Ultrastructural studies on the localization of IgG in the aortic endothelium and subendothelial intima of atherosclerotic and nonatherosclerotic rabbits

Immunoelectron microscopy with peroxidase-conjugated Fab fragments of anti-IgG was used for studying the localization of IgG in the aortic endothelium and subendothelial intima of atherosclerotic and nonatherosclerotic rabbits. Small amounts of IgG were found in the cell coat, in caveolae and vesicles, and also in intercellular clefts of endothelial cells from normocholesterolemic rabbits. Injured endothelial cells exhibited prominent accumulations of IgG in the cytoplasmic matrix, possibly due to leakage through plasma membrane defects. In atherosclerotic lesions from hypercholesterolemic rabbits, there was a striking increase in the amount of IgG-reactive material in the cell coat and vesicles of intact endothelial cells. Also in these animals, injured endothelial cells were characterized by a cytoplasmic IgG accumulation. There were prominent IgG depositions in the subendothelial zone of the lesions. IgG was adhering to collagen fibers, and also coating the surfaces of subendothelial foam cells. The pathophysiological significance of an interaction between such intimal IgG and phagocytes is discussed.     

Suggestive evidence for a direct innervation of mucosal mast cells

Mast cells are often observed near nerves and functional evidence suggests an innervation of these cells. In the present ultrastructural study, nerve terminals containing many small clear vesicles and a few large vesicles with dense matrix were observed in direct contact with the plasma membrane of mucosal mast cells in the rat ileum, strongly suggestive of a direct innervation.     

Plasma protein accumulation in injured endothelial cells. Immunofluorescent localization of IgG and fibrinogen in the rabbit aortic endothelium.

The relationship between endothelial cell injury in the rabbit aorta, and the distribution of the plasma proteins IgG, fibrinogen, and albumin were studied by immunofluorescence techniques. Normal rabbit aortae, as well as different types of experimentally induced atherosclerotic lesions, were studied. Injured endothelial cells were visualized by a dye exclusion test. Uninjured endothelial cells were found to be free from plasma proteins, except for a thin continuous lining of fibrinogen on the luminal side of the cells. In injured endothelial cells, there was an accumulation of both IgG and fibrinogen. Such cells were preferentially localized to branching-points, and the borders of atherosclerotic lesions. Albumin could neither be demonstrated in injured nor in uninjured cells. These observations indicate an uptake of IgG and fibrinogen by injured endothelial cells. The immunohistochemical method employed may be used to visualize injured endothelial cells.     

Modification of bronchial hyperreactivity after treatment with sodium cromoglycate during pollen season

Repeated bronchial histamine challenges before, during, and after the birch pollen season were performed in 22 allergic patients with bronchial hyperreactivity (BHR) treated for 6 wk with sodium cromoglycate (SCG), 20 mg, four times a day, or placebo in a double-blind, randomized group comparison. Clinical assessments of the asthmatic symptom score and peak expiratory flow revealed less symptoms and less use of bronchodilators in the SCG group. Responsiveness to histamine was significantly increased in the placebo group after 14 days with high pollen counts. After the season there was an immediate return to preseasonal value. There was no change in responsiveness in the SCG group, demonstrating significant protection against pollen-induced increase of BHR. The results support the hypothesis that inhibition of mediator release, which is demonstrated for SCG, leads to a reduction of the nonspecific BHR.     

