Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: a step toward a thermosensitive industrial biocatalyst

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10(6) and, presumably, cross-linked protein-polymer aggregates of MW > 10(7). Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 microM) was found to be slightly higher than that of the free enzyme (12 microM), and evaluation of V(max) testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from d-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of beta-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).     

Iron distribution in three central Amazon tree species from whitewater-inundation areas (várzea) subjected to different iron regimes

Trees inhabiting central Amazon floodplain forests are subjected to an annual flood-pulse lasting up to 10 months, leading to both oxygen shortage and accumulation of high levels of reduced iron. To understand the mechanisms underlying the adaptation to these conditions, cuttings from three tree species typical of várzea inundation forests (Salix martiana, Tabernaemontana juruana, and Laetia corymbulosa), were cultivated either aerobically or anaerobically under different iron regimes in greenhouse experiments. Although all species are considered to be non-deciduous, Laetia corymbulosa lost and formed new leaves continuously during the experimental period. Although relative growth rates (RGRs) of all species declined in response to hypoxic conditions, no marked changes in RGRs were apparent among different iron concentrations in the growth medium, ranging from 50 to 500 µM, supplied in ferrous form as FeSO₄. Whereas roots exhibited color changes due to the formation of iron precipitates, no visual symptoms of iron toxicity were observed in the leaves. Iron concentration increased in all organs of all species with increasing iron concentrations in the medium, except for leaves of S. martiana and T. juruana, suggesting an effective restriction of iron influx into the leaf symplast. Although the leaf iron concentration was at the upper limit of the critical range at high external iron levels, it is suggested that internal active transport rather than intracellular detoxification mechanisms contribute to the tolerance to supra-optimal iron levels. Anatomical traits such as suberization of peripheral cell walls and the formation of aerenchyma appear to be of minor importance for Fe tolerance.     

Characterization and chromosomal localization of the chicken avidin gene family

Chicken avidin is a biotin-binding protein expressed under inflammation in several chicken tissues and in the oviduct after progesterone induction. The gene encoding avidin belongs to a family that has been shown to include multiple genes homologous to each other. The screening and chromosomal localization studies performed to reveal the structure and organization of the complete avidin gene family is described. The avidin gene family is arranged in a single cluster within a 27-kb genomic region. The cluster is located on the sex chromosome Z on band q21. The organization of the genes was determined and two novel avidin-related genes, AVR6 and AVR7, were cloned and sequenced.     

Responses of Nine Trifolium repens L. Populations to Ultraviolet-B Radiation: Differential Flavonol Glycoside Accumulation and Biomass Production

This study aimed to quantify and identify flavonoids involvedin the response of nine populations of white clover (Trifoliumrepens L.) to ultraviolet-B radiation (UV-B). Plants were grownfor 12 weeks in controlled environment rooms with or withoutsupplemental UV-B radiation of 13.3 kJ m^-2d^-1. Methanol–waterextractable flavonoids were quantified using high performanceliquid chromatography (HPLC). Two major peaks showed significantenhancement in the HPLC chromatogram in response to supplementalUV-B. The structures of the compounds responsible were identifiedby¹H and¹³C nuclear magnetic resonance (NMR) spectroscopy tobe the flavonols quercetin-3-O-ß- D -xylopyranosyl-(1 2)-ß- D -galactopyranoside and kaempferol-3-O-ß-D -xylopyranosyl-(1 2)-ß- D -galactopyranoside. Withsupplemental UV-B, quercetin glycoside levels increased on averageby 200% while the kaempferol glycoside response was much smaller.Significant differences in flavonol accumulation were foundamong T. repens populations, both constitutively and in responseto UV-B. Stress-adapted populations displayed particularly highflavonol levels under UV-B. There was an inverse correlationbetween plant productivity and quercetin accumulation. Furthermore,higher quercetin accumulation under UV-B was correlated withtolerance against UV-B-induced growth reduction. In conclusion,within-species comparisons in T. repens lend support to a distinctrole for ortho -dihydroxylated flavonoids in the adaptationto UV-B stress and suggest particular advantages in this UV-B-inducedbiochemical adaptation for populations characterized by lowhabitat and plant productivity. Copyright 2000 Annals of BotanyCompany Ultraviolet-B, Trifolium repens, white clover, HPLC, NMR, flavonoids, flavonols, quercetin, kaempferol, biomass, genetic variation, intraspecific     

Identification of immunologically related proteins in sieve-tube exudate collected from monocotyledonous and dicotyledonous plants

