Analysis of ß-Subunit-dependent GABAA Receptor Modulation and Behavioral Effects of Valerenic Acid Derivatives.

Valerenic acid (VA)-a ß2/3-selective GABA type A (GABAA) receptor modulator-displays anxiolytic and anticonvulsive effects in mice devoid of sedation, making VA an interesting drug candidate. Here we analyzed ß-subunit-dependent enhancement of GABA-induced chloride currents (IGABA) by a library of VA derivatives and studied their effects on pentylenetetrazole (PTZ)-induced seizure threshold and locomotion. Compound-induced IGABA enhancement was determined in oocytes expressing α1ß1γ2S, α1ß2γ2S, or α1ß3γ2S receptors. Effects on seizure threshold and locomotion were studied using C57BL/6N mice and compared with saline-treated controls. ß2/3-selective VA derivatives such as VA-amide (VA-A) modulating α1ß3γ2S (VA-A: Emax = 972 ± 69%, n = 6, P < 0.05) and α1ß2γ2S receptors (Emax = 1119 ± 72%, n = 6, P < 0.05) more efficaciously than VA (α1ß3γ2S: VA: Emax = 632 ± 88%, n = 9 versus α1ß2γ2S: VA: Emax = 721 ± 68%, n = 6) displayed significantly more pronounced seizure threshold elevation than VA (saline control: 40.4 ± 1.4 mg/kg PTZ versus VA 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ versus VA-A 3 mg/kg: 57.9 ± 1.9 mg/kg PTZ, P < 0.05). Similarly, VA's methylamide (VA-MA) enhancing IGABA through ß3-containing receptors more efficaciously than VA (Emax = 1043 ± 57%, P < 0.01, n = 6) displayed stronger anticonvulsive effects. Increased potency of IGABA enhancement and anticonvulsive effects at lower doses compared with VA were observed for VA-tetrazole (α1ß3γ2S: VA-TET: EC50 = 6.0 ± 1.0 µM, P < 0.05; VA-TET: 0.3 mg/kg: 47.3 ± 0.5 mg/kg PTZ versus VA: 10 mg/kg: 49.0 ± 1.8 mg/kg PTZ, P < 0.05). At higher doses (≥10 mg/kg), VA-A, VA-MA, and VA-TET reduced locomotion. In contrast, unselective VA derivatives induced anticonvulsive effects only at high doses (30 mg/kg) or did not display any behavioral effects. Our data indicate that the ß2/3-selective compounds VA-A, VA-MA, and VA-TET induce anticonvulsive effects at low doses (≤10 mg/kg), whereas impairment of locomotion was observed at doses ≥10 mg/kg.

Identification of the putative binding pocket of valerenic acid on GABAA receptors using docking studies and site-directed mutagenesis.

BACKGROUND AND PURPOSE: ß2/3-subunit-selective modulation of GABAA receptors by valerenic acid (VA) is determined by the presence of transmembrane residue ß2/3N265. Currently, it is not known whether ß2/3N265 is part of VA's binding pocket or is involved in the transduction pathway of VA's action. The aim of this study was to clarify the localization of VA's binding pocket on GABAA receptors. EXPERIMENTAL APPROACH: Docking and a structure-based three-dimensional pharmacophore were employed to identify candidate amino acid residues that are likely to interact with VA. Selected amino acid residues were mutated, and VA-induced modulation of the resulting GABAA receptors expressed in Xenopus oocytes was analysed. KEY RESULTS: A binding pocket for VA at the ß(+) /α(-) interface encompassing amino acid ß3N265 was predicted. Mutational analysis of suggested amino acid residues revealed a complete loss of VA's activity on ß3M286W channels as well as significantly decreased efficacy and potency of VA on ß3N265S and ß3F289S receptors. In addition, reduced efficacy of VA-induced IGABA enhancement was also observed for α1M235W, ß3R269A and ß3M286A constructs. CONCLUSIONS AND IMPLICATIONS: Our data suggest that amino acid residues ß3N265, ß3F289, ß3M286, ß3R269 in the ß3 subunit, at or near the etomidate/propofol binding site(s), form part of a VA binding pocket. The identification of the binding pocket for VA is essential for elucidating its pharmacological effects and might also help to develop new selective GABAA receptor ligands.

GATA3 inhibits lysyl oxidase-mediated metastases of human basal triple-negative breast cancer cells.

