Thermostability of a nuclear-targeted luciferase expressed in mammalian cells. Destabilizing influence of the intranuclear microenvironment

Protein denaturation and aggregation are most likely the cause for the noxious effects of heat shock. There are some indications that the nucleus is one of the most sensitive cellular compartments. To test the possibility that the intranuclear microenvironment might be detrimental to the heat stability of proteins, we compared the in situ thermal stability of a reporter protein localized in the nucleus or in the cytoplasm. A recombinant firefly (Photynus pyralis) luciferase carrying a point mutation in the C-terminal domain remains in the cytoplasm (cyt-luciferase). A nuclear localization sequence was fused to the N-terminal domain of cyt-luciferase; the resulting nuc-luciferase was efficiently targeted to the cell nucleus. In both cases, decreased luciferase activity and solubility were found in lysates from heat-shocked cells. These characteristics were taken as an indication of thermal denaturation in situ. The heat-inactivated luciferases were partially reactivated during recovery after stress, indicating the capacity of both the cytoplasmic and nuclear compartments to reassemble proteins from an aggregated state. Although both the nuc- and the cyt-luciferases were heat inactivated at similar rates in vitro, nuc-luciferase was more susceptible to thermal denaturation in situ compared to cyt-luciferase. This observation suggests that the microenvironment of an intracellular compartment may modulate the thermal stability of proteins. The local concentration might be one element of this microenvironment affecting the heat-stability of proteins. In cells made thermotolerant by a priming shock, the thermal inactivation of the recombinant luciferases occurred at a slower rate during a second challenging stress. However, this decreased thermal sensitivity was less pronounced for the nuc-luciferase (threefold) than for the cyt-luciferase (sevenfold). The nuclear luciferase might become a useful tool to investigate the action of molecular chaperones in the nucleus.     

Identification and cloning of three novel human G protein-coupled receptor genes GPR52, PsiGPR53 and GPR55: GPR55 is extensively expressed in human brain

OBJECTIVES: To examine the psychoeducational profile associated with the chromosome 22q11.2 microdeletion (DiGeorge/velocardiofacial syndrome). STUDY DESIGN: Thirty-three patients (aged 6 to 27 years) with a 22q11.2 microdeletion underwent psychoeducational testing as part of a comprehensive evaluation. Nonparametric statistics were used to compare verbal and performance IQ, academic achievement scores, and receptive versus expressive language scores. Post hoc comparisons were made of IQ subtest scores and of language versus verbal IQ. RESULTS: Full-scale IQ ranged from the normal to the moderately retarded range. Mean verbal IQ was significantly higher than mean performance IQ. In a similar manner, mean reading and spelling scores were superior to the mean mathematics score, although achievement scores typically were in the range of verbal IQ. In addition, many children showed clinically significant language impairments, with mean language scores lower than mean verbal IQ. CONCLUSIONS: The IQ and academic profiles are reminiscent of a "nonverbal learning disability," although achievement was not discrepant from IQ. The coincidence of language impairment with a relative strength in reading belies a unique neuropsychologic profile. Educational programming for these children must address both verbal and nonverbal deficits.     

Impaired pituitary hormonal response to exhaustive exercise in overtrained endurance athletes

The effects of 3,5-diiodo-L-thyronine (3,5-T2, 2.5-10 microg/100 g BW) on cold tolerance, energy expenditure and oxidative capacity of four metabolically very active tissues (brown adipose tissue, skeletal muscle, liver and heart) were determined in hypothyroid, cold-exposed rats. Hypothyroid rats survived cold for only 3-4 days. 3,5-T2 improved survival dose dependently; with 10 microg/100 g BW the rats survived 3 weeks (limit of observation). This effect was paralleled by an increased energy expenditure of the whole animal for the entire 3 weeks. Similar effects were observed in hypothyroid rats treated with 3,3',5-triiodo-L-thyronine (T3). 3,5-T2 stimulated the specific oxidative capacity (expressed as cytochrome oxidase activity per milligram protein) of all four tissues dose dependently. When the oxidative capacity was expressed as total activity (cytochrome oxidase activity times organ weight), the percentage increases were of the same order. T3 exerted similar effects, but the changes in total activity were much greater than in specific activity, indicating an effect on the tissue trophism. The effect of 3,5-T2 on cold tolerance thus mimics the effect of T3, but via different cellular mechanisms. T3 seems to act primarily on the trophism of the tissues, while 3,5-T2 may act directly on mitochondria without an effect on tissue trophism.     

