We have designed an economical non-invasive movement detector for small animal studies and used it for monitoring and quantifying itch in mice. The system is based on a sensitive force transducer ...positioned below a recording platform holding a lightweight polystyrene recording box in which an animal is placed. A programmed micro-controller is used to discriminate between non-specific movement, grooming behaviour, and scratching movements made by the animal's hind limb. Following sub-dermal injection of histamine receptor agonists into the neck of a mouse, dose-related scratching occurred which was detected and quantified. There was 91% correlation between bouts of scratching as counted manually from playback of the video recording and recorded by the detector. The detector was also able rapidly to count the individual scratch movements of the hind limb that comprise a bout, with 95% accuracy in comparison with manual counting during slow motion playback of video tape, something that is impossible for an unaided observer to achieve because individual scratch movements are too fast to discriminate by eye. Separate detectors were used for the efficient non-invasive study of four animals simultaneously, and this number could easily be increased by adding more platforms. The system could also be modified to record the animal's position within the box, which would be of value in studies involving exploratory behaviour.
In summary, the non-invasive multichannel repetitive movement detector will be very useful for accurate measurement of scratching during pruritus studies in small animals, with considerable savings in staff time and effort. It should therefore be a valuable tool for helping to investigate pruritus and in the evaluation of anti-pruritic drugs.
The location and sequence of androgen responsive elements (AREs) in the 5'-flanking DNA of the androgen-regulated rat probasin (PB) gene were determined. The DNA- and steroid-binding domains of the ...rat androgen receptor glutathione-S-transferase (GST)-AR1 and the DNA-binding domain and hinge region alone (GST-AR2) were expressed in Escherichia coli as isopropyl-B-D-thioglactopyranoside-induced fusion proteins with GST and purified using glutathione affinity chromatography. Band shift assays indicated that the AR1 peptide was at least five times more effective than AR2 in binding to PB 5'-flanking DNA (-426 to +28), although both gave qualitatively similar patterns and were displaced by anti-AR antibodies. DNase I footprinting experiments revealed two putative AREs: one between positions -236 and -223 (ARE-1) and the other between -140 and -117 (ARE-2). Hormonal regulation of PB was determined by cotransfecting reporter constructions containing the PB 5'-flanking region (-426 to +28) linked to the bacterial chloramphenicol acetyl transferase (CAT) gene with androgen, glucocorticoid, or progesterone receptor expression vectors into human prostatic carcinoma cells (PC-3). PB-CAT gene expression was more effectively induced by androgens than by glucocorticoids or progestins. Both 5'- and 3'-deletion mapping of the PB 5'-flanking DNA revealed that ARE-1 and ARE-2 were required for androgen regulation. A single base mutation in either ARE resulted in a more than 95% loss of androgen induction of CAT. In comparable transfection experiments, the PB hormone-responsive elements showed a greater induction by androgens than did mouse mammary tumor virus or tyrosine aminotransferase elements. Thus, the preferential androgen regulation of the PB gene involves the participation of two different cis-acting DNA elements that bind AR.
The aim of the present study was to determine the effect of estradiol-17 beta (E2), in its positive feedback mode for gonadotropin release, on the serotonin transporter (SERT) in female rat brain. ...Levels of SERT mRNA were determined by in situ hybridization and SERT-binding sites were measured by quantitative 3Hparoxetine receptor autoradiography. The injection of estradiol benzoate (EB) in acutely ovariectomized rats increased significantly (approximately 50%) the numbers of cells that expressed SERT mRNA in the dorsal raphe nucleus and the density of SERT-binding sites in lateral septum (90%), basolateral amygdala (20%), ventral nucleus of thalamus (250%) and ventromedial hypothalamic nucleus (250%). SERT-binding sites in EB-treated rats were significantly lower in periaqueductal central grey (15%). These findings indicate that effects on SERT gene expression may be involved in the E2-induction of the gonadotropin surge. Together with our previous findings, they also suggest that the sex differences in depression and the apparent psychotropic effect of E2 may be due to the action of E2 on the serotonin transporter as well as 5-HT2A receptors.
