Diversity and expression of cyanobacterial hupS genes in pure cultures and in a nitrogen-limited phototrophic biofilm

A PCR was developed for conserved regions within the cyanobacterial small subunit uptake hydrogenase (hupS) gene family. These primers were used to PCR amplify partial hupS sequences from 15 cyanobacterial strains. HupS clone libraries were constructed from PCR-amplified genomic DNA and reverse-transcribed mRNA extracted from phototrophic biofilms cultivated under nitrate-limiting conditions. Partial hupS gene sequences derived from cyanobacteria, some of which were not previously known to contain hup genes were used for phylogenetic analysis. Phylogenetic trees constructed with partial hupS genes were congruent with those based on 16S rRNA genes, indicating that hupS sequences can be used to identify cyanobacteria expressing hup. Sequences from heterocystous and nonheterocystous cyanobacteria formed two separate clusters. Analysis of clone library data showed a discrepancy between the presence and the activity of cyanobacterial hupS genes in phototrophic biofilms. The results showed that the hupS gene can be used to characterize the diversity of natural populations of diazotrophic cyanobacteria, and to characterize gene expression patterns of individual species and strains.     

Aging and Curvature Discrimination from Static and Dynamic Touch

Two experiments evaluated the ability of 30 older and younger adults to discriminate the curvature of simple object surfaces from static and dynamic touch. The ages of the older adults ranged from 66 to 85 years, while those of the younger adults ranged from 20 to 29 years. For each participant in both experiments, the minimum curvature magnitude needed to reliably discriminate between convex and concave surfaces was determined. In Experiment 1, participants used static touch to make their judgments of curvature, while dynamic touch was used in Experiment 2. When static touch was used to discriminate curvature, a large effect of age occurred (the thresholds were 0.67 & 1.11/m for the younger and older participants, respectively). However, when participants used dynamic touch, there was no significant difference between the ability of younger and older participants to discriminate curvature (the thresholds were 0.58 & 0.59/m for the younger and older participants, respectively). The results of the current study demonstrate that while older adults can accurately discriminate surface curvature from dynamic touch, they possess significant impairments for static touch.     

姜黄色素及其提制方法

来源于姜科姜黄属植物姜黄根茎的姜黄是国内外食品行业允许使用的重要天然色素之一,具有重要的开发利用价值。姜黄素的主要提取方法可分为有机溶剂提取法和水提取法;精制方法主要包括专一性溶剂法、酸碱法、酶水解法和柱层析法等。文中对各种方法进行了简要介绍和评述。     

Molecular identification and differentiation of Brevibacterium species and strains

Amplified ribosomal DNA restriction enzyme analysis (ARDRA), pulsed field gel electrophoresis (PFGE) and ribotyping were used to differentiate among 24 strains of Brevibacterium linens, Brevibacterium casei and Brevibacterium epidermidis obtained from type culture collections or isolated from various smear ripened cheeses. ARDRA was applied to the 16S rDNA. B. linens was shown to be a quite heterogenic group with 2 to at least 4 copies of rrn operons per strain with aberrant nucleotide sequences. AccI gave genus specific restriction patterns and was used to separate Brevibacterium from Corynebacterium species. The expected species specificity of TaqI applied to B. linens type culture strains, but not to all strains isolated from cheese. By AvaI restriction, B. casei and B. linens were differentiated from B. epidermidis and the orange pigmented Arthrobacter casei, a new species of coryneform bacteria; by XmnI restriction, B. linens and B. epidermidis were differentiated from B. casei. One of 4 B. linens genotypes could not be distinguished from B. casei by this method. Here, the typical orange B. linens pigments were used for classification, which was confirmed by partial sequencing of the 16S rDNA.     

Biochemical and dynamic studies of collagen from human normal skin and keloid tissue

Keloids are exuberant scars, in which collagen, fibronectin and glycosaminoglycans are overdeposited. Biochemical analysis of the collagen isolated from normal skin and keloid tissue by pepsin treatment, indicated an increase in the type III and GAG content. Viscosity measurements of collagen from normal skin and keloid tissue were used in the present study to establish the interaction between collagen and GAG. Physico-chemical properties such as intrinsic viscosity, reduced viscosity and hydrated volume were computed from viscosity measurements. These measurements were also used to determine the denaturation temperature of collagen which was further confirmed by DSC measurements. Chondroitinase has been used in this study to probe the influence of GAG on the physico-chemical characteristic of keloid collagen.     

