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In this study, 30 strains of filamentous, non-heterocystous cyanobacteria from different habitats and different geographical regions assigned to diverse oscillatorian genera but here collectively referred to as members of the Phormidium group have been characterized using a polyphasic approach by comparing phenotypic and molecular characteristics. The phenotypic analysis dealt with cell and filament morphology, ultrastructure, phycoerythrin content, and complementary chromatic adaptation. The molecular phylogenetic analyses were based on sequences of the 16S rRNA gene and the adjacent intergenic transcribed spacer (ITS). The sequences were located on multiple branches of the inferred cyanobacterial 16S rRNA tree. For some, but not all, strains with identical 16S rDNA sequences, a higher level of discrimination was achieved by analyses of the less conserved ITS sequences. As shown for other cyanobacteria, no correlation was found between position of the strains in the phylogenetic tree and their geographic origin. Genetically similar strains originated from distant sites while other strains isolated from the same sampling site were in different phylogenetic clusters. Also the presence of phycoerythrin was not correlated with the strains' position in the phylogenetic trees. In contrast, there was some correlation among inferred phylogenetic relationship, original environmental habitat, and morphology. Closely related strains came from similar ecosystems and shared the same morphological and ultrastructural features. Nevertheless, structural properties are insufficient in themselves for identification at the genus or species level since some phylogenetically distant members also showed similar morphological traits. Our results reconfirm that the Phormidium group is not phylogenetically coherent and requires revision.

Dried herbarium specimens of cyanobacteria (exsiccata) deposited over 100 years ago were analysed and characterized using combined morphological and molecular approaches. Six representative coccoid and filamentous cyanobacteria from two historic collections and a 15-year-old air-dried environmental sample were studied. Morphological features observed by light and electron microscopy were correlated with the results of 16S rRNA gene sequencing. Historic identifications achieved by means of classical morphology could thus be confirmed by extracted, amplified and sequenced 16S rRNA gene fragments. The results of this study open the possibility of providing genotypic characterizations to botanical type specimens, thus reconciling the botanical and bacteriological approaches to the taxonomic treatment of these micro-organisms.

The carotenoid composition of 16 marine red algae from polar and temperate habitats was investigated. With the exception of Delesseria lancifolia, all showed a simple carotenoid pattern with one major xanthophyll, either lutein or zeaxanthin. Delesseria lancifolia exhibited a more complex pattern with three major xanthophylls. They were identified according to their retention times in high-performance liquid chromatography (HPLC) and their spectral characteristics as violaxanthin, antheraxanthin and zeaxanthin. Although these pigments are part of the xanthophyll cycle in higher plants and brown algae, we found no evidence for a short-term light-induced conversion among these pigments in Delesseria lancifolia. The pigment stoichiometry was unchanged after several days in different irradiances.

The High-Output Polymer Screening (HOPS) combines process-relevant automated reactor systems and rapid polymer characterization with on-line polymerization monitoring and automated data acquisition (electronic notebook) in order to make effective use of advanced data mining tools. This has led to the development of fingerprint technology based upon correlations between spectroscopic data and polymerization process conditions, catalyst compositions, as well as polymer end-use properties. Infrared spectroscopic fingerprints proved to be very useful for accelerating polymer analyses including characterization of polymer molecular architectures as well as non-destructive testing of the mechanical, thermal and other end-use polymer properties. Such spectroscopic fingerprints represent important components of effective on-line quality control systems. With ATR-FT-IR probes on-line monitoring of catalytic olefin copolymerization was performed in solution to measure in real time copolymerization kinetics, catalyst productivities, catalyst deactivation as well as copolymerization parameters and copolymer sequence distributions. Monomer consumption and comonomer incorporation were monitored simultaneously. Advanced fingerprint technology can reduce significantly the need for time- and money consuming polymer testing and can also stimulate the search for new catalyst systems and polymeric materials.

Wadden Sea inhabiting diatoms often show a patchy distribution on sediment surfaces with areas of higher and lower cell densities. The factors causing patchiness have not been clearly worked out and are under debate. Even the mode of reaching the sediment surface is almost completely unknown. The observations indicate that trails of individual diatoms might be used by others for upward directed migration and offer another plausible explanation for the existence of surfaces areas of higher and lower cell densities in general.

The composition of the diatom community on the bryozoans Electra pilosa, Membranipora membranacea, Flustra foliacea, and Alcyonidium gelatinosum from the German Bight was studied by light and scanning electron microscopy. In total, members of 26 diatom genera were found, with Cocconeis, Tabularia, Licmophora, Amphora, and Navicula being the most abundant. The amount and the composition of the diatom covering seem to be typical for single bryozoan species. Electra pilosa and Membranipora membranacea showed a rather dense covering with 71–547 cells/mm2 and 77–110 cells/mm2, respectively. The most prominent genus on Electra pilosa was Cocconeis, reaching up to 58% of all diatoms in one sample, followed by Navicula, Tabularia and Amphora. The most abundant genera on Membranipora membranacea were Tabularia and Licmophora, making up almost 70% of all diatoms in one sample, followed by Navicula, Cocconeis and Amphora. The diatom composition was very stable on all Electra samples, but varied on Membranipora samples. With

