Probing ligands to reaction centers to limit the photocycle in photosynthetic bacterium Rubrivivax gelatinosus.

Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view. SIGNIFICANCE: A crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)-1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.

Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus.

The administration of leptin to leptin-deficient humans, and the analogous Lepob/Lepob mice, effectively reduces hyperphagia and obesity. But common obesity is associated with elevated leptin, which suggests that obese humans are resistant to this adipocyte hormone. In addition to regulating long-term energy balance, leptin also rapidly affects neuronal activity. Proopiomelanocortin (POMC) and neuropeptide-Y types of neurons in the arcuate nucleus of the hypothalamus are both principal sites of leptin receptor expression and the source of potent neuropeptide modulators, melanocortins and neuropeptide Y, which exert opposing effects on feeding and metabolism. These neurons are therefore ideal for characterizing leptin action and the mechanism of leptin resistance; however, their diffuse distribution makes them difficult to study. Here we report electrophysiological recordings on POMC neurons, which we identified by targeted expression of green fluorescent protein in transgenic mice. Leptin increases the frequency of action potentials in the anorexigenic POMC neurons by two mechanisms: depolarization through a nonspecific cation channel; and reduced inhibition by local orexigenic neuropeptide-Y/GABA (gamma-aminobutyric acid) neurons. Furthermore, we show that melanocortin peptides have an autoinhibitory effect on this circuit. On the basis of our results, we propose an integrated model of leptin action and neuronal architecture in the arcuate nucleus of the hypothalamus.

Phosphorylation stabilizes the N-termini of alpha-helices.

The role of phosphorylation in stabilizing the N-termini of alpha-helices is examined using computer simulations of model peptides. The models comprise either a phosphorylated or unphosphorylated serine at the helix N-terminus, followed by nine alanines. Monte Carlo/stochastic Dynamics simulations were performed on the model helices. The simulations revealed a distinct stabilization of the helical conformation at the N-terminus after phosphorylation. The stabilization was attributable to favorable electrostatic interactions between the phosphate and the helix backbone. However, direct helix capping by the phosphorylated sidechain was not observed. The results of the calculations are consistent with experimental evidence on the stabilization of helices by phosphates and other anions.

Analysis of synaptic transmission in the neuromuscular junction using a continuum finite element model.

There is a steadily growing body of experimental data describing the diffusion of acetylcholine in the neuromuscular junction and the subsequent miniature endplate currents produced at the postsynaptic membrane. To gain further insights into the structural features governing synaptic transmission, we have performed calculations using a simplified finite element model of the neuromuscular junction. The diffusing acetylcholine molecules are modeled as a continuum, whose spatial and temporal distribution is governed by the force-free diffusion equation. The finite element method was adopted because of its flexibility in modeling irregular geometries and complex boundary conditions. The resulting simulations are shown to be in accord with experiment and other simulations.

Longitudinal variation in the activities of mucosal enzymes in the small intestine of suckling rats.

The extent of positional variation in mucosal enzyme activity along the small intestine was investigated in 14-day-old suckling rats. Samples were taken from ten equally spaced sites along the intestine in 11 rat pups and the activities of the enzymes alkaline phosphatase, neutral aminopeptidase, gamma-glutamyl transferase, lactase and sucrase were measured. All the enzymes except sucrase were subject to considerable positional variation. Alkaline phosphatase and aminopeptidase activities were distributed throughout the intestine, with a broad maximum in the distal intestine. Lactase was also broadly distributed but with greatest activity in the mid intestine. gamma-glutamyl transferase exhibited a novel profile, with a very high proportion of the total activity (78%) present in the distal intestine. Sucrase was essentially absent throughout the intestine.

Review of literature showing that undernutrition affects the growth rate of all processes in the brain to the same extent.

The growth of the brain is certainly less affected by undernutrition than the growth of the rest of the body (brain sparing). We inquire whether a similar phenomenon occurs within the brain: whether there are differences in sensitivity to undernutrition amongst the brain's component growth processes. We conclude from our review of the literature that, contrary to popular belief, undernutrition depresses the growth rate of various processes within the brain to the same extent. The much quoted "selective" effect on the cerebellum is not an example of an especially sensitive process. It results from expressing deficits as "% of age control values" and disappears when proper comparisons of depression in rates of growth are made. The one growth process which does at first sight appear to be truly especially sensitive is myelin synthesis, in that undernutrition depresses its rate more than that of other growth processes in brain. However, undernutrition also results in fewer fibres being myelinated, and when rate of myelin synthesis is expressed per myelinated fibre the special sensitivity disappears.

