Lipid-mediated interactions between intrinsic membrane proteins: dependence on protein size and lipid composition.

The present study is an application of an approach recently developed by the authors for describing the structure of the hydrocarbon chains of lipid-bilayer membranes (LBMs) around embedded protein inclusions ( Biophys. J. 79:2867-2879). The approach is based on statistical mechanical integral equation theories developed for the study of dense liquids. First, the configurations extracted from molecular dynamics simulations of pure LBMs are used to extract the lateral density-density response function. Different pure LBMs composed of different lipid molecules were considered: dioleoyl phosphatidylcholine (DOPC), palmitoyl-oleoyl phosphatidylcholine (POPC), dipalmitoyl phosphatidylcholine (DPPC), and dimyristoyl phosphatidylcholine (DMPC). The results for the lateral density-density response function was then used as input in the integral equation theory. Numerical calculations were performed for protein inclusions of three different sizes. For the sake of simplicity, protein inclusions are represented as hard smooth cylinders excluding the lipid hydrocarbon core from a small cylinder of 2.5 A radius, corresponding roughly to one aliphatic chain, a medium cylinder of 5 A radius, corresponding to one alpha-helix, and a larger cylinder of 9 A radius, representing a small protein such as the gramicidin channel. The lipid-mediated interaction between protein inclusions was calculated using a closed-form expression for the configuration-dependent free energy. This interaction was found to be repulsive at intermediate range and attractive at short range for two small cylinders in POPC, DPPC, and DMPC bilayers, whereas it oscillates between attractive and repulsive values in DOPC bilayers. For medium size cylinders, it is again repulsive at intermediate range and attractive at short range, but for every model LBM considered here. In the case of a large cylinder, the lipid-mediated interaction was shown to be repulsive for both short and long ranges for the DOPC, POPC, and DPPC bilayers, whereas it is again repulsive and attractive for DMPC bilayers. The results indicate that the packing of the hydrocarbon chains around protein inclusions in LBMs gives rise to a generic (i.e., nonspecific) lipid-mediated interaction which favors the association of two alpha-helices and depends on the lipid composition of the membrane.

Lipid-mediated interactions between intrinsic membrane proteins: a theoretical study based on integral equations.

This study of lipid-mediated interactions between proteins is based on a theory recently developed by the authors for describing the structure of the hydrocarbon chains in the neighborhood of a protein inclusion embedded in a lipid membrane [Lagüe et al., Farad. Discuss. 111:165-172, 1998]. The theory involves the hypernetted chain integral equation formalism for liquids. The exact lateral density-density response function of the hydrocarbon core, extracted from molecular dynamics simulations of a pure dipalmitoylphosphatidylcholine bilayer based on an atomic model, is used as input. For the sake of simplicity, protein inclusions are modeled as hard repulsive cylinders. Numerical calculations were performed with three cylinder sizes: a small cylinder of 2.5-A radius, corresponding roughly to an aliphatic chain; a medium cylinder of 5-A radius, corresponding to a alpha-helical polyalanine protein; and a large cylinder of 9-A radius, representing a small protein, such as the gramicidin channel. The calculations show that the average hydrocarbon density is perturbed over a distance of 20-25 A from the edge of the cylinder for every cylinder size. The lipid-mediated protein-protein effective interaction is calculated and is shown to be nonmonotonic. In the case of the small and the medium cylinders, the lipid-mediated effective interaction of two identical cylinders is repulsive at an intermediate range but attractive at short range. At contact, there is a free energy of -2k(B)T for the 2.5-A-radius cylinder and -9k(B)T for the 5-A-radius cylinder, indicating that the association of two alpha-helices of both sizes is favored by the lipid matrix. In contrast, the effective interaction is repulsive at all distances in the case of the large cylinder. Results were obtained with two integral equations theories: hypernetted chain and Percus-Yevick. For the two theories, all results are qualitatively identical.

