Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit

Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coil, and purified the AHAS regulatory subunit of Ambidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.

Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus

The 12 cysteine residues in the flavivirus NS1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the intramolecular disulfide bond arrangement of NS1 of Murray Valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. Disulfide linkages were identified by separating tryptic-digested NS1 by reverse-phase high pressure liquid chromatography and analysing the resulting peptide peaks by protein sequencing, amino acid analysis and/or electrospray mass spectrometry. The pairing arrangements between the six amino-terminal cysteines were identified as follows: Cys(4)-Cys(15), Cys(55)-Cys(143) and Cys(179)-Cys(223). Although the pairing arrangements between the six carboxyterminal cysteines were not determined, we were able to eliminate several cysteine-pairing combinations. Furthermore, we demonstrated that all three putative N-linked glycosylation sites of NS1 are utilized and that the Asn(207) glycosylation site contains a mannose-rich glycan.

A novel variant genotype C of hepatitis B virus identified in isolates from Australian Aborigines: complete genome sequence and phylogenetic relatedness

There have been no reports of DNA sequences of hepatitis B virus (HBV) strains from Australian Aborigines, although the hepatitis B surface antigen (HBsAg) was discovered among them. To investigate the characteristics of DNA sequences of HBV strains from Australian Aborigines, the complete nucleotide sequences of HBV strains were determined and subjected to molecular evolutionary analysis. Serum samples positive for HBsAg were collected from five Australian Aborigines. Phylogenetic analysis of the five complete nucleotide sequences compared with DNA sequences of 54 global HBV isolates from international databases revealed that three of the five were classified into genotype D and were most closely related in terms of evolutionary distance to a strain isolated from a healthy blood donor in Papua New Guinea. Two of the five were classified into a novel variant genotype C, which has not been reported previously, and were closely related to a strain isolated from Polynesians, particularly in the X and Core genes. These two strains of variant genotype C differed from known genotype C strains by 5.9-7.4% over the complete nucleotide sequence and 4.0-5.6 % in the small-S gene, and had residues Arg(122), Thr(127) and Lys(160) characteristic of serotype ayw3, which have not been reported previously in genotype C. In conclusion, this is the first report of the characteristics of complete nucleotide sequences of HBV from Australian Aborigines. These results contribute to the investigation of the worldwide spread of HBV, the relationship between serotype and genotype and the ancient common origin of Australian Aborigines.

No evidence of a role for activating CDK2 mutations in melanoma

Inactivation of p16(INK4a) and/or activation of cyclin-dependent kinase-4 (CDK4) are strongly associated with both susceptibility and progression in melanoma. Activating CDK4 mutations prevent the binding and inhibition of CDK4 by p16(INK4a). A second, more indirect role for CDK4 is in late G(1), where It may sequester the inhibitors p27(KIP1) or p21(CIP1) away from CDK2, and in doing so upregulate the CDK2 activity necessary for cells to proceed completely through G(1) into S phase. As the pivotal residues around the most predominant R24C activating CDK4 mutation are invariant between CDK2 and CDK4, we speculated that the pivotal arginine (position 22 in CDK2), or a nearby residue, may be mutated in some melanomas, resulting in the diminution of its binding and inhibition by p27(KIP1) or p21(CIP1). However, except for a silent polymorphism, we detected no variants within this region of the CDK2 gene in 60 melanoma cell lines. Thus, if CDK2 activity is dysregulated in melanoma it is likely to occur by a means other than mutations causing loss of direct inhibition. We also examined the expression of the CDK2 gene in melanoma cell lines, to assess its possible co-regulation with the gene for the melanocyte-lineage antigen pmel17, which maps less than 1 kb away in head to head orientation with CDK2 and may be transcribed off the same bidirectional promoter. However, expression of the genes is not co-regulated. (C) 2001 Lippincott Williams & Wilkins.

