DNA homology and adsorption specificity of Pseudomonas aeruginosa virulent bacteriophages

Summary The DNA homology and adsorption specificity of newly isolated virulent bacteriophages of P. aeruginosa have been studied. On the basis of this analysis all phages were divided into four groups: k, m, mnP78-like and mnF82-like bacteriophages. DNA's of k as well as m phages were shown to possess different restriction patterns although they have an extensive homology. Unlike other groups, k phages were characterized by the presence of T4 DNA ligase-repaired, single-chain breaks.Abbreviations kbp kilobase pairs - EM electron microscopy     

New expanded bed adsorbents for the recovery of DNA

A 20–40 m pellicular high density (3.7 g cm^–3) expanded bed material has been designed for the capture of DNA and other large macromolecules. Anion exchangers fashioned out of these supports exhibited dramatically enhanced DNA binding capacities over commercial anion exchange adsorbents (6 mg ml^–1, c.f. 50 g ml^–1 at 10% breakthrough), due to a combination of small particle and fuzzy surface architecture created through the coupling of polyethylene imine chains.     

Ultrastructure of Sporothrix schenckii treated with iodine-potassium iodide solution

The ultrastructural changes produced by iodine-potassium iodide solution on yeast cells of Sporothrix schenckii were investigated by transmission electron microscopy in order to clarify the mechanism of oral potassium iodide therapy for sporotrichosis. Yeast cells were dipped with solutions containing various concentrations of iodine. The rate of germination decreased markedly between the range of iodine concentrations from 0.63 g/ml to 5.0 g/ml. No significant ultrastructural changes were seen at the concentration of the iodine of 1.25 g/ml (80% germination) or less. In the concentration of 2.5 g/ml (50% germination), normal cells and degenerated cells coexisted. When the cells were treated with 5.0 g of iodine per ml (0% germination) or more, their interior structures were completely destroyed. It is assumed that iodine treatment of the organism causes rapid destruction in the whole cell.     

Rethinking neurasthenia: The illness concepts of Shenjing Shuairuo among Chinese undergraduates in Hong Kong

Neurasthenia is both a Western disease construct and a popular Chinese illness concept (shenjing shuairuo, SJSR). Using a self-report questionnaire, we examined 148 Hong Kong Chinese undergraduates' concept of its epidemiology, symptomatology, etiology and treatment. Notwithstanding that fatigue is thesine qua non of neurasthenia in Western nosology, subjects believed that SJSR was compatible with a diversity of symptoms which fell, on factor analysis, into the neurotic, psychotic, somatic and dysfunctional subgroups. Contrary to the popular portrayal of SJSR as a physical or chronic fatigue disorder, the most common perceived symptoms were anxiety, insomnia, depression and fright. Logically, psychological etiology and remedy were highly emphasized. The perceived high prevalence, non-aggressive nature and symptomatic diversity of SJSR attested to the notion that it might camouflage and destigmatize psychiatric labels of insanity. The contextual study of neurasthenia illustrates how in its search for legitimacy an originally Western concept adapts, transforms, and acquires distinctive local meanings in a non-Western culture.     

Plastid Sequence Evolution: A New Pattern of Nucleotide Substitutions in the Cucurbitaceae

Nucleotide substitutions (i.e., point mutations) are the primary driving force in generating DNA variation upon which selection can act. Substitutions called transitions, which entail exchanges between purines (A=adenine, G=guanine) or pyrimidines (C=cytosine, T=thymine), typically outnumber transversions (e.g., exchanges between a purine and a pyrimidine) in a DNA strand. With an increasing number of plant studies revealing a transversion rather than transition bias, we chose to perform a detailed substitution analysis for the plant family Cucurbitaceae using data from several short plastid DNA sequences. We generated a phylogenetic tree for 19 taxa of the tribe Benincaseae and related genera and then scored conservative substitution changes (e.g., those not exhibiting homoplasy or reversals) from the unambiguous branches of the tree. Neither the transition nor (A+T)/(G+C) biases found in previous studies were supported by our overall data. More importantly, we found a novel and symmetrical substitution bias in which Gs had been preferentially replaced by A, As by C, Cs by T, and Ts by G, resulting in the GACTG substitution series. Understanding this pattern will lead to new hypotheses concerning plastid evolution, which in turn will affect the choices of substitution models and other tree-building algorithms for phylogenetic analyses based on nucleotide data.     

Transposome Insertional Mutagenesis and Direct Sequencing of Microbial Genomes

Preformed transposase-transposon complexes called Transposomes have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0×10⁴and 1.0×10⁷kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species. Transposon integration sites were examined by direct genome sequencing of chromosomal DNA. Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon. The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences. Mutant phenotypes were rapidly correlated with genomic insertions sites.     

