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The atomic structure of the bacterial light-harvesting complex helps to explain how the energy of sunlight is efficiently absorbed and transferred between bacteriochlorophyll molecules towards the reaction centre.
Irreversible photoinactivation of photosystem II (PSII) results in the degradation of the reaction center II D1 protein. In Synechocystis PCC 6714 cells, recovery of PSII activity requires illumination. The rates of photoinactivation and recovery of PSII activity in the light are similar in cells grown in minimal (MM) or glucose-containing medium (GM). Reassembly of PSII with newly synthesized proteins requires degradation of the D1 protein of the photoinactivated PSII. This process may occur in darkness in both types of cells. The degraded D1 protein is, however, only partially replaced by newly synthesized protein in MM cells in darkness while a high level of D1 protein synthesis occurs in darkness in the GM cells. The newly synthesized D1 protein in darkness appears to be assembled with other PSII proteins. However, PSII activity is not recovered in such cells. Illumination of the cells in absence but not in the presence of protein synthesis inhibitors allows recovery of PSII activity.
A supercomplex containing the photosystem I (PSI) and chlorophyll a/b light-harvesting complex I (LHCI) has been isolated using a His-tagged mutant of Chlamydomonas reinhardtii. This LHCI-PSI supercomplex contained approximately 215 chlorophyll molecules of which 175 were estimated to be chlorophyll a and 40 to be chlorophyll b, based on P700 oxidation and chlorophyll a/b ratio measurements. Its room temperature long wavelength absorption peak was at 680 nm, and it emitted chlorophyll fluorescence maximally at 715 nm (77 K). The LHCI was composed of four or more different types of Lhca polypeptides including Lhca3. No LHCII proteins or other phosphoproteins were detected in the LHCI-PSI supercomplexes suggesting that the cells from which they were isolated were in State 1. Electron microscopy of negatively stained samples followed by image analysis revealed the LHCI-PSI supercomplex to have maximal dimensions of 220 A by 180 A and to be approximately 105 A thick. An averaged top view was used to model in x-ray and electron crystallographic data for PSI and Lhca proteins respectively. We conclude that the supercomplex consists of a PSI reaction center monomer with 11 Lhca proteins arranged along the side where the PSI proteins, PsaK, PsaJ, PsaF, and PsaG are located. The estimated molecular mass for the complex is 700 kDa including the bound chlorophyll molecules. The assignment of 11 Lhca proteins is consistent with a total chlorophyll level of 215 assuming that the PSI reaction center core binds approximately 100 chlorophylls and that each Lhca subunit binds 10 chlorophylls. There was no evidence for oligomerization of Chlamydomonas PSI in contrast to the trimerization of PSI in cyanobacteria.
Cytotoxic necrotizing factor 1 (CNF-1) is an exotoxin of Escherichia coli that constitutively activates the GTPases Rho, Rac, and CDC42. Stimulation of Rho was shown to enhance myosin light-chain (MLC) phosphorylation via Rho kinase-mediated inhibition of MLC phosphatase in endothelial cells. Here we report that 3 h after CNF stimulation of endothelial cells, RhoA was activated and MLC phosphorylation was increased in a Rho/Rho-kinase-dependent manner, but no decrease in MLC phosphatase activity could be detected. Despite continuous RhoA activation, MLC phosphatase activity was doubled after 24 h of CNF stimulation, and this coincided with decreased MLC phosphorylation and cell spreading. Rac was also activated at 3 to 24 h but did not contribute to MLC phosphorylation, and its amount gradually decreased in the CNF-stimulated cells. CDC42Hs was not activated above control values by CNF. These results suggest that CNF can induce specific decoupling (Rho kinase from MLC phosphatase) and deactivation events in Rho GTPase signaling, potentially reflecting cellular protection mechanisms against permanently active Rho GTPases.
