The time course of non-photochemical quenching in phycobilisomes of Synechocystis sp. PCC6803 as revealed by picosecond time-resolved fluorimetry

As high-intensity solar radiation can lead to extensive damage of the photosynthetic apparatus, cyanobacteria have developed various protection mechanisms to reduce the effective excitation energy transfer (EET) from the antenna complexes to the reaction center. One of them is non-photochemical quenching (NPQ) of the phycobilisome (PB) fluorescence. In Synechocystis sp. PCC6803 this role is carried by the orange carotenoid protein (OCP), which reacts to high-intensity light by a series of conformational changes, enabling the binding of OCP to the PBs reducing the flow of energy into the photosystems. In this paper the mechanisms of energy migration in two mutant PB complexes of Synechocystis sp. were investigated and compared. The mutant CK is lacking phycocyanin in the PBs while the mutant ΔPSI/PSII does not contain both photosystems. Fluorescence decay spectra with picosecond time resolution were registered using a single photon counting technique. The studies were performed in a wide range of temperatures — from 4 to 300 K. The time course of NPQ and fluorescence recovery in darkness was studied at room temperature using both steady-state and time-resolved fluorescence measurements. The OCP induced NPQ has been shown to be due to EET from PB cores to the red form of OCP under photon flux densities up to 1000 μmol photons m^− 2 s^− 1. The gradual changes of the energy transfer rate from allophycocyanin to OCP were observed during the irradiation of the sample with blue light and consequent adaptation to darkness. This fact was interpreted as the revelation of intermolecular interaction between OCP and PB binding site. At low temperatures a significantly enhanced EET from allophycocyanin to terminal emitters has been shown, due to the decreased back transfer from terminal emitter to APC. The activation of OCP not only leads to fluorescence quenching, but also affects the rate constants of energy transfer as shown by model based analysis of the decay associated spectra. The results indicate that the ability of OCP to quench the fluorescence is strongly temperature dependent. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

The variation of aneuploidy frequency in the developing and adult human brain revealed by an interphase FISH study.

Despite the lack of direct cytogenetic studies, the neuronal cells of the normal human brain have been postulated to contain normal (diploid) chromosomal complement. Direct proof of a chromosomal mutation presence leading to large-scale genomic alterations in neuronal cells has been missing in the human brain. Large-scale genomic variations due to chromosomal complement instability in developing neuronal cells may lead to the variable level of chromosomal mosaicism probably having a substantial effect on brain development. The aim of the present study was the pilot assessment of chromosome complement variations in neuronal cells of developing and adult human brain tissues using interphase multicolor fluorescence in situ hybridization (mFISH). Chromosome-enumerating DNA probes from the original collection (chromosomes 1, 13 and 21, 18, X, and Y) were used for the present pilot FISH study. As a source of fetal brain tissue, the medulla oblongata was used. FISH studies were performed using uncultured fetal brain samples as well as organotypic cultures of medulla oblongata tissue. Cortex tissues of postmortem adult brain samples (Brodmann area 10) were also studied. In cultured in vitro embryonic neuronal brain cells, an increased level of aneuploidy was found (mean rate in the range of 1.3-7.0% per individual chromosome, in contrast to 0.6-3.0% and 0.1-0.8% in uncultured fetal and postmortem adult brain cells, respectively). The data obtained support the hypothesis regarding aneuploidy occurrence in normal developing and adult human brain.     

Patterning of chick brain vesicles as revealed by peanut agglutinin and cholinesterases.

Differentiation of individual rhombomeres of the chicken hindbrain directly follows the emergence of primary brain vesicles. Immediately after the constriction of the prosencephalon at HH9, a series of vesicles of decreasing size is established almost simultaneously between HH9 and HH10, including mesencephalon, four preotic (R2-R5) and one postotic (R6/R7) rhombomeres. Thereby, the cranial neural tube is ventrally embedded in a mesodermal PNA-binding matrix that particularly accumulates underneath vesicular constriction sites, as demonstrated for the segregation of the prosencephalon at HH9 and the cerebellar rhombomere R1 from R2 at HH13. The subsequent period of hindbrain differentiation is analyzed by cholinesterase (AChE, BChE) and peanut lectin histochemistry, by the BrdU and the neurite-specific G4 antibodies. Preotically, differentiation of two pairs of rhombomeres (R4 + R5, R2 + R3) starts in R4, immediately followed by R2. The caudal rhombomeres of both pairs are delayed (R5, R3). Then the postotic rhombomere is subdivided, whereby R7 differentiates before R6. Thus, the development in the direct vicinity of the otic vesicle is delayed (R5, R6). R7 is the last rhombomere that is demarcated caudally. Based on these findings, we postulate two processes that may regulate rhombomere formation in the chicken embryo: (a) an early rostrocaudal wave establishing the major brain vesicles, (b) a superimposed pairwise segmentation emanating rostrally and caudally from the otic vesicle. The segregation of the cerebellar rhombomere is a late step.     

