The genus Theissenia: T. pyrenocrata, T. cinerea sp. nov., and T. eurima sp. nov

The diagnosis of Theissenia is emended to include taxa that lack a definite central perithecial columella and taxa that feature ascospores with a germ slit. Theissenia cinerea is described as a new species that lacks a perithecial columella; T. eurima is described as a new species with ascospores having a germ slit. The type species, T. pyrenocrata, is redescribed and compared with the new species. The taxonomic position of Theissenia is discussed.     

Segmentation and the pairing hypothesis

The effect of stimulus contiguity and response contingency on responding in chain schedules was examined in two experiments. In Experiment 1, four pigeons were trained on two simple three-link chain schedules that alternated within sessions. Initial links were correlated with a variable-interval 30s schedule, and middle and terminal links were correlated with interdependent variable-interval 30s variable-interval 30s schedules. The combined duration of the interdependent schedules summed to 60s. The two chains differed with respect to signaling of the schedule components: a two-stimulus chain had one stimulus paired with the initial link and one stimulus paired with both the middle and the terminal link, while a three-stimulus chain had a different stimulus paired with the each of the three links. The results showed that the two-stimulus chain maintained lower initial-link responding than the three-stimulus chain. In Experiment 2, four pigeons were exposed to three separate conditions, the two- and three-stimulus chains of Experiment 1 and a three-stimulus chain that had a 3s delay to terminal-link entry from the middle-link response that produced it. The two-stimulus chain maintained lower initial-link responding than the three-stimulus chain, as in Experiment 1, and a similar initial-link responding was maintained by the two-stimulus chain and the three-stimulus chain with the delay contingency. The results demonstrate that a stimulus noncontiguous with food can maintain responding that is sometimes greater than a stimulus contiguous with food, depending on the response contingency for terminal-link entry. The results are contrary to the pairing hypothesis of conditioned reinforcement.     

Structure of replicating DNA molecules of Bacillus subtilis bacteriophage phi 29.

We isolated phi 29 DNA replicative intermediates from extracts of phage-infected Bacillus subtilis, pulsed-labeled with [3H]thymidine, by velocity sedimentation in neutral sucrose followed by CsCl equilibrium density gradient centrifugation. During a chase, the DNA with a higher sedimentation coefficient in neutral sucrose and a lower sedimentation rate in alkaline sucrose than that of viral phi 29 DNA was converted into mature DNA. The material with a density higher than that of mature phi 29 DNA consisted of replicative intermediates, as analyzed with an electron microscope. We found two major types of molecules. One consisted of unit-length duplex DNA with one single-stranded branch at a random position. The length of the single-stranded branches was similar to that of one of the double-stranded regions. The other type of molecules was unit-length DNA with one double-stranded region and one single-stranded region extending a variable distance from one end. Partial denaturation of the latter molecules showed that replication was initiated with a similar frequency from either DNA end. These findings suggest that phi 29 DNA replication occurs by a mechanism of strand displacement and that replication starts non-simultaneously from either DNA end, as in the case of adenovirus.     

Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipoproteins of dimyristoylphosphatidylcholine

The structure of the exchangeable apolipoprotein, apolipophorin-III from Locusta migratoria, apoLp-III, is described as a bundle of five amphipathic alpha-helices. To study the interaction of each of the helices of apoLp-III with a lipid surface, we designed five single-Trp mutants, each containing a Trp residue in a different alpha-helix. The Trp residues were located in the nonpolar domains of the amphipathic alpha-helices. The kinetics of the spontaneous interaction of the mutants with dimyristoylphosphatidylcholine (DMPC) indicated that all mutants behaved as typical exchangeable apolipoproteins. Circular dichroism in the far-UV indicated that all proteins have a high and similar helical content in the lipid-bound state. The interaction of the Trp residues with the lipid surface was investigated in recombinant lipoprotein particles made with DMPC. The properties of the Trp residues were investigated by fluorescence spectroscopy. These studies showed major changes in the spectroscopic properties of the Trp residues upon binding to lipid. These changes are observed with all single-Trp mutants, indicating that a major conformational change, which affects the properties of all helices, takes place upon binding to lipid. The position of the fluorescence maximum, the quenching efficiency of acrylamide as determined by steady-state and time-resolved fluorescence, and the fluorescence lifetimes of the single-Trp mutants suggest that helices 1, 4, and 5 interact with the nonpolar domains of the lipid. The properties of the Trp in helices 2 and 3 suggest that these helices adopt a different binding configuration than helices 1, 4, and 5. Helices 2 and 3 appear to be interacting with the polar headgroups of the phospholipids or constitute a different domain that does not interact with the lipid surface.     

