Ultrastructal morphology of some prokaryotic microorganisms associated with the hindgut of cockroaches.

Scanning electron microscopy and transmission electron microscopy have been used to visualize the morphology and ultrastructure of two types of microorganisms in the hindgut of the cockroach Blaberus posticus. Both organisms, designated as either short or long rods, are attached to chitinous projections from the gut wall. Micrographs suggest that the organisms are prokaryotic with a cell wall complex characteristic of gram-negative bacteria. However, certain differences were noted between the cell wall complex of the two types. Two forms of the long-rod type were noted, with one form appearing to be a "degenerate" or "transitional" cell. In the degenerate cells, vesicles are observed that often are contiguous with the cytoplasmic membrane. There are indications that the long-rod type may divide by longitudinal fission. Neither the short- nor long-rod type has been cultivated in its respective recognizable form.     

Discovery of three novel coccidian parasites infecting California sea lions (Zalophus californianus), with evidence of sexual replication and interspecies pathogenicity

Enteric protozoal infection was identified in 5 stranded California sea lions (Zalophus californianus). Microscopically, the apical cytoplasm of distal jejunal enterocytes contained multiple stages of coccidian parasites, including schizonts with merozoites and spherical gametocytes, which were morphologically similar to coccidians. By histopathology, organisms appeared to be confined to the intestine and accompanied by only mild enteritis. Using electron microscopy, both sexual (microgametocytes, macrogamonts) and asexual (schizonts, merozoites) coccidian stages were identified in enterocytes within parasitophorous vacuoles, consistent with apicomplexan development in a definitive host. Serology was negative for tissue cyst-forming coccidians, and immunohistochemistry for Toxoplasma gondii was inconclusive and negative for Neospora caninum and Sarcocystis neurona. Analysis of ITS-1 gene sequences amplified from frozen or formalin-fixed paraffin-embedded intestinal sections identified DNA sequences with closest homology to Neospora sp. (80%); these novel sequences were referred to as belonging to coccidian parasites "A," "B," and "C." Subsequent molecular analyses completed on a neonatal harbor seal (Phoca vitulina) with protozoal lymphadenitis, hepatitis, myocarditis, and encephalitis showed that it was infected with a coccidian parasite bearing the "C" sequence type. Our results indicate that sea lions likely serve as definitive hosts for 3 newly described coccidian parasites, at least 1 of which is pathogenic in a marine mammal intermediate host species.     

Polyclonal Antisera To Distinguish Strains and Form Variants of Photorhabdus (Xenorhabdus) luminescens

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode strain NLH-E87.3) and against the primary and secondary forms of P. luminescens PSH (obtained from nematode strain DH-SH1). In double-diffusion tests all form variants of strain PE reacted with the antiserum against the primary form, but each variant produced a different diffusion pattern. The primary and secondary forms of strain PSH were also serologically different. Antiserum 9226 reacted with almost all P. luminescens strains tested, but it reacted differently with each strain in the double-diffusion test, showing that the strains were serologically different. The specificity of the antisera was increased by cross-absorption. After cross-absorption the antiserum against the strain PSH primary or secondary form was specific for that form and did not react with the other form. Using the cross-absorbed antisera in immunofluorescence cell-staining tests, we could distinguish primary and secondary form cells in a mixed strain PSH culture.     

Influence of heat and sodium dodecyl sulfate on the endopeptidase I from Bacillus sphaericus 9602

Endopeptidase I from Bacillus sphaericus is a stable enzyme which retains its activity at 37 degrees C in the presence of sodium dodecyl sulfate. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed two forms of the enzyme: an active, fast-running form, for the enzyme preheated at 37 degrees C and a denatured, slow-running form, for the enzyme preheated at 100 degrees C. Such behavior is similar to that of the "heat-modifiable" outer membrane proteins from gram-negative bacteria. In the absence of sodium dodecyl sulfate, endopeptidase I aggregated in an enzymatically active dimer, with an apparent molecular weight of 90,000 daltons, which could be the native form of the enzyme.     

The ultrastructural characteristics of the corticotropic cells of the rat adenohypophysis

Ultrastructural characteristics of corticotropic cells have been studied on serial semithin and ultrathin sections of the hypophysis from intact rats by the immunofluorescence and electron microscopic methods. It is shown that the population of corticotropic cells is heterogeneous and consists of degranulated, moderate granulated, hypergranulated cells and transitional forms among them. Ultrastructurally corticotropic cells correspond to Siperstein-Nakayama's ACTH cells. Kurosumi-Kobayashi "corticotrophs" fail to react with the antiserum to ACTH, so they have not regard for its production.     

Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity.

We previously reported that virus-specific particles with polycytidylate [poly(C)]-dependent RNA polymerase activity accumulated at 30 degrees C in reovirus-infected cells. These particles sedimented heterogeneously from 300 to 550S and traversed through a 40% glycerol cushion to the pellet in 3 h at 190,000 x g. In the present report, we found that smaller particles with poly(C)-dependent RNA polymerase activity remained in the glycerol cushion. These smaller, enzymatically active particles, when purified, sedimented at 15 to 1S. They were spherical or triangular with a diameter of 11 to 12 nm. They were comprised mostly, and likely solely, of one reovirus protein, sigma NS. No particles with poly(C)-dependent RNA polymerase activity were found in mock-infected cells. Chromatography on the cation exchanger, CM-Sephadex, ascertained that sigma NS was the poly(C)-dependent RNA polymerase and showed its existence in two forms. In one form, it was enzymatically active and eluted from the column at 0.5 M KCl. In the enzymatically inactive state, it did not bind to the column. Our results suggest that the enzymatically active form of sigma NS carries a greater net positive charge than the inactive form. They also suggest that both forms of sigma NS are associated with a particle which has poly(C)-dependent RNA polymerase activity.     

A novel type of phosphofructokinase from plants

A phosphofructokinase (PFK) has been purified to homogeneity from carrot roots as a large aggregated form (molecular weight greater than 5 million). The purified plant PFK, seemingly the cytosolic form, differed from its mammalian counterpart in a lower subunit molecular weight (60,000 verses 80,000), in being only sluggishly activated by fructose-2,6-bisphosphate, and in immunological properties. Similar to liver PFK, the purified carrot PFK could be dissociated by addition of 5 mM ATP to small and intermediate forms (respective molecular mass values of 2.4 X 10(5) and 6 X 10(5) Da). These small and intermediate forms could partially reassociate to the original large form in the presence of 5 mM Fru-6-P. Alkaline pH also effected the dissociation of the large and intermediate forms to the small form of PFK. All forms were present in significant amounts in freshly prepared carrot root extracts. The different forms of PFK showed characteristic pH activity profiles with pH optima of 8.6 (small form), 5.5 and 9.0 (intermediate form), and 7.0 and 8.5 (large forms). As alkaline pH (greater than or equal to approximately 8.5) dissociated the large and intermediate enzyme forms to yield the small form, it was concluded the "true" pH optima of the intermediate and large forms are pH 5.5 and 7.0, respectively. The pH optimum displayed by the intermediate and large forms in the alkaline region (pH 8.5-9.0) was considered to be due to their dissociation during assay. The different forms of PFK also had dissimilar regulatory properties, each showing a characteristic response to ATP, citrate, and Pi, but all were sensitive to inhibition by phosphoenolpyruvate and NADPH. Leaf cytosolic PFK, partially purified from spinach, showed similar properties. The results suggest that metabolite-dependent aggregation-disaggregation is a mechanism whereby plants regulate the activity of cytosolic PFK and the accompanying rate of glycolytic carbon flux.     

A previously undescribed mangabey from Southern Tanzania

A new form ofCercocebus galeritus is described. The new mangabey was discovered recently in the Uzungwe Mountains of Tanzania. It inhabits a restricted area of lowland and transitional rain forest on the steep, dissected, southeast-facing scarp slopes. In general appearance (size, shape, hair patterns, posture, gait), it closely resembles other forms of this species:C. g. galeritus from the Tana River gallery forests in Kenya andC. g. agilis from Gabon and Zaire. The pelage is intermediate in color between these two subspecies. Areas of bare skin differ markedly from the otherC. galeritus, particularly the almost cream-colored face. Vocalizations are typical forC. galeritus. Preliminary field observations indicate that these mangabeys form multimale groups with a tendency to move as dissociated subparties, as is the case forC. g. galeritus. Although no formal classification is proposed, the evidence suggests that the new mangabey should joinC. g. galeritus,C. g. agilis andC. g. chrysogaster as a fourth distinct form within the polytypic speciesC. galeritus.     

