Degradation pattern of photosystem II reaction center protein D1 in intact leaves. The major photoinhibition-induced cleavage site in D1 polypeptide is located amino terminally of the DE loop.

Photoinhibition-induced degradation of the D1 protein of the photosystem II reaction center was studied in intact pumpkin (Cucurbita pepo L.) leaves. Photoinhibition was observed to cause the cleavage of the D1 protein at two distinct sites. The main cleavage generated an 18-kD N-terminal and a 20-kD C-terminal degradation fragment of the D1 protein. this cleavage site was mapped to be located clearly N terminally of the DE loop. The other, less-frequent cleavage occurred at the DE loop and produced the well-documented 23-kD, N-terminal D1 degradation product. Furthermore, the 23-kD, N-terminal D1 fragment appears to be phosphorylated and can be detected only under severe photoinhibition in vivo. Comparison of the D1 degradation pattern after in vivo photoinhibition to that after in vitro acceptor-side and donor-side photoinhibition, performed with isolated photosystem II core particles, gives indirect evidence in support of donor-side photoinhibition in intact leaves.     

D1 protein degradation during photoinhibition of intact leaves a modification of the D1 protein precedes degradation

Illumination of intact pumpkin leaves with high light led to severe photoinhibition of photosystem II with no net degradation of the D1 protein. Instead, however, a modified form of D1 protein with slightly slower electrophoretic mobility was induced with corresponding loss in the original form of the D1 protein. When the leaves were illuminated in the presence of chloramphenicol the modified form was degraded, which led to a decrease in the total amount of the D1 protein. Subfractionation of the thylakoid membranes further supported the conclusion that the novel form of the D1 protein was not a precursor but a high-light modified form that was subsequently degraded.     

The molecular size of glycans liberated by hydrazinolysis from semliki forest virus proteins

The glycans of well characterized, [6-³H]galactose-labelled glycopeptides, GC-4 from bovine IgG₁ as well as GP-V-2 and GP-V-5 from α₁-acid glycoprotein, were liberated by hydrazinolysis. Molecular weights close to the expected values were observed by gel filtration. Desialated glycans of Semliki Forest virus proteins were likewise liberated by hydrazinolysis and subjected to gel filtration. Metabolically labelled [1-³H]galactose-oligosaccharides of the mixed viral proteins revealed an apparent molecular weight of 1800. The bi-antennary glycan liberated from the reference glycopeptide GC-4 was of 1750 daltons. A mixture of [2-³H]mannose-labelled E₁-and E₂-proteins of the virus contained L-type glycans of 1800 daltons (formerly called A-type), and M-type glycans of 1200 daltons (formerly called B-type). A fraction of the E₃-glycans isolated by affinity chromatography on Concanavalin A-Sepharose showed an average molecular weight of 2150, a value intermediate between the three- and four-antennary glycans liberated from the reference glycopeptides GP-V-5 and GP-V-2. The rest of the E₃-glycans were of 1850 daltons, a value close to the bi-antennary GC-4 glycan. We suggest that the comparatively large size of the E₃-glycans and the exposed position of E₃-proteins on the viral surface may be interrelated.     

Daily and annual variations of free fatty acid, glycerol and leptin plasma concentrations in goats (Capra hircus) under different photoperiods

The aim of the study was to differentiate the impact of lighting conditions and feeding times on the regulation of lipid metabolism of goats under different photoperiods throughout the year. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60 degrees N (the longest light period 18 h, the shortest 6 h). Ambient temperature and feeding regime were kept constant. Blood samples were collected six times a year at 2-h intervals for 2 days, first in light/dark (LD) conditions and then after 3 days in constant darkness (DD). Significant daily variations were detected in the concentrations of plasma free fatty acids (FFA) and glycerol throughout the year. The nocturnal decrease and morning rise of FFA levels were related to the photoperiod, while the trough levels of glycerol were associated with the concentrate meal times. In DD conditions, FFA and glycerol rhythms were unstable. A significant seasonal variation was detected in the overall FFA and glycerol levels suggesting decreased lipogenesis in winter, increased lipolysis in spring and high lipogenesis in summer and fall. There was no significant daily rhythm in serum leptin levels, nor did the profiles in LD and DD conditions differ. The leptin level was slightly lower in early fall than in the other seasons, paralleling a small decrease of body mass in the goats after the grazing season. The daily or annual variations of FFA and glycerol levels were not clearly related to leptin concentrations. The results suggest that lipid metabolism of goats is regulated by light even in constant temperature and feeding conditions; however, no significant contribution of leptin levels could be shown.     

Sleep fragmentation in mentally retarded people decreases with increasing daylength in spring

We studied the sleep-wake behavior of mentally retarded people from late winter to early summer at 60 degrees N. During this time the daylength increased 8 h 51 min. The data were collected by observing the sleep-wake status of 293 subjects at 20-min intervals for five randomized 24h periods (= recording days). The intervals during which the individual recording days of the same order (1st, 2nd, etc.) were carried out, were called recording periods. Consequently, there were five recording periods, each containing 293 individual recording days. Even though there was overlap among the recording periods, the median daylength from one period to another increased approximately by 100 min. In the initial statistical analysis, the number of wake-sleep transitions was found to differ significantly among the five recording periods (Friedman test, p < 0.001). The mean ranks in the Friedman test suggested that the number of wake-sleep transitions was highest during the 1st and lowest during the 5th recording period. In further statistical analyses using a program for mixed effects regression analysis (MIXOR 2.0) it was found that the increase in daylength during the study period was associated with a simultaneous decrease of approximately 0.5 wake-sleep transitions in the whole study population (p < 0.001). The decrease in the number of wake-sleep transitions was significant only in the subgroups of subjects with a daylength change of more than 350 min between the 1st and 5th recording days (Wilcoxon tests, p < 0.005). This suggests that after a marked prolongation of the natural photoperiod, the reduction in sleep episodes was more probable than after smaller changes in daylength. It is concluded that the sleep of mentally retarded people living in a rehabilitation center at a northern latitude is more fragmented in winter than in early summer and that the change is related probably to the simultaneous increase in the length of the natural photoperiod. The sleep quality of persons living in institutional settings might be improved by increasing the intensity and/or duration of daily artificial light exposure during the darker seasons.     