Successful prediction of immediate effects of undernutrition throughout the brain growth spurt on capillarity and synapse-to-neuron ratio of cerebral cortex in rats.

Based on the hypothesis that undernutrition depresses the growth rate of all processes in brain contemporaneous with it to the same extent (Peeling & Smart, 1994), specific predictions were made regarding the effect of undernutrition on two quite different facets of anatomical development within visual cortex. It was predicted that severe undernutrition during the suckling period would leave capillarity unaffected, but would result in a deficit in synapse-to-neuron ratio of similar magnitude to that in brain weight. At birth rat pups were fostered and either well fed to 30 days or undernourished to the same age by underfeeding their foster mothers. Rats were killed at 30 days by perfusion with glutaraldehyde. Visual cortex was dissected out for quantitative histological study. The number of capillary profiles per unit area of section and the area of each profile were assessed with an image analysis system. Quantitative characteristics of the neuron and synapse populations were estimated by light and electron microscopy respectively. Undernutrition resulted in a 21% deficit in brain weight, and a 22% deficit in the number of synapses per neuron. Capillarity, expressed as the fraction of section area occupied by capillary lumen, appeared completely indifferent to nutrition. However, fewer capillary profiles were found per unit area of section in growth-restricted samples, and the profiles which were present were, on average, larger. Neuron density was elevated by 19% in the undernourished group. These findings are in good agreement with the hypothesis.

A prospective study of the development of laterality: neonatal laterality in relation to perinatal factors and maternal behavior.

As part of an investigation of the development of laterality, 150 neonates were tested for bilateral asymmetries in behavior. Palmar and plantar grasping, and head-turning preference were measured, and observations were made of the way mothers held their babies and laid them in their cribs. Overall, right head-turning predominated. However, the side on which the baby had been lain pre-test markedly affected head-turning behavior: 120 of 147 babies turned predominantly to the right after lying on the right, whereas only 75 did so after lying on their left side (p < .001). There was no predominant asymmetry in the population in palmar or plantar grasping. These findings are explored in relation to perinatal factors and maternal behavior.

Effects of urogastrone-epidermal growth factor and age at administration on five enzymes in the small intestine of suckling rats.

Suckling rats were given urogastrone-epidermal growth factor (EGF: 1,000 micrograms/kg body weight) or vehicle by gavage at one of three stages of development: 8 to 10, 11 to 13 or 14 to 16 days of age. Intubation was carried out at 8-hourly intervals over these periods. Fourteen to 16 h after the last intubation the rats were killed; that is, at 11, 14 and 17 days respectively. Samples of proximal and distal small intestine (SI) were taken for enzyme analysis. Five enzymes were assayed; sucrase, lactase, gamma-glutamyl transferase, alkaline phosphatase and neutral amino-peptidase, and their activities expressed per g protein. Treatment with EGF had no effect on body weight or on the length of the small intestine at any age. The nature of the effects on enzyme activities depended on the specific enzyme concerned, the site within the small intestine and the timing of the treatment. Lactase was increased by EGF at both sites only on day 14, whereas gamma-glutamyl transferase was increased in proximal samples at 11 and 14 days, and in distal samples at 17 days. Nor was the outcome always to increase activity. On day 11 alkaline phosphatase was increased in proximal SI, but decreased in distal SI; and so too was aminopeptidase N decreased in distal SI at 11 days. Sucrase showed no response at all. The pattern is complex. Certainly it does not indicate accelerated functional maturation.

Analysis of walking locomotion in adult female rats undernourished as sucklings.

Suckling rats were undernourished from birth by underfeeding their mothers. They were refed from 3 weeks of age. Detailed analyses were made of the walking locomotion of previously undernourished and well-fed control female rats at 10 weeks, using electronic, computer, and TV techniques. Previously undernourished rats were found to deploy shorter stride times resulting in greater forward velocities. The shorter stride times were attained by reductions in stance time, while swing time was unaffected. No changes in stride length or width were observed, and the relationships between stride time and velocity, swing, or stance times were unaffected.

Urogastrone-epidermal growth factor and aspects of sexual maturation in female rats as a function of age at treatment.

