[Future family physicians - reasons for their specialty choice and crucial professional skills]. / Zukünftige Hausärztinnen und Hausärzte - Gründe für die Berufswahl und berufliche Kernkompetenzen.

PURPOSE: The aim of the present study was to investigate the motivation of young physicians to work in family medicine/general practice and the skills to be acquired during residency. METHODS: As part of a prospective study on career determinants in young physicians starting in 2001, 84 future family physicians at the end of their residency were asked about their motivation for specialty choice and about core competencies in general practice. Content analysis was applied to assign the answers given to open questions to inductively defined categories. RESULTS: The 254 answers concerning the motivation for specialty choice of general medicine or general internal medicine, and the 375 answers concerning core competencies of a family physician were assigned to eleven categories. The most frequently named motives fall into the categories <>, <>, <>, <> and <>. The most frequently named core competencies fall into the categories <>, <> and <>. CONCLUSION: Motivation for working in general practice and the core competencies to be acquired stand for a patient centered conception of the medical profession. They also imply personal responsibility and latitude in medical practice.

Large-scale purification of functional recombinant human aquaporin-2.

The homotetrameric aquaporin-2 (AQP2) water channel is essential for the concentration of urine and of critical importance in diseases with water dysregulation, such as nephrogenic diabetes insipidus, congestive heart failure, liver cirrhosis and pre-eclampsia. The structure of human AQP2 is a prerequisite for understanding its function and for designing specific blockers. To obtain sufficient amounts of AQP2 for structural analyses, we have expressed recombinant his-tagged human AQP2 (HT-AQP2) in the baculovirus/insect cell system. Using the protocols outlined in this study, 0.5 mg of pure HT-AQP2 could be obtained per liter of bioreactor culture. HT-AQP2 had retained its homotetrameric structure and exhibited a single channel water permeability of 0.93+/-0.03x10(-13) cm3/s, similar to that of other AQPs. Thus, the baculovirus/insect cell system allows large-scale expression of functional recombinant human AQP2 that is suitable for structural studies.

Surface tongue-and-groove contours on lens MIP facilitate cell-to-cell adherence.

The lens major intrinsic protein (MIP, AQP0) is known to function as a water and solute channel. However, MIP has also been reported to occur in close membrane contacts between lens fiber cells, indicating that it has adhesive properties in addition to its channel function. Using atomic force and cryo-electron microscopy we document that crystalline sheets reconstituted from purified ovine lens MIP mostly consisted of two layers. MIP lattices in the apposing membranes were in precise register, and determination of the membrane sidedness demonstrated that MIP molecules bound to each other via their extracellular surfaces. The surface structure of the latter was resolved to 0.61 nm and revealed two protruding domains providing a tight "tongue-and-groove" fit between apposing MIP molecules. Cryo-electron crystallography produced a projection map at 0.69 nm resolution with a mirror symmetry axis at 45 degrees to the lattice which was consistent with the double-layered nature of the reconstituted sheets. These data strongly suggest an adhesive function of MIP, and strengthen the view that MIP serves dual roles in the lens.

Purification and electron microscopic characterization of the membrane subunit (IICB(Glc)) of the Escherichia coli glucose transporter.

The glucose transporter of the bacterial phosphotransferase system mediates sugar transport across the cytoplasmic membrane concomitant with sugar phosphorylation. It consists of a cytoplasmic subunit IIA(Glc) and the transmembrane subunit IICB(Glc). IICB(Glc) was purified to homogeneity by urea/alkali washing of membranes and nickel-chelate affinity chromatography. About 1.5 mg highly pure IICB(Glc) representing 77% of the total activity present in the membranes was obtained from 8g (wet weight) of cells. IICB(Glc) was reconstituted into lipid bilayers by temperature-controlled dialysis to yield small 2D crystals and by a rapid detergent-dilution procedure to yield densely packed vesicles. Electron microscopy and digital image processing of the negatively stained 2D crystals revealed a trigonal lattice with a unit cell size of a = b = 14.5 nm. The unit cell morphology exhibited three dimers of IICB(Glc) surrounding the threefold symmetry center. Single particle analysis of IICB(Glc) in proteoliposomes obtained by detergent dialysis also showed predominantly dimeric structures.

Surface analysis of the photosystem I complex by electron and atomic force microscopy.

