Immunization of mice with Plasmodium TCTP delays establishment of Plasmodium infection.

Translationally controlled tumour protein (TCTP) may play an important role in the establishment or maintenance of parasitemia in a malarial infection. In this study, the potential of TCTP as a malaria vaccine was investigated in two trials. In the initial vaccine trial, Plasmodium falciparum TCTP (PfTCTP) was expressed in Saccharomyces cerevisiae and used to immunize BALB/c mice. Following challenge with Plasmodium yoelii YM, parasitemia was significantly reduced during the early stages of infection. In the second vaccine trial, the TCTP from P. yoelii and P. berghei was expressed in Escherichia coli and used in several mouse malaria models. A significant reduction in parasitemia in the early stages of infection was observed in BALB/c mice challenged with P. yoelii YM. A significantly reduced parasitemia at each day leading up to a delayed and reduced peak parasitemia was also observed in BALB/c mice challenged with the nonlethal Plasmodium chabaudi (P.c.) chabaudi AS. These results suggest that TCTP has an important role for parasite establishment and may be important for pathogenesis.

Molecular typing of Salmonella enterica serovar Sofia in Australia by pulsed-field gel electrophoresis and repetitive element PCR typing.

AIMS: In this study, we used two molecular fingerprinting methods to investigate the genetic and clonal relationship shared by Australian Salmonella Sofia isolates. METHODS AND RESULTS: A total of 84 Australian Salm. Sofia isolates from various states in Australia were typed using pulsed-field gel electrophoresis (PFGE) (XbaI and SpeI) and repetitive element PCR (REP1R-I primer). The previous problem of DNA degradation of Salm. Sofia strains was solved by modifying the lysis solution used to treat the bacterial plugs, allowing Salm. Sofia to be subtyped using PFGE. Molecular typing of isolates resulted in the generation of eight XbaI, six SpeI and five REP1 pattern profiles. Individual typing methods showed low discrimination index values (<0·5), indicating the poor discriminatory ability of the methods. However, the combination of the typing methods was able to improve the discrimination of isolates, further dividing them into 16 subtypes and raising the index value to 0·721. CONCLUSIONS: The combination of typing methods was shown to be the best approach to fingerprint Salm. Sofia. The Australian Salm. Sofia isolates only showed limited genetic diversity and probably share a clonal relationship. A majority of the Salm. Sofia isolates were not geographically restricted with the predominant pattern subtype observed amongst the isolates from various states. SIGNIFICANCE AND IMPACT OF THE STUDY: We have successfully devised a PFGE protocol that counteracts DNase activity of Salm. Sofia, enabling typing of this serovar.

A single amino acid substitution affects substrate specificity in cysteine proteinases from Fasciola hepatica.

The trematode Fasciola hepatica secretes a number of cathepsin L-like proteases that are proposed to be involved in feeding, migration, and immune evasion by the parasite. To date, six full cDNA sequences encoding cathepsin L preproproteins have been identified. Previous studies have demonstrated that one of these cathepsins (L2) is unusual in that it is able to cleave substrates with a proline in the P2 position, translating into an unusual ability (for a cysteine proteinase) to clot fibrinogen. In this study, we report the sequence of a novel cathepsin (L5) and compare the substrate specificity of a recombinant enzyme with that of recombinant cathepsin L2. Despite sharing 80% sequence identity with cathepsin L2, cathepsin L5 does not exhibit substantial catalytic activity against substrates containing proline in the P2 position. Molecular modeling studies suggested that a single amino acid change (L69Y) in the mature proteinases may account for the difference in specificity at the S2 subsite. Recombinant cathepsin L5/L69Y was expressed in yeast and a substantial increase in the ability of this variant to accommodate substrates with a proline residue in the P2 position was observed. Thus, we have identified a single amino acid substitution that can substantially influence the architecture of the S2 subsite of F. hepatica cathepsin L proteases.

