Cross-protection against four species of chicken coccidia with a single recombinant antigen.

A cDNA clone, SO7', from an Eimeria tenella cDNA library was inserted into the high-expression vector pJC264 and was expressed in Escherichia coli as a fusion protein, CheY-SO7', with a molecular mass of approximately 36 kDa. By using the purified recombinant antigen to immunize young chicks, it was demonstrated that a single dose, without adjuvant, not only protected against severe coccidiosis induced by infection with E. tenella but also protected chicks challenged with the heterologous species Eimeria acervulina, E. maxima, and E. necatrix. By using rabbit antiserum raised against recombinant CheY-SO7', Western blot (immunoblot) analysis of sporulated oocysts of all seven major species of chicken coccidia showed that all species tested contained proteins characteristic of the B class of antigens, of which CheY-SO7' is representative. It seems likely that a single B antigen could protect chickens against severe coccidiosis caused by infection with any of these Eimeria species. Although chicks exposed to prolonged, natural infection develop antibodies to B antigen, active immunization of young chicks with a protective dose of CheY-SO7' does not elicit a humoral antibody response, suggesting that the partial protection results from cell-mediated effector mechanisms. In addition, the cross-protective nature of the immunity indicates that the response to B antigen is different from that induced by natural infection, which elicits a species-specific immunity. To date, the protection induced by B antigen immunization, although remarkable for a single recombinant protein, is not sufficient to compete with prophylactic chemotherapy.

Activity of the anthelmintic benzimidazoles against Giardia lamblia in vitro.

In vitro growth of the protozoan parasite Giardia lamblia was highly sensitive to certain anthelmintic benzimidazoles. Albendazole and mebendazole were 30- to 50-fold more active than metronidazole and 4- to 40-fold more active than quinacrine. Thiabendazole, a noncarbamate benzimidazole, was less active. Since lack of intestinal absorption makes mebendazole an attractive new antigiardial agent, its in vitro activity was further characterized. At low concentrations (0.05 micrograms/ml) mebendazole had a static effect on G. lamblia growth; however, lethal activity was observed at a concentration fivefold lower (0.3 micrograms/ml) than necessary for the cidal agent metronidazole. Two observations are consistent with a microtubule target for mebendazole. First, attachment of cells to the culture tube, mediated by the ventral disk and flagella, was rapidly disrupted by mebendazole treatment. Second, the characteristic cell structure was grossly distorted by treatment. No mebendazole-resistant G. lamblia were detected in a population of 10(8) cells.

Evidence for unusually short tubulin mRNA leaders and characterization of tubulin genes in Giardia lamblia.

We have cloned alpha- and beta-tubulin cDNAs from Giardia lamblia and have used these to determine the gene copy number in the organism and to isolate alpha- and beta-tubulin genomic clones. Studies of the gene organization demonstrate that two copies of beta-tubulin are linked in a head-to-head configuration. The DNA from these two copies and that from one alpha-tubulin copy has been sequenced upstream of the translation initiation codon, and analyzed for consensus to typical eukaryotic promoter sequences. Characterization of the alpha- and beta-tubulin mRNAs in this parasite by primer extension and S1 nuclease mapping has revealed an unusually short (6 nucleotides) 5' untranslated region.

Heat shock and stress response in Giardia lamblia.

The heat shock response was studied as a model for control of gene expression and protein synthesis in Giardia lamblia. Cultured trophozoites were metabolically labelled with [35S]methionine, and proteins were analysed by SDS-polyacrylamide gel electrophoresis and fluorography. A temperature shift from 37 degrees C to 43 degrees C resulted in the depression of normal protein synthesis, and the enhanced synthesis of four major heat shock proteins of 100, 83, 70 and 30 kDa. This response resembles that seen in other organisms of wide phylogenetic diversity. An examination of the kinetics of induction and recovery from heat shock suggests that the individual heat shock proteins are independently regulated. In vitro translation of messenger RNA isolated from heat shocked cells further indicates that regulation occurs at both transcriptional and translational levels. The response of G. lamblia to other stresses including cysteine deprivation, exposure to oxygen, ethanol, hydrogen peroxide, and the chemotherapeutic drugs metronidazole and quinacrine was also investigated. The induction of two or more of the heat shock proteins was generally observed; however, certain treatments inhibited synthesis of all proteins including heat shock proteins.

