A novel endogenous antimalarial: Fe(II)-protoporphyrin IX alpha (heme) inhibits hematin polymerization to beta-hematin (malaria pigment) and kills malaria parasites.

The polymerization of hemoglobin-derived ferric-protoporphyrin IX [Fe(III)PPIX] to inert hemozoin (malaria pigment) is a crucial and unique process for intraerythrocytic plasmodia to prevent heme toxicity and thus a good target for new antimalarials. Quinoline drugs, i.e., chloroquine, and non-iron porphyrins have been shown to block polymerization by forming electronic pi-pi interactions with heme monomers. Here, we report the identification of ferrous-protoporphyrin IX [Fe(II)PPIX] as a novel endogenous anti-malarial. Fe(II)PPIX molecules, released from the proteolysis of hemoglobin, are first oxidized and then polymerized to hemozoin. We obtained Fe(II)PPIX on preparative scale by electrochemical reduction of Fe(III)PPIX, and the reaction was monitored by cyclic voltammetry. Polymerization assays at acidic pH were conducted with the resulting Fe(II)PPIX using a spectrophotometric microassay of heme polymerization adapted to anaerobic conditions and the products characterized by infrared spectroscopy. Fe(II)PPIX (a) did not polymerize and (b) produced a dose-dependent inhibition of Fe(III)PPIX polymerization (IC(50) = 0.4 molar equiv). Moreover, Fe(II)PPIX produced by chemical reduction with thiol-containing compounds gave similar results: a dose-dependent inhibition of heme polymerization was observed using either L-cysteine, N-acetylcysteine, or DL-homocysteine, but not with L-cystine. Cyclic voltammetry confirmed that the inhibition of heme polymerization was due to the Fe(II)PPIX molecules generated by the thiol-mediated reduction of Fe(III)PPIX. These results point to Fe(II)PPIX as a potential endogenous antimalarial and to Fe(III)PPIX reduction as a potential new pharmacological target.     

A novel gene expression index (GEI) with software support for comparing microarray gene signatures

This study was aimed to examine the validity of commonly used statistical tests for comparison of expression data from simulated and real gene signatures as well as pathway-characterized gene sets. A novel algorithm based on 10 sub-gradations (5 for up- and 5 for down-regulation) of fold-changes has been designed and testified using an Excel add-in software support. Our findings showed the limitations of conventional statistics for comparing the microarray gene expression data. However, the newly introduced Gene Expression Index (GEI) appeared to be more robust and straightforward for two-group comparison of normalized data. The software automation simplifies the task and the results are displayed in a comprehensive format including a color-coded bar showing the intensity of cumulative gene expression.     

Development of a touch-down multiplex PCR method for simultaneously rapidly detecting three novel insertion/deletions (indels) within one gene: an example for goat GHR gene

Abstract

The multiplex polymerase chain reaction (PCR) method is fast and accuracy for screening several polymorphism loci in a single reaction in large samples. Therefore, this study aimed to identify three novel insertion/deletion (indel) loci in goat growth hormone receptor (GHR) gene by using multiplex PCR method and the genotypes in three indel loci are identified in 918 individuals from three Chinese goat breeds, as well as to evaluate its associations with growth traits. After validating the accuracy by taking the conventional PCR method, the concordance between these two methods was 100%. Moreover, P14 locus polymorphism was significantly associated with six growth traits of Hainan black goats. Briefly, an economic multiplex PCR method is presented to simultaneously and accurately detect three indel polymorphisms within goat GHR without the use of any special equipment, thus improving the experimental efficiency and fast obtaining the experimental result for indel genotyping.     

Retrotransposon-based insertion polymorphisms (RBIP) for high throughput marker analysis

Two assays based upon PCR detection of a polymorphic PDR1 retrotransposon insertion in Pisum sativum have been developed. Both methods involve PCR with primers derived from the transposon and flanking DNA. The first method uses a dot assay for PCR product detection which could be fully automated for handling thousands of samples. The second method, which is designed to handle lower numbers, requires a single PCR and gel lane per sample. Both methods yield co-dominant markers, with presence and absence of the transposon insertion independently scorable, and both could in principle be applied to any transposable element in any plant species.     

A general method for gene isolation in tagging approaches: amplification of insertion mutagenised sites (AIMS)

A polymerase chain reaction (PCR) based procedure for the isolation of genes in transposon or T-DNA tagging approaches has been developed. The method can be generally applied and allows the rapid isolation of putative gene sequences even in the presence of high numbers of insertion sequences. The technique has been used successfully for the isolation of the maize Bx1 gene tagged by a Mutator element.     

A novel tri-primer PCR method (TP-PCR) for rapid construction of fpg gene

A novel tri-primer polymerase chain reaction method (TP-PCR) was developed for the construction of a fused fpg gene, in which no endonuclease and ligase were used. Instead, two templates and three specifically designed primers were applied. Results showed that pheB and gfp genes, which encodes the catechol 2, 3-dioxygenase and the green fluorescent protein (GFP), respectively, were successfully fused into an fpg gene through the rapid TP-PCR system, indicating that TP-PCR method could be a useful tool for DNA fragment fusion in which no proper endonuclease sites were available.     