Effects of electromagnetic fields on the reproductive success of American kestrels.

Reduced reproductive success of birds nesting near power lines has been documented but never directly attributed to electromagnetic fields (EMFs). Laboratory studies have identified EMF effects on embryonic development, but reproductive success of wild birds is dependent on additional factors, including fertility, egg size, hatching, and fledging success. We tested whether EMFs affect reproductive success of birds. Captive American kestrels (Falco sparverius) were bred for one season per year for 2 yr under either controlled or EMF conditions. EMF exposure was equivalent to that experienced by wild reproducing kestrels and was weakly associated with reduced egg laying in 1 yr only. In both years fertility was higher, but hatching success was lower in EMF pairs than control pairs. Fledging success was higher in EMF pairs than control pairs in 1995 only. Egg composition and embryonic development were examined in 1 yr only, but hatchlings were measured in both years. EMF eggs were larger, with more yolk, albumen, and water, but had thinner egg shells than control eggs. Late-term EMF embryos were larger and longer than control embryos, although hatchlings were similar in body mass and size. EMF exposure affected reproductive success of kestrels, increasing fertility, egg size, embryonic development, and fledging success but reducing hatching success.

Mild autosomal dominant hypophosphatasia: in utero presentation in two families.

We describe four pregnancies in two families in which mild hypophosphatasia, apparently transmitted as an autosomal dominant trait, manifested in utero as severe long bone bowing. Postnatally, there was spontaneous improvement of the skeletal defects. Recognition of this presentation for hypophosphatasia by family investigation and assessment of the fetal skeleton for degree of ossification and chest size using ultrasonography is important. The prognosis for this condition is considerably better than for more severe forms of hypophosphatasia and for many other disorders that cause skeletal defects with long bone bowing in utero.

Juvenile rheumatoid arthritis and del(22q11) syndrome: a non-random association.

Del(22q11) is a common microdeletion syndrome with an extremely variable phenotype. Besides classical manifestations, such as velocardiofacial (Shprintzen) or DiGeorge syndromes, del(22q11) syndrome may be associated with unusual but probably causally related anomalies that expand its phenotype and complicate its recognition. We report here three children with the deletion and a chronic, erosive polyarthritis resembling idiopathic cases of juvenile rheumatoid arthritis (JRA). Patient 1, born in 1983, initially presented with developmental delay, facial dysmorphism, velopharyngeal insufficiency, and severe gastro-oesophageal reflux requiring G tube feeding. From the age of 3 years, he developed JRA, which resulted in severe restrictive joint disease, osteopenia, and platyspondyly. Patient 2, born in 1976, had tetralogy of Fallot and peripheral pulmonary artery stenosis. She developed slowly, had mild dysmorphic facial features, an abnormal voice, and borderline intelligence. JRA was diagnosed at the age of 5 years. The disorder followed a subacute course, with relatively mild inflammatory phenomena, but an extremely severe skeletal involvement with major osteopenia, restrictive joint disease (bilateral hip replacement), and almost complete osteolysis of the carpal and tarsal bones with phalangeal synostoses, leading to major motor impairment and confinement to a wheelchair. Patient 3, born in 1990, has VSD, right embryo-toxon, bifid uvula, and facial dysmorphism. She developed JRA at the age of 1 year. She is not mentally retarded but has major speech delay secondary to congenital deafness inherited from her mother. In the three patients, a del(22q11) was shown by FISH analysis. These observations, and five other recently published cases, indicate that a JRA-like syndrome is a component of the del(22q11) spectrum. The deletion may be overlooked in those children with severe, chronic inflammatory disorder.

Protein inclusion in lipid membranes: a theory based on the hypernetted chain integral equation.