High-output polymer screening (HOPS) combines automated polymerization with online reaction monitoring, rapid polymer characterization and novel fingerprint technology useful in polymer preparation as well as polymer processing and polymer additive development. Originally, HOPS was introduced to develop polymerization catalysts and polyolefin materials more effectively. In comparison to conventional high-throughput screening, focusing on ultrahigh speed of catalyst screening using arrays of miniaturized reactors, output-oriented, process-relevant HOPS is aiming at generating and exploiting high information density (useful information/experiment). Catalyst systems for olefin polymerization are evaluated in automated workstations with multiparallel as well as semi- and fully automated, upgraded lab reactors. Automated polymerizations under standardized conditions afford large families of well-characterized polymers which serve as calibration samples for data analysis. Data analysis, using multivariate calibration, is the key to basic correlations between spectroscopic information and catalyst and polymer properties as well as reaction parameters and processing conditions. IR spectroscopic fingerprints are used to measure chemical copolymer composition, density, molecular weight as well as thermal and even mechanical properties. This fingerprint technology can be applied in online quality control and facilitates transfer from lab results into pilot and production plants. Fingerprint methods are important components of rapid online analysis and can reduce the need for time- and money-consuming polymer testing. Fingerprint technology combines spectroscopic analysis by means of “cheap” spectrometers with multivariate calibration.

A novel amino acid based dimethacrylate monomer (lysineurethanedimethacrylate, LUDM) was prepared by the addition of hydroxyethylmethacrylate (HEMA) to lysinediisocyanate (LDI). The structure was confirmed by FT-IR and 1H and 13C NMR spectroscopy as well as FAB-MS. Photopolymerized LUDM exhibited low volume shrinkage upon polymerization, good mechanical properties (Young's modulus: 3740 MPa) and high thermal stability. Osteoblast adhesion and growth on polymerized LUDM samples evidenced the biocompatibility. Further improvement of the mechanical properties was obtained by using Ca-hydroxyapatite as inorganic filler varying between 10 and 30 wt%. The Young's and flexural moduli increased with increasing filler content ranging from 3740 to 5250 MPa and from 2020 to 3690 MPa, respectively. The mechanical properties and the good biocompatibility of the lysine-based methacrylate networks make them interesting materials for medical applications, e.g. bone cements, and tissue engineering.

The melt miscibility of stereoregular polypropene with branched ethene homopolymers, prepared by Ni- and Pd-based catalysts, was investigated by means of transmission electron microscopy, atomic force microscopy, and differential scanning calorimetry. The number of branched C atoms per 1000 C atoms, referred to as degree of branching (DB), was varied from 6 to 112. Miscibility increases with increasing DB. Blends of branched ethene homopolymers and poly(ethene-co-1-octene) with isotactic (i-PP) and syndiotactic polypropylene (s-PP) were compared and show slightly improved miscibility for s-PP. For DBs of 98 and 112 partial miscibility with polypropene was found. The miscibility of polyethenes with longer branches resemble that of polyethene/1-octene copolymers, whereas short branched polyethenes behave more like polyethene/1-butene copolymers.

As a consequence of developing fully automated reactors for organic and organometallic synthesis and polymerizations combined with rapid on-line analysis, databases, and data mining, the analysis of polymers with respect to composition and properties has been speeded up. High-throughput evaluation of olefin copolymers requires fast measurements and high accuracy without tedious sample preparation such as pressing KBr pellets. This has been achieved by using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR spectroscopy) in conjunction with multivariate calibration in order to determine the composition of olefin copolymers such as ethene/propene, ethene/1-hexene and ethene/1-octene copolymers.

Recently [Marquardt et al. (2000) Gene 255: 257–265], we isolated a gene encoding a polypeptide of the light-harvesting complex of Photosystem I (LHC I) of the red alga Galdieria sulphuraria. By screening a G. sulphuraria cDNA library with a DNA probe coding for the conserved first transmembrane helix of this protein we isolated four additional genes coding for LHC I polypeptides. The deduced preproteins had calculated molecular masses of 24.6–25.6 kDa and isoelectric points of 8.09–9.82. N-terminal sequencing of a LHC I polypeptide isolated by gel electrophoresis allowed us to determine the cleavage site of the transit peptide of one of the deduced polypeptides. The mature protein has a calculated molecular mass of 20.6 kDa and an isoelectric point of 7.76. The genes were amplified from nuclear G. sulphuraria DNA by polymerase chain reaction (PCR) using oligonucleotides annealing in the regions of the start and stop codons as primers. All genomic sequences were 80–300 base pairs longer than the PCR products obtained from the respective cDNA clones, pointing to the existence of 1–5 introns per gene. The G. sulphuraria genes form a homogeneous gene family with overall pairwise amino acid identities of 46.0–56.6%. Homology to two diatom, one cryptophytic and two higher plant light-harvesting polypeptides was lower with pairwise identities of 21.1–34.1%. Only one diatom polypeptide showed a higher degree of identity of up to −39.3%.

