Microtubule-acting drugs lead to the nonpolarized delivery of the influenza hemagglutinin to the cell surface of polarized Madin-Darby canine kidney cells.

The synchronized directed transfer of the envelope glycoproteins of the influenza and vesicular stomatitis viruses from the Golgi apparatus to the apical and basolateral surfaces, respectively, of polarized Madin-Darby canine kidney (MDCK) cells can be achieved using temperature-sensitive mutant viruses and appropriate temperature shift protocols (Rindler, M. J., I. E. Ivanov, H. Plesken, and D. D. Sabatini, 1985, J. Cell Biol., 100:136-151). The microtubule-depolymerizing agents colchicine and nocodazole, as well as the microtubule assembly-promoting drug taxol, were found to interfere with the normal polarized delivery and exclusive segregation of hemagglutinin (HA) to the apical surface but not with the delivery and initial accumulation of G on the basolateral surface. Immunofluorescence analysis of permeabilized monolayers of influenza-infected MDCK cells treated with the microtubule-acting drugs demonstrated the presence of substantial amounts of HA protein on both the apical and basolateral surfaces. Moreover, in cells infected with the wild-type influenza virus, particles budded from both surfaces. Viral counts in electron micrographs showed that approximately 40% of the released viral particles accumulated in the intercellular spaces or were trapped between the cell and monolayer and the collagen support as compared to less than 1% on the basolateral surface of untreated infected cells. The effect of the microtubule inhibitors was not a result of a rapid redistribution of glycoprotein molecules initially delivered to the apical surface since a redistribution was not observed when the inhibitors were added to the cells after the HA was permitted to reach the apical surface at the permissive temperature and the synthesis of new HA was inhibited with cycloheximide. The altered segregation of the HA protein that occurs may result from the dispersal of the Golgi apparatus induced by the inhibitors or from the disruption of putative microtubules containing tracks that could direct vesicles from the trans Golgi apparatus to the cell surface. Since the vesicular stomatitis virus G protein is basolaterally segregated even when the Golgi elements are dispersed and hypothetical tracks disrupted, it appears that the two viral envelope glycoproteins are segregated by fundamentally different mechanisms and that the apical surface may be incapable of accepting vesicles carrying the G protein.

Intracellular transport of a variant surface glycoprotein in Trypanosoma brucei.

Trypanosome variant surface glycoproteins (VSGs) have a novel glycan-phosphatidylinositol membrane anchor, which is cleavable by a phosphatidylinositol-specific phospholipase C. A similar structure serves to anchor some membrane proteins in mammalian cells. Using kinetic and ultrastructural approaches, we have addressed the question of whether this structure directs the protein to the cell surface by a different pathway from the classical one described in other cell types for plasma membrane and secreted glycoproteins. By immunogold labeling on thin cryosections we were able to show that, intracellularly, VSG is associated with the rough endoplasmic reticulum, all Golgi cisternae, and tubulovesicular elements and flattened cisternae, which form a network in the area adjacent to the trans side of the Golgi apparatus. Our data suggest that, although the glycan-phosphatidylinositol anchor is added in the endoplasmic reticulum, VSG is nevertheless subsequently transported along the classical intracellular route for glycoproteins, and is delivered to the flagellar pocket, where it is integrated into the surface coat. Treatment of trypanosomes with 1 microM monensin had no effect on VSG transport, although dilation of the trans-Golgi stacks and lysosomes occurred immediately. Incubation of trypanosomes at 20 degrees C, a treatment that arrests intracellular transport from the trans-Golgi region to the cell surface in mammalian cells, caused the accumulation of VSG molecules in structures of the trans-Golgi network, and retarded the incorporation of newly synthesized VSG into the surface coat.

Actin microfilaments play a critical role in endocytosis at the apical but not the basolateral surface of polarized epithelial cells.

