Plasmodium falciparum produces prostaglandins that are pyrogenic, somnogenic, and immunosuppressive substances in humans.

Plasmodium falciparum causes the most severe form of human malaria, which kills approximately 1.5-2.7 million people every year, but the molecular mechanisms underlying the clinical symptoms and the host-parasite interaction remain unclear. We show here that P. falciparum produces prostaglandins (PGs) D2, E2, and F2alpha. After incubation with 1 mM arachidonic acid (AA), cell homogenates of P. falciparum produced PGs as determined by enzyme immunoassay and gas chromatography-selected ion monitoring. PG production in the parasite homogenate was not affected by the nonsteroidal antiinflammatory drugs aspirin and indomethacin, and was partially heat resistant, whereas PG biosynthesis by mammalian cyclooxygenase was completely inhibited by these chemicals and by heat treatment. Addition of AA to the parasite cell culture markedly increased an ability of the parasite cell homogenate to produce PGs and of parasitized red blood cells to accumulate PGs in the culture medium. PGD2 and PGE2 accumulated in the culture medium at the stages of trophozoites and schizonts more actively than at the ring stage. These findings are the first evidence of the direct involvement of a malaria parasite in the generation of substances that are pyrogenic and injurious to the host defenses. We will discuss a possible contribution of the parasite-produced PGs to pathogenesis and host-parasite interaction of P. falciparum.

Identification of a novel prostaglandin f(2alpha) synthase in Trypanosoma brucei.

Members of the genus Trypanosoma cause African trypanosomiasis in humans and animals in Africa. Infection of mammals by African trypanosomes is characterized by an upregulation of prostaglandin (PG) production in the plasma and cerebrospinal fluid. These metabolites of arachidonic acid (AA) may, in part, be responsible for symptoms such as fever, headache, immunosuppression, deep muscle hyperaesthesia, miscarriage, ovarian dysfunction, sleepiness, and other symptoms observed in patients with chronic African trypanosomiasis. Here, we show that the protozoan parasite T. brucei is involved in PG production and that it produces PGs enzymatically from AA and its metabolite, PGH(2). Among all PGs synthesized, PGF(2alpha) was the major prostanoid produced by trypanosome lysates. We have purified a novel T. brucei PGF(2alpha) synthase (TbPGFS) and cloned its cDNA. Phylogenetic analysis and molecular properties revealed that TbPGFS is completely distinct from mammalian PGF synthases. We also found that TbPGFS mRNA expression and TbPGFS activity were high in the early logarithmic growth phase and low during the stationary phase. The characterization of TbPGFS and its gene in T. brucei provides a basis for the molecular analysis of the role of parasite-derived PGF(2alpha) in the physiology of the parasite and the pathogenesis of African trypanosomiasis.

Use of a dual priming oligonucleotide system to detect multiple sexually transmitted pathogens in clinical specimens.

AIMS: To evaluate a new dual priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) assay for detection of six sexually transmitted pathogens, including Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma urealyticum and Trichomonas vaginalis. METHODS AND RESULTS: Using 130 clinical specimens, the results obtained by the multiplex PCR, previously established in-house PCR and COBAS Amplicor PCR assays were compared. The specimens frequently contained multiple pathogens (34/130 specimens). The multiplex PCR assay had an overall sensitivity of 96% and specificity of 100% compared to the in-house PCR assay at >20 microg ml(-1) of DNA concentrations in samples and there was no cross-reaction with nonpathogenic Neisseria species that cause the majority of false-positive results with the COBAS Amplicor PCR assay. CONCLUSIONS: The DPO-based multiplex PCR assay detected the six sexually transmitted pathogens in clinical specimens with a high sensitivity and specificity, although its sensitivity was dependent on the DNA content of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: It is the first report about the new DPO-based technique to detect multiple sexually transmitted pathogens in a single assay, which has considerable potential to diagnose the infections accurately and rapidly.

Cloning and expression analysis of YY1AP-related protein in the rat brain.

YY1AP-related protein (YARP) is a structural homolog of YY1AP, a transcriptional coactivator of the multifunctional transcription factor YY1. We cloned a rat YARP cDNA that encoded a 2256 amino acid protein with 93% homology to the human counterpart. Northern blots revealed significant expression of the YARP gene in the rat brain. In situ hybridization demonstrated its expression in neurons throughout the brain, including pyramidal cells in the cerebral cortex and hippocampus and granule cells in the dentate gyrus. YARP was coexpressed with YY1 in these same neuronal cells. However, there was no evidence of YARP expression in glia. In the developing rat brain, the level of YARP mRNA ( approximately 10 kb) peaked at embryonic day 18 and promptly declined thereafter to reach the steady-state level found in adulthood, by 14 days after birth. These results suggest that YARP functions at a late stage of neurogenesis during perinatal development of the rat brain, as well as in mature neurons.