A theory for describing the structure of the hydrocarbon chains around a protein inclusion embedded in a lipid bilayer is developed on the basis of the hypernetted chain integral equation formalism for liquids. The exact lateral density-density response function of the hydrocarbon core, which is extracted from a molecular dynamics simulation of a pure lipid bilayer, is used as input to the theory. Numerical calculations show that the average lipid order is perturbed over a distance of 25 to 30 A around a hard repulsive cylinder of 5 A radius representing an alpha-helical polyleucine protein inclusion. The lipid-mediated protein-protein interaction is calculated and is shown to be non-monotonic, being repulsive at an intermediate range but attractive at short range. It is found that the lipid matrix contributes a free energy well of 8 kBT to the association of two cylindrical inclusions.

The role of hen's weight and recent experience on dyadic conflict outcome.

This study simultaneously varied experiences of recent victory or defeat, 2-h familiarity with the meeting place, and hen weight in order to understand their combined effects on the establishment of dyadic dominance relationships between hens not previously acquainted with each other. Three kinds of encounters were arranged: (i) a previous winner unfamiliar with the meeting place met a previous loser familiar with the meeting place (n=28 dyads); (ii) a previous winner met a previous loser, both unfamiliar with the meeting place (n=27); (iii) a previous winner familiar with the meeting place encountered a previous loser unfamiliar with the meeting place (n=28). The weight asymmetry was combined with these three types of encounters by selecting hens showing various weight differences, in favour of the recent loser in 54 dyads and of the recent winner in 29 dyads. Results indicate that recent victory or defeat experience significantly affected the outcome. Even an important weight asymmetry, or familiarity with the meeting place were not sufficient for a hen recently defeated to overcome an opponent that was previously victorious. A 2-h period of familiarization with the meeting place did not provide any significant advantage over unfamiliarity. Although a significant relationship was found to exist between comb and wattles areas and the initial and final statuses, examination of partial correlations indicates that the influence was from initial status to final status, rather than from comb and wattles to final status. These results suggest that more importance should be attributed to recent social experience in comparison to intrinsic factors in determining dyadic dominance in the hen.

The role of recent experience and weight on hen's agonistic behaviour during dyadic conflict resolution.

Recent victory or defeat experiences and 2-h familiarity with the meeting place were combined with size differences in order to better understand their effects on the behaviour leading to the establishment of dyadic dominance relationships between hens not previously acquainted with each other. Three kinds of encounters were videotaped: (i) A previous winner unfamiliar with the meeting place met a previous loser familiar for 2 h with the meeting place (n=12 dyads); (ii) as in (i) but both were unfamiliar with the meeting place (n=12); (iii) as in (i) but the previous winner was familiar with the meeting place while the previous loser was unfamiliar (n=13). The weight asymmetry was combined with these three types of encounters by selecting hens of various weight differences: In 29 dyads the recent loser was heavier than the recent winner and in eight dyads it was the reverse. Recent experience had a major influence upon both agonistic behaviour and dominance outcome. Hens that were familiar with the meeting site initiated attacks more frequently than their unfamiliar opponent but did not win significantly more often. Recent experience and site familiarity could be used to identify 80% of future initiators. Once the first aggressive behaviour had been initiated, it led to victory of its initiator in 92% of cases. Weight was not found to influence agonistic behaviour nor dominance outcome. However, hens with superior comb and wattles areas won significantly more initial meetings than opponents with smaller ones. In the final encounters, victory also went more frequently to the bird showing larger comb and wattles, which happened also to be the previous dominant in a majority of cases. The use of higher-order partial correlations as an ex post facto control for comb and wattles indicates that they were not influential upon agonistic behaviour nor on dominance outcome, but were simply co-selected with the selection of victorious and defeated birds in the first phase of the experiment designed to let hens acquire recent victory/defeat experience.

Apparent digestibility of protein and amino acids in broiler chickens fed a corn-soybean diet supplemented with microbial phytase.

