A model for the 77 K excited state dynamics in Chlamydomonas reinhardtii in state 1 and state 2

The regulatory mechanism of state transitions was studied in Chlamydomonas reinhardtii (C.r.) wild type (WT) as well as mutant strains deficient in the photosystem I (PSI) or the photosystem II (PSII) core. Time-resolved fluorescence measurements were obtained on instantly frozen cells incubated beforehand in the dark in aerobic or anaerobic conditions which leads to state 1 (S1) or state 2 (S2). WT data contains information on the light-harvesting complex (LHC) connected to PSI and PSII. The mutants' data contain information on either LHCII-LHCI-PSI or LHCII-PSII, plus information on LHC antennas devoid of a PS core. In a simultaneous analysis of the data from all strains under S1 or S2 conditions a unified model for the excited state dynamics at 77 K was created. This yielded the completely resolved LHCII-LHCI-PSI and LHCII-PSII dynamics and quantified the state transitions. In WT cells the fraction of light absorbed by LHCII connected to PSII decreases from 45% in S1 to 29% in S2, while it increases from 0% to 16% for LHCII connected to PSI. Thus (16/45 =) 36% of all LHCII is involved in the state transition. In the mutant strains deficient in the PSI core, the red most species peaking at 716 nm disappears completely, indicating that this far red Chl pigment is located in the PSI core. In the mutant strain deficient in the PSII core, red shifted species with maxima at 684 and 686 nm appear in the LHCII antenna. LHCII-684 is quenched and decays with a rate of (310 ps)^? 1.     

Tryptophan fluorescence of human hemoglobin. I. Significant change of fluorescence intensity and lifetimes in the T − R transition

The fluorescence spectra and fluorescence lifetimes due to tryptophan residues in HbA, Hb Chesapeake, NES-des-Arg Hb and Hb Kempsey were determined at room temperature. The fluorescence intensity and apparent fluorescence lifetimes decrease when the deoxy or T structure in HbA changes to the oxy or R structure, while no significant difference was observed in Hb Kempsey. The difference of fluorescence behavior was ascribed to the quaternary conformational transition of T- and R-states.     

Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating <Emphasis Type="Italic">Brevundimonas diminuta</Emphasis> in drinking water

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100–200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 × 10³ colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5′ 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (<4 h) enumeration of B. diminuta and may be viable alternatives to plating when validating drinking water filtration systems.     

A residue in helical conformation in the native state adopts a β-strand conformation in the folding transition state despite its high and canonical Φ-value

Delineating structures of the transition states in protein folding reactions has provided great insight into the mechanisms by which proteins fold. The most common method for obtaining this information is Φ-value analysis, which is carried out by measuring the changes in the folding and unfolding rates caused by single amino acid substitutions at various positions within a given protein. Canonical Φ-values range between 0 and 1, and residues displaying high values within this range are interpreted to be important in stabilizing the transition state structure, and to elicit this stabilization through native-like interactions. Although very successful in defining the general features of transition state structures, Φ-value analysis can be confounded when non-native interactions stabilize this state. In addition, direct information on backbone conformation within the transition state is not provided. In the work described here, we have investigated structure formation at a conserved β-bulge (with helical conformation) in the Fyn SH3 domain by characterizing the effects of substituting all natural amino acids at one position within this structural motif. By comparing the effects on folding rates of these substitutions with database-derived local structure propensity values, we have determined that this position adopts a non-native backbone conformation in the folding transition state. This result is surprising because this position displays a high and canonical Φ-value of 0.7. This work emphasizes the potential role of non-native conformations in folding pathways and demonstrates that even positions displaying high and canonical Φ-values may, nevertheless, adopt a non-native conformation in the transition state.     

