Isolation and characterisation of a novel rabbit sulfotransferase isoform belonging to the SULT1A subfamily

Sulfotransferases (SULTs) catalyse the sulfonation of both endogenous and exogenous compounds including hormones, catecholamines. drugs and xenobiotics. While in most occasions, sulfonation is a detoxication pathway. in the case of certain drugs and carcinogens. it leads to metabolic activation. Since, the rabbit has been extensively used for both pharmacological and toxicological studies, the purpose of this study was to further characterise the sulfotransferase system of this animal. In the present study, a novel sulfotransferase isoform (GenBank Accession no. AF360872) was isolated from a rabbit liver cDNA lambdaZAP 11 library. The full-length sequence of the clone was 1138 bp long and contained a coding region of 888 bp encoding a cytosolic protein of 295 amino acids (deduced molecular weight 34,193 Da). The amino acid sequence of this novel SULT isoform showed >70% identity with members of the SULT1A subfamily of sulfotransferases from other species. Upon expression of the encoded rabbit sulfotransferase in Escherchia coli (E. coli), it was shown that the enzyme was capable of sulfonating both p-nitrophenot (K-m and V-max values of 0.15 muM and 897.5 nmol/min/mg protein. respectively) and dopamine (K-m and V-max values of 175.3 muM and 151.1 nmol/min/mg protein, respectively). Based on the sequence data obtained and substrate specificity, this new rabbit sulfotransferase was named rabSULT1A1. Immunoblotting was used to demonstrate that rabSULT1A1 protein is expressed in liver, duodenum, jejunum, ileum, colon and recturm. (C) 2002 Elsevier Science Ltd. All rights reserved.

The human sulfotransferase SULT1A1 gene is regulated in a synergistic manner by Sp1 and GA binding protein

Human sulfotransferase SULT1A1 is an important phase II xenobiotic metabolizing enzyme that is highly expressed in the liver and mediates the sulfonation of drugs, carcinogens, and steroids. Until this study, the transcriptional regulation of the SULT1A subfamily had been largely unexplored. Preliminary experiments in primary human hepatocytes showed that SULT1A mRNA levels were not changed in response to nuclear receptor activators, such as dexamethasone and 3-methylcolanthrene, unlike other metabolizing enzymes. Using HepG2 cells, the high activity of the TATA-less SULT1A1 promoter was shown to be dependent on the presence of Sp1 and Ets transcription factor binding sites (EBS), located within - 112 nucleotides from the transcriptional start site. The homologous promoter of the closely related SULT1A3 catecholamine sulfotransferase, which is expressed at negligible levels in the adult liver, displayed 70% less activity than SULT1A1. This was shown to be caused by a two-base pair difference in the EBS. The Ets transcription factor GA binding protein (GABP) was shown to bind the SULT1A1 EBS and could transactivate the SULT1A1 promoter in Drosophila melanogaster S2 cells. Cotransfection of Sp1 could synergistically enhance GABP-mediated activation by 10-fold. Although Sp1 and GABP alone could induce SULT1A3 promoter activity, the lack of the EBS on this promoter prevented a synergistic interaction between the two factors. This study reports the first insight into the transcriptional regulation of the SULT1A1 gene and identifies a crucial difference in regulation of the closely related SULT1A3 gene, which accounts for the two enzymes' differential expression patterns observed in the adult liver.

TFEC is a macrophage-restricted member of the microphthalmia-TFE subfamily of basic helix-loop-helix leucine zipper transcription factors

The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed,vith all other known MiT subfamily members (Mitf, TFE3, TFEB), Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU,I under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage.

Species of Trifoliovarium Yamaguti, 1940 (Digenea : Lecithasteridae) from Australian waters, with a description of T-draconis n. sp and a cladistic study of the subfamily Trifoliovariinae Yamaguti, 1958

Three species of Trifoliovarium are described from marine fishes from Moreton Bay, Queensland: T. triacanthi (Parukhin, 1964) n. comb. (syns Hysterolecitha triacanthi Parukhin, 1964; T. triacanthi Bilqees, 1973; T. triacanthusi Gupta & Ahmad, 1976) from Tripodichthys angustifrons; T. ovarilobulus (Wang, 1989) n. comb. (syn. Hysterolecithia[sic]ovarilobulus) from Paramonacanthus japonicus and Pelates quadrilineatus; and T. draconis n. sp. from Callionymus sublaevis and C. belcheri. A list of the species of the subfamily Trifoliovariinae is given along with a key. A cladistic study of the subfamily based on 23 characters is presented, the results of which indicate the monophyly of the genus Assitrema and the paraphyly of Trifoliovarium.