Treatment with cytochalasin D, a drug that acts by inducing the depolymerization of the actin cytoskeleton, selectively blocked endocytosis of membrane bound and fluid phase markers from the apical surface of polarized MDCK cells without affecting the uptake from the basolateral surface. Thus, in MDCK cell transformants that express the VSV G protein, cytochalasin blocked the internalization of an anti-G mAb bound to apical G molecules, but did not reduce the uptake of antibody bound to the basolateral surface. The selective effect of cytochalasin D on apical endocytosis was also demonstrated by the failure of the drug to reduce the uptake of 125I-labeled transferrin, which occurs by receptor-mediated endocytosis, via clathrin-coated pits, almost exclusively from the basolateral surface. The actin cytoskeleton appears to play a critical role in adsorptive as well as fluid phase apical endocytic events, since treatment with cytochalasin D prevented the apical uptake of cationized ferritin, that occurs after the marker binds to the cell surface, as well as uptake of Lucifer yellow, a fluorescent soluble dye. Moreover, the drug efficiently blocked infection of the cells with influenza virus, when the viral inoculum was applied to the apical surface. On the other hand, it did not inhibit the basolateral uptake of Lucifer yellow, nor did it prevent infection with VSV from the basolateral surface, or with influenza when this virus was applied to monolayers in which the formation of tight junctions had been prevented by depletion of calcium ions. EM demonstrated that cytochalasin D leads to an increase in the number of coated pits in the apical surface where it suppresses the pinching off of coated vesicles. In addition, in drug-treated cells cationized ferritin molecules that were bound to microvilli were not cleared from the microvillar surface, as is observed in untreated cells. These findings indicate that there is a fundamental difference in the process by which endocytic vesicles are formed at the two surfaces of polarized epithelial cells and that the integrity and/or the polymerization of actin filaments are required at the apical surface. Actin filaments in microvilli may be part of a mechanochemical motor that moves membrane components along the microvillar surface towards intermicrovillar spaces, or provides the force required for converting a membrane invagination or pit into an endocytic vesicle within the cytoplasm.

The production of post-Golgi vesicles requires a protein kinase C-like molecule, but not its phosphorylating activity.

We have recently described a system that recreates in vitro the generation of post-Golgi vesicles from purified Golgi fractions obtained from virus-infected MDCK cells in which the vesicular stomatitis virus-G envelope glycoprotein had been allowed to accumulate in vivo in the TGN. Vesicle formation, monitored by the release of the viral glycoprotein, was shown to require the activation of a GTP-binding ADP ribosylation factor (ARF) protein that promotes the assembly of a vesicle coat in the TGN, and to be regulated by a Golgi-associated protein kinase C (PKC)-like activity. We have now been able to dissect the process of post-Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and another of vesicle scission, neither of which requires an ATP supply. The first stage can occur at 20 degrees C, and includes the GTP-dependent activation of the ARF protein, which can be effected by the nonhydrolyzable nucleotide analogue GTP gamma S, whereas the second stage is nucleotide independent and can only occur at a higher temperature of incubation. Cytosolic proteins are required for the vesicle scission step and they cannot be replaced by palmitoyl CoA, which is known to promote, by itself, scission of the coatomer-coated vesicles that mediate intra-Golgi transport. We have found that PKC inhibitors prevented vesicle generation, even when this was sustained by GTP gamma S and ATP levels reduced far below the K(m) of PKC. The inhibitors suppressed vesicle scission without preventing coat assembly, yet to exert their effect, they had to be added before coat assembly took place. This indicates that a target of the putative PKC is activated during the bud assembly stage of vesicle formation, but only acts during the phase of vesicle release. The behavior of the PKC target during vesicle formation resembles that of phospholipase D (PLD), a Golgi-associated enzyme that has been shown to be activated by PKC, even in the absence of the latter's phosphorylating activity. We therefore propose that during coat assembly, PKC activates a PLD that, during the incubation at 37 degrees C, promotes vesicle scission by remodeling the phospholipid bilayer and severing connections between the vesicles and the donor membrane.

Polarized delivery of viral glycoproteins to the apical and basolateral plasma membranes of Madin-Darby canine kidney cells infected with temperature-sensitive viruses.

