Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of β-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities. Received: 9 January / 5 March 1998     

Respiratory response to CO2 by controlled alveolar hypercapnia

A method of assessing the respiratory response to a hypercapnic stimulus after an increase in alveolar pCO₂ in accordance with an assigned program is suggested. The results are independent of the metabolic level, resistance to respiration, and other factors. Unlike the widely used rebreathing method, this new method enables the ventilatory sensitivity to CO₂ to be compared at rest, during muscular work, when the resistance to respiration is changed, and so on. It can also be used for both clinical and experimental investigations.Respiratory Physiology Group, I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Chernigovskii.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 1, pp. 6–7, January, 1980.     

Hydration during exercise

Summary Five young unacclimatised subjects were exposed for 4 h at 34 C (10 C dew-point temperature and 0.6 m · s^–1 air velocity), while exercising on a bicycle ergometer: 25 min work — 5 min rest cycles for 2 hours followed by 20 min work — 10 min rest cycles for two further hours. 5 experimental sessions were carried out: one without rehydration (NO FLUID) resulting in 3.1% mean loss of body weight ( M_b), and four sessions with 20 C fluid ingestion of spring water (WATER), hypotonic (HYPO), isotonic (ISO) and hypertonic (HYPER) solutions to study the effects of fluid osmolarity on rehydration. Mean final rehydration (±SE) after fluid intake was 82.2% (±1.2). Heart rate was higher in NO FLUID while no difference among conditions was found in either M_b or hourly sweat rates. Sweating sensitivity was lowest in the dehydration condition, and highest in the WATER one. Modifications in plasma volume and osmolarity demonstrated that NO FLUID induced hyperosmotic hypovolemia, ISO rehydration rapidly led to plasma isoosmotic hypervolemia, while WATER led to slightly hypoosmotic normovolemia.It is concluded that adequate rehydration through ingestion of isotonic electrolyte-sucrose solution, although in quantities much smaller than evaporative heat loss, rapidly restored and expanded plasma volume. While osmolarity influenced sweating sensitivity, the plasma volume changes ( PV) within the range –6% PV+4% had little effect on temperature adjustments in our conditions.     

The effects of subdiaphragmatic vagotomy on circadian corticosterone rhythmicity in rats with continuous or restricted food access

Plasma corticosteroid circadian periodicity was determined in three groups of individual adult male rats. Categories were: postsubdiaphragmatic vagotomy, sham-operated, and controls. Blood was sampled every 4 hours over a 48-hour period with ad lib feeding and after a 13-day period on daytime (0930–1330) restriction of food and water availability. Plasma corticosterone circadian periodicity was normal under ad lib conditions in control, sham-operated, and vagotomized animals. Under food-restricted conditions, both the sham-operated and vagotomized animals exhibited the 12-hour shift in the circadian peak of plasma corticosterone levels that we have previously described in normal animals under such conditions. There were no differences between groups in the total amount of food and water consumed or the percentage of nocturnal food intake on an ad lib feeding schedule. Both vagotomized and sham-operated animals manifested reductions in food intake under conditions of food restriction, although vagotomized consumed less than sham-operated animals. The present data indicate that an intact vagus nerve is not necessary for the establishment of circadian periodicity of plasma corticosterone levels or the shift in the periodicity of corticosteroid secretion produced by a restricted feeding regimen.     

Electrical self-stimulation of single and multiple loci: long term observations

Rats with chronic hypothalamic electrodes were allowed continuous access to self-stimulation, food and water in a 3 lever chamber. A prolonged burst of self-stimulation, with little food and water intake, was followed by bursts of activity on all 3 levers. A 12 hr light-dark cycle imposed a diurnal periodicity on all behaviors except in animals self-stimulating at the highest daily rates where self-stimulation was equal in dark and light. Under constant light conditions there was a 30 min daily shift in the peak periodicity of all behaviors. Increasing the current by 10 μA led to another continuous self-stimulation session for one day, with a subsequent decline and stabilization after three days. Reduction of the current to its original setting abolished self-stimulation for one day, but within 5 days rates returned to control values. Animals with electrodes in the septal, anterior and posterior hypothalamic areas allowed continuous access to self-stimulation on all 3 electrodes displayed similar behavior in that after a long initial self-stimulation session alternating on all 3 electrodes, periodic bursts of activity occurred on all three electrodes. The diurnal periodicity of self-stimulation seemed to be determined by the current intensity. The similarity of self-stimulation to normal drive mechanisms is discussed.     

