Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

Energetics of byssus attachment and feeding in the green-lipped mussel Perna canaliculus.

In most animals, significant increases in metabolic rate are due to activity and to feeding (known as apparent specific dynamic action). We determined the energetic costs of activity and feeding in adult green-lipped mussels (Perna canaliculus). Maximal metabolic rate was determined, using closed-chamber respirometry, during byssus re-attachment, during specific dynamic action after 16 h of feeding with Isochrysis galbana, and for the two activities combined, in 23 mussels. Metabolic rate was significantly elevated above rest by about 1.9-fold during byssus attachment (17.1 ± 1.53 μg O(2) h(-1) g(-1) whole mussel wet weight at rest, increased to 27.9 ± 0.91 μg O(2) h(-1) g(-1)), and by 2.2-fold after feeding (31.4 ± 1.20 μg O(2) h(-1) g(-1)). Combined feeding and byssus attachment led to a still higher metabolic rate (34.0 ± 1.23 μg O(2) h(-1) g(-1)). Behavior was also significantly altered, with mussels being almost continuously open during attachment and after feeding (90%-99% of the time); however, the time spent open during the day decreased, reaching a minimum of 52% ± 9% 3 days after feeding, and remained low (67%-82%) for the following 45-day starvation period. Significant diurnal differences were observed, with mussels continuously (92%-100%) open at night. The key findings from this study are that green-lipped mussels (1) have an aerobic scope of approximately 2-fold; (2) reach a higher metabolic rate during feeding than during activity, and the two combined can raise the metabolic rate higher still; (3) display a marked diurnal behavior.

Does the choice of bottle nipple affect the oral feeding performance of very-low-birthweight (VLBW) infants?

BACKGROUND: There is a continuous debate regarding the best bottle nipple to be used to enhance the bottle-feeding performance of a preterm infant. Aim: To verify that feeding performance can be improved by using the bottle nipple with the physical characteristics that enhance infants' sucking skills. METHODS: Ten "healthy" VLBW infants (941+/-273 g) were recruited. Feeding performance was monitored at two time periods, when taking 1-2 and 6-8 oral feedings/d. At each time and within 24 h, performance was monitored using three different bottle nipples offered in a randomized order. Rate of milk transfer (ml/min) was the primary outcome measure. The sucking skills monitored comprised stage of sucking, suction amplitude, and duration of the generated negative intraoral suction pressure. RESULTS: At both times, infants demonstrated a similar rate of milk transfer among all three nipples. However, the stage of sucking, suction amplitude, and duration of the generated suction were significantly different between nipples at 1-2, but not 6-8 oral feedings/d.CONCLUSION: We did not identify a particular bottle nipple that enhanced bottle feeding in healthy VLBW infants. Based on the notion that afferent sensory feedback may allow infants to adapt to changing conditions, we speculate that infants can modify their sucking skills in order to maintain a rate of milk transfer that is appropriate with the level of suck-swallow-breathe coordination achieved at a particular time. Therefore, it is proposed that caretakers should be more concerned over monitoring the coordination of suck-swallow-breathe than over the selection of bottle nipples.

Maturation of oral feeding skills in preterm infants.

AIM: Safe and successful oral feeding requires proper maturation of sucking, swallowing and respiration. We hypothesized that oral feeding difficulties result from different temporal development of the musculatures implicated in these functions. METHODS: Sixteen medically stable preterm infants (26 to 29 weeks gestation, GA) were recruited. Specific feeding skills were monitored as indirect markers for the maturational process of oral feeding musculatures: rate of milk intake (mL/min); percent milk leakage (lip seal); sucking stage, rate (#/s) and suction/expression ratio; suction amplitude (mmHg), rate and slope (mmHg/s); sucking/swallowing ratio; percent occurrence of swallows at specific phases of respiration. Coefficients of variation (COV) were used as indices of functional stability. Infants, born at 26/27- and 28/29-week GA, were at similar postmenstrual ages (PMA) when taking 1-2 and 6-8 oral feedings per day. RESULTS: Over time, feeding efficiency and several skills improved, some decreased and others remained unchanged. Differences in COVs between the two GA groups demonstrated that, despite similar oral feeding outcomes, maturation levels of certain skills differed. CONCLUSIONS: Components of sucking, swallowing, respiration and their coordinated activity matured at different times and rates. Differences in functional stability of particular outcomes confirm that maturation levels depend on infants' gestational rather than PMA.

