An estimator for the proportional hazards model with multiple longitudinal covariates measured with error

In many longitudinal studies, it is of interest to characterize the relationship between a time-to-event (e.g. survival) and several time-dependent and time-independent covariates. Time-dependent covariates are generally observed intermittently and with error. For a single time-dependent covariate, a popular approach is to assume a joint longitudinal data-survival model, where the time-dependent covariate follows a linear mixed effects model and the hazard of failure depends on random effects and time-independent covariates via a proportional hazards relationship. Regression calibration and likelihood or Bayesian methods have been advocated for implementation; however, generalization to more than one time-dependent covariate may become prohibitive. For a single time-dependent covariate, Tsiatis and Davidian (2001) have proposed an approach that is easily implemented and does not require an assumption on the distribution of the random effects. This technique may be generalized to multiple, possibly correlated, time-dependent covariates, as we demonstrate. We illustrate the approach via simulation and by application to data from an HIV clinical trial.     

Photosystem-II repair and chloroplast recovery from irradiance stress: relationship between chronic photoinhibition,light-harvesting chlorophyll antenna size and photosynthetic productivity in Dunaliella salina (green algae)

High-light (HL) grown Dunaliella salina cells exhibit lower pigment content, a highly truncated chlorophyll (Chl) antenna size, and accumulation of photodamaged PS II centers in the chloroplast thylakoids (chronic photoinhibition). In HL-grown cells, the rate of photosynthesis saturated at higher irradiances and the quantum yield was lower compared to that of normally-pigmented low-light (LL) grown cells. In spite of these deficiencies, the light-saturated rate of photosynthesis for the HL-cells, when measured on a per chlorophyll basis, was 3 times greater than that of the LL-grown cells. To delineate the effect of photoinhibition from the Chl antenna size on quantum yield and rate of photosynthesis, HL-acclimated cells were switched to LL-conditions. Repair of photodamaged PS II, estimated from the recovery of functional PS II centers and from the increase in the quantum yield of photosynthesis, occurred with a half-time of 1 h. Chlorophyll accumulation in the cells occurred with a half-time of 4 h. The differential kinetics in repair versus Chl accumulation provided a window of opportunity, within about 2–3 h after the HLLL shift, when cells exhibited a high quantum yield of photosynthesis, a small Chl antenna size and a light-saturated rate that was 6–9 times greater than that of the normally pigmented LL-grown cells. The work provides insight on the temporal sequence of events at the chloroplast and thylakoid membrane levels, leading from a chronic photoinhibition of PS II to repair and recovery. It is suggested that it is possible to maximize photosynthetic productivity and light utilization in mass microalgal cultures by minimizing the light-harvesting Chl antenna size of the photosystems.     

13C shieldings, 18O isotope effects on 13C shieldings, and 57Fe-13C spin couplings of the Fe-C-O unit in superstructured hemoprotein models: Comparison with hemoproteins, C-O vibrational frequencies, and X-ray structural data

¹³C NMR spectra of several carbon monoxide (99.7% ¹³C and 11.8% ¹⁸O enriched) hemoprotein models with varying polar and steric effects of the distal organic superstructure and constraints of the proximal side are reported. This enables the ⁵⁷Fe-¹³C(O) coupling constants ( ), ¹³C shieldings ((¹³C)), and ¹⁸O isotope effects on¹³ C shieldings (¹¹³C(¹⁸O/¹⁶O)) to be measured and hence comparisons with hemoproteins, C-O vibrational frequencies and X-ray structural data to be made. Negative polar interactions in the binding pocket and inhibition of Fe//CO back-donation or positive distal polar interactions with amide NH groups appear to have little direct effect on couplings. Similarly, the axial hindered base 1,2-dimethylimidazole has a minor effect on the values despite higher rates of CO desorption being observed for such complexes. On the contrary,¹³ C shieldings vary widely and an excellent correlation was found between the infrared C-O vibrational frequencies ((C-O)) and¹³ C shieldings and a reasonable correlation with¹⁸ O isotope effects on ¹³C shieldings. This suggests that (¹³C), (C-O) and^{1 13} C(¹⁸O/¹⁶O) are accurate monitors of the multiple mechanisms by which steric and electronic interactions are released in superstructured heme model compounds. The ¹³C shieldings of heme models cover a 4.0 ppm range which is extended to 7.0 ppm when several HbCO and MbCO species at different pH values are included. The latter were found to obey a similar linear (¹³ (¹³C) versus (C-O) relationship, which proves that both heme models and heme proteins are homogeneous from the structural and electronic viewpoint. Our results suggest that (C-O), (¹³C) and ¹¹³C(¹⁸O/¹⁶O) measurements reflect similar interaction which is primarily the modulation of back-bonding from the Fe d to the CO * orbital by the distal pocket polar interactions. The lack of correlation between^{1 13} C(¹⁸O/¹⁶O) and crystallographic CO bond lengths (r(C-O)) reflects significant uncertainties in the X-ray determination of the carbon and oxygen positions.     