Lipomatous tumors: a correlative cytologic and histologic study of 27 tumors examined by fine needle aspiration cytology

A correlative cytologic and histologic study of 12 benign lipomatous tumors and 15 liposarcomas (well-differentiated, myxoid, round cell, and pleomorphic) is presented. In two cases the fine needle aspiration material was embedded in Epon for light and electron microscopic examination. Good correlation was found between the histologic and cytologic findings in the fine needle aspiration material. Pitfalls in the cytologic diagnosis of regressively changed lipoma, intramuscular lipoma, angiolipoma, hibernoma, and lipoblastoma, which may lead to an erroneous diagnosis of liposarcoma, are illustrated. The cytologic appearances of the liposarcomas varied with histologic type, although in all of these tumors the main criterion was the presence of atypical multivacuolated lipoblasts with characteristically scalloped nuclei. Staining of the aspirated material with Alcian blue at varying pH levels for characterization of the glycosaminoglycan content may help in the distinction of myxoid liposarcomas from myxoid chondromatous tumors and chordomas. May-Grünewald-Giemsa staining is considered the most useful staining method, while fat staining is considered of limited or no value in the cytologic diagnosis of lipomatous tumors. Epon embedding of fine needle aspirates for light and electron microscopic examination seems to be a useful diagnostic technique.     

Rearrangement of chromosome No. 3 in a case of preleukemia with thrombocytosis

The clinical and cytogenetic findings of a patient with the preleukemic syndrome and a structural rearrangement involving both chromosomes No. 3 are described. The karyotypic abnormality consisted of an insertion of a part of the long arm of one chromosome No. 3 into the other, i.e., ins(3;3)(q27;q21q27). A prominent feature of the bone marrow was a marked megakaryocytic hyperplasia. The platelet count temporarily exceeded 1000 × 10⁹/liter. The findings of the present case, together with similar observations by others, suggest that the long arm of chromosome No. 3 may contain a region involved in the regulation of megakaryopoiesis.     

Immunochemical Properties of the Major Outer Membrane Protein of Vibrio cholerae

Antisera to the major outer membrane protein of Vibrio cholerae (molecular weight, 48,000) raised in rabbits (i) agglutinated several strains of V. cholerae and (ii) immunoprecipitated outer membrane proteins prepared from both the biotypes and serotypes of V. cholerae. Antibodies of all isotypes to the major outer membrane protein were detected in immune human sera by enzyme-linked immunosorbent assay. These results suggest that the major outer membrane protein was the common outer membrane antigen of V. cholerae which was immunogenic in humans.     

Lytic transglycosylase contributes to the survival of lipooligosaccharide-deficient,colistin-dependent Acinetobacter baumannii

ObjectivesThe phenomenon of colistin dependence in Acinetobacter baumannii has been described in a situation in which colistin is now considered as the last resort for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. In this study, we aimed to reveal a gene associated with colistin dependence in A. baumannii.MethodsThe colistin-dependent A. baumannii H08-391D strain was isolated from a patient, and target gene-inactivation mutants were constructed. We investigated the effects of target gene on colistin dependence with quantitative real-time PCR and endotoxin assay. Also, we observed the change of cell morphology by electron microscopy.ResultsThe expression of ACICU_02898, encoding a soluble lytic transglycosylase associated with cell-wall degradation and recycling, was increased by eight-to 42-fold in colistin-dependent mutants, and deletion of ACICU_02898 in a colistin-dependent strain led to colistin susceptibility (MIC = 8 mg/L). Endotoxin activity was significantly low in a colistin-dependent derivative ACICU_02898-inactivated mutant and a complemented mutant. In addition, the ACICU_02898-inactivated mutant showed a highly reduced growth rate. The colistin-dependent derivative and ACICU_02898-inactivated mutant showed clearly distinguished absorption profiles in the red/green fluorescence dot blot with regard to their membrane potential. Electron microscopy revealed that the deletion mutant cells were elongated compared to the colistin-susceptible wild-type strain and colistin-dependent strain.ConclusionsA colistin-dependent A. baumannii strain exhibited a deficiency in its outer membrane integrity and high expression of lytic transglycosylase was required for survival. This study reveals why the colistin-dependent mutant can tolerate high antibiotic concentrations.     

