Population data and sequence analysis of a 'new' microsatellite locus HumHUU (D16S3433).

This study reports sequence characteristics and population genetic data on a 'new' STR locus HumHUU (D16S3433) located in the non-coding region of chromosome 16q. Based on a population sample of 306 non-related Polish individuals 205 genotypes and 15 alleles with length range of 157-211bp were distinguished. No deviation from HWE was observed. The sequence analysis of each D16S3433 allele revealed a tetranucleotide repeat motif with a basic sequence structure (AAAA)(0-1)(AAAG)(11-22)(AAAAG)(AAAA)(AG)(AAAAAAG). The power of discrimination is 0.9538, showing a high degree of polymorphism. The presented results demonstrate that the D16S3433 is a useful genetic marker for forensic purposes and paternity testing.

Vaccine potential of inclusion bodies containing cysteine proteinase of Fasciola hepatica in calves and lambs experimentally challenged with metacercariae of the fluke.

Despite intensive research efforts, progress in the development of effective anti-Fasciola hepatica vaccine has not been satisfactory. However, it has been found that cysteine proteinases of F. hepatica are very important candidates for a vaccine antigen because of their role in fluke biology and in the host-parasite relationship. In our previous experiments we found that recombinant cysteine proteinase which we have cloned from adult F. hepatica (CPFhW) can protect rats against the liver fluke infection when administered intramuscularly or when given intranasally in the form of cDNA. In the present experiments we aimed to evaluate the protectivity of the mucosal vaccination in calves and lambs with inclusion bodies containing recombinant CPFhW using different vaccination doses and various sites of antigen delivery. Female calves vaccinated intranasally with two doses of 300 microg of the recombinant CPFhW showed 54.2% protection against the subsequent challenge of 400 metacercariae (mc). Flukes which developed in vaccinated calves showed a reduction of reproductive potential. Male Corriedale lambs vaccinated at the age of 4 months demanded three doses of the antigen to gain 56.5% of protection to a challenge with 250 mc of F. hepatica. Vaccinated animals showed significantly lower blood eosinophil counts. No correlation was found between serum and mucosal IgG or IgA reacting with F. hepatica ES antigens and the protection level.

Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Characterization of changes in the short unique segment of pseudorabies virus BUK-TK900 (Suivac A) vaccine strain.

Mutant strains of pseudorabies virus (PRV) of reduced virulence, such as Bartha or BUK-TK900, have been used for vaccination purposes for many years. In contrast to the Bartha strain, BUK-TK900 has not been well characterised at the molecular level. The detailed analysis of this vaccine strain was urged by the fact of the isolation in Poland of field strains which were suspected to originate from BUK-TK900. We characterised changes in the U(S) region of this strain, focusing our attention on gE and gI genes. The only deletion, about 300 bp, found in BamHI 7 fragment (covering most of the U(S) region) was located in the 28 K (US2) gene. BUK-TK 900 produced small plaques on all cell lines tested in our laboratory (SK6, Vero, MDBK, 3T3). The plaque size was restored to about 70% of wild type virus plaque size when growing BUK-TK900 virus on 3T3 complementing cell line expressing PRV gE and up to 100% when cell line producing gE and gI was used. Both gE and gI genes from BUK-TK900 and from some derivative field isolates have been amplified by PCR reaction but no deletions in these genes have been found. Molecular weight of gene products differed from wild type proteins: gE was bigger than wild type gE while gI was smaller. Both proteins were correctly recognised by all tested polyclonal and monoclonal antibodies. Radioimmunoprecipitation study showed that BUK-TK900 gE and gI interact forming a complex. The whole ORF of BUK-TK900 gE was sequenced and only few point mutations were found; only two of them led to changes of amino acids in the polypeptide chain. These were: methionine at position 124 replaced by threonine and glutamine at position 162 replaced by arginine. The introduction of first of these mutations (Met to Thr) to PRV wild type strain NIA-3 resulted in 22% reduction of plaque size. This result confirms the importance of this domain of gE for its function; it was found previously by others that deletion of amino acids 125 and 126 reduced virulence and neurotropism of PRV. More changes were found in BUK-TK900 gI sequence. Over 80% of these changes were located in the terminal 1/3rd of the sequence. Some of these mutations may have significant effect on the secondary structure of gI glycoprotein. The change of the secondary structure may be responsible for the decrease of gI stability and the observed reduction of gI molecular mass.

The immune response of rats to vaccination with the cDNA or protein forms of the cysteine proteinase of Fasciola hepatica.