Unusual ribosomal RNA of the intestinal parasite Giardia lamblia.

The anaerobic protozoan Giardia lamblia is a common intestinal parasite in humans, but is poorly defined at molecular and phylogenetic levels. We report here a structural characterization of the ribosomal RNA (rRNA) and rRNA genes of G. lamblia. Gel electrophoresis under native or non-denaturing conditions identified two high molecular weight rRNA species corresponding to the 16-18S and 23-28S rRNAs. Surprisingly, both species (1300 and 2300 nucleotides long, respectively) were considerably shorter than their counterparts from other protozoa (typically 1800 and 3400 nucleotides), and from bacteria as well (typically 1540 and 2900 nucleotides long). Denaturing polyacrylamide gel electrophoresis identified a major low molecular RNA of 127 nucleotides and several minor species, but no molecules with the typical lengths of 5.8S (160 nucleotides) and 5S (120 nucleotides) rRNA. The G. lamblia 1300, 2300, and 127 nucleotide RNAs are encoded within a 5.6 kilobase pair tandemly repeated DNA, as shown by Southern blot analysis and DNA cloning. Thus, the rRNA operon of this eukaryotic organism can be no longer than a typical bacterial operon. Sequence analysis identified the 127 nucleotide RNA as homologous to 5.8S RNA, but comparisons to archaebacterial rRNA suggest that Giardia derived from an early branch in eukaryotic evolution.

Effects of diethylstilbestrol on Propionibacterium acnes immunomodulation: inhibition of macrophage activation and antitumor activity.

Exposure of mice to diethylstilbestrol (DES) inhibited Propionibacterium acnes-induced antitumor activity in vivo against the B16F10 subcutaneous tumor. The inhibitory effect was associated with inhibition by DES of the characteristic P. acnes induced splenomegaly and changes in splenic and peritoneal macrophages (M phi) cell populations. The characteristic P. acnes induced reductions in M phi alkaline phosphodiesterase I (APD) ectoenzyme activity and in total RNA synthesis, proposed biochemical markers of tumoricidal M phi, were partially or completely reversed in DES-treated mice. As predicted from these in vivo and in vitro results, DES treatment significantly decreased P. acnes activation of M phi antitumor activity in vitro against B16F10 melanoma and Lewis lung carcinoma cells. These data suggest a macrophage activation defect may be involved in the reduced resistance that DES-treated animals exhibit to a variety of neoplastic and microbial challenges.

Development and use of a rat albumin cDNA clone to evaluate the effect of chronic ethanol administration on hepatic protein synthesis.

A rat albumin cDNA probe (pBR alb 149) was developed in order to investigate the molecular mechanisms responsible for changes in hepatic protein synthesis after chronic administration of ethanol to rats. Rats fed a diet for up to 1 year in which 36% of calories were from ethanol, developed fatty livers but not cirrhosis. Cell-free protein synthesis with liver membrane-bound polysomes of ethanol-fed rats was increased as compared to control membrane-bound polysomes, whereas protein synthesis with free polysomes was unchanged. Total RNA extracted from liver membrane-bound polysomes and translated in a rabbit reticulocyte mRNA-dependent system showed a marked increase in albumin synthesis in the ethanol-fed group. Analysis of RNA molecules separated according to molecular weight by gel electrophoresis and hybridized with recombinant-cloned albumin cDNA demonstrated an increase in full-sized albumin mRNA species in ethanol-fed animals. Therefore, chronic ethanol administration appears to increase albumin synthesis by increasing the steady-state level of biologically active albumin mRNA in liver membrane-bound polysomes. Despite development of fatty liver, the protein synthesis machinery functions normally.

Extraction of vanadium(V) chelates with N-benzohydroxamic acid (BHA) and ammonium thiocyanate and its application in steel and rock analyses.

