Production of in vivo-biotinylated rotavirus particles.

Although inserting exogenous viral genome segments into rotavirus particles remains a hard challenge, this study describes the in vivo incorporation of a recombinant viral capsid protein (VP6) into newly assembled rotavirus particles. In vivo biotinylation technology was exploited to biotinylate a recombinant VP6 protein fused to a 15 aa biotin-acceptor peptide (BAP) by the bacterial biotin ligase BirA contextually co-expressed in mammalian cells. To avoid toxicity of VP6 overexpression, a stable HEK293 cell line was constructed with tetracycline-inducible expression of VP6-BAP and constitutive expression of BirA. Following tetracycline induction and rotavirus infection, VP6-BAP was biotinylated, recruited into viroplasms and incorporated into newly assembled virions. The biotin molecules in the capsid allowed the use of streptavidin-coated magnetic beads as a purification technique instead of CsCl gradient ultracentrifugation. Following transfection, double-layered particles attached to beads were able to induce viroplasm formation and to generate infective viral progeny.

Quantification of feline herpesvirus 1 DNA in ocular fluid samples of clinically diseased cats by real-time TaqMan PCR.

A fluorogenic PCR was established for the quantification of feline herpesvirus 1 (FeHV-1) DNA in ocular fluid samples of clinically diseased cats. The new assay was specific for FeHV-1 and sensitive. The 100% detection rate ranged from 0.6 to 6 50% tissue culture infective doses per sample. When spiked samples with known quantities of virus were used, infectious virus titers and quantification of viral DNA by PCR correlated to each other in a linear fashion (R(2) = 0.9858) over a range of 4 orders of magnitude. Within this range, it was possible to calculate the FeHV-1 DNA content from a given infectious dose, and vice versa. The new diagnostic procedure was applied to ocular fluid samples from cats experimentally infected with FeHV-1 and specific FeHV-1-free cats. A good correlation between virus titer and quantitative PCR was observed, although only early in infection. In a second stage, the titer of infectious virus collapsed, while the PCR signal remained high. A constantly decreasing PCR signal accompanied by negative virus isolation was characteristic for a final stage of the infection. Finally, clinical samples from 20 cats that were suspected to suffer from FeHV-1 infection were analyzed. By comparing virus titers and quantitative PCR signals, it was possible to determine the current stage of the ongoing infection. Based on these findings, comparison of the results of consecutive samples allows the tracking of the course of the infection. Therefore, the new method combines the advantages of the two previously established conventional methods, qualitative PCR and virus isolation and titration.

Enhanced resolution of membranes in cultured cells by cryoimmobilization and freeze-substitution.

Investigations of cellular processes demand immediate arresting of the process at any given time and excellent retention of cellular material and excellent visibility of membranes. To achieve this goal we used cryofixation to arrest cellular processes instantly and tested diverse freeze-substitution protocols. Madin-Darby kidney cells and Vero cells were grown on carbon-coated sapphire disks. For cryofixation the sapphire disks covered with a cell monolayer were injected with the aid of a guillotine into liquid propane or ethane or a mixture of both cooled by liquid nitrogen. Freezing of the cryogen was prevented by using a partially insulated cylinder and by vigorous stirring that results in a substantial decrement of the freezing point of the cryogen. Cell monolayers can be cryofixed successfully using the guillotine in a safety hood at ambient temperature and humidity or at 37 degrees C and 45% humidity. The freezing unit can also be placed in a laminar flow for working under biohazard conditions. For visualizing cell membranes at high contrast and high resolution, cells were substituted in the presence of various concentrations of glutaraldehyde and osmium tetroxide and the temperature was raised to diverse final temperatures. Substitution for 4 hours at -90 degrees C in anhydrous acetone containing 0.25% anhydrous glutaraldehyde and 0.5% osmium tetroxide followed by a temperature rise of 5 degrees C/hour to 0 degrees C and final incubation for 1 hour at 0 degrees C resulted in high contrast and excellent visibility of subcellular components at the level of the membrane bilayer. The high spatial and temporal resolution makes this methodology an excellent tool for studying cell membrane-bound processes, such as virus-cell interactions.

[Bovine herpes mammillitis: clinical symptoms and serologic course]. / Bovine Herpes-Mammillitis: klinische Symptome und serologischer Verlauf.