The intracellular route followed by viral envelope glycoproteins in polarized Madin-Darby canine kidney cells was studied by using temperature-sensitive mutants of vesicular stomatitis virus (VSV) and influenza, in which, at the nonpermissive temperature (39.5 degrees C), the newly synthesized glycoproteins (G proteins) and hemagglutinin (HA), respectively, are not transported out of the endoplasmic reticulum. After infection with VSV and incubation at 39.5 degrees C for 4-5 h, synchronous transfer of G protein to the plasma membrane was initiated by shifting to the permissive temperature (32.5 degrees C). Immunoelectron microscopy showed that under these conditions the protein moved to the Golgi apparatus and from there directly to a region of the lateral plasma membrane near this organelle. G protein then seemed to diffuse progressively to basal regions of the cell surface and, only after it had accumulated in the basolateral domain, it began to appear on the apical surface near the intercellular junctions. The results of these experiments indicate that the VSV G protein must be sorted before its arrival at the cell surface, and suggest that passage to the apical domain occurs only late in infection when tight junctions are no longer an effective barrier. In complementary experiments, using the temperature-sensitive mutant of influenza, cultures were first shifted from the nonpermissive temperature (39.5 degrees C) to 18.5 degrees C, to allow entrance of the glycoprotein into the Golgi apparatus (see Matlin, K.S., and K. Simons, 1983, Cell, 34:233-243). Under these conditions HA accumulated in Golgi stacks and vesicles but did not reach the plasma membrane. When the temperature was subsequently shifted to 32.5 degrees C, HA rapidly appeared in discrete regions of the apical surface near, and often directly above, the Golgi elements, and later diffused throughout this surface. To ensure that the anti-HA antibodies had access to lateral domains, monolayers were treated with a hypertonic medium to dilate the intercellular spaces. Some labeling was then observed in the lateral plasma membranes soon after the shift, but this never increased beyond 1.0 gold particle/micron, whereas characteristic densities of labeling in apical surfaces soon became much higher (approximately 10 particles/micron). Our results suggest that the bulk of HA follows a direct pathway leading from the Golgi to regions of the apical surface close to trans-Golgi cisternae.

Viral glycoproteins destined for apical or basolateral plasma membrane domains traverse the same Golgi apparatus during their intracellular transport in doubly infected Madin-Darby canine kidney cells.

Madin-Darby canine kidney (MDCK) cells can sustain double infection with pairs of viruses of opposite budding polarity (simian virus 5 [SV5] and vesicular stomatitis virus [VSV] or influenza and VSV), and we observed that in such cells the envelope glycoproteins of the two viruses are synthesized simultaneously and assembled into virions at their characteristic sites. Influenza and SV5 budded exclusively from the apical plasma membrane of the cells, while VSV emerged only from the basolateral surfaces. Immunoelectron microscopic examination of doubly infected MDCK cells showed that the influenza hemagglutinin (HA) and the VSV G glycoproteins traverse the same Golgi apparatus and even the same Golgi cisternae. This indicates that the pathways of the two proteins towards the plasma membrane do not diverge before passage through the Golgi apparatus and therefore that critical sorting steps must take place during or after passage of the glycoproteins through this organelle. After its passage through the Golgi, the HA accumulated primarily at the apical membrane, where influenza virion assembly occurred. A small fraction of HA did, however, appear on the lateral surface and was incorporated into the envelope of budding VSV virions. Although predominantly found on the basolateral surface, significant amounts of G protein were observed on the apical plasma membrane well before disruption of the tight junctions was detectable. Nevertheless, assembly of VSV virions was restricted to the basolateral domain and in doubly infected cells the G protein was only infrequently incorporated into the envelope of budding influenza virions. These observations indicate that the site of VSV budding is not determined exclusively by the presence of G polypeptides. Therefore, it is likely that, at least for VSV, other cellular or viral components are responsible for the selection of the appropriate budding domain.

Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis.

A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn-78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with alpha-2-macroglobulin at the low temperature. Thus, the process of lysosomal maturation appears to involve the progressive delivery of lysosomal enzymes to various types of endosomes that may have already received some of the lysosomal membrane proteins. Although endolyn-78 would be one of the proteins added early to endosomes, other lysosomal membrane proteins may be added only to multivesicular endosomes that represent very advanced stages of maturation.

