Gonadotrophin-releasing hormone pulse generator activity in the hypothalamus of the goat.

Pulsatile release of gonadotrophin-releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.

In vivo results for interstitial laser application in thyroid gland.

OBJECTIVE: Aim of this study was to evaluate the potential of denaturation of hormone active tissue in the thyroid gland by laser induced interstitial thermotherapy (LITT) as a treatment of autonomous hyperthyroidism. MATERIALS AND METHODS: An interstitial thyroid laser application (Nd:YAG 1064 nm, 5W, 2 min) was performed in 5 pigs. During laser application, the laryngeal recurrent nerve was controlled electro-physiologically. Postoperatively, TSH, total T(3) (TT(3)) and free T(4) (FT(4)) were measured regularly. After a follow-up period of up to 6 weeks, pigs were sacrificed and the thyroid glands were evaluated histological. RESULTS: A malfunction of the nerve due to laser treatment was not detected. During the first postoperative week there was a decrease of both FT(4) and TSH whereas TT(3) showed an extreme decline of its plasma levels reaching nearly the detection limit. All values showed a recovery to their initial levels during an interval of 10 days and than increased to levels sometimes higher than baseline. The coagulation zones were demarcated clearly towards normal tissue with increasing fibrosis of the treated areas. CONCLUSION: Interstitial thyroid ablation using a Nd:YAG laser is a minimal invasive, safe and effective procedure. Further evaluation including long term follow-up in humans is needed to confirm these results.

KiSS-1 neurones are direct targets for leptin in the ob/ob mouse.

Leptin is an adipocyte-derived hormone that acts on the hypothalamus to influence feeding, metabolism and reproduction, but the cellular and molecular targets for the action of leptin in the brain have yet to be fully elucidated. Kisspeptins are encoded by the Kiss1 gene, which is expressed in the hypothalamus and has been implicated in the neuroendocrine regulation of gonadotrophin-releasing hormone secretion. We tested the hypothesis that kisspeptin-expressing neurones are targets for leptin. First, we examined whether leptin regulates the expression of Kiss1 by comparing levels of KiSS-1 mRNA in the arcuate nucleus among groups of mice having different circulating levels of leptin: (i) wild-type (WT); (ii) leptin-deficient ob/ob; and (iii) ob/ob mice treated with leptin. All mice were castrated to control for endogenous concentrations of gonadal steroids. KiSS-1 mRNA was significantly reduced in ob/ob compared to WT mice and levels of KiSS-1 mRNA in ob/ob mice treated with leptin were increased, but not fully restored to that found in WT animals. Second, we performed double-label in situ hybridisation for KiSS-1 mRNA and the leptin receptor (Ob-Rb) mRNA and found that almost one-half (approximately 40%) of KiSS-1 mRNA-expressing cells in the arcuate nucleus expressed Ob-Rb mRNA. These results demonstrate that KiSS-1 neurones are direct targets for regulation by leptin and suggest that the reproductive deficits associated with leptin-deficient states may be attributable, in part, to diminished expression of Kiss1.

A role for kisspeptins in the regulation of gonadotropin secretion in the mouse.

Kisspeptins are products of the KiSS-1 gene, which bind to a G protein-coupled receptor known as GPR54. Mutations or targeted disruptions in the GPR54 gene cause hypogonadotropic hypogonadism in humans and mice, suggesting that kisspeptin signaling may be important for the regulation of gonadotropin secretion. To examine the effects of kisspeptin-54 (metastin) and kisspeptin-10 (the biologically active C-terminal decapeptide) on gonadotropin secretion in the mouse, we administered the kisspeptins directly into the lateral cerebral ventricle of the brain and demonstrated that both peptides stimulate LH secretion. Further characterization of kisspeptin-54 demonstrated that it stimulated both LH and FSH secretion, at doses as low as 1 fmol; moreover, this effect was shown to be blocked by pretreatment with acyline, a potent GnRH antagonist. To learn more about the functional anatomy of kisspeptins, we mapped the distribution of KiSS-1 mRNA in the hypothalamus. We observed that KiSS-1 mRNA is expressed in areas of the hypothalamus implicated in the neuroendocrine regulation of gonadotropin secretion, including the anteroventral periventricular nucleus, the periventricular nucleus, and the arcuate nucleus. We conclude that kisspeptin-GPR54 signaling may be part of the hypothalamic circuitry that governs the hypothalamic secretion of GnRH.

