S-adenosylhomocysteine Hydrolase Gene Research Articles

Antiparasitic activity of FLLL-32 against four Babesia species, B. bovis, B. bigemina, B. divergens and B. caballi, and one Theileria species, Theileria equi in vitro, and Babesia microti in mice.

Introduction: FLLL-32, a synthetic analog of curcumin, is a potent inhibitor of STAT3's constitutive activation in a variety of cancer cells, and its anticancer properties have been demonstrated both in vitro and in vivo. It is also suggested that it might have other pharmacological activities including activity against different parasites. Aim: This study therefore investigated the in vitro antiparasitic activity of FLLL-32 against four pathogenic Babesia species, B. bovis, B. bigemina, B. divergens, and B. caballi, and one Theileria species, Theileria equi. In vivo anti-Babesia microti activity of FLLL-32 was also evaluated in mice. Methods: The FLLL-32, in the growth inhibition assay with a concentration range (0.005-50μM), was tested for it's activity against these pathogens. The reverse transcription PCR (RT-PCR) assay was used to evaluate the possible effects of FLLL-32 treatment on the mRNA transcription of the target B. bovis genes including S-adenosylhomocysteine hydrolase and histone deacetylase. Results: The in vitro growth of B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi was significantly inhibited in a dose-dependent manner (in all cases, p < 0.05). FLLL-32 exhibits the highest inhibitory effects on B. bovis growth in vitro, and it's IC50 value against this species was 9.57μM. The RT-PCR results showed that FLLL-32 inhibited the transcription of the B. bovis S-adenosylhomocysteine hydrolase gene. In vivo, the FLLL-32 showed significant inhibition (p < 0.05) of B. microti parasitemia in infected mice with results comparable to that of diminazene aceturate. Parasitemia level in B. microti-infected mice treated with FLLL-32 from day 12 post infection (pi) was reduced to reach zero level at day 16 pi when compared to the infected non-treated mice. Conclusion: The present study demonstrated the antibabesial properties of FLLL-32 and suggested it's usage in the treatment of babesiosis especially when utilized in combination therapy with other antibabesial drugs.

S-Adenosyl Homocysteine Hydrolase (SAHH) Accelerates Flagellar Regeneration in Dunaliella salina

S-adenosylhomocysteine hydrolase (SAHH) is an enzyme, which catalyzes the hydrolysis of S-adenosylhomocysteine (SAH) which is formed after the donation of the methyl group of S-adenosylmethionine (SAM) to a methyl acceptor in methylation reaction. As a potent regulator of methylation, SAHH plays a critical role in methylation reaction in the cells. Here we cloned the SAHH gene from unicellular green alga Dunaliella salina (dsSAHH) and investigated its effects on flagellar regeneration of D. salina, and found that dsSAHH was upregulated both at the protein and the transcription levels during pH shock-triggered flagellar regeneration of D. salina. The flagellar regeneration was accelerated when dsSAHH was overexpressed, but it was inhibited by SAHH inhibitor 3-deazaadenosine (DZA). Moreover, a receptor for activated C kinase 1 from D. salina (dsRACK1), which was identified to interact with dsSAHH, was increased when dsSAHH was overexpressed in D. salina as shown by real-time PCR. The findings of this study suggest that dsSAHH may participate in the regulation of flagellar regeneration of D. salina.

Determining gene flow and the influence of selection across the equatorial barrier of the East Pacific Rise in the tube-dwelling polychaete Alvinella pompejana

BackgroundComparative phylogeography recently performed on the mitochondrial cytochrome oxidase I (mtCOI) gene from seven deep-sea vent species suggested that the East Pacific Rise fauna has undergone a vicariant event with the emergence of a north/south physical barrier at the Equator 1-2 Mya. Within this specialised fauna, the tube-dwelling polychaete Alvinella pompejana showed reciprocal monophyly at mtCOI on each side of the Equator (9°50'N/7°25'S), suggesting potential, ongoing allopatric speciation. However, the development of a barrier to gene flow is a long and complex process. Secondary contact between previously isolated populations can occur when physical isolation has not persisted long enough to result in reproductive isolation between genetically divergent lineages, potentially leading to hybridisation and subsequent allelic introgression. The present study evaluates the strength of the equatorial barrier to gene flow and tests for potential secondary contact zones between A. pompejana populations by comparing the mtCOI gene with nuclear genes.ResultsAllozyme frequencies and the analysis of nucleotide polymorphisms at three nuclear loci confirmed the north/south genetic differentiation of Alvinella pompejana populations along the East Pacific Rise. Migration was oriented north-to-south with a moderate allelic introgression between the two geographic groups over a narrow geographic range just south of the barrier. Multilocus analysis also indicated that southern populations have undergone demographic expansion as previously suggested by a multispecies approach. A strong shift in allozyme frequencies together with a high level of divergence between alleles and a low number of 'hybrid' individuals were observed between the northern and southern groups using the phosphoglucomutase gene. In contrast, the S-adenosylhomocysteine hydrolase gene exhibited reduced diversity and a lack of population differentiation possibly due to a selective sweep or hitch-hiking.ConclusionsThe equatorial barrier leading to the separation of East Pacific Rise vent fauna into two distinct geographic groups is still permeable to migration, with a probable north-to-south migration route for A. pompejana. This separation also coincides with demographic expansion in the southern East Pacific Rise. Our results suggest that allopatry resulting from ridge offsetting is a common mechanism of speciation for deep-sea hydrothermal vent organisms.

