Ultrastructural study of the regressing prothoracic glands of blattarian insects

Summary The prothoracic glands, source of the molting hormone ecdysone, regress within a few days after the final molt, a process which was analyzed with electron microscopic methods in the cockroaches Leucophaea and Blaberus. This strictly timed event is accompanied by drastic alterations in cellular fine structure. Early signs of breakdown appear in groups of nuclei whose substance becomes segregated into patches of contrasting electron density characteristic of pyknosis.The most conspicuous change in the cytoplasm of parenchymal cells concerns the appearance of large, heterogeneous inclusion bodies in which various cellular elements become segregated. These compartments seem to represent autophagic vacuoles within which the gradual degradation of much of their contents takes place, presumably under the influence of lysosomal enzymes. Undigested swirls of membranous character may remain sequestered within these packets for some time.At advanced stages of cellular atrophy, plasma membranes and nuclear envelopes have gradually disappeared, and masses of protoplasm undergoing autolysis become invaded by a greater number of hemocytes than are present in nymphal glands. These phagocytic elements appear to engulf debris of parenchymal cells as well as some degenerating connective tissue elements. After the completion of the regressive process, the axial band of musculature characteristic of the nymphal gland persists on its own. Whether or not some parenchymal cells (or possibly their precursors) capable of reactivation persist in the proximity of this muscle is unknown.The resorption of the prothoracic gland in the newly emerged insect is the result of physiological autolysis and seems to be aided by the activity of phagocytic hemocytes.Dedicated to Professor W. Bargmann on his 60th birthday in friendship and admiration.This study was supported by Research Grants AM-03984, NB-02145 and NB-05219 from the U.S.P.H.S.I wish to express my thanks to Mrs. S. Wurzelmann, Mrs. C. Jones, Mrs. C. Grubman, and Mr. S. Brown for their excellent technical assistance.     

Locust collagen: morphological and biochemical characterization

Natural-abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide toxin II from the sea anemone Anemonia sulcata have been analysed and compared with corresponding spectra reported recently for a closely related polypeptide anthopleurin A. The spectra contain many resolved one-carbon and two-carbon resonances from carbonyl, aromatic and methyl carbons, many of which have been assigned to individual carbons in the molecule on the basis of their chemical shifts, including their pH dependence, and by comparison with the 13C NMR spectrum of anthopleurin A. Analysis of the effects of pH on the spectrum yields estimates for the pKa values of a number of functional groups in the molecule, as follows: side-chain carboxylates of the two aspartic acid residues 2 and 3.1; COOH-terminal carboxylic acid, 3.5; imidazolium moieties of the two histidine residues, 6.7 and 7.6 NH2-terminal ammonium, 8. The similarity between the pKa values of these functional groups in toxin II and those of corresponding groups in anthopleurin A, together with the close agreement between chemical shifts of conserved carbons, indicates that many local interactions are nearly identical in the two molecules, and thus supports the thesis that their overall conformations in solution are similar. However, the local interactions involving one of the aspartic acid residues are altered in toxin II. Together with other data, this leads to a proposal for the site in these two molecules which is responsible for their cardiac stimulatory activity.     

Cockroach collagen: isolation, biochemical and biophysical characterization

Collagen fibrils from the mesenteric connective sheath of the adult cockroach Periplaneta americana were extracted by enzymatic digestion with pepsin and were purified. Chromatographic studies and sodium dodecylsulfate electrophoresis revealed the presence of a single chain. It was demonstrated that the structure of this collagen could be represented by the formula (alpha)3. The amino acid composition is typical of collagens (one-third glycine, and a high imino acid content) and similar to that of type II. The carbohydrate content was high (8.8%), and the cyanogen bromide pattern was different from that of known collagens. The chains were linked by the stable intermolecular bond dihydroxylysinonorleucine. The banding patterns of the segment-long-spacing crystallites and of the reconstituted fibrils were similar to type I collagen. The molecular weight (Mr 280,000) and length (285 nm) were typical, but the denaturation temperature was high (38.5 degrees C). It was concluded that cockroach mesenteric collagen showed the characteristic features of invertebrate mesodermal collagens, except that of the thermal stability of the triple-helical structure.     