Molecular cloning of a structural homolog of YY1AP, a coactivator of the multifunctional transcription factor YY1.

YY1 is a multifunctional transcription factor that activates or represses gene transcription depending on interactions with other regulatory proteins that include coactivator YY1AP. Here, we describe the cloning of a novel homolog of YY1AP, referred to as YARP, from the human neuroblastoma cell line SK-N-SH. The cloned cDNA encoded a 2240 amino acid protein that contained a domain which was 97% homologous to an entire YY1AP sequence of 739 amino acids. Two splice variants, YARP2 and YARP3, were also cloned. Northern blotting demonstrated the YARP mRNA (approximately 10 kb), which was increased 1.7-fold after dibutyryl cAMP-induced neural differentiation of the cells. Presence of YARP mRNA was also confirmed in human tissues such as the heart, brain and placenta. Bioinformatic analysis predicted various functional motifs in the YARP structure, including nuclear localization signals and domains associated with protein-protein interactions (PAH2), DNA-binding (SANT), and chromatin assembly (nucleoplasmin-like), outside the YY1AP-homology domain. Thus, we propose that YARP is multifunctional and plays not only a role analogous to YY1AP, but also its own specific roles in DNA-utilizing processes such as transcription.

Genomic imprinting in Dicer1-hypomorphic mice.

To address the function of RNA interference (RNAi) in transcriptional silencing in mammals, we analyzed genomic imprinting in Dicer1-hypomorphic mice, in which Dicer1 expression was significantly reduced. We did not observe any abnormality in the allelic expression of imprinted genes in these mice or their offspring, suggesting that reduced expression of Dicer1 did not significantly affect the maintenance and reprogramming of imprinting.

Efficacy of gemtuzumab ozogamicin on ATRA- and arsenic-resistant acute promyelocytic leukemia (APL) cells.

Acute promyelocytic leukemia (APL) cells express a considerable level of CD33, which is a target of gemtuzumab ozogamicin (GO), and a significantly lower level of P-glycoprotein (P-gp). In this study, we examined whether GO was effective on all-trans retinoic acid (ATRA)- or arsenic trioxide (ATO)-resistant APL cells. Cells used were an APL cell line in which P-gp was undetectable (NB4), ATRA-resistant NB4 (NB4/RA), NB4 and NB4/RA that had been transfected with MDR-1 cDNA (NB4/MDR and NB4/RA/MDR, respectively), ATO-resistant NB4 (NB4/As) and blast cells from eight patients with clinically ATRA-resistant APL including two patients with ATRA- and ATO-resistant APL. The efficacy of GO was analyzed by (3)H-thymidine incorporation, the dye exclusion test and cell cycle distribution. GO suppressed the growth of NB4, NB4/RA and NB4/As cells in a dose-dependent manner. GO increased the percentage of hypodiploid cells significantly in NB4, NB4/RA and NB4/As cells, and by a limited degree in NB4/MDR and NB4/RA/MDR cells. Similar results were obtained using blast cells from the patients with APL. GO is effective against ATRA- or ATO-resistant APL cells that do not express P-gp, and the mechanism of resistance to GO is not related to the mechanism of resistance to ATRA or ATO in APL cells. Leukemia (2005) 19, 1306-1311. doi:10.1038/sj.leu.2403807; published online 26 May 2005.

Purification, characterization and molecular cloning of an acidic amino acid-specific proteinase from Streptomyces fradiae ATCC 14544.