Guilt by association: the nuclear envelope proteome and disease

The discovery that many inherited diseases are linked to interacting nuclear envelope proteins has raised the possibility that human genetic studies could be assisted by a fusion with proteomics. Two principles could be applied. In the first, the proteome of an organelle associated with a genetically variable disease is determined. The chromosomal locations of the genes encoding the organellar proteins are then determined. If a related disease is linked to a large chromosomal region that includes a gene identified in the organelle, then that gene has an increased likelihood of causing the disease. Directly sequencing this allele from patient samples might speed identification compared with further genetic linkage studies as has been demonstrated for multiple diseases associated with the nuclear envelope. The second principle is that if an organelle has been implicated in the pathology of a particular disorder, then comparison of the organelle proteome from control and patient cells might highlight differences that could indicate the causative protein. The distinct, tissue-specific pathologies associated with nuclear envelope diseases suggest that many tissues will have a set of disorders linked to this organelle, and there are numerous as yet unmapped or partially mapped syndromes that could benefit from such an approach.     

Changes in DNase and DNA-polymerase activities in different tissues of chick with age.

The presence of acid and alkaline DNases in nuclei of chick brain cells has been demonstrated. The activities of these two DNases along with those of DNA-polymerases were assessed in chicken tissues with known varied cell proliferative capacities (eg., spleen, kidney and brain) at different ages. The results indicate that the acid and alkaline DNases are probably the 'house keeping' enzymes with a constitutive role in DNA repair process, the former with a DNA-repair process that is linked to cell proliferation (DNA synthesis) while the latter with the basal DNA-repair operations that must go on at all times without any regard to the cell division process. Chicken brain, unlike that of rat, possesses significant levels of aphidicolin sensitive DNA-polymerase(s) that are considered to be more replication oriented enzymes suggesting that the replication potential of adult and old avian brain cells may be different from that of a mammalian brain.     

Myeloperoxidase of the leukocyte of normal blood. Nature of the prosthetic group of myeloperoxidase

The absorption spectra of alkaline pyridine hemochrome of myeloperoxidase in its native, acid, and modified forms were similar to those of heme a, and the molar extinction coefficient of myeloperoxidase heme was very similar to that of heme a, assuming that myeloperoxidase contains only one heme. The anaerobic titration of myeloperoxidase with dithionite showed that one electron was consumed per molecule of the enzyme for its conversion to its reduced form. The EPR spectrum of myeloperoxidase indicated that the enzyme contains both high-spin heme and non-heme iron. Carbonyl reagents, such as borohydride, hydrazine, and benzhydrazide, reacted with myeloperoxidase, causing blue shifts in its absorption spectrum. The heme was labeled with a tritium of boro[3H]hydride, suggesting that the reagents reacted with a formyl group on the porphyrin ring of the myeloperoxidase heme. When hydrazine was added to cyanide complex I of myeloperoxidase the complex was converted to the hydrazine-enzyme compound. Myeloperoxidase reacted with bisulfite to form a compound with an absorption spectrum similar to that of cyanide complex I. Borohydride-treated myeloperoxidase formed only one cyanide complex, while the native enzyme formed two different cyanide complexes, I (Kd = 0.3 muM) and II (approximate Kd = 0.1 mM). The EPR spectrum indicated that cyanide complex I of myeloperoxidase still contained high-spin heme. The results suggested that cyanide complex I and the bisulfite compound of myeloperoxidase were adducts between the nucleophilic reagents and the formyl group of myeloperoxidase heme. Based on these results, we concluded that one of the two iron atoms in a myeloperoxidase molecule exists in a formyl-heme moiety similar to heme a and the other exists as a non-heme iron.     

Redox regulation of yeast flavin-containing monooxygenase

The flavin-dependent monooxygenase from yeast (yFMO) oxidizes biological thiols such as cysteine, cysteamine, and glutathione. The enzyme makes a major contribution to the pools of oxidized thiols that, together with reduced glutathione from glutathione reductase, create the optimum cellular redox environment. We show that the activity of yFMO, as a soluble enzyme or in association with the ER membrane of microsomal fractions, is correlated with the redox potential. The enzyme is active under conditions normally found in the cytoplasm, but is inhibited as GSSG accumulates to give a redox potential similar to that found in the lumen of the ER. Site-directed mutations show that Cys 353 and Cys 339 participate in the redox regulation. Cys 353 is the principal residue in the redox-sensitive switch. We hypothesize that it may initiate formation of a mixed disulfide that is partially inhibitory to yFMO. The mixed disulfide may exchange with Cys 339 to form an intramolecular disulfide bond that is fully inhibitory.     

Molecular characterization of the ZKT gene encoding a protein with PDZ, K-Box, and TPR motifs in Arabidopsis

The ZKT gene from Arabidopsis encodes a polypeptide of 335 amino acid residues, with a calculated molecular mass of 37.4 kDa. ZKT is a member of a novel protein family present in the plant kingdom, which contains a PDZ, a K-box, and a TPR motif. A BLAST search indicated that the ZKT gene is a single gene in Arabidopsis and that ZKT homologs are present in soybean and rice but not in animals. The level of ZKT mRNA decreased after wounding. Antisera from rabbit immunized with recommbinant ZKT protein recognized a protein of 37 kDa in Arabidopsis. Western analysis with anti-ZKT antibody indicated that the level of ZKT protein does not change after wounding. The ZKT protein has consensus sequence motifs for phosphorylation. Immunoprecipitation with anti-ZKT antibody and western analysis with anti-phosphoamino acid antibody indicated that the ZKT protein is phosophorylated at the threonine and serine residues after wounding. These results suggest that the ZKT protein may act as a molecular adaptor regulated by phosphorylation in wound responses.     