Cloning and sequencing of immunoglobulin variable-region genes using degenerate oligodeoxyribonucleotides and polymerase chain reaction

A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5'-oligodeoxyribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from gamma and mu heavy chains and from kappa light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5'-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from gamma and mu isotypes and the universal light-chain primer was used to amplify three separate kappa light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/kappa, IgG2b/kappa and IgM/kappa isotypes.     

Enzymatic and chemical methods for the generation of pure hyaluronan oligosaccharides with both odd and even numbers of monosaccharide units

Hyaluronan oligosaccharides display physiological activities not associated with the polymer and are widely used to characterize hyaluronan-binding proteins. They can also be used as biocompatible starting blocks for chemical derivatization. Here we present methods for generating milligram quantities of unusual odd- and even-numbered oligosaccharides, greatly increasing the diversity of reagents for use in such studies. These methods are based upon protocols from the 1960s, at which time it was very difficult to assess the stereochemical purity of the products. To address this, products were analyzed with modern high-field nuclear magnetic resonance spectroscopy. Alkaline beta-elimination conditions previously used to remove reducing-terminal N-acetylglucosamine residues in fact introduce a significant ( approximately 30%) level of stereoisomerism in the products by alkali-catalyzed keto-enol tautomerizations. Milder alkaline conditions were used to overcome this problem, reducing the contamination to <5%. The elimination by-products from this reaction were isolated and characterized, allowing the mechanism of alkaline degradation of hyaluronan to be investigated for the first time. beta-Glucuronidase was used to remove nonreducing-terminal glucuronic acid residues from oligosaccharides. Odd-numbered oligosaccharides with terminal glucuronic acid residues isolated from hyaluronidase digests are shown to originate from acid-catalyzed acetal hydrolysis during boiling denaturation and also have significant levels of stereochemical impurities.     

Thermophilic archaeal enzymes and applications in biocatalysis

Thermophilic enzymes have advantages for their use in commercial applications and particularly for the production of chiral compounds to produce optically pure pharmaceuticals. They can be used as biocatalysts in the application of 'green chemistry'. The thermophilic archaea contain enzymes that have already been used in commercial applications such as the L-aminoacylase from Thermococcus litoralis for the resolution of amino acids and amino acid analogues. This enzyme differs from bacterial L-aminoacylases and has similarities to carboxypeptidases from other archaeal species. An amidase/γ-lactamase from Sulfolobus solfataricus has been used for the production of optically pure γ-lactam, the building block for antiviral carbocyclic nucleotides. This enzyme has similarities to the bacterial signature amidase family. An alcohol dehydrogenase from Aeropyrum pernix has been used for the production of optically pure alcohols and is related to the zinc-containing eukaryotic alcohol dehydrogenases. A transaminase and a dehalogenase from Sulfolobus species have also been studied. The archaeal transaminase is found in a pathway for serine synthesis which is found only in eukaryotes and not in bacteria. It can be used for the asymmetric synthesis of homochiral amines of high enantioselective purity. The L-2-haloacid dehalogenase has applications both in biocatalysis and in bioremediation. All of these enzymes have increased thermostability over their mesophilic counterparts.     

Review of methodologies for extracting plant-available and amorphous Si from soils and aquatic sediments

There is a variety of methodologies used in the aquatic sciences and soil sciences for extracting different forms of Si from sediments and soils. However, a comparison of the published extraction techniques is lacking. Here we review the methodologies used to extract different Si fractions from soils and sediments. Methods were classified in those to assess plant-available Si and those to extract Si from amorphous silica and allophane. Plant-available Si is supposed to comprise silicic acid in soil solution and adsorbed to soil particles. Extraction techniques for plant-available Si include extractions with water, CaCl₂, acetate, acetic acid, phosphate, H₂SO₃, H₂SO₄, and citrate. The extractants show different capabilites to desorb silicic acid, with H₂SO₃, H₂SO₄ and citrate having the greater extraction potential. The most common extractants to dissolve amorphous silica from soils and aquatic sediments are NaOH and Na₂CO₃, but both also dissolve crystalline silicates to varying degrees. In soils moreover Tiron is used to dissolve amorphous silica, while oxalate is used to dissolve allophanes and imogolite-type materials. Most techniques analyzing for biogenic silica in aquatic environments use a correction method to identify mineral derived Si. By contrast, in the soil sciences no correction methods are used although pedologists are well aware of the overestimation of amorphous silica by the NaOH extraction, which is most commonly used to extract silica from soils. It is recommended that soil scientists begin to use the techniques developed in the aquatic sciences, since it seems impossible to extract amorphous Si from soils completely without dissolving some of the crystalline silicates.     

