380 research outputs found
Tyrosine phosphorylation in Escherichia coli
No full textThe phosphorylation on tyrosine of a protein in Escherichia coli both in vivo and in vitro was revealed by recognition by anti-phosphotyrosine antibodies, labelling with [gamma-32P]ATP, and phosphoamino acid analysis. This protein, which we name TypA, is the product of the o591 reading frame as revealed by N-terminal sequencing and antibody cross-reactivity. Inactivation of typA altered the patterns of protein synthesis during both exponential growth and carbon starvation. These alterations included the disappearance of an acidic isoform of both the universal stress protein UspA and carbon starvation protein Csp15, and increased synthesis of the histone-like protein H-NS. The sequence of TypA from strain K-12 differs from that of an enteropathogenic strain in six amino acid residues and the protein is three residues shorter. We propose that TypA interacts with global regulatory networks and that its phosphorylation may be relevant to pathogenesis.</p
Glutamate spillover drives endocannabinoid production and inhibits GABAergic transmission in the Substantia Nigra pars compacta
No full textEndocannabinoids (eCBs) modulate synaptic transmission in the brain, but little is known of their regulatory role in nigral dopaminergic neurons, and whether transmission to these neurons is tonically inhibited by eCBs as seen in some other brain regions. Using whole-cell recording in midbrain slices, we observed potentiation of evoked IPSCs (eIPSCs) in these neurons after blocking CB1 receptors with rimonabant or LY-320,135, indicating the presence of an eCB tone reducing inhibitory synaptic transmission. Increased postsynaptic calcium buffering and block of mGluR1 or postsynaptic G-protein coupled receptors prevented this potentiation. Increasing spillover of endogenous glutamate by inhibiting uptake attenuated eIPSC amplitude, while enhancing the potentiation by rimonabant. Group I mGluR activation transiently inhibited eIPSCs, which could be prevented by GDP-β-S, increased calcium buffering or rimonabant. We explored the possibility that the dopamine-derived eCB N-arachidonoyl dopamine (NADA) is involved. The eCB tone was abolished by preventing dopamine synthesis, and enhanced by l-DOPA. It was not detected in adjacent non-dopaminergic neurons. Preventing 2-AG synthesis did not affect the tone, while inhibition of NADA production abolished it. Quantification of ventral midbrain NADA suggested a basal level that increased following prolonged depolarization or mGluR activation. Since block of the tone was not always accompanied by attenuation of depolarization-induced suppression of inhibition (DSI) and vice versa, our results indicate DSI and the eCB tone are mediated by distinct eCBs. This study provides evidence that dopamine modulates the activity of SNc neurons not only by conventional dopamine receptors, but also by CB1 receptors, potentially via NADA
Acute action of rotenone on nigral dopaminergic neurons--involvement of reactive oxygen species and disruption of Ca2+ homeostasis
No full textAbstract
Rotenone is a toxin used to generate animal models of Parkinson’s disease; however, the mechanisms of toxicity in substantia nigra pars compacta (SNc) neurons have not been well characterized. We have investigated rotenone (0.05–1 lm) effects on SNc neurons in acute rat midbrain slices, using whole-cell patch-clamp recording combined with microfluorometry. Rotenone evoked a tolbutamide-sensitive outward current (94 ± 15 pA) associated with increases in intracellular [Ca2+] ([Ca2+]i) (73.8 ± 7.7 nm) and intracellular [Na+] (3.1 ± 0.6 mm) (all with 1 lm). The outward current was not affected by a high ATP level (10 mm) in the patch pipette but was decreased by Trolox. The [Ca2+]i rise was abolished by removing extracellular Ca2+, and attenuated by Trolox and a transient receptor potential M2 (TRPM2) channel blocker, N-(p-amylcinnamoyl) anthranilic acid. Other effects included mitochondrial
depolarization (rhodamine-123) and increased mitochondrial reactive oxygen species (ROS) production (MitoSox), which was also abolished by Trolox. A low concentration of rotenone (5 nm) that, by itself, did not evoke a [Ca2+]i rise resulted in a large (46.6 ± 25.3 nm) Ca2+ response when baseline [Ca2+]i was increased by a ‘priming’ protocol that activated voltage-gated Ca2+ channels. There was also a positive correlation between ‘naturally’ occurring variations in baseline [Ca2+]i and the rotenone-induced [Ca2+]i rise. This correlation was not seen in non-dopaminergic neurons of the substantia nigra pars reticulata (SNr). Our results show that mitochondrial ROS production is a key element in the effect of rotenone on ATP-gated K+ channels and TRPM2-like channels in SNc neurons, and demonstrate, in these neurons (but not in the SNr), a large potentiation of rotenone-induced [Ca2+]i rise by a small
