Structure of adsorbed fibrinogen obtained by scanning force microscopy

It is shown that scanning force microscopy (SFM), operated in the attractive mode, can be used to obtain high resolution pictures of adsorbed fibrinogen molecules on solid surfaces, without the need for staining or special microscope grids. SFM also reveals the three-dimensional structure of the adsorbed molecules. Two forms of adsorbed fibrinogen are demonstrated on hydrophobic silicone dioxide surfaces; a trinodular about 60 nm long and a globular with about a 40 nm diameter. Polymeric networks formed after storage of the surface with adsorbed fibrinogen in PBS for 11 days are also shown. The SFM-results for the trinodular structure suggest the existence of loops or peptide chains extending outside the basic structure of the fibrinogen molecule.     

Prebiotic sugar synthesis: Hexose and hydroxy acid synthesis from glyceraldehyde catalyzed by iron(III) hydroxide oxide

Summary Iron(III) hydroxide oxide [Fe(OH)O] efficiently catalyzed the condensation of 25 MM dl-glyceraldehyde to ketohexoses at 25°C (pH 5–6). At 16 days the yields were sorbose (15.2%), fructose (12.9%), psicose (6.1%), tagatose (5.6%), and dendroketose (2.5%) with 19.6% of triose unreacted. Analysis at 96 days showed no decomposition of hexoses. Under these conditions Fe(OH)O also catalyzed the isomerization and rearrangement of glyceraldehyde to dihydroxyacetone and lactic acid, respectively. In these reactions, about 10% of the glyceraldehyde was oxidized to glyceric acid with concurrent reduction of the iron(III) to iron(II). The partial reduction of Fe(OH)O did not noticeably reduce its ability to catalyze hexose synthesis. The relationship of these results to prebiotic sugar synthesis is discussed.     

The Benzodiazepine/GABA Receptor Complex: Molecular Size in Brain Synaptic Membranes and in Solution

Abstract: The molecular size of the benzodiazepine (BZ) receptor in the synaptic membrane of brain cortex (bovine or rat) was determined by an improved version of the radiation inactivation method to be 220,000. An identical size was found simultaneously for the associated γ-aminobutyric acid (GABA) receptor and for the component binding β-carboline esters. It is proposed that all three activities reside in a single protein or protein complex in the membrane. The size in solution, after extraction into Triton X-100 medium from exhaustively washed membranes, was estimated by sedimentation constant (9.4S) and by gel filtration (∼230,000 apparent MW), again with the BZ and GABA binding activities behaving identically. This size applies to the component that undergoes photoaffinity labelling by [³H]flunitrazepam in the membrane, and contains a 51,000 M_r polypeptide as the BZ-binding subunit. It is concluded that a protein complex or oligomer of 200,000–220,000 MW carries a class of BZ-binding sites and an associated class of GABA_A sites.     

The old REH theory remains unsatisfactory and the new REH theory is problematical - a reply to holmquist and jukes

Summary In response to criticism of REH theory (Fitch 1980), Holmquist and Jukes (1981) have mostly avoided the criticism or misunderstood it. Since they themselves state in their response that Amino acid sequence data alone cannot be used to estimate total nucleotide substitutions, they agree with the criticism. Most of their paper treats the newer theory (here designated as the REHN theory) which attempts to use the nucleotide sequences encoding proteins to better estimate total nucleotide substitutions (Holmquist and Pearl 1980). Since I made no criticism of REHN theory, their comments are frequently beside the point of my original criticism of REH theory. Nevertheless, it is shown here that REHN theory is also unsatisfactory in that: One, the varions are now more clearly defined but in such a way as to preclude the same codon from suffering a nucleotide substitution in more than one evolutionary interval. Two, the set of codons that accepts silent substitutions is identical to the set that accepts amino acid changing nucleotide substitutions. Three, the uncertainty in the REH estimate is considerable in that alternative excellent fits to the same observatuonal data may give alternative REH values that differ significantly even before stochastic variation and selective bias are considered. Four, the fit of their model to data is an irrelevancy where there are zero degrees of freedom.     