The nuclear:organelle distribution of chloroplast ribosomal proteins genes. Features of a cDNA clone encoding the cytoplasmic precursor of L11.

The majority of chloroplast ribosomal proteins are encoded in the nuclear genome. In order to characterize these proteins through their mRNA, we have previously constructed a spinach cDNA expression library and raised antisera to several spinach chloroplast ribosomal proteins. Here we describe the immuno isolation of cDNA clones encoding protein L11 and its chloroplast-targeting presequence. The cytoplasmic precursor form of L11 is 224 amino acid residues long (Mr 23,662); the mature L11 and the transit sequence are predicted to be of approximately 159 and approximately 65 residues, respectively. The predicted chloroplast L11 is significantly longer than the E coli L11, but similar (in size) to archaebacterial and yeast cytoplasmic L11. In sequence it is closer to E coli L11 (54% identity) than to the archaebacterial (32%) or yeast (23%) proteins. These results and the conservation of the contexts of the 3 methyl modified residues found in E coli L11 are discussed in the light of the endosymbiont theory and nuclear relocation of the rp/KAJL gene cluster.

Immunological approaches for the control of fasciolosis.

The immunological relationship between liver flukes and their mammalian hosts is being unravelled by in vivo and in vitro studies. Vaccine studies in cattle and sheep with purified antigens (fatty acid binding protein, FABP; glutathione S-transferase, GST; cathepsin L, CatL; hemoglobin) have shown that high reductions in worm burdens (31-72%) and egg production (69-98%) can be achieved, raising the realistic possibility that immunological control of Fasciola infection is a commercially achievable goal. Combination vaccines may also be feasible since a cocktail of CatL and hemoglobin elicits a significant 72% protection in cattle. Analysis of immune responses to Fasciola during infection in ruminants suggests that chronic infection correlates with a type 2 helper T cell response, implying that type 1 helper T cell responses are down-regulated in fasciolosis. Recent results studying the resistance of Indonesian Thin Tail (ITT) sheep to F. gigantica have shown that this breed exhibits high innate (or rapidly acquired) resistance to infection and acquires a higher level of resistance after a primary challenge. Initial studies suggest that the resistance of ITT sheep to F. gigantica may be determined by a major gene. Merino sheep also acquire resistance to F. gigantica. In contrast, ITT and Merino sheep do not exhibit resistance to F. hepatica. These results suggest that there are fundamental differences between these two species of Fasciola in the biology of their interaction with the sheep immune system. In vitro studies on immune mechanisms of killing of juvenile fluke have shown that juvenile larvae of F. hepatica are susceptible to antibody-dependent killing by activated rat macrophages in vitro which is mediated by nitric oxide. Future studies on the immune effector mechanisms expressed by resistant sheep which control infection by F. gigantica will lead to new knowledge which may allow the design of more effective vaccines for fasciolosis.

Evaluation of antigens of Fasciola gigantica as vaccines against tropical fasciolosis in cattle.

Vaccine trials were conducted in Brahman cross cattle evaluating the efficacy of 4 native antigens purified from adult Fasciola gigantica flukes, and 1 recombinant F. gigantica antigen, as vaccines against tropical fasciolosis. The antigens tested were native glutathione S-transferase, cathepsin L, paramyosin, fatty acid binding protein (FABP), and a recombinant FABP expressed in E. coli, and were formulated in 1 or more of several adjuvants (Quil A, Squalene Montanide 80, MF59-100, Auspharm, NAGO, polylactoglycolide microspheres, Algammulin, DEAE, Freund's). Vaccination induced low, moderate or high antibody titres to the various antigens which were dependent on the adjuvant. Low but significant reductions in fluke burdens (31%, P < 0.026) and fluke wet weight (36%, P < 0.041) were only observed in cattle vaccinated with the native FABP in Freund's adjuvant. There was no correlation between total antibody titres to FABP and protection. The protection observed in cattle vaccinated with native FABP of F. gigantica supports the notion that this class of proteins is a useful target for protection of animals against Fasciola and extends the efficacy of FABPs to the tropical liver fluke. This is the first report of vaccination of cattle against F. gigantica with a purified protein.