The Antarctic psychrophile, Chlamydomonas subcaudata, is deficient in state I–state II transitions

State I-State II transitions were monitored in vivo and in vitro in the Antarctic, psychrophillic, green alga, Chlamydomonas subcaudata, as changes in the low-temperature (77 K) chlorophyll fluorescence emission maxima at 722 nm (F722) relative to 699 nm (F699). As expected, the control mesophillic species, Chlamydomonas reinhardtii, was able to modulate the light energy distribution between photosystem II and photosystem I in response to exposure to four different conditions: (i) dark/anaerobic conditions, (ii) a change in Mg2+ concentration, (iii) red light, and (iv) increased incubation temperature. This was correlated with the ability to phosphorylate both of its major light-harvesting polypeptides. In contrast, exposure of C. subcaudata to the same four conditions induced minimum alterations in the 77 K fluorescence emission spectra, which was correlated with the ability to phosphorylate only one of its major light-harvesting polypeptides. Thus, C. subcaudata appears to be deficient in the ability to undergo a State I-State II transition. Functionally, this is associated with alterations in the apparent redox status of the intersystem electron transport chain and with higher rates of photosystem I cyclic electron transport in the psychrophile than in the mesophile, based on in vivo P700 measurements. Structurally, this deficiency is associated with reduced levels of Psa A/B relative to D1, the absence of specific photosystem I light-harvesting polypeptides [R.M. Morgan et al. (1998) Photosynth Res 56:303-314] and a cytochrome b6/f complex that exhibits a form of cytochrome f that is approximately 7 kDa smaller than that observed in C. reinhardtii. We conclude that the Antarctic psychrophile, C. subcaudata, is an example of a natural variant deficient in State I-State II transitions.     

Microenvironmental influences of apoptosis in vivo and in vitro

The apoptosis program of physiological cell death elicits a range of non-phlogistic homeostatic mechanisms—“recognition, response and removal”—that regulate the microenvironments of normal and diseased tissues via multiple modalities operating over short and long distances. The molecular mechanisms mediate intercellular signaling through direct contact with neighboring cells, release of soluble factors and production of membrane-delimited fragments (apoptotic bodies, blebs and microparticles) that allow for interaction with host cells over long distances. These processes effect the selective recruitment of mononuclear phagocytes and the specific activation of both phagocytic and non-phagocytic cells. While much evidence is available concerning the mechanisms underlying the recognition and responses of phagocytes that culminate in the engulfment and removal of apoptotic cell bodies, relatively little is yet known about the non-phagocytic cellular responses to the apoptosis program. These responses regulate inflammatory and immune cell activation as well as cell fate decisions of proliferation, differentiation and death. Here, we review current knowledge of these processes, considering especially how apoptotic cells condition the microenvironments of normal and malignant tissues. We also discuss how apoptotic cells that persist in the absence of phagocytic clearance exert inhibitory effects over their viable neighbors, paying particular attention to the specific case of cell cultures and highlighting how new cell-corpse-clearance devices—Dead-Cert^® Nanoparticles—can significantly improve the efficacy of cell cultures through effective removal of non-viable cells in the absence of phagocytes in vitro.     

Normal and superconducting state specific heat of Mg1 − xAlxB2 (0 < x < 0.2)

A theoretical analysis of specific heat C(T) behaviour of the Mg_1 ? xAl_xB₂ (0 < x < 0.2) in the temperature domain 0 ≤ T ≤ 300 K is presented. Calculations of C(T) have been made within the two-component scheme: one is the phonon and the other is the electronic contribution. Phonon specific heat is well estimated from the Debye and Einstein temperature for Mg_1 ? xAl_xB₂ obtained following an overlap repulsive potential. The interatomic potential of this model includes contributions from the long-range Coulomb attraction and the short-range overlap repulsion and the van der Waals attraction. The fermion constituent as the electronic specific heat is deduced using a suitable trial function above and below T_c. At the critical temperature, the discontinuity in specific heat is higher than that of other samples and the transition is sharper than for most samples. The theoretical results from boson and fermion terms are then compared with the experimental results.     