Electron microscopic analysis and biochemical characterization of a novel methanol dehydrogenase from the thermotolerant Bacillus sp. C1

Methanol dehydrogenase from the thermotolerant Bacillus sp. C1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing showed that the 5-fold view possesses mirror symmetry. The rectangular views can be divided into two separate classes, one of which has 2-fold rotational symmetry. It is concluded that methanol dehydrogenase is a decameric molecule, and a tentative model is presented. The estimated molecular weight is 430,000, based on a subunit molecular weight of 43,000. The enzyme contains one zinc and one to two magnesium ions per subunit. N-terminal amino acid sequence analysis revealed substantial similarity with alcohol dehydrogenases from Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium acetobutylicum, and Escherichia coli, which contain iron or zinc but no magnesium. In view of the aberrant structural and kinetic properties, it is proposed to distinguish the enzyme from common alcohol dehydrogenases (EC 1.1.1.1) by using the name NAD-dependent methanol dehydrogenase.     

Electron microscopic characterization of chick embryonic skeletal muscle proteoglycans

In this article, proteoglycans from embryonic chick leg muscle are quantitatively and qualitatively compared with day 8 high density cell culture cartilage proteoglycans by electron microscopy of proteoglycan-cytochrome c monolayers. The visualized proteoglycan profiles were separated into four categories according to shape, size, and complexity. The two major categories were further characterized by lengths of core proteins, lengths of side projections, and distance between side projections. Two large proteoglycans are identifiable in spread leg muscle preparations. One group has a core protein (mean length of 205 nm) from which extend long thin side projections that we interpret to be groups of chondroitin sulfate glycosaminoglycans with a mean length of 79 nm. This large chondroitin sulfate proteoglycan is the only type found in muscle cultures as determined both biochemically in the past and now by electron microscopy and is referred to as muscle proteoglycan. The second large proteoglycan has a mean core protein length of 250 nm and side projections that are visibly shorter (mean length of 38 nm) and thicker than those of the muscle proteoglycan. This group is referred to as the mesenchymal proteoglycan since its biosynthetic origin is still uncertain. We compare these two profiles with the chick cartilage chondroitin sulfate proteoglycan that has a mean core protein length of 202 nm and side projections with a mean length of 50 nm. The data presented here substantiate the earlier biochemical characterization of these noncartilage proteoglycans and establish the unique structural features of the muscle proteoglycan as compared with the similar profiles of the cartilage and mesenchymal proteoglycans.     

Electron microscopic and biochemical characteristics of nuclei and nucleoli isolated from rat liver

Rat liver nuclei were freed of cytoplasmic contamination by washing with Triton-X-100 and subsequent centrifugation through 2.2 M sucrose. Electron microscopic examination showed that the outer membranes of the nuclei had been removed, but that the nuclei otherwise resembled the nuclei of intact liver. Morphological studies, chemical estimations of DNA, RNA, and protein and the estimation of cytoplasmic "marker" enzymes suggested that contamination of nuclei by cytoplasmic components was limited. These nuclei were obtained in yields of about 70% and were suitable for the isolation of nucleoli. Nucleoli were isolated by the breaking of the nuclei by ultrasound and subsequent differential centrifugation. In ultrastructural appearance, the isolated nucleoli resembled nucleoli in intact tissue. However, at high magnifications the "granular" component of isolated nucleoli appeared to consist of tightly twisted fibers. The nucleoli could be obtained in yields of at least 30%, and the values for the chemical composition of the isolated nucleoli agreed with values previously reported.     

