Structural analysis of UBL5, a novel ubiquitin-like modifier

UBL5 is a widely expressed human protein that is strongly conserved across phylogeny. Orthologs of UBL5 occur in every eukaryotic genome characterized to date. The yeast ortholog of UBL5, HUB1, was reported to be a ubiquitin-like protein modifier important for modulation of protein function. However, unlike ubiquitin and all other ubiquitin-like modifiers, UBL5 and its yeast ortholog HUB1 both contain a C-terminal di-tyrosine motif followed by a single variable residue instead of the characteristic di-glycine found in all other ubiquitin-like modifiers. Here we describe the three-dimensional structure of UBL5 determined by NMR. The overall structure of the protein was found to be very similar to ubiquitin despite the low approximately 25% residue similarity. The signature C-terminal di-tyrosine residues in UBL5 are involved in the final beta sheet of the protein. This is very different to the di-glycine motif found in ubiquitin, which extends beyond the final beta sheet. In addition, we have confirmed an earlier report of an interaction between UBL5 and the cyclin-like kinase, CLK4, which we have determined is specific and does not extend to other cyclin-like kinase family members.     

Lesions in phycoerythrin chromophore biosynthesis in Fremyella diplosiphon reveal coordinated light regulation of apoprotein and pigment biosynthetic enzyme gene expression

We have characterized the regulation of the expression of the pebAB operon, which encodes the enzymes required for phycoerythrobilin synthesis in the filamentous cyanobacterium Fremyella diplosiphon. The expression of the pebAB operon was found to be regulated during complementary chromatic adaptation, the system that controls the light responsiveness of genes that encode several light-harvesting proteins in F. diplosiphon. Our analyses of pebA mutants demonstrated that although the levels of phycoerythrin and its associated linker proteins decreased in the absence of phycoerythrobilin, there was no significant modulation of the expression of pebAB and the genes that encode phycoerythrin. Instead, regulation of the expression of these genes is coordinated at the level of RNA accumulation by the recently discovered activator CpeR.     

Oxidative stress responses during cassava post-harvest physiological deterioration

A major constraint to the development of cassava (Manihot esculenta Crantz) as a crop to both farmers and processors is its starchy storage roots' rapid post-harvest deterioration, which can render it unpalatable and unmarketable within 24–72 h. An oxidative burst occurs within 15 min of the root being injured, that is followed by the altered regulation of genes, notably for catalase and peroxidase, related to the modulation of reactive oxygen species, and the accumulation of secondary metabolites, some of which show antioxidant properties. The interactions between these enzymes and compounds, in particular peroxidase and the coumarin, scopoletin, are largely confined to the vascular tissues where the visible symptoms of deterioration are observed. These, together with other data, are used to develop a tentative model of some of the principal events involved in the deterioration process. Abbreviations: ACMV, African cassava mosaic virus; AFLP, amplified fragment length polymorphism; CAT, catalase; cDNA, complementary deoxyribonucleic acid; CIAT, International Centre for Tropical Agriculture; Cu/ZnSOD, copper/zinc superoxide dismutase; DAB, 3,3-diaminobenzidine tetrahydrochloride; DPPH, 1,1-diphenyl-2-picrylhydrazyl; FeSOD, iron superoxide dismutase; FW, fresh weight; GUS, -glucuronidase; HPTLC, high-performance thin-layer chromatography; HR, hypersensitive response; IEF-PAGE, isoelectric focusing polyacrylamide gel electrophoresis; MAS, marker-assisted selection; MeJa, methyl jasmonate; MnSOD, manganese superoxide dismutase; NADPH, nicotinamide adenine dinucleotide phosphate (reduced form); NBT, nitroblue tetrazolium; PAL, phenylalanine ammonia-lyase; PCD, programmed cell death; PCR, polymerase chain reaction; POX, peroxidase; PPD, post-harvest physiological deterioration; QTL, quantitative trait loci; ROS, reactive oxygen species; RT, room temperature; SAR, systemic acquired resistance; SDS, sodium dodecyl sulfate; SOD, superoxide dismutase     

