Estimation of the photosynthesis/irradiance (P/I) curve parameters from light and dark bottle experiments

Light and dark bottle experiments, carried out in three systemsin the Netherlands, were used to estimate the parameters ofmodels relating the oxygen production rate to incident lightintensity. The maximum production rate (P_M) and the light saturationconstant (I_S) were estimated using both gross and nett oxygenproduction data. In the latter case, the community oxygen consumptionrate (R_ox was also estimated. Eight models were compared withrespect to goodness of fit, as has been accomplished previouslyby Jassby and Platt (Limnol. Oceanogr., 21, 540–547, 1976)and many others. This study, however, emphasizes the problemof parameter correlation and the consequential usefulness ofthese type of experiments to identify the P/I curve parameters.The results show that at a 90% level of confidence, the modelscannot be distinguished with respect to goodness of fit. However,the models do show distinct differences in parameter correlation.Parameter correlation was shown to be related to the shape ofthe curve. P/I curves with high convexity were shown to be lesssensitive for parameter correlation. In particular, models showinglow convexity suffer from over-paraineterisation, which meansthat on the basis of the observed production rates it is difficultto discriminate between the parameters. Also, alternative modelformulations, using P_M and the initial slope (), were investigated.These formulations produced less parameter correlation. However,for models with low convexity, showing high parameter correlationanyway, the reduction is limited. The use of nett oxygen productiondata does not show a significant difference in fit at a 90%confidence level. However, measured R_ox from dark bottle experimentstends to be higher than the values found by estimating R_ox fromnett oxygen production.     

Spectroscopic and functional characterization of an environmentally sensitive fluorescent actin conjugate

Rabbit skeletal muscle F-actin has been selectively labeled at a cysteine residue with the environmentally sensitive fluorophore 6-acryloyl-2-(dimethylamino)naphthalene. The fluorescent actin conjugate behaves similarly to native actin with respect to the polymerization kinetics, critical monomer concentration, and ability to form F-actin paracrystals. Upon polymerization to F-actin, the absorption of the actin conjugate is red-shifted, whereas the fluorescence emission is blue-shifted 740 wavenumbers and is accompanied by a decrease in the fluorescence bandwidth of 470 wavenumbers. These large shifts in the spectral properties of 6-propionyl-2-(dimethylamino)naphthalene (Prodan) in actin provide a simple method for obtaining a spectral discrimination between the G- and F-actin populations during the polymerization reaction. Steady-state fluorescence techniques were used to study the environment of the fluorophore in the monomeric and polymeric forms of actin. Fluorescence emission spectral analysis and quenching and polarization studies of G-actin-Prodan indicated that the fluorophore lies immobile on the protein surface but with one of its faces in full contact with the solvent. In F-actin, the fluorophore has a limited exposure to the solvent and is located in a dielectric environment similar to those seen for Prodan in polar, aprotic solvents or buried within a protein matrix [Macgregor, R. B., Jr., & Weber, G. (1986) Nature (London) 318, 70-73]. Additionally, our results demonstrate that the Prodan molecule conjugated to F-actin is completely immobile during its fluorescence lifetime, exhibits an increase in the resonance energy transfer (RET) from tryptophan residues compared to that observed in G-actin, and shows evidence of homologous RET within the polymer.     

Immunofluorescence localization of Z-DNA in chromosomes: quantitation by scanning microphotometry and computer-assisted image analysis

