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.     

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.     

The daily rhythms of melatonin and free fatty acids in goats under varying photoperiods and constant darkness

The purpose of the study was to explore parallel and divergent features of the daily rhythms of melatonin and plasma free fatty acids (FFA) in goats exposed to different lighting conditions. From these features, we attempted to analyze whether the endogenous melatonin rhythm plays any role in the maintenance of the FFA rhythm. Seven Finnish landrace goats were kept under artificial lighting that simulated the annual changes of photoperiod at 60°N (longest photoperiod, 18 h; shortest, 6 h). The ambient temperature and feeding regimen were kept constant. Blood samples were collected 6 times a year at 2 h intervals for 2 d, first in the prevailing light-dark (LD) conditions and then after 3 d in constant darkness (DD). In LD conditions, the melatonin levels always increased immediately after lights-off and declined around lights-on, except in winter (18 h darkness), when the low daytime levels were restored clearly before lights-on. The FFA levels also displayed a consistent rhythmicity, with low levels at night and a transient peak around lights-on. In DD conditions, the melatonin profiles were very similar to those found in the habitual LD conditions, but the rhythm tended to advance. The FFA rhythm persisted also in DD, and the morning peak tended to advance. There was an overall parallelism between the two rhythms, with one significant exception. In winter in LD conditions, the morning rise in FFA levels coincided with lights-on and not with the declining phase of melatonin, whereas in DD conditions, the FFA peak advanced several hours and coincided with the declining phase of melatonin. From this finding and comparisons of the calculated rhythm characteristics, i.e., phase-shifts, phase differences, and correlations, we conclude that the daily rhythm of FFA levels is most probably generated by an endogenous oscillator, primarily adjusted by dawn, whereas the melatonin rhythm in this species is regulated by an oscillator primarily adjusted by dusk. The results did not exclude a modulatory effect of melatonin on the daily FFA profiles, but melatonin secretion, alone, does not explain the patterns sufficiently.     

Fatty Chains of Different Lipid Classes of Semliki Forest Virus and Host Cell Membranes

Semliki Forest virus was grown in BHK-21 cells. The major classes of phospho-and glycolipids of the virus were analyzed for the compositions of fatty acids, aldehydes, and sphingosine bases, and the major glycerophospholipids were analyzed for the relative proportions of alkenyl-acyl, alkyl-acyl, and diacyl forms. All viral lipid classes proved to be mixtures of several molecular species. Each class contained a characteristic mixture of fatty chains, which was different in all other classes. All viral lipid classes resembled their counterparts of the host plasma membrane and also those of the endoplasmic reticulum. The gangliosides of the virus and the plasma membrane proved to be similar even at the level of individual molecular species. The number of certain lipid molecules in an average virion was less than the number of the protein molecules.     

Characterisation of aerobically grown non-spore-forming bacteria from paper mill pulps containing recycled fibres

A total of 179 non-spore-forming bacteria aerobically growing on Nutrient Agar, Plate Count Agar or in specific enrichment conditions for salmonella, campylobacteria, listeria, yersinia or staphylococci, were isolated from 16 untreated paper mill pulps. After phenotypical screening the isolates were characterised by automated ribotyping and partial sequencing of the 16S rRNA gene. They could be divided into seven taxonomical classes representing 63 taxa (species): actinobacteria (11 species), bacilli (7), flavobacteria (3) alphaproteobacteria (10), betaproteobacteria (5), gammaproteobacteria (25) and sphingobacteria (2). Most of the gammaproteobacteria were enterobacteria, mainly species of the genera Enterobacter (7 species, 7 samples/3 mills) and Klebsiella (5 species, 6 samples/3 mills). Other commonly occurring bacteria were most closely related to Microbacterium barkeri (7 samples/3 mills), Cloacibacterium normanense (6 samples/2 mills), Pseudoxanthomonas taiwanensis (5 samples/2 mills) and Sphingobacterium composti (5 samples/1 mill). Sporadic isolates of Listeria innocua, L. monocytogenes, Enterococcus casseliflavus and Staphylococcus warneri were detected, from which only L. monocytogenes is considered to be a food pathogen. No isolates of the genera Campylobacter, Salmonella or Yersinia were detected. The detected bacteria may be harmful in process control, but the load of food pathogens with recycled fibres to paper machines is insignificant. Faecal contamination of the pulp samples was not indicated.     

Sterol-induced upregulation of phosphatidylcholine synthesis in cultured fibroblasts is affected by the double-bond position in the sterol tetracyclic ring structure.

We have examined how a specific enrichment of cultured fibroblasts with various sterols (cholesterol, lathosterol, 7-dehydrocholesterol, allocholesterol and dihydrocholesterol) regulate synthesis de novo of phosphatidylcholine, cholesterol and cholesteryl (or steryl) esters in human skin fibroblasts. When human skin fibroblasts were incubated for 1 h with 130 microM cholesterol/CyD complexes, the mass of cellular free cholesterol increased by 100 nmol.mg-1 protein (from 90 nmol.mg-1 to 190 nmol.mg-1 protein). A similar exposure of cells to different sterol/CyD complexes increased the cell sterol content between 38 and 181 nmol sterol per mg cell protein. In cholesterol-enriched cells, the rate of phosphatidylcholine synthesis was doubled compared to control cells, irrespective of the type of precursor used ([3H]choline, [3H]palmitic acid, or [14C]glycerol). Enrichment of fibroblasts with 7-dehydrocholesterol, allocholesterol, or dihydrocholesterol also upregulated phosphatidylcholine synthesis, whereas cells enriched with lathosterol failed to upregulate their phosphatidylcholine synthesis. The activity of membrane-bound CTP:phosphocholine cytidylyltransferase, the rate-limiting enzyme, was increased by 47 +/- 4% in cholesterol-enriched cells whereas its activity was unchanged in lathosterol-enriched cells. Sterol enrichment with all tested sterols (including lathosterol) down-regulated acetate-incorporation into cholesterol, and upregulated sterol esterification in the sterol-enriched fibroblasts. Using 31P-NMR to measure the lamellar-to-hexagonal (Lalpha-HII) phase transition in multilamellar lipid dispersions, lathosterol-containing membranes underwent their transition at significantly higher temperatures compared to membranes containing any of the other sterols. In a system with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and either cholesterol or lathosterol (70:30 mol/mol), differential scanning calorimetry also revealed that the Lalpha-HII-transition occurred at a higher temperature with lathosterol compared to either cholesterol, allocholesterol, or dihydrocholesterol. These findings together suggest that there may exist a correlation between the propensity of a sterol to stabilize the Lalpha-HII-transition and its capacity to upregulate the activity of CTP:phosphocholine cytidylyltransferase in cells.