The distribution pattern of α2,3- and α2,6-linked sialic acids affects host cell preference in Toxoplasma gondii.

Tachyzoites of Toxoplasma gondii, an obligate intracellular parasite, actively invade almost all types of nucleated cells. However, T. gondii tachyzoites preferentially infect particular types of animal tissue cells. The mechanism underlying the host cell preference of T. gondii is not yet known. In this study, we found that enzymatic removal of α2,3- but not α2,6-linked sialic acids on the surface of Vero cells decreased T. gondii tachyzoite adhesion or invasion to the treated cells. Although Chinese hamster ovary (CHO) cells express only α2,3-linked sialic acid, a genetically modified CHO cell line constructed by transfection with the α2,6-sialiltransferase gene contains subpopulations with a variety of expression patterns of α2,3- and α2,6-linked sialic acids. When T. gondii tachyzoites were added to the modified CHO cells, the tachyzoites preferentially attached to cells belonging to a subpopulation of cells that highly expressed α2,3-linked sialic acids. Additionally, multiple regression analysis performed to analyse the relationship between the amount of α2,3-linked/α2,6-linked sialic acids and parasite-expressed fluorescence intensity suggested that more tachyzoites adhered to individual α2,3-linked sialic acid rich-cells than to α2,3-linked sialic acid-poor/null cells. The results of confocal laser microscopy confirmed this finding. These results indicate that the host cell preference of T. gondii was, at least partially, affected by the distribution pattern of α2,3-, but almost never α2,6-linked sialic acids on host cells.

A method for the purification of Shiga-like toxin 1 subunit B using a commercially available galabiose-agarose resin.

We describe a procedure for the affinity purification of Shiga toxin 1 subunit B (SLTB) using a commercial galabiose-agarose resin. Recombinant SLTB was purified to 99% homogeneity in a single-step protocol, from the periplasmic extracts of Vibrio cholerae 0395 N1/pSBC54. SDS-PAGE of the affinity purified SLTB showed one band of 8 kDa MW. SLTB purified by this procedure retained its chemical and biological activity as demonstrated by re-binding to the galabiose-agarose resin, and receptor-mediated binding and uptake in Vero cells. The galabiose-agarose resin could isolate roughly 1mg of SLTB/mL of gel. The resin was stable over 3 years and 500 cycles/year of usage. Hence, this method is a straightforward approach to the large-scale preparation of SLTB at a reasonable cost.

Assessing the contribution of the herpes simplex virus DNA polymerase to spontaneous mutations.

BACKGROUND: The thymidine kinase (tk) mutagenesis assay is often utilized to determine the frequency of herpes simplex virus (HSV) replication-mediated mutations. Using this assay, clinical and laboratory HSV-2 isolates were shown to have a 10- to 80-fold higher frequency of spontaneous mutations compared to HSV-1. METHODS: A panel of HSV-1 and HSV-2, along with polymerase-recombinant viruses expressing type 2 polymerase (Pol) within a type 1 genome, were evaluated using the tk and non-HSV DNA mutagenesis assays to measure HSV replication-dependent errors and determine whether the higher mutation frequency of HSV-2 is a distinct property of type 2 polymerases. RESULTS: Although HSV-2 have mutation frequencies higher than HSV-1 in the tk assay, these errors are assay-specific. In fact, wild type HSV-1 and the antimutator HSV-1 PAAr5 exhibited a 2-4 fold higher frequency than HSV-2 in the non-HSV DNA mutatagenesis assay. Furthermore, regardless of assay, HSV-1 recombinants expressing HSV-2 Pol had error rates similar to HSV-1, whereas the high mutator virus, HSV-2 6757, consistently showed significant errors. Additionally, plasmid DNA containing the HSV-2 tk gene, but not type 1 tk or LacZ DNA, was shown to form an anisomorphic DNA structure. CONCLUSIONS: This study suggests that the Pol is not solely responsible for the virus-type specific differences in mutation frequency. Accordingly, it is possible that (a) mutations may be modulated by other viral polypeptides cooperating with Pol, and (b) the localized secondary structure of the viral genome may partially account for the apparently enhanced error frequency of HSV-2.

Detection of rubella-virus-induced apoptosis in Vero cell cultures with hematoxylin and eosin staining.

