Electron cryomicroscopy methods.

Electron cryomicroscopy methods comprise a rapidly expanding field providing insights into the structure and function of biological macromolecules and their supramolecular assemblies. The 3.8 A resolution structure of the membrane protein aquaporin, a view of the herpesvirus capsid at 8.5 A and the 10 A resolution structure of the spliceosomal U1 small nuclear ribonucleoprotein complex are three outstanding examples emphasizing the versatility of this technique.

The isolated perfused liver. a new model using autologous blood and porcine slaughterhouse organs.

INTRODUCTION: Current models of isolated and perfused livers are limited by nonphysiologic perfusates or the need for the use of high numbers of laboratory animals. The present study was performed in order to rectify these difficulties. METHODS: To establish a new isolated perfused liver model, a perfusion circuit was developed using normothermic, autologous hemoperfusion and organs obtained from a slaughterhouse. RESULTS: Stable organ function was maintained over 220 min. The organs displayed physiologic values for measured variables, including oxygen consumption which varied from 5.2+/-1.5 ml/min at 40 min to 5.2+/-2.4 ml/min at 220 min, and bile production (0.15-0.31 ml/min, respectively). DISCUSSION: The present studies demonstrate a new approach for experimental liver perfusion by combining the optimal perfusion medium of autologous blood and slaughterhouse organs as source material.

Assessment of electron crystallographic data obtained from two-dimensional crystals of biological specimens.

Over the past few years, an increasing number of electron crystallographic studies using two-dimensional crystals have shed light on the structure of biologically important macromolecules. Steady progress in the development of specimen-preparation techniques and image-processing tools enable researchers to achieve resolutions in the range of 5-10 A almost routinely. However, reaching near-atomic resolution remains a formidable task that is likely to require several years. Without doubt, this process will become far less time-consuming as methods are improved further. However, the immediate future is more likely to be dominated by structures solved to an intermediate level of resolution. Since the reliability of such structures is more difficult to assess than that of density maps at near-atomic resolution and as the popularity of electron cryo-microscopy increases it becomes more important to define standardized criteria for the evaluation of electron crystallographic data. This article discusses some of the relevant issues with the aim of stimulating further discussion about the assessment and presentation of electron crystallographic data.

Expression, two-dimensional crystallization, and electron cryo-crystallography of recombinant gap junction membrane channels.

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa alpha(1) connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of approximately 7.5 A revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

Electron cryo-crystallography of a recombinant cardiac gap junction channel.

Gap junctions in the heart play an important functional role by electrically coupling cells, thereby organizing the pattern of current flow to allow co-ordinated muscle contraction. Cardiac gap junctions are therefore intimately involved in normal conduction as well as the genesis of potentially lethal arrhythmias. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated 2D crystals of a recombinant, C-terminal truncated form of connexin 43 (Cx43; alpha 1), the principal cardiac gap junction protein. The projection map at 7 A resolution revealed that each 30 kDa connexin subunit has a transmembrane alpha-helix that lines the aqueous pore and a second alpha-helix in close contact with the membrane lipids. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by approximately 30 degrees. We are now recording images of tilted, frozen-hydrated 2D crystals, and a preliminary 3D map has been computed at an in-plane resolution of approximately 7.5 A and a vertical resolution of approximately 25 A. As predicted by our model, the two apposing connexons that form the channel are staggered with respect to each other for certain connexin molecular boundaries within the hexamer. Within the membrane interior each connexin subunit displays four rods of density, which are consistent with an alpha-helical conformation for the four transmembrane domains. Preliminary studies of BHK hamster cells that express the truncated Cx43 designated alpha 1 Cx263T demonstrate that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of alpha 1 Cx263T channels. The comparison of the 3D structures in the presence and absence of oleamide may provide an opportunity to explore the conformational changes that are associated with oleamide-induced blockage of dye transfer. The structural details revealed by our analysis will be essential for delineating the molecular basis for intercellular current flow in the heart, as well as the general molecular design and functional properties of this important class of channel proteins.

Three-dimensional structure of a recombinant gap junction membrane channel.

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

Synthesis, assembly and structure of gap junction intercellular channels.

Gap junction membrane channels assemble as dodecameric complexes, in which a hexameric hemichannel (connexon) in one plasma membrane docks end to end with a connexon in the membrane of a closely apposed cell. Steps in the synthesis, assembly and turnover of gap junction channels appear to follow the general secretory pathway for membrane proteins. In addition to homo-oligomeric connexons, different connexin polypeptide subunits can also assemble as hetero-oligomers. The ability to form homotypic and heterotypic channels that consist of two identical or two different connexons, respectively, adds even greater versatility to the functional modulation of gap junction channels. Electron cryocrystallography of recombinant gap junction channels has recently provided direct evidence for alpha-helical folding of at least two of the transmembrane domains within each connexin subunit. The potential to correlate the structure and biochemistry of gap junction channels with recently identified human diseases involving connexin mutations makes this a particularly exciting area of research.