Quaternary structure built from subunits combining NMR and small-angle x-ray scattering data

A new principle in constructing molecular complexes from the known high-resolution domain structures joining data from NMR and small-angle x-ray scattering (SAXS) measurements is described. Structure of calmodulin in complex with trifluoperazine was built from N- and C-terminal domains oriented based on residual dipolar couplings measured by NMR in a dilute liquid crystal, and the overall shape of the complex was derived from SAXS data. The residual dipolar coupling data serves to reduce angular degrees of freedom, and the small-angle scattering data serves to confine the translational degrees of freedom. The complex built by this method was found to be consistent with the known crystal structure. The study demonstrates how approximate tertiary structures of modular proteins or quaternary structures composed of subunits can be assembled from high-resolution structures of domains or subunits using mutually complementary NMR and SAXS data.     

Reaction kinetics of a two-photon excitation microparticle based immunoassay--from modelling to practice

Real time observation of reaction kinetics is one of the key features of the newly developed microparticle based two-photon excitation fluorescence immunoassay system (TPX). By observing binding reactions at the surface of individual microparticles during the incubation of an assay, the binding constants of an assay become apparent. This paper describes the use of the new system in quantifying the reaction parameters of human thyroid stimulating hormone (hTSH) assay. A mechanistic reaction model for the assay is presented. The reaction model is further shown to precisely predict the behaviour of the assay kinetics over a wide range of analyte concentrations.     

Longitudinal Genome-Wide Association of Cardiovascular Disease Risk Factors in the Bogalusa Heart Study

Cardiovascular disease (CVD) is the leading cause of death worldwide. Recent genome-wide association (GWA) studies have pinpointed many loci associated with CVD risk factors in adults. It is unclear, however, if these loci predict trait levels at all ages, if they are associated with how a trait develops over time, or if they could be used to screen individuals who are pre-symptomatic to provide the opportunity for preventive measures before disease onset. We completed a genome-wide association study on participants in the longitudinal Bogalusa Heart Study (BHS) and have characterized the association between genetic factors and the development of CVD risk factors from childhood to adulthood. We report 7 genome-wide significant associations involving CVD risk factors, two of which have been previously reported. Top regions were tested for replication in the Young Finns Study (YF) and two associations strongly replicated: rs247616 in CETP with HDL levels (combined P = 9.7×10⁻²⁴), and rs445925 at APOE with LDL levels (combined P = 8.7×10⁻¹⁹). We show that SNPs previously identified in adult cross-sectional studies tend to show age-independent effects in the BHS with effect sizes consistent with previous reports. Previously identified variants were associated with adult trait levels above and beyond those seen in childhood; however, variants with time-dependent effects were also promising predictors. This is the first GWA study to evaluate the role of common genetic variants in the development of CVD risk factors in children as they advance through adulthood and highlights the utility of using longitudinal studies to identify genetic predictors of adult traits in children.     

Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy

Measurements of early tumor responses to therapy have been shown, in some cases, to predict treatment outcome. We show in lymphoma-bearing mice injected intravenously with hyperpolarized [1-(13)C]pyruvate that the lactate dehydrogenase-catalyzed flux of (13)C label between the carboxyl groups of pyruvate and lactate in the tumor can be measured using (13)C magnetic resonance spectroscopy and spectroscopic imaging, and that this flux is inhibited within 24 h of chemotherapy. The reduction in the measured flux after drug treatment and the induction of tumor cell death can be explained by loss of the coenzyme NAD(H) and decreases in concentrations of lactate and enzyme in the tumors. The technique could provide a new way to assess tumor responses to treatment in the clinic.     

Characterization of aerobic bacterial and fungal microbiota on surfaces of historic Scottish monuments

Twenty samples were taken from the inner or outer surfaces of stone monuments of six historic Scottish buildings and ruins. Biofilms developing on mineral substrates were analysed by in situ scanning electron microscopy and cultivation. Various methods were used to characterize the isolates including automated ribotyping, RAPD and sequencing of the 16S rRNA gene for bacteria, and stereomicroscopy and sequencing of the Internal Transcribed Spacers (ITS) for fungi. Most samples contained microbes between 10(5) and 10(7)cfug(-1) substrate. Actinobacteria belonging to the genus Streptomyces (17 samples/5 monuments) or Arthrobacter (12/3) and Pseudomonas (9/3) were frequently detected. Most streptomycetes were in terms of their 16S rRNA gene sequence most closely related to S. microflavus (10/3) or to the undescribed species S. "vulgaris" (8/3). Indoor and outdoor biofilms exhibited significant differences in their microbiota, as shown by both microscopy and isolation studies. Pigmented coccoid Arthrobacter species were typical for the outdoor samples, whereas Pseudomonas species were common in the indoor samples. Based on the low phylogenetic relationship to a known species (type strain), potential novel pigmented bacterial species belonging to the genera Arthrobacter, Brevundimonas, Cryseobacterium, Deinococcus and Dyadobacter were detected from the outdoor samples and to Pseudomonas from the indoor samples. Hyaline fungal species of Acremonium (10/4) mainly occurred in indoor samples, whereas pigmented species of Cladosporium (8/3), Penicillium (6/3) and Phialophora (6/2) were found outdoors. Using in situ microscopy diatom algae were also detected.