Our previous experiments have shown that intramuscular injection of Sprague-Dawley rats with a pcDNA 3.1 vector carrying cDNA encoding for a cysteine proteinase (CP) of F. hepatica may induce a high level of protection against subsequent infection with F. hepatica metacercariae (mc). The aim of the present study is to compare the immune response of Sprague-Dawley rats vaccinated intranasally with plasmid containing cDNA of CP of the fluke and intramuscularly or intraperitoneally with the recombinated enzyme protein to challenge with fluke metacercariae. In addition, protection following intranasal DNA vaccination was evaluated. Two experiments were carried out. In the first experiment rats were vaccinated twice with 50microg of cDNA containing plasmid or with 100microg protein of recombinated CP. Three weeks after the second vaccination rats were challenged orally with 25 mc. On days 0, 21, 42 and 63 after the challenge blood samples were collected for the evaluation of white blood cell, eosinophil and specific antibody responses. During the second experiment groups of five male and female rats were vaccinated twice intranasally with CPcDNA then challenged with 30 mc and dissected 5 weeks later. Results obtained in the experiments suggested that intranasal immunisation of rats with CPcDNA seems to favour a Th2 regulated antibody response. Intramuscular or intraperitoneal injections of CP protein stimulate both Th1 and Th2-dependent antibodies. Mean worm burdens found in rats vaccinated intranasally 5 or 10 weeks after the challenge were reduced by 61-75% in comparison with the challenge controls which suggests that intranasal vaccination with CPcDNA may protect hosts against F. hepatica infection.

Fingerprinting of bacterial genomes by amplification of DNA fragments surrounding rare restriction sites.

A method for generating limited representations of total bacterial DNA, without prior knowledge of the DNA sequence, has been developed. This method consists of three steps: digestion with two restriction enzymes, ligation of two oligonucleotide adapters corresponding to the restriction sites, and selective PCR amplification of the ligation products. The method relies on the use of two restriction enzymes with considerable differences in cleavage frequency of the investigated DNA and the ligation of two different oligonucleotides, each corresponding to one of the two cohesive ends of DNA fragments. Three subsets of DNA fragments are generated during digestion and subsequent ligation: terminated with the same oligonucleotide on both 5' ends of DNA fragments (two subsets) and terminated with two different oligonucleotides. Suppression PCR allows only the third subset of DNA fragments to be amplified exponentially. The method allows bacterial species strain differentiation on the basis of the different DNA band patterns obtained after electrophoresis in polyacrylamide gels stained with ethidium bromide and visualized in UV light.

Expression of bovine leukemia virus protein p24 in Escherichia coli and its use in the immunoblotting assay.

The gag gene encoded protein, p24 of bovine leukemia virus (BLV), was cloned and expressed as thioredoxin-6xHis-p24 protein in Escherichia coli. The bacterial cells carrying plasmid pT7THis-p24 expressed the protein of 38 kDa that was detected by immunoblotting analysis using anti-p24 monoclonal antibodies and sera from BLV infected cattle and sheep. The purified p24 fusion protein was shown to be sensitive and specific for detection of BLV antibodies in the infected cattle.

Application of FISH and Q-PCR techniques in breakpoint diagnostics in three cases of marker chromosomes derived from chromosome 15.

BACKGROUND: The goal of the study was a search for effective methods of diagnosing additional marker chromosomes. MATERIAL AND METHODS: Three cases of extra structurally abnormal chromosomes (ESACs) were diagnosed, the ESACs having been derived from chromosome 15 by cytogenetic techniques, the fluorescence in situ hybridisation (FISH) technique and the quantitative--polymerase chain reaction (Q-PCR). An application of a set of commercially available probes, specific for the 15q11.2-q12 regions (PWACR-Prader-Willi/Angelman Critical Region) allowed for a description of the breaking points. RESULTS: The presence of PWACR region was confirmed in one case and excluded in the other two. It was also attempted to apply the Q-PCR technique for a more accurate determination of the size of the region involved in chromosomal aberration, what would allow for a more reliable prognosing of the clinical outcome. In one of the patients, the breaking point was localized as distal to D15S144 locus, while it was proximal to D15S11 locus in the two remaining cases. CONCLUSIONS: The obtained results demonstrate a possibility of using the Q-PCR method in diagnosing unbalanced chromosome aberrations.

The quantitative PCR technique resolves ambiguities concerning a small rearrangement of human chromosome 6q12-13.

