High-calcium diet modulates effects of long-term prolactin exposure on the cortical bone calcium content in ovariectomized rats

High physiological prolactin induced positive calcium balance by stimulating intestinal calcium absorption, reducing renal calcium excretion, and increasing bone calcium deposition in female rats. Although prolactin-induced increase in trabecular bone calcium deposition was absent after ovariectomy, its effects on cortical bones were still controversial. The present investigation, therefore, aimed to study the effect of in vivo long-term high physiological prolactin induced by either anterior pituitary (AP) transplantation or 2.5 mg/kg prolactin injection on cortical bones in ovariectomized rats. Since the presence of prolactin receptors (PRLR) in different bones of normal adult rats has not been reported, we first determined mRNA expression of both short- and long-form PRLRs at the cortical sites (tibia and femur) and trabecular sites (calvaria and vertebrae) by using the RT-PCR. Our results showed the mRNA expression of both PRLR isoforms with predominant long form at all sites. However, high prolactin levels induced by AP transplantation in normal rats did not have any effect on the femoral bone mineral density or bone mineral content. By using (45)Ca kinetic study, 2.5 mg/kg prolactin did not alter bone formation, bone resorption, calcium deposition, and total calcium content in tibia and femur of adult ovariectomized rats. AP transplantation also had no effect on the cortical total calcium content in adult ovariectomized rats. Because previous work showed that the effects of prolactin were age dependent and could be modulated by high-calcium diet, interactions between prolactin and these two parameters were investigated. The results demonstrated that 2.0% wt/wt high-calcium diet significantly increased the tibial total calcium content in 9-wk-old young AP-grafted ovariectomized rats but decreased the tibial total calcium content in 22-wk-old adult rats. As for the vertebrae, the total calcium contents in both young and adult rats were not changed by high-calcium diet. The present results thus indicated that the adult cortical bones were potentially direct targets of prolactin. Moreover, the effects of high physiological prolactin on cortical bones were age dependent and were observed only under the modulation of high-calcium diet condition.     

N-terminal region of chitinase I of Bacillus circulans KA-304 contained new chitin-biding domain

Chitinase I (CHI1) of Bacillus circulans KA-304 forms protoplasts from Schizophyllum commune mycelia when the enzyme is combined with α-1,3-glucanase of B. circulans KA-304. CHI1 consists of an N-terminal unknown region and a C-terminal catalytic region classified into the glycoside hydrolase family-19 type. An N-terminal region-truncated mutant of CHI 1 (CatCHI1), which was expressed in Escherichia coli Rosetta-gami B (DE3), lost colloidal chitin- and powder chitin-binding activities. The colloidal chitin- and the powder chitin-hydrolyzing activities of CatCHI1 were lower than those of CHI1, and CatCHI1 was not effective in forming the protoplast. A fusion protein of the N-terminal region of CHI1 and green fluorescent protein (Nterm-GFP) was expressed in E. coli, and the fusion protein was adsorbed to colloidal chitin, powder chitin, and chitosan. Fluorescence microscopy analysis showed that Nterm-GFP bound to the S. commune cell-wall.     

Frequency of pyrethroid resistance in Aedes aegypti and Aedes albopictus (Diptera: Culicidae) in Thailand

Thirty‐two Aedes aegypti populations collected throughout Thailand and five populations of Aedes albopictus from southern Thailand were subjected to standard WHO contact bioassays to assess susceptibility to three commonly used synthetic pyrethroids: permethrin, deltamethrin, and lambda‐cyhalothrin. A wide degree of physiological response to permethrin was detected in Ae. aegypti, ranging from 56.5% survival (Lampang, northern Thailand) to only 4% (Kalasin in northeastern and Phuket in southern Thailand). All 32 populations of Ae. aegypti were found to have evidence of incipient resistance (62.5%) or levels of survival deemed resistant (37.5%) to permethrin. Four populations of Ae. albopictus were found with incipient resistance (97 – 80% mortality) and one with resistance (< 80%) to permethrin. The majority of Ae. aegypti populations (68.7%) was susceptible (> 98% mortality) to deltamethrin, with incipient resistance (observed 97–82% mortality) in other localities. In contrast, all populations of Ae. aegypti were completely susceptible (100% mortality) to the recommended operational dosage of lambda‐cyhalothrin. All five populations of Ae. albopictus were found completely susceptible to both deltamethrin and lambda‐cyhalothrin. Evidence of defined incipient or resistance to synthetic pyrethroids mandates appropriate response and countermeasures to mitigate further development and spread of resistance. In light of these findings, we conclude that routine and comprehensive susceptibility monitoring of dengue mosquito vectors to synthetic pyrethroids should be a required component of resistance management policies and disease control activities.     