Characterization of Japanese flounder karyotype by chromosome bandings and fluorescence in situ hybridization with DNA markers

The chromosomes of Japanese flounder, Paralichthys olivaceus, were examined by conventional differential staining methods including G-, Q-, C-, silver (Ag)-, fluorochrome, and replication R-bandings and by fluorescence in situ hybridization (FISH) with 5S and 18S rDNAs and telomeric DNA as probes. Replication R-banding substantially made it possible to identify 24 homologous pairs by their RBG-banding pattern and relative length. Both rDNA loci were mapped to chromosome 1, where 5S and 18S rDNA loci were located at the centromeric region and secondary constriction, respectively. C-banding revealed that both rDNA loci were heterochromatic, and 18S rDNA loci were positive for chromomycin A₃ but negative for 4′,6-diamidino-2-phenylindole (DAPI) staining. Telomeric FISH signals were observed at all chromosome ends and at the interstitial region of some chromosomes. The observed results were discussed in relation to the karyotype evolution in the order Pleuronectiformes.     

Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5'-phosphate decarboxylase: a role for substrate destabilization?

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed. To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis.     

The discovery of state transitions in photosynthesis 40 years ago

In the late 1960s, I identified an aspect of the kinetics of chlorophyll fluorescence in algal cells that I was unable to explain in terms of photochemical quenching. I proposed a novel regulatory mechanism for the distribution of light energy to photosystems I and II, which is now known by the term of “state transitions.” I also examined the “cation-dependent redistribution of light energy” to photosystems I and II and the “energy-dependent quenching” of chlorophyll fluorescence. At that time, financial constraints prevented me from measuring the emission and action spectra of chlorophyll fluorescence at liquid-nitrogen temperature and the light quality-dependent changes in the yield of chlorophyll fluorescence at room temperature. The financial problems were solved by constructing several pieces of electronic equipment using skills obtained by repairing radios when I was a high-school and college student.     

The transition state of coupled folding and binding for a flexible β-finger

Flexible and fully disordered protein regions that fold upon binding mediate numerous protein-protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a β-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the β-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the β-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the β-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.     

Decay kinetics and quantum yields of fluorescence in photosystem I from Synechococcus elongatus with P700 in the reduced and oxidized state: are the kinetics of excited state decay trap-limited or transfer-limited?

Transfer and trapping of excitation energy in photosystem I (PS I) trimers isolated from Synechococcus elongatus have been studied by an approach combining fluorescence induction experiments with picosecond time-resolved fluorescence measurements, both at room temperature (RT) and at low temperature (5 K). Special attention was paid to the influence of the oxidation state of the primary electron donor P700. A fluorescence induction effect has been observed, showing a approximately 12% increase in fluorescence quantum yield upon P700 oxidation at RT, whereas at temperatures below 160 K oxidation of P700 leads to a decrease in fluorescence quantum yield ( approximately 50% at 5 K). The fluorescence quantum yield for open PS I (with P700 reduced) at 5 K is increased by approximately 20-fold and that for closed PS I (with P700 oxidized) is increased by approximately 10-fold, as compared to RT. Picosecond fluorescence decay kinetics at RT reveal a difference in lifetime of the main decay component: 34 +/- 1 ps for open PS I and 37 +/- 1 ps for closed PS I. At 5 K the fluorescence yield is mainly associated with long-lived components (lifetimes of 401 ps and 1.5 ns in closed PS I and of 377 ps, 1.3 ns, and 4.1 ns in samples containing approximately 50% open and 50% closed PS I). The spectra associated with energy transfer and the steady-state emission spectra suggest that the excitation energy is not completely thermally equilibrated over the core-antenna-RC complex before being trapped. Structure-based modeling indicates that the so-called red antenna pigments (A708 and A720, i.e., those with absorption maxima at 708 nm and 720 nm, respectively) play a decisive role in the observed fluorescence kinetics. The A720 are preferentially located at the periphery of the PS I core-antenna-RC complex; the A708 must essentially connect the A720 to the reaction center. The excited-state decay kinetics turn out to be neither purely trap limited nor purely transfer (to the trap) limited, but seem to be rather balanced.     

Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes

In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation. A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed. One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age. Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively). These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children. The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells. Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo. All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.     

