The complete genome sequence of the murine respiratory pathogen Mycoplasma pulmonis.

Mycoplasma pulmonis is a wall-less eubacterium belonging to the Mollicutes (trivial name, mycoplasmas) and responsible for murine respiratory diseases. The genome of strain UAB CTIP is composed of a single circular 963 879 bp chromosome with a G + C content of 26.6 mol%, i.e. the lowest reported among bacteria, Ureaplasma urealyticum apart. This genome contains 782 putative coding sequences (CDSs) covering 91.4% of its length and a function could be assigned to 486 CDSs whilst 92 matched the gene sequences of hypothetical proteins, leaving 204 CDSs without significant database match. The genome contains a single set of rRNA genes and only 29 tRNAs genes. The replication origin oriC was localized by sequence analysis and by using the G + C skew method. Sequence polymorphisms within stretches of repeated nucleotides generate phase-variable protein antigens whilst a recombinase gene is likely to catalyse the site-specific DNA inversions in major M.pulmonis surface antigens. Furthermore, a hemolysin, secreted nucleases and a glyco-protease are predicted virulence factors. Surprisingly, several of the genes previously reported to be essential for a self-replicating minimal cell are missing in the M.pulmonis genome although this one is larger than the other mycoplasma genomes fully sequenced until now.

Interactions between mycoplasma lipoproteins and the host immune system.

Mycoplasmas typically have a number of distinct lipoproteins anchored on the outer face of the plasma membrane. These surface antigens have a potent modulin activity and are preferential targets of the host immune response. However, the variation of some of these lipoproteins provides mycoplasmas with an effective means of evading the host immune defence system.

Conformation, pore-forming activity, and antigenicity of synthetic peptide analogues of a spiralin putative amphipathic alpha helix.

Current models depict spiralin as a bitopic transmembrane protein with the transbilayer domain being an amphipathic alpha helix. However, though secondary structure prediction methods suggest a helical conformation for the hypothetical transmembrane segment of spiralin, no potential transmembrane helices could be detected in this protein using the method of Von Heijne (Von Heijne, G. (1992) J. Mol. Biol. 225, 487-494). Therefore, we have reconsidered the spiralin topological model by investigating the properties of the chemically synthesized peptides SM-BC3 (LNAVNTYATLAKAVLDAIQN-NH2) and SC-R8A2 (LNAVNTYATLASAVLEAIKN-NH2), corresponding to the hypothetical transmembrane segments of spiralins of two distinct spiroplasma species. The hydrophobic moment plot method suggests that these spiralin amino acid stretches are class G amphipathic alpha helices (i.e., helices localized on the surface of a globular protein domain). Circular dichroism spectra showed that both peptides have little ordered structure in aqueous solutions but adopt a mainly helical conformation in the presence of 25% trifluoroethanol or in detergent micelles (up to 74% alpha helix). Both peptides formed concentration- and voltage-dependent pores in planar lipid bilayers with a unitary conductance of 130 pS in 1 M KCl and with mean numbers of monomers per conducting aggregates of 6 for SC-R8A2 and 9 for SM-BC3. However, the two peptides displayed a haemolytic activity only at high concentrations (> 250 microM) and reacted with antibodies raised against membrane-bound spiralin. Together with previously published results, these data suggest that spiralin is a monotopic membrane protein anchored at the surface of the spiroplasma cell and that the 20-residue amphipathic segment is most probably a class G helix containing a B-cell epitope.

Purification and characterization of spiralin, the main protein of the Spiroplasma citri membrane.

The membrane proteins from Spiroplasma citri have been resolved into 16 components by SDS-polyacrylamide gel electrophoresis. By this technique it was also shown that the molecular weights of these proteins ranged from 13000 to 160 000. One of the proteins, which had an apparent molecular weight of 26 000 was the most abundant and represented more than 22% of total membrane protein. We have designated this protein spiralin. None of the proteins contained carbohydrate. Spiralin has been isolated by a procedure which involves removal of some membrane proteins with the neutral detergent Tween 20, selective solubilization of the Tween residue in DOC and fractionation of the DOC-soluble material by agarose-suspension electrophoresis. The homogeneity of spiralin was demonstrated by analytical polyacrylamide gel electrophoresis under different conditions and by crossed immunoelectrophoresis. Spiralin appeared to bind less DOC than the other membrane proteins of S. citri. This observation does not imply, however, that the binding of DOC to spiralin is weak. Spiralin was neither soluble in detergent-free buffers nor in Tween 20, which indicated that it is an intrinsic membrane protein. The amino-acid composition of spiralin was quite different from that of the membrane. Spiralin lacked methionine, histidine and tryptophan, and had a low content of glycine, leucine, tyrosine and phenylalanine, but a high content of threonine, alanine and valine.

