequisites for down-regulation of the receptor. Like other G protein-coupled receptors, the beta 2AR molecule spans the plasma membrane seven times, and the cytoplasmic carboxyl-terminal domain has been proposed to contain molecular determinants for each of these regulatory processes. We replaced four serine and threonine residues located within a 10-amino acid segment of this domain of beta 2AR and thereby prevented agonist-promoted phosphorylation, sequestration, and rapid desensitization of the adenylyl cyclase response. In contrast, these mutations did not affect functional coupling to the stimulatory G protein Gs or long-term down-regulation. These findings thus define a small, hitherto unappreciated region of the receptor molecule that may selectively subserve its rapid regulation. In addition, with the demonstration that beta 2AR does not have to be phosphorylated or sequestered in order to enter the down-regulation pathway, the results suggest that the classical receptor endocytosis model may not be appropriate for beta 2AR regulation.     

Altered Ca2+ signalling in human neutrophils from inflammatory sites.

OBJECTIVES--To determine whether the intracellular store release of Ca2+ in neutrophils from patients with rheumatoid arthritis, other joint disease and active leg ulceration was different from normal neutrophils. METHODS--The release into the cytosol of Ca2+ from stores within individual neutrophils was determined using ratiometric imaging of fura2. The size of the elevated Ca2+ 'cloud' and its concentration were quantified in neutrophils from the circulation of patients with rheumatoid arthritis, other joint diseases, and leg ulcers and from the joints of those with joint disease. RESULTS--In neutrophils isolated from both the synovial fluid of patients with rheumatoid arthritis and other joint conditions, and also arising from leg ulcers, the amount of the cell cytosol occupied by elevated Ca2+ was significantly increased compared with neutrophils from healthy subjects; for neutrophils from rheumatoid, non-rheumatoid joints and leg ulcers p values were 0.0006, < 0.0001, 0.016 respectively (Student's t test). There was also a significant increase in Ca2+ release from circulating neutrophils from patients with rheumatoid arthritis (p = 0.09), but not in circulating neutrophils from patients with leg ulcers or non-rheumatoid joint conditions. CONCLUSIONS--It is proposed that the increased release of free Ca2+ into the cytosol of neutrophil at inflammatory sites results in increased oxidase activation.     

mTor is required for hypertrophy of Pten-deficient neuronal soma in vivo

The mechanisms that regulate mammalian cell size during development and homeostatic maintenance are poorly understood. The tumor suppressor Pten is required for correct maintenance of mammalian neuronal soma size. Selective inactivation of Pten in postnatal granule neurons of the cerebellum and dentate gyrus in mouse causes cell-autonomous hypertrophy as well as more complex phenotypes, including progressive macrocephaly, seizures, and premature death. To determine the contribution of mTor signaling to Pten-mediated growth regulation in the mammalian nervous system, we treated Pten conditional knockout mice with CCI-779, a specific mTor inhibitor. mTor inhibition decreased the seizure frequency and death rate in Pten mutant mice, prevented the increase in Pten-deficient neuronal soma size in young mice, and reversed neuronal soma enlargement in adult mice. mTor inhibition did not decrease the size of wild-type adult neurons. Thus, mTor is required for neuronal hypertrophy downstream of Pten deficiency, but is not required for maintenance of normal neuronal soma size. mTOR inhibitors may be useful therapeutic agents for diseases in brain resulting from PTEN deficiency such as Lhermitte-Duclos disease or glioblastoma multiforme.     

The reactivity of Bw4+ HLA-B and HLA-A alleles with KIR3DL1: implications for patient and donor suitability for haploidentical stem cell transplantations