The effect of epidermal growth factor (EGF) on sexual maturation of female rats was studied. A within-litter experimental design was employed, so that in each litter each female received four daily injections of EGF (E, 500 ng/g body weight s.c.) or vehicle (V), at one of three ages: days 0-3 (E1, V1), days 8-11 (E2, V2), days 16-19 (E3, V3). Body weight, pinna detachment, incisor eruption, eye opening, auditory startle, visual placing, vaginal opening and first cytological oestrus were assessed. Neonatal treatment with EGF (E1) delayed pinna detachment and the appearance of the auditory startle, but accelerated eye opening. Also, E1, but not E2 and E3, resulted in lower body weight at weaning. Treatment E3 advanced sexual maturation, as indicated by vaginal opening and first cytological oestrus, by 5-6 days. E1 and E2 had no such effect. Hence the sensitive period for the effect of EGF on female rat sexual maturation is later than that for effects on other developmental characteristics and body weight. In a second experiment, ovary and uterus weights were found not to differ between E3 and V3 females killed on the day of vaginal opening of the E3 rats, suggesting that the effect of EGF may be specifically on the perineal epithelium and not on sexual maturation generally.

Vulnerability of developing brain to undernutrition: claims of selective sensitivity of the cerebellum found to be misleading on theoretical and empirical grounds.

The growing rat brain acquires most of its weight, cells, and other material during the suckling period. Undernutrition at this stage can diminish brain growth. Nutritionally induced depressions of cerebellar growth may be smaller or larger than corresponding depressions in other brain regions, but when the resulting deficits are transformed to '% of age control values', the cerebellar deficits appear specially large. The ubiquitous '% transformation' and its misrepresentation of cerebellar deficits have nurtured the impression that cerebellum has a special sensitivity to insult. The sensitivities of cerebellum and forebrain growth in rats were compared empirically. Data came from a large set constructed, some years ago, from measurements made of regional DNA contents. The animals had been raised from birth to various ages in small (well-fed) or large (underfed) litters. Growth curves were fitted, from which rate curves were derived, and these showed the depression in rate of DNA accumulation to be the same for each region, throughout suckling. The widely held extra-sensitivity of cerebellum is explained as an artifact of the % transformation, the timing of cerebellar growth, and the timing of restriction which experimenters usually choose. Problems of inferring uniformity, or otherwise, of growth rate depressions from incomplete, noisy, and badly sampled growth data are discussed and illustrated with computer simulations.

Critical periods in brain development.

The growth of the brain after organogenesis can be described as occurring in two somewhat overlapping phases: a phase of neuronal multiplication followed by one of glial proliferation, during and after which occur myelination and dendritic and axonal arborization. Within this gross chronology is a finer-grained chronology, with, for instance, different neuronal populations dividing at different times. The course of brain development can be affected by a variety of factors, the nature and extent of the perturbation dependent on the timing of the treatment with respect to stage of brain development. Growth processes completed before treatment are unaffected. Only those processes occurring at the time of the treatment are affected, plus some later-occurring processes, as a result of a cascade of effects. These concepts are examined briefly with reference to ionizing radiation, hormones and environmental stimulation and more fully with respect to nutrition. Undernutrition appears to depress the rate of all brain growth processes contemporaneous with it to the same extent. Whether the effects produced are likely to be permanent is discussed, together with the possibility that there may be mechanisms that attenuate or compensate for adverse effects.

Effects of maternal undernutrition and uterine artery ligation on placental lipase activities in the rat.

Lipase activities measured at pH 4 and 8 were determined in the placentas of rats subjected to undernutrition or uterine artery ligation. The fetal lipid content following maternal undernutrition was also determined. The placental lipase activity (per gram placenta) measured at pH 8 (placental lipoprotein lipase) remained unchanged, whilst the activity of the intracellular lipase (per gram placenta) measured at pH 4 was significantly less than in the control group (21% lower in maternal undernutrition and 16% lower after uterine artery ligation). Although placentas and fetuses were significantly lighter in both situations as compared to controls (well-nourished or sham operated, respectively), fetal lipid content in maternal undernutrition was only reduced to a degree concomitant with the decrease in fetal weight. We therefore conclude that the intracellular lipase may not have an important role in the provision of free fatty acids in undernutrition.

Results from a signal detection analysis approach to the study of lasting effects of early life undernutrition on the behaviour of rats.

Rats were undernourished from birth to 45 days of age, first by underfeeding their mothers (50% of ad libitum diet) and then, from weaning at 25 days, by feeding the pups a restricted diet. They were fed ad libitum from 45 days till the start of behavioural testing at 29 weeks. Eleven well-fed control (C) and 10 previously undernourished (PU) male rats were trained to discriminate a brief decrement in light intensity. A response during this period was rewarded with food (a 'hit'); a response during an equivalent period in the absence of the signal (a 'false alarm') was penalised by delaying the onset of the next signal. Both groups of rats learned an easy discrimination equally well, but when the discrimination was made more difficult the PU rats required longer test sessions that C rats, made both more hits and more false alarms and had a higher responsivity index, indicating that the PU rats were more highly motivated than the C rats under this condition. Session duration increased on the more difficult discrimination; consequently time of day of testing changed somewhat and more for some rats than others. This may have been a confounding factor on the difficult discrimination and was specifically investigated in a follow-up experiment with the same rats. The results suggest that time may have been a conditioned stimulus and that the influence of this factor differed between C and PU rats.

