Chlorophyll b is involved in long-wavelength spectral properties of light-harvesting complexes LHC I and LHC II.

Chlorophyll (Chl) molecules attached to plant light-harvesting complexes (LHC) differ in their spectral behavior. While most Chl a and Chl b molecules give rise to absorption bands between 645 nm and 670 nm, some special Chls absorb at wavelengths longer than 700 nm. Among the Chl a/b-antennae of higher plants these are found exclusively in LHC I. In order to assign this special spectral property to one chlorophyll species we reconstituted LHC of both photosystem I (Lhca4) and photosystem II (Lhcb1) with carotenoids and only Chl a or Chl b and analyzed the effect on pigment binding, absorption and fluorescence properties. In both LHCs the Chl-binding sites of the omitted Chl species were occupied by the other species resulting in a constant total number of Chls in these complexes. 77-K spectroscopic measurements demonstrated that omission of Chl b in refolded Lhca4 resulted in a loss of long-wavelength absorption and 730-nm fluorescence emission. In Lhcb1 with only Chl b long-wavelength emission was preserved. These results clearly demonstrate the involvement of Chl b in establishing long-wavelength properties.     

Interactions between sediment and water in a shallow and hypertrophic lake: a study on phytoplankton collapses in Lake Søbygård,Denmark

Short-term changes in phytoplankton and zooplankton biomass have occurred 1–3 times every summer for the past 5 years in the shallow and hypertrophic Lake Søbygård, Denmark. These changes markedly affected lake water characteristics as well as the sediment/water interaction. Thus during a collapse of the phytoplankton biomass in 1985, lasting for about 2 weeks, the lake water became almost anoxic, followed by rapid increase in nitrogen and phosphorus at rates of 100–400 mg N M^–2 day^–1 and 100–200 mg P m^–1 day^–1. Average external loading during this period was about 350 mg N m^–2 day^–1 and 5 mg P m^–2 day^–1, respectively.Due to high phytoplankton biomass and subsequently a high sedimentation and recycling of nutrients, gross release rates of phosphorus and nitrogen were several times higher than net release rates. The net summer sediment release of phosphorus was usually about 40 mg P m^–2 day^–1, corresponding to a 2–3 fold increase in the net phosphorus release during the collapse. The nitrogen and phosphorus increase during the collapse is considered to be due primarily to a decreased sedimentation because of low algal biomass. The nutrient interactions between sediment and lake water during phytoplankton collapse, therefore, were changed from being dominated by both a large input and a large sedimentation of nutrients to a dominance of only a large input. Nitrogen was derived from both the inlet and sediment, whereas phosphorus was preferentially derived from the sediment. Different temperature levels may be a main reason for the different release rates from year to year.     

Interaction of topotecan,DNA topoisomerase I inhibitor,with double-stranded polydeoxyribonucleotides. III. Binding at the minor groove

Interaction of topotecan (TPT) with calf thymus DNA, coliphage T4 DNA, and poly(dG-dC). poly(dG-dC) was studied by optical (linear flow dichroism, UV-vis spectroscopy) and quantum chemical methods. The linear dichroism (LD) signal of TPT bound to DNA was shown to have positive sign in the range 260-295 nm. This means that the plane of quinoline fragment (rings A and B) of TPT molecule form an angle lower 54 degrees with the long axis of DNA, and hence TPT molecule can not intercalate between DNA base pairs. TPT was established to bind to calf thymus DNA as readily as to coliphage T4 DNA whose all cytosines in the major groove were glycosylated at the 5th position. Consequently, the DNA major groove does not participate in TPT binding. TPT molecule was shown to compete with distamycin for binding sites in the minor groove of DNA and poly(dG-dC). poly(dG-dC). Thus, it was demonstrated for the first time that TPT binds to DNA at its minor groove.     

Macrorestriction Analysis of Caenorhabditis Elegans Genomic DNA

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a ``macrorestriction mapping' procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is <220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located ~400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.     

EDTA-assisted heavy-metal uptake by poplar and sunflower grown at a long-term sewage-sludge farm

Little information is available concerning the efficacy of chelates applied to biosolids (sewage-sludge)-treated soil for heavy-metal removal. The purpose of the experiment was to determine the availability to sunflower (Helianthus annuus L.) and hybrid poplar (Populus deltoides Marsh. × P. nigra L.) seedlings, of non-essential (Cd, Ni, Pb) and essential heavy metals (Cu, Fe, Mn, Zn) in field soil injected with biosolids since 1976 and treated with ethylenediamine-tetraacetic acid (EDTA) in 2001. Sunflower was grown at two densities, 20000 and 60000 plants/ha, and poplar at 10000 plants/ha. The tetrasodium salt of EDTA was applied at rates of 0, 0.5, 1, and 2 g EDTA salt per kg surface (25-cm depth) soil. The EDTA did not affect uptake by poplar of the three non-essential (Cd, Ni, Pb) and four essential (Cu, Fe, Mn, Zn) heavy metals. For sunflower, the 1.0 g/kg rate of chelate addition resulted in maximal removal of the three non-essential heavy metals (Cd, Ni, Pb). Uptake of the essential heavy metals by sunflower was little affected by the EDTA. At the 20000 plants/ha density, leaves of sunflower grown with 1.0 g EDTA Na₄2H₂O per kg soil accumulated more Cd, Ni, and Pb than leaves of sunflower grown without the EDTA salt. At this density, concentrations of Cd in leaves of sunflower without EDTA and with 1.0 g/kg EDTA salt were 2.2 and 6.5 g/g, respectively; for Ni, they were 6.7 and 19.2 g/g, respectively; and for Pb, they were 15.6 and 46.9 g/g, respectively. At the 60000 plants/ha density, stems of sunflower grown with 1.0 g EDTA Na₄2H₂O per kg soil accumulated more Cd, Ni, and Pb than stems of sunflower grown without the EDTA salt. At this density, concentrations of Cd in stems of sunflower without EDTA and with 1.0 g/kg EDTA salt were 0.6 and 4.6 g/g, respectively; for Ni, they were 1.7 and 17.6 g/g, respectively; and for Pb, they were 5.2 and 42.8 g/g, respectively. Removal of the non-essential heavy metals by sunflower was greater at the higher plant density (60000 plants/ha) compared to the lower one (20000 plants/ha).     

