Review: origin of complex algae by secondary endosymbiosis: a journey through time.

Secondary endosymbiosis-the merging of two eukaryotic cells into one photosynthetic cellular unit-led to the evolution of ecologically and medically very important organisms. We review the biology of these organisms, starting from the first proposal of secondary endosymbiosis up to recent phylogenetic models on the origin of secondarily evolved protists. In addition, we discuss the organelle character of the symbionts based on morphological features, gene transfers from the symbiont into the host and re-import of nucleus-encoded plastid proteins. Finally, we hypothesize that secondary endosymbiosis is more than enslaving a eukaryotic, phototrophic cell, but reflects a complex interplay between host and symbiont, leading to the inseparability of the two symbiotic partners generating a cellular entity.

Aging effects of plasma polymerized ethylenediamine (PPEDA) thin films on cell-adhesive implant coatings.

Thin plasma polymer films from ethylenediamine were deposited on planar substrates placed on the powered electrode of a low pressure capacitively coupled 13.56 MHz discharge. The chemical composition of the plasma polymer films was analyzed by Fourier Transform Infrared Reflection Absorption Spectroscopy (FT-IRRAS) as well as by X-ray photoelectron spectroscopy (XPS) after derivatization of the primary amino groups. The PPEDA films undergo an alteration during the storage in ambient air, particularly, due to reactions with oxygen. The molecular changes in PPEDA films were studied over a long-time period of 360 days. Simultaneously, the adhesion of human osteoblast-like cells MG-63 (ATCC) was investigated on PPEDA coated corundum blasted titanium alloy (Ti-6Al-4V), which is applied as implant material in orthopedic surgery. The cell adhesion was determined by flow cytometry and the cell shape was analyzed by scanning electron microscopy. Compared to uncoated reference samples a significantly enhanced cell adhesion and proliferation were measured for PPEDA coated samples, which have been maintained after long-time storage in ambient air and additional sterilization by γ-irradiation.

Influence of age and sex in exhaled breath samples investigated by means of infrared laser absorption spectroscopy.

Breath gas analysis provides insight into human metabolism of healthy and ill individuals. As an innovative and non-invasive method, it opens up options to improve diagnostics, monitoring and treatment decisions. Mid-infrared laser absorption spectroscopy is utilized to detect CH(4), H(2)O, CO(2), NH(3) and CH(3)OH in exhaled human breath. An off-line approach using breath sampling by means of Tedlar bags is applied. The breath gas samples are measured within the population-based epidemiological Study of Health in Pomerania (SHIP-TREND) performed at the University of Greifswald. The study covers about 5000 adult subjects aged 20-79 years within 3 years. Besides breath gas analysis many other examinations are conducted. It is expected to find associations between distinct concentration levels of species in the exhaled breath and diseases assessed in this study. The study will establish reference values for exhaled breath components and serve as background population for case-control studies. In the long run, morbidity and mortality follow-ups will be conducted, which will answer the question whether end-expiratory breath gas components predict future diseases and death. As first results, we present data from 45 dialysis patients (23 males, 22 females) which were recruited in a preliminary study in preparation for SHIP-TREND.

Floral induction and determination: where is flowering controlled?

Flowering is controlled by a variety of interrelated mechanisms. In many plants, the environment controls the production of a floral stimulus, which moves from the leaves to the shoot apex. Apices can become committed to the continuous production of flowers after the receipt of sufficient amounts of floral stimulus. However, in some plants, the commitment to continued flower production is evidently caused by a plant's commitment to perpetually produce floral stimulus in the leaves. Ultimately, the induction of flowering leads to the specification of flowers at the shoot apex. In Arabidopsis, floral specification and inflorescence patterning are regulated largely by the interactions between the genes TERMINAL FLOWER, LEAFY and APETALA1/CAULIFLOWER.

Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.

