Ancient conserved domain protein-1 binds copper and modifies its retention in cells

The ancient conserved domain protein (ACDP) family are a recently identified group of homologous mammalian proteins. Some family members have been suggested to have roles in the metabolism of metals. We investigated the capacity of ACDP-1 to bind metals. Using immobilised metal affinity chromatography and isothermal titration calorimetry we determined that ACDP-1 is a high affinity copper binding protein able to bind copper at nanomolar concentrations. In addition the promoter of ACDP-1 contains metal response elements and the cellular expression of ACDP-1 alters cellular retention of copper. However, cellular expression of ACDP-1 does not alter cellular resistance to the toxicity of copper or other metals. As our findings place the subcellular localisation of ACDP-1 in the cytoplasm it is possible that ACDP-1 represent a novel copper chaperone or storage protein.     

Structural requirements of the RNA precursor for the biosynthesis of the branched RNA-linked multicopy single-stranded DNA of Myxococcus xanthus

A precursor RNA molecule (pre-msdRNA) of approximately 375 bases is considered to form a stable secondary structure which serves as a primer as well as a template to synthesize the branched RNA-linked multicopy single-stranded DNA (msDNA) of Myxococcus xanthus. When 3-base mismatches were introduced into the stem structure immediately upstream of the branched rG residue to which msDNA is linked by a 2',5'-phosphodiester linkage, the production of msDNA was almost completely blocked. However, if additional 3-base substitutions were made on the other strand to resume the complementary base pairing, msDNA production was restored, being consistent with the proposed model of msDNA synthesis. We also found that the branched rG residue of pre-msdRNA could not be replaced with either rC or rA, while the 5' end (dC) of msDNA which is linked to the branched rG could be substituted with a dG residue. Together with several other mutations, the structural requirements of pre-msdRNA are discussed with respect to the mechanism of msDNA biosynthesis.     

Identification of 10-methylsphinganine in cerebrosides of the guinea pig harderian gland

A large amount of branched long chain bases was detected in the cerebrosides of guinea pig Harderian gland. The long chain bases of cerebrosides were analyzed by GLC as trimethylsilyl derivatives. The branched long chain bases were separated into four peaks (I, II, III, IV) according to the number of carbon atoms and the position of branching. In the present work, the structures of long chain bases in the four peaks were analyzed by GLC and GC-MS after conversion of them to aldehydes, alcohols, and fatty acids. Furthermore the main component of long chain bases (Peak II) was isolated by HPLC as N-acetyl derivatives and analyzed by NMR. The structures of branched long chain bases in Peaks I, II, III, and IV are as follows. Branched long chain bases of Peak I are 2-amino-10- (main component), 2-amino-9-, and 2-amino-8-methylhexadecane-1,3-diol. Branched long chain bases of Peak II also consist of a mixture of 2-amino-10-, 2-amino-9-, and 2-amino-8-methyl-heptadecane-1,3-diol. The branched long chain base of Peak III is 2-amino-10-methyl-octadecane-1,3-diol, while that of Peak IV is 2-amino-16-methyloctadecane-1,3-diol. Among these branched long chain bases, 10-methylsphinganines are dominant though the chain lengths are different. These branched long chain bases, in which the substituted positions exist in the middle part of aliphatic chain (10-, 9-, or 8-methylsphinganine) are novel long chain bases in mammals.     

Developmental patterning of carbohydrate antigens during early embryogenesis of the chick: expression of antigens of the poly-N-acetyllactosamine series