Eccentric endurance training in subjects with coronary artery disease: a novel exercise paradigm in cardiac rehabilitation?

This study evaluated the effects of 8 weeks of eccentric endurance training (EET) in male subjects (age range 42-66 years) with coronary artery disease (CAD). EET was compared to concentric endurance training (CET) carried out at the same metabolic exercise intensity, three times per week for half an hour. CET ( n=6) was done on a conventional cycle ergometer and EET ( n=6) on a custom-built motor-driven ergometer. During the first 5 weeks of the training program the metabolic load was progressively increased to 60% of peak oxygen uptake in both groups. At this metabolic load, mechanical work rate achieved was 97 (8) W [mean (SE)] for CET and 338 (34) W for EET, respectively. Leg muscle mass was determined by dual-energy X-ray absorptiometry, quadriceps strength with an isokinetic dynamometer and muscle fibre composition of the vastus lateralis muscle with morphometry. The leg muscle mass increased significantly in both groups by some 3%. Strength parameters of knee extensors improved in EET only. Significant changes of +11 (4.9)%, +15 (3.2)% and +9 (2.5)% were reached for peak isometric torque and peak concentric torques at 60 degrees s(-1) and 120 degrees s(-1), respectively. Fibre size increased significantly by 19% in CET only. In conclusion, the present investigation showed that EET is feasible in middle-aged CAD patients and has functional advantages over CET by increasing muscle strength. Muscle mass increased similarly in both groups whereas muscle structural composition was differently affected by the respective training protocols. Potential limitations of this study are the cautiously chosen conditioning protocol and the restricted number of subjects.

Distribution and regulation of galanin-like peptide (GALP) in the hypothalamus of the mouse.

Galanin-like peptide (GALP) is a newly discovered molecule whose expression in the brain is confined to the arcuate nucleus and median eminence. In the rat, cellular levels of GALP mRNA are reduced by fasting and reversed by peripheral administration of leptin. The purpose of this investigation was 1) to clone and map the distribution of GALP mRNA in the brain of the mouse; 2) to compare the pattern and magnitude of GALP mRNA expression in the leptin-deficient obese (ob/ob) mouse with that of wild-type controls; and 3) to examine the effects of leptin delivered into the brain on the expression of GALP mRNA in the ob/ob mouse. We report the sequence of a mouse GALP cDNA and show that GALP mRNA is expressed in the arcuate nucleus, median eminence, infundibular stalk, and the neurohypophysis of this species. The expression of GALP mRNA in the brain was markedly reduced in the ob/ob mice, compared with wild-type animals. Intracerebroventricular infusion of leptin to ob/ob mice increased both the number of GALP mRNA-expressing neurons and their content of GALP mRNA, compared with vehicle-treated controls. These observations demonstrate that GALP mRNA is induced by leptin through a direct action on the brain.

A reassessment of leptin's role in triggering the onset of puberty in the rat and mouse.