Solution structure determination by NMR spectroscopy of a synthetic peptide corresponding to a putative amphipathic alpha-helix of spiralin: resonance assignment, distance geometry and simulated annealing.

Spiralin is the major protein of the plasma membrane of several spiroplasmas. Neither the function of this protein nor the crystallographic structure is known. Analysis of the primary structure of spiralin from Spiroplasma melliferum BC3 suggests the presence of an amphipathic peptide in the 143-162 region (Chevalier, C., Saillard, C. and Bové, J.M. (1990) J. Bacteriol. 172, 6090-6097). The structure of a synthetic peptide, H2N-L-N-A-V-N-T-Y-A-T-L-A-K-A-V-L-D-A-I-Q-N-amide, corresponding to this fragment has been examined by 1H-NMR spectroscopy. This 20 amino acid peptide adopts a random coil structure in solution, but the addition of trifluoroethanol stabilizes a structure exhibiting alpha-helical character. The 1H-NMR spectrum has been fully assigned in CF3CD2OD/H2O (30:70, v/v) and the three-dimensional structure has been elucidated using NMR-derived distance information. The calculated structures have been obtained by dynamical simulated annealing or distance geometry followed by simulated annealing. Both sets of structures have been energy-minimized using CHARMm potential. The resulting structures are very similar in terms of constraint violations and energies. It is demonstrated that whereas the first three residues exhibit a large flexibility, the remaining sequence is helical.

Correlation between anti-bacterial activity and pore sizes of two classes of voltage-dependent channel-forming peptides.

Anti-bacterial activities were compared for two series of voltage-dependent pore-formers: (i) alamethicin (Alm) and its synthetic analogs (Alm-dUL) where alpha-amino-isobutyric acid residues (Aibs) were replaced by leucines and selected key residues substituted and (ii) homologous voltage sensors of the electric eel sodium channel (repeats S4L45 (III) and S4L45 (IV)). Spiroplasma melliferum, a bacterium related to the mycoplasmas, was used as a target cell. The data show that with respect to growth inhibition, cell deformation and plasma membrane depolarization, the highest efficient peptide remained natural Alm although the minimal inhibitory concentrations of its Leu analogs were within the same range as the parent molecule, except for Alm-dUL P14A. Thus, as for the pore-forming activity observed in artificial membranes and for the toxicity towards mammalian cells, proline-14 proved to be a critical residue for the anti-bacterial activity of alamethicin. Regarding the sodium voltage sensors, their anti-bacterial efficiency was at least 10 times lower although they promoted spiroplasma cell agglutination. The anti-bacterial activities of the peptides were correlated with their pore-forming properties, especially with the apparent and mean number of monomers per conducting aggregate () when both peptide families were considered and, secondly, with mean open times (tau(o)) within each family. This suggests that although they may form 'raft-like' structures, the mechanism underlying anti-bacterial activity of Alm and its active analogs, as well as the S4L45 voltage sensors with the S. melliferum plasma membrane, is predominantly through pore-formation according to the 'barrel-stave' mechanism.

Effects on mollicutes (wall-less bacteria) of synthetic peptides comprising a signal peptide or a membrane fusion peptide, and a nuclear localization sequence (NLS) -- a comparison with melittin.