Comparative folate metabolism in humans and malaria parasites (part I): pointers for malaria treatment from cancer chemotherapy

New inhibitors are urgently needed to overcome the burgeoning problem of drug resistance in the treatment of Plasmodium falciparum infection. Targeting the folate pathway has proved to be a powerful strategy for drug development against rapidly multiplying systems such as cancer cells and microorganisms. Antifolates have long been used for malaria treatment but, despite their success, much less is known about parasite folate metabolism than about that of the human host. In this article, we focus on folate enzymes used clinically as anticancer drug targets, in addition to those that have potential to be used as drug targets, for which there are inhibitors at various stages of development. We discuss how this information could lead to the identification of new targets in malaria parasites.     

A novel magnetic resonance-based method to measure gene expression in living cells

In unicellular and multicellular eukaryotes, elaborate gene regulatory mechanisms facilitate a broad range of biological processes from cell division to morphological differentiation. In order to fully understand the gene regulatory networks involved in these biological processes, the spatial and temporal patterns of expression of many thousands of genes will need to be determined in real time in living organisms. Currently available techniques are not sufficient to achieve this goal; however, novel methods based on magnetic resonance (MR) imaging may be particularly useful for sensitive detection of gene expression in opaque tissues. This report describes a novel reporter gene system that monitors gene expression dynamically and quantitatively, in yeast cells, by measuring the accumulation of inorganic polyphosphate (polyP) using MR spectroscopy (MRS) or MR spectroscopic imaging (MRI). Because this system is completely non-invasive and does not require exogenous substrates, it is a powerful tool for studying gene expression in multicellular organisms, as well.     

Cloning and uterus/oviduct-specific expression of a novel estrogen-regulated gene (ERG1)

The steroid hormone estrogen profoundly influences growth and differentiation programs in the reproductive tract of cycling and pregnant mamals. It is thought that estrogen exerts its cellular effects by regulating the expression of specific target genes. We utilized a messenger RNA differential display method to identify the genes whose expression is modulated by estrogen in the preimplantation rat uterus. Here we report the cloning of a novel gene (ERG1) that is tightly regulated by estrogen in two key reproductive tissues, the uterus and oviduct. Spatio-temporal analyses reveal that ERG1 mRNA is expressed in a highly stage-specific manner in the uterus and oviduct, and its expression is restricted to the surface epithelium of both of these tissues. Nucleotide sequence analysis of the full-length ERG1 cDNA indicates that it has an open reading frame of 1821 nuceotides encoding a putative protein of 607 amino acids with a single transmembrane domain and a short cytoplasmic tail. The extracellular part of the protein contains several distinct structural motifs. These include a zona pellucida binding domain, which is present in a number of proteins such as the zona pellucida sperm binding proteins, and uromodulin, In addition, there is a repeat of a motif called CUB domain, which exists in a number of genes involved in development and differentiation such as bone morphogenetic protein 1 (BMP1). Although the precise function of ERG1 eludes us presently, its unique pattern of expression in the uterus and oviduct and its regulation by estrogen, a principal reproductive hormone, lead us to speculate that this novel gene plays an important role in events during the reproductive cycle and early pregnancy.     

Blasticidin S-resistance gene (bsr): a novel selectable marker for mammalian cells.

Blasticidin S is a microbial antibiotic that inhibits protein synthesis in both prokaryotes and eukaryotes. The blasticidin S-resistance gene (bsr), isolated from Bacillus cereus K55-S1 strain, was inserted into pSV2 plasmid vector and introduced into cultured mammalian cells by transfection. The bsr gene was integrated into the genome and conferred blasticidin S resistance on HeLa cells. The transfection frequency of the bsr gene was as high as that of the aminoglycoside phosphotransferase gene, the so-called neo gene, which is a representative selectable marker for mammalian cells. Transfectants in which several copies of bsr had been integrated into the genome were highly resistant to blasticidin S. Furthermore, blasticidin S killed the cells more rapidly than G418, which is conventionally used as a selective drug for the neo gene. Thus bsr is concluded to be useful as a drug-resistance marker for mammalian cells.     

Evolution of gametocyte sex ratios in malaria and related apicomplexan (protozoan) parasites

'Survival of the fittest' is usually interpreted to mean that natural selection favours genes that maximize their transmission to the next generation. Here, we discuss recent applications of this principle to the study of gametocyte sex ratios in malaria and other apicomplexan parasites. Sex ratios matter because they are an important determinant of fitness and transmission success -- and hence of disease epidemiology and evolution. Moreover, inbreeding rates can be estimated from gametocyte sex ratios. The sex ratio is also an excellent model trait for testing the validity of important components of what is being marketed as 'Darwinian medicine'.