Arrangement of rhodopsin transmembrane alpha-helices.

Rhodopsins, the photoreceptors in rod cells, are G-protein-coupled receptors with seven hydrophobic segments containing characteristic conserved sequence patterns that define a large family. Members of the family are expected to share a conserved transmembrane structure. Direct evidence for the arrangement of seven alpha-helices was obtained from a 9A projection map of bovine rhodopsin. Structural constraints inferred from a comparison of G-protein-coupled receptor sequences were used to assign the seven hydrophobic stretches in the sequence to features in the projection map. A low-resolution three-dimensional structure of bovine rhodopsin and two projection structures of frog rhodopsin confirmed the position of the three least tilted helices, 4, 6 and 7. A more elongated peak of density for helix 5 indicated that it is tilted or bent, but helices 1, 2 and 3 were not resolved. Here we have used electron micrographs of frozen-hydrated two-dimensional frog rhodopsin crystals to determine the structure of frog rhodopsin. Seven rods of density in the map are used to estimate tilt angles for the seven helices. Density visible on the extracellular side of the membrane suggests a folded domain. Density extends from helix 6 on the intracellular side, and a short connection between helices 1 and 2, and possibly a part of the carboxy terminus, are visible.

An alpha-carbon template for the transmembrane helices in the rhodopsin family of G-protein-coupled receptors.

A model for the alpha-carbon positions in the seven transmembrane helices in the rhodopsin family of G-protein-coupled receptors is presented. The model incorporates structural information derived from the analysis of approximately 500 sequences in this family. The location, relative to the centre of the lipid bilayer, of each of the seven helical sequence segments and their probable lengths are deduced from sequence analysis, along with the orientation, relative to the centre of the helix bundle, of each helical segment around its axis. The packing of the helices in the model is guided by the density in a three-dimensional map of frog rhodopsin determined by electron cryo-microscopy. The model suggests which of the residues that are highly conserved in this family of receptors interact with each other. Helices III, V and VI are predicted to protrude more than the others from the central lipid core towards the aqueous phase on the intracellular side of the membrane. This feature could be a property of the receptor structure in some but not all of the conformations that it adopts, since recent studies suggest that relative movement occurs between these helices on photoactivation of rhodopsin. Results from other techniques, including the creation of metal-binding sites and disulphide bridges, site-directed spin-labelling studies, the substituted-cysteine accessibility method and other site-directed mutagenesis studies, are discussed in terms of the model.

[A new method of ex vivo whole blood perfusion of isolated mammalian organs, exemplified by the kidney of swine]. / Eine neue Methode zur Ex-vivo-Vollblut-Perfusion isolierter Warmblüterorgane, dargestellt an der Niere von Schweinen.

A new method for the ex vivo perfusion of organs from large mammals is described. Gas exchange and dialysis are carried out simultaneously with a low-flux polysulfon dialysis module. The dialysate (e.g. Tyrode solution) is aerated with a mixture of oxygen and carbon dioxide to ensure gas exchange with the blood. Dialysis is carried out in a closed thermostatically controlled system. Monitoring of ultrafiltration is maintained by continuously weighing the blood reservoir and adjusting an afferent and efferent blood pump. Initial results obtained with isolated pig kidneys demonstrate the suitability of the new method for use as a model for the replacement of animal experiments. Theoretically, clinical application in the area of in vivo regional organ perfusion may also be possible.

Projection structure of a gap junction membrane channel at 7 A resolution.

Electron cryo-microscopy and image analysis of frozen-hydrated, two-dimensional crystals of gap junction membrane channels formed by recombinant alpha 1 connexin (Cx43) reveal a ring of transmembrane alpha-helices that lines the aqueous pore and a second ring of alpha-helices in close contact with the membrane lipids.

Low resolution structure of bovine rhodopsin determined by electron cryo-microscopy.

The visual pigment rhodopsin is a member of the G protein-coupled receptor family. Electron cryo-microscopy was used to determine the three-dimensional structure of bovine rhodopsin from tilted two-dimensional crystals embedded in vitrified water. The effective resolution in a map obtained from the 23 best crystals was about 9.5 A horizontally and approximately 47 A normal to the plane of the membrane. Four clearly resolved tracks of density in the map correspond to four alpha-helices oriented nearly perpendicular to the plane of the membrane. One of these helices appears to be more tilted than anticipated from the projection structure published previously. The remaining three helices are presumably more highly tilted, given that they form a continuous "arc-shaped" feature and could not be resolved to the same extent. The overall density distribution in the low resolution map shows an arrangement of the helices in which the "arc-shaped" feature is extended by a fourth, less tilted helix. The band of these four tilted helices is flanked by a straight helix on the outer side and a pair of straight helices on its inner side.

