Lysosomal accumulation and recycling of lipopolysaccharide to the cell surface of murine macrophages, an in vitro and in vivo study.

In this study, we detailed in a time-dependent manner the trafficking, the recycling, and the structural fate of Brucella abortus LPS in murine peritoneal macrophages by immunofluorescence, ELISA, and biochemical analyses. The intracellular pathway of B. abortus LPS, a nonclassical endotoxin, was investigated both in vivo after LPS injection in the peritoneal cavity of mice and in vitro after LPS incubation with macrophages. We also followed LPS trafficking after infection of macrophages with B. abortus strain 19. After binding to the cell surface and internalization, Brucella LPS is routed from early endosomes to lysosomes with unusual slow kinetics. It accumulates there for at least 24 h. Later, LPS leaves lysosomes and reaches the macrophage cell surface. This recycling pathway is also observed for LPS released by Brucella S19 following in vitro infection. Indeed, by 72 h postinfection, bacteria are degraded by macrophages and LPS is located inside lysosomes dispersed at the cell periphery. From 72 h onward, LPS is gradually detected at the plasma membrane. In each case, the LPS present at the cell surface is found in large clusters with the O-chain facing the extracellular medium. Both the antigenicity and heterogenicity of the O-chain moiety are preserved during the intracellular trafficking. We demonstrate that LPS is not cleared by macrophages either in vitro or in vivo after 3 mo, exposing its immunogenic moiety toward the extracellular medium.     

Characterization of the neutral aminopeptidase activity associated to the mouse thymocyte-activating molecule

We previously characterized a dimeric, Mr = 115,000, developmentally regulated mouse T cell-activating molecule (THAM). We show in this report that the THAM-specific mAb H194-112 exhibits strong reactivity with several nonlymphoid tissues including polarized enterocytes from small intestine, kidney cortical tubuli, and liver bile ductuli, as well as kidney glomeruli and lung alveoloar pneumocytes. Both the tissue distribution and the structural features of THAM made it likely that this molecule belongs to the ectoenzyme family. This was confirmed by the following experimental evidence: 1) the H194-112+ molecules from enterocyte brush borders (BB) or M14.T thymoma cells were recognized by several antisera specific for intestinal aminopeptidase N (AP-N); 2) mAb H194-112 was found to immunodeplete the AP-N activity from both thymic or enterocyte BB detergent extracts; 3) the hydrophilic, Mr = 115,000 form obtained by papain treatment of thymoma or enterocyte BB could be immunopurified on H194-112 column and exhibited, after hypotonic elution, strong enzymatic activity on the AP-N substrates (i.e., leucyl or alanyl beta-derivatives); 4) mAb H194-112 was found to inhibit the AP-N activity when assayed on alanyl but not leucyl beta-naphthylamide substrate; and 5) preincubation of AP-N with mAb H194-112 prevented the inhibiting effects of bestatin and D,L-methionyl hydroxamate on AP-N activity. These data add a new member to the list of functional ectoenzymatic markers of lymphoid cells (i.e., CD10, CD13, CD26, CD55, and CD73). In view of the known immunomodulating properties of bestatin, one may speculate that the T cell-activating effects of mAb H194-112 is related to an impairment of a surface enzymatic function regulating lymphoid cell activation.     

Meiofauna associated with a Pacific coral reef in Costa Rica

The meiofauna of two coral reef habitats at Isla del Naño, Costa Rica was studied over a one year period. The dominant groups were: Foraminifera (21.2%), Copepoda (19.7%), Nematoda (19.1%), Gastropoda (16.5%), Polychaeta (7.2%) and Bivalvia (6.6%). The highest diversity was found in coarse, heterogeneous sands with the highest percentage of carbonates. The meiofauna showed a high degree of horizontal aggregation, which is a characteristic pattern for macro- and meiofauna in sediments of variable composition. No vertical variation in distribution was evident, probably due to the deep location of the Redox Potential Discontinuity layer. The total densities of organisms found in this study (99 to 575 ind/10 cm²) are low compared with densities in similar non-reefal sands (7 to 6116), and from fine sediments (80 to 17 000), but are comparable to densities found in other reef areas (39 to 609.5 ind/10 cm²). This is the first report on meiofauna from the eastern Pacific, and the first time that foraminiferans are the dominant group.     

Tyrosination-detyrosination of tubulin and microtubules during the development of chick erythrocytes

The protein composition of nuclear matrices containing different amount of DNA was examined. It was found that, in matrices containing 2% to 80% of total DNA, the quantity of DNA-bound proteins remains relatively constant varying from 10% to 15% of total nuclear proteins. Electrophoretic patterns do not differ substantially, but autoradiograms with in vitro ¹²⁵I labelled proteins show quantitative variations in the actin content. Application of radioimmunoassay (RIA) enabled to determine the exact content of actin in GAT nuclei and nuclear matrices – 5 g/ml in nuclei, of which 50% are bound to DNA and 3001o being a component of the protein part of the nuclear matrix. These results are supported by electron microscopic data, where immunogold technique was performed on thin sections and spread material. The applied methods suggest that part of the nuclear actin is tightly bound (resistant to 2 M NaCI) to DNA and represents a component of the internal nuclear matrix.     

Further characterization of an early expressed glycoprotein of the rabbit small intestinal brush border. Its interaction with some hydrolases

Mouse 23 B 921 monoclonal antibody recognized a rabbit brush-border antigen with an apparent molecular mass 140 kDa (140-kDa Ag) which, unlike most hydrolases, is expressed in the poorly differentiated crypt cells of the small intestine. Immunoelectron microscopy of brush-border vesicles showed that the 23 B 921 bound to an epitope localized on the outside of the membrane. As is the case with hydrolases the external domain of the 140-kDa Ag constitutes the main part of the molecule, which can be released by papain treatment of brush-border vesicles. The presence of a small hydrophobic domain, anchoring the molecule into the membrane and responsible for its amphipatic character, was shown by its affinity for Triton-X114 micelles. The topological organization in the membrane of 140-kDa Ag seemed to be identical to that of hydrolases. Unlike hydrolases, however, the native structure of the antigen was found from its sensitivity to proteolysis to be very dependent on its integration into the lipid bilayer. Nevertheless, detergent-extracted 140-kDa Ag can be purified by immunoaffinity chromatography although it cannot be stocked for more than 48 h even in the presence of protease inhibitors. The carbohydrate moiety of 140-kDa Ag, bearing the human blood group A determinant, amounts to 20% of the total molecular mass. The existence of some privileged relationship was established between 140-kDa Ag and hydrolases: in the membrane, hydrolases protect the 140-kDa Ag from papain action; after detergent extraction, 140-kDa Ag is strongly associated with several hydrolases particularly disaccharidases.     

A molecular, genetic and immunological approach to the functioning of colicin A, a pore-forming protein

We have constructed, by recombinant DNA techniques, one hybrid protein, colicin A-beta-lactamase (P24), and two modified colicin As, one (P44) lacking a large central domain and the other (PX-345) with a different C-terminal region. The regulation of synthesis, the release into the medium and the properties of these proteins were studied. Only P44 was released into the medium. This suggests that both ends of the colicin A polypeptide chain might be required for colicin release. None of the three proteins was active on sensitive cells in an assay in vivo. However, P44 was able to form voltage-dependent channels in phospholipid planar bilayers. Its lack of activity in vivo is therefore probably caused by the inability to bind to the receptor in the outer membrane. PX-345 is a colicin in which the last 43 amino acids of colicin A have been replaced by 27 amino acids encoded by another reading frame in the same region of the colicin A structural gene; it was totally unable to form pores in planar bilayers at neutral pH but showed a very slight activity at acidic pH. These results confirm that the C-terminal domain of colicin A is involved in pore formation and indicate that at least the 43 C-terminal amino acid residues of this domain play a significant role in pore formation or pore function. Fifteen monoclonal antibodies directed against colicin A have been isolated by using conventional techniques. Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A. In addition, the altered forms of the colicin A polypeptide were used to map the epitopes of ten monoclonal antibodies reacting specifically with colicin A. Some of the antibodies did not bind to colicin A when it was pre-incubated at acidic pH suggesting that colicin A undergoes conformational change at about pH 4. The effects of monoclonal antibodies on activity in vivo of colicin A were investigated. The degree of inhibition observed was related to the location of the epitopes, with monoclonal antibodies reacting with the N terminus giving greater inhibition. The monoclonal antibodies directed against the C-terminal region promoted an apparent activation of colicin activity in vivo.     