The physical origin and functional significance of the near infra-red light scattering changes observable upon flash illumination of diluted suspensions of magnetically oriented, permeabilised frog retinal rods has been reinvestigated with particular attention paid to the degree with which transducin remains attached to the membrane. In the absence of GTP, the so called "binding" signal is shown to include two components of distinctive origins, widely different kinetics, and whose relative amplitudes depend on the dilution of the suspension and resulting detachment of transducin from the disc membrane. The fast component is a consequence of the fast interaction between photoexcited rhodopsin (R*) and the transducin remaining on the membrane. Its kinetics monitors a structural modification of the discs caused by a change in electrostatic interaction between closely packed membranes upon the formation of R*-T complexes. The slow component monitors the slow rebinding to the membrane and possible subsequent interaction with excess R* of T-GDP which, in spite of its low solubility, had eluted into solution given the high dilution of the permeated rods. In the presence of GTP, the so called "dissociation" signal includes a fast, anisotropic "release" component that specifically monitors the release into the interdiscal space of T alpha-GTP formed from the membrane-bound pool, and a slower isotropic "loss" component monitoring the leakage from the permeated rod of the excess T alpha-GTP which did not interact with the cGMP phosphodiesterase. The amplitudes of both components depend exclusively on the membrane bound T-GDP pool. The kinetics of the "loss" component is limited by the size and degree of permeation of the rod fragments, rather than by the dissociation rate of T alpha-GTP from the membrane.
The combined processes of immunoglobulin (IG) gene rearrangement and somatic hypermutation allow for the creation of an extremely diverse antibody repertoire. Knowledge of the germline sequence of the IG genes is required so that hypermutation and the affinity matured humoral response can be properly studied. Variable region genes can be arranged into subgroups; in humans, there are 11 IGLV subgroups and six IGKV subgroups. The rhesus macaque (Macaca mulatta) is a relevant non-human primate model for human immunological systems. A number of macaque IGHV, IGHD and IGHJ genes have already been reported, but only one light-chain germline gene has been published so far. Here we report the isolation of new macaque IGKV genes by polymerase chain reaction (PCR) amplification from macaque genomic DNA using primers based on the human sequences. Twenty-eight IGKV1, 22 IGKV2 and 12 IGKV3 germline genes for the macaque were found, the open reading frames of which exhibit high homology to their human counterparts (>96, >99 and >96%, respectively).
An important goal for the treatment of human B cell malignancies lies in the induction of an active immune response directed against the tumoral clone, and more particularly against antigenic determinants of the monoclonal immunoglobulin (Ig). The presence of idiotype-reactive T-cells in patients with multiple myeloma has been previously reported, and strategies to increase their responsiveness towards the malignant plasma cells are being actively explored. Light chain (LC) myeloma is often an aggressive form of the disease, regarding which antitumoral immunity has not yet been studied. Here, we investigated in an experimental murine model a secreted monoclonal LC that may behave as a strong tumor antigen after immunization of animals with mitomycin C-treated malignant plasma cells producing monoclonal Ig. Non-dividing plasma cells were utilized as a cell suspension to immunize the mice intraperitoneally (i.p.). The immunized mice produced anti-Ig LC antibodies, mounted a cell-mediated response mainly directed against the monoclonal LC determinants, and survived tumor challenge with significant frequency. These results suggest that plasma cells are capable of presenting antigenic determinants derived from a secreted monoclonal LC in an MHC class I context, and of predominantly inducing a monoclonal Ig-specific T-cell response which can contribute to tumor rejection.