Discovery of mechanisms that impede the aggressive and metastatic phenotype of human basal triple-negative-type breast cancers (BTNBCs) could provide novel targets for therapy for this form of breast cancer that has a relatively poor prognosis. Previous studies have demonstrated that expression of GATA3, the master transcriptional regulator of mammary luminal differentiation, can reduce the tumorigenicity and metastatic propensity of the human BTNBC MDA-MB-231 cell line (MB231), although the mechanism for reduced metastases was not elucidated. We demonstrate through gene expression profiling that GATA3 expression in 231 cells resulted in the dramatic reduction in the expression of lysyl oxidase (LOX), a metastasis-promoting, matrix-remodeling protein, in part, through methylation of the LOX promoter. Suppression of LOX expression by GATA3 was further confirmed in the BTNBC Hs578T cell line. Conversely, reduction of GATA3 expression by small interfering RNA in luminal BT474 cells increased LOX expression. Reconstitution of LOX expression in 231-GATA3 cells restored metastatic propensity. A strong inverse association between LOX and GATA3 expression was confirmed in a panel of 51 human breast cancer cell lines. Similarly, human breast cancer microarray data demonstrated that high LOX/low GATA3 expression is associated with the BTNBC subtype of breast cancer and poor patient prognosis. Expression of GATA3 reprograms BTNBCs to a less aggressive phenotype and inhibits a major mechanism of metastasis through inhibition of LOX. Induction of GATA3 in BTNBC cells or novel approaches that inhibit LOX expression or activity could be important strategies for treating BTNBCs.

The contractile deceleration point: an important physiologic landmark on oesophageal pressure topography.

BACKGROUND: This study aimed to correlate oesophageal bolus transit with features of oesophageal pressure topography (OPT) plots and establish OPT metrics for accurately measuring peristaltic velocity. METHODS: About 18 subjects underwent concurrent OPT and fluoroscopy studies. The deglutitive Contractile Front Velocity (CFV) in OPT plots was subdivided into an initial fast phase (CFV(fast)) and subsequent slow phase (CFV(slow)) separated by a user-defined deceleration point (CDP). Fluoroscopy studies were analyzed for the transition from the initial rapidly propagated luminal closure associated with peristalsis to slow bolus clearance characteristic of phrenic ampullary emptying and to identify the pressure sensors at the closure front and at the hiatus. Oesophageal pressure topography measures were correlated with fluoroscopic milestones of bolus transit. Oesophageal pressure topography studies from another 68 volunteers were utilized to develop normative ranges for CFV(fast) and CFV(slow). KEY RESULTS: A distinct change in velocity could be determined in all 36 barium swallows with the fast and slow contractile segments having a median velocity of 4.2 cm s(-1) and 1.0 cm s(-1), respectively. The CDP noted on OPT correlated closely with formation of the phrenic ampulla making CFV(fast) (mean 5.1 cm s(-1)) correspond closely to peristaltic propagation and CFV(slow) (mean 1.7 cm s(-1)) to ampullary emptying. CONCLUSIONS & INFERENCES: The deceleration point in the CFV on OPT plots accurately demarcated the early region in which the CFV reflects peristaltic velocity (CFV(fast)) from the later region where it reflects the progression of ampullary emptying (CFV(slow)). These distinctions should help objectify definitions of disordered peristalsis, especially spasm, and improve understanding of impaired bolus transit across the oesophagogastric junction.

Ventilation, sensible heat loss, broiler energy, and water balance under harsh environmental conditions.

Air velocity (AV) is one of the main environmental factors involved in thermoregulation, especially at high ambient temperatures. To elucidate the effect of AV on performance and thermoregulation of 4- to 7-wk-old broiler chickens, an experiment was conducted using 4 different AV (0.8, 1.5, 2.0, and 3.0 m/s) at constant ambient temperatature (35 +/- 1.0 degrees C) and RH (60 +/- 2.5%). BW, feed intake, and fecal and urinary excretions were monitored in individuals and were used to calculate the amount of energy expended for maintenance. Infrared thermal imaging radiometry was used to measure surface temperatures for the calculation of heat loss by radiation and convection. Brachial vein blood was collected for plasma osmolality and arginine vasotocin analysis. Broilers performed optimally at an AV of 2.0 m/s. Energy expenditure for maintenance was significantly higher under these conditions, suggesting the ability to direct a sufficient amount of energy to control body temperature, while maintaining relatively high growth rates. Convective heat loss increased significantly with increasing AV, whereas radiative heat loss was not affected. Sensible heat loss, expressed as a percentage of energy expenditure for maintenance, was significantly higher at 2.0 m/s compared with 0.8 m/s but significantly lower than that of 3.0 m/s. The high level of heat loss observed at 3.0 m/s probably affected body water balance, as supported by significantly higher plasma osmolality, arginine vasotocin concentration, and the hyperthermic status of these birds. It can be concluded that AV of 2.0 m/s enables broilers to maintain proper performance together with efficient thermoregulation and water balance under harsh environmental conditions.