This report describes a striking temporal and spatial patterning of specific carbohydrate sequences in the developing chick embryo. By using oligosaccharide sequence-specific monoclonal antibodies as immunohistochemical reagents in conjunction with neuraminidase, it was possible to visualize the occurrence, as well as the changes in distribution, of oligosaccharides of the poly-N-acetyllactosamine series. These were (a) long-chain unbranched sequences reactive with anti-i Den, (b) long-chain branched sequences reactive with anti-I Step and (c) short-chain branched sequences reactive with anti-I Ma and (d) their sialylated forms. The salient observations with serial sections of embryos from the unincubated to the 17th stage were as follows. (1) A pronounced anteroposterior patterning appeared during neuroectodermal development, such that the long-chain unbranched and long-chain branched sequences, which were abundant on the ectoderm of the earlier stages, were replaced by short-chain branched sialo-oligosaccharides in the developing brain and anterior neural tube. (2) A striking anteroposterior and mediolateral patterning developed in the subectodermal extracellular spaces. The long-chain linear and short-chain non-sialylated sequences demarcated regions favourable for migration of the lateral plate mesoderm. (3) A distinction was made between the dorsal and ventral routes of the trunk neural crest in that the extracellular matrix of the dorsal route only was associated with long-chain linear and short-chain sialylated branched sequences. (4) A circumscribed perinotochordal distribution of the short-chain sialylated branched sequences was observed in the region of the future centra of the vertebrae. (5) An abundance of long-chain linear and long-chain sialylated branched structures was detected in primordial germ cells which permitted their identification during migration. These observations suggest that oligosaccharides of the poly-N-acetyllactosamine series may have roles as short-range, region-specific information factors during morphogenetic events that take place in the developing embryo, and they open the way to the search for recognition proteins (e.g. endogenous lectins) specific for each of these oligosaccharide structures.     

Evolution of a recombinant (gucoamylase-producing) strain of Fusarium venenatum A3/5 in chemostat culture

Fusarium venenatum JeRS 325 is a transformant of strain A3/5 which produces Aspergillus niger glucoamylase (GAM) under the control of a Fusarium oxysporum trypsin-like protease promoter. The evolution of JeRS 325 was studied in glucose-limited chemostat cultures grown on NaNO3 or (NH4)2SO4 as the nitrogen source. Thirteen mutants which were more highly branched and four mutants which were more sparsely branched than the parental strain were isolated from the NaNO3 chemostat. The highly branched mutants detected in this chemostat did not displace the sparsely branched population. The mutants isolated from the NaNO3 chemostat complemented representative strains previously isolated from glucose-limited chemostat cultures of F. venenatum A3/5 grown on (NH4)2SO4, but showed little complementation between themselves. By contrast, a highly branched mutant isolated from the (NH4)2SO4 chemostat culture displaced the sparsely branched mycelial population. None of the mutants isolated from the NaNO3 or (NH4)2SO4 chemostats produced as much GAM as JeRS 325. Southern blot analysis showed that all except one mutant had lost copies of both the glucoamylase and the acetamidase (the selectable marker) genes. However, specific GAM production was not necessarily correlated with the extent of glaA gene loss observed. Further, 10 of the mutants had lost the ability to grow on acetamide as the sole nitrogen source, although they retained copies of the amdS gene. In competition studies, mutants which could not utilize acetamide displaced mutants which could. The presence of foreign DNA in JeRS 325 resulted in a reduced specific growth rate (compared to A3/5), but the presence of the foreign DNA did not prevent the evolution of the strain or the isolation of mutants which had improved growth rates.     

Synthesis of 3'-3'-linked oligonucleotides branched by a pentaerythritol linker and the thermal stabilities of the triplexes with single-stranded DNA or RNA

Synthesis of 3'-3'-linked oligonucleotides branched by a pentaerythritol linker is described. The branched oligonucleotides were synthesized on a DNA/RNA synthesizer using a controlled pore glass (CPG) with a pentaerythritol linker carrying 4,4'-dimethoxytrityl (DMTr) and levulinyl (Lev) groups. The stability of the triplexes between the branched oligonucleotides and the target single-stranded DNA or RNA was studied by thermal denaturation. The oligonucleotides with the pentaerythritol linker formed thermally stable triplexes with the single-stranded DNA and RNA. Furthermore, the branched oligonucleotides containing 2'-O-methylribonucleosides, especially the oligonucleotide composed of 2'-deoxyribonucleosides and 2'-O-methylribonucleosides, stabilized the triplexes with the single-stranded DNA or RNA. Thus, the branched oligonucleotide containing 2'-O-methylribonucleosides may be a candidate for a novel antisense molecule by the triplex formation.     