The effect of microbial phytase supplementation on CP and amino acid (AA) digestibility was investigated in a 28-d trial using 360 sexed, day-old broiler chickens fed corn-soybean meal diets. The experimental design was a completely randomized one with a 3 x 2 factorial arrangement of treatments. The variables included P and Ca levels and phytase: P and Ca levels were: normal P-normal Ca [0.45% available P (Pa), 1.0% Ca], low P-normal Ca (0.35% Pa, 1.0% Ca), and low P-low Ca (0.35 Pa and 0.6% Ca); and phytase at 0 and 600 U/kg diet. Phytase supplementation increased body weight gain (P < 0.014) and feed intake (P < 0.004) at 19 d in male chickens; in females, phytase increased (P < 0.012) only body weight gain at 19 d. The low P-normal Ca diet reduced (P < 0.05) feed intake and body weight gain in both sexes at 7, 14, and 19 d, compared to the normal P-normal Ca diet; the reduction of Ca in the low P diet prevented the above depression, resulting in body weight gain and feed intake to a level comparable to that of the normal P-normal Ca diet. Microbial phytase supplementation had no effect (P < 0.065) on the apparent ileal digestibility (AID) of CP or any AA except Met and Phe in male broiler chickens. In females, adding phytase increased the AID of all AA except Lys, Met, Phe, and Pro. The low P-normal Ca diet reduced (P < 0.05) the AID of Phe, Asp, and Ser in male chickens and reduced the AID of all the AA except Met and Pro in females compared to the normal P-normal Ca diet. The reduction of Ca in the low P diet prevented the depression of the AID of the AA caused by the low P-normal Ca diet, resulting in AID of AA having a level comparable to that of the normal P-normal Ca diet in both sexes. Phytase supplementation did not have any effect (P > 0.05) on apparent "fecal" digestibility (AFD) of CP or any of the AA in male chickens; however, in female chickens it increased the AFD of Thr, Asp, Glu, and Ser. In summary, phytase supplementation increased growth performance in both sexes; increased AID and AFD of most of the AA, particularly in female chickens. The optimum growth performance and AA digestibilities were obtained with the lowest input of resources, in the low P-low Ca diet supplemented with microbial phytase.

Efficacy of supplemental microbial phytase at different dietary calcium levels on growth performance and mineral utilization of broiler chickens.

A 3-wk feeding trial with 240 sexed, day-old broiler chickens was conducted to determine the efficacy of microbial phytase at different levels of dietary Ca on performance and utilization of minerals in broiler chickens fed a low-P corn-soybean diet. The experimental design was a 3 x 2 factorial arrangement of treatments; Ca at 0.6, 1.0, and 1.25% and phytase at 0 and 600 phytase U/kg diet. Phytase supplementation, regardless of Ca level, increased (P < or = 0.005) feed intake, (P < or = 0.0001) body weight, and (P < or = 0.025) feed efficiency at 21 d; the optimum levels of body weight, feed intake, and feed efficiency were obtained with low (0.6%) dietary Ca plus phytase. Retentions of P, Ca, and N were increased (P < or = 0.05) by phytase supplementation. Although maximum retentions of P and N were obtained at the 1.0 and 1.25% Ca levels, respectively, they were not significantly different from the values obtained at 0.6% Ca. The increasing level of dietary Ca decreased plasma P ( P < or = 0.05) and Cu (P < or = 0.06). Phytase supplementation had the opposite effect; i.e., increased plasma P (P < or = 0.03) and Cu (P < or = 0.02). The maximum level of plasma P was obtained with phytase at the 1.0% Ca level, but this value was not significantly different from the value obtained with phytase at the 0.6% Ca level. Phytase supplementation increased (P < 0.04) the ash content of both tibia head and shaft but had no effect on mineral contents in the ash. The optimum level of ash content was observed with the 0.06% Ca diet plus phytase. The results show that microbial phytase supplementation to a low P diet improved growth performance and mineral utilization in broiler chickens. Dietary Ca levels had a significant effect on the response to phytase; the optimum growth performance and mineral utilization were achieved at the low (0.6%) level of dietary Ca supplemented with phytase.

