Isolation of synthetic lethal mutants of ras1 in Schizosaccharomyces pombe

Ras proteins are membrane-associated guanine nucleotide-binding proteins that serve as molecular switches for signal transduction pathways in a diverse array of organisms. Various cellular factors are known to interact with Ras proteins. In order to find the novel cellular factors that are associated with Ras function, we have constructed synthetic lethal mutants of the ras1+ gene in Schizosaccharomyces pombe and used them to identify the genes that are functionally dependent on the Ras1. We first constructed S. pombe strains in which chromosomal ras1+ gene is placed under the nmt1 promoter that is regulated by thiamine. This strain shows ras1+ phenotype in the absence of thiamine, whereas it shows ras1- phenotype in the presence of thiamine. Second, we mutated the constructed strains with ultraviolet light (UV) and selected two synthetic lethal mutants that could not grow when Ras1 function was repressed (ras1-). One of the mutants, KSC3, showed a swollen cell shape, aberrant deposition of septum materials, and aberrant nuclei. The other mutant, KSC4, showed sensitivity to hyper-osmolarity when Ras1 function is absent. These mutants, however, grow normally when Ras1 is expressed (ras1+). These two novel synthetic lethal mutants of ras1 provide the means to isolate the corresponding genes that function in association with Ras1 in S. pombe. Screening of a genomic library of S. pombe complementing the mutant phenotype allowed us to identify several novel genes associated with Ras1 of S. pombe.     

Exoglycosidase matrix-mediated sequencing of a complex glycan pool by capillary electrophoresis

This paper discusses oligosaccharide sequencing by consecutive enzymatic digestion of carbohydrates using an exoglycosidase array, followed by capillary electrophoresis separation of the digests. Because of the high resolving power and good reproducibility of capillary electrophoresis, multistructure sequencing of a complex glycan pool can be performed in most instances requiring no prior isolation of the individual oligosaccharides. High sensitivity laser-induced fluorescence detection enables acquisition of complete sequence information from several picomoles of glycoproteins. Comparison of the migration times of the exoglycosidase digest fragments to the maltooligosaccharide ladder, enables calculation of migration shifts, due to cleavage based on the actual exoglycosidases used. The particular sequence of each oligosaccharide in a glycan pool can be proposed with high confidence based on the migration time shifts of the various oligosaccharide structures. However, possible combinations of various sequence fragments may have very similar charge to hydrodynamic volume ratios, resulting in electrophoretic co-migration when a mixture of different oligosaccharides is sequenced together. Then, capillary electrophoresis separations of the resulting fragments should be evaluated after each digestion step. In the instances of complex separation profiles when multiple peaks are present, the evaluation of peak shifts can get very complicated and solved only with the aid of a software program. Data about the monosaccharide composition of the glycan pool provides useful information in designing the digestion enzyme matrix.     

The physical and physiological workload of refuse collectors

In order to secure a safer and healthy work situation, the heavy physical loads imposed on 23 refuse collectors (aged 26-54) working in the city of Haarlem, in The Netherlands, were studied in a series of three experiments between 1984 and 1987. The aims were respectively (1) to study the load for workers collecting dustbins or polythene bags; (2) to introduce changes to reduce the load to avoid exceeding the overload criteria by individual refuse collectors; and (3) to investigate the effects of interventions to improve the efficiency of refuse collecting. The maximal isometric lifting force (Fmax) and the maximal aerobic power (VO2max) of 23 refuse collectors were measured in the laboratory. Fmax was measured with an isometric dynamometer pulling with one arm from the floor; the mean value was 912 (+/- 127)N. VO2max was measured running on a treadmill; the mean value was 43.3 (+/- 0.8)ml O2 per kg body mass per min. The physical load on the oxygen transport system was measured through work analysis and by a continuous registration of the heart rate over three working days. Criteria for overload were set at a mean external load of 20% Fmax and a mean energy expenditure of 30% VO2max and an energy expenditure of 50% VO2max or more for a maximum of 60 min per day. Replacement of dustbins by polythene bags resulted in a 70% increase in the total amount of refuse collected, an increase in throwing frequency, but a lower mean load per throw, and no significant differences in the mean heart rate over the working day. When polythene bags were used the mean values did not exceed the overload criteria, but 39% of the individual collectors did have a workload that was too high with respect to one of the criteria. In the last experiment the collectors were advised to reduce their work load by (a) lifting no more than two bags at a time; (b) reducing their walking pace; and (c) taking more breaks. Although compliance with the recommendations was good, and the weight lifted and the walking speed decreased, the physiological load remained the same. This may have been caused by a 15% increase in the total amount of refuse that had to be collected at that time.     