Impaired drug-binding capacities of in vitro and in vivo glycated albumin

Albumin, the major circulating protein in blood, can undergo increased glycation in diabetes. One of the main properties of this plasma protein is its strong affinity to bind many therapeutic drugs, including warfarin and ketoprofen. In this study, we investigated whether or not there were any significant changes related to in vitro or in vivo glycation in the structural properties and the binding of human albumin to both therapeutic drugs. Structural parameters, including redox state and ketoamine contents of in vitro and in vivo glycated purified albumins, were investigated in parallel with their affinity for warfarin and ketoprofen. High-performance liquid chromatography was used to determine the free drug concentrations and dissociation constants according to the Scatchard method. An alternative method based on fluorescence spectroscopy was also used to assess drug-binding properties. Oxidation and glycation levels were found to be enhanced in albumin purified from diabetic patients or glycated with glucose or methylglyoxal, after determination of their ketoamine, free thiol, amino group and carbonyl contents. In parallel, significant impairments in the binding affinity of in vitro and in vivo glycated albumin, as indicated by the higher dissociation constant values and confirmed by higher free drug fractions, were observed. To a lesser extent, this alteration also significantly affected diabetic albumin affinity, indicated by a lower static quenching in fluorescence spectroscopy. This work provides useful information supporting in vivo diabetic albumin could be the best model of glycation for monitoring diabetic physiopathology and should be valuable to know if glycation of albumin could contribute to variability in drugs response during diabetes.     

Development of fluorescence quenching in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> upon prolonged illumination at 77 K

Low-temperature fluorescence measurements are frequently used in photosynthesis research to assess photosynthetic processes. Upon illumination of photosystem II (PSII) frozen to 77 K, fluorescence quenching is observed. In this work, we studied the light-induced quenching in intact cells of Chlamydomonas reinhardtii at 77 K using time-resolved fluorescence spectroscopy with a streak camera setup. In agreement with previous studies, global analysis of the data shows that prolonged illumination of the sample affects the nanosecond decay component of the PSII emission. Using target analysis, we resolved the quenching on the PSII-684 compartment which describes bulk chlorophyll molecules of the PSII core antenna. Further, we quantified the quenching rate constant and observed that as the illumination proceeds the accumulation of the quencher leads to a speed up of the fluorescence decay of the PSII-684 compartment as the decay rate constant increases from about 3 to 4 ns^??1. The quenching on PSII-684 leads to indirect quenching of the compartments PSII-690 and PSII-695 which represent the red chlorophyll of the PSII core. These results explain past and current observations of light-induced quenching in 77 K steady-state and time-resolved fluorescence spectra.     

Serological identification and molecular characterization of B (A) 02 subtype in patients and blood donors from Eastern China

This study was designed to identify the rare?type?ABO?blood?groups, B(A) 02, from Eastern China. Three samples with discordant serological results during routine blood type identification and four samples from one sample’ family were selected. All of them were detected by serological method. The exon 6 and 7 of the ABO genes were amplified by PCR and sequenced. They were typed as AsubB by serology and as BO by genotype. In AsubB samples, nt 700C>G mutation was detected in B gene, which was previously defined as B(A)02 alleles. In these seven samples, six showed B(A)02/O01 and one showed B(A)02/O02.B(A)02 allele was found to be more common in this study than B(A)04 which is considered to be more frequent than B(A)02. The careful identification of rare blood types is important for the safety of clinical blood transfusion.     

Somatic Embryogenesis and in vitro Flowering in <Emphasis Type="Italic">Saposhnikovia divaricata</Emphasis>

Efficient somatic embryogenesis (SE) and in vitro flowering and fruiting were achieved in Saposhnikovia divaricata (Turcz.) Schischk. Friable embryogenic callus developed from the root, internode, and leaf explants on Murashige and Skoog medium (MS) with 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D), and subsequently developed into somatic embryos on MS medium containing 4–5% sucrose, 1.74 μM naphthaleneacetic acid (NAA), 4.44 μM 6-benzylaminopurine (BA), and 1.90 μM abscisic acid (ABA). Then the mature embryos were separated and transferred onto MS with 3% sucrose and 0.6% agar for further development and conversion to plantlets. In vitro flowering and fruiting were obtained when the subcultures were carried out for over 15 months. Paclobutrazol (PP333) or ethephon (ETH) at low levels promoted flowering significantly. Also, abnormal rootless somatic embryos of S. divaricata could form flowers and fruits in vitro.