Plasmodesmata provide symplastic continuity linking individual plant cells. However, specialized cells may be isolated, either by the absence of plasmodesmata or by down regulation of the cytoplasmic flux through these channels, resulting in the formation of symplastic domains. Maintenance of these domains may be essential for the co-ordination of growth and development. While cells in the center of the meristem divide slowly and remain undifferentiated, cells on the meristem periphery divide more frequently and respond to signals determining organ fate. Such symplastic domains were visualized within shoot apices of Arabidopsis, by monitoring fluorescent symplastic tracers (HPTS: 8-hydroxypyrene 1,3,6 trisulfonic acid and CF: carboxy fluorescein). Tracers were loaded through cut leaves and distributed throughout the whole plant. Confocal laser scanning microscopy on living Arabidopsis plants indicates that HPTS moves via the vascular tissue from leaves to the apex where the tracer exits the phloem and moves symplastically into surrounding cells. The distribution of HPTS was monitored in vegetative apices, and just prior to, during, and after the switch to production of flowers. The apices of vegetative plants loaded with HPTS had detectable amounts of tracer in the tunica layer of the meristem and in very young primordia, whereas the corpus of the meristem excluded tracer uptake. Fluorescence signal intensity decreased prior to the onset of flowering. Moreover, at approximately the time the plants were committed to flowering, HPTS was undetectable in the inflorescence meristem or young primordia. Later in development, after several secondary inflorescences and mature siliques appeared, inflorescence apices again showed tracer loading at levels comparable to that of vegetative apices. Thus, analysis of fluorescent tracer movement via plasmodesmata reveals there is distinct temporal and spatial regulation of symplastic domains at the apex, dependent on the developmental stage of the plant.

Photoinduction of flower identity in vegetatively biased primordia.

Far-red light and long photoperiods promote flowering in Arabidopsis. We report here that when 30-day-old vegetative plants were induced with a continuous light treatment enriched in far-red light, flowers developed directly from previously initiated primordia. Specifically, plants induced with our continuous incandescent-enriched (CI) treatment produced an average of two primary-axis nodes with a leaf/flower phenotype, indicating that approximately two leaf/paraclade primordia per plant produced an individual flower from tissue that typically would differentiate into a paraclade (secondary inflorescence). Assays for APETALA1::beta-glucuronidase activity during the CI photoinduction treatment indicated that the floral meristem identity gene APETALA1 was transcriptionally activated in primordia with a leaf/paraclade bias and in primordia committed to leaf/paraclade development. APETALA1::beta-glucuronidase activity levels were initially highest in young primordia but were not correlated strictly with primordium fate. These results indicate that primordium fate can be modified after primordium initiation and that developing primordia respond quantitatively to floral induction signals.

Floral determination and expression of floral regulatory genes in Arabidopsis.

The expression of the floral regulators LEAFY, APETALA1 and AGAMOUS-LIKE8 was examined during light treatments that induced flowering in Arabidopsis, and was compared to time points at which floral determination occurred. Extension of an 8-hour day by either continuous red- or far-red-enriched light induced LEAFY and AGAMOUS-LIKE8 expression within 4 hours. The 4 hours of additional light was sufficient for floral determination only in the far-red-enriched conditions, while 12-16 hours of additional light was required for floral determination in the red-enriched conditions. These results indicate that the induction of floral regulatory genes and induction of flower formation can be uncoupled under certain circumstances. Expression of LEAFY and AGAMOUS-LIKE8 in the shoot apex at the time of floral determination is also consistent with genetic data indicating that these genes are involved in the first steps of the transition from vegetative to reproductive development. In contrast to LEAFY and AGAMOUS-LIKE8, APETALA1 expression was first observed 16 hours after the start of photoinduction. Since this time point was always after floral determination, APETALA1 is an indicator of floral determination.

Regulatory sequences for the transcription of the laminin B2 gene in astrocytes.

Astrocytes synthesize only the B2 chain of laminin and that this chain is sufficient to stimulate neurite outgrowth. In this study, we have examined laminin B1 and B2 promoter constructs in various cell types in order to understand the transcriptional regulation of laminin B2 gene in astrocytes. Comparison of nuclear factor binding by Southwestern analysis with the highly active B2 promoter fragment revealed different patterns of nuclear factor binding. In HepG2 cells, two proteins of 105 and 98 kDa were identified while, in primary astrocytes, human U251 and rat C6 glioma cells, a greater number of nuclear proteins ranging from 43 to 212 kDa were detected. The laminin B1 promoter construct was inactive in transient transfection experiments in astrocytes yet active in the HepG2 hepatoma cells which synthesize both the B1 and B2 chains. In contrast, the laminin B2 promoter construct was active in both astrocytes and HepG2 cells. These results are consistent with the lack of laminin B1 mRNA expression in astrocytes and suggest that the differential regulation of the laminin B1 and B2 gene is controlled at the transcriptional level. Delineation of the 5'-flanking regions responsible for basal levels of B2 laminin promoter activity revealed a silencer-like segment between -830 and -224 which reduced promoter activity. Deletion analysis further revealed that B2 laminin promoter possesses a highly active short promoter (-94 to +106) and basal transcriptional activity resides within -61 to +106. DNase 1 footprinting, gel-shift competition assays and site-directed mutagenesis of a highly active short promoter revealed that this region contained binding sites for cell-type nuclear factors. The shortest construct containing only residues -21 to +106 was inactive in HepG2 and U251 glioma cells. In primary astrocytes, however, this construct showed a high level of transcriptional activity. Deletion of 47 bp (+59 to +106) in 5'-UTR completely blocked promoter activity in astrocytes confirming that this downstream region is important for transcriptional activity in primary astrocytes. Together, these results suggest that astrocytes may utilize mutually exclusive transcription factors and regulatory sequences, in addition to common factors in the control of the laminin B2 promoter.