Karyotype analysis, performed on the basis of chromosome banding pattern, is a standard method used for identification of chromosomal aberrations, both numerical and structural. The application of classic cytogenetic techniques fails, however, to solve all diagnostic problems in certain types of chromosome aberrations. In this study, quantitative polymerase chain reaction technique (Q-PCR) application was applied to verify a cytogenetic diagnosis, which assumed that a difference observed in the banding pattern of homologous chromosome 6q12-13 region of a foetus had resulted from an inversion and/or duplication of the region in question. The obtained results indicate a possibility to use the Q-PCR method in the diagnostics of unbalanced chromosomal aberrations.

A plant-derived edible vaccine against hepatitis B virus.

The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein.

Cloning of complementary DNA encoding the pB1 component of the 54-kilodalton glycoprotein of boar seminal plasma.

Complementary DNA (cDNA) encoding a protein component pB1 (also pAIF-1 and DQH) of the 54-kilodalton glycoprotein of boar seminal plasma was cloned and its nucleotide sequence was determined (Gene Bank accession no. AF047026). The pB1 precursor protein is a 130-amino-acid-long polypeptide containing a 25-amino-acid-long signal peptide. The amino acid sequence of the pB1 is homologous to that of SFP1_BOVIN (named also BSP-A1/A2, PDC-109/ major protein and SVSp109), SFP3_BOVIN (BSP-A3), SFP4 BOVIN (BSP-30 KD), and SP1_HORSE (HSP-1) seminal plasma proteins. The homology extends also for the signal peptide of SFP1_BOVIN protein. All these seminal plasma proteins contain two fibronectin type-II domains that differ from those found in other proteins such as colagenases, fibronectins, and mannose receptors. The first domain located in the N-terminal region of pB1 is four amino acids shorter than those present in other proteins. High homology is also observed between 3' noncoding regions of the nucleotide sequences of cDNAs of pB1_PIG and SFP1_BOVIN (Gene Bank accession nos. AF047026 and P02784, respectively).

Fragments of LINE-1 retrotransposons flanked by inverted telomeric repeats are present in the bovine genome. Homology with human LINE-1 elements.

In the bovine genome we found two intrachromosomal DNA fragments flanked by inverted telomeric repeats (GenBank Accession Nos. AF136741 and AF136742). The internal parts of the fragments are homologous exclusively to the human sequences and to the consensus sequence of the L1MC4 subfamily of LINE-1 retrotransposons which are widespread among mammalian genomes. We found that distribution of homologous human sequences within our fragments is not random, reflecting a complicated pattern of insertion mechanisms of and maintenance of retrotransposons in mammalian genomes. One of the possible explanations of the origin of LINE-1 truncated elements flanked by inverted telomeric repeats in the bovine genome is that extrachromosomal DNA fragments may be modified by telomerase and subsequently, transferred into chromosomal DNA.

Multiple sclerosis: the frequency of allelic forms of tumor necrosis factor and lymphotoxin-alpha.

The cytokines LTa and TNF have been implicated as major mediators of tissue injury in multiple sclerosis (MS). In this study we have assessed the frequency of specific polymorphisms for these genes in MS (n = 53) and controls (n = 81) using a highly sensitive, two stage nested polymerase chain reaction (PCR), with the second stage using mutation-specific primers. Genomic DNA was extracted from blood cells and the results confirmed by direct dideoxy chain termination sequencing. The frequency of the -308 G to A mutation in the TNF promoter region in normal controls was 15% and in MS was 24%. For LTa gene the exon 3 polymorphism allele A was detected in 36% of controls and 34% of the MS patients. In MS, the combined genotype TNF G + A and LTa C + C was present 6 times more frequently (12%) than in controls (2%), and patients with this genotype showed the highest EDSS scores. We found the TNF and LTa polymorphisms to occur independently from the HLA class II DR2 allele distribution in MS. Whilst the G - A polymorphism in TNF gene promoter has been studied previously in MS, with conflicting results, this is the first study that has addressed the exon 3 polymorphism in LTa in MS. The results indicate that this polymorphism is not linked with the higher genetic predisposition for MS, but that combined TNF G + A and LTa C + C genotype might contribute to development of the disease.

Isolation and characterization of a ColE1-like plasmid from Enterobacter agglomerans with a novel variant of rom gene.