Bovine herpes mammillitis was diagnosed in a dairy herd with udder and teat skin lesions. Clinical symptoms seen in 6 cows consisted of round dry areas at the teats as well as large red and painful areas with crust formation at the teats, the teat basis and the udder. Diagnosis was verified by demonstrating numerous virus particles with the typical herpes structure and by BHV-2 serum neutralization test. Prevalence of BHV-2 in the herd was determined by using BHV-2 SNT at 7 occasions during a period of 15 months. The relatively low BHV-2 SNT-titres as well as the seasonal increase of BHV-2 titres and seroprevalence in the month of September were indicative of a chronic and latent BHV-2 infection in the herd.

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli.

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.

Structural analysis of F18 fimbriae expressed by porcine toxigenic Escherichia coli.

The F18 fimbriae expressed by porcine toxigenic Escherichia coli strains are 1- to 2-mm-long filaments that mediate the adhesion of the bacteria to enterocytes. The backbone of these fimbriae is built from a major structural 15.1-kDa protein, FedA. The structure of isolated negatively stained F18 fimbriae imaged by dark-field scanning transmission electron microscopy (STEM) was resolved to approximately 2 nm. Analyzing their helical symmetry showed the axially repeating units to alternate in a "zigzag" manner around the helical axis with an axial rise of 2.2 nm. Two repeating units give rise to the observed 4.3-nm helical repeat, which is practically identical to the pitch of the one-start helix formed. Additionally, an axially repeating pattern with a 27-nm spacing was found on rotary-shadowed fimbriae. Mass-per-length determination of unstained F18 fimbriae by STEM revealed the axially repeating unit to have a molecular mass of 25.4 kDa, indicating that it is a FedA monomer, with the difference in mass arising from the minor subunits, FedE and FedF. The presence of the latter two proteins might cause the observed 27-nm axial pattern.

Novel entry pathway of bovine herpesvirus 1 and 5.

Herpesviruses enter cells by a yet poorly understood mechanism. We visualized the crucial steps of the entry pathway of bovine herpesvirus 1 (BHV-1) and BHV-5 by transmission and scanning electron microscopy, employing cryotechniques that include time monitoring, ultrarapid freezing, and freeze substitution of cultured cells inoculated with virus. A key step in the entry pathway of both BHV-1 and BHV-5 is a unique fusion of the outer phospholipid layer of the viral envelope with the inner layer of the plasma membrane and vice versa resulting in "crossing" of the fused membranes and in partial insertion of the viral envelope into the plasma membrane. The fusion area is proposed to function as an axis for driving the virus particle into an invagination that is concomitantly formed close to the fusion site. The virus particle enters the cytoplasm through the opened tip of the invagination, and the viral envelope defuses from the plasma membrane. There is strong evidence that the intact virus particle is then transported to the nuclear region.

Reevaluation of the effect of lysoyzme on Escherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution.

Lysozyme is able to lyse Gram-positive bacteria acting as muramidase on the peptidoglycan polymer. Gram-negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido-glycan is protected by the outer membrane and thus that Gram-negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180-187] found that lysozyme per se is able to kill some Gram-negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram-negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified.

Green fluorescent protein as a reporter for retrovirus and helper virus-free HSV-1 amplicon vector-mediated gene transfer into neural cells in culture and in vivo.

Green fluorescent protein (GFP) is an effective marker for retrovirus and herpes virus vector-mediated gene transfer into various central nervous system-derived cells, both proliferative and non-proliferative, in culture and in vivo. Retrovirus vectors were used to stably transduce several rat and human glioma lines, and a multipotent mouse neural progenitor line in culture. Implantation of selected pools of transduced glioma cells into rodent brain allowed clear visualization of the tumor and the invading tumor edge. Helper virus-free HSV-1 amplicon vectors successfully transferred gfp into non-dividing primary neural cells in culture and in the rat brain. This study describes the versatility of GFP for: (i) labelling of glioma cells in experimental brain tumor models and neural progenitor cells by retrovirus vectors, and (ii) efficient, non-toxic delivery of genes to post mitotic cells of the nervous system using helper-virus free HSV-1 amplicon vectors.

Morphometric analysis of parathyroids in hypophysectomized rats in normocalcaemic and short-term induced hypocalcaemic conditions.