Anti-Z-DNA polyclonal and monoclonal immunoglobulins raised against left-handed polynucleotides show various degrees of specificity for base sequence and substitution. Class 1 IgGs recognize all Z-DNA with equal affinity; class 2 IgGs show a preference for d(G-C)n sequences and class 3 IgGs for d(G-C)n sequences with substitutions at the C5 position of the pyrimidine. These antibodies served as probes for the localization of Z-DNA in polytene and metaphase chromosomes and in interphase chromatin by indirect immunofluorescence. A quantitative assessment of the binding of anti-Z-DNA IgGs to polytene chromosomes of Chironomus and Drosophila was made by scanning microphotometry and by computer-assisted image analysis of double immunofluorescence and DNA-specific dye fluorescence images. The three classes of antibodies bind to most of the bands in acid fixed polytene chromosomes of C. thummi; however, preferential binding of one class of antibody over another can be observed in certain regions. These differences can be quantitated by arithmetic division or subtraction of the normalized digital images. If a class 2 antibody is first bound at saturating concentrations the binding of class 1 antibody is reduced throughout most bands by 40-50%. However, the telomeres of the three large chromosomes bind greater than 10 times as much class 1 antibody as class 2 antibody, indicating that the Z-DNA tracts in these regions are comprised largely of alternating sequences containing the A X T basepair, e.g., A-C. High-resolution image analysis of class 1 and class 2 immunofluorescence patterns and the total DNA distribution from polytene chromosomes of D. melanogaster show that the two antibody distributions are very similar in a large majority of the bands, but they often deviate from the mean DNA distribution profile. Z-DNA sequences of both G-C and A-C type are detectable at all levels of ploidy from 2n to 2(13)n and in species as diverse as insects and man. We conclude that the vast majority of polytene chromosome bands (genes) contain one or a few DNA sequences with potential for undergoing the B----Z transition and contain both alternating purine-pyrimidine G-C and A-C tracts or mixed sequences. Highly heterochromatic bands and telomeres have more Z potential sequences than do other bands.     

Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans

Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and D. simulans for two common allozyme forms, as well as for several other less common variants. Parallel latitudinal clines in the frequencies of the common EST6-F and EST6-S allozymes in these species have previously been interpreted in terms of a shared amino acid polymorphism that distinguishes the two variants and is subject to selection. Here we compare the sequences of four D. simulans Est-6 isolates and show that overall estimates of nucleotide heterozygosity in both coding and 5' flanking regions are more than threefold higher than those obtained previously for this gene in D. melanogaster. Nevertheless, the ratio of replacement to exon silent-site polymorphism in D. simulans is less than the ratio of replacement to silent divergence between D. simulans and D. melanogaster, which could be the result of increased efficiency of selection against replacement polymorphisms in D. simulans or to divergent selection between the two species. We also find that the amino acid polymorphisms separating EST6- F and EST6-S in D. simulans are not the same as those that separate these allozymes in D. melanogaster, implying that the shared clines do not reflect shared molecular targets for selection. All comparisons within and between the two species reveal a remarkable paucity of variation in a stretch of nearly 400 bp immediately 5' of the gene, indicative of strong selective constraint to retain essential aspects of Est-6 promoter function.      

Specificity and kinetics of alpha-synuclein binding to model membranes determined with fluorescent excited state intramolecular proton transfer (ESIPT) probe

Parkinson disease is characterized cytopathologically by the deposition in the midbrain of aggregates composed primarily of the presynaptic neuronal protein α-synuclein (AS). Neurotoxicity is currently attributed to oligomeric microaggregates subjected to oxidative modification and promoting mitochondrial and proteasomal dysfunction. Unphysiological binding to membranes of these and other organelles is presumably involved. In this study, we performed a systematic determination of the influence of charge, phase, curvature, defects, and lipid unsaturation on AS binding to model membranes using a new sensitive solvatochromic fluorescent probe. The interaction of AS with vesicular membranes is fast and reversible. The protein dissociates from neutral membranes upon thermal transition to the liquid disordered phase and transfers to vesicles with higher affinity. The binding of AS to neutral and negatively charged membranes occurs by apparently different mechanisms. Interaction with neutral bilayers requires the presence of membrane defects; binding increases with membrane curvature and rigidity and decreases in the presence of cholesterol. The association with negatively charged membranes is much stronger and much less sensitive to membrane curvature, phase, and cholesterol content. The presence of unsaturated lipids increases binding in all cases. These findings provide insight into the relation between membrane physical properties and AS binding affinity and dynamics that presumably define protein localization in vivo and, thereby, the role of AS in the physiopathology of Parkinson disease.     