Effects of linear and branched polyethylene glycol on PEGylation of recombinant hirudin: Reaction kinetics and in vitro and in vivo bioactivities

To improve the therapy efficacy of recombinant hirudin variant-2 (HV2), its PEGylation was investigated using linear mPEG-succinimidyl carbonate (mPEG-SC) and branched mPEG₂-N-hydroxysuccinimide (mPEG₂-NHS). The reaction mixtures of PEGylation were analyzed by RP-HPLC and the mono-PEG-HV2 products were purified by anion exchange chromatography (IEC). Effects of linear and branched PEG on the hydrolysis kinetics of the PEG reagent, the PEGylation kinetics of HV2 and the in vitro and in vivo bioactivity of mono-PEG-HV2 were investigated. The RP-HPLC and IEC analyses showed that linear and branched PEG-HV2 with identical molecular weight had different chromatographic behaviors. The reaction kinetics showed that branched mPEG₂-NHS displayed higher hydrolysis rate but lower PEGylation rates than linear mPEG-SC. Consequently, HV2 conjugated with mPEG₂-NHS required a greater molar ratio of PEG to HV2 than that of mPEG-SC to achieve the identically desired yield of mono-PEG-HV2. The in vitro and in vivo bioactivities of mono-PEG-HV2 showed that branched PEG-HV2 had higher therapeutic efficacy than linear PEG-HV2 with identical molecular weight. The in vivo bioactivity of mono-B-PEG40k-HV2 (mono-PEG-HV2 derived from 40 kDa branched mPEG₂-NHS) had a markedly longer duration in rabbits than did unmodified HV2, which showed its potential to be developed as a candidate antithrombotic drug.     

Studies on the subtrate specificity of the fatty acid 5-desaturase by use of methyl branched isomers of eicose-8,11,14-trienoic acid and the metabolism of these acids in rat liver.

Seven radioactive methyl branched isomers of eicosa-8,11,14-trienoic acid (20:3) in which the methyl branch was located at carbons 2, 5, 10, 13, 17, 18, and 19, were injected into rats to determine how methyl branching influenced desaturation at the 5-position when compared with the conversion of 20:3 to arachidonate and also to determine how the radioactive substrates and the respective desaturated products were incorporated into liver lipids. When the methyl branch was at positions 18 or 19 these isomers were desaturated to the same extent as 20:3 was converted to arachidonate. These two methyl branched isomers and their respective desaturation products were incorporated into total liver lipids, triacylglycerol (TG) and phosphatidyl choline (PC) in the same way as was 20:3 and arachidonate. When the methyl branch was at carbons 17 and 13 there was a decline in the amount of desaturated product produced. Negligible amounts of desaturated product were produced when the methyl branch was at positions 10 and 5. A small amount of desaturated product was produced when the methyl branch was at the 2-position. When the methyl branch was at positions 5, 10, 13 and 17 a greater percentage of the recovered radioactivity was found in the TG fractions and a smaller percentage was found in the PC fractions when compared with the radioactive distribution pattern obtained after injecting 20:3. Significant amounts of these four methyl branched isomers were esterified at the 1-position of PC.When the isomeric branched substrates were incubated with rat liver microsomes only the 19-methyl branched isomer was desaturated at a rate equal to that found for the conversion of 20:3 to arachidonate. As the methyl branch was moved towards the site of desaturation, the rate of desaturation at the 5-position declined showing that methyl branching between positions 2 through 18 alters the substrate so that these isomers are not desaturated as well as is 20:3.     

Leucine auxotrophy specifically alters the pattern of trichothecene production in a T-2 toxin-producing strain of Fusarium sporotrichioides

The biosynthetic pathway for trichothecenes in the filamentous fungus Fusarium sporotrichioides NRRL 3299 has been further characterized. Experiments using the techniques of mutational analysis and the incorporation of radiolabeled precursors indicated that leucine is a direct precursor to the isovalerate moiety present in the trichothecene, T-2 toxin. Analysis of trichothecene production in a UV-induced leucine auxotroph also revealed the existence of a branched biosynthetic pathway which results in the coproduction of T-2 toxin and the T-2 toxin analogs neosolaniol, 8-isobutyryl-neosolaniol, and 8-propionyl-neosolaniol. Leucine limitation imposed by the leucine auxotroph simultaneously led to underproduction of T-2 toxin and overproduction of these T-2 toxin analogs, which are produced in small amounts by the wild-type parent. Furthermore, it was shown that the ratio of T-2 toxin to T-2 toxin analogs produced by the leucine auxotroph can be modulated by the concentration of leucine in the medium. These results suggest that the four trichothecenes mentioned above are derived from a common intermediate and that there is competition for this intermediate among the branched pathways leading to these four cometabolites.     