Benzothiadiazole enhances the elicitation of rosmarinic acid production in a suspension culture of Agastache rugosa O. Kuntze

Callus and suspension cultures were established from the leaves of Agastache rugosa. The suspension cell growth was maximum at 15 days after inoculation. The cellular content of rosmarinic acid increased slowly and reached maximum (0.42 mg g^–1 dry wt) during the stationary phase of culture, after 18 days of inoculation. The addition of yeast extract preparation (MW <10000) at 50 g ml^–1 elevated the rosmarinic acid content up to 5.7-fold of that found in non-elicited suspension cells. The elicitation of yeast extract preparation was further 2-fold enhanced by the presence of benzothiadiazole, a synthetic activator of plant systemic acquired resistance, as compared to yeast extract alone. These results showed that benzothiadiazole can be used as a tool for enhancing secondary metabolite accumulation in cell cultures.     

Protein kinase C β from Friend erythroleukemia cells is associated with chromatin and DNA

Certain protein kinase C (PKC) isotypes are localized to the nucleus during cellular proliferation in murine erythroid cells, as well as in human promyelocytic leukemia and erythroleukemia cells. Because the structure of these PKC isotypes contains a conserved cysteine-rich region that contains the zinc finger DNA binding motif, we tested the hypothesis that selected PKC isotypes found in Friend erythroleukemia cells can bind to DNA. Cell lysates from murine Friend erythroleukemia cells, which express , I, and II PKC, expressed greater amounts of the isoforms than the isoform of PKC in their nuclei, and PKC I was found in the chromatin of these cells. Lysates of these cells were tested for their ability to bind to a DNA-cellulose columm. Bound proteins were eluted with a step gradient of increasing KCl concentrations, and eluant fractions were then subjected to immunoblot analysis using isotype-specific antibodies to the and I isotypes of PKC. DNA binding was detected for the PKC I isotype, which is present in the nucleus, but not for the more abundant PKC isotype, which resides primarily in the cytoplasm. These results demonstrate that PKC can associate with DNA, and that this association is isotype specific in Friend erythroleukemia cells. (Mol Cell Biochem151: 107–111, 1995)     

Conservation status of parrot populations in an Atlantic rainforest area of southeastern Brazil

A census of four species of syntopic parrots was carried out using distance sampling methods on São Sebastião island, SE Brazil. Most of the 33593 ha island is covered by mature and secondary Atlantic rainforest. Almost 80% of these forests are within the Ilhabela Park. Although the species counted have marked differences in size and weight, density (individuals/km²) and estimated population size in 23500 ha of well-preserved forests were similar: Amazona farinosa (13.82±5.94; 3247±1395), Pionus maximiliani (15.79±7.04; 3712±1654), Brotogeris tirica (15.05±4.87; 3537±1143) and Pyrrhura frontalis (13.06±5.53; 3068±1298). Encounter rates of Forpus crassirostris and Pionopsitta pileata were very low, which suggests that there is only a small population of these species on the island. The São Sebastião forests still support healthy populations of parrots. Although woodpecker population estimates on the island are large enough to provide nesting sites for parrots, competition for holes with other secondary cavity nesters such as toucans, flycatchers and tytiras, and the selective cutting of dead trees for canoe construction, which is a common practice on the island, may limit hole availability for parrots.     

Angiogenesis and myogenesis as two facets of inflammatory post-ischemic tissue regeneration