In order to investigate the effect of primary amphipathic peptides on mollicutes (wall-less bacteria), we have synthesised five molecules (P1, P2, P3, JM123, and JM133) comprising a 16 to 18-residue hydrophobic sequence and the nuclear localization sequence (NLS) PKKKRKV of simian virus 40 large-T antigen, C-terminated by a cysteamide group. The hydrophobic cluster was in P1 the signal sequence of the heavy chain of Caiman crocodilus immunoglobulin G and in JM123 the fusion peptide of human immunodeficiency virus 1 glycoprotein gp41 in which phenylalanine7 was replaced by a tryptophan residue. The homologues P2, P3, and JM133 were obtained by slight alterations of these sequences. Circular dichroism spectroscopy revealed that, in liposomes, P-series peptides were mainly under the form of beta-sheets whereas JM-series peptides displayed a high proportion of turns. These peptides proved to be bactericidal for some mollicutes, notably Acholeplasma laidlawii, but were much less potent than melittin. Furthermore, their antibiotic activity was independent of the average thickness of the plasma membrane hydrophobic core whilst that of melittin was inversely related to the thickness. Melittin and the synthetic peptides abolished spiroplasma cell motility and helicity, but only melittin and P-series peptides split the cells into globular forms displaying an average diameter of ca. 1 microm. In contrast to melittin, the synthetic peptides agglutinated spiroplasmas, suggesting that their polycationic NLS was exposed on the cell surface. P-series peptides decreased, though less efficiently than melittin, A. laidlawii and Spiroplasma melliferum membrane potential (delta psi) and transmembrane pH gradient (delta pH), at concentrations much lower than their minimal inhibitory concentrations whilst JM-series peptides had no effect on delta psi and delta pH in the same conditions. Actually, the bactericidal activity of these peptides towards mollicutes was proportional to their ability to collapse the electrochemical transmembrane potential.

Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family.

Dermaseptins are a family of cationic (Lys-rich) antimicrobial peptides that are abundant in the skin secretions of the arboreal frogs Phyllomedusa bicolor and P. sauvagii. In vitro, these peptides are microbicidal against a wide variety of microorganisms including Gram-positive and Gram-negative bacteria, yeasts, protozoa and fungi. To date, 6 dermaseptin B mature peptides, 24-34 residues long, 2 dermaseptin B cDNAs and 2 gene sequences have been identified in P. bicolor. To assess dermaseptin related genes further, we screened a P. bicolor genomic library with 32P-labeled cDNAs coding either for prepro-dermaseptins B1 or B2 (adenoregulin). A gene sequence was identified that coded a novel dermaseptin B, termed Drg3, which exhibits 23-42% amino acids identities with other members of the family. Analysis of the cDNAs coding precursors for several opioid and antimicrobial peptides originating from the skin of various amphibian species revealed that the 25-residue preproregion of these preproforms are all encoded by conserved nucleotides encompassed by the first coding exon of the Drg3 gene. Synthetic dermaseptin Drg3 exhibited a bactericidal activity towards several species of mollicutes (wall-less eubacteria), firmicutes (Gram-positive eubacteria), and gracilicutes (Gram-negative eubacteria), with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 microM. Experiments performed on Acholeplasma laidlawii cells revealed that this peptide is membranotropic and that if efficiently depolarizes the plasma membrane.

Membrane permeabilisation and antimycoplasmic activity of the 18-residue peptaibols, trichorzins PA.

The membrane permeabilisation properties of six linear natural 18-residue peptaibols, termed trichorzins PA, have been assessed on liposomes and on mollicutes (trivial name, mycoplasmas), a class of parasitic bacteria characterized by a small genome, the lack of a cell wall, a minute cell size, and the incorporation in their plasma membrane of exogenously supplied cholesterol. The trichorzins PA used in this study (PA II, PA IV-VI, PA VIII, and PA IX) differ between them by amino acid or amino alcohol substitutions at positions 4, 7, and 18, and form slightly amphipathic alpha-helices. They proved bactericidal for mollicutes belonging to the genera Acholeplasma, Mycoplasma, and Spiroplasma, with minimal inhibitory concentrations (3.12

Plasma disappearance of albumin and impact of capillary thickness in idiopathic dilated cardiomyopathy and after heart transplantation.