A mixed-ligand complex of vanadium(V) with N-benzohydroxamic acid and thiocyanate formed at various acidities can be extracted into methyl isobutyl ketone, and used for photometric determination of trace amounts of vanadium in materials such as alloy steels and rocks. The absorption maximum of the violet mixed-ligand complex is at 535 nm. The values for the simple complex are 505 nm and molar absorptivity 7.4 x 10(3)l.mole(-1).cm(-1).

Evidence for supercoiled hepatitis B virus DNA in chimpanzee liver and serum Dane particles: possible implications in persistent HBV infection.

In chimpanzee hepatitis B virus (HBV) carriers, the mechanism of viral persistence has been examined by analyzing viral DNA molecules in liver and serum. Chimpanzee liver DNA contained two extrachromosomal HBV DNA molecules migrating on hybridization blots at 4.0 kb and 2.3 kb. There was no evidence for integration of HBV DNA into the host genome. The extrachromosomal molecules were distinct from Dane particle DNA and were converted to linear 3.25 kb full-length double-stranded HBV DNA on digestion with Eco RI. Nucleases S1 and Bal 31 converted "2.3 kb" HBV DNA to 3.25 kb via an intermediate of "4.0 kb" apparent length. The HBV DNA molecule that migrated at 2.3 kb represents a supercoiled form I of the HBV genome, and the molecule that migrated at 4.0 kb represents a full-length "nicked," relaxed circular form II. Evidence for supercoiled HBV DNA in serum Dane particles was obtained by production of form II molecules upon digestion with nuclease S1 or Bal 31. It is proposed that most Dane particles represent interfering noninfectious virus containing partially double-stranded DNA circles and that particles containing supercoiled HBV DNA may represent infectious hepatitis B virus.

Characterization of viral genomes in the liver and serum of chimpanzee long-term hepatitis B virus carriers: a possible role for supercoiled HBV-DNA in persistent HBV infection.

In chimpanzee hepatitis B virus (HBV) carriers, the molecular mechanism for viral persistence has been examined by analyzing the properties of viral DNA molecules in liver and serum. Two extrachromosomal HBV-DNA molecules migrating on Southern blots at 4.0 kb and 2.3 kb were observed in chimpanzee liver DNA. There was no evidence for integration of HBV sequences into the host genome. The HBV-DNA molecule which migrated at 4.0 kb position represents a full-length "nicked," relaxed circular form, and the DNA molecules migrating at 2.3 kb position represents a supercoiled form of the HBV genome. Evidence for supercoiled HBV-DNA in serum was obtained by production of the relaxed circular intermediate upon digestion of Dane particle DNA with specific nucleases S1 and Bal 31. A possible role of these two extrachromosomal HBV-DNA molecules in the biology of hepatitis B virus infection and the mechanism for viral persistence are discussed.

Tumorigenicity in nude mice of a human hepatoma cell line containing hepatitis B virus DNA.

The human hepatocellular carcinoma cell line PLC/PRF/5, which synthesizes and secretes hepatitis B surface antigen, was grown under optimal conditions in tissue culture, using Eagle's minimal essential medium supplemented with 10% fetal bovine serum and 10(-11) M triiodothyronine on collagen rafts. Injection s.c. of the PLC/PRF/5 cell line into athymic BALB/c nude mice resulted in the growth of a well-circumscribed, moderately differentiated hepatocellular carcinoma. The intervals until tumor appearance and tumor "take" rates were dependent on inoculum dose. Four to 5 x 10(6) cells induced tumor growth in 29% of 14 injected mice within 29 to 40 days, while 7 to 13 X 10(6) cells induced tumors in all 15 mice within 10 to 12 days after inoculation. Hepatitis B surface antigen was detected in the nude mouse serum and tumor tissue, and its concentration roughly correlated with tumor weight. A low level of antibody against hepatitis B surface antigen was detected in five tumor-bearing animals, as well as in one mouse which did not produce a tumor. Hepatitis B core antigen and its antibody and hepatitis B e antigen and its antibody were not detected in 26 mice, using immunohistochemical and radioimmunoassay methods. alpha-Fetoprotein, carcinoembryonic antigen, and alpha-antitrypsin were detected in nude mice tumors, using the peroxidase-antiperoxidase technique. Finally, hepatitis B virus DNA, identified in the nude mouse tumor by molecular hybridization techniques, was compared to PLC/PRF/5 cell line hepatitis B virus DNA.

