bluuebunny/crossref_metadata_2025 huggingface dataset:
Origin and prevalence of allergy to the Asian beetle Harmonia axyridis
Authors: Graue Hayley , Quinn Tim , Barnes Charles , Herndon Betty
Published: June 2021

The imported Asian lady beetle Harmonia axyridis has been added to the list of allergens derived from insects. These pests hibernate in warm structures during winter, and some exposed humans respond to this beetle antigen with positive skin prick tests and show elevated immunoglobulin E. One report suggests the allergen is a secreted stress chemical, beta caryophyllene.

To study occurrence and source of the allergies, lady beetles were collected and stored at ‐70°C. A local allergy clinic provided 48 unselected serum samples without patient data. These sera were tested for IgE and IgG reactivity by ELISA to both beetle protein extract and the purified stress protein caryophyllene.

Crude lady beetle protein extract was prepared as a sterile filtrate of homogenized beetles containing 1.5 mg/mL protein. Caryophyllene was diluted to 1 mg/mL. Antibody capture ELISA utilized incubation of serum dilutions on plates coated with the beetle antigens followed by peroxidase coupled anti‐IgG or IgE. Peroxidase substrate (DAB) was read at 450 nm. IgG titers for crude protein vs. stress chemical were unrelated p=0.46, but serum IgE titers of the two antigens showed a correlation (p=0.005). Three samples of the 48 showed IgG and/or IgE reactivity against beetle extract but not caryophyllene, suggesting broad insect protein allergy.

Allergy against Asian lady beetles was not high in this urban population; 2% were highly reactive and 6% somewhat reactive, which is lower than agrarian reports. The data overall suggest that insect stress chemical exposure may contribute to the human allergic response to lady beetles over‐wintering in human habitations.
Gender disparity of hepatic lipid homeostasis regulated by the circadian clock
Authors: YANG XIAOXIA , ZHANG YU‐KUN , KLAASSEN CURTIS , WAN YU‐JUI
Published: June 2021

The current study is to identify specific patterns of circadian rhythms for lipid homeostasis in both female and male mouse livers, and to clarify gender disparity in coupling peripheral circadian clock to lipid metabolic outputs by nuclear receptors. Profiling the diurnal hepatic expression of genes encoding circadian clocks, nuclear receptors, and lipid metabolic enzymes was performed. Hepatic lipid levels including cholesterol, triglyceride, and nonesterified fatty acids were monitored over a 24 h period. The Cosinor analysis revealed that several genes encoding nuclear receptors and enzymes involved in the lipid metabolic pathway were rhythmically expressed in phase with the peripheral clocks, which were correlated with the diurnal changes of hepatic lipid levels. Gender disparity was observed for circadian characteristics including mesor and amplitude values, accompanied with advances in acrophases in female mouse livers. Accordingly, gender differences were also observed in diurnal lipid homeostasis. The identification of cycling patterns for lipid metabolic pathways in both genders may shed light on the development of gender‐based treatment for human diseases related to the coordination of cellular clock and control of lipid homeostasis. This work is supported by grants from National Institutes of Health [CA53596, AA14147] and COBRE [P20 RR021940].
Experimental alloxan‐induced diabetes model in rats: lack of vascular dysfunction despite oxidative stress
Authors: Capellini Verena Kise , Baldo Caroline Floreoto , Celotto Andrea Carla , Viaro Fernanda , Batalhão Marcelo Eduardo , Cárnio Evelin Capellari , Rodrigues Alfredo José , Evora Paulo Roberto Barbosa
Published: June 2021

Increased oxidative stress and reduced nitric oxide (NO) bioavailability are key features of diabetes mellitus that may result in vascular dysfunctions. We assessed whether an experimental model of alloxan‐diabetic Wistar rats promotes oxidative stress, reduces NO bioavailability and causes vascular dysfunction. After 1 week of alloxan injection (40 mg/Kg), rats with glycemia > 200 mg/dl were considered diabetics (D). The weekly glycemia was significantly higher in D rats compared to control (C) (p < 0.001). After 5 weeks of alloxan injection, plasma levels of malondialdehyde were increased in D compared to C rats (p = 0.002), however, plasma levels of nitrite and nitrate did not differ between the groups; this was accompanied by a significant reduction in endothelial NO synthase (NOS) immunostaining in endothelium (p = 0.001) and by a significant increase in inducible NOS immunostaining in adventitia of thoracic aorta from D rats (p < 0.001). Moreover, the vascular reactivity study, performed in organ chambers, showed that the responsiveness of thoracic aortic rings to acetylcholine, sodium nitroprusside and phenylephrine was similar in both groups. Thus, these findings demonstrated that despite the development of diabetes and the increase in oxidative stress the 5 weeks duration of diabetes did not reduce NO bioavailability nor promoted vascular dysfunction in rats. Support: CAPES; FAEPA‐HC/FMRP.
REGULATION OF RENAL AQUAPORINS AND SALT TRANSPORTERS IN LOW SODIUM DIET RATS WITH EXPERIMENTALLY INDUCED HEART FAILURE
Authors: Lütken Sophie Constantin , Praphaphimon Pariwat , Kwon Tae‐Hwan , Bie Peter , Frøkiær Jørgen , Nielsen Søren
Published: June 2021