Leptin is an adipocyte-derived hormone that has been implicated to serve as a metabolic signal to the reproductive axis. The role of leptin in pubertal maturation, however, has been a much-debated topic. We have previously reported that leptin serves as a permissive signal to the onset of puberty in the female rat. In an attempt to further understand the mechanics of leptin during pubertal maturation in rodent species, we had three experimental objectives: first, to describe the temporal relationship of leptin with development in the male and female rat; second, to seek evidence for an increase in responsiveness of the neuroendocrine axis to leptin by assessing for possible changes in leptin receptor expression during pubertal developmental in the female rat; and, third, to reevaluate the possible role of leptin as a permissive signal to the onset of puberty in the mouse. We found that serum leptin levels remain relatively constant during the prepubertal and postpubertal stages of both sexes. In addition, we could not detect any significant developmental changes in leptin receptor gene expression in the hypothalamus of the female rat. Lastly, we corroborated our findings in the female rat that leptin reversed the delay in pubertal maturation secondary to food restriction but did not advance the onset of puberty in female mice. Together, these results suggest that leptin is not a metabolic trigger for the onset of puberty in the rodent; instead, leptin is one of several permissive factors, whose presence may be necessary but alone is not sufficient to initiate sexual maturation in these species.

Dragon's Blood incense: misbranded as a drug of abuse?

An unknown red substance was being sold and used with other drugs of abuse in Virginia (often being used in conjunction with marihuana). The red substance was identified as Dragon's Blood incense from Daemonorops draco. In bioassays, Dragon's Blood incense exhibited a low, but measurable cytotoxicity in in vitro cell lines. Dragon's Blood incense or Volatilized Dragon's Blood had no adverse effect on mouse motor performance based on the inclined screen and rotorod tests. delta(9)-Tetrahydrocannibinol (THC) produced a dose-related decline in mouse performance on the rotorod test. The combination of Dragon's Blood incense or Volatilized Dragon's Blood with delta(9)-THC did not contribute further to the impairment of the mice on the rotorod. This data suggests that the abuse potential for Dragon's Blood incense alone or in combination with marihuana is minimal.

Effects of intrauterine and early postnatal growth restriction on hypothalamic somatostatin gene expression in the rat.

In the human, intrauterine growth retardation (IUGR) can result in persistent postnatal growth failure, which may be attributable, in part, to abnormal GH secretion. Whether putative alterations in GH secretion are the result of abnormalities intrinsic to the pituitary or reflect changes in the production of GH-releasing hormone or somatostatin (SS) is unknown. We tested the hypothesis that growth failure associated with IUGR or early postnatal food restriction (FR) is caused by a central defect in hypothalamic SS gene expression. Both models displayed persistent growth failure postnatally without any catch-up growth. We measured levels of SS mRNA levels in rats experimentally subjected to IUGR or FR. SS mRNA levels were measured by semiquantitative in situ hybridization throughout development. Levels of SS mRNA in the periventricular nucleus were significantly higher in both male and female IUGR rats in the juvenile and adult stages compared with matched controls (p < or = 0.05). FR was associated with higher SS mRNA levels only in neonatal female rats (p < or = 0.05). These results suggest that intrauterine malnutrition induces a persistent increase in the expression of SS mRNA in the periventricular nucleus, whereas early postnatal FR results in only a transient increase in SS gene expression. Because IGF-I levels were normal in juvenile IUGR and FR rats, central dysregulation of SS neurons does not appear to be the cause of early postnatal growth failure in either model. However, these observations are consistent with the hypothesis that nutritional stress at critical times during development can have persistent and potentially irreversible effects on organ function.

2-Methylbutyryl-coenzyme A dehydrogenase deficiency: a new inborn error of L-isoleucine metabolism.

An 4-mo-old male was found to have an isolated increase in 2-methylbutyrylglycine (2-MBG) and 2-methylbutyrylcamitine (2-MBC) in physiologic fluids. In vitro oxidation studies in cultured fibroblasts using 13C- and 14C-labeled branched chain amino acids indicated an isolated block in 2-methylbutyryl-CoA dehydrogenase (2-MBCDase). Western blotting revealed absence of 2-MBCDase protein in fibroblast extracts; DNA sequencing identified a single 778 C>T substitution in the 2-MBCDase coding region (778 C>T), substituting phenylalanine for leucine at amino acid 222 (L222F) and absence of enzyme activity for the 2-MBCDase protein expressed in Escherichia coli. Prenatal diagnosis in a subsequent pregnancy suggested an affected female fetus, supporting an autosomal recessive mode of inheritance. These data confirm the first documented case of isolated 2-MBCDase deficiency in humans.