In order to facilitate the detection of apoptotic cells (Apo C) in Rubella virus (RV) infected cultures in settings of low resources, we compared hematoxylin and eosin staining (H&E) with the conventional TUNEL technique, and confirmed our findings with DNA electrophoresis and transmission electron microscopy. H&E allowed to distinguish Apo C from non-apoptotic cells. The proportion of Apo C in infected cultures was proportional to the multiplicity of infection (MOI). At a MOI of 10, the percent of Apo C at 3, 4 and 5 days post infection (pi) were 26, 45 and 47%, respectively, which were significantly reduced when the caspase inhibitor z-VAD-fmk was present in the supernatant. By the TUNEL assay, the percent of Apo C in RV-infected cultures were lower (0.8, 1.2 and 1.2% at 3, 4 and 5 days pi, respectively). Our results have shown that H&E staining is an easy, rapid, economic and reproducible method to detect Apo C in RV infected Vero cells cultures. It is possible that H&E makes evident early stages of apoptosis, when an apoptotic cell shows chromatin condensation, nuclear and cytoplasmic contraction (but is still attached to the monolayer), while TUNEL detects later stages of apoptosis because it needs an extensive DNA fragmentation, when apoptotic cells are about to or have already detached from the substratum.

Molecular evolution in the hypervariable regions of fetuin: comparison between human and African green monkey fetuin.

Sequences of fetuin cDNA and its deduced amino acid residues from the African green monkey cell line Vero were found to differ by 7.3% and 12.9%, respectively, from the corresponding human sequences. Most amino acid substitutions were clustered within a small segment of the third domain (D3). Calculations of nonsynonymous and synonymous nucleotide substitution rates suggest that this small segment was mutated under positive selection. cDNAs encoding alpha1-antitrypsin, beta-actin and the sequences of intron 4 of alpha1-antitrypsin gene in human liver and Vero cells were also investigated. The results substantiated the positive selection imposed on the D3 segment.

Effect of bafilomycin A1 on the growth of Japanese encephalitis virus in Vero cells.

We studied the effect of bafilomycin A1 (Baf-A1), a novel and highly specific inhibitor for vacuolar-type proton (V-H+) pump, on the growth of Japanese Encephalitis virus (JEV) in Vero cells. Viral fluorescence microscopic study showed that Baf-A1 induced the complete disappearance of acidified compartments such as endosomes and lysosomes in Vero cells by the treatment with 0.1 microM Baf-A1 for 1 h at 37 degrees C. In proportion to the disappearance of acidified compartments, virus growth was inhibited when Baf-A1 was present from 1 h before infection to the end of incubation in a dose-dependent manner, or added within as early as 5 min after infection. Conversely, the virus growth was recovered in correlation with the reappearance of acidified compartments after removal of Baf-A1. These results suggest that a low pH condition, which is regulated by Baf-A1-sensitive V-H+ pumps, is essential for the early stage of JEV growth.

Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin.

A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells. The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D. This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown. The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines. The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional. Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor. From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and pore formation in the cell membrane.

Nuclear colocalization of the Ro 60 kDa autoantigen and a subset of U snRNP domains.

The Ro 60 kDa protein is an RNA binding molecule present both in the cytoplasm and in the nucleus. Cytoplasmic Ro 60 kDa is complexed to other proteins and to certain RNAs denoted hYRNAs. This RNA-protein complex is also known as the Ro/SSA antigen recognized by sera from patients with certain autoimmune disorders. Components interacting with the nuclear Ro 60 kDa protein fraction in mammalian cells have not been identified. To look for an association with previously known nuclear structures, rabbit antisera to the amino- and carboxy-terminal parts of the Ro 60 kDa protein were used in immunomorphological studies on HeLa cells. A strong speckled nuclear pattern and a weak cytoplasmic staining were detected. Double immunofluorescence staining with affinity purified anti-Ro 60 kDa antibodies and monoclonal antibodies recognizing the Sm and RNP antigens of the U snRNPs, displayed colocalization. Another U snRNP containing nuclear compartment, the coiled bodies, did not contain any Ro 60 kDa protein. Cells infected with a toga virus demonstrated redistribution of both U snRNP antigens and the Ro 60 kDa protein with retained colocalization. These results indicate a role for the nuclear fraction of the Ro 60 kDa protein in RNA processing.

A 205 kDa protein from non-neuronal cells in culture contains tubulin binding epitopes.