In vitro analog of classical conditioning of feeding behavior in aplysia.

The feeding behavior of Aplysia californica can be classically conditioned using tactile stimulation of the lips as a conditioned stimulus (CS) and food as an unconditioned stimulus (US). Moreover, several neural correlates of classical conditioning have been identified. The present study extended previous work by developing an in vitro analog of classical conditioning and by investigating pairing-specific changes in neuronal and synaptic properties. The preparation consisted of the isolated cerebral and buccal ganglia. Electrical stimulation of a lip nerve (AT4) and a branch of the esophageal nerve (En2) served as the CS and US, respectively. Three protocols were used: paired, unpaired, and US alone. Only the paired protocol produced a significant increase in CS-evoked fictive feeding. At the cellular level, classical conditioning enhanced the magnitude of the CS-evoked synaptic input to pattern-initiating neuron B31/32. In addition, paired training enhanced both the magnitude of the CS-evoked synaptic input and the CS-evoked spike activity in command-like neuron CBI-2. The in vitro analog of classical conditioning reproduced all of the cellular changes that previously were identified following behavioral conditioning and has led to the identification of several new learning-related neural changes. In addition, the pairing-specific enhancement of the CS response in CBI-2 indicates that some aspects of associative plasticity may occur at the level of the cerebral sensory neurons.

Effectiveness of an early postoperative feeding protocol

Background. The increasing emphasis on medical outcomes and cost containment has made it imperative to identify patient populations in which aggressive nutritional care can improve quality of care. The aim of this prospective study was to implement a standardized early jejunal feeding protocol for patients undergoing small and large bowel resection, and to evaluate its effect on patient outcome and cost.^ Methods. Treatment patients (n = 81) who met protocol inclusion criteria had a jejunal feeding tube inserted at the time of surgery. Feeding was initiated at 10 cc/hour within 12 hours after bowel resection and progressed if hemodynamically stable. The control group (n = 159) received usual care. Outcome measures included postoperative length of stay, total direct cost, nosocomial infection rate and health status (SF-36) scores.^ Results. By postoperative day 4, the use of total parenteral nutrition (TPN) was significantly greater in the control group compared to the treatment group; however, total nutritional intake was significantly less. Multiple regression analysis indicated an increased likelihood of infection with the use of TPN. A reduction of 3.5 postoperative days (p =.013) with 4.3 fewer TPN days per patient (p =.001) and a 9.6% reduction in infection rate (p =.042) was demonstrated in the treatment group. There was no difference in health status scores between groups at discharge and 3 months post-discharge.^ Conclusion. These positive outcomes and an average total cost savings of $4,145 per treatment patient indicate that the treatment protocol was effective. ^

Effect of continuous gastric enteral feeding on tonometric measurement of gastric intramucosal carbon dioxide levels in healthy volunteers

Purpose. The aim of this research was to evaluate the effect of enteral feeding on tonometric measurement of gastric regional carbon dioxide levels (PrCO2) in normal healthy volunteers. Design and methods. The sample included 12 healthy volunteers recruited by the University Clinical Research Center (UCRC). An air tonometry system monitored PrCO2 levels using a tonometer placed in the lumen of the stomach via orogastric intubation. PrCO2 was automatically measured and recorded every 10 minutes throughout the five hour study period. An oral dose of famotidine 40 mg was self-administered the evening prior to and the morning of the study. Instillation of Isocal® High Nitrogen (HN) was used for enteral feeding in hourly escalating doses of 0, 40, 60, and 80 ml/hr with no feeding during the fifth hour. Results . PrCO2 measurements at time 0 and 10 minutes (41.4 ± 6.5 and 41.8 ± 5.7, respectively) demonstrated biologic precision (Levene's Test statistic = 0.085, p-value 0.774). Biologic precision was lost between T130 and T140 40 when compared to baseline TO (Levene's Test statistic = 1.70, p-value 0.205; and 3.205, p-value 0.042, respectively) and returned to non-significant levels between T270 and T280 (Levene's Test statistic = 3.083, p-value 0.043; and 2.307, p-value 0.143, respectively). Isocal® HN significantly affected the biologic accuracy of PrCO2 measurements (repeated measures ANOVA F 4.91, p-value <0.001). After 20 minutes of enteral feeding at 40 ml/hr, PrCO2 significantly increased (41.4 ± 6.5 to 46.6 ± 4.25, F = 5.4, p-value 0.029). Maximum variance from baseline (41.4 ± 6.5 to 61.3 ± 15.2, F = 17.22, p-value <0.001) was noted after 30 minutes of Isocal® HN at 80 ml/hr or 210 minutes from baseline. The significant elevations in PrCO2 continued throughout the study. Sixty minutes after discontinuation of enteral feeding, PrCO2 remained significantly elevated from baseline (41.4 ± 6.5 to 51.8 ± 9.2, F = 10.15, p-value 0.004). Conclusion. Enteral feeding with Isocal® HN significantly affects the precision and accuracy of PrCO2 measurements in healthy volunteers. ^