In the preceding paper are described the isolation and physical characterization of seven narrowly disperse fractions of calf thymus DNA in the molecular weight range 0.3 to 1.3 X 10(6) daltons. Herein, we have determined by light scattering the molecular weights and root mean square radii of these fractions in a solvent comprising 0.2 M NaCl, 2 mM EDTA, 2mM Na-PO4,pH7. Measurements were made in a modified Wippler-Scheibling photometer to a 20 degree lower limit of scattering angle on solutions rendered virtually dust-free by procedures described. The optical anisotropies of the DNA fractions were measured permitting the experimental molecular weights and root mean square radii to be corrected to their true values. From these values, with appropriate polydispersity corrections, we calculate a Kratky-Porod persistence length, a, of 54.0 +/- 5.6 nm which is invariant over the molecular range examined. From the sedimentation coefficients (preceding paper) and the theory of Yamakawa and Fujii, we calculate a to be 66 nm, a value found to apply equally well to several DNA samples of various origins whose sedimentation rates are known in themolecular weight range from about 4 X 10(4) to 10(8) daltons. Similarly, from the intrinsic viscosities and the theory of Yamakawa and Fujii, we calculate a to be 59 nm, which again adequately applies to a number of DNA samples whose viscosities have been measured by other workers in the molecular wieght range 3 X 10(5) to 10(8) daltons. The Flory-Mandelkern paramerter, beta, was found to vary with molecular weight in the manner predicted by the theory of Yamakawa and Fujii. The average value of a from the three sets of measurements is 60 +/- 6nm, which we believe applies to double-stranded DNA molecules, independent of chain length, over the whole range of molecular weights from which reliable data exist.
High-molecular weight-hindered amine light stabilizers (HMW-HALSs) are of utmost importance in modern polyolefin stabilization technology and in-depth knowledge about their chemical composition, particularly the oligomers, is essential for development of new and more efficient stabilizers. In the present study, the applicability of temperature-programmed packed capillary LC coupled to miniaturized ELSD and positive mode ESI-TOF-MS for analysis of HMW-HALSs is demonstrated through extensive characterization of two state-of-the-art stabilizers, i.e., HALS-1 and HALS-2. Both stabilizers were individually separated on a 320 microm i.d. x 35 cm long Hypersil 3 microm ODS-100 column using a temperature program from 30 to 120 degrees C and a quaternary mixture of ethylacetate, acetonitrile, triethylamine (TEA) and acetic acid (45.0:44.9:10.0:0.1 (v/v/v/v)) as the mobile phase. The effect of using various amounts of ethylacetate, acetonitrile and triethylamine in the mobile phase on the chromatographic separation is demonstrated. Furthermore, the LC-ESI-TOF-MS analyses revealed that HALS-1 (oligomeric) was highly complex and consisted of at least five different mass series, while HALS-2, which was assumed to be monomeric, contained two different mass series. Chemical structures for nearly all species of both stabilizers are proposed.
The non-linear response is generally the main limitation to the general quantitative use of evaporative light-scattering detection (ELSD). In the particular case of triacylglycerol (TG) analysis, we present a preliminary paper dealing with the use of post-column additives as a means of monitoring the response of such a detector. As TG can form molecular association complexes (ligand-ligate associations) with either cholesterol, urea or silver nitrate, we report the influence of the concentration of each of these chemical compounds in the liquid phase directed towards the ELSD system. The results show that the response coefficient b of the calibration curve either decreases from 1.25-1.30 to 0.51 or increases from 1.25-1.30 to 1.78 according to the nature and concentration of post-column additive. The use of cholesterol as additive, at a discrete concentration, may lead to a linear response curve (b = 1), i.e. to the direct proportionality of ELSD response versus the TG concentration, making quantitative analysis of such solutes easier. On the other hand, to improve sensitivity, the addition of silver nitrate may be chosen for an increase in b value.
Radiation-induced stress, either from visible or UV light, is strongest at midday. We found that, in the absence of stress or time cues, Euglena gracilis Z was the most resistant to UV-C and UV-B at subjective midday, whether judged from immediate or reproductive survival. The circadian UV-resistance rhythms were free-running in stationary cultures under 1-h light/1-h dark cycles or continuous darkness, indicating that cell-cycle dependent DNA susceptibility to UV was not involved. We moreover examined what was the primary cause of the circadian UV resistance, estimated as the immediate cell survival. The half-maximal lethal dose (LD(50)) of UV-C at subjective midday (the most resistant phase) was 156 J/m(2), which is approximately 3-fold that at subjective midnight. The same was true for UV-B, except the LD(50) was approximately 13-fold that of UV-C. Temperature during UV irradiation had little effect, indicating that survival was not mediated via enzymatic reactions. Non-enzymatic antioxidants were added 5 min before UV irradiation. Dimethylsulfoxide (a hydroxyl radical scavenger) increased survival after UV-B, but had little effect after UV-C; conversely, sodium ascorbate increased survival after UV-C, but not after UV-B. These findings suggest that circadian rhythms of resistance to UVs involve a common mechanism for maximizing non-enzymatic antioxidative capacity at subjective midday, but the specific antioxidants differ.