Cerebellar granule cell membranes and glutamate mediates transactivation of glutamine synthetase promoter.

Potential region of glutamine synthetase promoter driving astrocyte-specific transactivation, mediated by cerebellar granule cell membrane and glutamate has been identified by deletion analysis of promoter and transient transfection. The promoter region from -420 to -765 was found to be potentially important for this transactivation. These results provided further evidence for importance of neuronal-glial and glutamate-glial interactions in regulation of glial gene expression.

Hepatocellular carcinoma cell type specific interaction of P105 and P98 nuclear factors with laminin B1 and B2 genes.

Laminin is a major glycoprotein specific to basement membranes. Hepatocellular carcinoma tissue synthesize and secrete abundant laminin. By DNA-protein interaction assays, we have identified nuclear factors specific to hepatocellular carcinoma cells. The comparison of nuclear factor binding by Southwestern analysis with B1 and B2 laminin promoters revealed different patterns of nuclear factor binding in different cells types. In hepatocellular carcinoma, HepG2 cells, a specific pair of proteins (P105 and P98) consisting of 105 and 98 kDa were identified as common nuclear factors for both B1 and B2 laminin promoters, while in completely diverse human glioma cells (U251), various different and greater number of nuclear proteins ranging from 212 to 68 kDa were detected to interact separately with laminin B1 and B2 genes.

Hormone-mediated positive and negative regulation of a silencer region containing laminin B2 promoter in astrocytes.

Primary astrocytes synthesize only B2 chain (200 Kda) of laminin, which is one of the major components of basement membranes in the central nervous system (CNS). In CNS, B2 laminin functions as a potent neurite growth factor. Laminin B2 promoter contain no TATA or CAAT boxes but is GC rich. By deletion analysis and transient transfection assays of B2 laminin promoter, we have identified a silencer-like activity in the upstream region of the promoter. Thyroid hormone, insulin and phorbol ester mediated the induction of the promoter activity. Induction was repressed by the treatment of astrocytes with synthetic glucocorticoid hormone, dexamethasone. Hormone-mediated regulation through specific positive and negative responsive elements in transactivation of this silencer-containing TATA-less laminin B2 gene has been postulated.

Specification of chimeric flowering shoots in wild-type Arabidopsis.

Within wild-type Arabidopsis populations, a subset of the plants were found to have a single chimeric shoot on their primary shoot axes. The chimeric shoots were located below the lowest primary-axis flower; and they exhibited features of both flowers and paraclades (lateral flowering shoots). Morphological analyses of chimeric shoots indicated that they developed from single primordia. In each chimeric shoot, the side furthest from the apical meristem was specified as 'flower'--while the side closest to the meristem was specified as 'paraclade'--suggesting that a stimulus from outside the apical meristem can directly induce primordia to develop as flowers. It is concluded that the development of the teratological chimeric shoots resulted from the overlap of the vegetative and floral specification processes within single primordia.

Transcriptional regulation of a seed-specific carrot gene, DC8.

Many late embryogenesis abundant (Lea) protein genes in plants are regulated by abscisic acid (ABA). The RNA level of a carrot gene, DC8, increases in response to ABA in developing seeds. However, DC8 cannot be induced by ABA in adult tissues. We used chimeric genes made of various DC8 promoter fragments fused to beta-glucuronidase (GUS) to analyze the transcriptional regulation of DC8. DC8:GUS expression was measured in electroporated carrot protoplasts and in stably transformed carrots. The region of the DC8 promoter from -170 to -51 contained ABA-responsive sequences that required a 5' upstream region for high levels of expression in embryogenic callus protoplasts. 505 bp of the DC8 promoter conferred GUS expression in stably transformed somatic and zygotic embryos. DC8:GUS was expressed only in tissues formed in the seed. This includes cells in the embryo, the endosperm and the germinating seedlings. Gel retardation and competition experiments were performed to analyze the embryo nuclear protein-DNA binding activities in vitro. No binding activity was detected on the putative ABA-responsive region; however the 5' upstream regions located between -505 and -301 interacted with embryo nuclear factors. An additional site of DNA-protein interaction was located between positions -32 and +178. The nuclear proteins that bind these sequences were found in the embryo nuclei only, not in the nuclei from leaves or roots.