Erythropoiesis regulation during the development of ascites syndrome in broiler chickens: a possible role of corticosterone.

The ascites syndrome in broiler chickens is attributed to metabolic burdening, which results from intensive genetic selection for rapid growth coupled with exposure to extreme environmental conditions, such as low ambient temperature. These conditions impose on the broilers difficulties in fulfilling tissue demands for oxygen, and the birds exhibit a decrease in blood oxygen saturation and high hematocrit values. It is unknown whether the increase in hematocrit results from alteration in erythropoiesis or from fluid exudation out of the blood system to the abdominal cavity. The present study was conducted to examine the association between abnormal stress response and erythropoiesis process in ascitic broilers. Ascitic chickens revealed a uniquely continuous stress response: expressing an increase (P < or = 0.05) in plasma corticosterone concentration 2 to 3 wk before death. At 5 wk of age, ascitic broilers exhibited an increase (P < 0.05) in hematocrit, blood cell count, and packed cells and blood volumes, with no significant change in plasma volume. These results confirm an accelerated erythropoiesis process in ascitic birds. Increased blood cell production in ascitic birds was matched by an increase (P < 0.05) in the proportion of immature red blood cells (23%) in comparison with broilers that remained healthy (7%), and by decreased (P < 0.05) hemoglobin content relative to red blood cells. We conclude that continually increased corticosterone concentrations, as an inducer of erythropoiesis proliferation and differentiation arrest, in ascitic chickens, resulted in increased production of red blood cells (partially immature) with decreased hemoglobin content; this decrease in hemoglobin might have contributed to enhanced development of hypoxemia and to aggravation of the syndrome.

Association between weight gain, blood parameters, and thyroid hormones and the development of ascites syndrome in broiler chickens.

The present study examined the association between thyroid hormones and the development of ascites on one hand and the ability to predict ascites from growth rate and hematocrit on the other hand. Ascites syndrome was induced in broiler chickens in two trials by exposing the chicks to low ambient temperature (Ta) and by supplying a pellet form of diet. Weight gain, hematocrit, hemoglobin, and plasma thyroxin (T4) and triiodothyronine (T3) concentrations were measured weekly for each bird, and comparisons were made between birds that eventually died from ascites and those that did not. Mortality from ascites amounted to 24.3 and 24.2% in Trials 1 and 2, respectively. Weight gain did not differ between ascitic and healthy chickens up to approximately 2 wk before death but was significantly lower in the ascitic broilers 1 to 2 wk before death. Hematocrit was significantly higher in broilers with ascites with the exception of ascitic broilers that died at the age of 7 wk (Trial 1). In ascitic broilers, T4 and T3 concentrations declined significantly during the week of death. The present findings raise the question of whether the association between low levels of thyroid hormones and the development of ascites is one of the physiological responses in the syndrome cascade, or whether the failure to maintain thyroid hormones concentration is one of the triggers of the syndrome initiation. This question requires further investigation. It can be concluded that a high rate of weight gain is not always a good predictor of ascites development. Hematocrit and thyroid hormones can provide a good indication but only during the last week of life, and not in all cases. None of these parameters, however, can predict the development of ascites at an early age.

Thermoregulation in naked neck chickens subjected to different ambient temperatures.

1. Heterozygous (Na/na) naked neck chickens and their normally feathered (na/na) sibs, were exposed to constant ambient temperatures (Ta) ranging between 15 and 35 degrees C and 12h: 12h diurnal high:low temperatures of 15 degrees C:35 degrees C. 2. No significant effect of genotype was obtained in weight gain and food intake. However, the naked neck birds tended to gain somewhat more weight at high Ta and consume more food at low Ta. 3. At 35 degrees C Na birds showed better regulation of body temperature (Tb) and demonstrated considerably higher radiation from the neck. 4. The greater food intake of the naked neck chickens at 15 degrees C was associated with significantly higher packed cell volume, haemoglobin concentration, heart and liver size. These appear to involve both higher heat production and haemodynamic changes to accommodate the higher oxygen demands of the naked neck chickens at low Ta. 5. The results indicate the ability of the naked neck chickens, on the one hand to thermoregulate at low Tas and, on the other their slightly better capacity to maintain Tb at high Tas. However, no genotype advantage was obtained under diurnal cyclic temperature conditions.