The mature, functional sieve-tube system in higher plants is dependent upon protein import from the companion cells to maintain a functional long-distance transport system. Soluble proteins present within the sieve-tube lumen were investigated by analysis of sieve-tube exudates which revealed the presence of distinct sets of polypeptides in seven monocotyledonous and dicotyledonous plant species. Antibodies directed against sieve-tube exudate proteins from Ricinus communis L. demonstrated the presence of shared antigens in the phloem sap collected from Triticum aestivum L., Oryza sativa L., Yucca filamentosa L., Cucurbita maxima Duch., Robinia pseudoacacia L. and Tilia platyphyllos L. Specific antibodies were employed to identify major polypeptides. Molecular chaperones related to Rubisco-subunit-binding protein and cyclophilin, as well as ubiquitin and the redox proteins, thioredoxin h and glutaredoxin, were detected in the sieve-tube exudate of all species examined. Actin and profilin, a modulator of actin polymerization, were also present in all analyzed phloem exudates. However, some proteins were highly species-specific, e.g. cystatin, a protease-inhibitor was present in R. communis but was not detected in exudates from other species, and orthologs of the well-known squash phloem lectin, phloem protein 2, were only identified in the sieve-tube exudate of R. communis and R. pseudoacacia. These findings are discussed in terms of the likely roles played by phloem proteins in the maintenance and function of the enucleate sieve-tube system of higher plants. Received: 12 February 1998 / Accepted: 16 March 1998     

Construction and characterisation of a gridded chicken cosmid library with four-fold genomic coverage

Gridded genomic libraries are crucial for the positional candidate gene approach. For this purpose we constructed a gridded genomic library from a female chicken using the vector sCos 1. About 110 000 cosmid clones were grown and replicated in 384-well plates. An average insert size of 39 kb was calculated from the analysis of 68 randomly selected clones. No chimerism could be observed from 31 in situ hybridisations. One replica of the library (number 125) has been transferred to the Resource Centre/Primary Database (RZPD) of the German Human Genome Project (DHGP). The whole library was gridded onto four nylon filters at high density for efficient identification of cosmid clones by colony hybridisation. Twenty-two probes were used for screening the library and each of them gave at least one positive signal. This result is in good agreement with a four-fold coverage of the genome as estimated from the insert length and number of recombinant clones. This library provides a powerful tool for rapid physical mapping and complex analysis of the chicken genome.     

Peptide labeling with photoactivatable trifunctional cadaverine derivative and identification of interacting partners by biotin transfer

A new photoactivatable trifunctional cross-linker, cBED (cadaverine-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate), was synthesized by chemical conversion of sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-(p-azidobenzamido) hexanoamido]ethyl-1,3′-dithiopropionate) with cadaverine. This cross-linker was purified by reversed-phase high-performance liquid chromatography (RP–HPLC) and characterized using matrix-assisted laser desorption/ionization time-of-flight (MALDI–TOF) analysis. cBED is based on sulfo-SBED that has a photoactivatable azido group, a cleavable disulfide bond for label transfer methods, and a biotin moiety for highly sensitive biotin/avidin detection. By ultraviolet (UV) light, the azido group is converted to a reactive nitrene, transforming transient bindings of interacting structures to covalent bonds. In contrast to the sulfo-N-hydroxysuccinimide (sulfo-NHS) moiety of sulfo-SBED, which attaches quite unspecifically to amino groups, cBED includes a cadaverine moiety that can be attached by transglutaminase more specifically to certain glutamine residues. For instance, thymosin β₄ can be labeled with cBED using tissue transglutaminase. By high-resolution HPLC/ESI–MS (electrospray ionization–mass spectrometry) and tandem MS (MS/MS) of the trypsin digest, it was established that glutamine residues at positions 23 and 36 were labeled, whereas Q39 showed no reactivity. The covalent binding of cBED to thymosin β₄ did not influence its G-actin sequestering activity, and the complex could be used to identify new interaction partners. Therefore, cBED can be used to better understand the multifunctional role of thymosin β₄ as well as of other proteins and peptides.     

<Emphasis Type="Italic">Mycosphaerella podagrariae</Emphasis>—a necrotrophic phytopathogen forming a special cellular interaction with its host <Emphasis Type="Italic">Aegopodium podagraria</Emphasis>

We present a new kind of cellular interaction found between Mycosphaerella podagrariae and Aegopodium podagraria, which is remarkably different to the interaction type of the obligate biotrophic fungus Cymadothea trifolii, another member of the Mycosphaerellaceae (Capnodiales, Dothideomycetes, Ascomycota) which we have described earlier. Observations are based on both conventional and cryofixed material and show that some features of this particular interaction are better discernable after chemical fixation. We were also able to generate sequences for nuclear ribosomal DNA (complete SSU, 5.8 S and flanking ITS-regions, D1–D3 region of the LSU) confirming the position of M. podagrariae within Mycosphaerellaceae.