increase in baseline [Ca2+]i
Acute effects of 6-hydroxydopamine on dopaminergic neurons of the rat substantia nigra pars compacta in vitro
No full text6-Hydroxydopamine (6-OHDA) is a neurotoxin which has been implicated in the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc) in Parkinson's disease (PD), and is frequently used to produce animal models of the disease. The aim of our study, conducted on midbrain slices obtained from young Wistar rats, was to determine the little known acute effects of this toxin (0.2-2.0 mM; 10-20 min exposure; 34 degrees C) on electrophysiological properties, intracellular Ca2+ levels and dendritic morphology of SNc neurons. Four experimental approaches were used: extracellular recording of firing frequency, whole-cell patch-clamping, ratiometric fura-2 imaging, and cell labeling with lucifer yellow (LY) or dextran-rhodamine. Extracellular recording revealed a concentration-dependent decrease in the tonic, pacemaker-like firing. In whole-cell recordings in voltage-clamp (V(hold) -60 mV), smaller doses (0.2-0.5 mM) induced an outward current (or cell membrane hyperpolarization in current-clamp), which could in some cells be reversed with tolbutamide (blocker of ATP-dependent K+ channels). A higher dose (1.0-2.0 mM) caused rapid reductions of cell membrane capacitance and membrane resistance. Toxin exposure gradually increased the intracellular Ca2+ level, which did not subsequently return to control. The increase in Ca2+ signal was not prevented by depletion of intracellular Ca2+ stores with thapsigargin (10 microM) or cyclopiazonic acid (30 microM), nor by removing extracellular Ca2+. Cell membrane current and Ca2+ responses were not prevented by blocking dopamine transporter (DAT). Cells loaded with LY or dextran-rhodamine showed signs of damage (cell membrane blebbing) in dendrites following toxin exposure (1 mM; 10-20 min). These results demonstrate that the oxidative and metabolic stress induced in SNc neurons by 6-OHDA results in rapid dose-dependent changes of cell membrane properties with morphological evidence of dendritic damage, as well as in disturbance of intracellular Ca2+ homeostasis
The contribution of biological sex to heat stress-mediated outcomes in growing pigs
No full textHeat stress (HS) negatively impacts a variety of production parameters in growing pigs; however, the impact of biological sex on the HS response is largely unknown. To address this, 48 crossbred barrows and gilts (36.8 ± 3.7 kg BW) were individually housed and assigned to one of three constant environmental conditions: (1) thermoneutral (TN) (20.8 ± 1.6 °C; 62.0 ± 4.7% relative humidity; n = 8/sex), (2) HS (39.4 ± 0.6 °C; 33.7 ± 6.3% relative humidity) for 1 d (HS1; n = 8/sex), or (3) or for 7 d (HS7; n = 8/sex). As expected, HS increased rectal temperature (Tr) following 1 d of HS (1.0 °C; P < 0.0001) and 7 d of HS (0.9 °C; P < 0.0001). By 7 d, heat-stressed gilts were cooler than barrows (0.4 °C; P = 0.016), despite identical heating conditions. There was a main effect of sex such that barrows had higher Tr than gilts (P = 0.031). Heat-stressed pigs on d 1 had marked reductions in feed intake and BW compared to TN (P < 0.0001). One day of HS resulted in negative gain to feed (G:F) in barrows and gilts and was reduced compared to TN (P < 0.0001). Notably, following 1 d of HS, the variability of G:F was greater in gilts than in barrows. Between 1 and 7 d of HS, G:F improved in barrows and gilts and were similar to TN pigs, even though HS barrows had higher Tr than gilts over this period. Heat stress for 1 and 7 d reduced empty gastrointestinal tract weight compared to TN (P < 0.0001). Interestingly, HS7 gilts had decreased gastrointestinal tract weight compared to HS1 gilts (2.43 vs 2.72 kg; P = 0.03), whereas it was similar between HS1 and HS7 barrows. Lastly, a greater proportion of gastrointestinal contents was in the stomach of HS1 pigs compared to TN and HS7 (P < 0.05), which is suggestive of decreased gastric emptying. Overall, HS barrows maintained an elevated Tr compared to HS gilts through the duration of the experiment but also maintained similar growth and production metrics compared to gilts, despite this higher temperature.This article is published as Rudolph, Tori E., Melissa Roths, Alyssa D. Freestone, Robert P. Rhoads, Sarah H. White-Springer, Lance H. Baumgard, and Joshua T. Selsby. "The contribution of biological sex to heat stress-mediated outcomes in growing pigs." animal 18 (2024): 101168. doi:10.1016/j.animal.2024.101168. Copyright 2024 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)
