Random mutagenesis of the cAMP chemoattractant receptor, cAR1, of Dictyostelium. Evidence for multiple states of activation

cAMP receptor 1 (cAR1) of Dictyostelium couples to the G protein G2 to mediate activation of adenylyl and guanylyl cyclases, chemotaxis, and cell aggregation. Other cAR1-dependent events, including receptor phosphorylation and influx of extracellular Ca2+, do not require G proteins. To further characterize signal transduction through cAR1, we performed random mutagenesis of the third intracellular loop (24 amino acids), since the corresponding region of other seven helix receptors has been implicated in the coupling to G proteins. Mutant receptors were expressed in car1(-) cells and were characterized for G protein-dependent and -independent signal transduction. Our results demonstrate that cAR1 is remarkably tolerant to amino acid substitutions in the third intracellular loop. Of the 21 positions where amino acid substitutions were observed, one or more replacements were found that retained full biological function. However, certain alterations resulted in receptors with reduced ability to bind cAMP and/or transduce signals. There were specific signal transduction mutants that could undergo cAMP-dependent cAR1 phosphorylation but were impaired either in coupling to G proteins, in G protein-independent Ca2+ influx, or in both pathways. In addition, there were general activation mutants that failed to restore aggregation to car1(-) cells and displayed severe defects in all signal transduction events, including the most robust response, cAMP-dependent cAR1 phosphorylation. Certain of these mutant phenotypes were obtained in a complementary study, where the entire region of cAR1 from the third to the seventh transmembrane helices was randomly mutagenized. Considered together, these studies indicate that the activation cycle of cAR1 may involve a number of distinct receptor intermediates. A model of G protein-dependent and -independent signal transduction through cAR1 is discussed.     

Severe manifestations in carrier females in X linked retinitis pigmentosa

The aim of this study was to assess the effect of menopause on circadian profile of blood pressure (BP) and heart rate (HR) in the normotensive pre- and postmenopausal women. Systolic BP (SBP), diagnostic BP (DBP) and HR were monitored every 30 min for 48 hrs using noninvasive ambulatory BP monitoring in 24 premenopausal and 40 postmenopausal women. Mean 48-hours, daytime (awake), and nighttime (sleeping) SBP, DBP and HR values were analyzed by reviewing the patients' diaries, and the nocturnal reduction rate (NRR) of SBP, DBP and HR were calculated according to the following formula. NRR (%9 = [(daytime mean-nighttime mean)/daytime mean] x 100. The study subjects were then divided into two groups according to the presence (dipper) or absence (nondipper) of a significant reduction in nocturnal BP (> 10%). Mean SBP, DBP and HR measured over 48 hours were similar between the premenopausal and the postmenopausal group. The NRR of DBP and HR in the postmenopausal group were significantly smaller than those in the premenopausal group (17.1 +/- 6.0% vs. 13.5 +/- 7.0%, 241.1 +/- 6.0% vs. 19.8 +/- 9.0%: p < 0.05). There tended to be higher prevalence of nondipper in the postmenopausal (37%) than in the premenopausal group (29%).     

Neurotransmitter modulation of gap junctional communication in the rat hippocampus

Increasing experimental evidence indicates that gap junctions can be modulated by neurotransmitters, in particular dopamine. To examine possible modulation of gap junctional communication in the rat hippocampus by neurotransmitters, we studied dye coupling and electrotonic transmission in the CA1 area in the presence of carbachol, a cholinergic agonist, and dopamine agonists. Carbachol markedly reduced dye coupling and the frequency of electrotonic potentials (spikelets). Spikelet amplitudes were decreased in the presence of carbachol. These effects were reversed by the cholinergic antagonist atropine, suggesting a muscarinic action of carbachol on gap junctional function. The non-specific dopamine agonist apomorphine, and the specific D1 receptor agonist SKF 38393, reduced dye coupling between pyramidal cells. Spikelet frequency was also decreased in the presence of dopamine agonists, but less than with carbachol. The specific D1 receptor antagonist, SCH 23390, reversed the effects of both dopamine agonists. These observations indicate that cholinergic and dopaminergic transmission can affect electrical and chemical (dye coupling) communication through gap junctions, and could therefore alter properties of neuronal assemblies, in addition to their effects on intrinsic membrane properties.     