The role of histamine H1, H2, H3 and H4 receptors in acute itch induced by histamine was investigated in female BalbC mice. Scratching was induced by intradermal injections of pruritogen into the ...back of the neck and ‘itch’ assessed by quantifying the scratching evoked.
Histamine (0.03–80 μmol), histamine‐trifluoromethyl‐toluidine (HTMT, H1 agonist, 0.002–2 μmol), clobenpropit (H4 agonist, H3 antagonist, 0.002–0.6 μmol) and to a lesser extent imetit (H3/H4 agonist, 0.03–3 μmol) all induced dose‐dependent scratching. Dimaprit (H2 agonist, 0.04–40 μmol) did not cause scratching.
Mepyramine (H1 antagonist, 20 mg kg−1, i.p.) reduced scratching evoked by histamine and HTMT, but not that caused by H3 or H4 agonists. Thioperamide (H3/H4 antagonist, 20 mg kg−1, i.p.) reduced scratching induced by histamine, H3 and H4 agonists, but not that caused by HTMT. The non‐sedating H1 antagonist, terfenadine, also significantly reduced the scratching induced by the H1 agonist, HTMT. Cimetidine (H2 antagonist, 20 mg kg−1, i.p.) did not affect histamine‐induced scratching.
These results indicate that activation of histamine H4 receptors causes itch in mice, in addition to the previously recognised role for H1 receptors in evoking itch. Histamine H4 receptor antagonists therefore merit investigation as antipruritic agents.
British Journal of Pharmacology (2004) 142, 374–380. doi:10.1038/sj.bjp.0705754
Estrogen increases serotonin transporter (SERT) mRNA and binding sites in female rat brain. In order to determine whether changes in SERT are gender- and steroid-specific we have now carried out ...studies on adult male Wistar rats which were either intact or castrated (under halothane anesthesia) and injected with arachis oil, estradiol benzoate (EB), testosterone propionate (TP) or the non-aromatizable androgen, 5α-dihydrotestosterone (5α-DHT). The number of SERT mRNA-expressing cells in the dorsal raphe (DR) nucleus was decreased by castration and increased by treatment (for ∼32 h) with EB or TP, but not 5α-DHT. Sex steroids had no effect on the number of SERT mRNA-expressing cells in the median raphe nucleus. The density of SERT sites, assessed by autoradiography of
3
H
paroxetine binding, was significantly reduced in arcuate nucleus and median raphe after castration, and increased in arcuate, basolateral amygdala and ventromedial hypothalamic nucleus by treatment with EB or TP, but not 5α-DHT. Estradiol, but not testosterone or 5α-DHT reduced the density of SERT sites in midbrain central grey. These data show that testosterone as well as estrogen affects SERT expression in male brain, and that the action of testosterone probably depends upon its enzymatic conversion, by aromatase, to estradiol. Our findings may have implications for sex steroid control of mood and behavior, and the action of neurotoxic derivatives of amphetamine, such as 3,4-methylenedioxymethamphetamine, in the human.
Animal and human studies have implicated oxytocin in affiliative and prosocial behaviors. We tested whether genetic variation in the oxytocin receptor (OXTR) gene is associated with conduct disorder ...(CD). Utilizing a family-based sample of adolescent probands recruited from an adolescent substance abuse treatment program, control probands and their families (total sample, n=1750), we conducted three tests of association with CD and 10 single nucleotide polymorphisms (SNPs) in the OXTR gene: (a) family-based comparison utilizing the entire sample; (b) within-Whites, case-control comparison of adolescent patients with CD and controls without CD; and (c) within-Whites case-control comparison of parents of patients and parents of controls. Family-based association tests failed to show significant results (no results P<0.05). While strictly correcting for the number of tests (α=0.002), adolescent patients with CD did not differ significantly from adolescent controls in genotype frequency for the OXTR SNPs tested; similarly, comparison of OXTR genotype frequencies for parents failed to differentiate patient and control family type, except a trend association for rs237889 (P=0.004). We concluded that in this sample, 10 SNPs in the OXTR gene were not significantly associated with CD.