Tissue-specific and non-tissue-specific heavy-chain isoforms of myosin in the brain as revealed by monoclonal antibodies.

Four types of monoclonal antibody (BM-1, BM-2, BM-3 and BM-4) each having distinctive tissue specificity were obtained by immunizing mice with purified bovine cerebrum myosin. Both BM-1 and BM-2 reacted most efficiently with cerebrum myosin and less efficiently with myosins from other limited nonmuscle tissues, the tissue specificity of BM-1 being much narrower than that of BM-2. BM-3 reacted more efficiently with several other nonmuscle myosins than with cerebellar or cerebral myosin. BM-4 recognized various nonmuscle and smooth muscle myosins with a nearly equal efficiency. Cerebral myosin as well a cerebellar myosin contained two or more electrophoretic variants of the heavy chains. BM-1 and BM-3 as well as BM-2 and BM-3 were found to recognize selectively these distinct heavy-chain isoforms. The antigenic sites of the three tissue-specific antibodies (BM-1, BM-2 and BM-3) were all localized near the head/tail junction of the myosin molecules, while that of non-tissue-specific antibody BM-4 was near the center of the tail. These and additional results indicate that mammalian brain tissues as well as several other nonmuscle tissues contain multiple heavy-chain isoforms of myosin, the levels of which differed considerably from one tissue to another.     

Directed motion of telomeres in the formation of the meiotic bouquet revealed by time course and simulation analysis

Chromosome movement is critical for homologous chromosome pairing during meiosis. A prominent and nearly universal meiotic chromosome reorganization is the formation of the bouquet, characterized by the close clustering of chromosome ends at the nuclear envelope. We have used a novel method of in vitro culture of rye anthers combined with fluorescent in situ hybridization (FISH) detection of telomeres to quantitatively study bouquet formation. The three-dimensional distribution of telomeres over time was used to obtain a quantitative profile of bouquet formation intermediates. The bouquet formed through a gradual, continuous tightening of telomeres over approximately 6 h. To determine whether the motion of chromosomes was random or directed, we developed a computer simulation of bouquet formation to compare with our observations. We varied the diffusion rate of telomeres and the amount of directional bias in telomere movement. In our models, the bouquet was formed in a manner comparable to what we observed in cultured meiocytes only when the movement of telomeres was actively directed toward the bouquet site, whereas a wide range of diffusion rates were permitted. Directed motion, as opposed to random diffusion, was required to reproduce our observations, implying that an active process moves chromosomes to cause telomere clustering.     

The time course of segmentation and cue-selectivity in the human visual cortex

Texture discontinuities are a fundamental cue by which the visual system segments objects from their background. The neural mechanisms supporting texture-based segmentation are therefore critical to visual perception and cognition. In the present experiment we employ an EEG source-imaging approach in order to study the time course of texture-based segmentation in the human brain. Visual Evoked Potentials were recorded to four types of stimuli in which periodic temporal modulation of a central 3° figure region could either support figure-ground segmentation, or have identical local texture modulations but not produce changes in global image segmentation. The image discontinuities were defined either by orientation or phase differences across image regions. Evoked responses to these four stimuli were analyzed both at the scalp and on the cortical surface in retinotopic and functional regions-of-interest (ROIs) defined separately using fMRI on a subject-by-subject basis. Texture segmentation (tsVEP: segmenting versus non-segmenting) and cue-specific (csVEP: orientation versus phase) responses exhibited distinctive patterns of activity. Alternations between uniform and segmented images produced highly asymmetric responses that were larger after transitions from the uniform to the segmented state. Texture modulations that signaled the appearance of a figure evoked a pattern of increased activity starting at ～143 ms that was larger in V1 and LOC ROIs, relative to identical modulations that didn't signal figure-ground segmentation. This segmentation-related activity occurred after an initial response phase that did not depend on the global segmentation structure of the image. The two cue types evoked similar tsVEPs up to 230 ms when they differed in the V4 and LOC ROIs. The evolution of the response proceeded largely in the feed-forward direction, with only weak evidence for feedback-related activity.     