We have isolated a novel acidic amino-acid-specific proteinase from Streptomyces fradiae ATCC 14544, using benzyloxycarbonyl-L-Phe-L-Leu-L-Glu-p-nitroanilide (Z-Phe-Leu-Glu-pNA) as a substrate. A proteinase, which we propose to call SFase, was purified from the culture filtrate by salting out, repeated S-Sepharose chromatography, and affinity chromatography (CH-Sepharose-Phe-Leu-D-Glu-OMe). The purified enzyme showed a single band having an apparent molecular weight of 19,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. When synthetic peptides were used as substrates, SFase showed high specificity for Z-Phe-Leu-Glu-pNA. Comparison with nitroanilides of glutamic acid and aspartic acid as substrates revealed that the reactivity was about 10-fold higher for a glutamyl bond than an aspartyl bond. SFase selectively hydrolyzed the -Glu-Ala-bond of two glutamyl bonds in the oxidized insulin B-chain within the initial reaction time until the starting material was completely digested. Diisopropylfluorophosphate and benzyloxycarbonyl-Phe-Leu-Glu chloromethylketone completely inhibited SFase, while metalloproteinase inhibitors, such as EDTA and o-phenanthrolin, did not inhibit the enzyme. The findings indicate that SFase can be classified as a serine proteinase, and is highly specific for a glutamyl bond in comparison with an aspartyl bond. To elucidate the complete primary structure and precursor of SFase, its gene was cloned from genomic DNA of the producing strain, and the nucleotide sequence was determined. Consideration of the N- and C-terminal amino-acid sequences of the mature protein of SFase indicates that it consists of 187 amino acids, which follows a prepropeptide of 170 residues. In comparison with the acidic amino-acid-specific proteinase from Streptomyces griseus (Svendsen, I., Jensen, M.R. and Breddam, K. (1991) FEBS Lett. 292, 165-167), SFase had 82% homology in the amino acid sequence. The processing site for maturation of SFase was a unique sequence (-Glu-Val-), so that the propeptide could be released by cleavage of the peptide bond between Glu and Val.

Inhibitory effects of N- and C-terminal truncated Escherichia coli recA gene products on functions of the wild-type recA gene.

The effects of the expression of Escherichia coli truncated RecA protein on the host recA functions were examined. The recA gene on a multicopy plasmid was manipulated to express the truncated RecA protein from its carboxyl (C) and amino (N) terminal ends where a maximum of four extra amino acid residues was added. The regulatory part of the recA gene was substituted by the lacUV5 promoter in the plasmid to facilitate the artificial control of recA expression. Enzyme-linked immunosorbent assay and Western blot analyses revealed great differences in accumulation of the truncated RecA proteins in the cell, depending on the location of the site of truncation. The expression of truncated proteins lacking 62, 77, 93 or 149 amino acid residues from the C-terminal end caused the host recA+ wild-type cell to become sensitive to ultraviolet irradiation and interfered with chromosomal recombination but did not interfere with the induction of lambda prophage. The expression of truncated RecA protein with 25 amino acid residues deleted from the C-terminal end caused the host cell to induce SOS functions constitutively. Truncated RecA proteins with 15 or 28 amino acid residues missing from the N-terminal end severely interfered with all of the host recA functions examined here. The effect of the loss of 41 amino acid residues from the N-terminal end of RecA was significant but less than the effect of proteins lacking 15 or 28 amino acid residues from the N-terminal end. A protein lacking 59 amino acid residues from the N-terminal end showed little interference with any measured recA functions, suggesting that the deletion of the region from around residues 41 to 59, which is rich in hydrophobic side-chains, influenced the ability of the truncated protein to interfere with the functions of wild-type RecA protein. We also constructed a mutant gene with an internal deletion whose product was missing a region from residues 184 to 204. That mutant RecA protein was stably accumulated in the cell. This protein had little effect on the function of host wild-type recA gene product. The possible function of the regions at the N and C termini are discussed.

C-terminal truncated Escherichia coli RecA protein RecA5327 has enhanced binding affinities to single- and double-stranded DNAs.

RecA5327 is a truncated RecA protein that is lacking 25 amino acid residues from the C-terminal end. The expression of RecA5327 protein in the cell resulted in the constitutive induction of SOS functions without damage to the DNA. Purified RecA5327 protein effectively promoted the LexA repressor cleavage reaction and ATP hydrolysis at a lower concentration of single-stranded DNA than that required for wild-type RecA protein. A DNA binding study showed that RecA5327 has about ten times higher affinity for single-stranded DNA than does the wild-type RecA protein. Moreover RecA5327 protein binds stably to double-stranded (ds) DNA in conditions where the wild-type RecA protein could not bind. The binding of RecA5327 protein to dsDNA was associated with the unwinding of dsDNA, suggesting that RecA5327 binds to dsDNA in the same manner as does the wild-type protein. The fact that RecA5327 does not bind stoichiometrically but forms short filaments on dsDNA suggests that it nucleates to dsDNA much more frequently than does the wild-type protein. The role of the 25 C-terminal residues, in the regulation of RecA binding to DNA, is discussed.