Heart failure (HF) is a common complication to myocardial infarction (MI) and is associated with increased plasma vasopressin levels and renal AQP2 protein abundance as well as sodium and water retention. Thus reduced sodium intake is often recommended to patients with HF. To examine the underlying renal molecular mechanisms in low‐output HF, male rats were subjected to ligation of the left anterior descending artery. The following MI and HF were confirmed with echocardiography at day seven. During the last seven days of experiment all the rats were fed a low sodium diet and treated with dDAVP (20 ng/h s.c.) or vehicle. The rats were divided into four groups: Sham (n=6); Sham+dDAVP (n=6); HF (n=5), HF+dDAVP (n=6). In dDAVP treated groups the protein abundance of AQP2, p(S256)‐AQP2 and renal sodium transporters increased. Surprisingly vehicle‐treated HF rats failed to increase or even decreased protein abundance of water and sodium transporters, the finding being most profound in inner medulla. In conclusion, the findings support that sodium restriction may be important as a powerful clinical tool against renal water and sodium retention associated with low‐output HF.Danish Medical Research Council
Dynamin 2 inhibits reactive oxygen species‐dependent EGF receptor transactivation by Angiotensin II in vascular smooth muscle cells
Authors: Si Shuyi , Xiao Chunling , He Xiaobo , Jiang Wei , Su Lianfang , Zuo Lian
Published: June 2021

Angiotensin II (Ang II) induces transactivation of the epidermal growth factor (EGF) receptor (EGF‐R), which serves as a scaffold for various signaling molecules in vascular smooth muscle cells (VSMC). This response, which is critical for the full expression of the Ang II receptor signaling repertoire, is dependent on reactive oxygen species (ROS) derived from NAD(P)H oxidase and caveolin‐enriched membrane fractions. The dynamin family of large GTPases has been suggested in the formation of nascent vesicles in endocytic pathways. However, the specific effects of Dynamin 2 in ROS‐dependent AT1 receptor (AT1R)‐mediated signaling remain unknown. Here we show that DN‐dynamin 2 and Dynamin 2 siRNA significantly inhibited Ang II‐induced EGF‐R transactivation. Detergent‐free cell fractionation demonstrates that EGF receptor (EGF‐R) is found basally in Caveolin‐enriched fractions (CF). Ang II stimulates AT1R movement into CF and egress of EGF‐R out of these microdomains. DN‐dynamin 2 dramatically blocks Ang II‐stimulated AT1R moving into and EGF‐R moving out of CF. These data suggest that Dynamin 2 specifically interferes with the ROS‐dependent signaling pathways which couple AT1R to the EGF‐R., thereby regulating the spatially‐ and temporally‐organized ROS‐dependent Ang II signaling in VSMCs.
Compensatory role for spinal glutamate in the regulation of renal sympathetic nerve activity in Wistar rats fed a low Na+ diet
Authors: Houghton Belinda L , Johns Edward J
Published: June 2021

Spinal V1 and glutamate receptor mediated RSNA are sensitized by high dietary Na+ intake. This study compared the RSNA responses to intrathecal (IT) vasopressin (AVP) and glutamate infusion between male Wistar rats fed a normal Na+ (NNa+, 0.3%) or low Na+ (LNa+, 0.03%) diet from weaning (4 weeks) to adulthood (11 weeks). Adult rats were anaesthetized (1ml IP chloralose/urethane) and instrumented with arterial and IV cannulae. The tip of the IT cannula was placed at spinal vertebra T13. RSNA was recorded from the left kidney. ?RSNA (%baseline) were measured after IT AVP (1, 5, 10, 20 μM; 10 μL/2min; NNa+=6, LNa+=4) or glutamate (10, 20, 50, 100mM; 10 μL/2min, NNa+=6, LNa+=5). Data are presented as mean ± SE (% baseline RSNA). P<0.05 indicated significance. There was no difference in absolute baseline RSNA ( μV/s, NNa+ 13±2, LNa+ 17±6, P=0.21) or MAP (mmHg, NNa+ 88±4, LNa+ 92±3, P=0.36). IT AVP elicited a dose dependent rise in RSNA in NNa+, while RSNA responses were not observed at any dose of IT AVP in LNa+ group (P<0.001). A dose dependent rise in RSNA to IT glutamate was observed in NNa+ and LNa+. In addition, augmented RSNA responses were observed in LNa+ at 10mM (NNa+: 98±1%, LNa+: 121±5%, P<0.01) and 50mM (NNa+: 109±2%, LNa+: 127±9% P<0.05). This suggests that LNa+ intake blunts spinal AVP mediated RSNA, but the sympathoexcitatory pathway may be compensated by an increased sensitivity to spinal glutamate. Funding: HRB RP/2004/17.
Dopamine inhibits N‐type channels in chemosensory afferents to reduce synaptic transmitter release under normoxic and chronic intermittent hypoxic (CIH) conditions
Authors: Kline David D. , Hendricks Gabriel , Hermann Gerlinda E. , Rogers Richard C. , Kunze Diana L.
Published: June 2021

Evoked synaptic currents elicited in second‐order neurons in the nucleus of the solitary tract (nTS) by activation of chemoafferent fibers are reduced after 10 days of CIH. The mechanism by which this occurs is unknown. One candidate is dopamine (DA) which is released from the presynaptic terminal and exerts a tonic presynaptic inhibition on glutamate release. We postulated that 1) tonic activation of D2 receptors inhibit presynaptic calcium currents to reduce transmitter release and 2) this occurs to a greater extent in CIH. Experiments were performed in isolated nodose‐petrosal cells to examine calcium currents and nTS slices to study presynaptic calcium fluorescence and synaptic currents. DA and quinpirole (Quin, D2 agonist) inhibited calcium currents by acting on the N‐type calcium channel under normoxic and CIH conditions. Quin also decreased the change in presynaptic terminal calcium fluorescence to solitary tract stimulation in nTS slices, an effect which occurred through inhibition of N‐type calcium channels. Yet in nTS slices from CIH animals, although the D2 antagonist sulpiride increased the CIH‐reduced amplitude of synaptic currents, EPSCs were not restored to normal levels. This indicates that while the DA inhibitory effect on calcium currents remains intact in CIH, most of the reduction in synaptic currents occurs via alternative mechanisms. NS52142 (RCR), HL085108 (DDK) and HL25830 (DLK).
Analysis of H,K‐ATPase‐mediated acid secretion along the mouse collecting duct
Authors: Lynch Irma Jeanette , Rudin Alicia , Xia Shen‐ling , Wingo Charles S.
Published: June 2021