BACKGROUND-The increased plasma disappearance of albumin has previously been described in decompensated congestive heart failure (CHF); this disappearance normalized after diuretic treatment. Cardiac transplantation (HTX) and current medical treatment affect microvascular structure and function. We investigated the plasma disappearance of albumin and the impact of microvascular thickness and electrostatic properties in patients with compensated CHF and after HTX. METHODS AND RESULTS-The fraction of intravascular albumin that passes to the extravascular space per unit time, as determined from the plasma disappearance of intravenously injected (131)I-labeled albumin, was increased to 7.8+/-1.7% in 16 patients with CHF compared with 18 controls (6.5+/-1.9%, P<0.05); these levels normalized after HTX (5.8+/-2.6%, P<0.01, n=17). The change in ratio between (131)I-albumin and simultaneously injected negatively charged glycosylated (125)I-albumin (selectivity index, >1/hour in controls) was lower in patients with HTX (0.993+/-0. 022/hour) than in controls (1.008+/-0.019/hour; P<0.05), which indicated a relatively increased plasma disappearance of negatively charged albumin in HTX patients. Capillary basement membrane thickness was evaluated semiquantitatively from skin biopsies and showed no difference in the 3 groups (control, CHF, and HTX patients). However, in all 3 study groups, subjects with thicker capillary basement membranes had lower albumin escape rates (6.1+/-1. 8%, n=32, versus 7.6+/-2.6% in subjects without thickening of capillary basement membranes, n=19; P<0.05). CONCLUSIONS-The plasma disappearance of albumin increased in patients with compensated CHF and it normalized after HTX. The present normalized capillary basement thicknesses in patients with CHF and the direct association between this parameter and plasma albumin disappearance indicate that previous compensatory microvascular basement membrane growth results in restricted permeability. Microvascular electrostatic properties did not relate to plasma albumin disappearance.

Altered peripheral vasodilator profile of nitroglycerin during long-term infusion of N-acetylcysteine.

OBJECTIVES: The aim of this study was to compare the short- and long-term effects of intravenous nitroglycerin plus placebo and nitroglycerin plus N-acetylcysteine on peripheral arteries, veins and microcirculation in humans. BACKGROUND: The thiol donor N-acetylcysteine may potentiate the hemodynamic response to nitrates in nitrate-tolerant and nontolerant patients. The vascular changes responsible for this effect are not clear. METHODS: Eight male volunteers were treated with nitroglycerin (0.1 microgram/kg per min) combined with N-acetylcysteine (2 g intravenously, followed by 5 mg/kg per h) or placebo for 23 h in a double-blind, randomized, crossover study. Venous volume, the diameter of the radial and temporal arteries, calf blood flow and subcutaneous blood flow were measured at baseline and repeated after 1 and 23 h of infusion. RESULTS: Prolonged coadministration of N-acetylcysteine and nitroglycerin potentiated the acute venodilator effect of nitroglycerin as estimated by changes in venous volume (nitroglycerin plus N-acetylcysteine, 4.45 +/- 0.36 ml/100 g; nitroglycerin plus placebo, 3.65 +/- 0.46 ml/100 g, mean +/- SEM, p < 0.05) and prevented development of tolerance as seen after 23 h of treatment with nitroglycerin plus placebo (4.35 +/- 0.25 vs. 3.47 +/- 0.41 ml/100 g, p < 0.05). N-acetylcysteine had no effect on nitroglycerin-induced changes in arterial diameters (p > 0.05) but significantly increased microcirculatory subcutaneous blood flow after 1 h (nitroglycerin plus N-acetylcysteine: 6.3 +/- 1.3 ml/100 g per min vs. nitroglycerin plus placebo: 3.5 +/- 0.3 ml/100 g per min, p < 0.05) and after 23 h (4.4 +/- 0.6 vs. 3.1 +/- 0.5 ml/100 g per min, p < 0.05). CONCLUSIONS: The results suggest that coadministration of nitroglycerin and N-acetylcysteine in humans 1) potentiates and preserves nitroglycerin-induced venodilation and 2) augments the effect of nitroglycerin on small resistance vessels (regulating subcutaneous blood flow) without affecting the response to nitroglycerin in middle-sized arteries. Both the development of nitrate tolerance and the administration of N-acetylcysteine significantly change the normal vasodilator profile of nitroglycerin in humans.

Orthotopic cardiac transplantation reverses abnormal reflex regulation of the microvasculature in the lower leg.

