Heterologous Production of Antimicrobial Peptides in Propionibacterium freudenreichii

Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P₄) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P₄ and P_pamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.     

Juvenile open angle glaucoma: fine mapping of the TIGR gene to 1q24.3–q25.2 and mutation analysis

Autosomal dominant juvenile open angle glaucoma (JOAG) is an early-onset form of primary open angle glaucoma (POAG), which has been linked to chromosome 1q21–q31. Recently, mutations in the trabecular meshwork inducible glucocorticoid response gene (TIGR), one of the candidate genes mapped in this region, were identified in glaucoma patients of several families. We screened for mutations of the TIGR gene in two German families with JOAG and in 100 unselected sporadic cases of POAG. In the first family we identified a Pro370Leu mutation and in the second family a Gly367Arg mutation cosegregating with the glaucoma phenotype. No pathogenic mutation was found in 100 sporadic cases but a Tyr347Tyr polymorphism was found in two patients. Furthermore, fluorescence in situ hybridization (FISH) analysis was used to map a TIGR-specific yeast artificial chromosome to 1q24.3–q25.2. Received: 19 June 1997 / Accepted: 12 August 1997     

Zur Innervation des glatten Penisretraktormuskels von Helix pomatia: Allgemeine Histologie und Histochemie des monoaminergen Nervensystems

Summary The penis retractor muscle of Helix pomatia is passed by thick nerve trunks, which probably are nerve nets. In the contracted muscle, they are folded meanderlike, in the extended they are pulled smooth. Four types of vesicles different in size and contents can be distinguished in the nerves.Varicose fibres accompany the muscle cells. Frequently the terminals are running in a groove of the muscle fibre. In certain axon types occur presynaptic accumulations of vesicles and thickenings of the terminal membrane. Terminal and muscle cell are separated by a cleft of 300 AE width. The muscle fibre membrane has no subsynaptic infoldings. Beside these axons thin, naked neurites are running through the connective tissue. They are characterized by a high content of neurotubuli.One part of the axons presumably possesses a monoaminergic transmitter. After the glutaraldehyde-dichromate-reaction they contain dense grana, whose diameters are mainly below 1000 AE. The nerve trunks fluoresce after exposure to paraformaldehyde vapour, excited with UV-light, green to green-yellow. The maximum of the excitation was determined at 413 nm and the maximum of emission at 496 respectively at 510 nm. It is concluded, that both, a catecholamine and 5-HT are responsible for the fluorescence. Extraction and paperchromatographic separation lead to the opinion, that the catecholamine is dopamine.

Auszug aus der Dissertation Histologische und histochemische Untersuchungen zur Innervation des glatten Penisretraktormuskels der Weinbergschnecke Helix pomatia L. (Göttingen, 1970). Auf Anregung von Herrn Prof. Dr. Fr.-W. Schlote, mit Unterstützung durch die Deutsche Forschungsgemeinschaft und die Akademie der Wissenschaften.     

Metabolism of polycyclic aromatic hydrocarbons in cultured human leukocytes under genetic control

Summary Metabolism of the polycyclic hydrocarbon benzo(a)pyrene(BP) to watersoluble products was measured in cultured human leukocytes from 35 healthy volunteers. The classification into 3 different groups with respect to degree of BP metabolism fits that obtained in a previous population and family study, where only the ratio of aryl hydrocarbon hydroxylase (AHH) activity of 3-methylcholanthrene (3MC) treated cultures to the activity of control cultures was measured. These data support previous observations that metabolism of polycyclic hydrocarbons is under genetic control.

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Zusammenfassung In der vorliegenden Arbeit wurde der Metabolismus des carcinogenen Kohlen wasserstoffes Benzpyren zu wasserlöslichen Produkten in Leukocytenkulturen von 35 gesunden Individuen untersucht. Hinsichtlich der Geschwindigkeit im Benzpyrenmetabolismus können 3 Gruppen unterschieden werden. Diese Einteilung ist in völliger Übereinstimmung mit der einer Populations-und Familienstudie, wo das Verhältnis der Aryl-Hydrocarbon-Hydroxylase-Aktivität von 3-Methylcholanthren-behandelten Leukocytenkulturen zu der von Kontrollkulturen bestimmt wurde. Diese Ergebnisse stützen die Beobachtung, daß der Metabolismus von carcinogenen Kohlenwasserstoffen unter genetischer Kontrolle ist.