Cloning and nucleotide sequence of the chlD locus

The nucleotide sequence of a Sau3A1 restriction nuclease fragment that complemented an Escherichia coli chlD::Mu cts mutant strain was determined. DNA and deduced amino acid sequence analysis revealed two open reading frames (ORFs) that potentially codes for proteins with amino acid sequence homology with binding protein-dependent transport systems. One of the ORFs showed a sequence that encoded a protein with properties that were characteristic of a hydrophobic inner membrane protein. The other ORF, which was responsible for complementing a chlD mutant, encoded a protein with conserved sequences in nucleotide-binding proteins and hydrophilic inner membrane proteins in active transport systems. A proposal that the chlD locus is the molybdate transport operon is discussed in terms of the chlD phenotype.     

用RACE结合cDNA文库筛选的方法获取新的锌指蛋白基因

大多数有重要功能的蛋白质都含相应的由保守氨基酸顺序组成的功能结构域。本文首先根据蛋白质功能结构域保守氨基酸序列设计简并引物,用PCR方法扩增出基因EST序列,再利用改进的快速扩增cDNA末端（RACE）方法从cDNA文库中扩增出基因非同源部位,然后以非同源序列为探针,筛选cDNA文库。利用此方法成功地从人骨髓cDNA文库中克隆到几个编码锌指蛋白并代表原有EST的新的全长cDNA。这一策略也应适用于筛选编码具有其他序列保守性功能结构域蛋白的基因。 Abstract:Most of the important functionally proteins contain the corresponding function domains that consist of conserved amino acid sequences.The study provided a method to identify novel genes that encode proteins containing important functionally domains with conserved sequences.First,primers were designed according to the sequence of the cDNA library vector and the ESTs that have been obtained by reverse PCR and degenerate primers encoding Zinc finger domain.The cDNA library DNA was used as template for PCR amplification.The amplified fragment that contains nonhomologous sequences of the cDNA was inserted into pGEM-T easy vector.The fragment was recovered and used as a probe for screening the cDNA library.Several cDNAs with full length that encode proteins with Zinc finger domain and represent the original ESTs have been successfully cloned from a human bone marrow cDNA library.This strategy can also be used in screening genes that encode proteins containing differential function domains with conserved sequences.     

N-acetoxy-2-acetylaminofluorene modification of a deoxyoligonucleotide duplex.

The carcinogen N-acetoxy-2-acetylaminofluorene was reacted with d (CCACGCACC) to form a covalent adduct with attachment at the single guanine. The sample was purified, mixed 1:1 with d (GGTGCGTGG) and studied by thermal denaturation experiments. The Tm for the mixture was 35 +/- 3 degrees C, consistent with duplex formation. The method of continuous variation shows that the modified oligomer, d (CCACGAAFCACC), forms a 1:1 duplex with d (GGTGCGTGG). Circular dichroism spectra also indicate the formation of a duplex and suggest that the modified duplex has a left-handed conformation. Addition of the intercalating drug ethidium alters the CD spectrum of the modified duplex, resulting in a CD spectrum similar to that of ethidium bound to right-handed DNA.     

The sense of consciousness

I propose that consciousness might be understood as the property of a system that functions as a sense in the biological meaning of that term. The theory assumes that, as a complex system, the sense of consciousness is not a fixed structure but implies structure with variations and that it evolved, as many new functions do, through the integration of simpler systems. The recognized exteroceptive and enteroceptive senses provide information about the organism's environment and about the organism itself that are important to adaptation. The sense of consciousness provides information about the brain and thus about the organism and its environment. It senses other senses and processes in the brain, selecting and relating components into a form that "makes sense"-where making sense is defined as being useful to the organism in its adaptation to the environment. The theory argues that this highly adaptive organizing function evolved with the growing complexity of the brain and that it might have helped resolve discrepancies created at earlier stages. Neural energies in the brain that are the input to the sense of consciousness, along with the processing subsystem of which they are a part, constitute the base of consciousness. Consciousness itself is an emergent effect of an organizing process achieved through the sense of consciousness. The sense of consciousness thus serves an organizing function although it is not the only means of organization in the brain. Its uniqueness lies in the character of the organization it creates with consciousness as a property of that organization. The paper relates the theory to several general conceptions-interactionism, epiphenomenalism and identity theory-and illustrates a number of testable hypotheses. Viewing consciousness as a property of a sense provides a degree of conceptual integration. Much of what we know about the evolution and role of the conventionally recognized senses should help us understand the evolution and role of the sense of consciousness, and of consciousness itself.     

Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon)

We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection.