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Crystallographic models of photosystem I (PS I) highlight a symmetrical arrangement of the electron transfer cofactors which are organized in two parallel branches (A, B) relative to a pseudo-C-2 symmetry axis that is perpendicular to the membrane plane. Here, we explore the electron transfer pathways of PS I in whole cells of the deuterated green alga Chlamydomonas reinhardtii using high-time-resolution electron paramagnetic resonance (EPR) at cryogenic temperatures. Particular emphasis is given to quantum oscillations detectable in the tertiary radical pairs P(700)(+)A(1A)(-) and P(700)(+)A(1B)(-) of the electron transfer chain. Results are presented first for the deuterated site-directed mutant PsaA-M684H in which electron transfer beyond the primary electron acceptor A(0A) on the PsaA branch of electron transfer is impaired. Analysis of the quantum oscillations, observed in a two-dimensional Q-band (34 GHz) EPR experiment, provides the geometry of the B-side radical pair. The orientation of the g tensor of P-700(+) in an external reference system is adapted from a time-resolved multifrequency EPR study of deuterated and N-15-substituted cyanobacteria (Link, G.; Berthold, T.; Bechtold, M.; Weidner, J.-U.; Ohmes, E.; Tang, J.; Poluektov, O.; Utschig, L.; Schlesselman, S. L.; Thurnauer, M. C.; Kothe, G. J. Am. Chem, Soc. 2001, 123, 4211-4222). Thus, we obtain the three-dimensional structure of the B-side radical pair following photoexcitation of PS I in its native membrane. The new structure describes the position and orientation of the reduced B-side quinone A(1B)(-) on a nanosecond time scale after light-induced charge separation. Furthermore, we present results for deuterated wild-type cells of C. reinhardtii demonstrating that both radical Pairs P(700)(+)A(1A)(-) and P(700)(+)A(1B)(-) participate in the electron transfer process according to a mole ratio of 0.71/0.29 in favor of P(700)(+)A(1A)(-). A detailed comparison reveals different orientations of A(1A)(-) and A(1B)(-) in their respective binding sites such that formation of a strong hydrogen bond from A(1)(-) to the protein backbone is possible only in the case of A(1A)(-). We suggest that this is relevant to the rates of forward electron transfer from A(1A)(-) or A(1B)(-) to the iron-sulfur center F-x, which differ by a factor of 10. Thus, the present study sheds new light on the orientation of the phylloquinone acceptors in their binding pockets in PS I and the effect this has on function.

The mechanism of in vivo iron chelation by 3-hydroxypyridin-4-ones (CP compounds) was studied in hypertransfused rats in which the major storage iron pools in hepatocytes and in the reticuloendothelial (RE) system have been labeled by selective radioiron probes. Both dimethyl-3- hydroxypyridin-4-one (CP 20 or L1) and diethyl-3-hydroxypyridine-4-one (CP 94) have an identical and very high (log beta 3 36) binding constant and selective affinity to iron(III), but the lipid solubility of CP 94 is considerably higher than that of CP 20. Both chelators induced an increase in the fecal excretion of hepatocellular iron with no effect on urinary excretion. In contrast, about one third to one half of the iron mobilized from RE cells was excreted in the urine. The chelating efficiency of CP 20 was comparable with that of deferoxamine (DF), whereas CP 94 was up to eight times more effective than DF. Unlike DF, which had no effect by the oral route, the oral and parenteral effectiveness of both CP compounds was identical. These findings indicate that: (1) lipid solubility is an important determinant of in vivo chelating efficiency; (2) urinary iron excretion induced by the CP compounds is derived from RE cells; (3) part of the iron mobilized from RE cells and all of the iron derived from hepatocytes is excreted through the bile; and (4) contrary to previous observations in cell cultures, there is no in vivo evidence for a diminishing chelating efficiency at the lowest doses used.

The ability of 3-hydroxypyridin-4-ones (CP), a family of bidentate orally effective iron chelators, to remove iron and to prevent iron- induced lipid peroxidation was studied in beating rat myocardial cells in culture. The iron (III) binding constant (log beta 3) of all CP compounds is 36, but their lipophilicity may be modified by altering the length of the R2 substituent on the ring nitrogen. There was a direct relation between lipid solubility and chelating efficiency. Although at high concentrations all CP compounds were more effective in iron mobilization than deferoxamine, the opposite was true for low concentrations. Further studies with 1,2-diethyl-3-hydroxypyridin-4-one (CP94), the most effective CP compound, have shown that iron mobilization is completed within 6 hours, that effective mobilization requires a drug: iron molar ratio exceeding 3:1 permitting the formation of a hexadentate complex, and that the beneficial effects of iron mobilization are manifested in a marked reduction in membrane lipid peroxidation as indicated by cellular malonaldehyde content. Our study represents the first demonstration of a direct interaction between myocardial cells and an orally effective iron chelator, and underlines the need for high molar concentrations for achieving an optimal therapeutic effect.