A review of fish-derived antioxidant and antimicrobial peptides: their production, assessment, and applications

Fishes are rich sources of structurally diverse bioactive compounds. In recent years, much attention has been paid to the existence of peptides with biological activities and proteins derived from foods that might have beneficial effects for humans. Antioxidant and antimicrobial peptides isolated from fish sources may be used as functional ingredients in food formulations to promote consumer health and improve the shelf life of food products. This paper presents an overview of the antioxidant and antimicrobial peptides derived from various fishes. In addition, we discuss the extraction of fish proteins, enzymatic production, and the techniques used to isolate and characterize these compounds. Furthermore, we review the methods used to assay the bioactivities and their applications in food and nutraceuticals.     

A rapid and efficient method of lysis of Listeria and other gram-positive bacteria using mutanolysin.

A rapid and simple procedure is described for cell lysis for preparation of nucleic acids and intact ribosomal RNA from Gram-positive bacteria. Commercial mutanolysin (purified from Streptomyces globisporus) was used for inducing lysis. Listeria, Lactobacillus and Lactococcus strains were very sensitive to mutanolysin when compared to lysozyme. Susceptibility to mutanolysin was improved by a preliminary treatment with acetone, and sodium dodecyl sulfate reduced the efficiency of lysis when used together with mutanolysin. The procedure was also effective for recovering plasmids from these bacteria.     

Estimating body weight and habitat type from extinct avian and avian‐like theropod footprints

The relationship between body weight and footprint area of modern avians was derived and used to estimate the body weights of non‐avian theropods taxa from the Triassic to Cretaceous and extinct avian taxa from the Cretaceous periods. Geometric information, such as the area and shape of fossil tracks of extinct avians and non‐avian theropods, was used to estimate body weight and habitat type. The percentage prediction and standard error of estimates indicated that the body weight estimated from track area is comparable with body weight estimated from body fossils bones. Therefore, this approach is useful when the fossilized track record is richer than the fossilized skeletal record. The data sets for avians and reptiles were combined and used to derive a body weight–area relationship that may be applicable to a broader range of organisms, such as plantigrade quadrupeds and digitigrade bipeds. Additionally, scatter plots of the relationship between habitat type and footprint shape of modern avians were used to infer the habitat type of extinct avians. This finding suggests that the pes of animals, living in areas characterized by fluctuating water levels, and under conditions facilitating the preservation of footprints, were similar in form to those of extant semi‐aquatic avians.     

一种简单、有效的适于PCR操作的放线菌DNA提取方法

目的:利用改良酶法发展了一种从微量(几百微升)发酵液中快速安全的提取放线菌基因组DNA的方法。方法:利用溶菌酶破壁,蛋白酶K和SDS除蛋白,成功提取较高质量的放线菌基因组DNA,所得的DNA可作为PCR反应的模板进行16SrRNA等基因有效扩增。结果:能从海绵和土壤分离的放线菌中成功提取基因组DNA。结论:该方法操作简单、费用低廉、不使用酚、氯仿等有毒害作用有机试剂,非常适于长期从事放线菌操作的研究人员。为大量放线菌菌株的快速鉴别、高通量筛选和系统分类研究创造了条件。     

Restriction endonuclease BglI as a tool for in vitro reconstruction and recombination of plasmid and bacteriophage genomes

The restriction endonuclease BglI produces different individual fragment ends from different cut sites. This property has allowed us to reconstruct efficiently several commonly used plasmid and bacteriophage genomes and a number of recombinant plasmids containing up to seven BglI restriction sites from their constituent BglI fragments. It is demonstrated that in vitro reconstitution from BglI fragments can be used to create, in a simple way, recombinant DNA molecules by recombining in vitro BglI fragments from different mutated or otherwise related genomes. Further applications of the method are discussed.     