Diverse phenotype in patients with complex I deficiency due to mutations in NDUFB11

Mitochondrial complex I deficiency is the most frequent mitochondrial disorder presenting in childhood and the mutational spectrum is highly heterogeneous. The NDUFB11 gene is one of the recently identified genes, which is located in the short arm of the X-chromosome. Here we report clinical, biochemical, functional and genetic findings of two male patients with lactic acidosis, hypertrophic cardiomyopathy and isolated complex I deficiency due to de novo hemizygous mutations (c.286C > T and c.328C > T) in the NDUFB11 gene. Neither of them had any skin manifestations. The NDUFB11 gene encodes a relatively small integral membrane protein NDUFB11, which is essential for the assembly of an active complex I. The expression levels of this protein was decreased in both patient cells and a lentiviral complementation experiment also supported the notion that the complex I deficiency in those two patients is caused by NDUFB11 genetic defects. Our findings together with a review of the thirteen previously described patients demonstrate a wide spectrum of clinical features associated with NDUFB11-related complex I deficiency. However, histiocytoid cardiomyopathy and/or congenital sideroblastic anemia could be indicative for mutation in the NDUFB11 gene, while the clinical manifestation of the same mutation can be highly variable.     

Outer Membrane Protein Composition in Disease Isolates of Haemophilus influenzae: Pathogenic and Epidemiological Implications

The outer membrane protein composition of 50 disease isolates of Haemophilus influenzae has been determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. All strains, including 28 strains of serotype b, one strain each of serotypes a, c, d, e, and f, and 17 untypable strains, had an outer membrane protein composition typical of gram-negative bacteria, i.e., these membranes contained two to three dozen proteins with four to six proteins accounting for most of their protein content. Variation in the mobility of these major outer membrane proteins from strain to strain was common but not universal; the observed patterns provided useful data and new insight into the epidemiology of type b disease. The basic findings can be summarized as follows: (i) All 50 strains possessed three proteins (one minor and two major) each having identical mobilities. The other proteins, both major and minor, varied in mobility. (ii) All type b strains possessed a fourth (major) protein of identical mobility. (iii) The 28 type b strains, on the basis of the mobility of the six major outer membrane proteins, could be divided into eight subtypes. Of all the other strains examined, both typable and untypable, only the serotype a strain belonged to one of these subtypes. (iv) The untypable strains showed considerable variation in the mobilities of their major outer membrane proteins. Of these 17 strains, 13 had an additional major outer membrane protein not present in encapsulated strains. (v) The outer membrane protein composition of a single strain remained unchanged after many passages on solid media, but varied with the growth phase. (vi) The outer membrane protein composition of isolates obtained from nine patients during an epidemic of type b meningitis varied, indicating that a single strain was not responsible for the epidemic. At least five different strains were responsible for these nine cases. (vii) Identical outer membrane protein compositions were observed in the following: in a type b strain and a mutant of this strain deficient in capsule production, indicating that the level of capsule synthesis is not obviously related to outer membrane protein composition; in type b strains isolated from different anatomic sites of patients acutely ill with meningitis, indicating that the strain associated with bacteremia is the same as that isolated from the cerebrospinal fluid; in type b strains isolated from siblings who contracted meningitis at about the same time, indicating infection with the same strain; and in type b strains isolated from the initial and repeat infection of a single patient, suggesting that reinfection was due to the same strain.     

Expression of Vibrio cholerae zonula occludens toxin and analysis of its subcellular localization