Electron microscopic and biochemical study of lipoprotein synthesis in the isolated perfused rat liver

The isolated perfused rat liver was used to study the 300-800 A electron-opaque bodies which had previously been described in the liver cell Golgi apparatus, smooth endoplasmic reticulum, and space of Disse. When the perfusion medium was enriched with linoleate, the number and electron opacity of these particles increased markedly. Sequential biopsies showed that they appeared first in the smooth surfaced terminal ends of the rough reticulum, the smooth endoplasmic reticulum proper, and the Golgi apparatus and later in the space of Disse. After 60 min of perfusion, particles of the same size and shape as those in the liver cells could be isolated in large numbers from the d < 1.006 fraction of the perfusate. Control livers perfused with an identical medium but without linoleate did not show these changes. Puromycin markedly depressed the production of 300-800 A particles by livers perfused with an oleate-rich medium; however, it did not interfere with the formation of large cytoplasmic droplets of neutral fat. In keeping with these findings, puromycin blocked the incorporation of oleate-(14)C into lipoprotein triglyceride isolated from the perfusate, but did not interfere with the appearance of the labeled fatty acid in tissue triglyceride. Puromycin also blocked the incorporation of leucine-(3)H into both tissue protein and perfusate lipoprotein. We concluded that the 300-800 A particles observed are, in all likelihood, very low density lipoproteins and that their formation is blocked by puromycin, presumably through interference with the synthesis of their apoprotein.     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1–3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29–43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of ≤ 0.3 μm and volume of ≤ 0.014 μm³) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18–0.20 × 0.20–0.30 μm in size, forming in situ spherical aggregates (microcolonies) of dividing cells. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Electron microscopic microprobe analysis of mineralized collagen fibrils and extracollagenous regions in Turkey leg tendon

Summary Bundles of tibia tendon from 19 week-old turkeys were deep frozen, freeze dried and embedded in styrol methacrylate or Epon. In the distal mineralized region, bundles of unmineralized collagen fibrils as well as mineralized regions consisting of round microcompartments with low contrast surrounded by a mineral sheath with high contrast were found. The inner regions with low contrast corresponded to the mineralized collagen fibrils, while the contrast-rich peripheral zones corresponded to the mineralized collagen-free ground substance. Using electron microscopic microprobe analysis, it was shown that the peripheral mineralized region, consisting mainly of closely packed needles, often contained 100% more mineral substance than the central, mineralized collagen zone, which consisted mainly of plate-like crystallites. Possible reasons for this difference in mineral content are discussed on the molecular level.The authors would like to express their gratitude to the Deutsche Forschungsgemeinschaft for financial support and to Fräulein Christine Dörnen for valuable technical assistance     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1-3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29-43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of < or =0.3 microm and volume of < or =0.014 microm3) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18-0.20 x 0.20-0.30 microm in size, forming spherical aggregates (microcolonies) of dividing cells in situ. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris

Summary Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding vesicles from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO₄, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO₄ (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO₄ for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).     

Electron microscopic morphometry

The fundamental concepts of morphometry, the principal correction factors for systematic errors and the basic principles of efficient sampling are outlined for quantitative morphology at the electron microscopic level of resolution. The important usefulness of correlating electron microscopic morphometry with complementary light microscopic morphometry is emphasized.     

Identification and biochemical characterization of two novel collagen binding MSCRAMMs of Bacillus anthracis