In the collecting duct (CD), the H,K‐ATPases function in cation reabsorption and H secretion. The purpose of this study was to evaluate H,K‐ATPase‐mediated H secretion along the CD. Intracellular pH (pHi) recovery to acute H loading was investigated in 1) common CD cell lines and 2) in the microperfused cortical, outer medullary, and inner medullary CD (CCD, OMCD, IMCD). pHi recovery rates (U/min) were greatest in the OMCD1 cell line (0.25 ± 0.01), followed by mpk‐CCDc14 (0.17 ± 0.01), mIMCD‐K2 (0.12 ± 0.01), and mIMCD‐3 (0.06 ± 0.02) cells. Na,H‐exchange inhibition (EIPA) diminished the majority of pHi recovery in these cells (65%, 97%, 74%, and 90%, respectively). In OMCD1, where EIPA‐insensitive pHi recovery was greatest, H,K‐ATPase activity was 0.10 ± 0.01 measured as EIPA‐ and bafilomycin A1 (H‐ATPase inhibitor)‐insensitive (EBI) pHi recovery. Sch28080 (HKa1 inhibitor) significantly inhibited this recovery by 84%. EBI pHi recovery was greatest in the microperfused CCD (0.10 ± 0.02) then in OMCD (0.04 ±0.01) and was minimal in IMCD (0.01 ± 0.00). EBI pHi recovery was 0.30 ± 0.03 and 0.26 ± 0.03 in A‐ and B‐type ICs and was Sch28080 or ouabain (HKα2 inhibitor) sensitive. These data suggest: 1) H,K‐ATPase mediated H secretion is greatest in the CCD, 2) both A‐ and B‐type ICs possess HKα1 and HKα2 H,K‐ATPase isoforms, and 3) the OMCD1 cell line best exhibits H,K‐ATPase activity. This work was funded by NIH Grant R01‐DK‐049750.
Role of AT2 receptor in long‐term blood pressure control in obese Zucker rat
Authors: Ali Quaisar , Siddiqui Athar , Hussain Tahir
Published: June 2021

Earlier, we reported that there was an increase in angiotensin II AT2 receptor expression in the renal proximal tubule, and selective activation of the AT2 receptor by agonist inhibits Na,K‐ATPase activity and increases urinary Na excretion in obese Zucker rats. We hypothesized that AT2 receptor has a protective role against blood pressure increase in obese Zucker rats. To test this hypothesis, we treated obese Zucker rats with the AT2 receptor antagonist PD123319 (30 μg/kg/min) or AT2 receptor agonist CGP42112A (1 μg/kg/min) using osmotic pumps. Age‐matched lean and vehicle treated obese Zucker rats served as controls. On day 15 of the treatment with PD and CGP, mean arterial blood pressure (MAP) was measured by cannulation of the left carotid artery. Control obese rats exhibited higher MAP (122±3) versus lean control rats (97±5). The PD‐treatment of obese rats further raised MAP by 13 mmHg and CGP‐treatment decreased the MAP by 11mmHg. Western blot analysis revealed that the PD‐treatment in obese rats caused ~3‐fold increase in the renin expression in the kidney cortex, but had no effect on the expression of the cortical AT1 and AT2 receptors. The present study suggests that the AT2 receptors play a significant role in long‐term blood pressure regulation in obese Zucker rats, and this regulation, in part, could be due to the ability of the AT2 receptors to keep the kidney renin expression low in obese rats (NIH R01‐DK‐61578).
Synthesis of aldosterone in the brain contributes to the hypertension in the Dahl salt sensitive rat
Authors: Gomez Sanchez Elise Peery , Gomez‐Sanchez Clara M , Gomez‐Sanchez Celso E
Published: June 2021

Despite low or normal plasma aldo levels, the salt‐induced hypertension in Dahl SS rats is mitigated by the same pharmacological maneuvers and brain ablations that prevent mineralocorticoid‐salt excess hypertension. We found that all enzymes required for de novo synthesis of aldo are expressed in rat and human brain and that aldo is synthesized in the normal rat brain in vivo and in vitro in quantities that suggest autocrine or paracrine functions. mRNA for aldo synthase was measured by real time RT‐PCR and aldo by an extraction/ELISA assay in brains. SS rat brains had significantly greater amounts of aldo synthase mRNA in the brain stem, hypothalamus, hippocampus and cerebellum and aldo concentrations compared to control brains, though SS plasma aldo levels tended to be lower. The central infusion of the specific aldo synthase enzyme inhibitor, FAD286, significantly and reversibly lowered the blood pressure of SS rats. Many hypertensive patients benefit from MR antagonists, even though plasma levels of aldo are normal to low. As MR are most abundant in the hippocampus, where they mediate trophic responses necessary for learning and memory, it is important to determine the relevance of this model to human hypertension and devise more specific methods of blood pressure control.