The use of chemical reagents in the detection of DNA mutations.

As the analysis of the human genome proceeds at an ever-increasing pace, many genes have been identified which are the site for mutations responsible for inherited diseases. The identification of the mutations within these genes has become a major application of molecular biology technologies, and to this end a number of mutation detection systems have been developed for use in diagnostic and research laboratories. The uses of these mutation detection systems are in the diagnosis of inherited disease (both prenatal and neonatal) and in an understanding of the function of the affected protein by cataloguing the range of mutations. Two of these mutation detection systems are reviewed here. Both rely on chemical modification of mismatched nucleotides, by either carbodiimide or hydroxylamine and osmium tetroxide. The methods are termed the carbodiimide (CDI) and the Chemical Cleavage of Mismatch (CCM) methods. The history and evolution of the methods is tracked, illustrating the way in which they developed, both as suitable technology became available (for example, the polymerase chain reaction) and as a result of a specific need. The current methodologies are briefly discussed, followed by a discussion of their applications, especially in the realm of disease mutation detection.

The sensitivity and specificity of two methods for detecting Fasciola infections in cattle.

Counts of Fasciola spp. eggs in faeces and measurements of antibody concentration to the excretory/secretory antigens of Fasciola spp. by ELISA were related to the numbers of flukes in the livers of 92 cattle killed in the abattoirs of Hanoi City, Vietnam. In this population, about 22% of the cattle had no flukes, another 22% had between 1 and 10 flukes, 44% between 11 and 100 flukes and 12% had more than 100 flukes in their livers. Of the 14 animals less than 2 years of age, only three were infected. At 2 years of age the mean number of flukes per liver was 10 whereas at 3 years and older, the mean varied between 60 and 80 flukes. Prevalence of infection was 78.3%. No eggs of Fasciola spp. were detected in the faeces of one third of infected cattle and 60% of the counts were less than 100 eggs per gram. The sensitivity of the egg counting method was 66.7% and specificity 100%, overall accuracy was 73.9%. Corresponding values for the ELISA method were 86.1, 70 and 82.6%, respectively. The positive and negative predictive values for the egg counting method were 100 and 45.5% and for the ELISA method were 91.2 and 58.3%, respectively.

Construction and immunogenicity of Salmonella vaccine vector expressing HIV-1 antigen and MCP3.

UNLABELLED: This study aims to determine the efficacy of Salmonella enterica serovar Typhimurium STM-1 bearing MCP-3 gene as a delivery vehicle for the HIV gag gene (in particular p24 gene) and HIV env gene. The STM1 delivery HIV-p24 vaccination was carried out in the form of a recombinant or a DNA vaccine whereas only a DNA vaccine was used for HIV env . Naked DNA vaccination was also tested and immune responses were evaluated following immunisation in mouse model. RESULTS: vaccination cellular immune responses induced by recombinant p24 STM1 (STM1/pHly-p24) were greater than those elicited by the p24 DNA vaccine in STM1 (STM1/VR-p24), (but statistically not significant) than those induced by oral vaccination. However, IgA responses induced by oral vaccination with either a recombinant or DNA vaccine of p24 in STM1 are higher than those induced by IP vaccination. In addition, the numbers of cells secreting IL4 are reduced after oral vaccination with STM1/VR-p24/MCP3. However, for the HIV p24 antigen, STM1/MCP3 preferentially induces IFNgamma-secreting splenocytes. CONCLUSIONS: This result confirms other studies that Salmonella was able to deliver HIV antigens to the immune system and induced specific immune responses to the HIV antigen and for the HIV p24 antigen, STM1/MCP3 induces secretion of IFNgamma.

Molecular basis of dihydropteridine reductase deficiency.