Chronic hepatitis in chimpanzee carriers of hepatitis B virus: morphologic, immunologic, and viral DNA studies.

Years after infection with hepatitis B virus, chimpanzees may have manifestations of the carrier state as described in man. In addition to serologic evidence for persistent viral infection, percutaneous liver biopsy specimens showed hepatitis B virus surface antigen in the cytoplasm and hepatitis B virus core antigen in the nucleus. Four carrier animals had portal inflammatory reaction as seen in human chronic persistent hepatitis. Viral DNA was demonstrated in nucleic acid extracts of liver biopsy specimens by molecular hybridization to cloned plasmic pA01 containing hepatitis B virus DNA sequences. Although a viral molecule of length greater than the putative virus was identified, it did not appear to represent integration of viral DNA into the host genome. The chimpanzee model may serve as a means to study the mechanism of hepatitis B viral persistence and progression to chronic liver disease.

Genetic variation during persistent reovirus infection: presence of extragenically suppressed temperature-sensitive lesions in wild-type virus isolated from persistently infected L cells.

Persistent reovirus infection of L cells was established with a serially passaged stock of temperature-sensitive (ts) mutant C(447) containing greater than 90% defective interfering particles. Within a month after establishment of the carrier culture, the ts mutant was replaced by virus that expressed the wild-type (ts(+)) temperature phenotype (R. Ahmed and A. F. Graham, J. Virol. 23:250-262, 1977). To determine whether the ts(+) phenotype of the virus was due to intragenic reversion or to the presence of an extragenic mutation suppressing the original ts defect, several clones were backcrossed to wild-type reovirus, and the progeny of each cross were screened for temperature sensitivity. The results indicated that the original tsC lesion had reverted. However, in two of the seven clones examined, new ts lesions were found. These new ts lesions appeared phenotypically as ts(+) due to the presence of extragenic suppressor mutations. Temperature-sensitive mutants representing three different groups were rescued from one suppressed clone, indicating that this ts(+) clone contained multiple ts lesions. Among the ts mutants rescued were the initial isolates of a new recombination group which we have designated H. Some of the ts mutants rescued from the suppressed clones are capable of interfering with the growth of wild-type reovirus and may play a role in maintaining the carrier state. The results of this study show that persistently infected L cells contain a genetically heterogeneous population of reovirus even though all virus clones express the ts(+) phenotype. It is thus critical to distinguish between genotype and phenotype when analyzing viruses that emerge during persistent infection.

Genetic variation during lytic reovirus infection: high-passage stocks of wild-type reovirus contain temperature-sensitive mutants.

Wild-type clones of reovirus serotypes 1 (Lang), 2 (Jones), and 3 (Dearing) were serially passaged in L cells at a high multiplicity of infection, and the virus population was examined at passage levels 2, 5, and 11 for the presence of temperature-sensitive (ts) mutants. By passage 11 all three serotypes contained ts mutants that were not present in the original wild-type stock. ts mutants representing three mutant groups were identified. The majority of these mutants were in group G. Our results show that high-passage stocks of reovirus consist of a genetically heterogeneous population.

Ribonuclease III cleavage of bacteriophage T3RNA polymerase transcripts to late T3 mRNAs.

In vitro transcription of T3 DNA by T3 phage-induced RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase; EC 2.7.7.6) yields eight discrete RNAs (designated I-VIII) with molecular weights of approximately 6.2, 4.7, 4, 2.8, 1.8, 0.9, 0.52, and 0.21 X 10(6), respectively. Comparison of the size of in vitro T3 RNA polymerase transcripts with in vivo late T3 mRNAs indicates that several late RNAs produced in T3-infected cells do not correspond to any of the in vitro RNAs, and no RNAs as large as the three largest in vitro RNA species, I, II, and III, are observed. Escherichia coli RNase III cleaves these three high molecular weight T3 RNA polymerase transcripts to discrete RNAs that comigrate in polyacrylamide gel electrophoresis with some of the late T3 RNAs.