Biosynthesis and intracellular sorting of growth hormone-viral envelope glycoprotein hybrids.

Various aspects of the biogenetic mechanisms that are involved in the insertion of nascent plasma membrane proteins into the endoplasmic reticulum (ER) membrane and their subsequent distribution through the cell have been investigated. For these studies chimeric genes that encode hybrid proteins containing carboxy-terminal portions of the influenza virus hemagglutinin (154 amino acids) or the vesicular stomatitis virus envelope glycoprotein (G) (60 amino acids) linked to the carboxy terminus of a nearly complete secretory polypeptide, growth hormone (GH), were used. In in vitro transcription-translation experiments, it was found that the insertion signal in the GH portion of the chimeras led to incorporation of the membrane protein segments into the ER membrane. Effectively, GH became part of the luminal segment of membrane proteins of which only very small segments, corresponding to the cytoplasmic portions of the G or HA proteins, remained exposed on the surface of the microsomes. When the chimeric genes were expressed in transfected cells, the products, as expected, failed to be secreted and remained cell-associated. These results support the assignment of a halt transfer role to segments of the membrane polypeptides that include their transmembrane portions. The hybrid polypeptide containing the carboxy-terminal portion of HA linked to GH accumulated in a juxtanuclear region of the cytoplasm within modified ER cisternae, closely apposed to the Golgi apparatus. The location and appearance of these cisternae suggested that they represent overdeveloped transitional ER elements and thus may correspond to a natural way station between the ER and the Golgi apparatus, in which further transfer of the artificial molecules is halted. The GH-G hybrid could only be detected in transfected cells treated with chloroquine, a drug that led to its accumulation in the membranes of endosome or lysosome-like cytoplasmic vesicles. Although the possibility that the chimeric protein entered such vesicles directly from the Golgi apparatus cannot be ruled out, it appears more likely that it was first transferred to the cell surface and was then internalized by endocytosis.

Realistic and interactive high-resolution 4D environments for real-time surgeon and patient interaction.

BACKGROUND: Remote consultations that are realistic enough to be useful medically offer considerable clinical, logistical and cost benefits. Despite advances in virtual reality and vision hardware and software, these benefits are currently often unrealised. METHOD: The proposed approach combines high spatial and temporal resolution 3D and 2D machine vision with virtual reality techniques, in order to develop new environments and instruments that will enable realistic remote consultations and the generation of new types of useful clinical data. RESULTS: New types of clinical data have been generated for skin analysis and respiration measurement; and the combination of 3D with 2D data was found to offer potential for the generation of realistic virtual consultations. CONCLUSION: An innovative combination of high resolution machine vision data and virtual reality online methods, promises to provide advanced functionality and significant medical benefits, particularly in regions where populations are dispersed or access to clinicians is limited. Copyright © 2016 John Wiley & Sons, Ltd.

Decay-accelerating factor in human skin is associated with elastic fibers.

Recently a complement inhibitor, decay-accelerating factor (DAF), has been found in association with uncharacterized fibers in the extracellular matrix of human dermis. Here we show by immunohistochemistry and immunoelectronmicroscopy that DAF is on the periphery of elastic fibers, and that it appears to be associated with some microfibrillar elements that cover the fibers. That DAF is a component of these microfibrils is also suggested by studies of lesional skin from anetoderma, a disease characterized by destruction of elastic fibers. In two patients we found a network of residual fine fibers in the dermis that stain with antibodies against DAF and fibrillin (one of the proteins known to be present in the microfibrils of elastin), but do not stain with antibodies to elastin. Western blot analysis of dermal extracts with monoclonal antibodies to DAF identified a 67 kDa molecule, slightly smaller than membrane DAF, and similar in size to soluble DAF found in secretions. It is possible that together with vitronectin, an inhibitor of the membrane attack complex recently identified in association with elastin, DAF prevents damage of elastic fibers by complement.

Treatment of broiler litter with organic acids.