Microtubule-associated proteins (MAPs) interact with tubulin in vitro and in vivo. Despite that there is a large amount of information on the roles of these proteins in neurons, the data on non-neuronal MAPs or MAPs-related proteins is scarce. There is an increasing number of microtubule-interacting proteins that have been identified in different cultured cell lines, and some of them share common functional epitopes with the most well-known MAPs, MAP-2 and tau. In a search for tubulin-interacting proteins in non-neuronal cells we identified a 205 kDa protein in the monkey kidney Vero cells in culture, on the basis of immunological studies and affinity chromatography. This protein interacts with the C-terminal moiety of beta-tubulin and cosediments with taxol assembled microtubules, but it was not recovered after successive cycles of assembly and disassembly. The presence of neuronal MAPs such as MAP-1, MAP-2 and tau was not detected in these cells. Interestingly, the studies showed that the 205 kDa protein contained a tubulin binding motif which was recognized by site-directed antibodies that also tag tubulin binding epitopes on MAP-2 and tau. This characteristic led us to designate this protein as MBD-205, a component that shares binding domains with these MAPs, rather than as a marker of the MAPs family. On the other hand, immunofluorescence experiments using site-specific antibodies, i.e. MAP-reacting monoclonal anti-idiotypic reagent MTB6.22 and a polyclonal antibody to the second tau repeat, revealed a MBD-205 co-localization with membrane structures and microtubule-organizing centers in Vero cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The rubella virus E2 and E1 spike glycoproteins are targeted to the Golgi complex.

Rubella virus (RV) has been reported to bud from intracellular membranes in certain cell types. In this study the intracellular site of targeting of RV envelope E2 and E1 glycoproteins has been investigated in three different cell types (CHO, BHK-21 and Vero cells) transfected with a cDNA encoding the two glycoproteins. By indirect immunofluorescence, E2 and E1 were localized to the Golgi region of all three cell types, and their distribution was disrupted by treatment with BFA or nocodazole. Immunogold labeling demonstrated that E2 and E1 were localized to Golgi cisternae and indicated that the glycoproteins were distributed across the Golgi stack. Analysis of immunoprecipitates obtained from stably transfected CHO cells revealed that E2 and E1 become endo H resistant and undergo sialylation without being transported to the cell surface. Transport of RV glycoproteins to the Golgi complex was relatively slow (t1/2 = 60-90 min). Coprecipitation experiments indicated that E2 and E1 form a heterodimer in the RER. E1 was found to fold much more slowly than E2, suggesting that the delay in transport of the heterodimer to the Golgi may be due to the slow maturation of E1 in the ER. These results indicate that RV glycoproteins behave as integral membrane proteins of the Golgi complex and thus provide a useful model to study targeting and turnover of type I membrane proteins in this organelle.

One step high yield affinity purification of shiga-like toxin II variants and quantitation using enzyme linked immunosorbent assays.

We have previously purified both shiga-like toxin (SLT) I and II using the toxins' affinity to P1 glycoprotein (P1gp) from hydatid cyst material (HCM). Binding of these toxins is based on their affinity for terminal Gal alpha 1-->4Gal disaccharide residues present in HCM. Although the binding specificity of SLT-II variants (v) differs from that of STL-II they are reported to recognize Gb3 and should bind to P1gp. Therefore we examined the usefulness of HCM to purify SLT-IIv of porcine (p) and human (h) origin. Toxins were purified from fermenter culture supernatants of Escherichia coli HB101 (pDLW5) (SLT-IIvp), and E. coli DH5 alpha (pJES210) (SLT-IIvh) utilizing HCM. SLT-IIvh and SLT-IIvp consisted of A and B subunits, as determined by SDS-PAGE. We obtained 0.16 mg SLT-IIvp and 0.12 mg SLT-IIvh/I of culture (yields > 65%). Various capture systems to detect shiga toxin, SLT-II, SLT-IIvp and SLT-IIvh by ELISA were examined. All toxins bound to HCM, and all except SLT-IIvp bound to the monoclonal antibody 4D1. Only SLT-IIvp bound to the glycolipid Gb4, and only shiga toxin bound significantly to Gb3. Similarities in the level of Gb4 expression in HeLa 229 (ATCC) and Vero cells may explain the lack of differential cytotoxicity between SLT-IIvp and SLT-IIvh on these cell lines.