The ultrastructure of parathyroid glands of hypophysectomized rats in normo- and hypocalcaemic conditions was qualitatively and quantitatively analysed. Parathyroid glands of both intact and hypophysectomized rats comprised cells of similar shape and staining intensity and of an equal number of cells in mitosis. Cell volume was not affected but the surface area of plasma membrane, RER and Golgi complex was increased in parathyroid cells after hypophysectomy. Parathyroid cells of both intact and hypophysectomized rats responded to hypocalcaemia by a drastic increase in surface area of the plasma membrane and a decrease in surface area of the Golgi complex. Since RER, Golgi complex and plasma membrane are concerned with secretion of parathyroid hormone, the data suggest that (1) the pituitary has either no effect on parathyroid cells, or a possibly stimulating effect can be compensated after hypophysectomy, or the pituitary even has a suppressive effect on parathyroid cells, and (2) the responsiveness of parathyroid cells to changes in serum calcium is unimpaired after hypophysectomy.

Cellular changes in rat parathyroids provoked by progesterone and testosterone.

Male rats kept on a standard diet were treated either with progesterone or testosterone by a single intramuscular injection of preparations which are slowly absorbed and metabolized. The rats were anaesthetized 24 h after application of the hormones, perfused with glutaraldehyde, and the parathyroid glands prepared for electron microscopy. Morphometric analysis revealed that both progesterone and testosterone provoked (1) an increment in nuclear and cell volume and a concomitant increment in cell surface area, and (2) an increment in surface area of rough endoplasmic reticulum by 42% and 49%, and of the Golgi complex by 85% and 63%, respectively. Previously, we had found that oestradiol treatment led to a similar response in parathyroid cells. The conclusion is thus drawn that male and female sex hormones induce membrane synthesis resulting in an enhanced capacity for parathyroid hormone secretion since RER and Golgi complex are concerned with this secretion. It is considered probable that sex hormones have the ability fundamentally to modulate secretory activity in parathyroid cells.

Morphometric analysis of compartments concerned with secretion of parathyroid hormone in male and female rats.

Parathyroid cells responded to estradiol application in rats by increment in surface area of the compartments concerned with parathyroid hormone secretion, i.e. the rough endoplasmic reticulum, Golgi complex and secretory granules. We thus compared the morphology of parathyroids of male rats with those of females of the same age and same weight, respectively. Morphometric analysis revealed that (i) the mean cell volume was significantly larger and, consequently, the number of parathyroid cells lower expressed per 1 mm3 tissue and (ii) the surface area of the Golgi complex was larger expressed per mean parathyroid cell volume and per 1 mm3 tissue in female rats than in males. Diversity in cell volume seems to depend on age rather than on sex, whereas diversity in the Golgi complex is considered to be sex related. The physiological implications remain to be clarified.

Adhesive fimbriae produced in vivo by Escherichia coli O139:K12(B):H1 associated with enterotoxaemia in pigs.

Two strains of E. coli O139:K12 (B):H1 were compared in vitro and in the intestinal environment. Both strains colonized the small intestines of experimentally inoculated pigs and exhibited in vivo a similar relationship to the microvillus border as enterotoxigenic E. coli (ETEC). Strain 107/86 grown on blood agar expressed numerous long flexible non-haemagglutinating fimbriae which were antigenically distinct from the known fimbriae of porcine ETEC. It adhered in vitro to porcine enterocyte brush border fragments. Strain 124/76 grown on blood agar was devoid of fimbriae and did not adhere to brush border fragments. However, fimbriae morphologically and antigenically indistinguishable from those of strain 107/86 were detected in the intestinal environment by direct immunofluorescence and by immuno electron microscopy.

Parathyroid cell polarity as revealed by cytochemical localization of ATPases, alkaline phosphatase and 5'-nucleotidase.

Activities of Ca2(+)-dependent ATPase, Mg2(+)-dependent ATPase, Na(+)-K(+)-dependent ATP-ase, alkaline phosphatase, and 5'-nucleotidase were demonstrated after incubation of 40-microns vibratome sections of bovine parathyroids and subsequent visualization by electron microscopy. Prior to sectioning, parathyroid tissue was fixed with 1% glutaraldehyde for localization of alkaline phosphatase, and with 2% formaldehyde and 1% glutaraldehyde for demonstration activities of ATPases and 5'-nucleotidase. The activities of the five enzymes were found at the apicolateral domain of the plasma membrane in parathyroid cells, i.e. at the site parathyroid cells face neighbouring parenchymal cells. Ca2(+)-ATPase activity was also seen on mitochondria, Golgi complex and RER. The presence of these plasma membrane associated enzymes at the apicolateral domain only indicate polarity in parathyroid cells. It further suggests that many processes including transmembrane transport take place at the apicolateral domain, the site of parathyroid cells opposing blood capillaries.