An epidemiologic study was initiated in 1987 to evaluate the long-term safety and efficacy of zidovudine in patients with advanced human immunodeficiency virus disease. Data from 886 patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex and CD4+ lymphocyte count less than 0.25 x 10(9)/L are reported. Eighteen-month survival was 67% for the cohort. Pretreatment factors associated with increased survival time included index diagnosis of AIDS-related complex, hematocrit of 0.35 or greater, CD4+ lymphocyte count of 0.15 x 10(9)/L or greater, high functional status, and time from diagnosis of AIDS to treatment of less than 60 days. By proportional hazards analysis, development of serious anemia was the most significant factor associated with early death. Receiving zidovudine for a high proportion of time significantly improved chances of survival even if anemia developed. Serious leukopenia occurring in 37% and serious anemia occurring in 32% of patients. Nonhematologic adverse events were uncommon and no previously unreported adverse events were seen.

Low-density parity check codes are a form of error correcting codes used in various wireless communication applications and in disk drives. While LDPC codes are desirable due to their ability to achieve near Shannon-limit communication channel capacity, the computational complexity of the decoder is a major concern. LDPC decoding consists of a series of iterative computations derived from a message-passing bipartite graph. In order to efficiently support the communication intensive nature of this application, we present a LDPC decoder architecture based on a network-on-chip communication fabric that provides a 1.2 Gbps decoded throughput rate for a 3/4 code rate, 1024-bit block LDPC code. The proposed architecture can be reconfigured to support other LDPC codes of different block sizes and code rates. We also propose two novel power-aware optimizations that reduce the power consumption by up to 30%.

Low density parity checks (LDPC) are a method of error detection and correction that are able to achieve near Shannon-limit channel communication. LDPC decoders involve a series of computations between two units, the check node and the bit node. In this paper we propose the use of an approximation unit to perform the check node operation. Additionally, we propose a ROM based look-up table (LUT) as a function approximation technique, to be used with an LDPC decoder. The paper shows that a ROM based LUT achieves better performance than using a piecewise linear approximation method to approximate the LDPC computation function. Furthermore, this paper shows that the ROM LUT method can gradually take over as the selected function approximation technique for computationally intensive demanding VLSI designs as the technology shifts to the nanometer era.

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Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKCmu/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKCmu/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKCmu partially colocalizes with PKC-eta in different cell types. Colocalization depends on the presence of the PKCmu pleckstrin homology domain. Coexpression of constitutively active PKC-eta with PKCmu leads to a significant enhancement of the PKCmu substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser(738/742) of PKCmu, whereas Ser(910) autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKCeta directly phosphorylates PKCmu on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKCeta-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKCmu/PKCeta affecting different arms of the p38/ERK/ JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKCmu for interaction with and activation by PKCeta as pleckstrin homology domain and activation loop.

We here describe the structural requirements for Golgi localization and a sequential, localization-dependent activation process of protein kinase C (PKC) involving auto- and transphosphorylation. The structural basis for Golgi compartment localization was analyzed by confocal microscopy of HeLa cells expressing various PKC-green fluorescent protein fusion proteins costained with the Golgi compartment-specific markers p24 and p230. Deletions of either the NH2-terminal hydrophobic or the cysteine region, but not of the pleckstrin homology or the acidic domain, of PKC completely abrogated Golgi localization of PKC. As an NH2-terminal PKC fragment was colocalized with p24, this region of PKC is essential and sufficient to mediate association with Golgi membranes. Fluorescence recovery after photobleaching studies confirmed the constitutive, rapid recruitment of cytosolic PKC to, and stable association with, the Golgi compartment independent of activation loop phosphorylatio n. Kinase activity is not required for Golgi complex targeting, as evident from microscopical and cell fractionation studies with kinase-dead PKC found to be exclusively located at intracellular membranes. We propose a sequential activation process of PKC, in which Golgi compartment recruitment precedes and is essential for activation loop phoshorylation (serines 738/742) by a transacting kinase, followed by auto- and transphosphorylation of NH2-terminal serine(s) in the regulatory domain. PKC activation loop phosphorylation is indispensable for substrate phosphorylation and thus PKC function at the Golgi compartment.

Here we show that human protein kinase C mu (PKC mu) activates the mitogen-activated protein kinase (MAPK). Transient expression of constitutive active PKC mu leads to an activation of Raf-1 kinase as demonstrated by in vitro phosphorylation of MAPK. PKC mu enhances transcriptional activity of a basal thymidine kinase promotor containing serum response elements (SREs) as shown by luciferase reporter gene assays. SRE driven gene activation by PKC mu is triggered by the Elk-1 ternary complex factor. PKC mu-mediated activation of SRE driven transcription can be inhibited by the MEK1 inhibitor PD98059. In contrast to the activation of the p42/ERK1 MAPK cascade, transient expression of constitutive active PKC mu does neither affect c-jun N-terminal kinase nor p38 MAPK.

International audience

International audience