This work was supported by Medical Research funds from the Department of Veterans Affairs and NIH grants HL27255 and HL75321.
Overexpression of a HIF prolyl‐4‐hydroxylase Transgene in the Renal Medulla Increases the Salt Sensitivity of Arterial Blood Pressure
Authors: Xia Min , Wang Zhengchao , Zhang Fan , Li Pin‐Lan , Li Ningjun
Published: June 2021

We have demonstrated that HIF prolyl‐4‐hydroxylase domain‐containing proteins (PHDs), a family of enzymes that promote HIF‐1α degradation, are abundantly present in the renal medulla and regulate sodium excretion through regulation of HIF‐1 α and its targeted genes, such as nitric oxide synthase, cyclooxygenase and heme oxygenase. The present study was designed to test the hypothesis that overexpression a PHD transgene in the renal medulla increases the salt sensitivity of blood pressure. Plasmids expressing PHD2, the predominant isoform of PHDs in the kidneys, were transfected into the renal medulla in uninephretomized rats. Three weeks after PHD2 transfection, the mRNA levels of PHD2 was increased by two folds in the renal medulla. Mean arterial pressure measured by a telemetry system was significantly raised in PHD2 transfected rats after high salt challenge compared with control animals (131.3 ± 6.7 vs. 110.7 ± 4.2 mmHg). There was no blood pressure change in PHD2 transfected rats that were maintained on the normal salt diet. Urinary sodium excretion in response to acute sodium loading or elevation of renal perfusion pressure were blunted by 30% and 50%, respectively, in PHD2 transfected animals. In conclusion, PHD2 in the renal medulla importantly participates in the regulation of renal medullary function and in the long‐term control of arterial blood pressure. (Supported by NIH grant DK54927 and HL89563)
Serum aldosterone regulation, blood pressure response to high salt intake and renal function and structure in adult female offspring are modified by salt overload during pregnancy
Authors: Rodrigues Edson Nogueira Alves , Oliveira Ivone B. , Furukawa Luzia NS. , Ferreira Daniele N. , Coelho Michella S. , Heimann Joel C.
Published: June 2021

Objective

To evaluate the effects of high salt intake during pregnancy on blood pressure (BP), renal function and structure and serum aldosterone levels in adult female offspring (off).

Methods

Female Wistar rats were fed normal (NS, 1.3%) or high (HS, 8% NaCl) salt diet during pregnancy. During lactation all dams received NS as well as the off from weaning until the 21st week of age. Starting on the 21st week of age, 50% of each off group was fed high (hs, 4% NaCl) salt diet during 15 weeks.

Results (mean ± SEM, p<0.05, n=5‐8/groups)

Tail‐cuff BP (TcBP, mmHg) was not different between HS (109±5) and NS (105±2) off groups. However, TcBP was higher in HShs (117±4) compared to NShs (106±2). Kidney weight (g/kg) was higher in HShs (3.7±0.1) compared to HS (3.3±0.1) but not in NShs (3.5±0.1) compared to NS (3.4±0.1). Twenty‐four hour urinary protein excretion (mg/100g/day) was higher in HShs (0.9±0.1) compared to HS (0.5±0.05) but not in NShs (0.7±0.03) compared to NS (0.6±0.09). Serum aldosterone (pg/ml) was lower in NShs (824±62) compared to NS (1128±11) but was not different between HShs (919±60) and HS (988±172) off groups.

Conclusions

HS during pregnancy is associated with BP and renal abnormalities in adult female off. Adult off from HS dams are hyperresponsive to salt overload possibly due to an alteration of the regulation of serum aldosterone.

Supported by CNPq.
BK channel function, venous tone and blood pressure regulation
Authors: Xu Hui , Kandlikar Sachin
Published: June 2021

Impaired large‐conductance Ca2+‐activated K+ (BK) channel function in smooth muscle cells (SMC) causes an increase in intracellular Ca2+ and vascular tone in arteries in hypertension. There have been few studies of BK channel function in venous SMC. Venomotor tone contributes to blood pressure (BP) regulation and increased venomotor tone occurs in human and experimental hypertension. Venous SMC BK channel function was studied using molecular, cellular and integrative approaches in normotensive rats. Unitary BK currents were recorded using inside‐out patches of myocytes from acutely isolated mesenteric veins (MV) SMCs. Currents were sensitive to voltage and [Ca2+]i. Whole cell recordings from MV SMCs revealed spontaneous transient outward currents (STOCs). STOCs were blocked by TEA (1 mM) indicating that BK channels are functional in MV SMCs. Immunocytochemical and Western blot techniques confirmed the presence of BK channel α‐ and β1‐subunits in MV SMCs. Ex vivo studies of the exposed mesentery of anesthetized rats revealed that local block of BK channels by iberiotoxin (IBTX, 100 nM) caused greater constriction of MV compared to mesenteric arteries (MA). Acute inhibition of BK channels with IBTX (0.3 μM/kg, iv) in vivo increased arterial BP and reduced venous capacitance, measured as an increase in mean circulatory filling pressure (MCFP) in conscious rats. Increased MCFP caused by IBTX was more sustained than the increase in arterial BP. These data show that MV SMCs express functional BK channels which regulate venous tone. BK channels play a larger role in regulation of MV than MA tone. These data also show for the first time that BK channels contribute to the regulation of venous tone in vivo, since acute blockade with IBTX increased MCFP. (Supported by P01 HL070687)
Reduction of heart rate, but not heart rate variability, in diabetic rats
Authors: Cupples Will , Lau Catherine , Obara Robert , Koeners Maarten
Published: June 2021