The human nuclear pore complex as revealed by cryo-electron tomography

Nuclear pore complexes (NPCs) are the sole passage through the nuclear envelope, connecting the cytoplasm to the nucleoplasm. These gigantic molecular machines, over 100 MDa in molecular weight, allow free diffusion of small molecules and ions while mediating selective energy-dependent nucleocytoplasmic transport of large macromolecules. Here, we applied cryo-electron tomography to human fibroblast cells, reconstructing their nuclear envelopes without applying any purification steps. From these reconstructions, we extracted subtomograms containing individual NPCs and utilized in silico subtomogram averaging procedures to determine the structure of the mammalian pore complex at a resolution of ～6.6?nm. Beyond revealing the canonical features of the human NPC, our analysis identified inner lateral channels and fusing bridge-like structures, suggesting alternative routes of peripheral nuclear passage. Finally, we concluded from our structural analysis that the human NPC is structurally distinct from that of lower eukaryotes in terms of dimension and organization but resembles its amphibian (frog) counterpart.     

Time course and cytokine dependence of human T-cell lymphotropic virus type 1 T-lymphocyte transformation as revealed by a microtiter infectivity assay.

Human T-cell lymphotropic virus type 1 (HTLV-1) enhances the growth of T lymphocytes, allowing the generation of T-lymphocyte cell lines. This report describes a limiting-dilution assay system which uses low input numbers of HTLV-1-producing cells for generation of T-lymphocyte cultures. The HTLV-1 transformants generated with this assay system produced high levels of HTLV-1 p24 antigen and required exogenous cytokines for maintenance. Clonal populations of CD4- or CD8-positive HTLV-1 transformants were generated with transformation efficiency rates as high as 78%. An exogenous cytokine is necessary for HTLV-1 T-lymphocyte transformation, and cytokine dependence is the most likely outcome of infection and transformation. HTLV-1 T-lymphocyte transformation can occur in the presence of cytokines other than interleukin-2 (IL-2), such as IL-4 or IL-7. IL-4- or IL-7-dependent HTLV-1 transformants underwent T-lymphocyte mitogenesis in response to their homologous cytokines but proliferated best in the presence of IL-2. Since the receptors for IL-2, IL-4, and IL-7 share the IL-2 gamma chain, this component may be the common element in the signaling pathway for HTLV-1-associated transformation.     

Heterogeneity of human alpha-fetoprotein as revealed by isoelectric focusing in urea-containing gels.

The heterogeneity of human alpha-fetoprotein has been studied by analytical isoelectric focusing in polyacrylamide gel slabs in the presence of 8 M urea. Six major isoelectric variants could be identified over a pH range of 6.0--6.2. Verification of their identity was achieved by crossed immunoelectrophoresis into agarose gel containing monospecific antiserum to human alpha-fetoprotein. Complete desialylation of the protein did not abolish the heterogeneity; a complex pattern of major alpha-fetoprotein bands persisted over a more alkaline pH range. We have been able to correlate the pattern of alpha-fetoprotein heterogeneity seen following extended agarose gel electrophoresis with that obtained during isoelectric focusing in the presence of urea. The quantity of certain alpha-fetoprotein charge isomers in various alpha-fetoprotein isolates may be important in considering certain biological functions of this protein.     

Cognitive association formation in human memory revealed by spatiotemporal brain imaging

Cognitive theory posits association by juxtaposition or by fusion. We employed the measurement of event-related brain potentials (ERPs) to a concept fusion task in order to explore memory encoding of these two types of associations between word pairs, followed by a memory test for original pair order. Encoding processes were isolated by subtracting fusion task ERPs corresponding to pairs later retrieved quickly from ERPs corresponding to pairs later retrieved slowly, separately for pairs fused successfully and unsuccessfully (i.e., juxtaposed). Analyses revealed that the encoding of these two types of associations yields different ERP voltage polarities, scalp topographies, and brain sources extending over the entire time course of processing.     