Experiments for treatment of contaminated broiler litter with citric, tartaric and salicylic acids were performed. At days 2 and 6 after the treatment, pH values (using a pH-meter), the ammonia concentrations (titration with 0.1 N HCl) and the microbial cells counts were determined in both experimental and control specimens of litter. The cost of acidification of litter was also determined. Our studies showed that the treatment of the contaminated litter with 5 per cent citric acid, 4 per cent tartaric acid and 1.5 per cent salicylic acid created an acid medium with pH under 5.0 and thus reduced the microbial counts to 2.2 x 10(3)colony forming units per gram manure litter. The treatment reduced the content of ammonia in the litter and in the air under the hygienic limits, i.e. 25-50 ppm. The cost of acidification of litter with these organic acids amounted to 0.1 $ per bird and 1.5 $ per 15 birds on one square metre in a growth period of 50 days.

Mechanism of formation of post Golgi vesicles from TGN membranes: Arf-dependent coat assembly and PKC-regulated vesicle scission.

We have developed an experimental system that utilizes purified Golgi fractions obtained from virus infected infected MDCK cells to reproduce in vitro the process of vesicle generation in the trans Golgi network, an important site for the sorting of proteins addressed to the plasma membrane, secretory vesicles, or lysosomes. Using an integrated biochemical and electron microscopic approach, we have shown that the formation of post Golgi vesicles carrying proteins destined to both plasma membrane domains of epithelial cells requires the activation of an ArF-like GTP-binding protein that serves to promote the assembly of the protein coat necessary to deform the donor membrane and generate a vesicle. The formation of the post Golgi vesicles also requires the participation of a Golgi membrane-associated Protein Kinase C, but not its phosphorylating activity. Other authors have shown that this is also the case for the PKC activation of the enzyme phospholipase D, which generates phosphatidic acid from phosphatidyl choline and may be involved in remodeling of membranes. We have been able to dissect the process of post Golgi vesicle generation into two sequential stages, one of coat assembly and bud formation, and a subsequent one of vesicle scission. The first stage can occur at 20 degrees C and requires the activation of the Arf protein necessary for coat assembly. The second stage does not require nucleotides or an energy supply, but requires cytosolic proteins, and in particular, an NEM sensitive membrane scission promoting activity that operates only at a higher temperature of incubation. Because various PKC inhibitors blocked vesicle scission without preventing bud formation, we propose that the PKC is required for the activation of a PLD in the TGN, which leads to remodeling of the donor membrane and the severing of connections between the emerging vesicles and the membranes.

[Use of cultured, virus-infected cells to study the biogenesis of polarity of epithelial cells]. / Utilisation de cultures de cellules infectées par des virus pour l'étude de la biogénèse de la polarité des cellules épithéliales.

The major characteristic of the eucaryote cell is the presence of specialized organelles in which macromolecular components responsible for various subcellular functions are segregated. The membranes of these organelles serve not only as divisions between the various cytoplasmic compartments, but also provide scaffolding within which the macromolecular complexes of the organelle assemble and become functionally integrated. It is obvious that because of the degree of complexity resulting from the existence of numerous compartments and membrane systems, the development of a genetic programme in a eucaryote cell requires not only the transcription of specific genes and translation in the cytoplasma of the resultant messenger RNA, but also the activity of mechanisms which ensure that each polypeptide reaches the site of its function, which may be in the cytosol, in a membrane, or in the luminal cavity of an organelle. In the special case of membrane proteins, such mechanisms must result not only in the specific distribution of polypeptides newly synthesized in the various types of cell membrane, but also the arrangement of them required in the lipid bi-layer necessary for their normal function.

[Evaluation of the specificity of skin allergy tests in cattle with IBR-IPV]. / Prouchvaniia vurkhu spetsifichnostta na kozhnoalergichnata reaktsiia pri zarazniia rinotrakheit-zarazniia pustulozen vulvovaginit po govedata.