OBJECTIVE: The aim was to investigate the effect of cardiac transplantation on reflex control of lower leg subcutaneous blood flow. METHODS: The reflex regulation of subcutaneous blood flow of the lower leg was studied in 11 patients following orthotopic cardiac transplantation, in 11 patients with severe congestive heart failure (New York Heart Association functional class III or IV), and in 11 healthy subjects. Four patients were studied before and after cardiac transplantation. Cause of heart failure was classified as idiopathic dilated cardiomyopathy in all heart failure patients and in all the cardiac transplant patients before transplantation. Blood flow was measured by the local 133xenon washout method in the supine position and during 45 degrees head up tilt. RESULTS: When performing head up tilt without activation of the local nervous venoarteriolar axon reflex in patients with congestive heart failure, the relative subcutaneous blood flow increased abnormally, by 50(SD 25)%, but in patients after cardiac transplantation a normal decrease was seen [-28(13)%, p < 0.001]. The responses in the transplant group were similar to those observed in normal controls with a decrease in blood flow [-32(15)%; NS]. Head up tilt with simultaneous activation of the local venoarteriolar axon reflex increased blood flow [31(22)%] in patients with heart failure as compared to the decrease in blood flow found in the transplants [-44(17)%, p < 0.001]. The decrease of blood flow was not significantly different between the transplant group and control subjects [-53(19)%; NS]. CONCLUSIONS: These results indicate that abnormal reflex regulation in severe congestive heart failure with peripheral vasodilation of the lower leg during orthostasis is reversed and even normalised after cardiac transplantation. The haemodynamic consequence may be a regaining of an oedema-protective mechanism that eliminates the stress (capillary hypertension) on the microcirculation seen in severe heart failure.

Plasma endothelin in congestive heart failure: effect of the ACE inhibitor, fosinopril.

OBJECTIVES: The study evaluates the influence of treatment with the angiotensin-converting enzyme inhibitor, fosinopril, on the plasma endothelin level in patients with congestive heart failure, and the relationship between plasma endothelin and clinical study parameters (bicycle exercise test, echocardiography, heart failure score and blood pressure). METHODS: Plasma endothelin was measured in 34 patients with moderately severe congestive heart failure at randomisation in the fosinopril/placebo-controlled study 'Fosinopril Efficacy and Safety Trial' and at the end of the 12-week study period. RESULTS: The patients had elevated pre-treatment plasma endothelin concentrations (3.5 +/- 1.2 pg/ml, mean +/- s.d., n = 34) compared with healthy volunteers (2.0 +/- 0.4 pg/ml, n = 21, P < 0.0001). Treatment with fosinopril for 12 weeks lowered plasma endothelin from 3.5 +/- 1.2 to 2.5 +/- 0.7 pg/ml (m = 18, P < 0.005), in contrast to the non-significant increase in the placebo-treated group 3.5 +/- 1.3 to 4.3 +/- 2.4 pg/ml, n = 16). A multiple regression analysis for baseline study parameters, demonstrated a significant relationship between plasma endothelin and exercise test duration and a composite heart failure score classification (r = 0.53, P < 0.001). CONCLUSIONS: Treatment of patients with congestive heart failure with the angiotensin-converting enzyme inhibitor, fosinopril, reduce the elevated plasma endothelin level to normal values. The relation between plasma endothelin and clinical parameters indicates that endothelin may play a pathophysiological role in the progression of congestive heart failure.

Characterization of the Pasteurella multocida skp and firA genes.

A 2.9-kb fragment of the Pasteurella multocida (Pm) genome encoding proteins p25 (25 kDa) and p28 (28 kDa) has previously been cloned and expressed in Escherichia coli (Ec). In the present paper, the nucleotide (nt) sequence of a 1.8-kb subfragment encoding the two proteins is described. The cloned fragment contains three open reading frames (ORFs). ORF1 is incomplete. ORF2 is homologous to the skp gene of Ec. ORF3 overlaps ORF2 and is highly homologous to the firA gene of Ec. The skp and firA genes are part of an operon governing the first steps of lipid A synthesis. Comparing the nt sequence with the N-terminal sequences of p25 and p28 revealed that the two proteins are encoded by ORF2 (skp). The preprotein p28 is converted into p25 by cleavage of a 23-amino-acid leader peptide. Though it serologically cross-reacts with porin H of Pm, p25 is not related to known bacterial porins.