Properties of dopaminergic neurons in organotypic mesencephalic-striatal co-cultures - evidence for a facilitatory effect of dopamine on the glutamatergic input mediated by α-1 adrenergic receptors
No full textOrganotypic cultures (OCs) have been widely used to investigate the midbrain dopaminergic system, but only a few studies focused on the functional properties of dopaminergic neurons and their synaptic inputs from dopaminergic and non-dopaminergic neurons also contained in such cultures. In addition, it is not clear whether the culturing process affects the intrinsic neuronal properties and the expression of specific receptors and transporters. We performed patch-clamp recordings from dopaminergic neurons in mesencephalic–striatal co-cultures obtained from transgenic mice expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. Some (10 ⁄ 44) GFP+ neurons displayed a bursting activity that renders the firing of these cells similar to that of the dopaminergic neurons in vivo. The culturing process reduced the hyperpolarization-activated current (Ih) and the expression of D2 receptors. Downregulation of D2 receptor mRNA and protein was confirmed with reverse transcriptase polymerase chain reaction and Western blotting. Immunocytochemistry revealed that many synaptic terminals, most likely originating from dopaminergic neurons, co-expressed the dopamine (DA) transporter and the vesicular glutamate transporter-2, suggesting a co-release of DA and glutamate. Interestingly, exogenous DA decreased glutamate release in young cultures [days in vitro (DIV) 26) DA increased glutamate release by acting on a-1 adrenoreceptors. The facilitatory effect of DA on glutamatergic transmission to midbrain dopaminergic neurons may be important in conditions when the expression of D2 receptors is compromised, such as long-term treatment with antipsychotic drugs. Our data show that midbrain OCs at DIV > 26 may provide a suitable model of such conditions
Heat stress alters hematological parameters in barrows and gilts
No full textThe purpose of this investigation was to establish the role biological sex plays in circulating factors following heat stress (HS). Barrows and gilts (36.8 ± 3.7 kg body weight) were kept in either thermoneutral (TN; 20.8 ± 1.6 °C; 62.0% ± 4.7% relative humidity; n = 8/sex) conditions or exposed to HS (39.4 ± 0.6 °C; 33.7% ± 6.3% relative humidity) for either 1 (HS1; n = 8/sex) or 7 (HS7; n = 8/sex) d. Circulating glucose decreased as a main effect of the environment (P = 0.03). Circulating non-esterified fatty acid (NEFA) had an environment × sex interaction (P < 0.01) as HS1 barrows had increased NEFA compared to HS1 gilts (P = 0.01) and NEFA from HS7 gilts increased compared to HS1 gilts (P = 0.02) and HS7 barrows (P = 0.04). Cortisol, insulin, glucagon, T3, and T4 were reduced as a main effect of environment (P ≤ 0.01). Creatinine was increased in HS1 and HS7 animals compared to TN (P ≤ 0.01), indicative of decreased glomerular filtration rate. White blood cell populations exhibited differential patterns based on sex and time. Neutrophils and lymphocytes had an environment × sex interaction (P ≤ 0.05) as circulating neutrophils were increased in HS1 barrows compared to TN and HS7 barrows, and HS1 gilts (P ≤ 0.01) and HS7 barrows had less neutrophils compared to TN barrows (P = 0.01), whereas they remained similar in gilts. In contrast, barrow lymphocyte numbers were similar between groups, but in HS7 gilts they were decreased compared to TN and HS1 gilts (P ≤ 0.04). In total, these data demonstrate that HS alters a host of circulating factors and that biological sex mediates, at least in part, the physiological response to HS.This article is published as Tori E Rudolph, Melissa Roths, Alyssa D Freestone, Sarah H White-Springer, Robert P Rhoads, Lance H Baumgard, Joshua T Selsby, Heat stress alters hematological parameters in barrows and gilts, Journal of Animal Science, Volume 102, 2024, skae123, https://doi.org/10.1093/jas/skae123
Long-duration transit and food and water deprivation alter behavioral activities and aggressive interactions at the feed bunk in beef feedlot steers