The subfamily Aephnidiogeninae Yamaguti, 1934 (Digenea: Lepocreadiidae), its status and that of the genera Aephnidiogenes Nicoll, 1915, Holorchis Stossich, 1901, Austroholorchis n g, Pseudaephnidiogenes Yamaguti, 1971, Pseudoholorchis Yamaguti, 1958 and N

The status of all of the putative member genera of the subfamily Aephnidiogeninae is reconsidered, based mainly on the morphology of the terminal genitalia, Aephnidiogenes Nicoll, 1915 is the only genus retained in the Aaephnidiogeninae. Aephnidiogenes major Yamaguti, 1934 from Diagramma labiosum from the southern Great Barrier Reef is redescribed with particular reference to the terminal genitalia, and is shown to lack a true cirrussac, a condition considered to be diagnostic of the Aephnidiogeninae. Holorchis Stossich, 1901 is placed in the subfamily Lepidapedinae. Holorchis pycnoporus Stossich, 1901 from Pagellus acarne from off Spanish Sahara and from Diplodus vulgaris from off Italy and H. legendrei Dollfus, 1946 from Sparodon durbanensis and D. sargus from off eastern Cape Province, South Africa and from Pagellus erythrinus from the Adriatic Sea and Italy are studied and illustrated. The terminal genitalia of H. pycnoporus are found to be enigmatic, but those of H. legendrei are found to fit clearly into the 'Lepidapedon-like' pattern. A new genus Austroholorchis is erected in the Lepidapedinae, with A. sprenti (Gibson, 1987) n. comb. as the type-species. Its diagnostic features are its ani, infundibuliform oral sucker and the position of the ovary at about mid-level of the uterus. A. sprenti is illustrated, its hosts in Queensland waters being Sillago maculata, S, analis and S. ciliata. A, levis n. sp. is described from Sillago bassensis from south-western Western Australia. The genus Pseudaephnidiogenes Yamaguti, 1971 is placed in the Lepidapedinae. P. rhabdosargi (Prudhoe, 1956) from Rhabdosargus sarba from off Natal, South Africa is illustrated and the terminal genitalia of P. rhabdosargi from R. sarba and from R. holubi from off eastern Cape Province and Pseudaephnidiogenes vossi Bray, 1985 from Caffrogobius nudiceps from off eastern Cape Province, South Africa are illustrated. The genus Pseudoholorchis Yamaguti, 1958 is placed in the subfamily Lepocreadiinae. The terminal genitalia of P. pulcher (Manter, 1954) from Latridopsis ciliaris from New Zealand are illustrated, The genus Neolepocreadium Thomas, 1960 is placed in the Lepocreadiidae.

A new subfamily, Bothriocrotoninae n. subfam., for the genus Bothriocroton Keirans, King & Sharrad, 1994 status amend. (Ixodida : Ixodidae), and the synonymy of Aponomma Neumann, 1899 with Amblyomma Koch, 1844

Evidence suggesting polyphyly of the traditionally recognised tick genus Aponomma Neumann, 1899 is summarized. Continued recognition of this genus in its current concept leaves a polyphyletic genus Aponomma and a paraphyletic genus Amblyomma Koch, 1844. To improve the correlation between our understanding of phylogenetic relationships in metastriate ticks and their classification, a few changes in classification are proposed. The members of the 'indigenous Australian Aponomma' group (sensu Kaufman, 1972), A. auruginans Schulze, 1936, A. concolor Neumann, 1899, A. glebopalma Keirans, King & Sharrad, 1994, A. hydrosauri (Denny, 1843) and A. undatum (Fabricius, 1775), are transferred to Bothriocroton Keirans, King & Sharrad, 1994, which is raised to full generic rank. The remaining members of Aponomma are transferred to Amblyomma. Uncertainty remains on relationships of Bothriocroton to other metastriate lineages and on the systematic position of the two species formerly included in the 'primitive Aponomma' group, A. elaphense Price, 1959 and A. sphenodonti Dumbleton, 1943.

Human SULT1A genes: Cloning and activity assays of the SULT1A promoters

The three human SULT1A sulfotransferase enzymes are closely related in amino acid sequence (>90%), yet differ in their substrate preference and tissue distribution. SULT1A1 has a broad tissue distribution and metabolizes a range of xenobiotics as well as endogenous substrates such as estrogens and iodothyronines. While the localization of SULT1A2 is poorly understood, it has been shown to metabolize a number of aromatic amines. SULT1A3 is the major catecholamine sulfonating form, which is consistent with it being expressed principally in the gastrointestinal tract. SULT1A proteins are encoded by three separate genes, located in close proximity to each other on chromosome 16. The presence of differential 5′-untranslated regions identified upon cloning of the SULT1A cDNAs suggested the utilization of differential transcriptional start sites and/or differential splicing. This chapter describes the methods utilized by our laboratory to clone and assay the activity of the promoters flanking these different untranslated regions found on SULT1A genes. These techniques will assist investigators in further elucidating the differential mechanisms that control regulation of the human SULT1A genes. They will also help reveal how different cellular environments and polymorphisms affect the activity of SULT1A gene promoters.