VO2 kinetics in the horse during moderate and heavy exercise

The horse is a superb athlete, achieving a maximal O2 uptake (approximately 160 ml . min-1 . kg-1) approaching twice that of the fittest humans. Although equine O2 uptake (VO2) kinetics are reportedly fast, they have not been precisely characterized, nor has their exercise intensity dependence been elucidated. To address these issues, adult male horses underwent incremental treadmill testing to determine their lactate threshold (Tlac) and peak VO2 (VO2 peak), and kinetic features of their VO2 response to "square-wave" work forcings were resolved using exercise transitions from 3 m/s to a below-Tlac speed of 7 m/s or an above-Tlac speed of 12.3 +/- 0.7 m/s (i.e., between Tlac and VO2 peak) sustained for 6 min. VO2 and CO2 output were measured using an open-flow system: pulmonary artery temperature was monitored, and mixed venous blood was sampled for plasma lactate. VO2 kinetics at work levels below Tlac were well fit by a two-phase exponential model, with a phase 2 time constant (tau1 = 10.0 +/- 0.9 s) that followed a time delay (TD1 = 18.9 +/- 1.9 s). TD1 was similar to that found in humans performing leg cycling exercise, but the time constant was substantially faster. For speeds above Tlac, TD1 was unchanged (20.3 +/- 1.2 s); however, the phase 2 time constant was significantly slower (tau1 = 20.7 +/- 3.4 s, P < 0.05) than for exercise below Tlac. Furthermore, in four of five horses, a secondary, delayed increase in VO2 became evident 135.7 +/- 28.5 s after the exercise transition. This "slow component" accounted for approximately 12% (5.8 +/- 2.7 l/min) of the net increase in exercise VO2. We conclude that, at exercise intensities below and above Tlac, qualitative features of VO2 kinetics in the horse are similar to those in humans. However, at speeds below Tlac the fast component of the response is more rapid than that reported for humans, likely reflecting different energetics of O2 utilization within equine muscle fibers.     

Island colonization and evolution of the insular woody habit in Echium L. (Boraginaceae)

Numerous island-inhabiting species of predominantly herbaceous angiosperm genera are woody shrubs or trees. Such "insular woodiness" is strongly manifested in the genus Echium, in which the continental species of circummediterranean distribution are herbaceous, whereas endemic species of islands along the Atlantic coast of north Africa are woody perennial shrubs. The history of 37 Echium species was traced with 70 kb of noncoding DNA determined from both chloroplast and nuclear genomes. In all, 239 polymorphic positions with 137 informative sites, in addition to 27 informative indels, were found. Island-dwelling Echium species are shown to descend from herbaceous continental ancestors via a single island colonization event that occurred < 20 million years ago. Founding colonization appears to have taken place on the Canary Islands, from which the Madeira and Cape Verde archipelagos were invaded. Colonization of island habitats correlates with a recent origin of perennial woodiness from herbaceous habit and was furthermore accompanied by intense speciation, which brought forth remarkable diversity of forms among contemporary island endemics. We argue that the origin of insular woodiness involved response to counter-selection of inbreeding depression in founding island colonies.     

Human nasal blood vessels after permanent tracheostomy. Electron-microscopic study

The influence of Dopamine on microcirculation during hemorrhagic shock was investigated by means of the pancreas chamber technique (HEISIG, 1967). Parameter of microcirculation was the corpuscular flow velocity measured with the "flying spot" method. Simultaneously arterial and venous blood pressure, heart rate, blood gases and pH-status were registered. While a Dopamine-dosage of 5 mug/kg/min did not markedly effect perfusion of the pancreas, application of 10 as well as 20 mug/kg/min Dopamine caused a significant improvement of microcirculation. There was no difference between 10 and 20 mug/kg/min Dopamine. The increase of capillary perfusion can certainly not be explained by the small rise of blood pressure that was found under treatment with Dopamine. The results support the statement of the existence of Dopamine-specific receptors in pancreatic vessels. Compared to a group of animals treated with 5 mug/kg/min Dopamine and an untreated control group, survival rate of animals treated with 10 as well as 20 mug/kg/min Dopamine was significantly increased.