The spectrum of mutations causing dihydropteridine reductase is reviewed. A total of 12 point mutations have been described that map in the DHPR cDNA, resulting in amino acid substitutions, insertions and premature terminations. A further two mutations are described which result in aberrant splicing of DHPR transcripts. The application of the mutation identification to diagnostics and clinical treatment is discussed.

A ribosomal protein is encoded in the chloroplast DNA in a lower plant but in the nucleus in angiosperms. Isolation of the spinach L21 protein and cDNA clone with transit and an unusual repeat sequence.

The distribution of chloroplast ribosomal protein genes between the organelle DNA and the nuclear DNA is highly conserved in land plants, but a notable exception is rpl21. This gene has been found in the completely sequenced chloroplast genome of a lower plant but not in that of two higher plants. We describe the purification and characterization of the spinach chloroplast ribosomal protein L21 and the isolation and nucleotide sequence of a cDNA clone that encodes its cytoplasmic precursor. The mature protein, identified by NH2-terminal sequencing, has 201 residues (Mr 22,766) and is thus substantially larger than either its Escherichia coli (103 residues) or the lower plant homologue (116 residues). The extra length is in peptide extensions at both amino and carboxyl termini. The COOH-terminal extension is unusual in that it comprises seven Ala-Glu repeats, a feature not found in any other ribosomal proteins described so far. The cDNA clone also encodes a 55-residue long transit peptide (with a high proportion of the polar residues, threonine and serine), to target the L21 protein into chloroplasts. The identification of rpl21 as a nuclear gene in a higher plant (spinach) and chloroplast gene in a lower plant (liverwort) suggests an organelle-to-nucleus gene relocation during the evolution of the former.

Ribosomal protein L35: identification in spinach chloroplasts and isolation of a cDNA clone encoding its cytoplasmic precursor.

We describe the isolation of spinach chloroplast ribosomal protein L35 and characterization of a cDNA clone encoding its cytoplasmic precursor. This protein was only recently identified in ribosomes, but the sequences of four L35 genes have now been reported and confirm its presence in eubacteria, chloroplasts, and cyanelles. Using N-terminal sequence data, oligonucleotides were designed and a cDNA library was screened. The nucleotide sequence of the cDNA clones shows that the spinach L35 protein is encoded as a precursor of 159 residues, comprising a mature protein of 73 residues and a transit peptide of 86 residues. The cleavage site for forming the mature protein is deduced to be Thr-Val-Phe-Ala decreases Ala-Lys-Gly-Tyr. The L35 protein in the photosynthetic organelle of the protozoan Cyanophora paradoxa is encoded in the organelle DNA [Bryant & Stirewalt (1990) FEBS Lett. 259, 273-280]. The corresponding gene has not been found in the chloroplast DNA of a lower plant (liverwort) and two higher plants. Our results demonstrate that the L35 protein in a higher plant (spinach) is encoded in the nucleus. This finding, in light of the endosymbiont hypothesis, suggests an organelle to nucleus transfer of the L35 gene at the evolutionary beginnings of land plants.

Identification and in vitro expression of mutations causing dihydropteridine reductase deficiency.

Six mutations resulting in the recessive inherited disorder dihydropteridine reductase deficiency are reported, five of which are previously unknown. Two are nonsense mutations, resulting in premature termination of the protein, with the remaining four being missense mutations. The mutations found lie in the middle to 3' end of the dihydropteridine reductase reading frame, with the exception of one mutation which lies at codon 23, which is the only mutation found in more than one patient. The mutation pattern can be described as heterogeneous. The wild type and several of the mutant DHPR cDNA's were expressed in E. coli and the proteins purified and examined by a variety of techniques, including calculation of kinetic constants. One mutation (Gly23-->Asp) results in completely inactive protein, while a second (Trp108-->Gly) has substantial activity but does not completely dimerize. Both this mutant and a third, His158-->Tyr, are extremely susceptible to in vitro protease digestion, indicating that their three-dimensional structure has been altered. The protein studies underline the heterogeneous nature of DHPR mutations, in that the effects of different amino acid substitutions on the DHPR enzyme are varied.