The relationship between assessments of jet lag and some of its symptoms

The power of the symptoms of jet lag in predicting the amount of jet lag measured at the same and different times of the day has been investigated. A total of 85 subjects was studied for 6 days after a flight from the UK to Australia (10 time zones to the east). At 08:00, 12:00, 16:00, 20:00, and 24:00h, the subjects recorded their jet lag and fatigue. At 08:00h, they also assessed their sleep. At 12:00 and 16:00h, they assessed their attitude to a meal, as well as their motivation, commitment, and irritability. On retiring, they recorded bowel activity. Assessments were by visual analog scales. Jet lag was treated as the dependent variable and the symptoms as covariates in ANCOVAs. Fatigue was a powerful predictor of jet lag, provided it was measured at the same time, and some aspects of sleep predicted jet lag measured on retiring or rising. The other symptoms predicted jet lag less powerfully and/or at a wider range of times. It is concluded that, even though jet lag at any time of the day can be predicted from contemporaneous assessments of fatigue and that it can be predicted on retiring or rising from some aspects of changed sleep, jet lag is predicted less reliably from other symptoms, including aspects of mental performance. These findings question exactly what causes jet lag at a particular time of day, and so are relevant to studies which use this measurement to investigate the problems associated with time-zone transitions, and ways to ameliorate them.     

The use of actimetry to assess changes to the rest-activity cycle

The endogenous circadian oscillator (the body clock) is slow to adjust to altered rest-activity patterns. As a result, several negative consequences arise during night work and after time-zone transitions. The process of adjustment can be assessed by measurements of the sleep electroencephalogram (EEG), core temperature or melatonin secretion, for example, but these techniques are very difficult to apply in field studies, and make very great demands upon both experimenters and subjects. We have sought to establish if the activity record, measured conveniently and unobtrusively by a monitor attached to the wrist, can be treated in ways that enable estimates to be made of the disruption caused by changes to the rest-activity cycle, and the process of adjustment to them. In Part A, we describe the calculation and assessment of a series of “activity indices” that measure the overall activity pattern, activity when out of bed or in bed, or the activity in the hours adjacent to going to bed or getting up. The value of the indices was assessed by measuring changes to them in subjects undergoing night work or undergoing time-zone transitions. In both cases, there is a large body of literature describing the changes that would be expected. First, night workers (working 2 to 4 successive night shifts) were investigated during rest days and night shifts. The indices indicated that night work was associated with lower activity when the subjects were out of bed and higher activity when in bed. Some indices also measured when subjects took an afternoon nap before starting a series of night shifts and gave information about the process of adjustment to night work and recovery from it. Second, in studies from travelers crossing six or more time zones to the east or west, the indices indicated that there were changes to the rest-activity cycle immediately after the flights, both in its overall profile and when activity of the subjects in bed or out of bed was considered, and that adjustment took place on subsequent days. By focusing on those indices describing the activity records during the last hour in bed (LHIB) and the first hour out of bed (FHOB), some evidence was found for incomplete adjustment of the body clock, and for differences between westward and eastward flights. In Part B, the battery of indices are applied to the activity records of long-haul pilots, whose activity patterns showed a mixture of effects due to night work and time-zone transitions. Actimetry was performed during the flights themselves and during the layover days (which were either rest or work days). The indices indicated that all pilots had disrupted rest-activity cycles caused by night flights, and that there were added problems for those who had also undergone time-zone transitions. Rest days were valuable for normalizing the activity profile. For those pilots who flew to the west, adjustment was by delay, though not all aspects of the rest-activity cycle adjusted immediately; for those who flew to the east, some attempted to advance their rest-activity cycle while others maintained home-based activity profiles. The indices indicated that the activity profile was disrupted more in those pilots who attempted to advance their rest-activity cycle. We conclude that objective estimates of the disruption caused to the rest-activity cycle and the circadian system can be obtained by suitable analysis of the activity record.     

Measurement of, and some reasons for, differences in eating habits between night and day workers

A questionnaire was designed to assess the following: why working people chose to eat or not to eat at a particular time of day; the factors that influenced the type of food eaten; and subjective responses to the meal (hunger before, enjoyment during, satiety afterward). Self-assessments were done every 3 h during a typical week containing work and rest days, by one group of 50 day workers and another group of 43 night workers. During the night work hours compared to rest days, night workers evidenced a significantly altered food intake, with a greater frequency of cold rather than hot food (p < 0.001). The type and frequency of meals were influenced significantly more (p < 0.05) by habit and time availability and less by appetite. This pattern continued into the hours immediately after the night shift had ended. In day workers food intake during work hours, compared to rest days, was also influenced significantly more often (p < 0.05) by time availability than hunger, but less so than with night workers. Moreover, day workers were less dependent than night workers upon snacks (p = 0.01), and any significant differences from rest days did not continue beyond work hours. Not only did night workers change their eating habits during work days more than did day workers but also they looked forward to their meals significantly less (p < 0.001) and felt more bloated after consuming them (p < 0.05), such effects being present to some extent during their rest days also. These findings have clear implications for measures designed to ease eating problems that are commonly problematic in night workers.     