Two-dimensional (2D) crystals of the photosystem I (PSI) reaction center from Synechococcus sp. OD24 were analyzed by electron and atomic force microscopy. Surface relief reconstructions from electron micrographs of freeze-dried unidirectionally shadowed samples and topographs recorded with the atomic force microscope (AFM) provided a precise definition of the lumenal and stromal PSI surfaces. The lumenal surface was composed of four protrusions that surrounded an indentation. One of the protrusions, the PsaF subunit, was often missing. Removal of the extrinsic proteins with the AFM stylus exposed the stromal side of the PSI core, whose surface structure could then be imaged at a resolution better than 1.4 nm. This interfacial surface between core and extrinsic subunits, had a pseudo-2-fold symmetry and protrusions that correlated with the surface helices e and e' or were at the sites of putative alpha-helix-connecting loops estimated from the 4 A map of the complex. The molecular dissection achieved with the AFM, opens new possibilities to unveil the interfaces between subunits of supramolecular assemblies.

2D crystallization of membrane proteins: rationales and examples.

The difficulty in crystallizing channel proteins in three dimensions limits the use of X-ray crystallography in solving their structures. In contrast, the amphiphilic character of integral membrane proteins promotes their integration into artificial lipid bilayers. Protein-protein interactions may lead to ordering of the proteins within the lipid bilayer into two-dimensional crystals that are amenable to structural studies by electron crystallography and atomic force microscopy. While reconstitution of membrane proteins with lipids is readily achieved, the mechanisms for crystal formation during or after reconstitution are not well understood. The nature of the detergent and lipid as well as pH and counter-ions is known to influence the crystal type and quality. Protein-protein interactions may also promote crystal stacking and aggregation of the sheet-like crystals, posing problems in data collection. Although highly promising, the number of well-studied examples is still too small to draw conclusions that would be applicable to any membrane protein of interest. Here we discuss parameters influencing the outcome of two-dimensional crystallization trials using prominent examples of channel protein crystals and highlight areas where further improvements to crystallization protocols can be made.

Purified lens major intrinsic protein (MIP) forms highly ordered tetragonal two-dimensional arrays by reconstitution.

Lens major intrinsic protein (MIP) is the founding member of the MIP family of membrane channel proteins. Its isolation from ovine lens fibre cell membranes and its two-dimensional crystallization are described. Membranes were solubilized with N-octyl-beta-D-glucoside and proteins fractionated by sucrose gradient centrifugation containing decyl-beta-D-maltoside. MIP was purified by cation exchange chromatography, and homogeneity was assessed by mass analysis in the scanning transmission electron microscope. Purified MIP reconstituted into a lipid bilayer at a low lipid-to-protein ratio formed highly ordered tetragonal two-dimensional crystals. The square unit cell had a side length of 6.4 nm, and exhibited in negative stain four stain-excluding elongated domains surrounding a central stain-filled depression. Projection maps of freeze-dried crystals exhibited a resolution of 9 A, and revealed a monomer structure of MIP consisting of distinct densities. Despite significant differences in the packing of tetramers in the crystals, the projection map of the MIP monomer was similar to that of aquaporin-1 (AQP1), the first member of the MIP family which had its structure resolved to 6 A. Our protocols for the purification and reconstitution of MIP establish the feasibility for future work to visualize structure elements which determine the diverse functional properties of the MIP family members.

Structural Analysis of Photosystem II: Comparative Study of Cyanobacterial and Higher Plant Photosystem II Complexes

Oxygen evolving photosystem II (PSII-OEC) complexes and PSII core complexes were isolated from spinach and the thermophilic cyanobacterium Synechococcus sp. OD24 and characterized by gel electrophoresis, immunoblotting, and absorbance spectroscopy. The mass of the core complexes was determined by scanning transmission electron microscopy (STEM) and found to be 281 ± 65 kDa for spinach and 313 ± 52 kDa for Synechococcus sp. OD24. The mass of the spinach PSII-OEC complex was 327 ± 64 kDa. Digital images of negatively stained PSII-OEC and PSII core complexes were recorded by STEM and analyzed by single particle averaging. All monomeric complexes showed similar morphologies and were of comparable length (14 nm) and width (10 nm). The averages revealed a pseudo-twofold symmetry axis, which is a prominent structural element of the monomeric form. Difference maps between the averaged projections of the oxygen evolving complexes and the core complexes from both species indicated where the 33-kDa extrinsic manganese stabilizing protein is bound. A symmetric organization of the PSII complex, with the PsbA and the PsbD proteins in the center and symmetrically arranged PsbB and PsbC proteins at the periphery of the monomeric complex, is proposed.

Increase in circulating insulin induced by atrial natriuretic peptide in normal humans.