ANALYSIS OF SEROTONERGIC MODULATION OF NEURONS INVOLVED IN TWO DEFENSIVE BEHAVIORS IN APLYSIA CALIFORNICA (CYCLIC-AMP, AROUSAL, CALCIUM, POTASSIUM)

Sensitization is a simple form of learning which refers to an enhancement of a behavioral response resulting from an exposure to a novel stimulus. While sensitization is found throughout the animal world, little is known regarding the underlying neural mechanisms. By taking advantage of the simple nervous system of the marine mollusc Aplysia, I have begun to examine the cellular and molecular mechanisms underlying this simple form of learning. In an attempt to determine the generality of the mechanisms of neuromodulation underlying sensitization, I have investigated and compared the modulation of neurons involved in two defensive behaviors in Aplysia, the defensive inking response and defensive tail withdrawal.^ The motor neurons that produce the defensive release of ink receive a slow decreased conductance excitatory postsynaptic potential (EPSP) in response to sensitizing stimuli. Using electrophysiological techniques, it was found that serotonin (5-HT) mimicked the physiologically produced slow EPSP. 5-HT produced its response through a reduction in a voltage-independent conductance to K('+). The 5-HT sensitive K('+) conductance of the ink motor neurons was separate from the fast K('+), delayed K('+), and Ca('2+)-activated K('+) conductances found in these and other molluscan neurons. 5-HT was shown to produce a decrease in K('+) conductance in the ink motor neurons through an elevation of cellular cAMP.^ The mechanosensory neurons that participate in the defensive tail withdrawal response are also modulated by sensitizing stimuli through the action of 5-HT. Using electrophysiological techniques, it was found that 5-HT modulated the tail sensory neurons through a reduction in a voltage-dependent conductance to K('+). The serotonin-sensitive K('+) conductance was found to be largely a Ca('2+)-activated K('+) conductance. Much like the ink motor neurons, 5-HT produced its modulation through an elevation of cellular cAMP. While the actual K('+) conductance modulated by 5-HT in these two classes of neurons differs, the following generalizations can be made: (1) the effects of sensitizing stimuli are mimicked by 5-HT, (2) 5-HT produces its effect through an elevation of cellular cAMP, and (3) the conductance to K('+) is modulated by 5-HT. ^

BIOCHEMICAL PROPERTIES OF GABA (B) RECEPTORS (BACLOFEN, ADENYLATE CYCLASE, CYCLIC-AMP, PHOSPHOLIPASE, PROTEIN KINASE C)