The effects of supplemental microbial phytase on the performance and utilization of dietary calcium, phosphorus, copper, and zinc in broiler chickens fed corn-soybean diets.

A 3-wk feeding trial with 180 sexed day-old broiler chickens was conducted to study the efficacy of microbial phytase (Natuphos 1000) on growth performance, relative retention of P, Ca, Cu, and Zn, and mineral contents of plasma and bone. Treatments involved a normal P level corn-soybean diet, a low-P diet, and a low-P plus phytase (600 phytase units/kg) diet. Phytase supplementation increased (P < or = 0.05) body weight in male and female chickens by 13.2 and 5.8%, respectively, at 21 d. The improvements yielded body weights comparable to those obtained on the normal P diet. Phytase supplementation overcame (P < or = 0.05) the depression of feed intake observed on the low-P diet. Treatments had no effect on feed:gain ratio. Phytase supplementation of the low-P diet increased (P < or = 0.05) the relative retention of total P, Ca, Cu, and Zn by 12.5, 12.2, 19.3, and 62.3 percentage units, respectively, in male chickens. Microbial phytase increased the plasma P by 15.7% and reduced (P < or = 0.05) the Ca concentration by 34.1%, but had no effect on plasma concentrations of Cu or Zn. Phytase supplementation increased the percentage ash in both head and shaft portions of dry, fat-free tibia bone to a level comparable to that of the normal-P diet. Phytase supplementation had no effect on the concentration of any of the minerals measured in whole tibia ash but did increase (P < or = 0.05) the DM percentage of P and Ca min tibia head of male chickens by 0.65 and 1.4 percentage units, respectively. These results show that microbial phytase supplementation of a low-P diet increased growth and relative retention of total P, Ca, Cu, and Zn and improved bone mineralization in broiler chickens.

The role of individual differences and patterns of resolution in the formation of dominance orders in domestic hen triads.

This research compares the role of initial individual characteristics to that of patterns of resolution in which successive dominance relationships are established during the formation of triads in the domestic hen. Combining weight and comb size with prior victory or defeat in the site of encounter, we created three levels of asymmetries of characteristics for triads of hens. The effects of these asymmetries were then examined on the resultant hierarchies and on the order of conflict resolution within triads under two different conditions of assembly. In one condition (simultaneous triad), the three hens were simultaneously introduced to each other and could thus freely choose their opponent. In the other condition (step-assembled triad), the hen predicted to occupy the highest rank was left on standby and introduced once the other two hens had settled dominance. This condition disrupts the normal process of hierarchy formation by imposing the first sequence of dominance settlement. We found that the structure of triadic hierarchies can be predicted from individual characteristics existing prior to hierarchy formation. No difference in the resultant structures were found between conditions of introduction, though different paths of conflict resolution were followed indicating that individual differences had a more determining role on the resultant hierarchies than patterns of resolution. In addition to demonstrating that individual differences determine resultant triadic structures, the present results also show that the same end structures can be reached by following resolution paths that are not necessarily of the Double Dominance and Double Subordinance types as prescribed by Chase's model. It is also found that in the simultaneous condition hens select each other to form pairs. Therefore, individuals do not meet at random but choose each other as opponents. The two hens predicted from individual differences to occupy the highest ranks first settle dominance, followed by settlement between the winner of the previous encounter and the bystander.

Coherent use of information by hens observing their former dominant defeating or being defeated by a stranger.