To search for possible metabolic interactions of alpha-human-atrial natriuretic peptide (alpha hANP), we evaluated in 20 normal subjects blood levels of alpha hANP, glucose, insulin, cortisol, electrolytes, catecholamines, free fatty acids, carnitine and amino acids, blood pressure (BP), and heart rate before, during, and after a 45-min infusion of synthetic alpha hANP. Group A [n = 10] was studied on liberal and Group B on three consecutive sodium (Na) intakes of 17, 140, and 310 mM/day. Plasma alpha hANP was slightly but not significantly higher following 5 days on "normal" or high than on low Na+ intakes. alpha hANP infused at 0.1 microgram/kg/min produced on all Na+ intakes comparable percentage increases in plasma insulin (+34 to 63%, p less than 0.001), norepinephrine (+76 to 155%, p less than 0.001) and heart rate (p less than 0.001), and a similar fall in diastolic BP (p less than 0.001). Plasma glucose tended to be decreased slightly and cortisol was reduced; epinephrine, dopamine, and potassium levels were not significantly modified. As evaluated in group A, serum free fatty acids were increased (p less than 0.01), plasma free carnitine levels were reduced (p less than 0.001), and amino acids were not consistently altered. These findings indicate that in normal humans alpha hANP may, on various sodium intakes, modulate insulin secretion and/or metabolism and elicit a possibly baroreflex-mediated sympathetic activation and lipolysis.

Cardiovascular, endocrine and renal effects of atrial natriuretic peptide in essential hypertension.

Plasma concentrations of human atrial natriuretic peptide (hANP) and effects of synthetic alpha-hANP on blood pressure (BP), on endocrine and metabolic variables, and on renal function were investigated in 10 patients with essential hypertension. Alpha-human atrial natriuretic peptide was given intravenously as a 50-micrograms bolus followed by a 45-min infusion at 0.1 microgram/kg per min. The following effects were observed: (1) a marked rise in plasma alpha-hANP, (2) a progressive fall in BP (from 181/127 to 165/109 mmHg) and plasma volume, (3) a probably baroreflex-mediated sympathetic activation, evidenced by raised heart rate and plasma norepinephrine levels, (4) an increase in serum free fatty acids and circulating insulin (+45%), (5) an enhanced diuresis (+770%) and excretion of sodium (+665%), chloride (+524%), phosphate (+518%), other electrolytes, amino acids and free water clearance, (6) biphasic responses in the glomerular filtration rate (GFR) and p-aminohippurate (PAH) clearance, with initial increases (+40 and 30%, respectively) followed by a rapid return to (GFR), or even a fall below (PAH clearance) control values, and (7) a marked rise in the filtration fraction. Plasma antidiuretic hormone and urinary prostaglandin E2, F2 alpha and dopamine levels were not modified during alpha-hANP infusion, while plasma renin increased. Discontinuation of alpha-hANP was followed by rises in plasma aldosterone, the aldosterone:renin ratio and cortisol. Compared with previously studied normal subjects, in the hypertensive patients alpha-hANP caused a distinctly greater diuresis and electrolyte excretion but lowered BP only slightly more. In essential hypertension, as in normal man, alpha-hANP circulates in the blood and may exert a wide spectrum of cardiovascular, metabolic, endocrine and renal actions.

Plasma levels and cardiovascular, endocrine, and excretory effects of atrial natriuretic peptide during different sodium intakes in man.

Endogenous plasma concentrations of human atrial natriuretic peptide (alpha hANP) as well as effects of synthetic alpha hANP on some cardiovascular, endocrine, and renal excretory parameters were investigated in 10 normal subjects during sodium (Na+) intakes of 17, 140, and 310 mmol/day, respectively. Plasma hANP was slightly but not significantly higher after 5 days of normal or high sodium intake than after 5 days of low sodium intake [54 +/- 13, 46 +/- 8, and 37 +/- 5 (mean +/- SEM) pg/ml, respectively]. alpha hANP infused at 0.1 microgram/kg X min during all Na+ intakes produced a similar fall in diastolic blood pressure (P less than 0.001) and rise in heart rate (P less than 0.001), a comparable percent increase in plasma norepinephrine (P less than 0.001), and a reduction in plasma cortisol and aldosterone (P less than 0.01-0.001) despite raised renin activity (P less than 0.05-0.001) and unchanged plasma electrolytes. alpha hANP-induced plasma volume contraction, diuresis, and natriuresis were greater during high than low Na+ intake (P less than 0.01-0.001). Therefore, in normal man different Na+ intakes are accompanied by marked modifications in renal excretory responsiveness to alpha hANP. Regardless of sodium intake, alpha hANP can promote BP reduction and hemoconcentration, elicit reflex (?) sympathetic activation, and depress basal circulating aldosterone and cortisol levels in the face of an activated renin system.