Further evidence for the reliance of catalysis by rabbit muscle pyruvate kinase upon isomerization of the ternary complex between enzyme and products

Isothermal calorimetry has been used to examine the effect of thermodynamic non-ideality on the kinetics of catalysis by rabbit muscle pyruvate kinase as the result of molecular crowding by inert cosolutes. The investigation, designed to detect substrate-mediated isomerization of pyruvate kinase, has revealed a 15% enhancement of maximal velocity by supplementation of reaction mixtures with 0.1 M proline, glycine or sorbitol. This effect of thermodynamic non-ideality implicates the existence of a substrate-induced conformational change that is governed by a minor volume decrease and a very small isomerization constant; and hence, substantiates earlier inferences that the rate-determining step in pyruvate kinase kinetics is isomerization of the ternary enzyme product complex rather than the release of products.     

Crystallization of AcpA, a respiratory burst-inhibiting acid phosphatase from Francisella tularensis

Francisella tularensis is a highly infectious bacterial pathogen that is classified as a Category A Pathogen by the Centers for Disease Control and Prevention. Here, we report crystallization of a recombinant form of F. tularensis AcpA, a unique and highly expressed acid phosphatase that is thought to play a role in intracellular survival by inhibiting the host respiratory burst. Three crystal forms have been obtained, with form III being the most suitable for high-resolution structure determination. Form III crystals were grown in the presence of PEG 1500 and the competitive inhibitor sodium orthovanadate (5 mM). The space group is C222(1) with unit cell parameters a=112.1 A, b=144.4 A, c=123.9 A. The asymmetric unit is predicted to contain two protein molecules and 43% solvent. A 1.75-A native data set was recorded at beamline 8.3.1 of the Advanced Light Source. To our knowledge, this is the first report of high-resolution crystals of any F. tularensis protein.     

ICln, a novel integrin alphaIIbbeta3-associated protein, functionally regulates platelet activation

A critical role for the conserved alpha-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin alpha(IIb)beta(3). To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with alpha(IIb)beta(3) by surface plasmon resonance. The affinity of this interaction was 82.2 +/- 24.4 nm in a cell free assay. ICln co-immunoprecipitates with alpha(IIb)beta(3) in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 microm to 5 mm), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10-100 microm) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.     

Lack of evidence for a marked endogenous component determining food intake in humans during forced desynchrony

In an attempt to investigate the relative importance of endogenous and exogenous factors in determining food intake, 14 healthy subjects were studied while living in an Isolation Unit (where external time cues were absent) for eighteen 28 h "days" (equal to 21 solar days). The subjects were free to spend their waking time as they chose, and they had a free choice of what they ate and when they ate it. The only restrictions were that no naps were allowed in the "daytime," that some time was required to perform a variety of tests at regular intervals throughout the 18.67 h waking periods, and that any food preparation had to be performed by the subjects themselves. Core (rectal) temperature and activity were monitored throughout, and the subjects answered a questionnaire on their eating habits at 3 h intervals during the waking periods. The questionnaire investigated reasons for eating or not eating a meal during the previous 3 h and, if a meal had been eaten, its type, the factors influencing that choice, and the subjects' subjective responses (hunger before, enjoyment during, and satiety after) to it. The results were analyzed (two-way ANOVA) in terms of both the imposed day length (the exogenous component) and the free-running period of the temperature rhythm (the endogenous component). Results indicated that by far the main reason for eating/not eating was hunger/lack of hunger rather than factors such as food availability and time-pressure. There were statistically significant effects of time within the imposed waking periods upon the type of meal eaten--"breakfast" tending to be a snack, "lunch" a small hot meal, and the "evening meal" a large hot meal. Hot meals (whether small or large) were associated with more hunger before the meal, more enjoyment of the meal, and a greater degree of satiety afterward than were cold meals. These effects suggest that the individuals adjusted their eating habits to fit in with the imposed wake times. By contrast, the effect of circadian phase upon food intake, the type of meal eaten, and subjective responses to the meal was much weaker, and either statistically nonsignificant (P > 0.10) or only marginally so (0.10 > P > 0.05). For example, a large hot meal was chosen as readily for an "evening meal," and subjective responses to it were the same, at whatever circadian phase it was eaten. We conclude that food intake during forced desynchronization is dominated by the waking schedule rather than by circadian influences; some of the implications of these findings when eating habits and the metabolism of food are concerned, particularly in night workers, are considered briefly.