An allergic and serologic investigation was carried out by the skin allergic test (SAT) and the virus neutralization test (VNT) to demonstrate the presence of infectious bovine rhinotracheitis -- infectious pustular vulvovaginitis (IBR-IPV) in cattle divided into three groups according to age, treated with the live attenuated vaccine MK25 against Aujeszky's disease. It was found that this vaccine induced the production of specific antibodies against the virus of IBR-IPV, which could be demonstrated through VNT (beta-variant) in low titers (2-2.5 log10), while through VNT (alfa-variant) no specific antibodies could be found. On the other hand, the virus of Aujeszky's disease did not usually lead or on very rare occasions (massive virus doses and continuous antigenic challenge) led to allergization of the body of cattle for IBR-IPV as well as to a positive SAT. Out of a total of 30 investigated calves only one proved positive by SAT, and 2 others were suspected of being carriers of infection. The problem is discussed of the specificity of SAT and its diagnostic value in IBR-IPV under some epizootiologic circumstances.

[Breed, line and sex differences of phagocytic activity in relation to broiler poultry resistance to Marek's disease virus]. / Porodni, lineini i polovi razliki na fagotsitnata aktivnost vuv vruzka s ustoichivostta na ptitsite broilerno napravlenie kum virusa na bolestta na Marek.

The phagocytic activity was studied of 421 poulets and cocks of lines 66 and 77 of the Cornish breed, and lines 88 and 99 of the White Plymouth rock breed. Breed and linear differences were noticed in the changes of the phagocytic activity (phagocytic number and phagocytic index) with birds infected with the virus of Marek's disease. The rise of the phagocytic activity with lines 66 and 77 was slighter as compared with that of the White Plymouth Rock lines 88 and 99. Strongest were the changes in the phagocytic activity with birds that were strongly susceptible to Marek's disease, and slight est--with birds that were resistant to the disease. A trend of a better expressed phagocytic activity was noticed with the cocks of all investigated lines.

[Timing of sensitization and appearance of skin allergic reaction in infectious bovine rhinotracheitis--infectious pustular vulvovaginitis]. / Prouchvaniia vurkhu sroka na alergizirane na organizma i poiavata na kozhnoalergichna reaktsiia pri zarazniia rinotrakheit--zarazniia pustulozen vulvovaginit po goveda.

Allergic and serologic investigations were carried out on infectious bovine rhinotracheitis--infectious pustular vulvovaginitis with cattle artificially and spontaneously affected with the causative agent of the disease as well as with cattle that were treated with a live attenuated vaccine. It was found that in the case of experimental infection the allergization of the body and the positive skin allergic reaction set in as early as the 7th day of the disease while in the case of vaccination they developed two weeks later. In the course of a natural development of the disease (spontaneous abortions) allergization likewise set in more rapidly than in animals that were vaccinated. In all three groups of cattle the results of the serologic investigation corresponded in general terms to those obtained by the skin allergic reaction. Discussed are the factors that substantiate the differences in the time of allergization of the body and the development of a positive skin reaction.

Organization of microtubules in dendrites and axons is determined by a short hydrophobic zipper in microtubule-associated proteins MAP2 and tau.

Here we report that the microtubule-associated proteins MAP2 and tau share two separable functional domains. One is the microtubule-binding site which serves to nucleate microtubule assembly; the second is a short C-terminal alpha-helical sequence which can crosslink microtubules by means of a hydrophobic zipper interaction into dense stable parallel arrays characteristic of axons or dendrites. Thus, interactions between molecules of a single type are capable of drastically reorganizing microtubules and completely suppressing their dynamic properties.

[Effect of tuberculin and levamisole on the immune response after vaccinating calves against para-influenza and Salmonella infections]. / Vliianie na tuberkulina i levamizola vurkhu imunniia otgovor pri vaktsinatsiia na teleta sreshtu parainfluenchna i salmonelna infektsiia.

Studied was the effect of tuberculin and levamizole on the immune response following vaccination of calves against parainfluenza and Salmonella infections. It was found that in calves that had colostrum antibodies against parainfluenza the immunogenic stimulation with the two agents first led to a drop of the total protein and of the antibody titer of the blood serum; in later periods the antibody titer grew severalfold. The stimulation with tuberculin and levamizole in the case of Salmonella infection substantiated more abrupt humoral immune response as against the case of animals that were not stimulated. More effective was the stimulation when lemvamizole was applied on the 7th day following vaccination. It was established that both tuberculin and levamizole stimulated the phagocytic activity of the blood of calves vaccinated against a parainfluenza virus and did not influence essentially the same activity in calves vaccinated against Salmonella infection. The amount of erythrocyte-rosette forming cells (E-POK) also remained visibly unchanged by the effect of tuberculin and levamizole with the use of the two vaccines.