Genes for light-harvesting proteins ( lhc genes) of higher plants are well examined. However, little is known about the corresponding genes of algae, although this knowledge might give valuable information about the evolution of photosynthetic antennae. In the case of rhodophytes only two cDNA sequences from a single organism, Porphyridium cruentum , have been published. Here we describe an additional sequence from another species, the thermo-acidophilic red alga Galdieria sulphuraria . For the first time also a genomic sequence for a red algal lhc gene is presented. From a cDNA library of G. sulphuraria we isolated a clone containing an open reading frame for a protein of 302 amino acids with a deduced molecular mass of 33.86 kDa. It shares major structural features with eukaryotic light-harvesting polypeptides. A proposed cleavage site between transit peptide and mature protein gives rise to a transit peptide of 119 amino acids and a mature protein of 183 residues. Hydropathy analysis suggests that the mature protein consists of three transmembrane helices. Several amino acid residues supposed to bind chlorophyll a and chlorophyll b in higher plants are conserved. The protein shows up to 69% identity and 81% similarity to the Porphyridium polypeptides in the transmembrane helices 1 and 3. Using oligonucleotides annealing in the regions of the start and stop codons of the gene as primers, a DNA sequence was amplified from nuclear G. sulphuraria DNA by PCR. Compared with the cDNA clone, this sequence contains five additional intervening DNA strings of 50–74 bp length. Four of them show typical features of spliceosomal introns with GT–AG borders, and the fifth differs by starting with GC. Three of the supposed introns are located in similar positions as introns of higher plant light-harvesting proteins. Southern blotting and hybridization experiments indicate that G. sulphuraria contains at least three copies of this gene.

Thylakoid membranes of the cryptophyte Rhodomonas sp. were solubilized with the mild detergent dodecyl-β-maltoside and subjected to sucrose density gradient centrifugation. The resulting gradients showed six pigment-bearing bands which were characterized further by means of absorption and fluorescence emission (77K) spectroscopy, polyacrylamide gel electrophoresis and Western immunoblotting. Two of the bands showed characteristics of light-harvesting complexes, other bands could be attributed to photosystem II and photosystem I. Up to 10 different light-harvesting proteins could be identified, some of which are specific for photosystem I, others for photosystem II. The polypeptides of the light-harvesting complex of photosystem II show a higher chlorophyll c/a ratio than the antenna proteins of photosystem I. As in vascular plants, they represent the bulk of the membrane-intrinsic light-harvesting proteins.

We analyzed the effects of iron deficiency on the ultrastructure and biochemical composition of the photosynthetic apparatus of the marine alga Rhodella violacea (Kornmann) Wehrmeyer (Porphyridiale...

We investigated the localization, structure and function of the biliproteins of the oxygenic photosynthetic prokaryote Acaryochloris marina, the sole organism known to date that contains chlorophyll d as the predominant photosynthetic pigment. The biliproteins were isolated by means of sucrose gradient centrifugation, ion exchange and gel filtration chromatography. Up to six biliprotein subunits in a molecular mass range of 15.5–18.4 kDa were found that cross-reacted with antibodies raised against phycocyanin or allophycocyanin from a red alga. N-Terminal sequences of the α- and β-subunits of phycocyanin showed high homogeneity to those of cyanobacteria and red algae, but not to those of cryptomonads. As shown by electron microscopy, the native biliprotein aggregates are organized as rod-shaped structures and located on the cytoplasmic side of the thylakoid membranes predominantly in unstacked thylakoid regions. Biochemical and spectroscopic analysis revealed that they consist of four hexameric units, some of which are composed of phycocyanin alone, others of phycocyanin together with allophycocyanin. Spectroscopic analysis of isolated photosynthetic reaction center complexes demonstrated that the biliproteins are physically attached to the photosystem II complexes, transferring light energy to the photosystem II reaction center chlorophyll d with high efficiency.

Summary A Galdieria sulphuraria (Galdieri) Merola strain that retains its photosynthetic apparatus in the dark was treated with the inhibitor of carotenoid biosynthesis, norflurazon, under autotrophic and heterotrophic conditions. Under autotrophic conditions cultures were not able to grow. The chlorophyll content of the cells decreased constantly and a partial decomposition of their chloroplast structure could be observed. In the dark, under heterotrophic conditions, the inhibitor caused a nearly total loss of carotenoids, a reduction of the chlorophyll content per cell, a slight increase of the ratio of phycocyanin to chlorophyll and an accumulation of a pheophorbide a -like pigment. The ultrastructure of the algae, however, was not affected. The polypeptides of the photosystem II core-complex and of the light-harvesting complex of photosystem I were dramatically reduced, indicating that carotenoids are essential for their assembly. The core-complex of photosystem I was present. Its fluorescence maximum, however, was blue-shifted from 725 to 717nm. When excited at 580 nm norflurazon-treated cells exhibited a fluorescence maximum at 686 nm, indicating that phycobilisomes are energetically neither connected with photosystem II nor with photosystem I. Nevertheless, phycobilisomes of norflurazon-treated and control algae were not distinguishable.

Phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris marina, containing chlorophyll d as major pigment. In the electron microscope the biliprotein aggregates appear as rod-shaped structures of 26.0×11.3 nm, composed of four ring-shaped subunits 5.8 nm thick and 11.7 nm in diameter. Spectral data indicate that the aggregates contain two types of biliproteins: phycocyanin and an allophycocyanin-type pigment, with very efficient energy transfer from the phycocyanin- to allophycocyanin-type constituent. The chromophore-binding polypeptides of the pigments have apparent molecular masses of 16.2 and 17.4 kDa. They crossreact with antibodies against phycocyanin and allophycocyanin from a red alga.

The membrane-intrinsic light-harvesting complex of the red alga Galdieria sulphuraria (formerly Cyanidium caldarium ) could be isolated by gel-electrophoresis as a green band with an apparent molecular mass of about 20 kDa. The band had a long-wavelength absorption maximum at 672 nm and a fluorescence maximum (77 K) at 680 nm and reacted with an antibody against light-harvesting proteins of higher plant Photosystem I. Screening of thylakoid membranes with antisera directed against various chlorophyll a/b and chlorophyll a/c light-harvesting proteins indicated the existence of at least 4 distinct light-harvesting polypeptides with apparent molecular masses between 17 and 20 kDa. Isolation of Photosystem I and of a fraction enriched in Photosystem II showed that these polypeptides are exclusively bound to Photosystem I, thus forming a holocomplex which binds at least 205 molecules of chlorophyll a , and 33 and 37 molecules of zeaxanthin and β-carotene, respectively. Additionally, there is some evidence for the existence of a second Photosystem I pool without light-harvesting complexes. In-vitro translation experiments showed that at least two of the five polypeptides which constitute the membrane-intrinsic light-harvesting complex of Galdieria sulphuraria are translated from the poly(A)-enriched RNA fraction. They could be immunoprecipitated as preproteins being 3 to 4 kDa larger in size than the mature polypeptides.

The main chlorophyll a/c light harvesting complex of the diatom Cyclotella cryptica was isolated by sucrose density gradient centrifugation. It consisted of two polypeptides of Mrs 18000 and 22000. Both polypeptides and fragments thereof, obtained by formic acid treatment, were blocked at their N-ter-mini. An antiserum raised against the two subunits selectively immunolabeled the thylakoid within the chloroplasts. The subunits were nuclear encoded and could be immunoprecipitated from poly (A)+ RNA as precursor proteins in the Mr range of 20000 to 24000. The existence of minor chlorophyll protein complexes and their possible function in light climate adaptation processes was investigated in cells adapted to low light and high light conditions. Low light grown cells contained more fucoxanthin and less β-carotene relative to chlorophyll a than high light adapted cells. The xanthophyll cycle pigments diatoxanthin and diadinoxanthin increased five-fold relative to chlorophyll a under high light conditions. Western-immunoblotting experiments with antisera raised against several chlorophyll a/b and chlorophyll a/c antenna complexes demonstrated that, beside the dominating chlorophyll a/c light harvesting complex, minor antenna complexes might exist, which, in part, seem to react to the light climate applied.

Recent investigations have shown the existence of a light-harvesting complex (LHC) of photosystem I (PSI) with a fluorescence maximum at 680 nm in the red alga Porphyridium purpureum (Wolfe et al., Nature, 367 (1994) 566–568). We have attempted to determine whether the composition of this red algal LHC is invariable or subject to light acclimatization and whether the 680–690 nm fluorescence band of intact algae, which is usually ascribed to photosystem II (PSII), could be partially due to the LHC of PSI. For this purpose, we characterized PSI core complexes and PSI holocomplexes consisting of core and LHC from algae grown under red or green light conditions and calculated the in situ spectra of the photosystems by normalizing the thylakoid spectra of red and green light algae to an identical PSI or PSII concentration. The composition of the LHC seems to be independent of the light quality, since no significant difference was found in the antenna size and the pigment composition of the PSI holocomplexes from red and green light algae; both holocomplex preparations contained 150–160 chlorophylls, 21–22 zeaxanthins and 23–25 β-carotenes per reaction centre, while the core complexes contained approximately 90 chlorophylls, 15 carotenes and no zeaxanthin. Both holocomplex preparations had a fluoroscence maximum (77 K) at 718 nm. A short-wavelength emission band, as reported by Wolfe et al., was not found in the spectra of the isolated PSI complexes or in the PSI spectrum calculated from thylakoid spectra. This indicates that the LHC does not contribute to the fluorescence spectrum of intact cells, but transfers energy very effectively to the PSI core complex.

Twenty samples were taken from the inner or outer surfaces of stone monuments of six historic Scottish buildings and ruins. Biofilms developing on mineral substrates were analysed by in situ scanning electron microscopy and cultivation. Various methods were used to characterize the isolates including automated ribotyping, RAPD and sequencing of the 16S rRNA gene for bacteria, and stereomicroscopy and sequencing of the Internal Transcribed Spacers (ITS) for fungi. Most samples contained microbes between 10(5) and 10(7)cfug(-1) substrate. Actinobacteria belonging to the genus Streptomyces (17 samples/5 monuments) or Arthrobacter (12/3) and Pseudomonas (9/3) were frequently detected. Most streptomycetes were in terms of their 16S rRNA gene sequence most closely related to S. microflavus (10/3) or to the undescribed species S. "vulgaris" (8/3). Indoor and outdoor biofilms exhibited significant differences in their microbiota, as shown by both microscopy and isolation studies. Pigmented coccoid Arthrobacter species were typical for the outdoor samples, whereas Pseudomonas species were common in the indoor samples. Based on the low phylogenetic relationship to a known species (type strain), potential novel pigmented bacterial species belonging to the genera Arthrobacter, Brevundimonas, Cryseobacterium, Deinococcus and Dyadobacter were detected from the outdoor samples and to Pseudomonas from the indoor samples. Hyaline fungal species of Acremonium (10/4) mainly occurred in indoor samples, whereas pigmented species of Cladosporium (8/3), Penicillium (6/3) and Phialophora (6/2) were found outdoors. Using in situ microscopy diatom algae were also detected.