Reduction of heart rate is an early event in type 1 diabetes mellitus (DM) in a number of species including rats, rabbits, and humans. This reduction has been attributed to baroreflex dysfunction resulting from increased parasympathetic tone (McDowell et al AJP 1994; 266: H235). DM was induced by streptozotocin in Long Evans rats (intact, n=5 & DM n=7). Systolic pressure did not differ between the groups. 24h average heart rate declined from 362 ± 4 at control to 356 ± 3 at week 3 and 346 ± 4 min−1 at week 11 in intact rats and from 370 ± to 328 ± 6 and 300 ± 6 min−1 in DM (decline p < 0.00003 versus intact). However, heart rate variability, expressed as coefficient of variation, was unchanged. We had previously shown that spectral analysis of blood pressure from heart rate (~6 Hz) to circadian (0.0000115 Hz) readily detects the roughly fivefold increase in blood pressure (BP) spectral power induced by chemoreceptors‐sparing partial baroreceptor denervation. This increase occurs only in the interval from 0.08 Hz out to 0.0004 Hz. Here we used spectral analysis of inter‐beat interval (one hour record) and of BP to compare intact and DM rats at the three time points. Repeated measures ANOVA was used to compare intact and DM rats in the frequency band noted above and in standard very low frequency (0.02 ‐ 0.2 Hz), low frequency (0.2 ‐ 0.6 Hz), and high frequency (0.6 ‐ 3 Hz) bands. As expected there were no differences between groups in the control condition. However there were also no differences between the intact and DM rats at weeks 3 and 11. To the extent that this analysis probes arterial baroreflex is, we can detect no differences between intact and diabetic rats. Funded by Heart & Stroke Fdn of BC & Yukon
PKC modulation of neurovascular coupling
Authors: Vetri Francesco , Xu Hao Liang , Pelligrino Dale Alan
Published: June 2021

Protein kinase C (PKC) has been shown to phosphorylate several ion channels involved in neurovascular coupling. Therefore, we sought to assess whether PKC is involved in the regulation of neurally‐evoked pial arteriolar dilation. Using a closed cranial window technique for vessel diameter monitoring, we studied pial arteriolar dilations during sciatic nerve stimulation (SNS) in rats. Topical application of the PKC non‐isoform specific agonist phorbol 12,13‐dibutyrate (PdBu), was able to significantly reduce the dilatory response to SNS. We previously found that a substantial portion of SNS‐induced arteriolar dilation is mediated by the release of K+ from glia limitans large‐conductance Ca2+‐operated K+ (BKCa) channels, which, in turn, activate pial arteriolar smooth muscle inward rectifier K+ (Kir) channels. Both, BKCa and Kir channels can be phosphorylated by PKC. Thus, we compared BKCa and Kir ‐induced vasodilating function in the presence and absence of PdBu. The reactivity to topical applications of the BKCa channel opener, NS1619, and the Kir agonist, KCl, was found to be significantly decreased when the drugs were co‐applied with PdBu. However, responses to adenosine, SNAP, and hypercapnia were not affected by PdBu suffusion, ruling out the possibility of non‐specific effects. These results point to a significant involvement of PKC in regulating the gliovascular mechanisms underlying neurovascular coupling

This work was supported by grant 3‐2008‐462 from Juvenile Diabetes Research Foundation (JDRF) to F.V., NIH grant HL088259 to D.A.P. and American Heart Association grant 0635337N to H.L. Xu
Differential Anorexigenic Effects of Beta‐Melanocyte Stimulating Hormone in Lines of Chickens Divergently Selected for Low or High Body Weight
Authors: Smith Marissa L. , Cline Mark A. , Siegel Paul B. , Denbow D. Michael
Published: June 2021

The melanocortin system plays a key role in appetite and metabolic regulation in mammals and chicks. The present study was designed to investigate the appetite‐associated effects of beta‐melanocyte stimulating hormone (beta‐MSH) administered at 4 days post hatch in lines of chicks genetically selected from a common founder population for high (HWS) or low (LWS) juvenile body weight for 50 consecutive generations. The HWS chicks are hyperphagic and obese, while the LWS line contains some anorexic individuals. Centrally administered beta‐MSH dose‐dependently decreased food intake in HWS chicks whereas all doses (0.01, 0.1, and 1 nmol) caused a similar magnitude decrease in food intake in LWS chicks. Water intake was decreased in both lines. Central beta‐MSH did not affect whole blood glucose concentrations in either line. Plasma corticosterone was increased in LWS, but not HWS, in response to beta‐MSH. These data suggest that potential differential melanocortinergic appetite regulation between these lines may be due to either direct effects of beta‐MSH on food intake or secondary effects of beta‐MSH on appetite via alterations in the stress response.
NEDD4 mediated degradation of target proteins under simulated microgravity conditions
Authors: Fiedorowicz Katarzyna , Da Silva‐Azevedo Luis , Kuebler Wolfgang Michael , Zakrzewicz Andreas , Pries Axel Radlach
Published: June 2021

The effects of microgravity on human vascular endothelial cells may contribute to altered cardiovascular functions in space. Since endothelial function critically depends on protein synthesis and degradation, we addressed the effects of microgravity on the ubiquitin‐ligase NEDD4 in endothelial cells. Human umbilical vein endothelial cells or EAhy926 cells were exposed to simulated microgravity in the Rotating Wall Vessel (RWV) for 24 h, 48 h and 72 h. Controls were kept under normal gravity or exposed to shear stress using a cone and plate apparatus (6 dyn\cm2, 24 h). Gene expression and protein concentration were analysed by real‐time RT‐PCR and Western blotting, respectively. Our results show a two fold upregulation of NEDD4 under microgravity and no regulation by shear stress (relative to controls 1G, no shear). NEDD4 targets e.g. Cx43, Notch1, eps15, IGF1R, PLCgamma1 were strongly downregulated after 48 h and 72 h exposure to microgravity. These data suggest that NEDD4 expression is sensitive to gravity, which may be a mechanism to enhance protein degradation in endothelial cells.
Nitration of p190RhoGAP secondary to caveolae‐mediated endocytosis increases endothelial junctional permeability
Authors: SIDDIQUI MOHAMMAD RIZWAN , KOMAROVA YULIA , BROVKOVYCH VIKTOR , VOGEL STEPHEN M , MALIK ASRAR B
Published: June 2021