Dunaliella salina (Chlorophyta) with small chlorophyll antenna sizes exhibit higher photosynthetic productivities and photon use efficiencies than normally pigmented cells

The photon use efficiencies and maximal rates of photosynthesis in Dunaliella salina (Chlorophyta) cultures acclimated to different light intensities were investigated. Batch cultures were grown to the mid-exponential phase under continuous low-light (LL: 100 μmol photon m^-2 s^-1) or high-light (HL: 2000 μmol photon m^-2 s^-1) conditions. Under LL, cells were normally pigmented (deep green) containing ∼500 chlorophyll (Chl) molecules per photosystem II (PSII) unit and ∼250 Chl molecules per photosystem I (PSI). HL-grown cells were yellow-green, contained only 60 Chl per PSII and 100 Chl per PSI and showed signs of chronic photoinhibition, i.e., accumulation of photodamaged PSII reaction centers in the chloroplast thylakoids. In LL-grown cells, photosynthesis saturated at ∼200 μmol photon m^-2 s^-1 with a rate (P_max) of ∼100 mmol O₂ (mol Chl)^-1 s^-1. In HL-grown cells, photosynthesis saturated at much higher light intensities, i.e. ∼2500 μmol photon m^-2 s^-1, and exhibited a three-fold higher P_max (∼300 mmol O₂ (mol Chl)^-1 s^-1) than the normally pigmented LL-grown cells. Recovery of the HL-grown cells from photoinhibition, occurring prior to a light-harvesting Chl antenna size increase, enhanced P_max to ∼675 mmol O₂ (mol Chl)^-1 s^-1. Extrapolation of these results to outdoor mass culture conditions suggested that algal strains with small Chl antenna size could exhibit 2–3 times higher productivities than currently achieved with normally pigmented cells. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thylakoid Membrane Development and Differentiation: Assembly of the Chlorophyll a-b Light-Harvesting Complex and Evidence for the Origin of Mr=19, 17.5 and 13.4 kDa Proteins

Pea plants were grown under intermittent illumination (ImL)conditions. The low dosage of light given to ImL plastids limitedthe rate of chlorophyll (Chl) a and Chl b biosynthesis and,therefore, it retarded the rate of photosynthetic unit formationand thylakoid membrane development. Depending on the developmentalstage of the photosynthetic unit, ImL plastids had variableChl a/Chl b ratios (2.7 <Chl a/Chlb<20) and showed distinctintermediates in the assembly of the chlorophyll a–b light-harvestingcomplex (LHC) of photosystem-II (PSII). The results are consistentwith a step-wise increment in the PSII antenna size involvingthree distinct forms of the PSII unit: (i) a PSII-core formwith about 37 Chl a molecules; (ii) a PSIL_ß form containingthe PSII-core and the LHC-II-inner antenna with a total of about130 Chl (a + b) molecules, and (iii) the mature PSII_a form containingPSII_ß and the LHC-II-peripheral antenna with a totalof 210–300 Chl (a + b) molecules. The thylakoid membranecontained polypeptide subunits b, c and d (the Lhcb1, 2 and3 gene products, respectively) when only the LHC-II-inner waspresent. Polypeptide subunit a, (the apoprotein of the chlorophyll-proteinknown as CP29), along with increased amounts of b and c appearedlater in the development of thylakoids, concomitant with theassembly of the LHC-II-peripheral. The results suggest thatpolypeptide subunit d has priority of assembly over subunita. It is implied that, of all LHC-II constituent proteins, subunitd is most proximal to the PSII-core complex and that it servesas a linker in the transfer of excitation energy from the bulkLHC-II (subunits b and c) to the PSII-core. The work also addressesthe origin of low-molecular-weight proteins (M_r = 19, 17.5 and13.4 kDa) which co-isolate with intact developing plastids andwhose abundance decreases during plastid development. Aminoacid compositional and immunoblot analyses show a nuclear histoneorigin for these low-molecular-weight proteins and suggest co-isolationof histone-containing nuclear vesicles along with intact developingplastids. ¹Present address: Plant Physiology Research Group, The Universityof Calgary, Department of Biological Sciences, 2500 UniversityDrive N.W., Calgary, Alberta CANADA T2N 1N4.     

Assembly and composition of the chlorophyll a-b light-harvesting complex of barley (Hordeum vulgare L.): Immunochemical analysis of chlorophyll b-less and chlorophyll b-deficient mutants

The chlorina-f2 mutant of barley (Hordeum vulgare L.) contains no chlorophyll b in its light-harvesting antenna, whereas the chlorina-103 mutant contains approximately 10% of the chlorophyll b found in wild-type. The absolute chlorophyll antenna size for Photosystem-II in wild-type, chlorina-103 and chlorina-f2 mutant was 250, 58 and 50 chlorophyll molecules, respectively. The absolute chlorophyll antenna size for Photosystem-I in wild-type, chlorina-103 and chlorina-f2 mutant was 210, 137 and 150 chlorophyll molecules, respoectively. In spite of the smaller PS I antenna size in the chlorina mutants, immunochemical analysis showed the presence of polypeptide components of the LHC-I auxiliary antenna with molecular masses of 25, 19.5 and 19 kDa. The chlorophyll a-b-binding LHC-II auxiliary antenna of PS II contained five polypeptide subunits in wild-type barley, termed a, b, c, d and e, with molecular masses of 30, 28, 27, 24 and 21 kDa, respectively. The polypeptide composition of the LHC-II auxiliary antenna of PS II was found to be identical in the two mutants, with only the 24 kDa subunit d present at an equal copy number per PS II in each of the mutants and in the wild-type barley. This d subunit assembles stably in the thylakoid membrane even in the absence of chlorophyll b and exhibits flexibility in its complement of bound chlorophylls. We suggest that polypeptide subunit d binds most of the chlorophyll associated with the residual PS II antenna in the chlorina mutants and that is proximal to the PS II-core complex.Abbreviations CP chlorophyll-protein - LHC the chlorophyll a-b binding light-harvesting complex - LHC-II subunit a the Lhcb4/5 gene product - subunit b the Lhcb1 gene product - subunit c Lhcb2 the gene product - subunit d the Lhcb3 gene product - subunit e the Lhcb6 gene product - PMSF phenylmethane sulphonyl fluoride - RC reaction center - Q_A the primary quinone electron acceptor of Photosystem-II - P700 the reaction center of PS I     

Organization and Stability of Polypeptides Associated with the Chlorophyll a-b Light-Harvesting Complex of Photosystem-II

In the oxygen-evolving photosystem-II (PSII) of higher plantchioroplasts and green algae, most of the light-harvesting functionis performed by the chlorophyll (Chl) a-b-protein complex (LHC-II).On the average, the LHC-II contains about 210 Chl (a+b) moleculesper PSII reaction center. The polypeptide composition, copynumber and organization of assembly in the LHC-II complex arenot fully understood at present. This work utilized the chlorinaf2 mutant of barley (lacking Chl b and having a LHC-II antennaof only 13 Chl a molecules) to determine the organization andstability of assembly of proteins in the LHC-II. High-resolutionSDS-PAGE and immunoblot analysis showed the presence of fourmain constitutive polypeptides in the wild-type LHC-II (termedhere subunits a, b, c and d) with molecular masses in the range30–25 kDa. Of those, only subunit d (a 25 kDa polypeptide)was found to occur at an equal copy number per PSII reactioncenter in both wild-type and in the Chl b-less chlorina f2 mutant.All other subunits were either absent or existed in much loweramounts in the mutant. Subunit d is a polypeptide constituentof the major Chl-protein subcomplex (CPII) of the LHC-II. Itis stably incorporated in the thylakoid membrane in the absenceof Chl b and probably binds the 13 Chl a molecules in the residualLHC-II antenna of the chlorina f2 mutant. We propose that, ofall LHC-II polypeptides, subunit d is most proximal to the PSIIcore and may serve as a linker in the process of excitationenergy transfer from the bulk LHC-II to the PSII reaction centerin chloroplasts. (Received February 25, 1992; Accepted May 12, 1992)     