Atm knock-in mice harboring an in-frame deletion corresponding to the human ATM 7636del9 common mutation exhibit a variant phenotype

ATM, the gene mutated in the human immunodeficiency disorder ataxia-telangiectasia (A-T), plays a central role in recognizing ionizing radiation damage in DNA and in controlling several cell cycle checkpoints. We describe here a murine model in which a nine-nucleotide in-frame deletion has been introduced into the Atm gene by homologous recombination followed by removal of the selectable marker cassette by Cre-loxP site-specific, recombination-mediated excision. This mouse, Abm-Delta SRI, was designed as a model of one of the most common deletion mutations (7636de19) found in A-T patients. The murine Atm deletion results in the loss of three amino acid residues (SRI; 2556-2558) but produces near full-length detectable Atm protein that lacks protein kinase activity. Radiosensitivity was observed in Atm-Delta SRI mice, whereas the immunological profile of these mice showed greater heterogeneity of T-cell subsets than observed in Atm(-/-) mice. The life span of Atm-Delta SRI mice was significantly longer than that of Atm(-/-) mice when maintained under nonspecific pathogen-free conditions. This can be accounted for by a lower incidence of thymic lymphomas in Atm-Delta SRI mice up to 40 weeks, after which time the animals died of other causes. The thymic lymphomas in Atm-Delta SRI mice were characterized by extensive apoptosis, which appears to be attributable to an increased number of cells expressing Fas ligand. A variety of other tumors including B-cell lymphomas, sarcomas, and carcinomas not seen in Atm(-/-) mice were observed in older Atm-Delta SRI animals. Thus, expression of mutant protein in Atm-Delta SRI knock-in mice gives rise to a discernibly different phenotype to Atm(-/-) mice, which may account for the heterogeneity seen in A-T patients with different mutations.

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Lipophilic methotrexate conjugates with glucose-lipoamino acid moieties: Synthesis and in vitro antitumor activity

To obtain methotrexate (MTX) derivatives with a balanced hydrolipophilic character, we synthesized a series of conjugates in which the drug was linked to lipoamino acid (LAA)-glucose residues (LAAG-MTX). These conjugates displayed increased solubility in polar media compared with the corresponding LAA-MTX conjugates previously described. In vitro biological testing of LAAG-MTX indicated that the introduction of the sugar moiety decreased the biological activity of these MTX conjugates. The tetradecyl derivative 6b, however, was effective in inhibiting the dihydrofolate reductase activity in vitro and showed an inhibitory effect on human lymphoblastoid cell growth. (C) 2001 Wiley-Liss, Inc.

Chemistry, properties, and phylogenetic implications of the methylated carrageenans from red algae of the genus Areschougia (Areschougiaceae, Gigartinales, Rhodophyta)

The three Australian-endemic species comprising the genus Aresehougia have been examined to determine the structure of their nonfibrillar wall components. The polysaccharide extracted from the most widely distributed species, A. congesta (Turner) J. Agardh, was shown by compositional analyses, Fourier transform infrared (FTIR) spectroscopy, linkage analysis, and C-13-NMR spectroscopy to be a carrageenan composed predominantly of the repeating disaccharides 6'-O-methylcarrabiose 2,4'-disulfate, carrabiose 2,4-disulfate (the repeating unit of L-carrageenan), 4',6'-O-(1-carboxyethylidene)carrabiose 2-sulfate, and 6'-O-methylcarrabiose 2-sulfate. The carrageenan also contained small amounts of 4-linked Galp residues, some bearing methyl ether substitution at O-3 and some possibly bearing sulfate ester and/or glycosyl substitutions at O-3. The A. congesta carrageenan had unique rheological properties, its gels having some similarities to those of commercial iota -carrageenan but with the viscosity of commercial lambda -carrageenan. Polysaccharides from A. ligulata Harvey ex J. Agardh and A. stuartii Harvey were shown by constituent sugar and FTIR analyses to be sulfated galactans rich in mono-O-methylgalactose. The carrageenan structures of Areschougia spp. were consistent with those of the genera Rhabdonia, Erythroclonium, and Austroclonium, the other genera constituting the family Areschougiaceae.