Effects of estradiol on parathyroid cell activity.

Beta-estradiol-3-benzoate provoked an initial centrifugal membrane shift in rat parathyroid cells and, later, enlargement of the compartments concerned with parathyroid hormone secretion, which suggests that estradiol modulates not only transport and release of parathyroid hormone but also the capacity for its synthesis, packaging and storage.

Quantitative assessment of cellular changes provoked by microwave enhanced fixation of parathyroids.

Rat parathyroids fixed by microwave enhancement, i.e. microwave irradiation in the presence of glutaraldehyde for 8 s and postfixation with OsO4 after a delay of 5 min, were compared with parathyroids fixed by perfusion with glutaraldehyde followed by immersion in glutaraldehyde and finally in OsO4. Morphometric analysis revealed that microwave enhanced fixation led to a larger mean cell volume, to larger cell surface area, and to larger surface area in membranes of RER and secretory granules. Though it is not known by which method parathyroid cells are conserved closer to the living state it is obvious that microwave enhanced fixation retains more membranes but provokes centrifugal dislocation of membranes mimicking exocytosis.

Potency of microwave irradiation during fixation for electron microscopy.

Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.

Unimpaired membrane dynamics in parathyroid cells in insulin deficient rats.

The responsiveness of parathyroid cells in insulin deficient and short-term diabetic rats was investigated by morphometric analysis. Insulin deficiency was produced by intravenous injection of D-mannoheptulose and short-term (7 days) diabetes mellitus by intraperitoneal application of streptozotocin. Parathyroid glands were stimulated for parathyroid hormone secretion by decreasing the serum calcium concentration through intravenous infusion of EGTA. Parathyroid cells of controls, insulin deficient, and short-term diabetic rats responded to reduced serum calcium by a 45% increase of the cell surface area. This increase is assumed to be the result of the membrane-bound transport of parathyroid hormone from the Golgi complex and secretory granules to the plasma membrane and subsequent exocytic release of parathyroid hormone induced by the low serum calcium concentration. Therefore, the unimpaired increase in the cell surface area of parathyroid cells in insulin deficient and short-term diabetic rats indicates that insulin does not modulate the release of parathyroid hormone. It is also considered likely that synthesis of parathyroid hormone is not suppressed in short-term diabetes but that fat metabolism is disturbed leading to accumulation of lipid vacuoles.

Parathyroid ultrastructure after aldehyde fixation, high-pressure freezing, or microwave irradiation.

Parathyroid cell variants, commonly observed in parathyroid glands fixed by immersion in glutaraldehyde, are believed to be the result of cyclic changes in the course of parathyroid hormone secretion. Immersion of bovine parathyroid glands in a mixture consisting of 1% glutaraldehyde, 1.5% formaldehyde, and 2.5% acrolein, followed by post-fixation in 1% osmium tetroxide, resulted in high uniformity with only one cell variant, whereas the same fixation procedure led to disruption of cell membranes and formation of cell variants in rat parathyroids. Parathyroid glands of both cattle and rats prepared by high-pressure quick-freezing and subsequent freeze-substitution contained only one cell variant. Excellent preservation of the ultrastructure of bovine and rat parathyroids, also exhibiting only one cell variant, was achieved by microwave irradiation in the presence of 2.5% glutaraldehyde in Na-cacodylate followed by post-fixation with OsO4 in Na-cacodylate or s-collidine, both containing Ca2+ and Mg2+. Use of the appropriate buffer, as well as osmication, is essential for successful fixation utilizing microwave energy. The main effects are considered to be heating specimens within sufficient short periods and enhancement of subsequent osmium fixation. The results support the idea, arising after examination of perfusion-fixed parathyroid tissue, that parathyroid cell variants occur during improper aldehyde fixation rather than that they express functional diversity.

The genome of caprine herpesvirus 1: genome structure and relatedness to bovine herpesvirus 1.

Caprine herpesvirus 1 (CapHV-1) DNA was examined by electron microscopy, restriction site mapping and homology studies with bovine herpesvirus 1 (BHV-1) DNA. Although the restriction site maps differed significantly, we showed that the genome structures of CapHV-1 and BHV-1 were identical and that the DNAs shared a high degree of base sequence homology.