Complete nucleotide sequence of a plasmid isolated from Enterobacter agglomerans has been determined. The plasmid, called pPIGDM1, consists of 2495 base pairs. The analysis of its nucleotide sequence suggested that pPIGDM1 may be a ColE1-like replicon. We confirmed this hypothesis by constructing a pPIGDM1-derived plasmid harboring the cat gene (pBW4), which could be introduced into Escherichia coli cells, and demonstrating that pBW4 cannot replicate in the absence of the polA function and that its copy number is significantly decreased in the pcnB mutant. Like some other ColE1-type replicons (e.g., pBR322), pPIGDM1-derived plasmids can be amplified both by chloramphenicol method and in isoleucine-starved relA mutants but not in relA+ bacteria. Inactivation of the putative rom gene by insertion of an amplicillin-resistance gene resulted in significant increase in pPIGDM1-derived plasmid copy number in E. coli-despite the fact that amino acid sequence of the putative RNA 1 modulator (Rom) protein is only 55.7% identical to the ColE1 analog. The pPIGDM1-derived rom-like coding sequence is also homologous to the rom-like gene present in the Proteus vulgaris plasmid pPvul. We suggest to group all these gene products into a new family called ROMS (RNA one modulators). Since a pPIGDM1-derived plasmid is compatible with other ColE1-like replicons (pMB1-, p15A, RSF1030-, and CloDF13-derived) in E. coli, one may consider pPIGDM1 as a progenitor of new cloning vehicles compatible with most (if not all) of currently used plasmid vectors. Moreover, this plasmid may serve as a source of the new rom-like gene coding for a protein useful in investigation of RNA-protein interactions. A role for the pPIGDM1 plasmid in the host strain is not known.

Effect of substance P and its precursor alpha-protachykinin on intracellular free calcium concentration in human polymorphonuclear leukocytes.

The increase in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation. The undecapeptide substance P can influence various functions of human polymorphonuclear leukocytes, including chemotaxis, phagocytosis, and respiratory burst. In this study we investigated the ability of low-concentration (that can occur in vivo) substance P (10(-7) M) and its precursor alpha-protachykinin (3 x 10(-7) M) to increase the intracellular free calcium concentration in human polymorphonuclear leukocytes. Cells isolated from ten healthy donors were incubated with substance P or alpha-protachykinin in 1 mM calcium medium for 5 min and the intracellular free calcium concentration was monitored using the fluorescent calcium indicator Fura-2am. Polymorphonuclear leukocytes from 40% of donors responded to both agonists. The substance P- and alpha-protachykinin-induced increase in intracellular free calcium concentration was 59 +/- 13 nM and 58 +/- 12 nM and the extracellular calcium influx contributed to 87 +/- 8% and 54 +/- 8% of the calcium response, respectively. alpha-Protachykinin released almost all the calcium from intracellular stores, while substance P mobilized only 24 +/- 5% of this calcium pool. Finally, cells that responded to a single challenge with substance P and alpha-protachykinin were able to increase their intracellular free calcium concentration in response to each of three consecutive stimulations with these agonists. This may be an additional mechanism by which substance P and its precursor modify the function of human polymorphonuclear leukocytes.

[Etiology of chronic hepatitis as evaluated by liver biopsy and serum samples submitted to the Department of Immunopathology of the National Institute of Hygiene in 1993-1995]. / Etiologia przewleklych zapalen watroby na podstawie badan biopsji watroby i surowic nadeslanych do Zakladu Immunopatologii Panstwowego Zakladu Higieny w Latach 1993-1995.

The purpose of this study was to assess the relative incidence of chronic hepatitis in a population of patients with chronic liver disease and to determine the etiological spectrum of this syndrome with special reference to its defined histopathological forms. Histopathology aided by immunohistochemistry, and serology aided by the PCR method were employed in studies of liver biopsy specimens and serum samples, respectively. Out of 1150 patients with chronic liver disease examined, chronic hepatitis was diagnosed in 685 (60% of all cases examined). In this group, there were 308 males aged 18-74 yrs (mean 32 yrs), 153 females aged 18-71 yrs (mean 43 yrs), and 213 children aged 1-17 yrs (mean 8 yrs). Viral infections documented in these patients included HBV (50.4%), HCV (36.2%), HBV/HCV (7.2%) and HBV/HDV (0.7%); cryptogenic and autoimmune hepatitis (AIH) accounted for 2.9% and 2.6% all cases, respectively. In the group of minimal hepatitis (16.1%), HBV infection was documented in 66.4% of cases, HCV-in 29.1%, HBV/HCV-in 3.6% (one case of AIH was included into this group). In the group of mild hepatitis (44.2%), HBV infection accounted for 47.3% of cases, HCV-for 41.9%, HBV/HCV-for 9.9%, and 0.9% was diagnosed as cryptogenic. In the group of moderate hepatitis (19.6%), HBV infection accounted for 50% of cases, HCV-for 37.3%, and HBV/HCV-for 4.5%; cases of cryptogenic and AIH accounted for 3.7% and 4.5%, respectively. In the group of severe hepatitis (20.1%), HBV etiology was found in 44.9% of cases, HCV-in 28.3%, HBV/HCV-in 6.5% and HBV/HDV-in 3.6%; cryptogenic and AIH accounted for 6.5% and 8.0% of cases, respectively. There was a high incidence of low-titer autoantibodies (SMA, ANA and LKM) ranging from 75% in cryptogenic hepatitis and 51% in each HBV and HBV/HCV hepatitis to 46.3% in HCV hepatitis.