Vibrio cholerae elaborates zonula occludens toxin (Zot), a protein that increases the permeability of small intestinal mucosa by opening intercellular tight junctions. The zot gene is located, together with the genes encoding CT and Ace enterotoxins, within the genome of V. cholerae filamentous phage CTXф. Interestingly, Zot appears to be structurally and functionally related to the gene I product of other filamentous phages and it has been shown to be required for CTXф morphogenesis. In this study we described the cloning of zot in several expression plasmid systems and we examined the subcellular localization of Zot by using affinity purified anti-Zot antibodies. We found that Zot localizes in the V. cholerae cell envelope with M_r45 kDa which is consistent with the predicted primary translation product from the first methionine of zot (44.8 kDa). A second molecule, corresponding to the 33 kDa N-terminal region of Zot, was also detected. Both molecules are exposed at the bacterial cell surface. The production of the 33 kDa Zot, that might represent a processing product, was abolished in mutant ZotG59. N-terminal tagged 6xHis-Zot fusion protein retained the capability to reach the outer membrane and the 6xHis tag was not cleaved off during the translocation to the periplasm, whereas the presence of the tag partially blocked the formation of the 33 kDa molecule. Zot secretion and anchorage to the bacterial outer membrane was also observed in E. coli strains expressing Zot, suggesting that the toxin may be directed to the outer membrane via the same pathway in E. coli and V. cholerae. Zot cleavage might be due to a V. cholerae specific protease activity, since the 33 kDa protein was not efficiently produced in E. coli. On the basis of these data and Zot amino acid sequence analysis, we suggest that while the N-terminal part of the molecule is involved in the morphogenesis of CTXф, the C-terminal region might carry the domain(s) responsible for Zot enterotoxic activity.     

Mechanisms of respiration and phosphorylation in Ascaris muscle mitochondria

In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electron transport this process appears to be coupled to energy conservation at phosphorylation site I. The branchpoint from which electrons are taken from the main respiratory chain to either the alternative oxidase or fumarate reductase is likely to be on the oxygen side of the NADH dehydrogenase segment.Malate and succinate are the only substrates which appreciably support respiration in the mitochondrion of the nematode. Regardless of the presence or absence of oxygen malate is utilized by an oxidation-reduction reaction resulting in the formation of pyruvate, acetate, succinate, propionate and CO₂. In addition, aerobically, hydrogen peroxide is formed as the product of oxygen reduction. Succinate accumulation was found to be significantly higher in the anaerobic as compared to the aerobic incubation mixtures. This effect was accompanied by an increase in anaerobic malate consumption. ATP generation and the formation of pyruvate, acetate and propionate were found to be similar in the presence and absence of oxygen.In malate-supported respiration of intact Ascaris mitochondria reducing equivalents (NADH) are produced exclusively through pyruvate and acetate formation. These enzymatic reactions are functionally coupled to the electron transport-linked reductions of fumarate to succinate and oxygen to hydrogen peroxide, respectively. In accordance with the position of the redox potentials of the fumarate/succinate and O₂/H₂O₂ couples, anaerobic and aerobic respiration was found to be associated with relatively low energy conservation efficiencies. Thus one molecule of ATP was conserved per 2e^? transferred to fumarate or oxygen, respectively. No evidence could be obtained for a significant activity of energy conservation sites II and III and electron transfer through the alternative oxidase pathway was shown not to be coupled to phosphorylation.     

Isolation and Characterization of the Outer Membrane of Borrelia hermsii

The outer membrane of Borrelia hermsii has been shown by freeze-fracture analysis to contain a low density of membrane-spanning outer membrane proteins which have not yet been isolated or identified. In this study, we report the purification of outer membrane vesicles (OMV) from B. hermsii HS-1 and the subsequent identification of their constituent outer membrane proteins. The B. hermsii outer membranes were released by vigorous vortexing of whole organisms in low-pH, hypotonic citrate buffer and isolated by isopycnic sucrose gradient centrifugation. The isolated OMV exhibited porin activities ranging from 0.2 to 7.2 nS, consistent with their outer membrane origin. Purified OMV were shown to be relatively free of inner membrane contamination by the absence of measurable β-NADH oxidase activity and the absence of protoplasmic cylinder-associated proteins observed by Coomassie blue staining. Approximately 60 protein spots (some of which are putative isoelectric isomers) with 25 distinct molecular weights were identified as constituents of the OMV enrichment. The majority of these proteins were also shown to be antigenic with sera from B. hermsii-infected mice. Seven of these antigenic proteins were labeled with [³H]palmitate, including the surface-exposed glycerophosphodiester phosphodiesterase, the variable major proteins 7 and 33, and proteins of 15, 17, 38, 42, and 67 kDa, indicating that they are lipoprotein constituents of the outer membrane. In addition, immunoblot analysis of the OMV probed with antiserum to the Borrelia garinii surface-exposed p66/Oms66 porin protein demonstrated the presence of a p66 (Oms66) outer membrane homolog. Treatment of intact B. hermsii with proteinase K resulted in the partial proteolysis of the Oms66/p66 homolog, indicating that it is surface exposed. This identification and characterization of the OMV proteins should aid in further studies of pathogenesis and immunity of tick-borne relapsing fever.     