This study examines the role of observation during the formation of triads in female domestic hens. Results indicate that during hierarchy formation, a hen observing agonistic interactions and conflict settlement between its former dominant and a stranger uses this information when in turn confronted by the latter. Under a first condition (E, n = 15 triads), bystanders witnessed their prior dominant being defeated by a stranger before being introduced to them. In a second condition (C1, n = 16 triads), bystanders witnessed the victory of their prior dominant over a stranger. In a third condition (C2, n = 15 triads), bystanders witnessed two strangers establishing a dominance relationship before being introduced to their prior dominant and to a stranger the former had just defeated. The behavioural strategies of bystanders depended on the issue of the conflict they had witnessed. Bystanders of the E condition behaved as having no chance of defeating the stranger. They never initiated an attack against it, and upon being attacked, readily submitted in turn to the stranger. On the contrary, bystanders of the C1 condition behaved as having some chances against the stranger. They initiated attacks in 50% of cases, and won 50% of conflicts against the stranger. Under condition C2, bystanders first initiated contact with the strangers in only 27% of cases, which approximates the average of their chances for defeating the stranger. However, bystanders finally defeated the strangers in 40% of cases. These results suggest that bystanders of conditions E and C1 gained some information on the relationship existing between their prior dominant and the stranger and that they used it coherently, perhaps through transitive inference, thus contributing to the existence of transitive relationships within the triads. Alternate explanations are examined.

The effect of prior victory or defeat in the same site as that of subsequent encounter on the determination of dyadic dominance in the domestic hen.

We examined the effect of prior victory or defeat in the same site as that of a subsequent encounter on the outcome of dyadic encounter of domestic hens by placing them in two situations. In the first set of dyads, two unacquainted hens having experienced prior victory were introduced in the site where one had experienced victory. In the second set, two unacquainted hens having experienced defeat were introduced in the site where one had recently lost. Results indicate that victories are equally shared between individuals with prior victory experiences, while familiarity with the meeting site did not give any advantage. However, hens having previously lost were disadvantaged when the encounter occurred in the same site as that of their prior defeat. This demonstrates that previous social experience in a site is more important on the outcome of subsequent encounters for losers than winners. Losers seem to associate the site with the stressful effect of losing or being more easily dominated.

Domains of E1A that bind p105Rb, p130, and p300 are required to block nerve growth factor-induced neurite growth in PC12 cells.

Nerve growth factor (NGF) causes PC12 cells to cease division and undergo sympathetic neuron-like differentiation, including neurite outgrowth. We have tested whether differentiation and division share overlapping control mechanisms in these cells. To do this, we have perturbed the activity of proteins known to participate in cell-cycle regulation by introducing the E1A oncogene or its mutant forms via microinjection into PC12 cells. The E1A protein binds to several putative cell cycle control proteins, including p105Rb (the product of the retinoblastoma susceptibility gene), as well as others of unknown function such as p130, p107, and p300. Similar to previous results, we find that wild-type E1A abrogates NGF-induced neurite extension. However, NGF does cause neurite outgrowth in the presence of E1A mutants known to have greatly reduced binding to either p105Rb and p130 or p300. Our experiments suggest that p105Rb, p130, and p300 may participate either in E1A-mediated inhibition of differentiation or in the NGF signal transduction pathway. We also report here that NGF affects phosphorylation of p105Rb, suggesting that Rb mediates at least some of NGF's effects. Our results raise the possibility that putative cell-cycle control proteins may participate not only in NGF-induced cessation of division but also in differentiation.

Research note: eggshell quality in Japanese quail fed different fatty acids.

The purpose of the present study was to determine the effects of diets containing 3% palmitic acid (Diet PA), oleic acid (Diet OA), or linoleic acid (Diet LA) on the shell quality of eggs of Japanese quail. Each diet was fed to 10 hens maintained individually in wire quail laying cages. There was no difference (P greater than .05) in feed consumption between hens fed Diets OA and LA, but hens of both groups consumed less (P less than .05) feed than those fed Diet PA. Egg weight, shell weight, and thickness of shell plus membrane were not influenced by dietary treatment (P greater than .05). However, specific gravity of eggs from hens fed Diet OA was significantly higher (P less than .05) than that of eggs from those fed either Diet PA or Diet LA. Comparison of these data with hatchability data obtained in a previous experiment conducted under similar conditions suggests that the differences in hatchability would not be explained by differences in eggshell quality.