Leucine auxotrophy specifically alters the pattern of trichothecene production in a T-2 toxin-producing strain of Fusarium sporotrichioides.

The biosynthetic pathway for trichothecenes in the filamentous fungus Fusarium sporotrichioides NRRL 3299 has been further characterized. Experiments using the techniques of mutational analysis and the incorporation of radiolabeled precursors indicated that leucine is a direct precursor to the isovalerate moiety present in the trichothecene, T-2 toxin. Analysis of trichothecene production in a UV-induced leucine auxotroph also revealed the existence of a branched biosynthetic pathway which results in the coproduction of T-2 toxin and the T-2 toxin analogs neosolaniol, 8-isobutyryl-neosolaniol, and 8-propionyl-neosolaniol. Leucine limitation imposed by the leucine auxotroph simultaneously led to underproduction of T-2 toxin and overproduction of these T-2 toxin analogs, which are produced in small amounts by the wild-type parent. Furthermore, it was shown that the ratio of T-2 toxin to T-2 toxin analogs produced by the leucine auxotroph can be modulated by the concentration of leucine in the medium. These results suggest that the four trichothecenes mentioned above are derived from a common intermediate and that there is competition for this intermediate among the branched pathways leading to these four cometabolites.     

Biosynthesis of C-labeled branched polysaccharides by pestalotiopsis from C-labeled glucoses and the mechanism of formation

Biosynthesis of branched glucan by Pestalotiopsis from media containing D-(1-¹³C)glucose, D-(2-¹³C)glucose, D-(4-¹³C)glucose, D-(6-¹³C)glucose or a mixture of D-(1-¹³C)glucose and D-(2-¹³C)glucose was carried out to elucidate biosynthetic mechanism of branched polysaccharides. ¹³C NMR spectra of the labeled polysaccharides were determined and assigned. Analysis of ¹³C NMR spectra of glucitol acetates obtained from hydrolysates of the labeled branched polysaccharides indicated that transfer of labeling from C-1 to C-3 and C-6 carbons, from C-2 to C-1, C-3 and C-5 carbons, and from C-6 to C-1 carbon. From the results the percentages of routes via which the polysaccharide is biosynthesized are estimated. They show that the biosynthesis of the polysaccharide via the Embden-Meyerhof pathway and that from lipids and proteins are more active, and the pentose cycle is less active, than in the biosynthesis of cellulose and curdlan. As for the results, labeling at C-6 carbon in the branched polysaccharide cultured from D-(6-¹³C)glucose was low, compared to that of cellulose and curdlan.     

Inhibition of pre-mRNA splicing by synthetic branched nucleic acids

The cellular transformation of a precursor mRNA (pre-mRNA) into its mature or functional form proceeds by way of a splicing reaction, in which the exons are ligated to form the mature linear RNA and the introns are excised as branched or lariat RNAs. We have prepared a series of branched compounds (bRNA and bDNA), and studied the effects of such molecules on the efficiency of mammalian pre-mRNA splicing in vitro. Y-shaped RNAs containing an unnatural L-2'-deoxycytidine unit (L-dC) at the 3' termini are highly stabilized against exonuclease hydrolysis in HeLa nuclear extracts, and are potent inhibitors of the splicing pathway. A bRNA containing internal 2'-O-methyl ribopyrimidine units and L-dC at the 3' ends was at least twice as potent as the most potent of the bRNAs containing no 2' modifications, with an IC50 of approximately 5 micro M. Inhibitory activity was maintained in a branched molecule containing an arabino-adenosine branchpoint which, unlike the native bRNAs, resisted cleavage by the lariat- debranching enzyme. The data obtained suggest that binding and sequestering of a branch recognition factor by the branched nucleic acids is an early event, which occurs prior to the first chemical step of splicing. Probably, an early recognition element preferentially binds to the synthetic branched molecules over the native pre-mRNA. As such, synthetic bRNAs may prove to be invaluable tools for the purification and identification of the putative branchpoint recognition factor.     