Interaction of HIV-1 gp120 molecule fragments with human monocytes: different requirements for tumor necrosis factor-alpha and IL-6 production.

The HIV-1 gp120 recombinant protein fragment encompassing aa residues 410-511, that contains the CD4 binding region (rp120cd), and fragment aa 446-511, which lacks the sequence responsible for CD4 binding (rp120), were synthesized to study their ability to induce TNF synthesis in human monocytes. The rp120cd stimulated TNF alpha secretion by monocytes while the rp120 and full-length recombinant protein (FL gp120), used as control, failed to do so. However, FL gp120 stimulated peripheral blood mononuclear cells (PBMC) and lymphocytes for TNF production and this was inhibited by anti-CD4 MAb. The rp120cd also caused TNF secretion by PBMC that was not blocked by this antibody. Furthermore, FL gp120 but not rp120cd inhibited anti-CD4 mAb binding to CEM cells. Hence, FL gp120 may cause TNF release from lymphocytes by binding to CD4, while rp120cd interacts with monocytes but not lymphocytes and induces TNF production by a mechanism not involving CD4 binding. Unexpectedly, FL gp120 but not rp120cd stimulated IL-6 secretion and IL-6 mRNA synthesis in monocytes. The FL gp120-induced production of IL-6 by monocytes was inhibited by anti-CD4 monoclonal antibody (MAb). Thus, there may be different requirements for TNF induction in lymphocytes and monocytes stimulated with various preparations of gp120 and for the selective induction of cytokines in monocytes. The enhanced production of TNF in HIV infection and AIDS may involve distinct cellular sources and different mechanisms.

Bovine Alu-like sequences mediate transposition of a new site-specific retroelement.

We describe a new family of 3.1-kb repetitive sequences which is present in the bovine genome. The 5' and 3' ends of the unit are flanked with sequences homologous to the 5' and 3' halves of the bovine Alu-like monomer (BM), respectively. Distribution of the 5' ends of the family members in the genome is not random. They are close to the truncated bovine Alu-like dimer (BD) which, in some cases, is followed by 40-bp repeated sequences containing block A of the RNA polymerase III promoter. The ORFs found within the unit code for peptides homologous to amino-acid sequences characteristic for reverse transcriptases (RT). The family members may be considered as mutant mobile elements whose propagation in the genomes was accomplished by means of a process including site-specific recognition with BD. Because of this, we call this family the bovine dimer-driven family (BDDF).

Expression and characterization of the multiplied, recombinant preS1 antigen of hepatitis B virus.

The amino acid sequence encoded by the preS1 region of hepatitis B virus genome is expressed on the surface of virions and subviral particles. The preS1 region is involved in the recognition of specific receptors responsible for the attachment of HBV to the host cell. The cell receptor binding site was assigned to the preS1 (20-47 aa) fragment. In order to obtain a large quantity of preS1 binding domains of HBV the expression vector pWX4 was constructed. It contains four tandemly joined DNA sequences, each coding for preS1 (20-49 aa), fused with the 3' end of a DNA fragment coding for 450 aa of beta-galactosidase. E. coli cells transformed with this vector produce fusion protein beta-gal-preSlx4 in the form of inclusion bodies. Owing to the specific trypsin digestion, the preSlx4 domain was cleaved from the fusion protein. The resulting product, a 16 kDa protein, was isolated and purified by anion exchange chromatography. The presence of four Asp-Pro bonds in this sequence and the primary structure of the first 28 N-terminal amino acids were determined. Following the confirmation of the antigenic properties, the recombinant preS1 protein was used for detection of the anti-preS1 response in sera from HBV infected patients.