Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Successful immunotherapy with matrix metalloproteinase-derived peptides in adjuvant arthritis depends on the timing of peptide administration

We have recently found that matrix metalloproteinases (MMPs) are targets for T-cell and B-cell reactivity in experimental arthritis. In the present article, we investigate whether modulation of MMP-specific T-cell responses could influence the course of adjuvant arthritis (AA). Lewis rats were treated nasally with MMP peptides prior to or after AA induction. Administration of the MMP-10 or the MMP-16 peptide prior to AA induction reduced the arthritic symptoms. In contrast, administration of the MMP-10 peptide after AA induction aggravated the arthritic symptoms. The present study shows the possible usefulness of MMP peptides for immunotherapy. However, a clear understanding of proper timing of peptide administration is crucial for the development of such therapies.     

The biosynthesis of gibberellic acids by the transformants of orchid-associated <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

The genus Fusarium, including multiple strains in the Gibberella fujikuroi species complex (GFC), is well known for its production of diverse secondary metabolites. F. fujikuroi, associated with the “bakanae” disease of rice, is an active producer of gibberellins (GAs), a wide class of plant hormones. In addition to some members of the GFC, the GA biosynthetic gene cluster, or parts of it, occurs also in some isolates of the closely related species of F. oxysporum, which does not belong to the GFC. However, production of GAs has never been observed in any F. oxysporum strain. In this study, we report on the GA biosynthetic activity in an orchid-associated F. oxysporum strain by transforming a cosmid with the entire F. fujikuroi GA gene cluster. Southern and Northern blot analyses confirmed not only the integration of the entire gene cluster into the genome but also the active expression of the seven GA biosynthetic genes under nitrogen-limiting conditions. The transformants produced GAs at levels similar to those of F. fujikuroi. These data show that the regulatory network for expression of GA genes is fully active in the F. oxysporum background.     

Granule-bound starch synthase: structure, function, and phylogenetic utility

Interest in the use of low-copy nuclear genes for phylogenetic analyses of plants has grown rapidly, because highly repetitive genes such as those commonly used are limited in number. Furthermore, because low- copy genes are subject to different evolutionary processes than are plastid genes or highly repetitive nuclear markers, they provide a valuable source of independent phylogenetic evidence. The gene for granule-bound starch synthase (GBSSI or waxy) exists in a single copy in nearly all plants examined so far. Our study of GBSSI had three parts: (1) Amino acid sequences were compared across a broad taxonomic range, including grasses, four dicotyledons, and the microbial homologs of GBSSI. Inferred structural information was used to aid in the alignment of these very divergent sequences. The informed alignments highlight amino acids that are conserved across all sequences, and demonstrate that structural motifs can be highly conserved in spite of marked divergence in amino acid sequence. (2) Maximum-likelihood (ML) analyses were used to examine exon sequence evolution throughout grasses. Differences in probabilities among substitution types and marked among-site rate variation contributed to the observed pattern of variation. Of the parameters examined in our set of likelihood models, the inclusion of among-site rate variation following a gamma distribution caused the greatest improvement in likelihood score. (3) We performed cladistic parsimony analyses of GBSSI sequences throughout grasses, within tribes, and within genera to examine the phylogenetic utility of the gene. Introns provide useful information among very closely related species, but quickly become difficult to align among more divergent taxa. Exons are variable enough to provide extensive resolution within the family, but with low bootstrap support. The combined results of amino acid sequence comparisons, maximum-likelihood analyses, and phylogenetic studies underscore factors that might affect phylogenetic reconstruction. In this case, accommodation of the variable rate of evolution among sites might be the first step in maximizing the phylogenetic utility of GBSSI.      

Bacteria associated with the roots of epiphytic orchids

This work is the first to report the isolation and identification of bacteria colonizing the roots of tropical epiphytic orchids Acampe papillosa (Lindl.) Lindl. and Dendrobium moschatum (Buch.-Ham.) Swartz. and bacteria inhabiting inner layers of the aerial and substrate roots of A. papillosa. We showed by the example of this epiphyte that associative bacteria are present in large amounts on the aerial but not substrate roots. We isolated and identified bacteria from the substrate roots of D. moschatum and from its growth substrate (pine bark). The structure of the intercellular matrix of the associative bacteria was studied.     

Localization of Associative Cyanobacteria on the Roots of Epiphytic Orchids

This work is the first study of the localization of phototrophic microorganisms in the rhizoplane and velamen of epiphytic orchids, namely, on the aerial and substrate roots of Acampe papillosa and Dendrobium moschatum and on the aerial roots of Phalaenopsis amabilis and Dendrobium phalaenopsis. The composition of the bacterial community on the plant roots depended on the conditions of plant growth. Under conditions simulating the climate of moist tropical forests, the aerial roots proved to be populated with phototrophic microorganisms, among which cyanobacteria predominated. Interlaced fungal hyphae and filamentous cyanobacteria formed a sheath on the surface of the aerial roots. The nitrogen-fixing capacity of the sheath of the aerial roots was studied on the example of P. amabilis.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Bacteria associated with orchid roots and microbial production of auxin

Associative bacteria of terrestrial (Paphiopedilum appletonianum) and epiphytic (Pholidota articulata) tropical orchids were investigated. Microbial community of epiphytic plant differed from that of the terrestrial one. Streptomyces, Bacillus, Pseudomonas, Burkholderia, Erwinia and Nocardia strains populated Paphiopedilum roots, whereas Pseudomonas, Flavobacterium, Stenotrophomonas, Pantoea, Chryseobacterium, Bacillus, Agrobacterium, Erwinia, Burkholderia and Paracoccus strains colonized Pholidota roots. Endophytic bacteria populations were represented with less diversity: Streptomyces, Bacillus, Erwinia and Pseudomonas genera were isolated from P. appletonianum, and Pseudomonas, Bacillus, and Flavobacterium genera were isolated from Ph. articulata. Microorganisms produced indole-3-acetic acid (IAA). Variations in its biosynthesis among the strains of the same genus were also observed. The highest auxin level was detected during the stationary growth phase. Biological activity of microbial IAA was proved by treatment of kidney bean cuttings with bacterial supernatants, revealing considerable stimulation of root formation and growth.