Depletion of caveolin‐1, which increases endothelial nitric oxide synthase (eNOS) activity, may influence the function of endothelial adherens junctions (AJs), thereby increases the junctional permeability. We showed that stimulation of caveolae‐mediated endocytosis increased the activity of eNOS (Miniatis et al., Circ Res. 2006; 99:870‐7). We now show that activation caveolae‐mediated endocytosis augments the increase in endothelial permeability in response to activation of protease activated receptor‐1 (PAR‐1) suggesting a relationship between signals emanating from caveolae and disruption of the junctional barrier. Following endocytosis, eNOS was translocated to AJs, resulting in augmented NO production after PAR‐1 activation. Thus, we addressed the possibility that protein nitration of p190RhoGAP (which constitutively associated with the AJ protein p120‐catenin) occurring secondary to the NO production p190RhoGAP nitration might interfere with the GAP activity, and hence RhoA activation. We observed nitration of p190RhoGAP that led to inhibition of its GAP activity resulting in sustained activation of RhoA. We conclude that signals initiated by caveolae internalization regulate junctional endothelial permeability by activating eNOS and through NO‐mediated inhibition of p190RhoGAP activity.
New connections linking sorting & trafficking of VEGFR2 from Golgi with angiogenesis
Authors: Choudhury Amit , Manickam Venkatraman , Tiwari Ajit , Sierra Ana , Bhattacharya Resham , Pagano Richard E. , Mukhopadhyay Debabrata
Published: June 2021

Vascular endothelial growth factor receptor‐2 (VEGFR2) plays a key role in vasculogenesis and pathophysiological angiogenesis. Cell surface levels of VEGFR2 is regulated by endocytosis and secretory transport of newly synthesized receptors to the plasma membrane. The mechanism of VEGFR2 sorting and trafficking along the secretory pathway is not well defined. In the present study, using human endothelial cells we demonstrate that sorting and trafficking of VEGFR2 from the Golgi is regulated by syntaxin 6, a t‐SNARE involved in intra‐Golgi and post‐Golgi membrane fusion events. Loss of syntaxin 6 function results in altered sorting of VEGFR2 from the Golgi resulting in receptor down regulation. Our data support a model in which syntaxin 6 regulates delivery of VEGFR2 from the Golgi apparatus to the cell surface thereby regulating VEGF‐A‐induced endothelial cell proliferation, migration, and morphogenesis.

Grant Funding Source

NIH
Shp2 dependent FGF signaling in retinal development
Authors: Zhang Xin
Published: June 2021

From Drosophila to mammals, the retina develops in a conserved wave‐like pattern emanating from optic stalk to peripheral region. While Drosophila retinal differentiation is regulated by Hedgehog and Receptor Tyrosine Kinase (RTK) signaling in a sequential‐induction fashion, whether such mechanism applies to vertebrate retinal development remains controversial. Previous studies have also implicated Fibroblast Growth Factor (FGF) signaling in specifying retina versus retinal pigmented epithelium (RPE) fate during early eye development, but the molecular mechanism of such profound effect of FGF signaling is still lacking. Shp2 protein tyrosine phosphatase is a critical mediator of many RTK pathways, including FGF signaling. We have determined the mechanism of FGF signaling in retinal development by studying the genetic ablation of Shp2, providing compelling genetic evidence that Shp2 is dispensable for the retinal differentiation wave or maintenance of retinal cell fate. In contrast, our results suggest that Shp2 signaling functions in early patterning of the optic vesicle.
What fruit flies teach us about RNA silencing.
Authors: Zamore Phillip D
Published: June 2021

RNA silencing is remarkably similar between fruit flies and man. Like humans, flies have three distinct RNA‐guided mechanisms to silence genes: the microRNA (miRNA), the siRNA‐guided RNA interference (RNAi), and Piwi‐interacting RNA (piRNA) pathways (1). These three classes of small RNAs are produced by different pathways from different types of precursor RNAs, but each functions as a sequence‐specific guide for a member of the Argonaute family of proteins (2). Much of what we know about these three types of small silencing RNAs was learned through biochemical and genetic studies in the model organism, Drosophila melanogaster (3‐5). For each pathway, the fundamental mechanisms for small RNA biogenesis and function were discovered first in flies, then in man. In flies, each pathway serves a unique and largely non‐overlapping function. We know that miRNAs regulate the expression of protein coding genes, RNAi defends against viral infection and transposons in the soma, and piRNAs protect the germ line from selfish genetic elements such as transposons. New functions for small silencing RNAs continue to be discovered.

What are the lessons taught us by flies and how can we apply them to develop robust RNAi strategies for drug‐target discovery and human therapy? Finally, what discoveries in flies remain to be translated into improvements in triggering RNA silencing in mammals?
Small RNA metabolism and function in Arabidopsis
Authors: Chen Xuemei
Published: June 2021

The transcriptome of plants includes diverse species of 21‐24 nucleotide small RNAs, which are composed of microRNAs (miRNAs) and endogenous small interfering RNAs (siRNAs). miRNAs regulate target mRNAs through transcript cleavage or translational inhibition and play crucial roles in various developmental and physiological processes. Endogenous siRNAs trigger and maintain DNA and histone methylation leading to heterochromatin formation.