Effects of myosin light chain kinase inhibitors on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes

The effects of myosin light chain kinase inhibitors on muscarinic stimulation-activated nonselective cationic current (ICCh) in guinea-pig gastric antral myocytes were studied using the whole-cell patch-clamp technique. ICCh was induced by carbachol (CCh, 50 microM) at a holding potential of -30 mV or -60 mV. ML-7, a chemical inhibitor of myosin light chain kinase (MLCK), inhibited ICCh concentration dependently in a reversible manner (53 +/- 8.6% at 1 microM, mean +/- SE, n = 11). In addition, amplitudes of ICCh were only 37 +/- 2.7% of the daily control values following the addition of a peptide inhibitor of MLCK to the pipette solution. On the other hand, ML-7 had an inhibitory effect on voltage-operated Ca2+ channel current. The peak value of Ba2+ current at 0 mV was reduced to 35 +/- 7.4% (n = 9) by 3 microM of ML-7. As ICCh is known to have an intracellular Ca2+ dependence, we tried to exclude the possibility that ML-7 inhibited ICCh indirectly via suppression of Ca2+ current and the similar inhibitory effects of ML-7 on ICCh were confirmed under the following conditions: (1) clamp of membrane potential at -60 mV; (2) clamp of intracellular [Ca2+] to 1 microM by 10 mM BAPTA; (3) pre-inhibition of Ca2+ channel by verapamil. Different from the effects on ICCh, ML-7 barely inhibited the same cationic current induced by guanosine 5'-O-(3-thiotriphosphate) (GTP[gammaS], 0.2 mM) in the pipette solution. These results suggest that a Ca2+/calmodulin-MLCK-dependent pathway can modulate the activation of ICCh in guinea-pig gastric antral myocytes.     

Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model

Expression of parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) and protein was investigated throughout the developmental progression of endochondral bone formation in mouse and intramembranous bone formation in an in vivo model in rabbit, using in situ hybridization and immunohistochemistry. Endochondral bone formation was investigated in a developing embryo, newborn, and adult mouse. In fetal long bones through to newborn (day 7), PTHrP mRNA and protein were consistently expressed in chondrocytes within the proliferative, transitional, and hypertrophic zones. In addition, high levels of PTHrP were also detected in osteoblasts on the surface of trabecular bone surfaces. Similarly, at the adult stage (week 7), PTHrP mRNA and protein were consistently expressed in chondrocytes at epiphyseal ends of the subarticular cartilage, within cortical periosteum, as well as in osteoblasts located at the metaphyseal trabecular bone surfaces. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in preosteoblasts prior to trabecular bone formation (1-week bone harvest). As bone formed (2-, 3-, and 4-week bone tissue harvests), PTHrP mRNA and protein were highly expressed in actively synthesizing osteoblasts and in those osteocytes embedded within the superficial layers of the bone matrix. Lining osteoblasts and osteocytes buried deeply in the bone matrix displayed weak or no signal for PTHrP. The pattern of spatial and temporal expression of PTHrP demonstrated in cartilage cells and osteoblasts in the two systems suggests an important role of PTHrP in both endochondral and intramembranous bone formation.     

Resource pool management: Reactive versus proactive or let’s be friends

The consolidation of multiple workloads and servers enables the efficient use of server and power resources in shared resource pools. We employ a trace-based workload placement controller that uses historical information to periodically and proactively reassign workloads to servers subject to their quality of service objectives. A reactive migration controller is introduced that detects server overload and underload conditions. It initiates the migration of workloads when the demand for resources exceeds supply. Furthermore, it dynamically adds and removes servers to maintain a balance of supply and demand for capacity while minimizing power usage. A host load simulation environment is used to evaluate several different management policies for the controllers in a time effective manner. A case study involving three months of data for 138 SAP applications compares three integrated controller approaches with the use of each controller separately. The study considers trade-offs between: (i) required capacity and power usage, (ii) resource access quality of service for CPU and memory resources, and (iii) the number of migrations. Our study sheds light on the question of whether a reactive controller or proactive workload placement controller alone is adequate for resource pool management. The results show that the most tightly integrated controller approach offers the best results in terms of capacity and quality but requires more migrations per hour than the other strategies.     

Observed and perceived parental overprotection in relation to psychosocial adjustment in preadolescents with a physical disability: The mediational role of behavioral autonomy.

The purpose of this study was to test a mediational model of associations between parental overprotectiveness (OP), behavioral autonomy, and psychosocial adjustment in 68 families with 8- and 9-year-old preadolescents with spina bifida and a demographically matched sample of 68 families with able-bodied children. Measures included questionnaire and observational assessments of parental OP; parent and child reports of behavioral autonomy; and parent, child, and teacher reports of preadolescent adjustment. On the basis of both questionnaire and observational measures of OP, mothers and fathers of children with spina bifida were significantly more overprotective than their counterparts in the able-bodied sample, although this group difference was partially mediated by children's cognitive ability. Both questionnaire and observational measures of parental OP were associated with lower levels of preadolescent decision-making autonomy as well as with parents being less willing to grant autonomy to their offspring in the future. For the questionnaire measure of OP, and only for the spina bifida sample, the mediational model was supported such that parental OP was associated with less behavioral autonomy, which was, in turn, associated with more externalizing problems. (PsycINFO Database Record (c) 2010 APA, all rights reserved)