Inferior vena caval pressure increase contributes to sodium and water retention during PEEP in awake dogs.

UNLABELLED: This study compared the hemodynamic, renal, and hormonal effects of an experimentally induced increase in inferior vena caval pressure (IVCP) [to the same extent as during controlled mechanical ventilation (CMV) with positive end-expiratory pressure (PEEP)] with those of CMV with PEEP. Six volume-expanded conscious chronically tracheotomized dogs were studied under three conditions: CONTROL: 4 h of spontaneous breathing at 4 cmH2O continuous positive airway pressure (CPAP); CMV: CPAP for the 1st and 4th h and CMV with PEEP for the 2nd and 3rd h, resulting in a mean airway pressure of 20 cmH2O; and Increased IVCP: 4 h of CPAP, with IVCP increased during the 2nd and 3rd h by inflation of a chronically implanted cuff. Urine volume, sodium excretion, and fractional sodium excretion decreased during the 2nd and 3rd h of CMV and during increased IVCP compared with CONTROL. Glomerular filtration rate, mean arterial pressure, and antidiuretic hormone, atrial natriuretic peptide, and aldosterone plasma concentrations were not affected by CMV or Increased IVCP. Plasma renin activity decreased during CONTROL and Increased IVCP conditions but remained elevated during the 2nd and 3rd h of CMV. We conclude that, in conscious extracellular volume-expanded dogs, IVCP elevation contributes considerably to the water- and sodium-retaining effect of short-term CMV with PEEP.

Extracellular volume expansion inhibits antidiuretic hormone increase during positive end-expiratory pressure in conscious dogs.

1. This study in conscious dogs examined the effects of extracellular volume expansion on plasma antidiuretic hormone, atrial natriuretic peptide and aldosterone concentrations, plasma renin activity, and haemodynamic and renal responses during controlled mechanical ventilation with 20 cmH2O positive end-expiratory pressure. 2. Twenty experiments (10 controls, 10 expansion experiments with 0.5 ml min-1 kg-1 body weight of a balanced electrolyte solution given intravenously throughout) were performed in five trained, conscious, tracheotomized dogs over 4 h: first and fourth hour, spontaneous breathing; second and third hour, 20 cmH2O positive end-expiratory pressure. 3. In the control experiments positive end-expiratory pressure increased plasma antidiuretic hormone concentration from 1.4 +/- 0.2 to 10.0 +/- 3.3 pg/ml, plasma aldosterone concentration from 113 +/- 19 to 258 +/- 58 pg/ml and heart rate from 77 +/- 5 to 94 +/- 5 beats/min. Positive end-expiratory pressure did not change plasma atrial natriuretic peptide concentration (55 +/- 5 pg/ml), plasma renin activity (2.6 +/- 0.4 pmol of angiotensin I h-1 ml-1) and mean arterial pressure 103 +/- 3 mmHg). 4. In the expansion experiments, positive end-expiratory pressure did not change plasma antidiuretic hormone concentration (1.1 +/- 0.1 pg/ml), plasma aldosterone concentration (25 +/- 2 pg/ml), plasma atrial natriuretic peptide concentration (82 +/- 8 pg/ml), plasma renin activity (0.8 +/- 0.15 pmol of angiotensin I h-1 ml-1), heart rate (92 +/- 6 beats/min) and mean arterial pressure (111 +/- 4 mmHg). 5. In the control experiments, urine volume, sodium excretion and fractional sodium excretion remained in a low range during positive end-expiratory pressure, whereas potassium excretion increased.(ABSTRACT TRUNCATED AT 250 WORDS)

[Pneumoperitoneum in a premature, non-ventilated neonate with respiratory distress syndrome leading to respiratory failure. What is the etiology?]. / Pneumoperitoneum u nezralého, neventilovaného novorozence s RDS vedouci k respiracnímu selhání. Jaký muze mít puvod?

The authors describe the finding of extensive tensive pneumoperitoneum which developed during distension therapy (CNP) in an immature neonate. The absence of another extraalveolar cumulation of air made the authors use PO2 analysis in the peritoneal air by means of a transcutaneous electrode. Because the PO2 rose after connection of the electrode to the catheter draining the abdominal cavity, the authors assumed a respiratory aetiology of the pneumoperitoneum and did not consider laparatomy. They assume that the above test may be valuable in the differential diagnosis between respiratory and gastrointestinal pneumoperitoneum in those instances where it is not possible to assess the aetiology of pneumoperitoneum unequivocally.