A sorting signal for the basolateral delivery of the vesicular stomatitis virus (VSV) G protein lies in its luminal domain: analysis of the targeting of VSV G-influenza hemagglutinin chimeras.

When synthesized in polarized epithelial cells, the envelope glycoproteins hemagglutinin of influenza and G of vesicular stomatitis virus are targeted to the apical and basolateral plasma membranes, respectively. To determine which portions of these transmembrane proteins contain information necessary for their sorting, the behavior of two different G-hemagglutinin chimeric polypeptides, consisting of all or nearly all the luminal portion of the vesicular stomatitis virus G protein linked to C-terminal segments of influenza hemagglutinin that included its transmembrane and cytoplasmic domains, was studied in MDCK cells transformed with the corresponding cDNAs. Both chimeras were transported from the endoplasmic reticulum to the Golgi apparatus and from there to the cell surface with the same rapid kinetics as the intact G protein. By using a cell surface immunoprecipitation assay with monolayers cultured on permeable filters that allows the recovery of labeled protein molecules present in each cell surface domain, it was found that both chimeric proteins as well as the intact G protein were delivered almost exclusively to the basolateral surface. This polarized distribution of the polypeptides did not change during a subsequent 90-min chase period, although during this time a large fraction of the glycoprotein molecules underwent degradation. In addition, a small fraction of the cell surface-associated glycoprotein molecules shed their ectoplasmic segments into the basolateral compartment, apparently as a result of a proteolytic cleavage. Immunofluorescence on transverse frozen sections and immunoelectron microscopy revealed a prominent accumulation of the chimeric polypeptides in the lateral cell membranes, with lesser amounts on the basal and apical surfaces. These results indicate that information specifying the basolateral transport of the G glycoprotein is located within the first 426 N-terminal amino acids of its ectoplasmic portion.

Cell-free reconstitution of the transport of viral glycoproteins from the TGN to the basolateral plasma membrane of MDCK cells.

An in vitro system to study the transport of plasma membrane proteins from the TGN to the basolateral plasma membrane of polarized MDCK cells has been developed in which purified cell fractions are combined and transport between them is studied under controlled conditions. In this system, a donor Golgi fraction derived from VSV or influenza virus-infected MDCK cells, in which 35S-labeled viral glycoproteins were allowed to accumulate in the TGN during a low temperature block, is incubated with purified immobilized basolateral plasma membranes that have their cytoplasmic face exposed and are obtained by shearing-lysis of MDCK monolayers grown on cytodex beads. Approximately 15-30% of the labeled glycoprotein molecules are transferred from the Golgi fraction to the acceptor plasma membranes and are recovered with the sedimentable (1 g) beads. Transport is temperature, energy and cytosol dependent, and is abolished by alkylation of SH groups and inhibited by the presence of GTP-gamma-S, which implicates GTP-binding proteins and the requirement for GTP hydrolysis in one or more stages of the transport process. Endo H-resistant glycoprotein molecules that had traversed the medial region of the Golgi apparatus are preferentially transported and their luminal domains become accessible to proteases, indicating that membrane fusion with the plasma membrane takes place in the in vitro system. Mild proteolysis of the donor or acceptor membranes abolishes transport, suggesting that protein molecules exposed on the surface of these membranes are involved in the formation and consumption of transport intermediates, possibly as addressing and docking proteins, respectively. Surprisingly, both VSV-G and influenza HA were transported with equal efficiencies to the basolateral acceptor membranes. However, low concentrations of a microtubular protein fraction preferentially inhibited the transport of HA, although this effect was not abolished by microtubule depolymerizing agents. This system shows great promise for elucidating the mechanisms that effect the proper sorting of plasma membrane proteins in the TGN and their subsequent targeting to the appropriate acceptor membrane.