Blood levels and renal effects of atrial natriuretic peptide in normal man.

Since mammalian atria were recently found to contain vasoactive and natriuretic peptides, we investigated the following in normal humans: plasma human atrial natriuretic peptide concentrations, effective renal plasma flow (ERPF), glomerular filtration rate (GFR), urinary water and electrolyte excretion, blood pressure (BP), and catecholamine, antidiuretic hormone (ADH), angiotensin II, and aldosterone levels before, during, and after intravenous administration of the newly synthetized alpha-human atrial natriuretic peptide (alpha hANP). In 10 subjects alpha hANP given as an initial bolus of 50 micrograms followed by a 45-min maintenance infusion at 6.25 micrograms/min increased plasma alpha hANP from 58 +/- 12 to 625 +/- 87 (mean +/- SEM) pg/ml; caused an acute fall in diastolic BP (-12%, P less than 0.001) and a hemoconcentration (hematocrit +7%, P less than 0.01) not fully explained by a negative body fluid balance; increased GFR (+15%, P less than 0.05) despite unchanged or decreased ERPF (filtration fraction +37%, P less than 0.001); augmented (P less than 0.05- less than 0.001) urinary chloride (+317%), sodium (+224%), calcium (+158%), magnesium (+110%), phosphate excretion (+88%), and free water clearance (from -0.76 to +2.23 ml/min, P less than 0.001) with only little change in potassium excretion; and increased plasma norepinephrine (P less than 0.001) while plasma and urinary epinephrine and dopamine, and plasma ADH, angiotensin II, and aldosterone levels were unchanged. The magnitude and pattern of electrolyte and water excretion during alpha hANP infusion could not be accounted for by increased GFR alone. Therefore, in normal man, endogenous alpha hANP seems to circulate in blood. alpha hANP can cause a BP reduction and hemoconcentration which occur, at least in part, independently of diuresis and are accompanied by sympathetic activation. An increase in GFR that occurs in the presence of unchanged or even decreased total renal blood flow is an important but not sole mechanism of natriuresis and diuresis induced by alpha hANP in man.

Plasma kinetics of synthetic alpha-human atrial natriuretic peptide in man.

Atrial natriuretic peptides (ANPs) circulate in the blood stream and may modulate the regulation of blood pressure, sodium-fluid volume state and renal function. In man, alpha-human ANP (alpha hANP) is probably the major circulating form of ANP. To evaluate its plasma kinetics, we studied in 7 healthy men plasma alpha hANP concentrations under basal conditions and at short intervals during and up to 40 min after discontinuation of a 45-min constant alpha hANP infusion at a rate of 0.1 microgram/min/kg. From basal levels averaging 75 +/- 18 pg/ml (mean +/- SEM), plasma alpha hANP concentrations increased to 1,185 +/- 321 and 1,117 +/- 175 pg/ml at 30 and 40 min during the alpha hANP infusion, respectively. After discontinuing the latter, plasma alpha hANP decreased rapidly, following first-order kinetics, with a plasma half-life of 3.2 +/- 0.4 min. This finding is in line with the brief effects of intravenously applied alpha hANP and suggests that this system may be designed for rapid minute-to-minute adjustments.

Characterization of effector cells responsible for cell-mediated cytotoxicity in patients with carcinoma of the prostate.

In earlier experiments it has been shown that cellular cytotoxicity of patients with prostatic carcinoma (CaP) as measured by cytolysis of EB 33 target cells correlates with the extent of their tumor lesion: a high grade of cytotoxicity was observed only when CaP was confined to gland. In order to define a possible in vivo immunological host-tumor interaction, further characterization of the type of effector cell responsible for in vitro killing has been attempted. Effector cells could be either tumor-specific sensitized T cells, K cells, macrophages or NK cells. Antibody-dependent K-cell activity could most likely be excluded, since autologous and homologous serum did not affect the extent of EB 33 killing when added to the test medium. Further separation of effector cells by nylon wool columns resulted in increased cytolysis of EB 33 target cells. This can be due either to tumor-specific T-cell-mediated mechanisms or to an increased natural killer cell activity, as macrophages and B cells were significantly removed by this measure. Separation of T and NK cells is necessary to ascertain the effector cell responsible for the in vitro killing of tumor cells derived from human CaP.