Natural killer (NK)–cell alloreactivity can be exploited in haploidentical hematopoietic stem cell transplantation (HSCT). NK cells from donors whose HLA type includes Bw4, a public epitope present on a subset of HLA-B alleles, can be alloreactive toward recipients whose cells lack Bw4. Serologically detectable epitopes related to Bw4 also exist on a subset of HLA-A alleles, but the interaction of these alleles with KIR3DL1 is controversial. We therefore undertook a systematic analysis of the ability of most common HLA-B alleles and HLA-A alleles with Bw4 serologic reactivity to protect target cells from lysis by KIR3DL1-dependent NK cells. All Bw4^– HLA-B alleles failed to protect target cells from lysis. All Bw4⁺ HLA-B alleles with the exception of HLA-B*1301 and -B*1302 protected targets from lysis. HLA-A*2402 and HLA-A*3201 unequivocally protected target cells from lysis, whereas HLA-A*2501 and HLA-A*2301 provided only weak protection from lysis. KIR3DL1-dependent alloreactive NK clones were identified in donors with HLA-A*2402 but not in donors with HLA-B*1301 or -B*1302. These findings clarify the HLA types that donors and recipients need in haploidentical HSCT and other NK allotherapies in order to benefit from NK alloreactivity.     

Efficacy and safety of argatroban with or without glycoprotein IIb/IIIa inhibitor in patients with heparin induced thrombocytopenia undergoing percutaneous coronary intervention for acute coronary syndrome

Background There is limited experience with the use of argatroban in combination with glycoprotein IIb/IIIa (GPIIb/IIIa) inhibitor in acute coronary syndrome (ACS) patients with heparin-induced thrombocytopenia (HIT) undergoing percutaneous coronary intervention (PCI). Materials and methods This single-center, retrospective study evaluated the efficacy (composite of death, myocardial infarction, or urgent revascularization) and safety (evaluated by TIMI major bleeding) of the argatroban with or without a GPIIb/IIIa inhibitor during PCI. Among 102 consecutive ACS patients (71.6% unstable angina or NSTEMI and 28.4% STEMI) who received argatroban (239 ± 104 μg/kg bolus, followed by a 17 ± 11 μg/kg/min infusion) for confirmed or suspected HIT during PCI, 52 patients (51%) received a GPIIb/IIIa inhibitor simultaneously (86% integrilin, 10% tirofiban, 4% abciximab) and 50 patients (49%) did not. Results There was no difference between the groups in the efficacy endpoint, which occurred in nine patients (17.3%) who received GPIIb/IIIa inhibitor and in eight patients (16%) who did not (P = 0.70). TIMI major bleeding occurred in three (5.8%) patients in the GPIIa/IIIb inhibitor group versus 0 (0%) patients in the argatroban alone group (P = 0.085). Conclusion In patients with suspected or confirmed HIT undergoing PCI for ACS, argatroban with or without GPIIb/IIIa appears to provide adequate anticoagulation and is well tolerated with a low rate of bleeding.     

Evolution of a Second Gene for β-Galactosidase in Escherichia coli

Mutants of E. coli K12 with deletions of the beta-galactosidase gene (lacZ) can reacquire the ability to hydrolyze beta-galactosides during prolonged intense selection for growth on lactose. Full lactose competence is restored through a sequence of at least five mutations. Cell extracts of these derived strains hydrolyze o-nitrophenyl-beta-D-galactoside, the standard substrate for assay of beta-galactosidase. The enzyme responsible for this activity differs in its immunological, kinetic, and sedimentation characteristics from the lacZ beta-galactosidase of wild-type E. coli. Its genetic determinant, designated ebg-5, maps at 59 min on the E. coli chromosome, whereas the lac operon maps at 10 min. We suggest that a gene not involved in lactose utilization has been progressively changed into a form capable of specifying a beta-galactosidase and that this process is similar to that whereby genes with new functions are evolved by natural selection.     

Changing habits of practice

PURPOSE: The majority of health care, both for acute and chronic conditions, is delivered in the ambulatory setting. Despite repeated proposals for change, the majority of internal medicine residency training still occurs in the inpatient setting. Substantial changes in ambulatory education are needed to correct the current imbalance. To assist educators and policy makers in this process, this paper reviews the literature on ambulatory education and makes recommendations for change. METHODS: The authors searched the Medline, Psychlit, and ERIC databases from 2000 to 2004 for studies that focused specifically on curriculum, teaching, and evaluation of internal medicine residents in the ambulatory setting to update previous reviews. Studies had to contain primary data and were reviewed for methodological rigor and relevance. RESULTS: Fifty-five studies met criteria for review. Thirty-five of the studies focused on specific curricular areas and 11 on ambulatory teaching methods. Five involved evaluating performance and 4 focused on structural issues. No study evaluated the overall effectiveness of ambulatory training or investigated the effects of current resident continuity clinic microsystems on education. CONCLUSION: This updated review continues to identify key deficiencies in ambulatory training curriculum and faculty skills. The authors make several recommendations: (1) Make training in the ambulatory setting a priority. (2) Address systems problems in practice environments. (3) Create learning experiences appropriate to the resident's level of development. (4) Teach and evaluate in the examination room. (5) Expand subspecialty-based training to the ambulatory setting. (6) Make faculty development a priority. (7) Create and fund multiinstitutional educational research consortia.     

