Characterization and conservation of the inner E2 core domain structure of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Construction of a cDNA encoding the entire transacylase (E2b) precursor

A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex has been constructed from two overlapping incomplete cDNA clones which were isolated from a lambda ZAP library prepared from bovine liver poly(A)+ RNA. Nucleotide sequencing indicates that this bovine E2b cDNA insert (bE2-11) is 2701 base pairs in length with an open reading frame of 1446 base pairs. The bE2-11 cDNA insert encodes a leader peptide of 61 residues and a mature E2b polypeptide of 421 amino acid residues with a calculated monomeric molecular mass of 46,518 daltons. The molecular mass of the native E2b component isolated from bovine liver is 1,110,000 daltons as determined by sedimentation equilibrium. This value establishes the 24-subunit octahedral model for the quaternary structure of bovine E2b. The amino-terminal sequences of two tryptic fragments (A and B) of the E2b protein have been determined. Fragment A comprises residues 175 to 421 of the E2b protein and is the inner E2 core domain which contains the transacylase active site. Fragment B, produced by further tryptic cleavage of fragment, comprises residues 205 to 421, but does not have transacylase activity. Both fragments A and B confer the highly assembled 24-mer structure. The primary structure of the inner E2 core domain of bovine E2b (fragment A) is very similar to those of three other E2 proteins (human E2p, Escherichia coli E2p, and E. coli E2k). These similarities suggest that these E2 proteins are structurally and evolutionarily related.     

Resonance Raman spectra of horseradish peroxidase and bovine liver catalase compound I species. Evidence for predominant 2A2u pi-cation radical ground state configurations.

The nature of the porphyrin pi-cation radicals in the horseradish peroxidase and bovine liver catalase (BLC) compound I species have been investigated by studying their resonance Raman spectra. A variety of laser excitation and sample interrogation procedures have been employed in order to minimize previously documented problems arising from photoinduced conversions. With Soret band excitation, the spectra obtained for both species resemble that of a compound II-like photoproduct unless the samples are excited with residence times in the microsecond regime with very low (approximately 1 milliwatt) powers. When these precautions are taken, spectra attributable to the compound I species themselves are obtained. The spectrum for horseradish peroxidase compound I is similar to that reported by Paeng and Kincaid (Paeng, K.-J., and Kincaid, J. R. (1988) Am. Chem. Soc. 110, 7913-7915) using a similar approach. Both horseradish peroxidase and BLC compound I exhibit frequency shifts relative to their compound II species that are in the direction observed for model pi-cation radicals with predominant 2A2u character. The magnitudes of these shifts are smaller than those observed for heme models that lack aromatic axial ligands, but agree well with those observed on formation of the compound I analog of N alpha-acetyl microperoxidase-8 that has His as a proximal ligand. This observation is consistent with partial delocalization of the radical density onto the proximal His-170 and Tyr-357 ligands in horseradish peroxidase and BLC, respectively. The strong ligand field provided by these ligands on the proximal side and oxo ligand on the distal side of the heme group is apparently sufficient to reverse the 2A1u radical ground state preference observed for heme-like porphyrin species (e.g. octaethylporphyrins) with weak axial fields. Enhancement of several bands assigned to the Tyr-357 ligand has also been observed for BLC compound I with 406.7-nm excitation. This is attributed either to resonance with a tyrosinate----Fe(IV) charge transfer band or to the coupling provided by radical spin delocalization onto the tyrosinate ligand.     

Conformational properties of oxytocin in dimethyl sulfoxide solution: NMR and restrained molecular dynamics studies.

The conformation of oxytocin, the neurohypophyseal nonapeptide hormone, in solution in deuterated dimethyl sulfoxide has been determined by 1H-nmr. The structural determination is based on the experimental data set of nuclear Overhauser effect restraints. Obtained after the restrained molecular dynamics simulation on an initial structure of extended conformation, five resultant structures satisfy the experimental restraints well. These structures resemble that of the crystal structure of deamino-oxytocin, an analogue of oxytocin, in terms of a close correlation observed both at two beta-turn regions of the 20-membered tocin ring and at the tripeptide tail end. Based on this comparison and analysis of restrained molecular dynamics trajectories, we found that, although the turns are stabilized by the formation of hydrogen bonds, the oxytocin molecule possesses a slight twist in DMSO solution relative to the orientation of deamino-oxytocin in the crystalline state. Analyses of oxytocin conformation indicate that the tripeptide tail is more flexible than the tocin ring.     