Solubilization of thylakoid membranes of Cyclotella cryptica with dodecyl-beta maltoside followed by sucrose density gradient centrifugation or deriphate polyacrylamide gel electrophoresis resulted in the isolation of pigment protein complexes. These complexes were characterized by absorption and fluorescence spectroscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western immunoblotting using antisera against fucoxanthin chlorophyll a/c-binding proteins and the reaction center protein D2 of photosystem II. Sucrose density gradient centrifugation yielded four bands. Band 1 consisted of free pigments with minor amounts of fucoxanthin chlorophyll a/c-binding proteins. Bands 2, 3, and 4 represented a major fucoxanthin chlorophyll a/c-binding protein fraction, photosystem II, and photosystem I, respectively. Deriphate polyacrylamide gel electrophoresis gave rise to five bands, representing photosystem I, photosystem II, two fucoxanthin chlorophyll a/c-binding protein complexes, and a band mostly consisting of free pigments. In the Western immunoblotting experiments, the specific association of two fucoxanthin chlorophyll a/c-binding proteins, Fcp2 and Fcp4, to the photosystems could be demonstrated. In vivo experiments using antibodies against phosphothreonine residues and in vitro studies using [gamma-32P]ATP showed that fucoxanthin chlorophyll a/c binding-proteins of 22 kDa became phosphorylated.

The steady-state mRNA concentrations of two fcp genes encoding fucoxanthin chlorophyll a/c light-harvesting polypeptides of the centric diatom Cyclotella cryptica were investigated over a 4-day period by RNA dot-blotting experiments. Before and during the first day of the experiment, the cultures were grown under a 12-h light/12-h dark regime. On the following 3 days, the algae were kept in darkness. On the first day, the steady-state mRNA concentration of fcp2 followed a diurnal pattern, with a maximum occurring around noon, approximately 6 h after the onset of light. The gene fcp6 also had a diurnal pattern on the first day. Its maximum, however, occurred immediately after the onset of light. During the subsequent incubation period in darkness, the diurnal pattern of expression of both fcp genes continued, thus demonstrating that their steady-state mRNA concentrations oscillated in a circadian manner.

A method was established to investigate the steady state levels of mRNAs from genes encoding fucoxanthin chlorophyll a/c binding proteins (Fcp) of diatoms in situ. During the study, which was performed with Wadden Sea sediments from the German North Sea shore near Dangast, oxygenic photosynthesis was carried out mainly by pennate diatoms. Field samples were taken after tidal exposure from dawn up to late afternoon at 2-hourly intervals, and frozen in liquid nitrogen. In the laboratory, total RNA was isolated by isopycnic ultracentrifugation in caesium chloride gradients. Yields of approximately 10-300 micro g RNA per gram wet sediment were obtained. Defined amounts of total RNA were blotted onto nylon membranes and hybridised with probes against the fcp2 and 18S rDNA genes of Cyclotella cryptica. To estimate the steady state amount of fcp mRNAs, fcp signal intensities were normalized to the signal intensities obtained from hybridisation to an 18S rDNA gene probe. In the two time-course studies performed to demonstrate the applicability of the method, the steady state levels of fcp mRNA increased up to 12-fold with the onset of light, reaching a maximum 6-8 h after sunrise before they decreased again. Possible reasons for this time-course are discussed.

The centric diatom Cyclotella cryptica and two strains of the pennate diatom Phaeodactylum tricornutum were grown under low and high light intensities (300 lux and 3,000 lux) over 4–6 weeks. Growth was monitored by repetitive cell count. The culture media were replaced weekly to avoid morphological and biochemical alterations caused by nutrient depletion. The ultrastructure of the cells was examined by transmission electron microscopy. Alterations in the light-harvesting antenna systems were investigated by Western immunoblotting. Both diatoms reduced the plastid area, i.e. decreased the amount of thylakoid lamellae, under high light intensity. The thylakoids still ran in groups of three with parallel orientation within the chloroplasts. The girdle band lamellae were not affected. The amounts of storage compounds and vacuoles increased. SDS-PAGE of total cell protein followed by Western immunoblotting with antisera directed against subunits of the light-harvesting antenna systems of C. cryptica (cc-antiserum) and the cryptophyte Cryptomonas maculata (cmac-antiserum) revealed that both diatoms reduced the amount of antenna polypeptides under increased light intensity. The cc-antiserum immunodecorated two bands with relative molecular masses (M r) of 18,000 and 22,000 in C. cryptica. Both decreased under high light conditions to 67.2±6.1%. Five to seven bands in the M r range of 14,000–27,000 were recognized in P. tricornutum. They decreased to 83±5.3%. Furthermore, the immunolabeling pattern for both strains differed under the two light regimes. The cmac-antiserum immunodecorated two polypeptides with M r of 24,000 and 23,000 in C. cryptica, while both strains of P. tricornutum had five polypeptides in the M r range of 14,000–24,000 that showed some differences in staining intensities between the two strains and in response to the light intensity applied.