Fatty acid composition of an oral load affects chylomicron size in human subjects

This paper outlines the demographic characteristics of the Baiga tribe, one of the most primitive of the aboriginal tribal groups of Central India. The Baiga population has grown steadily since the first anthropological study of the tribe in the 1930s. Age at menarche, age at marriage, breast-feeding, and time interval between marriage and first conception are natural. There are more females than males. Sub-tribe endogamy is common; consanguineous marriage is favoured (34% of marriages are between first cousins) and marital distance is low (mean 7.1 km). Though the mean menarcheal age is high (15.2 years), mean age at first marriage is low (16.6 years), giving a long reproductive period.     

Partial activation of muscle phosphorylase by replacement of serine 14 with acidic residues at the site of regulatory phosphorylation

Phosphorylation of glycogen phosphorylase at residue Ser14 triggers a conformational transition that activates the enzyme. The N-terminus of the protein, in response to phosphorylation, folds into a 310 helix and moves from its location near a cluster of acidic residues on the protein surface to a site at the dimer interface where a pair of arginine residues form charged hydrogen bonds with the phosphoserine. Site-directed mutagenesis was used to replace Ser14 with Asp and Glu residues, analogs of the phosphoserine, that might be expected to participate in ionic interactions with the arginine side chains at the dimer interface. Kinetic analysis of the mutants indicates that substitution of an acidic residue in place of Ser14 at the site of regulatory phosphorylation partially activates the enzyme. The S14D mutant shows a 1.6-fold increase in Vmax, a 10-fold decrease in the apparent dissociation constant for AMP, and a 3-fold decrease in the S0.5 for glucose 1-phosphate. The S14E mutant behaves similarly, showing a 2.2-fold increase in Vmax, a 6-fold decrease in the apparent dissociation constant for AMP, and a 2-fold decrease in the S0.5 for glucose 1-phosphate. The ability of the mutations to enhance binding of AMP and glucose 1-phosphate and to raise catalytic activity suggests that the introduction of a carboxylate side chain at position 14 promotes docking of the N-terminus at the subunit interface and concomitant stabilization of the activated conformation of the enzyme. Like the native enzyme, both mutants show significant activity only in the presence of the activator, AMP. Full activation, analogous to that provided by covalent phosphorylation of the enzyme, likely is not achieved because of differences in the charge and the geometry of ionic interactions at the phosphorylation site.     

Syndecan-3, tenascin-C, and the development of cartilaginous skeletal elements and joints in chick limbs

Since the European frogs (Rana spp.) have fallen under the German endangered species regulation, Xenopus laevis (South African Clawed Frog) is being used increasingly in animal research and education. Optimal growth rates and homogeneity of groups have not necessarily been attained as little statistical analysis of growth data has been available. Following metamorphosis, an as yet not understood variability of growth is exhibited by X. laevis. In this study the effect of environmental factors on this variability was determined. Feeding, population density, background colouring, water temperature, the availability of hiding places, water level and water care were each examined separately. Development of body weight and body length were recorded. A definite correlation between the feeding programme, population density, cover and water care on the one hand and growth on the other were seen. Of lesser importance were water temperature, water level and background colouring. The observed variability of growth is assumed to also be of ethological origin.     

DEHYDRATION BY DESORPTION AND BY SPRAY DRYING OF DAIRY PROTEINS: INFLUENCE OF THE MINERAL ENVIRONMENT

A drying method by desorption in a water activity meter was used to simulate the conditions of spray drying and to determine the water transfer inside dairy concentrates towards the surface and from the surface to the drying air. The concentrates were also spray dried and solubility index of powders were determined during reconstitution. Whey protein concentrates (WPC) and native phosphocaseinate suspensions (NPCS) were used to study the effect of NaCl (420 mM), CaCl₂ (222 raM), sodium phosphate (173 mM) and sodium citrate (238 mM) on the water transfers. The decrease in water transfer during drying was explained by the high hygroscopicity of added mineral salts to WPC. NaCl addition to NPCS decreased the water transfer during drying, but increased the solubility index. Citrate and phosphate addition to NPCS increased the water transfer during drying and reconstitution. CaCl₂ increased the water transfer during drying but the solubility index was always low. Results are discussed as a function of the aqueous environment, of the nature of mineral salts, of the structure of dairy proteins and of protein solvation.