Laboratory investigation of a suspected outbreak caused by Providencia stuartii with intermediate resistance to imipenem at a long-term care facility

Background

Providencia stuartii survives well in natural environment and often causes opportunistic infection in residents of long-term care facilities (LTCFs). Clinical isolates of P. stuartii are usually resistant to multiple antibiotics. The bacterium is also naturally resistant to colistin and tigecycline. Treatment of infections caused by carbapenem-resistant P. stuartii is challenging.

Multi-component accumulation of L-[14C]glutamate by rat spinal cord slices

The accumulation of L-[¹⁴C]glutamate by 0.4 mm thick rat spinal cord slices could be described by 4 saturable systems and diffusion. Two of the saturable processes (K_m values 25 and 138 μM) appear to be homologous with the ‘high’ and ‘low’ affinity uptake systems previously demonstrated in the spinal cord in vitro. The remaining two systems were of higher affinity (K_m values 2.4 and 2.7 μM) and may represent uptake or binding processes within the tissue.     

The 97-kD alpha-actinin-like protein in chromaffin granule membranes from adrenal medulla: evidence for localization on the cytoplasmic surface and for binding to actin filaments

α-Actinin, a protein of the contractile apparatus known to be associated with the chromaffin granule membrane, has been partially purified and localized by one-dimensional gel electrophoresis by electrophoretic transfer onto nitrocellulose sheets and detection with antiserum raised against α-actinin purified from bovine skeletal red muscle. The α-actinin-like protein from granule membrane has a molecular weight ofM_r = 97 kD, close to the molecular weight of skeletal muscle α-actinin. and is antigenically related to skeletal muscle α-actinin.Chromaffin granule membrane proteins were separated using two-dimensional gel electrophoresis; electrophoretic maps revealed a high degree of complexity because of the large number of protein species and because of the microheterogeneity of some of these components. In addition, certain components did not focus well and were poorly separated. Nevertheless, the 97-kD α-actinin-like protein and actin were localized on two-dimensional gel electrophoregrams of chromaffin granule membrane proteins. They appeared as minor components, showing great variability depending on media used for preparing granules. Isolation of granules in classical sucrose gradient steps (0.32 M sucrose; 1.6 M sucrose, both buffered with HEPES) resulted in low recovery of the α-actinin-like protein because it detached from the membrane in low ionic strength buffers. Isolation of granules in buffered isotonic KCl media led to an enrichment of the α-actinin-like protein. When granules were prepared in buffered sucrose media containing 2 mM Mg²⁺, there was a marked increase of 97-kD α-actinin-like protein.Labelling experiments with Na[¹²⁵I]lactoperoxidase were carried out on intact granules and broken membranes; in both experiments, the α-actinin-like protein was not found to be labelled, while actin was radio-iodinated with no increase of the radioactivity when comparing intact granule experiment with broken membrane experiment. Mild digestion of intact granules withStreptomyces griseus pronase led to the disappearance of all protein species with a molecular weight above 70 kD and of some unidentified components with molecular weight below 70 kD. The 97-kD α-actinin-like protein and actin were digested. We concluded that α-actinin-like protein and actin are attached to the vesicle membrane facing towards the cytoplasmic side. In addition, the α-actinin-like protein is probably a peripheral membraneassociated protein rather than an integral membrane protein.Chromaffin granule membranes are known to stimulate actin polymerization and to bind actin filaments. Anti-α-actinin antiserum and monospecific anti-α-actinin Fab fragments were found to inhibit by 50% [³H]actin binding to granule membranes. Inhibition was dependent on the amount of antibody present in the incubation medium. Membranes isolated from intact granules which have previously been incubated with pronase in conditions to digest completely α-actinin-like protein as judged from two-dimensional gel electrophoresis, were characterized by a lower (50%) actin binding capacity that we attributed to pronase-resistant binding sites to G-actin. All these data showed that α-actinin-like protein plays a role in the binding of actin filaments to the chromaffin granule membrane, either stabilizing actin nuclei present in this membrane and/or cross-linking actin filaments emerging from the membrane.     