A 17-bp insertion and a Phe215----Cys missense mutation in the dihydrolipoyl transacylase (E2) mRNA from a thiamine-responsive maple syrup urine disease patient WG-34.

We have amplified the cDNA for the transacylase (E2) subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex from a thiamine-responsive MSUD cell line (WG-34) by the polymerase chain reaction. Sequencing of the amplified WG-34 cDNA showed a 17-bp insertion (AAATACCTTGTTACCAG) apparently resulting from an aberrant splicing of the E2 gene, and a missense (T----G) mutation that changes Phe215 to Cys in the E2 subunit. The existence of these two mutations was confirmed by probing the amplified E2 cDNA or genomic DNA with allele-specific oligonucleotides. The above results support the thesis that the thiamine-responsive MSUD patient (WG-34) is a compound heterozygote at the E2 locus. The implication of the E2 mutations for the thiamine-responsiveness observed in this patient is discussed.     

广东连州田心省级自然保护区珍稀濒危植物资源现状及优先保护初步研究

掌握保护区珍稀植物本底与受威胁状态,完善保护区珍稀植物的优先保护评价体系,可为保护区制定保护对策、科学管理提供依据。通过资料收集和实地考察,对连州田心省级自然保护区珍稀植物资源开展研究,结果表明,田心省级自然保护区共有珍稀植物35科63属94种,其中国家重点保护野生植物有25科33属46种,广东省重点保护野生植物有7科10属10种,中国植物红皮书稀有濒危植物20科25属25种,CITES公约附录植物5科23属46种。采用优先保护评价体系对41种代表性珍稀濒危植物进行排序,结果属Ⅰ级保护的有2种,占评价总数的4.88%;属Ⅱ级保护的有22种,占评价总数的53.66%;属Ⅲ级保护的有17种,占评价总数的41.46%。田心省级自然保护区生境良好,珍稀濒危植物种类多,数量丰富,具有较高的生态保护价值。该评价体系对41种植物的评价与分级结果更真实地反映了它们在田心保护区受威胁状态和优先保护顺序,对该区珍稀濒危植物的优先保护工作具有一定指导意义。     

Regulatory BC1 RNA and the fragile X mental retardation protein: convergent functionality in brain

Background

BC RNAs and the fragile X mental retardation protein (FMRP) are translational repressors that have been implicated in the control of local protein synthesis at the synapse. Work with BC1 and Fmr1 animal models has revealed that phenotypical consequences resulting from the absence of either BC1 RNA or FMRP are remarkably similar. To establish functional interactions between BC1 RNA and FMRP is important for our understanding of how local protein synthesis regulates neuronal excitability.

Methodology/Principal Findings

We generated BC1−/− Fmr1−/− double knockout (dKO) mice. We examined such animals, lacking both BC1 RNA and FMRP, in comparison with single knockout (sKO) animals lacking either one repressor. Analysis of neural phenotypical output revealed that at least three attributes of brain functionality are subject to control by both BC1 RNA and FMRP: neuronal network excitability, epileptogenesis, and place learning. The severity of CA3 pyramidal cell hyperexcitability was significantly higher in BC1−/− Fmr1−/− dKO preparations than in the respective sKO preparations, as was seizure susceptibility of BC1−/− Fmr1−/− dKO animals in response to auditory stimulation. In place learning, BC1−/− Fmr1−/− dKO animals were severely impaired, in contrast to BC1−/− or Fmr1−/− sKO animals which exhibited only mild deficits.

Conclusions/Significance

Our data indicate that BC1 RNA and FMRP operate in sequential-independent fashion. They suggest that the molecular interplay between two translational repressors directly impacts brain functionality.     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.