Recently Hippophae rhamnoides has been reported to render chromatin compaction and significantly inhibit radiation induced DNA strand breaks. To investigate the mechanism of action of RH-3, a preparation of Hippophae rhamnoides, in this connection, present study was undertaken. Chromatin compaction induced by RH-3 (100 g/ml or more) was maximum at alkaline pH but was completely negated by acidic pH (< 6) or presence of free radical scavengers like glycerol, DMSO etc. In a concentration dependent manner, RH-3 inhibited the intercalation of ethidium ions from Et Br into calf thymus DNA and also increased the precipitation of DNA-protein cross-links (DPC) in thymocytes. Chromatin compaction caused by RH-3 treatment did not permit the separation of proteins from DNA even after treatment with 2 M NaCl solution. SDS-PAGE profiles also revealed that RH-3 in a dose dependent manner compacted the chromatin organization, induced DPC and inhibited the extraction of both histone and non-histone matrix proteins from chromatin maximally at 80 g/ml. More than 80 g/ml of RH-3, though extracted low molecular weight histones but did not separate non-histone proteins. The RH-3 mediated DPCs were resistant even to 1% SDS, 4 M NaCl and 3.8 M hydroxyl amine hydrochloride but were prone to both urea (8 M) and guanidine hydrochloride (6 M) indicating covalent bonding between DNA and proteins (serine/threonine). RH-3 in a concentration dependent manner induced superoxide anions and the phenomenon was dependent upon nature of medium, presence of metal ions and pH. RH-3 at concentrations up to 100 g/ml in presence of 50 M copper sulfate inflicted significant damage to extraneously added 2-deoxyribose molecules and maximum TBARS were formed at a concentration of 100 g/ml. Higher concentrations of RH-3 more than 100 g/ml quenched free radicals and inhibited 2-deoxyribose degradation. RH-3 also induced strand breaks in plasmid DNA at concentrations lower than 100 g/ml but completely inhibited at concentrations higher than 250 g/ml, indicating bimodal function. Strand breaks induced by lower concentrations of RH-3 (up to 100 g/ml) were inhibited by antioxidants like GSH, DFR etc. RH-3, in a concentration dependent mode also inhibited the relaxation of supercoiled plasmid DNA (PBR322) by topoisomerase I.Present study indicated that RH-3 caused compaction of reversible (< 100 g/ml) and irreversible (> 100 g/ml) nature which was related to the magnitude of DNA-protein cross-links formed. Maintenance of chromatin organization, induction of hypoxia, hydrogen atom donation, free radical scavenging and blocking of cell cycle at G2-M phase by interfering with topoisomerase I activity seem to contribute towards the radioprotective efficacy of RH-3.     

Zur Dynamik feiner Pseudopodien von Hochmoor-Amöben

Zusammenfassung Es werden verschiedene Vorgänge an sehr zarten Pseudopodien beschrieben und mit Hilfe der an submikroskopischen Fibrillen (schraubig gebauten Mikrofibrillen und Mikroröhrchen der Elektronenmikroskopie) auftretenden Torsionsspannungen und Rotationen gedeutet.

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On the dynamics of slender pseudopodia of bog amebae

Summary Different processes which occur on very slender pseudopods are described. They are interpreted with the help of torsional forces and revolutions of the submicroscopic fibrils (helical microfibrils and microtubules in electron microscopy).

     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

DNA sequence patterns in human,mouse, and rabbit immunoglobulin kappa-genes

Summary DNA sequences of the human, mouse, and rabbit immunoglobulin kappa-gene (J-C regions) are compared with respect to various DNA patterns, including dyad symmetry pairings, runs of nucleotides, repeat clusters, and repeats that occur with unusually high frequency. The significant dyad symmetry pairings within each of the sequences emphasize the two control-enhancer elements of the J₅-C intron. Dyad symmetry pairs between the J-C region and a number of kappa variable (V)-gene domains suggest differences in the affinities between the V and J segments. It is the consensus heptamer rather than the consensus nonamer that embodies the longest V-J dyad symmetry combinations. In the rabbit there are long runs and repeat clusters of the sequences that identify regions of high duplication; these regions are absent in the human and mouse sequences. High-frequency oligonucleotides feature the consensus nonamer 5 to the J segments, especially in the mouse sequence.     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Export and activity of hybrid FhuA''-''Iut receptor proteins and of truncated FhuA'' proteins of the outer membrane of Escherichia coli

Summary The FhuA protein in the outer membrane of Escherichia coli serves as a multifunctional receptor for the phages T5, T1, 80, for colicin M, for ferrichrome (Fe³⁺-siderophore) and for the structurally related antibiotic, albomycin. To determine structural domains required for these receptor functions and for export, a fusion protein between FhuA and Iut (receptor for Fe³⁺-aerobactin and cloacin DF13) was constructed. In the FhuA-Iut hybrid protein, 24 amino acids of FhuA were replaced by 19 amino acids, 18 of which were from Iut. The number of plaque forming units of phage T5 and T1 on cells expressing FhuA-Iut was nearly as high as on cells expressing plasmid-encoded wild-type FhuA. However, 10⁷-fold higher concentrations of phage 80 and 10³ times more colicin M were required to obtain a zone of growth inhibition. Truncated FhuA proteins in which the last 24 amino acids at the carboxy-terminus were replaced by 16 (FhuA2) or 3 (FhuAT) amino acids could hardly be detected on polyacrylamide electrophoretograms of outer membrane proteins, due to proteolytic degradation. Sensitivity of cells expressing FhuA2 to phage T5 and T1 was reduced by several orders of magnitude and sensitivity to phage 80 and colicin M was totally abolished. In contrast, cells expressing FhuAT were nearly as sensitive to phage T5, T1, and 80 and to colicin M as cells containing FhuA-Iut. None of the constructs could grow on ferrichrome as sole iron source and none was sensitive to albomycin. Ferrichrome did not inhibit binding of T5 to TonB^– cells expressing FhuA-Iut (as it did in FhuA⁺ cells) due to the lack of ferrichrome binding. It is concluded that very small deletions (relative to the size of FhuA with 714 amino acids) at the C-terminal end render FhuA susceptible to proteolytic cleavage. The C-terminal alterations affect sensitivity to FhuA-specific agents very differently. Apparently, the C-terminus is a very important part of FhuA regarding stability and activity. Expression of active FhuA and partially inactive FhuA derivatives in the same cell revealed no negative complementation, suggesting a FhuA monomer as functional unit.     