Parallel-Stranded Oligonucleotides with Alternating d(A-isoG)/d(T·C) and d(A·G)/d(T·m5isoC) Sequences

Abstract

Parallel-stranded (ps) DNA hairpins with alternating d(A-isoG)/d(T·C) (designated as ps-t1) and d(A·G)/d(T·m⁵isoC) (ps-t2) sequences were studied by means of UV, CD and fluorescence spectroscopy. The thermostability of d(A·G)/d(T·m⁵isoC) sequence was close to that of aps d(G·A)/d(T·C). The stability of the ps d(A·isoG)/d(T·C) sequence was even higher than that of a related anti-parallel-stranded (aps) d(G·A)/d(T·C) sequence, being unique for ps DNAs studied so far.     

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinson''s disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson''s disease progression, particularly in the context of bioenergetic dysfunction.Parkinson''s disease (PD), the second most common neurodegenerative disorder, is characterized by impairment of the motor system and associated non-motor clinical manifestations.¹ Age² and exposure to environmental toxins³ constitute the most important non-genetic risk factors in the development of sporadic disease. Neuronal loss is progressive, primarily (but not exclusively) dopaminergic, and accompanied by the accumulation of intracellular proteinaceous inclusions known as Lewy bodies and Lewy neurites.⁴α-Synuclein (aSyn) is the main protein constituent of these inclusions⁵ and numerous findings attribute to it a central role in the pathogenesis of PD.^{6, 7, 8, 9} Both missense mutations (p.A30P, p.E46K, p.H50Q,¹⁰ p.G51D,¹¹ p.A53T, p.A53E¹²) and increased copy number (duplication¹³ or triplication¹⁴) of the SNCA gene encoding aSyn (PARK1/4 locus) cause early onset autosomal dominant PD. In addition, multiple genome-wide association studies have established that variations at the SNCA locus contribute significantly to the etiology of sporadic disease.^{15, 16, 17}The induced pluripotent stem cell (iPSC) technology offers a unique and valuable tool for defining the early mechanisms underlying PD and the development of early diagnostics and new therapeutics.^{18, 19, 20} Cell lines have been generated from fibroblasts obtained from patients with a variety of neurodegenerative diseases and neurons differentiated therefrom reproduce specific features of those diseases in vitro.²⁰ Comparisons between patient-derived and appropriately selected healthy control lines are feasible, but unfortunately phenotypic differences unrelated to the disease mechanisms arise due to the high clonal variability inherent in the generation of iPSCs and differences in the genetic background of the iPSC lines.^{21, 22, 23} Lines manipulated by single gene mutation have demonstrated the power of iPS technology for disease modeling^{18, 19, 20} with possible therapeutic potential.^{24, 25}We have examined in this study the effects of increased aSyn expression on the differentiation capacity and phenotypic signatures of two iPS clones derived from a patient with a triplication of the SNCA gene, and compared them with (i) lines generated by lentiviral infection of the patient cells by an shRNA construct targeting aSyn, and (ii) two control iPSC lines one from an unaffected age-matched sibling²⁶ and the other from an unrelated healthy individual.²⁷ All lines were differentiated by defined protocols into neurons that exhibited cardinal neuronal markers. These paradigms were used to assess differentiation capacity, cell survival, neurite outgrowth and electrophysiological properties. The results establish aSyn-dosage as an important modulator of developmental fitness of neuronal progenitor cells and support our previous findings from studies of PD patient fibroblasts²⁸ and neural-committed induced pluripotent stem cells (NiPSCs) (including the knockdown lines featured in this report)²⁹ exposed to toxins: (i) quantifiable reduction in viability under starvation and stress and (ii) decreased mitochondrial function and upregulated catabolism.     

The distribution and evolutionary history of the PRP8 intein

Background  

We recently described a mini-intein in the PRP8 gene of a strain of the basidiomycete Cryptococcus neoformans, an important fungal pathogen of humans. This was the second described intein in the nuclear genome of any eukaryote; the first nuclear encoded intein was found in the VMA gene of several saccharomycete yeasts. The evolution of eukaryote inteins is not well understood. In this report we describe additional PRP8 inteins (bringing the total of these to over 20). We compare and contrast the phylogenetic distribution and evolutionary history of the PRP8 intein and the saccharomycete VMA intein, in order to derive a broader understanding of eukaryote intein evolution. It has been suggested that eukaryote inteins undergo horizontal transfer and the present analysis explores this proposal.