Cyanobacteria are photosynthetic prokaryotes with two photosystems (PSI and PSII). As higher plants, they use water as primary electron donor of the photosynthetic electron transport chain. The organization of their thylakoid membranes, however, differs significantly from that in higher plants. Usually, cyanobacterial thylakoids are unstacked and covered with phycobilisomes (PBS) which serve as antenna complexes of PSII. Phycobilisomes are normally constructed of a three-cylindrical core unit containing allophycocyanin (AP) from which several peripheral rods radiate. These rods are composed of phycocyanin, either alone or in combination with phycoerythrin (PE) or phycoerythrocyanin (PEC). For a schematic representation of a phycobilisome, see Fig. 1 A.

The phycobiliproteins (PBP) are the major light-harvesting pigments characteristic of cyanobacteria, red algae and cryptophytes. These pigments fall into four general classes, based upon their long wavelength absorption maxima: allophycocyanin (APC), phycocyanin (PC), phycoerythrin (PE) and phycoerythrocyanin (PEC). In cyanobacteria and red algae, the PBP are aggregated in the form of phycobilisomes (PBS) which have been shown to be associated with photosystem II (PSII) forming a PSII-PBSsupercomplex at the cytoplasmic or stromal side of thylakoid membranes. In cryptophytes, however, the PBP (which can be either PE or PC, having no APC) do not form PBS and are located in the thylakoid lumen. Functionally, a generally accepted pathway of energy transfer in cyanobacteria and red algae is from PE and/or PEC via PC and APC to chlorophyll (chl) a with almost 100% efficiency. In cryptophytes, PBP might transfer excitation energy directly to chi a, or in conjunction with chi c overlaps the chi a absorption spectrum of the reaction centers (1). Acaryochloris marina is the sole oxygenic prokaryote known to date that contains chl d as the predominant photosynthetic pigment and PBP and chi a as minor pigments (2). Earlier studies showed that the native PBP aggregates contain PE, PC and APC, which do not form PBS in vivo, but rather resemble the peripheral rods of PBS of other cyanobacteria and red algae (3). In this communication we address the location of the PBP with regard to thylakoid membranes and on the energy transfer chain from PBP to the photochemical reaction center pigments.

Phycobiliproteins (PBP) are major antenna pigments of cryptophytes, rhodophytes and cyanobacteria. According to their chemical structure and spectral properties four major classes of PBP can be distinguished: phycoerythrin (PE), phycoerythrocyanin (PEC), phycocyanin (PC) and allophycocyanin (AP). In cyanobacteria and red algae PBP are organized as phycobilisomes (PBS), large complexes (molecular mass 5000-30000 kDa) that are attached to photosystem II at the cytoplasmic or stromal side of the thylakoid membranes. They are normally constructed of three-cylindrical core units from which several peripheral rods radiate. The core cylinders contain AP while the peripheral rods are composed of PC, either alone or with PE or PEC. The basic unit of the biliproteins is a heterodimer composed of an α- and a β-subunit with molecular masses between 15 and 22 kDa. They are aggregated in trimers of the structure (αβ)3,which in turn form hexamers by a tight face-to-face-association. For a review on PBS structure, see [1]. In cryptophytes PBP are located in the thylakoid lumen. Here, their supramolecular organization is not yet fully understood.

It has recently been found that the quaternary structure of photosystem I (PSI) of cyanobacteria differs significantly from that of higher plants. Isolated PSI from higher plants appears as the monomeric complex surrounded by approx. 8 LHC I molecules (1), whereas the reaction centers purified from cyanobacteria and a prochlorophyte exist in various oligomeric forms, the major ones being the monomeric and trimeric forms (2,3). The recent discovery of a new type of oxygenic prokaryote Acaryochloris marina that contains chlorophyll d as a major pigment along with minor contents of chlorophyll a and phycobiliproteins has drawn much attention with respect to the structure and function of photosynthetic systems (4,5,6,7). Recently we have isolated a PSI complex from A. marina, which uses chl d as the primary electron donor (designated P740 after its peak wavelength) absorbing infra-red light at 740 nm rather than visible red light of 700 nm utilized by a special pair of chl a (P700) in chl a-based PSI of cyanobacteria and higher plants (8). In this study, we investigated the quaternary structure of PSI by using HPLC and electron microscopy, and have identified the oligomeric formation of PSI complexes in this organism.