We have been studying the biogenesis, degradation, and function of small RNAs in Arabidopsis. We showed that plant small RNAs are 2′‐O‐methylated by the small RNA methyltransferase HEN1 on the 3' terminal ribose and that the methyl group protects small RNAs from a 3′‐5' exonuclease that degrades small RNAs and from a so‐called uridylation activity that adds a short U tail to small RNAs. Subsequently, animal siRNAs and piRNAs were found to be 2′‐O‐methylated by HEN1 homologs. We have also identified a family of exonucleases that degrades small RNAs and demonstrated that small RNA turnover is crucial for plant development. Finally, we studied the developmental function of one miRNA, miR172, and revealed that it plays a crucial role in the temporal regulation of floral stem cells. These findings in small RNA metabolism and function will be discussed.
Molecular Origami: Protein folding and misfolding in health and disease
Authors: Frydman Judith
Published: June 2021

Folding within the crowded cellular milieu often requires assistance from molecular chaperones, which prevent inappropriate interactions leading to aggregation and toxicity. The contribution of individual chaperones to protein folding and quality control remain elusive. The accumulation of misfolded proteins in intracellular amyloid inclusions, typical of many neurodegenerative disorders including Huntington's and Prion Disease, is thought to arise upon failure of the cellular protein Quality Control (QC) mechanisms. Our work aims to understand the role of chaperones in folding and in the formation of misfolded protein inclusions in the eukaryotic cytosol. Recently, we identified two intracellular compartments for the sequestration of misfolded cytosolic proteins. Soluble ubiquitinated misfolded proteins accumulate in a juxtanuclear compartment where proteasomes are concentrated. In contrast, terminally aggregated proteins are sequestered in a perivacuolar inclusion. Strikingly, disease‐associated Huntingtin and prion proteins are preferentially directed to the perivacuolar compartment. Our findings provide a framework for understanding the preferential accumulation of amyloidogenic proteins in inclusions linked to human disease.
High Content Screening Applications for RNA Therapeutics
Authors: Howell Bonnie , Keough Edward , Cerra Michael , Bahl Amit , Pak Irene , Crawford Randy , Krotzer Steven , Bailey Wendy , Bagchi Ansu , Sepp‐Lorenzino Laura
Published: June 2021

RNA interference (RNAi) is a post‐transcriptional gene silencing process mediated by short, interfering RNA molecules (siRNAs) targeting complementary mRNA. siRNAs prevent protein expression and create an opportunity to use RNAi for drug target identification, validation, and therapeutics for treating human diseases. Successful application requires efficient delivery approaches, a hurdle which can be overcome through packaging siRNA inside lipid nanoparticles (LNPs). To evaluate biological activities of LNPs, we developed innovative assays and microscopy techniques to monitor tissue biodistribution, cell internalization, endosomal escape, and gene silencing. In combination with fluorescent markers, we visualized Cy5‐labeled siRNA encapsulated in LNPs and quantified kinetics of cell uptake and endosomal escape in various cells. Some LNPs displayed defects in cell uptake whereas other internalized into endosomes and distributed rapidly to the cytosol. Use of endocytosis inhibitors reveal clathrin and non‐clathrin coated pits participate, in part, in internalization in hepatocytes. Multiplexed, image‐based assays were employed to evaluate potential cellular toxicities and bioinformatics tools were developed to visualize multiparameter HCS data and mine phenotypic signatures.
Pathway‐specific small molecule probes of cytokinesis
Authors: Eggert Ulrike S.
Published: June 2021

How cells regulate and execute cytokinesis, the final step in cell division, remain major unsolved questions in basic biology. Cytokinesis is a very rapid and dynamic process that involves the coordinated action of the cytoskeleton, cell cycle and membrane machineries of cells. Small molecules, which act quickly and afford high temporal control, are ideal tools to probe this complex process. The Rho pathway plays a key role in the regulation of cytokinesis, but the underlying mechanisms remain poorly characterized. To address this question, we have developed a high‐throughput phenotypic screen to discover Rho pathway‐specific small molecules using a combination of chemical genetics and RNAi. We screened ~50,000 natural product extracts and commercial compounds in Drosophila cells and identified several promising candidates. Small molecules that target the Rho pathway will be important tools to dissect the role of Rho signaling during cytokinesis. In addition, these compounds may catalyze therapeutic studies. This work was funded by DFCI and NIH.
Independent, combined, and duration‐dependent effects of diet and exercise on WAT inflammation and metabolic disturbances in obese C57/BL6 mice
Authors: Vieira Victoria Jeanne , Valentine Rudy J , Natao Nirav , Young Virginia , Woods Jeffrey A
Published: June 2021

White adipose tissue (WAT) inflammation causes insulin resistance (IR) and hepatic steatosis (HS). Exercise training (EX) may decrease inflammation in WAT thereby ameliorating these disturbances, even in the absence of fat loss.

This study compared low‐fat diet (LFD) and EX on WAT inflammation, IR, and HS in obese mice.

Mice (n=109) fed a 45% fat diet (HFD) for 6 wks were randomly assigned to EX (treadmill, 5d/wk; 40 min/d; 65%VO2max) or sedentary (SED) groups. Mice remained on HFD or switched to a 10% fat diet (LFD). After 6 or 12 wks, WAT and liver were weighed and relative fat (rFat) was calculated. HS was assessed by total liver TG, and IR by HOMA. Rt‐PCR was used to determine WAT gene expression (MCP‐1, F480, TNF‐α, IL1ra, leptin). Comparisons were made using 2‐way (EX, LFD, EXxLFD) ANOVA covarying for rFat.