Cloning of thefomAgene,encoding the major outer membrane porin ofFusobacterium nucleatumATCC10953

The major outer membrane protein, FomA, of the Gram-negative human oral pathogenFusobacterium nucleatumfunctions as a porin and is assumed to act as a receptor protein in coaggregation with other oral pathogenic bacteria such asStreptococcus sanguisandPorphyromonas gingivalis. We describe here the cloning offomAfromF. nucleatuminE. coli. Using pGEM3Zf(+), three recombinant plasmids were carrying parts of thefomAgene, but none of these contained regions upstream of the coding sequence. From these plasmids a clone was constructed which contained the wholefomAgene. The ATCC 10953fomAgene was cloned under the phosphate limitation-induciblephoEpromoter, using a vector derived from pACYC184. The protein was found to be incorporated into the outer membrane of the host in an apparently normal manner, as judged by heat-modifiability, trypsin-accessibility, and accessibility to antibodies to the protein in a whole cell enzyme-linked immunosorbent assay. The cloned FomA was found to exhibit pore-forming activity.     

Antibody response of infected mice to outer membrane proteins of Pseudomonas aeruginosa.

The antibody response to outer membrane proteins of Pseudomonas aeruginosa was studied in mice experimentally infected with P. aeruginosa 220. The infection consisted of an abscess established by subcutaneous injection of bacteria. Sera from these mice were analyzed by indirect radioimmunoprecipitation and immunoblot methods for the presence of antibodies to proteins of the isolated outer membrane. Sera from mice 14 days postinfection were shown to contain antibodies directed against proteins that comigrated with the major outer membrane proteins F (porin), H2, and I (lipoprotein). A 16,000-dalton protein that did not appear to be a major outer membrane protein also elicited a significant antibody response in some instances. It is concluded that mice, in response to infection, elicit an immunological response to outer membrane proteins of P. aeruginosa.     

Sequential removal of outer bilayer and apical plasma membrane from the surface epithelial syncytium of Schistosoma mansoni

The outer and inner bilayers of the apical membrane complex of Schistosoma mansoni were sequentially stripped from adult worms by two incubations in 0.1% digitonin solutions. Membrane removal was evaluated by electron microscopy of worms and bilayer material, using Con A-ferritin as a marker for the outer bilayer. Amounts of Con A removed by the digests were measured with a tritiated Con A marker.To measure the purity of the fractions membrane markers were characterised and quantitated for both bilayers. In the absence of the usual enzymatic markers for plasma membrane diazotised [¹²⁵I]-iodosulfanilic acid was used as a marker for the outer bilayer. Alkaline phosphatase and a Na⁺, Mg²⁺-ATPase were localised to the inner bilayer. From these results we can deduce that the inner bilayer is analogous to the typical, apical plasma membrane of other animal epithelia. The outer bilayer does not share these enzymatic similarities. The integrity of the syncytium after removal of the outer bilayer and the increased levels of lactate dehydrogenase in the supernatant after removal of the inner bilayer suggests that the outer bilayer is secondary in maintaining the permeability barrier of the apical membrane complex, with respect to soluble proteins. The possible significance of these results in terms of the destructive action of complement on the parasite are discussed.