Adsorption of proteins from the Spiroplasma citri cell membrane by magnesium lauroyl-sarcosinate crystals.

Interactions between the ionic detergent Sarkosyl (sodium lauroyl sarcosinate), Mg2+ ions, and the Spiroplasma citri cell membrane were analyzed microscopically and electrophoretically. Studies were performed under conditions where membrane proteins were apparently not released from the membrane by the detergent (molar ratio of MgCl2/Sarkosyl = 0.5). Although the S. citri membrane interfered with the crystallization phenomenon to some extent, the formation of Sarkosyl-Mg2+ crystals occurred regardless to the sequence of addition of the three components. Concomitantly the structure of the membrane disintegrated and membrane components were adsorbed to the crystal surfaces. The membrane protein fraction bound to the crystals was composed of the majority of the putatively intrinsic polypeptides, including the amphiphilic protein spiralin, and several extrinsic polypeptides. The polypeptide compositions of M-bands (crystal fractions loaded with membrane material) prepared from S. citri cells and from isolated S. citri membranes were similar, as shown by sodium dodecyl-sulfate electrophoresis and crossed immunoelectrophoresis. These results show that, the S. citri cell membrane, in contrast to bacterial membranes, is not protected from the effect of Sarkosyl by Mg2+ ions.

Highly selective extraction of spiralin from the Spiroplasma citri cell membrane with alkyl-N-sulfobetaines.

The extraction of proteins from the membrane of the mollicute (mycoplasma) Spiroplasma citri by sodium N-dodecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB12) and sodium N-tetradecyl-N,N-dimethyl-3-amino-1-propane sulfonate (SB14) was studied with electrophoretic methods. The membranes were prepared by osmotic lysis of the cells and depleted of the bulk of extrinsic proteins. It was possible to extract up to 35 and 45% of membrane proteins with SB12 and SB14, respectively. Maximal yield was obtained in both cases with detergent concentrations greater than or equal to 5 mumoles/mg of membrane protein. Spiralin, the major protein in the S. citri membrane, was highly selectively solubilized without the loss of antigenicity, with a yield of about 90% with SB12 and close to 100% with SB14, for a detergent concentration greater than or equal to 0.2 M. The degree of selectivity in favour of spiralin was higher with SB12 (purity approximately equal to 70%) than with SB14 (purity approximately equal to 50%). Treatment of the S. citri membrane with high concentrations of SB12 is a simple and fast procedure for partial purification of spiralin. This example shows that, in some cases, it should be possible to modulate the selectivity of the extraction of membrane proteins simply by varying the relative concentration of detergent.

Solubilization of Spiroplasma citri cell membrane proteins with the anionic detergent sodium lauroyl-sarcosinate (Sarkosyl).

1. Up to 90 per cent of the membrane proteins from Spiroplasma citri could be solubilized with the anionic detergent Sarkosyl (sodium lauroyl-sarcosinate). Maximal solubilization was obtained with 6 to 20 mumoles of of detergent per mg of membrane protein. The insoluble residue, comprising about 10 per cent of the membrane protein, contained mainly the protein spiralin, which is quantitatively the major one of this membrane. 2. Mg2+ ions completely prevented solubilization of the membrane proteins at a molar ratio of MgCl2/Sarkosyl greater than 0.5. 3. The selectivity of Sarkosyl was also tested at low detergent concentrations and in the presence of Mg2+ ions. Spiralin was the least soluble protein also under these conditions. Other proteins were not selectively solubilized. 4. An electrophoretical and immunoelectrophoretical approach was used to study the interaction between Sarkosyl and membrane proteins. The results indicated that Sarkosyl should be considered as a mild detergent which usually solubilizes membrane proteins without gross donformational changes. This hypothesis was supported by experiments with a membrane-bound enzyme in the presence of Sarkosyl.

Cloning and expression of two Pasteurella multocida genes in Escherichia coli.