Light-dependent reduction of target disulfides on certain chloroplast enzymes results in a change in activity. We have modeled the tertiary structure of four of these enzymes, namely NADP-linked glyceraldehyde-3-P dehydrogenase, NADP-linked malate dehydrogenase, sedoheptulose bisphosphatase, and fructose bisphosphatase. Models are based on x-ray crystal structures from non-plant species. Each of these enzymes consists of two domains connected by a hinge. Modeling suggests that oxidation of two crucial cysteines to cystine would restrict motion around the hinge in the two dehydrogenases and influence the conformation of the active site. The cysteine residues in the two phosphatases are located in a region known to be sensitive to allosteric modifiers and to be involved in mediating structural changes in mammalian and microbial fructose bisphosphatases. Apparently, the same region is involved in covalent modification of phosphatase activity in the chloroplast.
To ascertain the minimal structural requirements for formation of the subunit and core light-harvesting complex (LH1), the alpha- and beta-polypeptides of the LH1 from three purple photosynthetic bacteria were enzymatically or chemically truncated or modified. These polypeptides were then used in reconstitution experiments with bacteriochlorophyll a (BChla), and the formation of subunit and LH1 complexes was evaluated using absorbance and circular dichroism spectroscopies. Truncation or modification outside of the conserved core sequence region of the polypeptides had no effect on subunit or LH1 formation. However, the extent of formation and stability of the subunit and LH1 decreased as the polypeptide was shortened inside the core region within the N-terminal domain. This behavior was suggested to be due to the loss of potential ion-pairing and/or hydrogen-bonding interactions between the polypeptides. While the spectroscopic properties of the subunit complexes generated using truncated polypeptides were analogous to those obtained using native polypeptides, in some cases the resulting LH1 complex absorption was blue-shifted relative to the control. Thus, truncation within the N-terminal domain may have long-range effects on the immediate BChla binding environment, since the putative BChla binding site resides near the C-terminal end of the polypeptides. It was also demonstrated that the His located within the membrane-spanning domain on the N-terminal end of the beta-polypeptide is not participating in ligation of the BChla in the reconstituted subunit and therefore probably not in LH1.
The B800-850 light-harvesting complex (also called LH2) was isolated from photosynthetic membranes of Rhodospirillum molischianum DSM 119 using molecular sieve and ion-exchange chromatography. Its two bacteriochlorophyll a-binding polypeptides (alpha-subunit and beta-subunit) were purified with a reverse-phase HPLC system. The complete amino acid sequences of both subunits have been determined. The alpha- and beta-subunits consist of 56 and 45 amino acids, respectively, corresponding to molecular weights of 5939 and 5133. In contrast to the B800-850 complexes from other photosynthetic bacteria, the native B800-850 complex from Rs. molischianum is most likely an octamer of monomers with a stoichiometry of three bacteriochlorophyll a and 1.5 lycopenes per alpha,beta-subunit. Resonance Raman spectra provide evidence for a 5-coordinated Mg2+ in the BChl, and a carotenoid mainly in the all-trans configuration. A comparison between resonance Raman data from different photosynthetic bacteria indicates that the BChl a-binding site of the B800-850 complex from Rs. molischianum is more similar to the B870 complexes (also called LH1) than to the B800-850 complexes of other photosynthetic bacteria. Sequence similarities especially between the beta-subunits of the B800-850 complex of Rs. molischianum and the B870 and B800-850 complexes of other photosynthetic bacteria agree with this result and provide information on the mode of pigment binding in bacterial antenna complexes.