The human beta-globin locus is a complex genetic system widely used for analysis of eukaryotic gene expression. The locus consists of five functional beta-like globin genes, epsilon, (G)gamma, ...(A)gamma, delta, and beta, arrayed on the chromosome in the order that they are expressed during ontogeny. Globin gene expression is regulated, in part, by the locus control region, which physically consists of five DNaseI-hypersensitive sites located 6-22 Kb upstream of the epsilon -globin gene. During ontogeny two switches occur in beta-globin gene expression that reflect the changing oxygen requirements of the fetus. The first switch from embryonic epsilon - to fetal gamma-globin occurs at six weeks of gestation. The second switch from gamma- to adult delta- and beta-globin occurs shortly after birth. Throughout the locus, cis-acting elements exist that are dynamically bound by trans-acting proteins, including transcription factors, co-activators, repressors, and chromatin modifiers. Discovery of novel erythroid-specific transcription factors and a role for chromatin structure in gene expression have enhanced our understanding of the mechanism of globin gene switching. However, the hierarchy of events regulating gene expression during development, from extracellular signaling to transcriptional activation or repression, is complex. In this review we attempt to unify the current knowledge regarding the interplay of cis-acting elements, transcription factors, and chromatin modifiers into a comprehensive overview of globin gene switching.
Although 24-h urinary measure to estimate sodium and potassium excretion is the gold standard, it is not practical for large studies. We compared estimates of 24-h sodium and potassium excretion from ...a single morning fasting urine (MFU) using three different formulae in healthy individuals.
We studied 1083 individuals aged 35-70 years from the general population in 11 countries. A 24-h urine and MFU specimen were obtained from each individual. A subset of 448 individuals repeated the measures after 30-90 days. The Kawasaki, Tanaka, and INTERSALT formulae were used to estimate urinary excretion from a MFU specimen.
The intraclass correlation coefficient (ICC) between estimated and measured sodium excretion was higher with Kawasaki (0.71; 95% confidence interval, CI: 0.65-0.76) compared with INTERSALT (0.49; 95% CI: 0.29-0.62) and Tanaka (0.54; 95% CI: 0.42-0.62) formulae (P <0.001). For potassium, the ICC was higher with the Kawasaki (0.55; 95% CI: 0.31-0.69) than the Tanaka (0.36; 95% CI: -0.07 to 0.60; P <0.05) formula (no INTERSALT formula exists for potassium). The degree of bias (vs. the 24-h urine) for sodium was smaller with Kawasaki (+313 mg/day; 95% CI: +182 to +444) compared with INTERSALT (-872 mg/day; 95% CI: -728 to -1016) and Tanaka (-548 mg/day; 95% CI: -408 to -688) formulae (P <0.001 and P = 0.02, respectively). Similarly for potassium, the Kawasaki formula provided the best agreement and least bias. Blood pressure correlated most closely and similarly with the 24-h and Kawasaki estimates for sodium compared with the other two formulae.
In a diverse population, the Kawasaki formula is the most valid and least biased method of estimating 24-h sodium excretion from a single MFU and is suitable for population studies.
We analyze data from the fall 2020 pandemic response efforts at the University of Colorado Boulder, where more than 72,500 saliva samples were tested for severe acute respiratory syndrome coronavirus ...2 (SARS-CoV-2) using qRT-PCR. All samples were collected from individuals who reported no symptoms associated with COVID-19 on the day of collection. From these, 1,405 positive cases were identified. The distribution of viral loads within these asymptomatic individuals was indistinguishable from what has been previously observed in symptomatic individuals. Regardless of symptomatic status, ∼50% of individuals who test positive for SARS-CoV-2 seem to be in noninfectious phases of the disease, based on having low viral loads in a range from which live virus has rarely been isolated. We find that, at any given time, just 2% of individuals carry 90% of the virions circulating within communities, serving as viral "supercarriers" and possibly also superspreaders.
PDF