Galanin-like peptide (GALP) is a target for regulation by leptin in the hypothalamus of the rat.

Galanin-like peptide (GALP), which was recently isolated from the porcine hypothalamus, shares sequence homology with galanin and binds with high affinity to galanin receptors. To study the distribution and regulation of GALP-expressing cells in the brain, we cloned a 120 base-pair cDNA fragment of rat GALP and produced an antisense riboprobe. In situ hybridization for GALP mRNA was then performed on tissue sections throughout the forebrain of adult ovariectomized female rats. We found GALP mRNA-containing cells in the arcuate nucleus (Arc), caudal dorsomedial nucleus, median eminence and the pituitary. Because GALP mRNA in the Arc appeared to overlap with the known distribution of leptin receptor mRNA, we tested the hypothesis that GALP expression is regulated by leptin. Using in situ hybridization, we compared the number of GALP mRNA-containing cells among groups of rats that were fed ad lib or fasted for 48 h and treated with either leptin or vehicle. Fasting reduced the number of identifiable cells containing GALP mRNA in the Arc, whereas the treatment of fasted animals with leptin produced a 4-fold increase in the number of cells expressing GALP message. The presence of GALP mRNA in the hypothalamus and pituitary and its regulation by leptin suggests that GALP may have important neuroendocrine functions, including the physiological regulation of feeding, metabolism, and reproduction.

Using cognitive strategies to enhance bladder control and comfort.

Compromised urinary bladder syndrome (CUBS), a combination of frequency and incontinence, causes multiple discomforts for community-dwelling adults. A holistic intervention--audiotaped cognitive strategies--was designed to augment the effects of an educational program designed to treat CUBS. CUBS was operationalized with a voiding diary, and comfort related to bladder health was operationalized in a questionnaire. In this quasi-experimental design the outcomes were measured at four time points. Repeated measures multivariate analyses of variance and nonparametric analyses were conducted to assess differences between the two groups. Results indicated that the treatment group had more comfort and improved CUBS compared with the control group.

Empirical evidence for the nature of holistic comfort.

The nursing outcome of holistic comfort encompasses physical, psychospiritual, social, and environmental aspects of human nature. The primary purpose of this study was to test four propositions about the nature of comfort: (a) Comfort has equal proportions of state and trait characteristics; (b) comfort is sensitive to changes over time; (c) when subjects are exposed to an effective intervention, they demonstrate differences in comfort that increase in a linear way compared to a control group; and (d) the whole (total comfort) is greater than the sum of its parts (relief plus ease plus transcendence). A secondary purpose of this study was to present preliminary concurrent validity between two types of comfort instruments, a traditional questionnaire with a Likert-type format and visual analog scales. Findings were positive for all theoretical propositions and moderate concurrent validity between the Radiation Therapy Comfort Questionnaire and the visual analog scale for total comfort was demonstrated.

Treatment of pyruvate carboxylase deficiency with high doses of citrate and aspartate.

A patient with severe pyruvate carboxylase deficiency presented at age 11 weeks with metabolic decompensation after routine immunization. She was comatose, had severe lactic acidemia (22 mM) and ketosis, low aspartate and glutamate, elevated citrulline and proline, and mild hyperammonemia. Head magnetic resonance imaging showed subdural hematomas and mild generalized brain atrophy. Biotin-unresponsive pyruvate carboxylase deficiency was diagnosed. To provide oxaloacetate, she was treated with high-dose citrate (7.5 mol/kg(-1)/day(-1)), aspartate (10 mmol/kg(-1)/day(-1)), and continuous drip feeding. Lactate and ketones diminished dramatically, and plasma amino acids normalized, except for arginine, which required supplementation. In the cerebrospinal fluid (CSF), glutamine remained low and lysine elevated, showing the treatment had not normalized brain chemistry. Metabolic decompensations, triggered by infections or fasting, diminished after the first year. They were characterized by severe lactic and ketoacidosis, hypernatremia, and a tendency to hypoglycemia. At age 3(1/2) years she has profound mental retardation, spasticity, and grand mal and myoclonic seizures only partially controlled by anticonvulsants. The new treatment regimen has helped maintain metabolic control, but the neurological outcome is still poor.