Interactions with Actin Monomers,Actin Filaments,and Arp2/3 Complex Define the Roles of WASP Family Proteins and Cortactin in Coordinately Regulating Branched Actin Networks

Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.     

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.     

Crystal structure of MutS2 endonuclease domain and the mechanism of homologous recombination suppression

DNA recombination events need to be strictly regulated, because an increase in the recombinational frequency causes unfavorable alteration of genetic information. Recent studies revealed the existence of a novel anti-recombination enzyme, MutS2. However, the mechanism by which MutS2 inhibits homologous recombination has been unknown. Previously, we found that Thermus thermophilus MutS2 (ttMutS2) harbors an endonuclease activity and that this activity is confined to the C-terminal domain, whose amino acid sequence is widely conserved in a variety of proteins with unknown function from almost all organisms ranging from bacteria to man. In this study, we determined the crystal structure of the ttMutS2 endonuclease domain at 1.7-angstroms resolution, which resembles the structure of the DNase I-like catalytic domain of Escherichia coli RNase E, a sequence-nonspecific endonuclease. The N-terminal domain of ttMutS2, however, recognized branched DNA structures, including the Holliday junction and D-loop structure, a primary intermediate in homologous recombination. The full-length of ttMutS2 digested the branched DNA structures at the junction. These results indicate that ttMutS2 suppresses homologous recombination through a novel mechanism involving resolution of early intermediates.     

The size of the C-chain maltosaccharide of glycogen: evidence for the presence of only a single branch

Glycogen is found in mammals and yeast bound to glycogenin forming proteoglycogen. The branched polysaccharide is joined to the protein through the C-chain, a maltosaccharide considered to be 13 glucose units long and double branched as the other branched glycogen B-chains. We described before the isolation of c-glycogenin, the debranched C-chain bound to glycogenin, from muscle proteoglycogen. In this work, the size of the C-chain is analyzed for the first time. The maltosaccharide moiety of c-glycogenin was auto[14C]glucosylated by a short incubation with UDP-[14C]glucose, and the labeled maltosaccharide was released by heating in 2 M NaOH containing 0.1 M NaBH4 and analyzed by high-performance thin layer chromatography (HPTLC). The results indicate that the C-chain is about half the size of the B-chains, not long enough to be double branched.     

Curved helix segments can uniquely orient the topology of supertwisted DNA

To show that DNA containing sharp sequence-directed curvature can preferentially establish ends of supertwisted domains, a highly curved DNA from Crithidia fasciculata was cloned into two sites separated by 28% in pBR325. When this construct (pJGC2) was examined by electron microscopy, 63% of the supercoiled molecules were branched with three or more arms, and the remaining molecules appeared as linear interwound rods. The distance between the tips of two of the arms for the branched molecules measured within 2% of 28% of the DNA contour for 32% of the pJGC2 molecules, as contrasted with only 6.6% for the poorly branched pBR325 DNA. When one of the curved segments in pJGC2 was replaced by a highly curved fragment from SV40, similar results were obtained.     

Mismatch repair factor MSH2-MSH3 binds and alters the conformation of branched DNA structures predicted to form during genetic recombination

Genetic studies in Saccharomyces cerevisiae predict that the mismatch repair (MMR) factor MSH2-MSH3 binds and stabilizes branched recombination intermediates that form during single strand annealing and gene conversion. To test this model, we constructed a series of DNA substrates that are predicted to form during these recombination events. We show in an electrophoretic mobility shift assay that S. cerevisiae MSH2-MSH3 specifically binds branched DNA substrates containing 3' single-stranded DNA and that ATP stimulates its release from these substrates. Chemical footprinting analyses indicate that MSH2-MSH3 specifically binds at the double-strand/single-strand junction of branched substrates, alters its conformation and opens up the junction. Therefore, MSH2-MSH3 binding to its substrates creates a unique nucleoprotein structure that may signal downstream steps in repair that include interactions with MMR and nucleotide excision repair factors.