Kinetic analysis of P-700 photoconversion: Effect of secondary electron donation and plastocyanin inhibition

The kinetics of P-700 photoconversion under weak continuous actinic illumination were quantitatively analyzed to provide information on the relative absorption cross-section σ_PSI of the light-harvesting pigments associated with photosystem I and on the number of electrons stored between the two photosystems in dark-adapted chloroplasts. The theory of chemical kinetics for a system of monomolecular consecutive first-order reactions is reviewed briefly to provide support for the experimental approach taken. A complete inhibition of plastocyanin by cyanide eliminated all secondary electron donation to P-700⁺ and allowed the registration of the exponential (monomolecular) P-700 photoconversion at room temperature. The rate constant K_p-700 of the exponential kinetics was independent of the ionic (± Mg²⁺) and osmotic (± sucrose) strength of the chloroplast suspension medium, and of the oxidation-reduction state of photosystem II. The extent of plastocyanin inhibition in partially inhibited samples was greater under low ionic and low osmotic conditions. In dark-adapted chloroplast samples that were not cyanide treated, the number of electrons stored between the two photosystems was 3.9 ± 0.2 and independent of divalent cations. It is concluded that plastocyanin inhibition by cyanide is favored under low ionic and low osmotic conditions. The Mg²⁺ ion and redox state of photosystem II-independent photoconversion of P-700 does not support significant changes in the spillover of excitation from photosystem II to photosystem I in isolated chloroplasts.     

Frequency content asymmetry of the isokinetic curve between ACL deficient and healthy knee

The torque-time curve patterns of concentric isokinetic knee extension in anterior cruciate ligament (ACL) deficient patients usually present mid-range irregularities associated with the level of anterior tibial translation. The purpose of this study was to compare the smoothness in isokinetic torque production between the ACL deficient and the healthy knee. Thirty ACL deficient soccer players performed bilaterally five trials of maximum concentric knee extension-flexion at 60 degrees /s on a Biodex dynamometer. The three middle trials (a total of six curves) were retained and submitted to further data processing. Maximum frequency values contained within the 90%, 95% and 99% level of the signal power were calculated for each extension and flexion curve. The frequency content of the ACL deficient side proved to be statistically higher compared to the intact side at all levels of the power spectrum. The percentage differences in the frequency content were 18.8%, 10.6% and 40.0% for knee extension, and 49.5%, 24.5% and 16.3% for knee flexion, for the respective power levels. This indicated higher oscillations and, therefore, more unstable mechanical output of the injured knee. An overall biological interpretation of the present results is based on the notion that disturbed motion is generally connected to poor level of joint functionality.     

Plant 2-arylobenzofurans demonstrate a selective estrogen receptor modulator profile

We have isolated from the plant Onobrychis ebenoides three novel arylobenzofurans with binding affinity for the estrogen receptor. In this study, we evaluated these arylobenzofurans, namely ebenfuran I, ebenfuran II and ebenfuran III for their potential selective estrogen receptor modulator (SERM)-like properties. We examined their ability, (1) to induce the insulin growth factor binding protein-3 (IGFBP-3) in MCF-7 breast cancer cells, (2) to stimulate differentiation and mineralization of osteoblastic cell culture by histochemical staining for alkaline phosphatase, Alizarin Red-S staining and calcium levels in the supernatants and (3) to inhibit cell proliferation of cervical adenocarcinoma (Hela) cells by use of the MTT assay. An estrogen receptor mediated effect was investigated by carrying out chloramphenicol acetyl transferase (CAT) assay on transient MCF-7 transfectants. Estradiol and the "pure" antiestrogen ICI 182780 were included to serve as control samples of the estrogenic and antiestrogenic effect respectively. Our data reveal that ebenfuran II is a highly potent SERM, exhibiting antiestrogenic activity in breast cancer cells via the estrogen receptor, estrogenic effect on osteoblasts and no stimulatory effect on cervix adenocarcinoma cells. In conclusion, our study is the first to demonstrate that plant derived arylobenzofurans show a SERM profile and may be considered for the prevention and treatment of diseases such as breast cancer, cervical cancer and osteoporosis.