The cystine knot motif in toxins and implications for drug design

The cystine knot structural motif is present in peptides and proteins from a variety of species, including fungi, plants, marine molluscs. insects and spiders. It comprises an embedded ring formed by two disulfide bonds and their connecting backbone segments which is threaded by a third disulfide bond. It is invariably associated with nearby beta-sheet structure and appears to be a highly efficient motif for structure stabilization. Because of this stability it makes an ideal framework for molecular engineering applications. In this review we summarize the main structural features of the cystine knot motif, focussing on toxin molecules containing either the inhibitor cystine knot or the cyclic cystine knot. Peptides containing these motifs are 26-48 residues long and include ion channel blockers, haemolytic agents, as well as molecules having antiviral and antibacterial activities. The stability of peptide toxins containing the cystine knot motif, their range of bioactivities and their unique structural scaffold can be harnessed for molecular engineering applications and in drug design. Applications of cystine knot molecules for the treatment of pain. and their potential use in antiviral and antibacterial applications are described. (C) 2000 Elsevier Science Ltd. All rights reserved.

Solution structures by H-1 NMR of the novel cyclic trypsin inhibitor SFTI-1 from sunflower seeds and an acyclic permutant

SFTI-1 is a recently discovered cyclic peptide trypsin inhibitor from sunflower seeds comprising 14 amino acid residues. It is the most potent known Bowman-Birk inhibitor and the only naturally occurring cyclic one. The solution structure of SFTI-1 has been determined by H-1-NMR spectroscopy and compared with a synthetic acyclic permutant. The solution structures of both are remarkably similar. The lowest energy structures from each family of 20 structures of cyclic and acyclic SFTI-1 have an rmsd over the backbone and heavy atoms of 0.29 Angstrom and 0.66 Angstrom, respectively. The structures consist of two short antiparallel beta -strands joined by an extended loop containing the active site at one end. Cyclic SFTI-1 also has a hairpin turn completing the cycle. Both molecules contain particularly stable arrangements of cross-linking hydrogen bonds between the beta -strands and a single disulfide bridge, making them rigid and well defined in solution. These stable arrangements allow both the cyclic and acyclic variants of SFTI-1 to inhibit trypsin with very high potencies (0.5 nM and 12.1 nM, respectively). The cyclic nature of SFTI-1 appears to have evolved to provide higher trypsin inhibition as well as higher stability. The solution structures are similar to the crystal structure of the cyclic inhibitor in complex with trypsin. The lack of a major conformational change upon binding suggests that the structure of SFTI-1 is rigid and already pre-organized for maximal binding due to minimization of entropic losses compared to a more flexible ligand. These properties make SFTI-1 an ideal platform for the design of small peptidic pharmaceuticals or pesticides. (C) 2001 Academic Press.

Anthropogenic and Natural Organohalogen Compounds in Blubber of Dolphins and Dugongs (Dugong dugon) from Northeastern Australia

A range of organohalogen compounds (10 polychlorinated biphenyl [PCB] congeners, DDT and metabolites, chlordane-related compounds, the potential natural organochlorine compound Q1, toxaphene, hexachlorobenzene, hexachlorocyclohexanes, dieldrin, and several yet unidentified brominated compounds) were detected in the blubber of four bottlenose dolphins (Tursiops truncatus), one common dolphin (Delphinus delphis), and seven dugongs (Dugong dugon), as well as in adipose tissue of a green turtle (Chelonia mydas) and a python (Morelia spilota) from northeast Queensland (Australia). The green turtle and dugongs accumulated lower organohalogen levels than the dolphins. Lower levels in dugongs were expected because this species is exclusively herbivorous. Highest PCB and DDT levels recorded in dugongs were 209 and 173 mug/kg lipids, respectively. Levels of the nonanthropogenic heptachlorinated compound Q1 (highest level in dugongs was 160 mug/kg lipids) were estimated using the ECD response factor of trans-nonachlor. Highest organohalogen levels were found in blubber of dolphins for sumDDT (575-52,500 mug/kg) and PCBs (600-25,500 mug/kg lipids). Furthermore, Q1 was a major organohalogen detected in all samples analyzed, ranging from 450 -9,100 mug/kg lipids. The highest concentration of Q1 determined in this study represents the highest concentration reported to date in an environmental sample. Levels of chlordane-related compounds were also high (280-7,700 mug/kg, mainly derived from trans-nonachlor), but concentrations of hexachlorobenzene, hexachlorocyclohexanes, dieldrin, and toxaphene were relatively low and contributed little to the overall organohalogen contamination. Furthermore, a series of three major (BC-1, BC-2, and BC-3) and six minor (BC-4 through BC-9) unknown brominated compounds were observable by extracting m/z 79 and m/z 81 from the GC/ECNI-MS full scan run. Structural proposals were made for the two major recalcitrant compounds (referred to as BC-1 and BC-2). BC-2 appears to be a tetrabromo-methoxy-diphenylether (512 u) and BC-1 has 14 u (corresponding with an additional CH2 group) more relative to BC-1. In general the organohalogen pattern observed in blubber of dolphins was different compared to similar samples from other locations in the world, which is apparent from the fact that the four major abundant signals in the GC/ECD chromatogram. of D. delphis originated from the four unknown compounds Q1, BC-1, BC-2, and BC-3.