Discovery and characterization of orthosteric and allosteric muscarinic M2 acetylcholine receptor ligands by affinity selection-mass spectrometry

Screening assays using target-based affinity selection coupled with high-sensitivity detection technologies to identify small-molecule hits from chemical libraries can provide a useful discovery approach that complements traditional assay systems. Affinity selection-mass spectrometry (AS-MS) is one such methodology that holds promise for providing selective and sensitive high-throughput screening platforms. Although AS-MS screening platforms have been used to discover small-molecule ligands of proteins from many target families, they have not yet been used routinely to screen integral membrane proteins. The authors present a proof-of-concept study using size exclusion chromatography coupled to AS-MS to perform a primary screen for small-molecule ligands of the purified muscarinic M2 acetylcholine receptor, a G-protein-coupled receptor. AS-MS is used to characterize the binding mechanisms of 2 newly discovered ligands. NGD-3350 is a novel M2-specific orthosteric antagonist of M2 function. NGD-3366 is an allosteric ligand with binding properties similar to the allosteric antagonist W-84, which decreases the dissociation rate of N-methyl-scopolamine from the M2 receptor. Binding properties of the ligands discerned from AS-MS assays agree with those from in vitro biochemical assays. The authors conclude that when used with appropriate small-molecule libraries, AS-MS may provide a useful high-throughput assay system for the discovery and characterization of all classes of integral membrane protein ligands, including allosteric modulators.     

Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction

Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. monocytogenes.     

Gas holdup, foaming and oxygen transfer in a jet loop bioreactor with artificial foaming media and yeast culture

A concentric draft tube jet loop bioreactor (10.5 m3) was used to study the influence of aerated liquid height (above the draft tube) on the amount of surfactant addition allowable without foaming. Sodium lauryl sulfate (SLS) and defatted soybean flour in tap water were used as model artificial media. The amount of surfactant required to develop foaming and the maximum gas holdup achieved prior to foaming were notably influenced by aerated liquid height. Decreasing the aerated liquid height from 1.50 to 0.05 m increased the amount of SLS allowed without foaming from 2.2 to 12.1 g, the gas holdup in the riser from 0.18 to 0.31 and the gas holdup in the downcomer from 0.12 to 0.25. Similar behavior was observed for soybean flour. Decreasing the aerated liquid height from 1.45 to 0.05 m increased the amount of soybean flour allowed without foaming from 822 to 3200 g, the gas holdup in the riser from 0.17 to 0.26 and the gas holdup in the downcomer from 0.10 to 0.19. Data from a representative continuous yeast culture are reported to show that operation at low aerated liquid heights (0.5 m) can also be used to produce a culture broth with large gas holdup and oxygen transfer but without foaming.     

Endogenous oxidative DNA base modifications analysed with repair enzymes and GC/MS technique

GC/MS technique was used to identify endogenous levels of oxidatively modified DNA bases. To avoid possible artefact formation we used Fpg and Endo III endonucleases instead of acid hydrolysis to liberate the base products from unmodified DNA samples. Several different DNA preparations were used: (i) commercial calf thymus DNA, (ii) DNA isolated from rat liver, (iii) DNA isolated from human lymphocytes and (iv) nuclei isolated from rat liver. In all DNA samples used in our assays the most efficiently removed bases by Fpg protein are FapyG and FapyA although 8-oxoG was also detected in all preparations. The amount of 8-oxoG in human lymphocytes and in rat liver DNA was 3 and 2 per 10(7)bases, respectively. It is reasonable to postulate that the presented method is one of the techniques which should be used to reveal the enigma of endogenous, oxidative DNA damage.     

Isolation and transformation of rice aleurone protoplasts

Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.     

Structural and mechanical indicators of limb specialization in primates

The structural mechanics of femora and humeri from primates representing a wide spectrum of habitual locomotor activities were examined to determine how cross-sectional properties vary with functional specializations of the extremities. Average bending rigidities of the midshaft of humerus and femur were measured in 60 individuals of four nonhuman primate species (Macaca nemestrina, Macaca fascicularis, Presbytis cristata, Hylobates lar) using single-beam photon absorptiometry. Linear regression analyses of the loge transformed data were used to assess the relative usage of the forelimb and hindlimb in propulsion and weight bearing, and to evaluate deviations from generalized mammalian quadrupedalism. The results suggest that average bending rigidities of the humerus and femur in primates reflect the extent to which the forelimb and hindlimb are used differently in locomotion; deviations of average bending rigidity from geometric similarity indicate functional variations from generalized mammalian quadrupedalism and the ratio of humeral to femoral bending rigidity can be used to identify trends towards hindlimb or forelimb dominance in locomotion and can be employed in general to determine how the limb was used.