In vivo comparison of cytochrome aa3 redox state and tissue PO2 in transient anoxia.

We have determined, in vivo and simultaneously, the tissue PO2 (Ptio2) and the oxidation-reduction (redox) state of cytochrome aa3 (cyt aa3) of cat cerebral cortex during and after a short period of N2 breathing. Thirteen cats were anesthetized, ventilated mechanically with room air, subjected to a limited bilateral craniotomy, and then injected with 25 mg/kg of pyrenebutyric acid (PBA) intravenously. Ptio2 was measured from PBA generated fluorescence, emitted by monitored cerebral cortical cells. The cyt aa3 redox state was measured from differential absorption of monochromatic light at 605 vs. 590 nm reflected from the same cortical cells. In response to a 1.5-min N2 ventilation (phase I) the increase in PBA fluorescence signal, indicating a decline in Ptio2, lagged behind the cyt aa3 reduction. When the animal was ventilated with room air (phase II), rapid reoxidation, followed by hyperoxidation of cyt aa3 occurred. The decrease in PBA fluorescence signal, indicating an increase in Ptio2, was seen to lag behind cyt aa3 reoxidation. These results indicate that hysteresis exists in the relation between Ptio2 and cyt aa3 redox state. This may be the result of the situation that 1) low tissue O2 concentration is partially compensated by accumulation of reduced cyt aa3, and 2) following brief periods of anoxia, the affinity of cyt aa3 to O2 is increased.

Enhancement of cortical evoked potentials by high atmospheric pressures of helium.

Adult guinea pigs with electrodes chronically implanted in the optic chiasm (OC(, lateral geniculate nucleus (LGN), and visual cortex (CX) were compressed at 1 bar/min to 120 bars pressure in helium-oxygen. Body temperature was controlled to within +/- 1 degree C, CO2 was removed by a soda-lime absorbent. Electrical stimuli (50 microA, 0.05 msec) were delivered to the OC at 9.6/sec at 10 bar intervals, with pressure held constant. Pressure caused only negligible changes inthe amplitudes of pre- and postsynaptic components of potentials in the LGN. Amplitudes of evoked potentials in the CX increased approximately linearly with pressure, reaching values of up to 300% above normobaric. Latencies did not change independently of temperature. Excitability of nerve fibers changed very little, but intracortical synaptic transmission was substantially enhanced. Differences in the effects of elevated helium pressure at the LGN and CX may be attributed to differences in organization of local circuits.

In vivo comparison of cerebral tissue PO2 and cytochrome aa3 reduction-oxidation state in cats during hemorrhagic shock.

To assess the adequacy of oxygen availability and utilization within the cerebral cortex in vivo, we have measured the partial pressure of oxygen in tissue (PtO2), as well as the reduction oxidation state of cytochrome c oxidase (cyt aa3) during shock induced by slow or rapid hemorrhage in anesthetized cats. PtO2 was measured with pyrenebutyric acid-generated fluorescence in cerebral cortical cells. Cyt aa3 redox state was measured by the absorption of monochromatic light at 605 nm absorption peak of the enzyme reflected from the same cortical field. The PtO2 remained within the normal range until either 30 +/- 1.5 ml blood/kg was removed or the mean arterial pressure fell by 70 +/- 5% of base line. Beyond either point, the PtO2 fell rapidly to a low value approximating zero. By contrast, the reduction of cyt aa3 began early when as little as 5 ml blood/kg was removed. Thereafter, the shift toward reduction was progressive and continuous with a slow rate at first and a rapid rate later. This accelerated rate of cyt aa3 reduction preceded the rapid fall of PtO2. We concluded that, under these experimental conditions, cyt aa3 reduction is a much earlier and more sensitive indicator of perturbed intracellular aerobic metabolism due to hemorrhage that is PtO2.

Correspondence of brain and rectal temperatures of guinea pigs in helium environments.

Brain and rectal temperatures were measured in guinea pigs exposed to helium-oxygen environments at pressures as high as 50 bars. Very high correlations (r = .856, P less than 0.001) between the two values were observed, with an average difference of 0.47 degrees C (computed SD = 0.31). Brain temperature was usually higher than rectal. At a given ambient temperature, heat loss at 50 bars was more rapid than at 20 bars, which supports existing data for lower pressures. At 50 bars, normal equilibrium temperatures (approximately equal to 39 degrees C) could be maintained only if ambient temperature was in the vicinity of 35 degrees C. Lower ambient temperatures resulted in lower equilibrium temperatures. Between 33 and 39 degrees C, brain temperature can be predicted from rectal temperature, with an error of about 0.5 degree C.