Characterization of cocksfoot mottle sobemovirus genomic RNA and sequence comparison with related viruses.

The genome of cocksfoot mottle virus (CfMV) is a positive-sense ssRNA molecule of 4082 nucleotides as revealed by sequencing the entire genome. The 5'-untranslated region of the genome is 69 nucleotides and the 3'-untranslated region is 225 nucleotides in length. The coding region contains four open reading frames (ORFs). The organization of CfMV ORFs differs significantly from that of the previously sequenced sobemoviruses southern bean mosaic virus and rice yellow mottle virus. ORF1 encodes a protein having a calculated molecular mass of 12.3 kDa. The function of this protein is unknown. The next ORF codes for the putative VPg and serine protease. The ORF2a product consists of 568 amino acids, with a calculated molecular mass of 60.9 kDa. The replicase of CfMV is translated as part of a polyprotein by--1 ribosomal frameshifting in ORF2a. The calculated molecular mass of the transframe protein is 103.4 kDa. ORF3 encodes the 27.6 kDa coat protein. This has been verified by amino acid sequencing of the CfMV coat protein N terminus. Northern blots of total RNA from CfMV-infected barley leaves reveal the 4.1 kb genomic RNA band and one virus-specific band of 1.2 kb, which may represent a subgenomic RNA for coat protein synthesis.

The putative replicase of the cocksfoot mottle sobemovirus is translated as a part of the polyprotein by -1 ribosomal frameshift.

The polyprotein of cocksfoot mottle sobemovirus (CfMV) is encoded by two overlapping open reading frames (ORF). The ORF 2a codes for the putative VPg and serine protease and the ORF 2b codes for the putative replicase. The consensus signals for a -1 ribosomal frameshifting event are found at the very beginning of the overlapping region of these ORFs. The shifty heptanucleotide in CfMV is UUUAAAC, and the secondary structure after the shifty sequence is predicted to be a stem-loop. In vitro translation of the CfMV RNA in wheat germ extract produced proteins of several sizes, including one of 100 kDa. According to the nucleotide sequence data, no single ORF is capable of directing the synthesis of a 100-kDa protein. A chimeric beta-glucuronidase-CfMV cDNA containing the entire ORF 2a and 2b overlap region including frameshift signals was constructed. A trans-frame protein of 108 kDa was produced from this construct with an efficiency of 26-29% by in vitro translation in wheat germ extract. CfMV is the first sobemovirus in which the putative replicase is reported to be produced as a part of a polyprotein by a -1 frameshift event. The replicases of the sobemoviruses are related to the luteovirus subgroup II replicases, which are known to be produced by -1 ribosomal frameshift. The reported amino acid sequences of the putative replicases of sobemo- and subgroup II luteoviruses were compared to that of the putative replicase of CfMV. This comparison revealed more extensive homology between these groups than previously reported.

DNA base composition, aerotolerance and enzyme patterns of Leptotrichia buccalis.

The guanine plus cytosine content of the DNA of Leptotrichia buccalis varied from 28.4 to 29.5 mol% (three strains). Eleven strains examined grew well under anaerobic and microaerobic conditions, but slowly in air in the presence of CO2. When examined for preformed enzymes in the APIZYM Complete Research Kit, positive reactions were obtained for several glucosidases and carboxylic ester hydrolases, and for a few peptidases.

Adherence of Campylobacter jejuni and Campylobacter coli to porcine intestinal brush border membranes.

The adhesion of Campylobacter jejuni and C. coli to isolated porcine intestinal brush border membranes was studied by phase-contrast and electron microscopy. Approximately 45% of the cell population adhered to the brush borders, possibly in a specific manner. Pretreatment of the brush borders with trypsin or pronase, and competitive inhibition with L-rhamnose caused a slight reduction of the adhesion. Different forms of pretreatment of the bacterial cells reduced their ability to adhere, but also their motility.

Biological activity of lipopolysaccharides from oral Selenomonas.

Lipopolysaccharides (LPSs) were extracted by phenol-water from three oral strains of Selenomonas. The preparations were tested for the ability to induce a blastogenic response in cultures of spleen cells from normal and nude BALB/c mice, to activate guinea pig complement and the clotting enzyme system of Limulus polyphemus amoebocytes, and to kill Actinomycin-D treated mice. The capacity of the three LPSs was comparable to that of enterobacterial LPS.

A possible effect on the Shwartzman reaction in the rabbit caused by fentanyl-fluanisone tranquillization.