Smoke out.

This year the EPA will finalize rules that will make medical waste incineration nearly a thing of the past. The author reports on an array of alternative approaches.

Physician retraining in primary care.

Part of the health care provider restructuring consists of physician specialists retraining in primary care. The need is there, with additional pressures for qualified "gatekeepers" coming from the increase in managed care contracting.

The mglB sequence of Salmonella typhimurium LT2; promoter analysis by gene fusions and evidence for a divergently oriented gene coding for the mgl repressor.

The mglB gene of Salmonella typhimurium LT2 coding for the galactose-binding protein (GBP) was sequenced. We compared the deduced amino acid sequence with the GBP sequence of Escherichia coli K 12. The mature proteins differ in only 19 of 309 amino acid residues, corresponding to 94% homology. Analysis of the mglB control region by promoter-probe vectors revealed that two promoters, P1 and P2, constitute the mgl control region (Pmgl). P1 and P2 function in a synergistic way. P1 is the main promoter of the operon; its activity is 20 times the activity of P2. Both promoters are activated by the cyclic adenosine monophosphate catabolite activator protein (cAMP/CAP) complex. While P1 is inactive in the absence of the cAMP/CAP complex, there is residual activity of P2 under these conditions. Studies on the inducibility of the mglBAEC operon using multicopy plasmid promoter-probe vectors were hampered by the titration of the mgl repressor resulting in a partially constitutive expression of the mgl operon. The results indicate that only P1 is responding to induction by D-fucose. A weak promoter, PD, within the P1 region but divergent to it was found. PD is neither stimulated by the cAMP/CAP complex nor by D-fucose. We cloned the gene located downstream to PD and found it to strongly repress the expression of the mgl operon. We termed this gene mglD. The presence of D-fucose abolished the repression caused by the plasmid-encoded mglD gene product.

The use of deep frozen, stored bovine blood for in vitro feeding of tsetse flies.

A method of blood preparation is described which resulted in the successful rearing of Glossina p. palpalis with in vitro feeding. Cells of defibrinated bovine blood were washed in 0.89% NaCl solution prior to deep-freezing. Cells and serum were stored (--28 degrees C) for up to four months. For feeding, both components were mixed in the volumetric proportion 1 : 1. Adenosine triphosphate was added at a concentration of 10-3 M to stimulate uptake of blood. Survival rate and mean weight of puparia remained constant over three generations while productivity increased.

In vitro feeding in the rearing of tsetse flies (Glossina m. morsitans and G.p. palpalis, Diptera: Glossinidae).

The increasing demand for laboratory reared tsetse flies for research and biological control makes it necessary to develop effective and standardized tsetse fly feeding methods without using live animals for the daily blood uptake. The in vitro feeding technique, described in this paper, has been used for rearing G. m. morsitans by feeding them defibrinated equine blood through a silicone membrane. The results obtained for female longevity and productivity and mean weight of puparia are satisfactory. However, feeding defibrinated bovine blood results in significantly lighter puparia. A colony of G. p. palpalis feeding on defibrinated bovine blood is the only colony of this species that has been successfully maintained by in vitro feeding over several years. The survival rate of the females being fed defibrinated bovine, equine or porcine blood is equal. The number of larvae produced by females being fed defibrinated equine blood is significantly lower. Females younger than 50 days produce larvae which form a heavier puparia than females aged between 51 to 80 or 100 days, irrespective of blood source. Bovine blood used within the first 3 days after its collection leads to significantly higher mean weights of puparia than bovine blood used therafter. The increasing degree of haemolysis is most probably not the reason for this observation. A colony production model based on the performance of both species, G. m. morsitans and G. p. palpalis, fed in vitro, shows the importance of the first five age group periods (i.e. 45 to 50 days after emergence) for the overall performance of the flies. According to the results obtained, about 2,3 puparia per female are needed to maintain the same number of females in the colony. This level of production is reached in the fifth age group period. All larvae produced thereafter are available for colony expansion or experimental purposes. Rearing of both species with in vitro feeding is now a matter of routine.