(gamma)-Aminobutyric acid (GABA), a neurotransmitter in the mammalian central nervous system, influences neuronal activity by interacting with at least two pharmacologically and functionally distinct receptors. GABA(,A) receptors are sensitive to blockade by bicuculline, are associated with benzodiazepine and barbiturate binding sites, and mediate chloride flux. The biochemical and pharmacolocal properties of GABA(,B) receptors, which are stereoselectively activated by (beta)-p-chlorophenyl GABA (baclofen), are less well understood. The aim of this study was to define these features of GABA(,B) receptors, with particular emphasis on their possible relationship to the adenylate cyclase system in brain.^ By themselves, GABA agonists have no effect on cAMP accumulation in rat brain slices. However, some GABA agonists markedly enhance the cAMP accumulation that results from exposure to norepinephrine, adenosine, VIP, and cholera toxin. Evidence that this response is mediated by the GABA(,B) system is provided by the finding that it is bicuculline-insensitive, and by the fact that only those agents that interact with GABA(,B) binding sites are active in this regard. GABA(,B) agonists are able to enhance neurotransmitter-stimulated cAMP accumulation in only certain brain regions, and the response is not influenced by phosphodiesterase inhibitors, although is totally dependent on the availability of extracellular calcium. Furthermore, data suggest that inhibition of phospholipase A(,2), a calcium-dependent enzyme, decreases the augmenting response to baclofen, although inhibitors of arachidonic acid metabolism are without effect. These findings indicate that either arachidonic acid or lysophospholipid, products of PLA(,2)-mediated degradation of phospholipids, mediates the augmentation. Moreover, phorbol esters, compounds which directly activate protein kinase C, were also found to enhance neurotransmitter-stimulated cAMP accumulation in rat brain slices. Since this enzyme is known to be stimulated by unsaturated fatty acids such as arachidonate, it is proposed that GABA(,B) agonists enhance cAMP accumulation by fostering the production of arachidonic acid which stimulates protein kinase C, leading to the phosphorylation of some component of the adenylate cyclase system. Thus, GABA, through an interaction with GABA(,B) receptors, modulates neurotransmitter receptor responsiveness in brain. The pharmocological manipulation of this response could lead to the development of therapeutic agents having a more subtle influence than current drugs on central nervous system function. ^

Small-Scale Features of Marine Sediments and Their Importance to the Study of Deposit-Feeding

Techniques currently in use by sedimentologists for the study of marine sedment microfabric are of limited use for understandmg the relationship between sediment organic matter and mineral grains. In this article it is shown that by combining standard histological protocols for fixation and dehydration with petrological protocols for resin embedding and thin sectioning, very fine details of the sediment structure can be seen. Because of the ubiquitous presence of the organic matrix, organicmineral aggregates are not seen in situ. Other features of the sediment of importance to deposit-feeders, such as the presence of intact chloroplasts, can be observed through the use of epifluorescence illumination, while partially crossed polarizers help to delimit the grain boundaries. It is suggested that if these procedures can be combined with histological staining techniques, it may be possible to determine the potential food value of sedment on a scale equivalent to that perceived by infaunal deposit-feeders.

Feeding mastitis milk to organic dairy calves: effect on health and performance during suckling and on udder health at first calving

BackgroundInfection pathways of S. aureus udder infections in heifers are still not well understood. One hypothesis is that calves become infected with S. aureus via feeding mastitis milk. Especially on small-scale farms, pasteurisers are not economic. The purpose of this randomised comparative study was to investigate the influence of feeding milk containing S. aureus genotype B (SAGTB) on the health and development of calves and udder health of the respective heifers. Additionally, a method reducing the bacterial load to obtain safer feeding milk was tested. Thirty-four calves were fed mastitis milk from cows with subclinical SAGTB mastitis. One group was fed untreated milk (UMG). For the other group, milk was thermised at 61°C for one minute (heat treated milk group¿=¿HMG). After weaning, calves were followed up until first calving. A milk sample of these heifers was taken at first milking to compare udder health of both groups.ResultsThermisation of milk led to an effective reduction of S. aureus in the feeding milk. 78% of the analysed pools were free of S. aureus, a reduction of at least one log was obtained in the other pools.Quarter milk samples revealed that two heifers had a S. aureus intramammary infection, but caused by a genotype different from genotype B.During the suckling period, the UMG had a significantly higher incidence rate of 1.09 diarrhoea cases per 100 calf days at risk compared to 0.26 cases per 100 calf days in the HMG (p¿<¿0.05).ConclusionsUnder the conditions of this study, no effects of feeding milk containing SAGTB on udder health after first calving were observed. But a power analysis indicated that the sample size in the current setup is insufficient to allow for assessment on mastitis risk after SAGTB exposition, as a minimal number of 4 calves infected (vs. 0 in the HMG) would have shown significant effects. High bacterial load, however, was associated with an increased incidence rate of diarrhoea. Thus, thermisation as a minimal preventive measure before feeding mastitis milk to calves might be beneficial for maintaining calf health.