No full textThe objective of these experiments was to assess the effects of food and water deprivation and transit duration on the behavior of beef feedlot steers. In Experiment 1, 36 Angus-cross steers (353 ± 10 kg) were stratified to six pens and assigned one of three treatments (n = 12 steers/treatment): control (CON; stayed in home pens with ad libitum access to feed and water), deprived (DEPR; stayed in home pens but deprived of feed and water for 18 h), or transported (TRANS; subjected to 18-h transit event and returned to home pens). In Experiment 2, 60 Angus-cross steers (398 ± 5 kg; 6 steers/pen) were transported either 8 (8H) or 18 (18H) h. Four 8H pens (n = 24 steers) and six 18H pens (n = 36 steers) were used for behavioral analysis. In both experiments, the time to eat, drink, and lay down was recorded for each steer upon return to home pens. Total pen displacements from the feed bunk were also assessed for the two hours following feed access in both experiments. Data were analyzed using Proc Mixed of SAS 9.4, with treatment as a fixed effect. Steer was the experimental unit for behavioral activities, while pen was the experimental unit for bunk displacements. Displacements were analyzed as repeated measures with the repeated variable of time. In Experiment 1, time to eat and drink was similar across treatments (P ≥ 0.17). However, TRANS laid down in 16.5 min while DEPR did not lay down until 70.5 min post-arrival to pen (P < 0.01). Deprived steers had greater bunk displacements in the first 70 min post-feed access than CON or TRANS, though displacements among treatments from 100 to 120 min post-feed access were similar (Treatment × Time: P = 0.02). In Experiment 2, both 8H and 18H steers laid down approximately 25 min post-home pen arrival (P = 0.14). There was no effect of transit duration or duration by time on bunk displacements (P ≥ 0.20), though displacements were greater from 0 to 20 min than from 20 to 30 min post-feed access (Time: P = 0.04). Steers that were deprived of feed and water were highly motivated to access those resources, while transported steers prioritized laying down. Producers should consider these priorities when preparing to receive cattle from a long transit event.This is a pre-copyedited, author-produced version of an article accepted for publication in Journal of Animal Science following peer review. The version of record: Heiderscheit, Katie J., Alyssa D. Freestone, Aubree M. Beenken, Erin L. Deters, Joshua M. Peschel, and Stephanie L. Hansen. "Long-duration transit and food and water deprivation alter behavioral activities and aggressive interactions at the feed bunk in beef feedlot steers." Journal of Animal Science (2022). is available online at DOI: 10.1093/jas/skac060. Copyright 2022. The Author(s). Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). Posted with permission
The Apricot Breeding Programme among Some Turkish and Foreign Cultivars
No full textThe aim of this study was to breed new table apricot varieties by hybridization among Turkish and foreign apricot cultivars. Alyanak, Sakit-1, Sakit-2, Sakit-6, 07K11 local cultivars and Cafona, Canino, Fracasso, J. Foulon and P. de Colomer foreign cultivars were used in the breeding programme. The breeding programme started in 1989; 4173 hybrids were obtained. 370 genotypes were selected from all hybrids combinations. In 1995, the evaluation phase was achieved. Five new apricot varieties were registered as Dr. Kaska, Cagataybey, Cagribey, Sahinbey and Alatayildizi. Dr. Kaska has juicy and freestone fruits. Its fruits are sweet and aromatic. Cagataybey is freestone and its fruit is juicy, aromatic and more coloured. Cagribey is freestone, juicy and aromatic. Alatayildizi is also freestone and has good fruit quality. Sahinbey has a late harvest date
The apricot breeding programme among some Turkish and foreign cultivars
No full textThe aim of this study was to breed new table apricot varieties by hybridization among Turkish and foreign apricot cultivars. Alyanak, Sakit-1, Sakit-2, Sakit-6, 07K11 local cultivars and Cafona, Canino, Fracasso, J. Foulon and P. de Colomer foreign cultivars were used in the breeding programme. The breeding programme started in 1989; 4173 hybrids were obtained. 370 genotypes were selected from all hybrids combinations. In 1995, the evaluation phase was achieved. Five new apricot varieties were registered as Dr. Kaşka, Çagataybey, Çagribey, Şahinbey and Alatayildizi. Dr. Kaşka has juicy and freestone fruits. Its fruits are sweet and aromatic. Çagataybey is freestone and its fruit is juicy, aromatic and more coloured. Çagribey is freestone, juicy and aromatic. Alatayildizi is also freestone and has good fruit quality. Şahinbey has a late harvest date
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