Existence of a Free Form of a Specific Membrane Lipoprotein in Gram-Negative Bacteria

The existence of a free form of a specific lipoprotein of molecular weight 7,200 was examined in the envelopes of several gram-negative bacteria. When the envelope proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, distinct peaks were observed in Salmonella typhimurium, Serratia marcescens, and Pseudomonas aeruginosa at the same position as the free form of the lipoprotein of Escherichia coli. However, the peak was not observed in Proteus mirabilis. The protein at the peak in S. typhimurium was shown to contain little or no histidine as expected from the amino acid composition of the lipoprotein. Furthermore, antiserum against the highly purified lipoprotein from E. coli was shown to react with the proteins from S. typhimurium and S. marcescens and to form the specific immunoprecipitates. In contrast, the protein from P. aeruginosa did not react with the antiserum at all. Thus, it is concluded that S. typhimurium and S. marcescens have the free form of the lipoprotein in their envelopes as does E. coli. P. aeruginosa contains a protein of the same size as the lipoprotein, but it is not certain whether the protein is the same structural protein as the lipoprotein from E. coli. P. mirabilis may not have any free form of the lipoprotein, may have it in a very small amount, or may have a lipoprotein of different molecular weight serving the same function.     

Properties and characteristics of a bacteriocin-like substance produced by Propionibacterium acnes isolated from dental plaque

A cell-associated bacteriocinlike substance with an approximate molecular weight of 78,000 was isolated from an oral isolate of Propionibacterium acnes. The substance was bacteriostatic and was active against both gram-positive and gram-negative anaerobes. Lysozyme inhibited the activity of the bacteriocinlike substance at low ionic strength.     

Maturation in Action: CryoEM Study of a Viral Capsid Caught during Expansion

Bacteriophage HK97 maturation involves discrete intermediate particle forms, comparable to transitional states in protein folding, before reaching its mature form. The process starts by formation of a metastable prohead, poised for exothermic expansion triggered by DNA packaging. During maturation, the capsid subunit transitions from a strained to a canonical tertiary conformation and this has been postulated to be the driving mechanism for initiating expansion via switching hexameric capsomer architecture from skewed to 6-fold symmetric. We report the subnanometer electron-cryomicroscopy reconstruction of the HK97 first expansion intermediate before any crosslink formation. This form displays 6-fold symmetric hexamers, but capsid subunit tertiary structures exhibit distortions comparable to the prohead forms. We propose that coat subunit strain release acts in synergy with the first crosslinks to drive forward maturation. Finally, we speculate that the energetic features of this transition may result from increased stability of intermediates during maturation via enhanced inter-subunit interactions.     

A note on sequence-dependence of DNA structure

A circular dichroism study was conducted on the solution structure of several different oligonucleotides, whose X-ray structures have been solved. It is suggested that in aqueous solution the oligonucleotides can form structures that maintain geometrical elements which are typical of B-DNA, A-DNA, and their intermediate forms. It is shown that 5'GGATGGGAG:5'CTCCCATCC, which forms an A-DNA helix in the crystal state (McCall et al. 1986), in aqueous solution maintains an A-DNA like structure at temperatures below 10 degrees C. At temperatures between 10 degrees C and 25 degrees C it shows a tendency to form an intermediate structure between A-DNA and B-DNA. Also, it is shown that TFE does not cause a transition from B-DNA to A-DNA helix in short DNA fragments, but instead disrupts the helix.     

Thermal denaturation of the erythrocyte cytoskeleton alters the morphological changes associated with osmotic swelling

1. 1. The shape changes during osmotic swelling of human erythrocytes in a hypotonic medium at room temperature, at 45°C and at the denaturation temperature (49.5°C) of the cytoskeletal protein, spectrin, have been monitored by video microscopy.

2. 2. At room temperature the great majority of cells (which were discoid prior to injection of hypotonic medium) swelled to a spherical shape through an intermediate ellipsoidal form.

3. 3.At 49.5°C (where cells had cupped shapes prior to injection) the transition to the spherical form often involved a stomatocytic rather than ellipsoidal intermediate shape.

4. 4. The cupped form of the cells prior to injection did not account for the high incidence of cells swelling through a stomatocytic intermediate shape at 49.5°C.

5. 5. A theoretical treatment by Svetina and Zeks (1983) attributes the nature of the osmotic swelling transition shape to the difference in area between the outer and inner faces of the membrane. Our results would be consistent with the theoretical predictions if it is assumed that an increase in the area of the inner face of the membrane follows thermal denaturation of the cytoskeleton of cells in hypotonic medium.

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.     