高通量细菌鉴定方法研究进展

高通量细菌鉴定是微生物领域的重要研究课题,对于疾病诊断和环境监测具有重要意义。相比传统的表型鉴定方法,分子遗传学鉴定方法具有稳定性高、检测周期短以及成本较低等特点,成为了主流的鉴定方法。特别是下一代DNA测序技术、核酸分子检测基础上的细菌检测芯片、质谱技术基础上的蛋白质图谱分析为高通量、快速、准确乃至定量的细菌鉴定提供了可行性方案。     

Diversity of an ectomycorrhizal fungal community studied by a root tip and total soil DNA approach

Molecular methods based on soil DNA extracts are increasingly being used to study the fungal diversity of ectomycorrhizal (EM) fungal communities in soil. Contrary to EM root tip identification, the use of molecular methods enables identification of extramatrical mycelia in soil. To compare fungal diversity as determined by root tip identification and mycelial identification, six soil samples were analysed. Root tips were extracted from the six samples and after amplification, the basidiomycete diversity on the root tips was analysed by denaturing gradient gel electrophoresis (DGGE). The soil from the six samples was sieved, total soil DNA was extracted and after amplification, the basidiomycete diversity in the soil fractions was analysed by DGGE. Fourteen different bands were excised from the DGGE gel and sequenced; fungal taxon names could be assigned to eight bands. Out of a total of 14 fungal taxa detected in soil, 11 fungal taxa were found on root tips, of which seven were EM fungal taxa. To examine whether the sieving treatment would affect EM species diversity, two different sieve mesh sizes were used and in addition, the organic soil fraction was analysed separately. DGGE analysis showed no differences in banding pattern for the different soil fractions. The organic fraction gave the highest DGGE band intensities. This work demonstrates that there is a high correspondence between basidiomycete diversity detected by molecular analysis of root tips and soil samples, irrespective of the soil fraction being analysed.     

Specific Chaperones for the Type VII Protein Secretion Pathway

Mycobacteria use the dedicated type VII protein secretion systems ESX-1 and ESX-5 to secrete virulence factors across their highly hydrophobic cell envelope. The substrates of these systems include the large mycobacterial PE and PPE protein families, which are named after their characteristic Pro-Glu and Pro-Pro-Glu motifs. Pathogenic mycobacteria secrete large numbers of PE/PPE proteins via the major export pathway, ESX-5. In addition, a few PE/PPE proteins have been shown to be exported by ESX-1. It is not known how ESX-1 and ESX-5 recognize their cognate PE/PPE substrates. In this work, we investigated the function of the cytosolic protein EspG(5), which is essential for ESX-5-mediated secretion in Mycobacterium marinum, but for which the role in secretion is not known. By performing protein co-purifications, we show that EspG(5) interacts with several PPE proteins and a PE/PPE complex that is secreted by ESX-5, but not with the unrelated ESX-5 substrate EsxN or with PE/PPE proteins secreted by ESX-1. Conversely, the ESX-1 paralogue EspG(1) interacted with a PE/PPE couple secreted by ESX-1, but not with PE/PPE substrates of ESX-5. Furthermore, structural analysis of the complex formed by EspG(5) and PE/PPE indicates that these proteins interact in a 1:1:1 ratio. In conclusion, our study shows that EspG(5) and EspG(1) interact specifically with PE/PPE proteins that are secreted via their own ESX systems and suggests that EspG proteins are specific chaperones for the type VII pathway.     

One-year survey of a single Micronesian reef reveals extraordinarily rich diversity of <Emphasis Type="Italic">Symbiodinium</Emphasis> types in soritid foraminifera

Recent molecular studies of symbiotic dinoflagellates (genus Symbiodinium) from a wide array of invertebrate hosts have revealed exceptional fine-scale symbiont diversity whose distribution among hosts, regions and environments exhibits significant biogeographic, ecological and evolutionary patterns. Here, similar molecular approaches using the internal transcribed spacer-2 (ITS-2) region were applied to investigate cryptic diversity in Symbiodinium inhabiting soritid foraminifera. Approximately 1,000 soritid specimens were collected and examined during a 12-month period over a 40 m depth gradient from a single reef in Guam, Micronesia. Out of 61 ITS-2 types distinguished, 46 were novel. Most types found are specific for soritid hosts, except for three types (C1, C15 and C19) that are common in metazoan hosts. The distribution of these symbionts was compared with the phylotype of their foraminiferal hosts, based on soritid small subunit ribosomal DNA sequences, and three new phylotypes of soritid hosts were identified based on these sequences. Phylogenetic analyses of 645 host-symbiont pairings revealed that most Symbiodinium types associated specifically with a particular foraminiferal host genus or species, and that the genetic diversity of these symbiont types was positively correlated with the genetic diversity found within each of the three host genera. Compared to previous molecular studies of Symbiodinium from other locations worldwide, the diversity reported here is exceptional and suggests that Micronesian coral reefs are home to a remarkably large Symbiodinium assemblage.     

Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000