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This work was supported in part by NIH Research Grant GM 15597 and Grant Ke 217/1 Deutsche Forschungsgemeinschaft (DFG).     

Two-photon excitation and photoconversion of EosFP in dual-color 4Pi confocal microscopy

Recent years have witnessed enormous advances in fluorescence microscopy instrumentation and fluorescent marker development. 4Pi confocal microscopy with two-photon excitation features excellent optical sectioning in the axial direction, with a resolution in the 100 nm range. Here we apply this technique to cellular imaging with EosFP, a photoactivatable autofluorescent protein whose fluorescence emission wavelength can be switched from green (516 nm) to red (581 nm) by irradiation with 400-nm light. We have measured the two-photon excitation spectra and cross sections of the green and the red species as well as the spectral dependence of two-photon conversion. The data reveal that two-photon excitation and photoactivation of the green form of EosFP can be selectively performed by choosing the proper wavelengths. Optical highlighting of small subcellular compartments was shown on HeLa cells expressing EosFP fused to a mitochondrial targeting signal. After three-dimensionally confined two-photon conversion of EosFP within the mitochondrial networks of the cells, the converted regions could be resolved in a 3D reconstruction from a dual-color 4Pi image stack.     

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Supplementation with phytase is an effective way to increase the availability of phosphorus in seed-based animal feed. The biochemical characteristics of an ideal phytase for this application are still largely unknown. To extend the biochemical characterization of wild-type phytases, the catalytic properties of a series of fungal phytases, as well as Escherichia coli phytase, were determined. The specific activities of the fungal phytases at 37°C ranged from 23 to 196 U · (mg of protein)⁻¹, and the pH optima ranged from 2.5 to 7.0. When excess phytase was used, all of the phytases were able to release five phosphate groups of phytic acid (myo-inositol hexakisphosphate), which left myo-inositol 2-monophosphate as the end product. A combination consisting of a phytase and Aspergillus niger pH 2.5 acid phosphatase was able to liberate all six phosphate groups. When substrate specificity was examined, the A. niger, Aspergillus terreus, and E. coli phytases were rather specific for phytic acid. On the other hand, the Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila phytases exhibited considerable activity with a broad range of phosphate compounds, including phenyl phosphate, p-nitrophenyl phosphate, sugar phosphates, α- and β-glycerophosphates, phosphoenolpyruvate, 3-phosphoglycerate, ADP, and ATP. Both phosphate liberation kinetics and a time course experiment in which high-performance liquid chromatography separation of the degradation intermediates was used showed that all of the myo-inositol phosphates from the hexakisphosphate to the bisphosphate were efficiently cleaved by A. fumigatus phytase. In contrast, phosphate liberation by A. niger or A. terreus phytase decreased with incubation time, and the myo-inositol tris- and bisphosphates accumulated, suggesting that these compounds are worse substrates than phytic acid is. To test whether broad substrate specificity may be advantageous for feed application, phosphate liberation kinetics were studied in vitro by using feed suspensions supplemented with 250 or 500 U of either A. fumigatus phytase or A. niger phytase (Natuphos) per kg of feed. Initially, phosphate liberation was linear and identical for the two phytases, but considerably more phosphate was liberated by the A. fumigatus phytase than by the A. niger phytase at later stages of incubation.     

Chiral recognition of verapamil by cyclodextrins studied with capillary electrophoresis,NMR spectroscopy,and electrospray ionization mass spectrometry

Capillary electrophoresis (CE) allows the observation of the opposite affinities of the enantiomers of (±)‐verapamil [2‐isopropyl‐2,8‐bis(3,4‐dimethoxyphenyl)‐6‐methyl‐6‐azaoctannitrile, VP] toward β‐cyclodextrin (β‐CD) and heptakis(2,3,6‐tri‐O‐methyl)‐β‐CD (TM‐β‐CD). In addition, in the presence of β‐CD in the background electrolyte, longer migration times and lower separation factors were observed compared to TM‐β‐CD. The binding constants of (+)‐ and (−)‐VP with β‐CD and TM‐β‐CD determined using ¹³C NMR spectroscopy explain the results observed in CE. Electrospray ionization mass spectrometry (ESI‐MS) was used as an alternative technique for the characterization of VP‐CD complexes. Chirality 11:635–644, 1999. © 1999 Wiley‐Liss, Inc.