T-cell leukemias of adulthood]

9 adult patients suffering from different forms of T-cell-malignancies were investigated: 4 patients with T-ALL; 1-T-ALL-CLL mixed form (prolymphocytic); 2 T-CLL; 2 Sézary-syndrome. The clinical peculiarities of the different forms of leukemias were compared: involvement of lymph nodes and spleen, of the central nervous system and the skin was frequent; in contrast to the findings in Sézary-syndrome, bone marrow infiltration was prominent. Light and electron microscopic morphology of the malignant cells are described. In all cases a strong activity of acid phosphatase was demonstrated, in one patient prominent deposits of glycogen. The T-cell-quality of the respective malignant cell population as well as the B-T-cell distribution of the remaining "normal" lymphocytes were shown by the following cell markers: demonstration of T-cell-antigen, resp. membrane immunoglobulins with the aid of specific heterologous antisera conjugated with peroxidase, 125iodine or fluoresceine; complement consumtion or cytotoxicity with such antisera; spontaneous rosette formation with sheep red cells or with acrylic acid beads. Usually, there was a good coincidence in results obtained with the different markers. In two patients, however, T-cells demonstrated by anti-T-globulin were not able to form T-rosettes. Responsiveness of the malignant T-cells and also of the remaining "normal" blood lymphocytes to different mitogens usually was depressed, immunoglobulin levels in the blood mostly were normal. Taking all findings into consideration, T-cell-leukemias of the adult represent a special group of hematological malignancies; the different subgroups show similarities; transitional forms occur.     

Can helminth community patterns be amplified when transferred by predation from intermediate to definitive hosts?

Helminth communities in definitive hosts are formed by the acquisition of packets of larvae arriving each time an intermediate host is consumed. It is thus possible that associations between parasite species or other aspects of community structure get transferred from intermediate to definitive hosts. Earlier computer simulations showed that associations between 2 parasite species, in particular positive associations, could be transferred up the food chain. Here, we alter some of the assumptions of previous models and generate new simulations of several ways in which source infracommunities in intermediate hosts can be transferred to target infracommunities in definitive hosts. In particular, we introduced nonrandom selection of intermediate hosts by predatory definitive hosts, to mimic the phenomenon of host manipulation by parasites; this consisted in biasing predation toward intermediate hosts harboring a certain parasite species. Overall, our results show that positive covariances between 2 parasite species can not only be transferred but can also be amplified during transmission to definitive hosts; significant covariance between parasite species can even appear in the definitive hosts when none existed in the intermediate hosts. Negative covariance was not as readily transferred to definitive hosts and amplified, in part because of properties of the presence-absence covariance index. Amplification of covariance results from intermediate host manipulation as well as from other processes taking place during transmission. These results suggest that the patterns of association between helminth species in definitive hosts cannot be taken to reflect the processes acting inside those hosts: they may simply be inherited, with amplification, from intermediate hosts.     

The isolation of multiple forms of beta-endorphin from the intermediate pituitary of the Australian lungfish, Neoceratodus forsteri

The pituitary of the Australian lungfish, Neoceratodus forsteri, was screened immunohistochemically with heterologous antisera specific for either the C-terminal of mammalian beta-endorphin or the acetylated N-terminal of beta-endorphin. Immunopositive cells were only detected with the N-terminal specific antiserum; these cells were restricted to the intermediate pituitary. Acid extracts of the intermediate pituitary were fractionated by Sephadex gel filtration chromatography, CM cation exchange chromatography and reverse phase HPLC. Fractions were analyzed by radioimmunoassay (RIA) with a N-acetyl specific beta-endorphin RIA and by radioreceptor assay for the presence of opiate active forms of beta-endorphin. Both immunoreactive and opiate active forms of beta-endorphin were detected. Of the total beta-endorphin-related material isolated from the intermediate pituitary, approximately 97% was detected with the N-terminal specific RIA and approximately 3% was detected by the radioreceptor assay. The N-acetylated immunoreactive beta-endorphin could be separated into two forms. The major form had an apparent molecular weight of 3.2 Kda. This material had a net charge at pH 2.5 of +5. The minor form of immunoreactive beta-endorphin had an apparent molecular weight of 1.4 Kda and a net charge at pH 2.5 of +1. Neither immunoreactive form exhibited receptor binding activity in the radioreceptor assay. A single peak of opiate active beta-endorphin was detected. This material had an apparent molecular weight of 3.5 Kda and a net charge at pH 2.5 of +7.     

Interaction of human alpha 1-antichymotrypsin with chymotrypsin

Human alpha 1-antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time-course studies, performed on mixtures containing an excess of native inhibitor and kept at 0 degree C or at 25 degrees C, show that the equimolar complex dissociates spontaneously; this dissociation results in the release of inactive modified alpha 1-antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native alpha 1-antichymotrypsin.