Shwartzman's reaction was elicited in four Chinchilla rabbits. Two of the animals were tranquillized with fentanyl-fluanisone (Hypnorm: Janssen Pharmaceuticals). There was a reduced response in the tranquillized rabbits while the untranquillized animals developed the typical dose-responsive skin reaction. Fentanyl-fluanisone tranquillization may interfere with the development of the Shwartzman reaction in Chinchilla rabbits.

In vitro blastogenic response and immunoglobulin synthesis in murine spleen cells stimulated with lipopolysaccharides from Campylobacter jejuni and Campylobacter coli.

Lipopolysaccharides isolated from strains of Campylobacter jejuni and Campylobacter coli were mitogenic for spleen cells as measured by 3H-thymidine incorporation. The incorporation was dose dependent with an increase with increased concentration of LPS. Addition of LPS beyond 500 micrograms/ml gave inhibition of incorporation. Stimulation of the spleen cells for 3 days with LPS led to a polyclonal activation of immunoglobulin synthesis. The amount of immunoglobulins synthesised showed a maximum between the 9th and 11th day of incubation, and another maximum between the 15th and 17th day. The immunoglobulins produced were IgM antibodies. Specific antibodies against the LPS used for the stimulation were not detected.

Antigenicity of lipopolysaccharides from Campylobacter jejuni and Campylobacter coli in passive haemagglutination tests and enzyme-linked immunosorbent assays.

Passive haemagglutination tests and ELISA were used to study the serological activity of homologous and heterologous rabbit antisera against LPS prepared from various strains of C. jejuni/coli. In both test systems the homologous antisera exhibited serological activity against LPS. The heterologous antisera showed some degree of intra- and inter- species cross-reactivity. The cross-reacting was most pronounced in the ELISA. Erythrocytes sensitized with untreated LPS gave higher antibody-titres than erythrocytes sensitized with alkali-treated LPS in the haemagglutination tests.

Chemical studies of partially hydrolysed lipopolysaccharides from four strains of Campylobacter jejuni and two strains of Campylobacter coli.

Lipopolysaccharides (LPS) from four strains of Campylobacter jejuni and two strains of C. coli were partially hydrolysed with 1% acetic acid. Subsequent chloroform extraction led to the formation of a polysaccharide-containing aqueous layer, an interfacial material and a lipid A-containing chloroform layer. The polysaccharides contained the neutral sugars, amino sugars, 2-keto-3-deoxy-octonic acid, and part of the phosphorus present in the undegraded LPS. The lipid As were made up of glucosamine, phosphorus, ester- and amide-linked 3-hydroxytetradecanoic acid, and ester-linked n-tetradecanoic and n-hexadecanoic acid. The interfacial material was made up of lipid A and undegraded LPS. When chromatographed on Bio-GEl P-60, the degraded polysaccharides were eluted as two incompletely separated peaks (strains NCTC 11168, NCTC 11351, 11041 and 11101) or as one peak (strains NCTC 11392 and E 8035). All peaks appeared close to the total volume of the column. When the different fractions were re-chromatographed on Bio-GEl P-10, the peaks still appeared close to the total volume of the column. These findings indicate that LPS from C. jejuni and C. coli are devoid of long O-antigenic side-chains.

Chemical composition and biological activity of lipopolysaccharides prepared from type strains of Campylobacter jejuni and Campolybacter coli.

Lipopolysaccharides (LPS) were extracted from seven type strains of Campylobacter jejuni and one type strain of Campylobacter coli with 45 per cent aqueous phenol. The sugar components present in all LPS were glucose, galactose, L-glycero-D-manno-heptose, 2-keto-3-deoxy-octulosonic acid and glucosamine. All but one LPS contained galactosamine, and two strains contained in addition mannose. The fatty acids present were 3-hydroxy-tetradecanoic acid, n-hexadecanoic acid and trace amounts of n-tetradecanoic acid. The LPS preparations examined showed anti-complementary effect, and were able to gelatinize Limulus amoebocyte lysate. LPS of the C.jejuni strain tested (NCTC 11168) was found to be lethal for mice and to produce the local Shwartzman reaction in rabbits.

Isolation and chemical composition of lipopolysaccharide from Campylobacter jejuni.

Lipopolysaccharide (LPS) from three strains of Campylobacter jejuni was extracted by aqueous phenol. The sugar components present in all strains were glucose, galactose, L-glycero-D-manno-heptose and glucosamine. One strain contained, in addition, galactosamine. The fatty acids present were mainly 3-hydroxy-tetradecanoic acid and n-hexadecanoic acid. The LPS contained 2-keto-3-deoxy-octonate and phosphorus.