We studied the organization of the antenna system of maize (Zea mays L.) seedlings grown under intermittent light conditions for 11 d. These plants had a higher chlorophyll-a/b ratio, a higher ratio of carotenoids to chlorophyll and a lower ratio of chlorophyll to protein than plants grown in continuous light. We found all chlorophyll-protein complexes of maize to be present. However, the minor chlorophyll a/b-proteins CP29 and CP26, and to a greater extent CP24 and the major light-harvesting complex II were reduced relative to the photosystem (PS) II core-complex. Also the chlorophyll a/b-antennae of PSI were reduced relative to the reaction-centre polypeptides. When isolated by flatbed isoelectrofocussing, the chlorophyll-a/b complexes of PSII showed a higher chlorophyll-a/b ratio and a lower ratio of chlorophyll to protein than the same complexes from continuous light; additionally, they bound more carotenoids per protein than the latter. Thus the altered organization of the photosynthetic apparatus of plants from intermittent light is caused by two different factors: (i) the altered stoichiometry of chlorophyll-binding proteins and (ii) a different ratio of pigment to protein within individual chlorophyll-proteins.

The distribution of the photosynthetic pigments of the chlorophyll-binding proteins or photosystem-II membranes, isolated from dark-adapted maize leaves was determined. Most (80%) of a xanthophyll, violaxanthin, was found in the three minor chlorophyll-a/b proteins CP24, CP26 and CP29 whose function is unknown. Violaxanthin is the precursor of zeaxanthin, which is involved in dissipating excess excitation energy into heat [Demmig-Adams, B. (1991) Biochim. Biophys. Acta 1020, 1-24] under conditions of high transmembrane pH gradient [Gilmore, A. M. & Yamamoto, H. Y. (1992) Proc. Natl Acad. Sci. USA 89, 1899-1903]. We propose that a role for the minor photosystem-II chlorophyll-a/b proteins is the regulation of energy transfer to the reaction centre. It was also confirmed that the photosystem II reaction centre (D1-D2-cytochrome b559) contains beta-carotene as the only carotenoid. However, the two other chlorophyll-a-binding proteins of photosystem II, CP47 and CP43, bind not only beta-carotene, but also the xanthophyll lutein, previously thought to be restricted to chlorophyll-a/b proteins.

The influence of recombinant equine interferon-beta 1 (rEqIFN-beta 1) on mononuclear cells of peripheral blood (PBMC) and polymorphonuclear neutrophilic granulocytes (PMN) was tested under in vitro and ex vivo conditions. Treatment of equine PBMC with IFN in vitro enhanced the antibody-independent cytotoxicity (AICC) and antibody-dependent cytotoxicity (ADCC) while there was no significant effect on the cytotoxic capacity of PMN treated with rEqIFN-beta 1 in vitro. Ex vivo there was an increased capacity of AICC and ADCC upon single or multiple application of rEqIFN-beta 1 in PMN, only. Treatment with rEqIFN-beta 1 thus induced an increased cellular cytotoxicity in vitro and in vivo but in different populations of peripheral blood cells. In vivo rEqIFN-beta 1 causes a pronounced activation of PMN but not of PBMC as cytotoxic effector cells. This might be achieved indirectly, e.g., by cytokines produced by IFN-sensitive cells.

We wished to assay recombinant equine interferon-β1, (rEqIFN-β1) but could not obtain satisfactory results with previously described methods. Therefore, we developed a yield-reduction assay, using primary horse peripheral blood mononuclear cells (PBMC) with vesicular stomatitis virus (VSV) for challenge, which proved consistently satisfactory and highly sensitive. It is suggested that this method of assay may be useful for IFNs from other animals where problems are encountered.

In angiosperms, chlorophyll synthesis is light dependent. Thus growth under intermittent light conditions results in a limitation of chlorophyll available. Chlorophyll, however, seems to be necessary for the stabilisation of chlorophyll-binding apoproteins1,2 and there are many reports on a reduced antenna size in plants grown in intermittent light3,4. To have more detailed information about the antenna system of these plants we investigated the stoichiometry of chlorophyll-binding proteins of PSII and the pigment composition of the light-harvesting complexes. Since limitation of chlorophyll is characteristic also for an early stage of chloroplast development these data might serve to give an insight in PSII development even under normal growth conditions.

Two green fractions, thought to represent the chlorophyll-antennae of photosystems I (PSI) and II (PSII), were isolated from the red alga Porphyridium purpureum by solubilisation of the thylakoid membranes using the detergent N-lauryl-s-iminodipropionate and subsequent sucrose-density-gradient centrifugation. No release of pigments from the pigment-protein complexes was detected during isolation. The fractions were analyzed with respect to their chlorophyll-protein pattern, spectral properties and pigment composition. The supposed PSII antenna fraction contained both the major carotenoids of P. purpureum, β-carotene and zeaxanthin, and showed a long-wavelength absorption maximum at 672 nm and a low-temperature fluorescence maximum at 692-694 nm. Polypeptides of this fraction cross-reacted with antibodies raised against the PSII polypeptides D1, CP43 and CP47 from higher plants. The PSI fraction could perform P-700 photooxidation and showed a long-wavelength absorbance maximum at 679 nm and a low-temperature fluorescence maximum at 718 nm. It contained β-carotene as the only carotenoid. The fluorescence excitation spectrum of the fraction and measurements of the photochemical activity of a thylakoid preparation excited with light that is preferentially absorbed either by chlorophyll (433 nm) or by carotenoids (495 nm) indicate that β-carotene serves as a very efficient antenna-pigment in PSI. In contrast, only a small amount of energy transfer from the carotenoids to chlorophyll could be observed with the supposed PSII fraction.