Identification and characterisation of a cytochrome P450 gene and processed pseudogene from an arachnid: the cattle tick, Boophilus microplus

We isolated and sequenced the first known cytochrome P450 gene and pseudogene from an arachnid, the cattle tick, Boophilus microplus. Bath the gene and pseudogene belong to the family CYP4, but a new subfamily, CYP4W, had to be created for these genes because they are substantially different to other CYP4 genes. The gene, CPP4W1, has greatest homology with CYP4C1 from a cockroach, Blaberus discoidalis. The predicted molecular weight of the protein encoded by CYP4W1 (63 KDa) is greater than that of the other CYP4 genes. The pseudogene, CYP4W1P, is probably a processed pseudogene derived from the functional gene CYP4W1. This is only the third CYP processed pseudogene to be identified. The pseudogene is 98% identical to the functional gene, CYP4W1, therefore we hypothesise that this pseudogene evolved recently from the functional gene. The CYP4 genes from arthropods have diverged from each other more than those of mammals; consequently the phylogeny of the arthropod genes could not be resolved. (C) 1999 Elsevier Science Ltd. All rights reserved.

Plant cyclotides: A unique family of cyclic and knotted proteins that defines the cyclic cystine knot structural motif

Several macrocyclic peptides (similar to 30 amino acids), with diverse biological activities, have been isolated from the Rubiaceae and Violaceae plant families over recent years. We have significantly expanded the range of known macrocyclic peptides with the discovery of 16 novel peptides from extracts of Viola hederaceae, Viola odorata and Oldenlandia affinis. The Viola plants had not previously been examined for these peptides and thus represent novel species in which these unusual macrocyclic peptides are produced. Further, we have determined the three-dimensional struc ture of one of these novel peptides, cycloviolacin O1, using H-1 NMR spectroscopy. The structure consists of a distorted triple-stranded beta-sheet and a cystine-knot arrangement of the disulfide bonds. This structure is similar to kalata B1 and circulin A, the only two macrocyclic peptides for which a structure was available, suggesting that despite the sequence variation throughout the peptides they form a family in which the overall fold is conserved. We refer to these peptides as the cyclotide family and their embedded topology as the cyclic cystine knot (CCK) motif. The unique cyclic and knotted nature of these molecules makes them a fascinating example of topologically complex proteins. Examination of the sequences reveals they can be separated into two subfamilies, one of which tends to contain a larger number of positively charged residues and has a bracelet-like circularization of the backbone. The second subfamily contains a backbone twist due to a cis-Pro peptide bond and may conceptually be regarded as a molecular Moebius strip. Here we define the structural features of the two apparent subfamilies of the CCK peptides which may be significant for the likely defense related role of these peptides within plants. (C) 1999 Academic Press.

Hypoechinorhynchus robustus sp n. from Notolabrus parilus (Labridae) from Western Australia with a discussion on the validity of the hypoechinorhynchidae (Acanthocephala : Palaeacanthocephala)

Hypoechinorhynchus robustus sp. n. is described from Notolabrus parilus (Richardson) (Labridae) from Pt Peron, Western Australia. It has a proboscis with 30 hooks arranged in ten longitudinal rows: 5 rows of a small apical spine, a large anterior hook and a small posterior spine, 5 rows of a large anterior hook, a middle spine and a posterior spine. The new species is distinguished from other species of the genus by having a set of 5 small apical spines anterior to the large hooks on the proboscis, by having lemnisci that barely extend beyond the proboscis receptacle and testes which are more adjacent than tandem. H. robustus also has robust trunk spines anteriorly. Re-examination of Hypoechinorhynchus alaeopis Yamaguti, 1939 (type species) revealed trunk spines that had been overlooked previously. The Hypoechinorhynchidae is made a junior synonym of Arhythmacanthidae because there is considerable overlap between the two family diagnoses, particularly in that both families have a proboscis armature that changes abruptly from small basal spines to large apical (or subapical if present) hooks. The genus Hypoechinorhynchus is placed in the subfamily Arhythmacanthinae because it has trunk spines and a spherical proboscis with few hooks (relative to other arhythmacanthid genera). It is also proposed that Heterosentis magellanicus (Szidat, 1950) be returned to the genus Hypoechinorhynchus since it was transferred to Heterosentis primarily because it had trunk spines. The other hypoechinorhynchid genus contained only Bolborhynchoides exiguus (Achmerov et Dombrowskaja-Achmerova, 1941) Achmerov, 1959 and is relegated to incertae sedis.