Analyzing S-Adenosylhomocysteine Hydrolase Gene Sequences in Deuterostome Genomes

S-adenosylhomocysteine hydrolase (SAHH) gene sequences of sea-urchin, two amphioxus, sea-squirt and eight vertebrates are comparatively analyzed in the current analysis. Although SAHH protein sequences are highly conserved in these species, their nucleotide sequences are much different, ranging from 5,446 bp in amphioxus to 40,174 bp in zebra fish. The length divergence is mainly caused by distinct introns in some species. SAHH genes in amphioxus (or sea-urchin), sea-squirt and vertebrates are composed of eight, nine and ten exons, respectively. Sequence alignment shows that exon 3 in amphioxus and sea-urchin is similar to exons 3 + 4 in vertebrates, exon 5 in amphioxus and sea-urchin is similar to exons 5 + 6 in sea-squirt, and the two exons are fused into exon 6 in vertebrates. Furthermore, exon 7 in sea-squirt is similar to exons 7 + 8 in vertebrates, indicating that exon-fission and exon-fusion events have been taken place during the evolution of deuterostome SAHH genes. Active sites and NAD+-binding sites are located in exons 2 ∼ 7 in amphioxus, which are dispersed into much more exons along with the evolution of vertebrates. It is speculated that ten-exon organization of SAHH gene occurred after the separation of invertebrates and vertebrates. Synonymous and non-synonymous substitution analysis shows that negative selection plays a dominant role in the evolution of SAHH genes. Phylogenetic analysis shows that SAHH genes in amphioxus, sea-urchin and sea-squirt form a cluster and locate at the base of neighbor-joining tree, suggesting that they are the archetype of vertebrate SAHH genes.

S-Adenosylhomocysteine Hydrolase from Corynebacterium glutamicum: Cloning, Overexpression, Purification, and Biochemical Characterization

The S-adenosylhomocysteine hydrolase gene (sahase) was cloned from the Gram-positive soil bacterium Corynebacterium glutamicum (ATCC 13032) and sequenced. The sahase gene possesses an open reading frame, which consists of 1,434 nucleotides that encode 478 amino acids. The sahase gene from C. glutamicum was expressed in Escherichia coli Rosetta cells by inserting the 1,434-bp fragment downstream from the isopropyl-β-D-thiogalactopyranoside-inducible promoter of the pET28a+ expression vector. The recombinant S-adenosylhomocysteine hydrolase from C. glutamicum (CgrSAHase) was purified efficiently by a two-step procedure, tangential ultrafiltration and affinity chromatography. The molecular weight of the CgrSAHase, estimated by gel filtration, was about 210 kDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 52 ± 1 kDa. The Michaelis-Menten constants for the natural substrates of the enzyme, S-adenosylhomocysteine (SAH), adenosine, and homocysteine, were determined to be 12, 1.4, and 40 µM, respectively. The overexpression of CgrSAHase was achieved at high level (>40 mg protein/g wet cells). Because of its high capacity to synthesize SAH, this enzyme is of high biotechnological interest.

Studies of S-adenosylhomocysteine-hydrolase polymorphism in a Croatian population

Recently, a proven case of human S-adenosylhomocysteine-hydrolase (SAHH) deficiency was reported in a Croatian boy. As molecular analysis of the SAHH gene in this case revealed two different mutant alleles, we investigated the polymorphism of human SAHH in a total of 237 red blood samples from unrelated Croats using starch gel electrophoresis and an enzyme-specific staining procedure. From the relative enzymatic activity of SAHH--determined by densitometric assessment of electrophoretic patterns, and calculated on the basis of the protein concentration of the red blood cells-we detected three individuals as being heterozygous for an SAHH 0-allele. Moreover, a total of four different electromorphic SAHHs have been observed, giving allele frequencies calculated as SAHH 1 = 0.941, SAHH 2 = 0.032, SAHH 3 = 0.006, SAHH 4 = 0.015, and SAHH 0 = 0.006.