Ein einfaches Verfahren zur Reinigung des Vaccinevirus wird beschrieben. Das Virus wird auf der Kaninchenhaut zur Vermehrung gebracht, durch Zentrifugieren gewaschen und anschliesend in einer Emulsion aus Puffer und Frigen 113 gefallt. Aus dem Emulgat lassen sich mit Puffer kolloidhaltige, stabile Suspensionen eluieren mit Wasser kolloidfreie, instabile Fraktionen von sehr hohem Reinheitsgrad. Etwa ein Drittel des Ausgangsmaterials wird als gereinigtes Virus erhalten. Die einzelnen Reinigungsschritte werden elektronenmikroskopisch kontrolliert und an Hand von Partikelzahlungen und Trockengewichts-bestimmungen quantitative Angaben gemacht. Das gereinigte Virus besitzt normale biologische Qualitat; die alteren Werte der Literatur fur DNA- und Proteingehalt wurden bestatigt. Aus elektronenmikroskopischen Messungen resultierte fur den dehydratisierten Elementarkorper ein Volumen von 8, 37·10−15 cm3; seine Dichte ergibt sich zu 0,68g/cm3, die mittlere Dichte der Bauelemente zu 1,31g/cm3. Die mittlere Masse des dehydratisierten Elementarkorpers bestimmten wir zu 5,69·10−15g.

Antigenic materials prepared from vaccinia infected tissues or cell cultures have been studied by diffusion-in-gel methods. Using an immune serum produced by hyperimmunization of a rabbit and employed as a reference serum and a material prepared from vaccinia infected rabbit skin, employed as a reference antigen, 8 separate precipitinogenic factors were revealed. The immunological relation between these factors and those produced in vaccinia or variola infected CAM, vaccinia infected HeLa cells and vaccinia infected bovine embryonic skin-muscle tissue was analyzed.

Discussion and summaryThe reported experiments form part of a major study of the pattern of virus particle-associated and of soluble antigens, related to vaccinia infection. To study the antigenic composition of the vaccinia virus particle a purified virus material freed from soluble antigen was required. Principally, the method of purification described is a combination of the one used by Marquardt et al(7) and fraction-ation in a sucrose density gradient. Electron microscopy indicated that very little particu-late impurity was present in the virus material banded in the gradient and washed by centrifugation. In the purified material, containing about 2 mg of virus particles per ml, no hemagglutinin and precipitinogenic factors were demonstrable. Ultrasonic treatment immediately preceding the diffusion-in-gel tests was found important as it increased the reactivity of the antigenic preparations.The lowest concentration of vaccinial pre-cipitinogens necessary for giving a visible reaction in the double di...

Vaccinial immunoprecipitinogens, referred to as the e-f antigens were found to have some immunological and chemical properties attributed to the classical LS antigen of vaccinia virus. The e-f antigens were isolated and purified from vaccinia virus infected chorioallantoic membranes (CAM). The technique used for separation involved chromatography on DEAE-cllulose, gelfiltration on Sephadex G-200 and separation on hydroxylapatite. The amino acid composition of the antigens was compared with that of CAM and preparations of purified vaccinia virus. Sedimentation analysis indicated the relationship between the e-f antigens and a 6 S component with a molecular weight calculated to 96,600. Immunodiffusion studies suggested that the e-f antigens contained three immunoprecipitinogens, two of which were found to be thermo-resistant but sensitive to trypsin, while the third was a heat-labile antigen relatively more stable against trypsin digestion.

Vaccinia virus particles and soluble vaccinial antigens were separated. Preparations of soluble antigens contained nine different immunoprecipitating factors. Mechanical disintegration of vaccinia virus, freed from soluble antigens, released eight precipitinogens all of which corresponded immunologically to precipitinogens demonstrable among the soluble antigens. Purified virus was treated with pepsin and subsequently by alkaline hydrolysis. The release of different precipitinogenic factors by these treatments was studied in relation to alterations in the morphology of virus. The results suggested that there were specific precipitinogenic activities associated with the peripheral protein layer and the nucleoid of the vaccinia virion. Precipitinogens released from the peripheral protein layer showed properties of vaccinia LS antigen. Vaccinia hemagglutinin seemed to play no role in the development of the immunoprecipitates studied.

Purified vaccinia virus freed from demonstrable hemagglutinating activity was inactivated and, subsequently, disintegrated by treatment with pepsin. The preparations obtained were examined in the electron microscope and tested for hemagglutinating and precipitating activity as well as for their immunogenicity. The release of hemagglutinating activity from the virions was dependent upon the concentration of pepsin used. Precipitinogens and antigen capable of inducing complement fixing antibodies in rabbits were released by low concentrations of pepsin. Formation of neutralizing and especially hemagglutination inhibiting antibodies was induced by preparations obtained by treatment with higher concentrations of pepsin. Immunization with one mg, but not with 0.1 mg, of inactivated but undigested virions resulted in formation of hemagglutination inhibiting antibodies. The site of a hemagglutinin component in the vaccinia virion is discussed.

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