Evidence of non-maternally mediated acceleration of eye-opening in 'enriched' artificially reared rat pups.

Rat pups were artificially reared, without their mothers, from postnatal day 5. They were exposed thereafter to impoverished or enriched environmental conditions. The enrichment included gentling, social interaction and homing experience. Enriched rats showed accelerated eye-opening compared with their impoverished littermates (P less than 0.02). This is the first demonstration of stimulation-induced acceleration of development which cannot have been maternally mediated.

Growth and development of male and female rats treated with the Leydig cell cytotoxin ethane dimethane sulphonate during the suckling period.

The growth inhibiting effects of the Leydig cell cytotoxin ethane dimethane sulphonate (EDS) were investigated after treatment of both male and female neonatal rats. Pups were injected daily from day 4 to day 15 of age with EDS (50 mg/kg body weight sc), vehicle or water, or were not injected, according to a within-litter control experimental design. The rats were killed for necropsy at 40 and 80 days. The timing of attainment of the developmental milestones that appear during the suckling period was unaffected by treatment, but vaginal opening was 2 days later in EDS-treated females. After a delay of 9-10 days after the start of treatment the growth in body weight of both male and female EDS-treated rats was substantially and progressively depressed, such that by 80 days male and female EDS-treated rats showed body-weight deficits of 18 and 25%, respectively. The food intake of EDS-treated rats at 10 wk of age was less than that of controls, but was appropriate or more than appropriate for metabolic body size. As expected, testes were vestigial in EDS-treated males and the accessory male sexual organs were very small. Ovary and uterus weights were appropriate for body weight in EDS-treated females. At 80 days, liver weight was high for body weight in EDS-treated rats of both sexes. Possible mechanisms for the effect on body growth are discussed. Depressed food intake is discounted. Endocrine involvement, perhaps some anomaly of growth hormone release, or a more generalized toxic effect seem more likely.

Epidermal growth factor administered in pregnancy has little effect on placental, fetal and postnatal growth and development in rats.

Biosynthetic human epidermal growth factor (EGF) was injected daily into female rats (40 micrograms/rat/day) on days 14-20 of pregnancy in two experiments. Number in litter was significantly greater in EGF- than in vehicle-treated mothers in experiment 1 but not experiment 2. Number in litter affected fetal weight. When this factor was taken into account statistically, there was no significant effect of EGF treatment on fetal weight or placental weight, DNA and protein concentrations at 21 days of gestation; nor was birth weight affected by treatment. However, growth in body weight postnatally from 4 to 115 days was slightly but significantly depressed in the offspring of the EGF-treated mothers. A negative effect of additional EGF in pregnancy on subsequent milk production is postulated. The timing of attainment of developmental milestones in the offspring was not affected.

Vulnerability of developing brain to undernutrition.

Four questions are addressed: - 1. What are the immediate effects of undernutrition on developing brain likely to be and what are they? 2. Can any general statement be made about vulnerability of developing brain to undernutrition? 3. To what extent are the immediate effects recoverable on refeeding? 4. Do any attenuating or compensatory mechanisms operate? 1. IMMEDIATE EFFECTS: It is worth stating what is almost axiomatic, because it is often forgotten, that undernutrition is likely to affect only those processes which are contemporaneous with it (plus some that follow it). This is borne out by the available evidence. There appears to be little or no destruction of existing structures by undernutrition. 2. GENERAL STATEMENT ON IMMEDIATE EFFECTS: The novel general statement is examined that undernutrition affects the growth rate of all processes contemporaneous with it to the same extent. This is found to have considerable generality. The possible, though perhaps illusory exception of myelination is discussed. Uniform growth restriction gives rise to distorted brain growth through interaction of the timing of different brain growth processes with the timing of undernutrition. 3. EXTENT OF RECOVERY: Recovery is considered in terms of true catch-up: diminution in absolute deficit. The extent of recovery on refeeding varies from none, through partial, to complete, depending on the process under consideration and the timing and severity of the undernutrition. 4. POSSIBLE COMPENSATORY MECHANISMS: Intrinsic to the animal include altered cell cycle characteristics, delayed development, that all of these operate to a greater or lesser extent, during or after undernutrition, thus reducing the magnitude of effects and facilitating recovery.