Vertebrate slow skeletal muscle actin - conservation, distribution and conformational flexibility

The existence of a unique sarcomeric actin is demonstrated in teleosts that possess substantial amounts of slow skeletal muscle in the trunk. The slow skeletal isotype is conserved. There is one amino acid substitution between Atlantic herring slow skeletal actin and the equivalent in salmonids. Conversely, the intra-species variation is considerable; 13 substitutions between different herring skeletal isotypes (slow versus fast). The isomorphisms (non-conservative underlined: residues, 2, 3, 103, 155, 160, 165, 278, 281, 310, 329, 358, 360 and 363) are restricted to sub-domains 1 and 3 and include the substitution Asp-360 in 'slow' to Gln in 'fast' which results in an electrophoretic shift at alkaline pH. The musculature of the trunk facilitates the preparation of isoactins for biochemical study. Herring slow skeletal G-actin (Ca.ATP) is more susceptible to thermal, and urea, -induced denaturation and subtilisin cleavage than that in fast skeletal, but more stable than the counterpart in salmonids (one substitution, Gln354Ala) highlighting the critical nature of actin's carboxyl-terminal insert. Fluorescent spectra of G-actin isoforms containing the isomorphism Ser155Ala in complexation with 2'-deoxy 3' O-(N'-Methylanthraniloyl) ATP infer similar polarity of the nucleotide binding cleft. An electrophoretic survey detected two skeletal actins in some (smelt and mackerel) but not all teleosts. One skeletal muscle actin was detected in frog and bird.     

Behavioral responses of Anopheles species (Culicidae: Diptera) with varying surface exposure to pyrethroid‐treated netting in an excito‐repellency test system

Indoor Residual Spray (IRS) with insecticides has been a procedure used for decades to protect humans from biting mosquitoes and potential vectors of pathogens. The objective of this study was to determine the responses of three wild‐caught species of malaria vectors exposed to pyrethroids of three different surface coverage percents using an excito‐repellency test box. Each species was exposed to three insecticide‐treated surfaces at varying exposure levels (full coverage, 50%, and 25% of the maximum allowable by the test system) to a single standard field dose of either lambda‐cyhalothrin or alpha‐cypermethrin. Larger numbers of mosquitoes escaped the treated chambers in the direct contact test compared to the spatial repellent chambers in all three different treated surface exposures. No significant differences in the percent of escaped mosquitoes were detected in the 50% and full coverage surface coverage exposures, whereas the 25% coverage produced significantly lower avoidance responses for both compounds. This study found that varying levels of surface exposure with synthetic pyrethroids can impact the behavioral avoidance responses of Anopheles; however, it may also be possible to reduce the amount of coverage to achieve similar avoidance actions. This information may assist policy makers in designing more cost effective strategies involving residual insecticides to control mosquito vectors.     

Effect of estradiol and dihydrotestosterone on hypergravity-induced MAPK signaling and occludin expression in human umbilical vein endothelial cells

Female astronauts have been reported to have a higher incidence of post-flight orthostatic intolerance (POI) compared with that of their male counterparts. POI may result from increased permeability of the endothelial cell (EC) layer in the vasculature. The goal of this study has been to determine whether estradiol (E₂) and dihydrotesterone (DHT) alter human umbilical vein ECs (HUVECs) responses to short term (10 min) hypergravity (1–3 g) mimicking the g force experienced by astronauts during liftoff. E₂ and DHT rapidly (within 5 min) activated MAPK (mitogen-activated protein kinase) in HUVEC at 1 g in a receptor-dependent manner. Liftoff inhibited MAPK phosphorylation, and rapid E₂ and DHT activation of MAPK was blocked. Liftoff simulation or brief (5–90 min) treatment with E₂ or DHT at 1 g had no effect on the expression of the EC tight-junction protein occludin. However, 24-h pre-treatment of HUVECs with E₂ and DHT prior to liftoff simulation significantly increased occludin expression, and hypergravity exposure did not alter this increase. These data provide evidence for a possible protective effect of E₂ and DHT on EC function as indicated by increased occludin; this may help maintain the integrity of EC tight junction and could thus retard or reduce the incidence of POI.This work was supported by NASA grant NAG5-12874 to C.M.K. and R.S.K. and by American Heart Association Ohio Valley Affiliate grant 0555270B to C.M.K. The American Heart Association Ohio Valley supported W.K.S. with an affiliate post-doctoral fellowship (0425431B).     