The role of testosterone in the feedback control of male FSH and LH secretion

Summary Intramuscular administration of 250 mg testosterone oenanthate per week over a period of 21 weeks treatment rapidly and sustainedly suppressed serum LH as well as FSH levels in seven normal males, while serum testosterone rose by a factor of approximately two. These together with other data provide increasing evidence for a feedback control of FSH secretion by gonadal steroids in the male in addition to the already described but as yet undefined tubular testicular factor.     

The structuring of an allergy index based on IgE-mediated skin sensitivity to common environmental allergens

We computed skin-test sensitivity levels in 485 adults puncture-tested with eight standardized, high-quality inhalant allergens tested at single concentrations. In order to quantitate the "average" IgE-mediated skin sensitivity of each subject, we used both nonparametric and parametric statistical methods to generate two "allergy indices" (Allergy Index I and Allergy Index II) based on sensitivity end-point data from the subpopulations of individuals positive to six of the eight allergens. For the 192 skin test-positive subjects, Allergy Index I and Allergy Index II were significantly correlated with each other (rs = 0.98, p less than 0.001) and with the number of positive skin-test reactions (rs congruent to 0.9, p less than 0.001) as well as with log[total serum IgE] (r congruent to 0.4, p less than 0.01). In 102 ragweed-positive subjects, log[specific IgE to ragweed] was significantly correlated with ragweed-specific "ragweed indices I and II" (r congruent to 0.6, p less than 0.01). Furthermore, the average daily symptom scores reported by 14 ragweed-positive subjects during the ragweed pollination season were significantly correlated with ragweed indices I and II (p less than 0.05). We propose the use of Allergy Index II in epidemiologic and genetic studies of allergic phenotypes as well as in clinical decisions for diagnosis and immunotherapeutic intervention.     

Hypothalamic self-stimulation in the female rat: effects of oestrus and food deprivation.

Natural oestrus (147 cycles) had no overall effect in a longitudinal study of self-stimulation in a group of rats with hypothalamic electrodes (n = 20), though some individual animals showed significant increases in response rate at oestrus or at dioestrus. Animals were significantly more likely to show peak rates at prooestrus or oestrus if their electrodes were located in hypothalamic feeding areas; the effects in these rats were therefore probably indirect and non-sexual, caused by the decrease in food intake that accompanies oestrus. In ovariectomized rats, however, self-stimulation of feeding areas was usually unaffected by oestradiol injections, and increases in response rate reported after oestradiol injections in ovariectomized animals may therefore be a direct effect of the hormone; replicated study of the only animal showing unequivocal oestrogen-sensitive enhancement of self-stimulation showed that the effect was specific to oestrogenic (and not androgenic) steroids, and that the minimum effective dose was within the physiological range. These findings indicate both direct and indirect effects of oestrus on hypothalamic self-stimulation.     

Hyposmolarity-activated fluxes of taurine in astrocytes are mediated by diffusion

In order to obtain information about the mechanism responsible for swelling associated taurine release in astrocytes, the kinetics of taurine uptake in cultured astrocytes from mouse cerebral cortex was studied under isosmotic and hyposmotic (50% osmolarity) conditions. It was found that the V_max for the high affinity component of taurine uptake was unaffected by exposure of the astrocytes to hyposmotic conditions and that the K_m value was somewhat increased. Contrary to V_max, the non-saturable component of the uptake was greatly increased (2.5-fold) after exposure of the cells to hyposmotic media leading to cell swelling. In addition to the kinetic characterization of taurine uptake the actual intracellular taurine content after incubation (15 min) in isosmotic or hyposmotic media with different taurine concentrations (0–100 mM) under Na⁺-free conditions was determined. At taurine concentrations < 30 mM corresponding to the intracellular content in cells not exposed to taurine, exposure to hyposmotic media led to a decrease in the intracellular taurine content. At higher external taurine concentrations (> 30 mM) the intracellular taurine contents were dramatically increased after exposure to hyposmotic conditions. The increase in intracellular taurine seen under hyposmotic conditions at 100 mM external taurine could be significantly reduced by 100 μM DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonate). Altogether these results suggest that a diffusional process rather than the high affinity taurine carrier is involved in the swelling induced increase in astrocytic taurine influx and efflux.