Effect of genetic variation in the human S-adenosylhomocysteine hydrolase gene on total homocysteine concentrations and risk of recurrent venous thrombosis.

Hyperhomocysteinemia is an independent and graded risk factor for arterial vascular disease and venous thrombosis. It is still debated via which mechanism homocysteine (Hcy) causes vascular disease. S-adenosylhomocysteine hydrolase (AHCY) catalyses the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) to Hcy. As an increase in AdoHcy, a strong inhibitor of many methyltransferases, is observed in hyperhomocysteinemic individuals, AdoHcy may play a role in the development of cardiovascular diseases by inhibiting transmethylation reactions. We sequenced the entire coding region and parts of the untranslated regions (UTRs) of the AHCY gene of 20 patients with recurrent venous thrombosis in order to identify genetic variation within this gene. We identified three sequence variants in the AHCY gene: a C > T transition in the 5' UTR (-34 bp C > T), a missense mutation in exon 2, which mandates an amino-acid conversion at codon 38 (112 C > T; Arg38Trp) and a silent mutation in exon 4 (390 C > T; Asp130Asp). We studied the effect of the first two variants on total plasma Hcy and venous thrombosis risk in a case-control study on recurrent venous thrombosis. The two polymorphisms under study seem to have no evident effect on tHcy. The adjusted relative risk of venous thrombosis associated with the 112CT genotype compared with 112CC individuals was 1.27 (95% CI 0.55-2.94), whereas the -34CT genotype confers a risk of 1.25 (95% CI 0.44-3.52) compared with the wild-type genotype at this locus. However, the wide confidence intervals do not allow firm conclusions to be drawn.

Structure, evolution, and inhibitor interaction of S-adenosyl-L-homocysteine hydrolase from Plasmodium falciparum.

S-adenosylhomocysteine hydrolase (SAHH) is a key regulator of S-adenosylmethionine-dependent methylation reactions and an interesting pharmacologic target. We cloned the SAHH gene from Plasmodium falciparum (PfSAHH), with an amino acid sequence agreeing with that of the PlasmoDB genomic database. Even though the expressed recombinant enzyme, PfSAHH, could use 3-deaza-adenosine (DZA) as an alternative substrate in contrast to the human SAHH, it has a unique inability to substitute 3-deaza-(+/-)aristeromycin (DZAri) for adenosine. Among the analogs of DZA, including neplanocin A, DZAri was the most potent inhibitor of the PfSAHH enzyme activity, with a K(i) of about 150 nM, whether Ado or DZA was used as a substrate. When the same DZA analogs were tested for their antimalarial activity, they also inhibited the in vitro growth of P. falciparum parasites potently. Homology-modeling analysis revealed that a single substitution (Thr60-Cys59) between the human and malarial PfSAHH, in an otherwise similar SAH-binding pocket, might account for the differential interactions with the nucleoside analogs. This subtle difference in the active site may be exploited in the development of novel drugs that selectively inhibit PfSAHH. We performed a comprehensive phylogenetic analysis of the SAHH superfamily and inferred that SAHH evolved in the common ancestor of Archaea and Eukaryota, and was subsequently horizontally transferred to Bacteria. Additionally, an analysis of the unusual and uncharacterized AHCYL1 family of the SAHH paralogs extant only in animals reveals striking divergence of its SAH-binding pocket and the loss of key conserved residues, thus suggesting an evolution of novel function(s).

Expression, purification, and characterization of recombinant S-adenosylhomocysteine hydrolase from the thermophilic archaeon Sulfolobus solfataricus.