Resveratrol and estradiol rapidly activate MAPK signaling through estrogen receptors alpha and beta in endothelial cells

Vascular endothelial cells (EC) are an important target of estrogen action through both the classical genomic (i.e. nuclear-initiated) activities of estrogen receptors alpha and beta (ERalpha and ERbeta) and the rapid "non-genomic" (i.e. membrane-initiated) activation of ER that stimulates intracellular phosphorylation pathways. We tested the hypothesis that the red wine polyphenol trans-resveratrol activates MAPK signaling via rapid ER activation in bovine aortic EC, human umbilical vein EC, and human microvascular EC. We report that bovine aortic EC, human umbilical vein EC, and human microvascular EC express ERalpha and ERbeta. We demonstrate that resveratrol and estradiol (E(2)) rapidly activated MAPK in a MEK-1, Src, matrix metalloproteinase, and epidermal growth factor receptor-dependent manner. Importantly, resveratrol activated MAPK and endothelial nitric-oxide synthase (eNOS) at nm concentrations (i.e. an order of magnitude less than that required for ER genomic activity) and concentrations possibly achieved transiently in serum following oral red wine consumption. Co-treatment with ER antagonists ICI 182,780 or 4-hydroxytamoxifen blocked resveratrol- or E(2)-induced MAPK and eNOS activation, indicating ER dependence. We demonstrate for the first time that ERalpha-and ERbeta-selective agonists propylpyrazole triol and diarylpropionitrile, respectively, stimulate MAPK and eNOS activity. A red but not a white wine extract also activated MAPK, and activity was directly correlated with the resveratrol concentration. These data suggest that ER may play a role in the rapid effects of resveratrol in EC and that some of the atheroprotective effects of resveratrol may be mediated through rapid activation of ER signaling in EC.     

Long-term prolactin exposure differentially stimulated the transcellular and solvent drag-induced calcium transport in the duodenum of ovariectomized rats

Prolactin, having been shown to stimulate transcellular active and solvent drag-induced calcium transport in the duodenum of female rats, was postulated to improve duodenal calcium transport in estrogen-deficient rats. The aim of the present study was, therefore, to demonstrate the effects of long-term prolactin exposure produced by anterior pituitary (AP) transplantation on the duodenal calcium transport in young (9-week-old) and adult (22-week-old) ovariectomized rats. We found that ovariectomy did not alter the transcellular active duodenal calcium transport in young and adult rats fed normal calcium diet (1.0% w/w Ca) but decreased the solvent drag-induced duodenal calcium transport from 75.50 +/- 10.12 to 55.75 +/- 4.77 nmol.hr(-1).cm(-2) (P < 0.05) only in adult rats. Long-term prolactin exposure stimulated the transcellular active calcium transport in young and adult AP-grafted ovariectomized rats fed with normal calcium diet by more than 2-fold from 7.56 +/- 0.79 to 16.54 +/- 2.05 (P < 0.001) and 9.78 +/- 0.72 to 15.99 +/- 1.75 (P < 0.001) nmol.hr(-1).cm(-2), respectively. However, only the solvent drag-induced duodenal calcium transport in young rats was enhanced by prolactin from 95.51 +/- 10.64 to 163.20 +/- 18.03 nmol.hr(-1).cm(-2) (P < 0.001) whereas that in adult rats still showed a decreased flux from 75.50 +/- 10.12 to 47.77 +/- 5.42 nmol.hr(-1).cm(-2) (P < 0.05). Because oral calcium supplement has been widely used to improve calcium balance in estrogen-deficient animals, the effect of a high-calcium diet (2.0% w/w Ca) was also investigated. The results showed that stimulatory action of long-term prolactin on the transcellular active duodenal calcium transport in both young and adult rats was diminished after being fed a high-calcium diet. The same diet also abolished prolactin-enhanced solvent drag-induced duodenal calcium transport in young and further decreased that in adult AP-grafted ovariectomized rats. We concluded that the solvent drag-induced duodenal calcium transport in adult rats was decreased after ovariectomy. Long-term prolactin exposure stimulated the transcellular active duodenal calcium transport in both young and adult rats whereas enhancing the solvent drag-induced duodenal calcium transport only in young rats. Effects of prolactin were abolished by a high-calcium diet.     

Host feeding responses of Aedes aegypti (L.) exposed to deltamethrin

Escape responses of mated and unmated nulliparous Aedes aegypti mosquitoes were compared using three different concentrations of deltamethrin in the presence or absence of a live animal host using an excito-repellency (ER) test system. Both insecticide contact (excitation) and non-contact (repellency) test configurations were compared. For contact trials, mated mosquitoes showed similar escape movements among the three concentrations when host stimuli were absent. Significant differences in responses were seen between the lower concentrations of (LC(50) and LC(75) ) deltamethrin with and without hosts present (P<0.05). Presence or absence of host stimuli produced no significant differences in escape response for unmated females when exposed to the highest concentration (LC(90) ) of deltamethrin. Our findings indicate that as deltamethrin concentrations decrease to sublethal levels, mating status and host cues play a more significant role in escape behavior. Therefore, insemination can influence the outcome of feeding success and flight movement of nulliparous female Ae. aegypti in contact with deltamethrin and in the presence of live host stimuli. The ER assay system serves as a useful tool for observing excitation and repellency responses of Ae. aegypti to insecticides in the presence or absence of other environmental and biological cues that can affect mosquito behavior.