Change of tyrosine hydroxylase in the parkinsonian brain and in the brain of MPTP-treated mice as revealed by homospecific activity

Changes in homospecific activity (unit of enzyme activity per unit of enzyme protein; Rush, Kindler and Udenfriend, 1974. Biochem. Biophys. Res. Commun., 61, 38) of tyrosine hydroxylase (TH) in the striatum of the brain were examined in MPTP-treated mice and parkinsonian patients. After a single injection of MPTP to mice, TH activity was acutely inhibited onlyin situ without changes in in vitro TH activity (Vmax) and TH protein; TH homospecific activity (TH Vmax/TH protein) did not change. After repeated injection of MPTP to mice for 8 days, in situ TH activity, in vitro TH Vmax, and TH protein were decreased in parallel, and TH homospecific activity did not change The result indicates that the decreases in in situ TH activity and in TH Vmax are due to the decrease in TH protein by nerve degeneration of dopaminergic neurons in MPTP treated mice. However, when MPP⁺ was infused in the striatum of rats for 3 hours, in vitro TH activity (Vmax) was decreased without changes in TH protein. Thus, TH homospecific activity was decreased. The results indicate that MPP⁺ inactivates TH protein in the striatum after continued infusion. In contrast, the homospecific activity of TH in post-mortem parkinsonian striatum was increased 3-fold. The increase in homospecific activity of residual TH in parkinsonian brain suggests such molecular changes in TH molecules as result in a compensatory increase in TH activity.Special issue dedicated to Dr. Sidney Udenfriend.     

The brain of the Nemertodermatida (Platyhelminthes) as revealed by anti-5HT and anti-FMRFamide immunostainings

The taxa Nemertodermatida and Acoela have traditionally been considered closely related and classified as sister groups within the Acoelomorpha Ehlers 1984 (Platyhelminthes). Recent molecular investigations have questioned their respective position. In this study, the 5-HT and FMRFamide immunoreactivity (IR) in the nervous system of two nemertodermatids, Nemertoderma westbladi and Meara stichopi, is described. The 5-HT immunoreactive pattern differs in the two nemertodermatids studied. In M. stichopi, two loose longitudinal bundles of 5-HT-immunoreactive fibres and an basi-epidermal nerve net were observed. In N. westbladi the 5-HT-IR shows a ring-shaped commissural structure, different from the commissural brain of acoels. In both nemertodermatids, FMRFamide immunoreactive nerve fibres followed the 5-HT-immunoreactive fibres. It is demonstrated that the Nemertodermatida have neither a 'commissural brain' structure similar to that of the Acoela, nor a 'true', ganglionic brain and orthogon, typical for other Platyhelminthes. The question of the plesiomorphic or apomorphic nature of the nervous system in Nemertodermatida cannot yet be answered. The neuroanatomy of the studied worms provides no synapomorphy supporting the taxon Acoelomorpha.     

Heritability of human brain functioning as assessed by electroencephalography.

To study the genetic and environmental contributions to individual differences in CNS functioning, the electroencephalogram (EEG) was measured in 213 twin pairs age 16 years. EEG was measured in 91 MZ and 122 DZ twins. To quantify sex differences in the genetic architecture, EEG was measured in female and male same-sex twins and in opposite-sex twins. EEG was recorded on 14 scalp positions during quiet resting with eyes closed. Spectral powers were calculated for four frequency bands: delta, theta, alpha, and beta. Twin correlations pointed toward high genetic influences for all these powers and scalp locations. Model fitting confirmed these findings; the largest part of the variance of the EEG is explained by additive genetic factors. The averaged heritabilites for the delta, theta, alpha and beta frequencies was 76%, 89%, 89%, and 86%, respectively. Multivariate analyses suggested that the same genes for EEG alpha rhythm were expressed in different brain areas in the left and right hemisphere. This study shows that brain functioning, as indexed by rhythmic brain-electrical activity, is one of the most heritable characteristics in humans.     

Biochemical identification of the site of action of benzodiazepines in human brain by 3H-diazepam binding.

Benzodiazepines, a group of minor tranquillizers, bind in a selective, stereo-specific fashion to a receptor site in human brain. Diazepam, their main representative, is bound with an apparent dissociation constant of 7 nM. The potency of various benzodiazepines in displacing diazepam parallels closely their therapeutic and pharmacological potencies. The density of the receptor site varies 24 fold in human brain, with the highest level in cerebral cortex and cerebellum. The apparent affinity of the receptor site, however, is remarkably similar in twenty different brain regions. The characteristics of the benzodiazepine receptor site suggest that it represents the site of therapeutic action of the benzodiazepines in human brain.