Component-resolved diagnosis (CRD) of type I allergy with recombinant grass and tree pollen allergens by skin testing.

The diagnosis of Type I allergy is based on the measurement of allergen-specific IgE antibodies and on provocation with allergens, most frequently conducted by skin testing. Both forms of diagnosis are currently performed with allergen extracts that are difficult to standardize regarding their allergen contents, and which contain additional undefined nonallergenic components. We report the expression in Escherichia coli and purification of some of the most relevant timothy grass- and birch pollen allergens. Recombinant timothy grass- (rPhl p 1, rPhl p 2, rPhl p 5) and birch pollen (rBet v 1, rBet v 2) allergens were purified and used for the measurement of allergen-specific IgE and IgG subclass responses as well as for skin prick testing in 55 pollen allergic patients and 10 nonatopic individuals. Results obtained showed that the recombinant allergens allowed in vivo allergy diagnosis in 52 of 54 of the grass pollen and in 35 of 36 of the birch pollen allergic patients. Positive skin reactions were observed almost exclusively in patients containing detectable allergen-specific IgE antibodies but not in the nonatopic group; however, sensitivity to a given allergen as measured by skin reactivity was weakly correlated with the levels of allergen-specific IgE. Our results demonstrate that recombinant allergens can be used for component-resolved skin test diagnosis (CRD) of the patients' allergen sensitization profile, whereas allergen extracts at best allow to identify allergen-containing sources. CRD may thus represent the basis for novel forms of patient-tailored immunotherapy.

Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob mice.

The hormone leptin acts on the brain to regulate feeding, metabolism, and reproduction; however, its cellular targets and molecular mechanisms of action remain to be fully elucidated. The melanocortins, which are derived from the precursor proopiomelanocortin (POMC), are also implicated in the physiological regulation of body weight. POMC-containing neurons express the leptin receptor, and thus it is conceivable that the POMC gene itself may be part of the signaling pathway involved in leptin's action on the brain. Using in situ hybridization and computerized image analysis, we tested the hypothesis that the POMC gene is a target for regulation by leptin by comparing cellular levels of POMC mRNA in the hypothalamus among groups of leptin-deficient (ob/ob) mice, leptin-treated ob/ob mice, and wild-type controls. POMC mRNA levels were significantly reduced throughout the arcuate nucleus in vehicle-treated ob/ob mice relative to wild-type controls, whereas POMC mRNA levels in leptin-treated ob/ob mice were indistinguishable from wild-type controls. These observations suggest that one or more products of POMC serve as an integrative link between leptin and the central mechanisms governing body weight regulation and reproduction.

Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus.

Leptin is a protein product of the obese (ob) gene, which is secreted by adipocytes and functions as a satiety factor to regulate food intake. The expression of the leptin receptor in several hypothalamic nuclei suggests that multiple neuronal subtypes are targets for leptin's action. Products of the proopiomelanocortin (POMC) gene are known to affect feeding behavior, and POMC neurons share a similar distribution with leptin receptor mRNA in the arcuate nucleus. We used double label in situ hybridization and computerized image analysis to test the hypothesis that POMC neurons coexpress the leptin receptor. Quantitative analysis confirmed that POMC neurons in the hypothalamus express leptin receptor mRNA. Based on this observation, we infer that POMC neurons and the products of the POMC gene may be part of the signaling pathway mediating leptin's action on feeding and perhaps other physiological functions.