Historical PCDD Inputs and Their Source Implications from Dated Sediment Cores in Queensland (Australia)

Recent investigations have demonstrated the presence of an unidentified source of polychlorinated dibenzo-p-dioxins (PCDDs) in the coastal zone of Queensland (Australia). The present study provides new information on the possible PCDD sources and their temporal input to this environment. Two estuarine sediment cores were collected in northern Queensland for which radiochemical chronologies were established. Core sections from different depositional ages, up to three centuries, have been analyzed for 2,3,7,8-substituted PCDDs and polychlorinated dibenzofurans (PCDFs). Variations of PCDD concentrations in the sediment cores over several centuries of depositional history were relatively small, and elevated PCDD levels were still present in sediment slices from the early 17th century. PCDD/F isomer patterns and congener profiles in sediments deposited during the last 350 years were almost identical and correlated well to the characteristic profiles observed in surface sediments and soils from the entire Queensland coastline. Profiles were dominated by higher chlorinated PCDDs, in particular octachlorodibenzodioxin (OCDD), whereas PCDF concentrations were below or near the limit of detection. These results indicate the presence of a PCDD source prior to industrialization and production of commercial organochlorine products. Further, the present study demonstrates that PCDD input patterns have been similar along an extensive but localized area over at least several centuries, contributing relatively high concentrations of PCDDs to the coastal system of Queensland.

Schistosoma japonicum cathepsin D aspartic protease cleaves human IgG and other serum components

Recombinant cathepsin D aspartic protease of Schistosoma japonicum cleaved human IgG in vitro in a time and dose-dependent manner. Optimal cleavage was seen at pH 3.6-4.5; modest cleavage remained at pH 5.0, and no cleavage was detected above pH 5.0. Amino terminal sequencing of the major cleavage fragments of human IgG identified a Fab fragment from the VH1 domain, and 2 cleavage sites in the CH2 domain below the hinge region. The P1 and P1' residues at the 2 CH2 cleavage sites were Phe254-Leu255 and Leu325-Thr326, indicating a preference by the schistosome protease for bulky hydrophobic residues flanking the scissile bond. No cleavage of the immunoglobulin light chain was detected. In addition, the recombinant schistosome protease indiscriminately degraded the human serum proteins complement C3 and serum albumin into numerous small fragments. These results demonstrate specific cleavage of human IgG by the recombinant schistosome aspartic protease, and highlight the broad range digestive specificity of the enzyme which may play a role in the degradation of host serum proteins ingested as part of the schistosome bloodmeal.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites - In vivo assessment using phospho-specific antibodies

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1·Mre11·Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

Alpha-conotoxins: Nicotinic acetylcholine receptor antagonists as pharmacological tools and potential drug leads

Alpha-Conotoxins are small disulfide rich peptides from the venoms of marine cone snails. They target specific nicotinic acetylcholine receptor (nAChR) subtypes with high affinity and potency and are therefore valuable as neurophamacological probes and potential drug leads. This article gives a general overview of the chemical and biological features of alpha -conotoxins, including their pharmacology, binding interactions and structure. A detailed analysis of recently reported three-dimensional structures from members of different subfamilies of the alpha -conotoxins, including those with 3/5, 4/3, 4/6 and 4.7 spacings of their two intracysteine loops is given. The structures are generally well defined and represent useful frameworks for the display of amino acid residues to target molecules.