Management of patients with community-acquired pneumonia treated in hospital in Sweden

To investigate the management of patients with community-acquired pneumonia (CAP) treated in hospital in Sweden, a multicentre retrospective cohort study was performed with medical record review of 982 patients (mean age 63 y) at 17 departments of infectious diseases at hospitals in Sweden. Information on antimicrobial therapy, demographic characteristics, comorbid conditions, physical examination findings, and laboratory and microbiological test results were recorded. Outcome measures were in-hospital mortality and length of hospital stay (LOS). Cultures were obtained from blood in 80% and from sputum in 22% of the patients. A microbiological aetiology was determined for 23% of the patients, with Streptococcus pneumoniae as the dominating agent (9%). The initial antibiotic treatment was mostly given intravenously (78%). Penicillin (50%) or a cephalosporin (30%) was the most common choice. Both of these drugs were usually given as a single agent. The overall mortality was 3.5% and the mean LOS was 6.4 d. Thus, the outcome was favourable despite the empirical antibiotic treatment having a narrow spectrum compared with the broader approach recommended in most recent guidelines on the management of CAP. These findings suggest that a majority of patients who are hospitalized with moderately severe pneumonia can be treated initially with penicillin alone.     

A waterborne outbreak of Norwalk-like virus among snowmobilers-Wyoming, 2001

In February 2001, episodes of acute gastroenteritis were reported to the Wyoming Department of Health from persons who had recently vacationed at a snowmobile lodge in Wyoming. A retrospective cohort study found a significant association between water consumption and illness, and testing identified Norwalk-like virus (NLV) in 8 of 13 stool samples and 1 well. Nucleotide sequences from the positive well-water specimen and 6 of the positive stool samples were identical. This multistrain NLV outbreak investigation illustrates the importance of NLV as a cause of waterborne illness and should encourage monitoring for NLVs in drinking water.     

Controlled study of the effect of nicardipine and ceruletide on the sphincter of Oddi.

Although sphincter of Oddi dysfunction is a recognised cause of post cholecystectomy pain, the control mechanisms involved in sphincter of Oddi function are poorly understood. Pharmacological relaxation of the sphincter of Oddi may have a beneficial effect particularly in sphincter of Oddi dysfunction where basal sphincter pressure is high. The aim of this study was to investigate the effects of calcium channel blockade (nicardipine) and synthetic cholecystokinin (ceruletide) on sphincter of Oddi pressures. Nineteen patients (median age 49 years; range 21-75) attending for routine endoscopic retrograde cholangiopancreatographic (ERCP) examination were studied. No patients with evidence of sphincter of Oddi dysfunction were included in the study. Each patient was randomly allocated to receive a three minute intravenous infusion of nicardipine 3 mg (six) ceruletide 5 ng/kg (seven) or placebo (six). Endoscopic biliary manometry was done with recording of basal sphincter of Oddi pressures, sphincter of Oddi phasic wave amplitude and frequency before and after intravenous infusions. In the nicardipine group patients showed a decrease in both basal and phasic amplitude sphincter of Oddi pressure (mm Hg) from the preinfusion values (mean (SEM)) of 24.7 (3.6) and 112.3 (13.4) to 12.9 (2.9) (p less than 0.01) and 89.9 (12.4) (p less than 0.03) after infusion respectively. Ceruletide produced a decrease in sphincter of Oddi phasic wave frequency (c/min) from 3.4 (0.3) before infusion to 2.6 (0.5) after infusion (p less than 0.05). We conclude that nicardipine effectively decreases sphincter of Oddi pressure. This drug may therefore be of value in the treatment of sphincter of Oddi dysfunction where raised sphincter pressures are thought to be the primary pathogenic feature.