S-Adenosylhomocysteine hydrolase from Sulfolobus solfataricus was expressed in Escherichia coli by inserting the genomic fragment containing the gene encoding for S-adenosylhomocysteine hydrolase downstream the isopropyl-beta-d-thiogalactoside-inducible promoter of pTrc99A expression vector. An ATG positioned 25 bp upstream of the gene which is in frame with a stop codon was utilized as the initiation codon. This construct was used to transform E. coli RB791 and E. coli JM105 strains. The recombinant protein, purified by a fast and efficient two-step procedure (yield of 0.4 mg of enzyme per gram of cells), does not appear homogeneous on SDS-PAGE because of the presence of a protein contaminant corresponding to a "truncated" S-adenosylhomocysteine hydrolase subunit lacking the first 24 amino acid residues. The recombinant enzyme shows the same molecular mass, optimum temperature, and kinetic features of S-adenosylhomocysteine hydrolase isolated from S. solfataricus but it is less thermostable. To construct a vector which presents a correct distance between the ribosome-binding site and the start codon of S-adenosylhomocysteine hydrolase gene, a NcoI site was created at the translation initiation codon using site-directed mutagenesis. The expression of the homogeneous mutant S-adenosylhomocysteine hydrolase was achieved at high level (1.7 mg of mutant protein per gram of cells). The mutant S-adenosylhomocysteine hydrolase and the native one were indistinguishable in all physicochemical and kinetic properties including thermostability, indicating that the interactions involving the NH(2)-terminal sequence of the protein play a role in the thermal stability of S. solfataricus S-adenosylhomocysteine hydrolase.

Cloning of the S-adenosylhomocysteine hydrolase gene of Pneumocystis carinii.

Journal of Eukaryotic MicrobiologyVolume 43, Issue 5 p. 6S-6S Cloning of the S-Adenosylhomocysteine Hydrolase Gene of Pneumocystis carinii MARK E. LASBURY, MARK E. LASBURY Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorSTEVE BRADY, STEVE BRADY Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorGERALD McLAUGHLIN, GERALD McLAUGHLIN Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorMARILYN S. BARTLETT, MARILYN S. BARTLETT Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorJAMES W. SMITH, JAMES W. SMITH Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorCHAO-HUNG LEE, CHAO-HUNG LEE Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this author MARK E. LASBURY, MARK E. LASBURY Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorSTEVE BRADY, STEVE BRADY Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorGERALD McLAUGHLIN, GERALD McLAUGHLIN Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorMARILYN S. BARTLETT, MARILYN S. BARTLETT Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorJAMES W. SMITH, JAMES W. SMITH Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this authorCHAO-HUNG LEE, CHAO-HUNG LEE Dept. of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202Search for more papers by this author First published: September 1996 https://doi.org/10.1111/j.1550-7408.1996.tb04947.xCitations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article.Citing Literature Volume43, Issue5September 1996Pages 6S-6S RelatedInformation

Broad resistance to plant viruses in transgenic plants conferred by antisense inhibition of a host gene essential in S-adenosylmethionine-dependent transmethylation reactions.

S-Adenosylhomocysteine hydrolase (SAHH) is a key enzyme in transmethylation reactions that use S-adenosylmethionine as the methyl donor. Because of the importance of SAHH in a number of S-adenosylmethionine-dependent transmethylation reactions, particularly the 5' capping of mRNA during viral replication, SAHH has been considered as a target of potential antiviral agents against animal viruses. To test the possibility of engineering a broad type of resistance to plant viruses, we expressed the antisense RNA for tobacco SAHH in transgenic tobacco plants. As expected, transgenic plants constitutively expressing an anti-sense SAHH gene showed resistance to infection by various plant viruses. Among those plants, about half exhibited some level of morphological change (typically stunting). Analysis of the physiological change in those plants showed that they contained excess levels of cytokinin. Because cytokinin has been found to induce acquired resistance, there is also a strong possibility that the observed resistance was induced by cytokinin.

Molecular characterization in the dpy-14 region identifies the S-adenosylhomocysteine hydrolase gene in Caenorhabditis elegans

The region around dpy-14 on chromosome 1 of Caenorhabditis elegans has been extensively studied genetically, with regard to essential gene organization. This region was one of the first for which cloned DNA was available as a result of restriction fragment length polymorphism mapping. To examine the information content of the cloned DNA in this region, evolutionarily conserved sequences were identified by cross-species hybridization. Ten regions of conservation have been identified and characterized with regard to mRNA abundance and DNA sequence. cDNAs were obtained for seven of these conserved regions and sequence from the cDNAs were used to search the SWISS protein and EMBL nucleotide data banks. Two coding regions shared DNA identifies with existing sequences, the opa repeat family of Drosophila and the S-adenosylhomocysteine hydrolase gene. Of the three for which no corresponding cDNA were found, one corresponds to the snRNA U1-1. The other two did not detect transcripts on Northern analysis and are either conserved, but not coding, or code for low abundance transcripts. The density of conserved coding regions in this study was one per 15 kbp of genomic DNA, three times lower than that reported on chromosome 3 by the genome sequencing project.