Nerve growth factor acts through cAMP-dependent protein kinase to increase the number of sodium channels in PC12 cells.

cAMP-dependent protein kinase (PKA) and phospholipid-dependent protein kinase (PKC) play a role in nerve growth factor (NGF)-mediated differentiation. In PC12 cells, NGF causes neurite outgrowth and increases the number of voltage-gated Na+ channels. Neurite outgrowth involves in part activation of PKC. How NGF regulates Na+ channel number is unknown. Using patch-clamp techniques, we find that agents activating PKC, including phorbol esters and a ras oncogene product (p21) that induces neurites, caused little increase in channel number. In contrast, agents increasing intracellular cAMP were as effective as NGF. A specific protein inhibitor of the PKA catalytic subunit blocked increases by NGF or cAMP. Thus, NGF increases Na+ channel number in PC12 cells in part by activating PKA but apparently not PKC.

ATFresno: a phenotype linking ataxia-telangiectasia with the Nijmegen breakage syndrome.

This report describes twin girls with typical features of ataxia-telangiectasia, including increased alpha-fetoprotein, radio-resistant DNA synthesis, characteristic chromosome abnormality, and immunodeficiency. They have, in addition, microcephaly and mental retardation. Complementation studies were performed utilizing Sendai virus--mediated fusion of fibroblast cell lines. Complementation was observed with patients in ataxia-telangiectasia complementation groups A, C, and E but not with the cell line from a patient with the Nijmegen breakage syndrome, in which patients have microcephaly, radio-resistant DNA synthesis, chromosome aberrations, and immunodeficiency but lack ataxia and telangiectasia. These data suggest that the Nijmegen breakage syndrome and the patients described here are not genetically distinct entities but form a spectrum of one disorder.

Calcium-dependent inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells.

The inactivation of calcium channels in mammalian pituitary tumor cells (GH3) was studied with patch electrodes under voltage clamp in cell-free membrane patches and in dialyzed cells. The calcium current elicited by depolarization from a holding potential of -40 mV passed predominantly through one class of channels previously shown to be modulated by dihydropyridines and cAMP-dependent phosphorylation (Armstrong and Eckert, 1987). When exogenous calcium buffers were omitted from the pipette solution, the macroscopic calcium current through those channels inactivated with a half time of approximately 10 ms to a steady state level 40-75% smaller than the peak. Inactivation was also measured as the reduction in peak current during a test pulse that closely followed a prepulse. Inactivation was largely reduced or eliminated by (a) buffering free calcium in the pipette solution to less than 10(-8) M; (b) replacing extracellular calcium with barium; (c) increasing the prepulse voltage from +10 to +60 mV; or (d) increasing the intracellular concentration of cAMP, either 'directly' with dibutyryl-cAMP or indirectly by activating adenylate cyclase with forskolin or vasoactive intestinal peptide. Thus, inactivation of the dihydropyridine-sensitive calcium channels in GH3 cells only occurs when membrane depolarization leads to calcium ion entry and intracellular accumulation.

Variations in plasma corticosterone, estrone, estradiol-17 beta, and progesterone concentrations with forced renesting, molt, and body weight of captive female American kestrels.

Corticosterone, progesterone, estradiol-17 beta, and estrone were quantified in plasma collected weekly (April-September) from renesting and nonlaying female American kestrels (Falco sparverius) paired with males in captivity. Hormone levels and body weights for laying females were maximal during courtship and egg-laying periods, while those for non-layers showed no such distinct peaks. This demonstrated that these profiles were not controlled solely by photoperiod. Plasma corticosterone levels were elevated in all females during August and September when kestrels are preparing for migration. For laying females, body weight was positively correlated with plasma estrogen levels. A low spring body weight gain, and not stress, may have prevented the females from breeding in captivity. The photoperiodic control of molt did not appear to be mediated directly by the hormones studied, since there were no changes in hormone levels associated with the onset of molt.