Scorpidotrema longistipes n. g., n. sp. (Digenea: Opecoelidae) from Scorpis georgiana (Teleostei: Scorpididae) from southern Western Australia

Scorpidotrema longistipes n. g., n. sp. is described from the intestine of Scorpis georgiana Valenciennes (Scorpididae) from off Point Peron, Western Australia. The new genus is distinguished by the combination of a remarkably long and retractable ventral sucker peduncle, a possible uroproct, well-developed cirrus-sac and a uterine seminal receptacle. The subfamilial relationships of the new genus are troublesome. It incorporates features of the Opecoelinae, Stenakrinae and Plagioporinae. The absence of a canalicular seminal receptacle suggests a relationship with the Opecoelinae and Stenakrinae, whereas the well-developed cirrus-sac suggests a relationship with the Plagioporinae and Stenakrinae. The overall arrangement of the gonads is not similar to that of existing genera of Stenakrinae. It is concluded that the genus is best placed in the Stenakrinae although that subfamily may now be an artificial assemblage. This new genus forms part of a distinctive fauna of trematodes restricted to Australian southern temperate fishes.

Phylogeny, evolution and historical zoogeography of ticks: a review of recent progress

There has been much progress in our understanding of the phylogeny and evolution of ticks, particularly hard ticks, in the past 5 years. Indeed, a consensus about the phylogeny of the hard ticks has emerged. Our current working hypothesis for the phylogeny of ticks is quite different to the working hypothesis of 5 years ago. So that the classification reflects our knowledge of ticks, several changes to the nomenclature of ticks are imminent. One subfamily, the Hyalomminae, will probably be sunk, yet another, the Bothriocrotoninae n. subfamily, will be created. Bothriocrotoninae n. subfamily, and Bothriocroton n. genus, are being created to house an early-diverging ('basal') lineage of endemic Australian ticks that used to be in the genus Aponomma (ticks of reptiles). There has been progress in our understanding of the subfamily Rhipicephalinae. The genus Rhipicephalus is almost certainly paraphyletic with respect to the genus Boophilus. Thus, the genus Boophilus will probably become a subgenus of Rhipicephalus. This change to the nomenclature, unlike other options, will keep the name Boophilus in common usage. Rhipicephalus (Boophilus) microplus may still called B. microplus, and Rhipicephalus (Boophilus) annulatus may still be called B. annulatus, but the nomenclature will have been changed to reflect our knowledge of the phylogeny and evolution of these ticks. New insights into the historical zoogeography of ticks will also be presented.

The systematics and biology of the south African gall-inducing scale insect, Calycicoccus merwei brain (Hemiptera : Coccoidea : Eriococcidae)

The scale insect genus Calycicoccus Brain has a single described species, C. merwei Brain, which is endemic to southeastern South Africa. Females of C. merwei induce small, mostly conical galls on the foliage of their host tree, Apodytes dimidiata E. Meyer ex Arn. (Icacinaceae), which has a wider, mostly coastal distribution, than that currently known for the scale insect. Calycicoccus has been placed in the family Eriococcidae and may be related to the South American genus Aculeococcus Lepage. No other native eriococcid species have been described so far in South Africa, although the family is diverse in other Gondwanan regions. This paper summarizes the biology of C. merwei, redescribes the adult female, describes the adult male, the second-instar female and the first-instar nymphs for the first time, and reconsiders the phylogenetic relationships of the genus. The adult female is shown to have unusual abdominal segmentation, in that segment I is present both dorsally and ventrally, but a segment is absent ventrally on the middle abdomen. First-instar nymphs are sexually dimorphic; males have a larger and relatively narrower body, larger mouthparts, longer antennae and legs, and more thoracic dorsal setae compared with females. Molecular data from nuclear small-subunit ribosomal DNA (18S) and elongation factor 1 alpha (EF-1a) show C. merwei to have no close relatives among the Eriococcidae sampled to date. Instead, the Calycicoccus lineage is part of a polytomy near the base of the Eriococcidae. Molecular dating of the node suggests that the Calycicoccus lineage diverged from other eriococcids more than 100 Mya. These data support the placement of Calycicoccus as the only genus in the subfamily Calycicoccinae Brain.