The functional role of sequentially neuromodulated synaptic plasticity in behavioural learning

To survive, animals have to quickly modify their behaviour when the reward changes. The internal representations responsible for this are updated through synaptic weight changes, mediated by certain neuromodulators conveying feedback from the environment. In previous experiments, we discovered a form of hippocampal Spike-Timing-Dependent-Plasticity (STDP) that is sequentially modulated by acetylcholine and dopamine. Acetylcholine facilitates synaptic depression, while dopamine retroactively converts the depression into potentiation. When these experimental findings were implemented as a learning rule in a computational model, our simulations showed that cholinergic-facilitated depression is important for reversal learning. In the present study, we tested the model’s prediction by optogenetically inactivating cholinergic neurons in mice during a hippocampus-dependent spatial learning task with changing rewards. We found that reversal learning, but not initial place learning, was impaired, verifying our computational prediction that acetylcholine-modulated plasticity promotes the unlearning of old reward locations. Further, differences in neuromodulator concentrations in the model captured mouse-by-mouse performance variability in the optogenetic experiments. Our line of work sheds light on how neuromodulators enable the learning of new contingencies.     

NetSurfP-2.0: Improved prediction of protein structural features by integrated deep learning

The ability to predict local structural features of a protein from the primary sequence is of paramount importance for unraveling its function in absence of experimental structural information. Two main factors affect the utility of potential prediction tools: their accuracy must enable extraction of reliable structural information on the proteins of interest, and their runtime must be low to keep pace with sequencing data being generated at a constantly increasing speed. Here, we present NetSurfP-2.0, a novel tool that can predict the most important local structural features with unprecedented accuracy and runtime. NetSurfP-2.0 is sequence-based and uses an architecture composed of convolutional and long short-term memory neural networks trained on solved protein structures. Using a single integrated model, NetSurfP-2.0 predicts solvent accessibility, secondary structure, structural disorder, and backbone dihedral angles for each residue of the input sequences. We assessed the accuracy of NetSurfP-2.0 on several independent test datasets and found it to consistently produce state-of-the-art predictions for each of its output features. We observe a correlation of 80% between predictions and experimental data for solvent accessibility, and a precision of 85% on secondary structure 3-class predictions. In addition to improved accuracy, the processing time has been optimized to allow predicting more than 1000 proteins in less than 2 hours, and complete proteomes in less than 1 day.     

Quantitative profiling of PE, MMPE, DMPE, and PC lipid species by multiple precursor ion scanning: a tool for monitoring PE metabolism

We report a method for the simultaneous identification and quantification of phosphatidylethanolamine (PE), monomethyl-phosphatidylethanolamine (MMPE), dimethyl-phosphatidylethanolamine (DMPE), and phosphatidylcholine (PC) species in lipid extracts. The method employs a specific "mass-tag" strategy where DMPE, MMPE, and PE species are chemically methylated with deuterated methyliodide (CD(3)I) to produce PC molecules having class-specific mass offsets of 3, 6 and 9Da, respectively. The derivatized aminoglycerophospholipids release characteristic phosphorylcholine-like fragment ions having specific mass offsets that powers sensitive and quantitative analysis by multiple precursor ion scanning on a hybrid quadrupole time-of-flight mass spectrometer. Using the mass-tag strategy, we could for the first time determine the stoichiometric relationship between the biosynthetic intermediates MMPE and DMPE, and abundant PE and PC species in a single mass spectrometric analysis. We demonstrated the efficacy of the methodology by conducting a series of biochemical experiments using stable isotope labeled ethanolamine to survey the activities and substrate specificities of enzymes involved in PE metabolism in Saccharomyces cerevisiae. Finally, we benchmarked the mass-tag strategy by specific and sensitive profiling of intermediate MMPE and DMPE species in liver.     

The negatively charged amino acids in the lumenal loop influence the pigment binding and conformation of the major light-harvesting chlorophyll a/b complex of photosystem II

The major chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb), in addition to their primary light-harvesting function, play key roles in the organization of the granal ultrastructure of the thylakoid membranes and in various regulatory processes. These functions depend on the structural stability and flexibility of the complexes. The lumenal side of LHCIIb is exposed to broadly variable pH environments, due to the build-up and decay of the pH gradient during photosynthesis. Therefore, the negatively charged amino acids in the lumenal loop might be of paramount importance for adjusting the structure and functions of LHCIIb. In order to clarify the structural roles of these residues, we investigated the pigment stoichiometries, absorption, linear and circular dichroism spectra of the reconstituted LHCIIb complexes, in which the negatively charged amino acids in the lumenal loop were exchanged to neutral ones (E94G, E107V and D111V). The mutations influenced the pigment binding and the molecular architecture of the complexes. Exchanging E94 to G destabilized the 3(10) helix in the lumenal loop structure and led to an acquired pH sensitivity of the LHCIIb structure. We conclude that these amino acids are important not only for pigment binding in the complexes, but also in stabilizing the conformation of LHCIIb at different pHs.