Prenatal diagnosis of DHPR deficiency by direct detection of mutation.

Prenatal diagnosis was requested by a family carrying a 3 base-pair insertion in the dihydropteridine reductase (DHPR) coding region. A chorionic villus sample was obtained and fetal DNA was isolated directly from this. Diagnosis was performed by a polymerase chain reaction (PCR)-based technique, with a simple electrophoretic assay for the insertion. The fetus was found to be heterozygous for the insertion. This is the first time that prenatal diagnosis of DHPR deficiency has been performed by direct detection of the mutation.

A mitochondrial intergenic mutation affecting processing of specific yeast mitochondrial transcripts.

The mutation in the temperature-conditional mit- mutant h56, mapped previously to the var1 gene region of Saccharomyces cerevisiae mitochondrial DNA, results in a specific inhibition of var1 protein synthesis in cells incubated at the non-permissive temperature, 36 degrees C (1). We have now characterized the mutation present in mutant h56 by DNA sequencing and found it to be an A to T transversion located 109 nucleotides upstream of the var1 reading frame. Two spontaneous revertants of mutant h56 restore the parental strain sequence at residue -109, confirming that this single base change within the 5'-untranslated region of the var1 mRNA is responsible for defective synthesis of the var1 protein. A comparison of var1 transcripts in the parental and mutant strains has shown that the mutation specifically blocks formation of var1 mRNA at 36 degrees C and leads to accumulation of precursor transcripts. Expression of the oli1 gene, co-transcribed with the var1 gene in primary transcripts, is not affected. It is concluded that the mutation in mutant h56 alters the secondary structure of the precursor RNA, inhibiting an endonucleolytic cleavage required to generate the 5' end of var1 mRNA.

A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulent Plasmodium chabaudi adami DS malaria.

The ultimate malaria vaccine will require the delivery of multiple antigens from different stages of the complex malaria life cycle. In order to efficiently deliver multiple antigens with use of DNA vaccine technology, new antigen delivery systems must be assessed. This study utilized a bicistronic vector construct, containing an internal ribosome entry site, expressing a combination of malarial candidate antigens: merozoite surface protein 4/5 (MSP4/5) (fused to a monocyte chemotactic protein 3 chemoattractant sequence) and apical membrane antigen 1 (AMA-1) (fused to a tissue plasminogen activator secretion signal). Transfection of COS 7 cells with bicistronic plasmids resulted in production and secretion of both AMA-1 and MSP4/5 in vitro. Vaccination of BALB/c mice via intraepidermal gene gun and intramuscular routes against AMA-1 and MSP4/5 resulted in antibody production and significant in vitro proliferation of splenocytes stimulated by both AMA-1 and MSP4/5. Survival of BALB/c mice vaccinated with bicistronic constructs after lethal Plasmodium chabaudi adami DS erythrocytic-stage challenge was variable, although significant increases in survival and reductions in peak parasitemia were observed in several challenge trials when the vaccine was delivered by the intramuscular route. This study using a murine model demonstrates that the delivery of malarial antigens via bicistronic vectors is feasible. Further experimentation with bicistronic delivery systems is required for the optimization and refinement of DNA vaccines to effectively prime protective immune responses against malaria.

A mutation causing DHPR deficiency results in a frameshift and a secondary splicing defect.

In our analysis of mutations causing DHPR deficiency we identified a patient in whom there was an aberrant transcription pattern detected by PCR of DHPR cDNA. However, unlike the pattern observed as a result of most splicing mutations, there is some full length transcript. The mutation was located and is a single nucleotide deletion at position 570/571 of the DHPR cDNA sequence and results in a frameshift and premature termination after the addition of six amino acids. The mutation is present in a homozygous state in the patient and in a heterozygous state in both parents. The exon which is deleted at high frequency in the patient is the putative exon 4, which is remote from the mutation, and confirms our observation that exon 4 skipping is a relatively common event.