Cell wall-anchored proteins play critical roles in the pathogenesis of infections caused by Gram-positive bacteria. Through the analysis of the genome of Bacillus anthracis Ames strain, we identified two novel putative cell wall-anchored proteins, BA0871 and BA5258, which have sequence homology to CNA, a cell wall-anchored collagen adhesin of Staphylococcus aureus. The two proteins have similar domain organization to that of CNA, with typical signal peptide sequences, a non-repetitive A region followed by repeats, and a characteristic cell wall-anchoring region. They are expressed on the surface of B. anthracis. The A regions of the two proteins were predicted to adopt similar structural folds as CNA. Circular dichroism analysis of the recombinant A regions of the two proteins (rBA0871A and rBA5258A) indicate that their secondary structure compositions are similar to those of the A regions of CNA and other cell wall-anchored adhesins. We demonstrate through solid phase binding assays and surface plasmon resonance analyses that rBA0871A and rBA5258A specifically bound type I collagen in a dose-dependent and saturable manner. Their dissociation constants (KD) for collagen are 1.6-3.2 microm for rBA0871A and 0.6-0.9 microm for rBA5258A, respectively. We further demonstrate that BA0871 and BA5258 can mediate cell attachment to collagen when expressed on the surface of a heterologous host bacterium. To our knowledge these are the first two adhesins of B. anthracis described, which may have important implications for our understanding of the pathogenic mechanisms explored by this organism.     

Electron microscopic characterization of calcium-binding physodes in the green alga Mougeotia scalaris.

Effect of the covalently cross-linking agents glutardialdehyde and osmium tetroxide, and of adsorption of the vital dye, neutral red, to the matrix of the calcium-binding "vesicles" from the green alga Mougeotia scalaris has been analysed in situ, both in terms of structural preservation and of the calcium-binding capacity of the vesicles. Upon cell fixation in glutardialdehyde without OsO4, the vesicles appear to dissolve, but upon simultaneous fixation in glutardialdehyde with OsO4 (1% w/v), the vesicles retain a globular form, are evenly stained by osmium and appear to be surrounded by a membrane-like structure. This structure was also observed around the vesicles in cells preincubated for 10 min in 0.1 mM neutral red and then fixed in glutardialdehyde/OsO4 for 1 h. More detailed information of the matrix structure is obtained when simultaneous fixation of the Mougeotia cells was shortened to 15 min: a membrane-like structure was no longer observed around the vesicles. After cell treatment in the presence of neutral red, no calcium at all was found inside the vesicles. A small amount of calcium remained, when cells were fixed simultaneously and extensively in the absence of neutral red. However, calcium was found, to a considerable extent, inside the vesicles after short simultaneous fixation of the cells in the absence of neutral red. Based on the ultrastructural and elemental features presented here, the calcium-binding vesicles in Mougeotia appear to represent a member of the large family of (calcium-binding) physodes in lower plants (CaBP).     

Comparative biochemical characterization of nitrile-forming proteins from plants and insects that alter myrosinase-catalysed hydrolysis of glucosinolates

The defensive function of the glucosinolate-myrosinase system in plants of the order Capparales results from the formation of isothiocyanates when glucosinolates are hydrolysed by myrosinases upon tissue damage. In some glucosinolate-containing plant species, as well as in the insect herbivore Pieris rapae, protein factors alter the outcome of myrosinase-catalysed glucosinolate hydrolysis, leading to the formation of products other than isothiocyanates. To date, two such proteins have been identified at the molecular level, the epithiospecifier protein (ESP) from Arabidopsis thaliana and the nitrile-specifier protein (NSP) from P. rapae. These proteins share no sequence similarity although they both promote the formation of nitriles. To understand the biochemical bases of nitrile formation, we compared some of the properties of these proteins using purified preparations. We show that both proteins appear to be true enzymes rather than allosteric cofactors of myrosinases, based on their substrate and product specificities and the fact that the proportion of glucosinolates hydrolysed to nitriles does not remain constant when myrosinase activity varies. No stable association between ESP and myrosinase could be demonstrated during affinity chromatography, nevertheless some proximity of ESP to myrosinase is required for epithionitrile formation to occur, as evidenced by the lack of ESP activity when it was spatially separated from myrosinase in a dialysis chamber. The significant difference in substrate- and product specificities between A. thaliana ESP and P. rapae NSP is consonant with their different ecological functions. Furthermore, ESP and NSP differ remarkably in their requirements for metal ion cofactors. We found no indications of the involvement of a free radical mechanism in epithionitrile formation by ESP as suggested in earlier reports.