Comparison of the carbohydrate moieties of recombinant soluble Fcε receptor (sFcε RII/sCD23) expressed inSaccharomyces cerevisiae and Chinese hamster ovary cells. Different O-glycosylation sites are used by yeast and mammalian cells

Recombinant human soluble low affinity receptor for the Fc portion of IgE (sFcRII/sCD23) was produced inSaccharomyces cerevisiae or Chinese hamster ovary cells and subjected to carbohydrate analysis. Applied methods included analytical SDS-PAGE, reversed phase HPLC, methylation analysis and sequential degradation with exoglycosidases. The results revealed that sFcRII derived from Chinese hamster ovary cells is glycosylated exclusively at Ser-147, containing mainly the trisaccharide Sia(2–3)Gal(1–3)GalNAc, whereas the yeast derived glycoprotein was glycosylated at Ser-167 and contained only -mannosyl residues. It is shown here for the first time that different amino acids of a given protein can be O-glycosylated when expressed in yeast or Chinese hamster ovary cells.     

Polysaccharide-enriched fraction isolated from Duchesnea chrysantha protects against oxidative damage

Reactive oxygen species (ROS)-related oxidative damages have been implicated in a wide variety of the pathological processes such as atherosclerosis, inflammation and carcinogenesis. A polysaccharide-enriched fraction (PEF) was isolated from Duchesnea chrysantha, a herbaceous plant, and its antioxidant activity was demonstrated using several assay systems in vitro. The PEF effectively inhibited Cu²⁺-stimulated low density lipoprotein oxidation in a concentration-dependent way and retarded the conjugated diene formation with the enhancement of the lag phase during oxidation. An assay for DNA strand breaks showed that PEF strongly protected DNA against damage caused by UV or by OH or O₂ ^– generated in the metal-catalyzed oxidation system. PEF effectively inhibited Nitroblue Tetrazolium reduction mediated by O₂ ^– in a dose-dependent manner. It also competed with 2-deoxy-d-ribose in absorbing OH generated by -irradiation (600 Gy) and thus inhibited the formation malondialdehyde. In conclusion, these evidences indicate that PEF may act as an antioxidant to scavenge O₂ ^–, OH or LO₂ directly. Our findings suggest the possibility that it may play a role as a potential therapeutic antioxidant in treatment of oxidative damage-derived diseases.     

Nucleic acid metabolism in yeast VI. Utilisation of exogenous dAMP

Summary It is shown that mutants of Saccharomyces cerevisiae able to efficiently utilise exogenous dTMP can also utilise exogenous dAMP. Under extracellular conditions permissive for dTMP uptake label stemming from offered [8-³H]dAMP is incorporated preferentially into alkali-resistant, high molecular weight material (putative DNA): only about 30% of high molecular weight cell-bound dAMP label was found to be sensitive towards mild alkali hydrolysis. This putative RNA label can be minimised to practically zero when mM Ade is employed in a dAMP labelling assay. Exogenous dAMP at 10 M was found to be cytostatic similarly to M dTMP and similarly to inhibit effectively import of exogenous P_i. We conclude from our results that there exists a yeast cytoplasmic membrane permease able to import dAMP. A model of this hypothetical permease system is presented.Abbreviations S. cerevisiae Saccharomyces cerevisiae - TIP yeast cytoplasmic membrane permease importing dTMP under permisive conditions - AIP hypothetical yeast cytoplasmic membrane permease importing dAMP under permissive conditions - tlr symbol for the recessive genetic trait to utilise effeciently exogenous dTMP - tmp symbol for the recessive genetic trait leading to dTMP auxotrophy - dTMP 2-deoxythymidine 5-monophosphate - dAMP 2-deoxyadenosine 5-monophosphate - (d)NMP (deoxy)ribonucleoside 5-monophosphate - dThd 2-deoxythymidine - Thy thymine - dAdo 2-deoxyadenosine - Ado adenosine - Ade adenine - Ura uracil - P_i inorganic phosphate Apart from discrete abbreviations we followed the rules of nomenclature as recommended by the IUPAC-IUB commission of biochemical nomenclature (CBN)