The subunit light-harvesting 1 (LH 1) complexes isolated from photosynthetic bacteria Rhodospirillum rubrum using n-octyl-beta-glucoside were reassociated and adsorbed on a mica substrate using spin-coat methods with the aim of using this LH complex in a nanodevice. The near-IR absorption and fluorescence spectra of the LH 1 complexes indicated that the LH 1 complex on the mica was stable, and efficient energy transfer from a carotenoid to a bacteriochlorophyll a was observed. Atomic force microscopy of the reassociated LH 1 complexes, under air, showed the expected ringlike structure. The outer and inner diameters of the ringlike structure of the LH 1 complex were approximately 30 and 8 nm, respectively, and the ringlike structure protruded by 0.2-0.6 nm.
The genes that encode the immunoglobulin proteins made by B lymphocytes are made up of segments that are separately encoded in the germ-line genome and brought together by recombination during B-cell ontogeny. There are two types of immunoglobulin light chain, kappa and lambda, but only a single type is expressed in individual B cells. It is thought that kappa gene recombination precedes lambda gene recombination during B-cell ontogeny. We describe here unusual recombinations that have occurred in two lambda-producing B-cell lines and suggest that they are involved in the developmental switch from kappa to lambda gene expression in maturing B cells. These recombinations involve the J kappa-C kappa introns of V-J joined but nonfunctional kappa genes and a sequence that in the germ line occurs downstream of the C kappa exon (called RS, for recombining sequence).
Myosin II regulatory light chains have an important role in the organization and function of the contractile machinery at cytokinesis. Two recent reports provide new insights into these important proteins.
The mRNA molecules coding for three mouse immunoglobulin lambda-type light (L) chains (MOPC-104E lambda(1), RPC-20 lambda(1), MOPC-315 lambda(2)) programme the cell-free synthesis of precursors larger than the mature proteins. Radioactive amino acid-sequence analyses of each of the three precursors labelled with [(3)H]alanine, [(3)H]serine, [(3)H]glutamine, [(3)H]glutamic acid and [(3)H]threonine showed that an extra piece, at least 18 residues long, is linked to the N-terminus of the mature L-chains. The N-terminal extra-peptide segment may be 19 residues long, since analyses of precursors labelled with [(35)S]methionine indicated an additional N-terminal methionine residue which was recovered in low yields. Presumably this is the initiator methionine, which is known to be short lived in eukaryotes. The mature forms of MOPC-104E, RPC-20 and MOPC-315 lambda L-chains are blocked at the N-termini by pyrrolid-2-one-5-carboxylic acid (pyroglutamic acid). Sequence analyses of precursors labelled with [(3)H]glutamine and [(3)H]glutamic acid showed incorporation only of glutamine in a position that matches with the position of pyrrolid-2-one-5-carboxylic acid in the mature forms of all three precursors, and incorporation of glutamic acid in other positions. The data showed the absence of glutamine-glutamic acid interconversion, since the radioactive peaks obtained from either (3)H-labelled amino acid were discrete, and free from cross-contamination. These results prove that glutamine is the precursor amino acid of pyrrolid-2-one-5-carboxylic acid at the N-termini of the mature MOPC-104E lambda(1), RPC-20 lambda(1) and MOPC-315 lambda(2) L-chains. Thus the formation of pyrrolid-2-one-5-carboxylic acid by cyclization of glutamine is a post-translational event which occurs after, or concomitant with, cleavage of the extra piece from the precursor to yield the mature L-chain. The variable (V) regions (110 amino acid residues) of mouse lambda L-chains are quite similar: when compared with that of MOPC-104E lambda(1) chain, the V-region of RPC-20 lambda(1) chain differs in one residue, and the V-region of MOPC-315 lambda(2) chain differs in 11 residues. The partial sequence data show that the N-terminal extra pieces of the two lambda(1) L-chain precursors have, so far, identical partial sequences; the extra piece of the lambda(2) L-chain precursor differs from these in at least three out of 19 positions.