The zygosity in the human HL-A transplantation system

Summary If by serological methods of tissue typing one antigen only is detected in one or both of the loci LA and 4 of the histocompatibility system HL-A, the question arises whether it is homozygous or heterozygous. The probability thereof is calculated. It depends on the frequency of the known and unknown genes in the HL-A-system. If e.g. the HL-A type of an examinee is 1,3,8 this subject could be homozygous 1,3,8,8 or heterozygous (with an unknown antigen X) 1,3,8,X. The frequencies of homozygous and heterozygous individuals are calculated in all possible combinations of the LA and 4 loci. The results justify always for both the LA-and 4 locus the assumption of heterozygosity, with the one exception that the gene HL-A 2 is under consideration. There the calculations result in a value of 49% for homozygosity (2,2) and 51% for heterozygosity (2,X). Neither alternative is in this case essentially more probable.For transplantations generally heterozygosity can be assumed — with the exception of HL-A 2 — and therefore the minor grade of histocompatibility.National Blood Group Reference Laboratory — WHO (Director: Prof. Dr. P. Speiser)     

Time-Dependent Increase in Protein Phosphorylation Following One-Trial Enhancement in Hermissenda

Abstract: One-trial conditioning of the nudibranch mollusk Hermissenda produces short- and long-term changes in excitability (enhancement) of identified sensory neurons. To investigate the biochemical mechanisms underlying this example of plasticity, we have examined changes in protein phosphorylation at different times following the in vitro conditioning trial. Changes in the incorporation of ³²PO₄ into proteins were determined using two-dimensional polyacrylamide gel electrophoresis, autoradiography, and densitometry. Conditioning resulted in increases in levels of several phosphoproteins, five of which, ranging in apparent molecular mass from 22 to 55 kDa, were chosen for analysis. The increased phosphorylation of the 46- and 55-kDa phosphoproteins detected 2 h postconditioning was significantly greater than the level of phosphorylation detected in an unpaired control group, indicating that long-term enhancement is pairing specific. Statistically significant increases in phosphorylation as compared with the control group that received only light were detected immediately after conditioning (5 min) for the 55-, 46-, and 22-kDa phosphoproteins, at 1 h for the 55- and 46-kDa phosphoproteins, and at 2 h for the 55-, 46-, and 22-kDa phosphoproteins. The 46- and 55-kDa phosphoproteins are putative structural proteins, and the 22-kDa phosphoprotein is proposed to be a protein kinase C substrate previously identified in Hermissenda following multitrial classical conditioning. Time-dependent increases in protein phosphorylation may contribute to the induction and maintenance of different memory stages expressed in sensory neurons after one-trial conditioning.     

Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts.

A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or lactoferricin B-treated LPS micelles of S. montevideo or B. abortus mimicked the effects observed with intact cells, and this was confirmed by using micelle hybrids of B. abortus and S. montevideo LPSs. The results showed that LPS is the main cause of B. abortus' resistance to bactericidal cationic peptides, the OM-disturbing action of divalent cationic chelants, and OM permeability to hydrophobic substances. It is proposed that these three features are related to the ability of Brucella bacteria to multiply within phagocytes.