A library of cloned Pasteurella multocida (toxigenic strain 9222, serotype D2) genomic sequences was constructed in Escherichia coli by incorporating TaqI digestion fragments into the plasmid vector pUC19. Immunological screening with antibodies directed against porin H, the major protein of the P multocida outer membrane, allowed the identification of a recombinant plasmid containing a 2.9-kbp DNA insert. This plasmid encoded the synthesis of two polypeptides, p25 (25 kDa) and p28 (28 kDa) which were detected in the different compartments of the E coli transformant. The peptide p25 was more abundant in the periplasm whereas p28 was mainly found in the cell envelope and in the cytosol. Immunological analysis indicates that p25, in contrast to p28, is antigenically related to porin H of P multocida. The expression in E coli of the gene encoding p28 was enhanced by induction of the lac promoter.

Plasma clearance of polyfructosan and extracellular body fluid distribution in idiopathic dilated cardiomyopathy and after heart transplantation.

The total extracellular fluid volume and distribution in plasma and interstitial spaces, and the microvascular permeability properties were studied in 16 nonedematous patients with congestive heart failure (CHF) due to idiopathic dilated cardiomyopathy and 17 such patients who underwent heart transplantation (HT) by analyzing the 3-hour plasma disappearance curve of polyfructosan. Eighteen healthy subjects served as controls. Polyfructosan (3.5 kD) is an extracellular marker and inulin analog transported almost solely by diffusion. The initial capillary membrane plasma clearance (i.e., the permeability-surface area product), the interstitial plasma clearance determined at 10 minutes (clearance[10), and the extracellular volume were determined from the polyfructosan curves. I-131-albumin was used as a plasma volume reference. Permeability-surface area product was elevated in both patient groups (6.6 +/- 1.9 ml/ kg/min in the CHF group and 6.7 +/- 2.0 ml/kg/min in the HT group vs 5.1 +/- 1.3 ml/kg/min in controls, p <0.01 for both), whereas clearance(10) was normalized in the HT group (4.5 +/- 0.9 ml/kg/min in the HT group, 4.4 +/- 0.7 ml/kg/min in controls vs 5.0 +/- 0.9 ml/kg/ min in the CHF group, p <0.1 and p <0.05, respectively). The normalization of interstitial plasma clearance of polyfructosan was associated with time since HT (r = 0.49, p <0.05). Plasma volumes were similar in all 3 groups (41 +/- 8 ml/kg in controls, 44 +/- 13 in the CHF group and 39 +/- 8 in the HT group). In contrast, total extracellular volume was elevated in both patients groups (177 +/- 27 ml/kg in the CHF group and 173 +/-27 in the HT group vs 152 +/- 12 in controls, p <0.01). The results strongly suggest a microvascular permeability defect in both patient groups that perhaps plays a role in the extravascular distribution of the excess extracellular fluid volume.

Evidence of increased microvascular resistance and arteriolar hyalinosis in skin in congestive heart failure secondary to idiopathic dilated cardiomyopathy.

Does nervous microvascular stress from backward cardiac failure and abnormal baroreceptor-mediated vasodilation in the upright position alter the microvascular resistance and structure of the resistance vessels with time? The minimal vascular resistance and structure of the terminal arterioles were measured in skin at the dorsum of the foot in 14 healthy subjects, in 12 patients with short-term congestive heart failure (CHF) (New York Heart Association functional class greater than or equal to II for less than 1 year), and in 14 with long-term CHF (New York Heart Association functional class greater than or equal to II for greater than 1 year). Blood flow was measured by the local technetium-99m-pertechnetate washout method in a vascular bed paralyzed by histamine before, during and after 3 to 5 stepwise increases of external counter pressure. Minimal vascular resistance was calculated from the relation between blood flow and applied pressure. Minimal vascular resistance was significantly increased in both short-term (9.0 +/- 1.9 mm Hg.ml-1.100 g.min; p = 0.0003) and long-term (9.1 +/- 3.6 mm Hg.ml-1.100 g.min; p = 0.008) CHF patients compared with that in healthy control subjects (6.0 +/- 1.7 mm Hg.ml-1.100 g.min). Structural microangiopathy (increased hyalinosis of the basement membranes in the terminal arterioles) was found in skin biopsies in 21 of 24 patients with CHF in whom biopsies were obtained, but in none of the 14 control subjects (p less than 0.002). Multiple regression analysis demonstrated a weak but significant direct association between minimal vascular resistance and the degree of structural microangiopathy (p less than 0.03; r = 0.45).(ABSTRACT TRUNCATED AT 250 WORDS)