EX and LFD reduced rFat and leptin, which improved when combined. Improvements in IR and HS were explained by reduced rFat at 6 wks. By 12 wks, MCP‐1, F480, TNF‐α, and IL1ra were reduced by EX and LFD. Regression analyses revealed EX, rFat, and WAT inflammation predicted reductions in IR (R2=.75; p<0.01); rFat and LFD predicted improvements in HS (R2=.51; p<0.01). From 6 to 12 wks, EX attenuated increases in rFat, HS, and WAT inflammation, but LFD decreased IL1ra.

Unique metabolic consequences of SED and HFD are evident long‐term, highlighting the importance of both EX and LFD in preventing obesity‐related metabolic disturbances.

Grant Funding Source

NIH T32 DK59802
Leucine contents of isonitrogenous protein sources predict post prandial skeletal muscle protein synthesis in rats fed a complete meal
Authors: Norton Layne Eiseman , Wilson Gabriel J , Rupassara Indu , Garlick Peter J , Layman Donald K
Published: June 2021

Leucine (leu) is recognized as a nutrient signal for regulation of muscle protein synthesis (MPS), however previous research has been limited primarily to acute studies with free amino acids (J. Nutr. 133:1553S‐6S, 2005). It is unclear if isonitrogenous protein sources containing different levels of leu will produce differential MPS responses in animals adapted to meal feeding. This study examined 4 different protein sources: wheat (supplemented with lysine), soy, egg, and whey, (containing 6.8, 8.0, 8.8, and 10.9% leu respectively) on the potential to activate initiation factors and MPS. Male rats (250g) were trained for 14d to eat 3 meals/d consisting of 16/54/30% of energy from protein, carbohydrates and fats. Rats were sacrificed after 13hr fast or 90min after consuming their normal breakfast. Measurements included plasma insulin, glucose and amino acids, MPS via D5‐Phe incorporation, and phosphorylation of initiation factors 4E‐BP1 and S6K. Wheat and soy did not stimulate MPS above fasted levels whereas egg and whey significantly increased MPS with whey greater than egg. MPS responses were closely related with changes in plasma leu and phosphorylation of 4E‐BP1 and S6K. These findings suggest that leu content of protein sources is a critical factor in determining protein quality as it relates to changes in post prandial MPS responses. Supported by National Dairy Council, Egg Nutrition Center, Illinois CFAR.

Grant Funding Source

Egg Nutrition Center and National Dairy Council
Aspirin‐triggered lipoxins enhance resolution of myeloperoxidase‐mediated lung inflammation by promoting neutrophil apoptosis
Authors: Filep Janos G , Jozsef Levente , Pan Wanling , Wang Lili , Petasis Nicos A , Serhan Charles N , El Kebir Driss
Published: June 2021

We have reported that myeloperoxidase (MPO) prolongs carrageenan‐induced lung injury parallel with suppression of neutrophil apoptosis in a mouse model of inflammation. Since aspirin‐triggered 15‐epi‐lipoxin A4 (15‐epi‐LXA4) overrides MPO signaling in neutrophils in vitro, we investigated the impact of 15‐epi‐LXA4 on the resolution of lung inflammation. Treatment of mice with 15‐epi‐LXA4 accelerated the resolution of established inflammation when administered intravenously at the peak of inflammation evoked by carrageenan plus MPO. 15‐epi‐LXA4 produced decreases in bronchoalveolar neutrophil accumulation and increased the number of monocytes/macrophages. 15‐epi‐LXA4 reduced lung injury, tissue MPO content, attenuated edema formation and IL‐6 release. 15‐epi‐LXA4 augmented DNA fragmentation and caspase‐3 activity in bronchoalveolar neutrophils. Decreases in neutrophil number occurred parallel with increases in the percentage of apoptotic neutrophils and the percentage of macrophages containing apoptotic bodies. Co‐treatement with the pan‐caspase inhibitor zVAD‐fmk fully abolished the beneficial actions of 15‐epi‐LXA4. Our results indicate that aspirin‐triggered 15‐epi‐LXA4 overrides the anti‐apoptosis signal of MPO in neutrophils, thereby demonstrating a hitherto unrecognized mechanism by which aspirin could promote resolution of inflammation. (Support: CIHR MOP‐64283).
Evolution and function of a novel clathrin isoform
Authors: Brodsky Frances M. , Esk Christopher , Hoshino Sachiko , Vassilopoulos Stéphane
Published: June 2021

Clathrin forms a lattice‐like intracellular membrane coat to organize adaptor molecules that interact with transmembrane protein cargo. This process facilitates protein sorting at the plasma membrane, on endosomes and in the trans‐Golgi network (TGN). The CHC17 isoform of the clathrin heavy chain is ubiquitously expressed in vertebrate tissues and plays a key role in receptor‐mediated endocytosis and lysosome biogenesis. A CHC17 ortholog is present in all eukaryotes where it performs related functions. During vertebrate evolution, a second isoform of clathrin heavy chain CHC22 arose by gene duplication. CHC22 is expressed highly in human skeletal muscle and adipocytes and at lower levels in other human cell types. CHC22 function is restricted primarily to the endosome‐TGN region of the cell, where it influences retrograde transport from endosomes to the TGN. In muscle cells and adipocytes, CHC22 regulates the formation of the storage compartment from which the GLUT4 glucose transporter is released upon insulin stimulation. The CHC22 gene was found to be a pseudogene in mice, revealing species‐restricted differences in membrane traffic components involved in glucose homeostasis. CHC22 appears to function by competition with CHC17 for clathrin adaptors. When introduced as a transgene into mice, it alters membrane traffic pathways involved in GLUT4 function and induces diabetic symptoms. Consistent with this observation, CHC22 distribution is altered in human patients with type 2 diabetes. This novel and species‐restricted function of CHC22 clathrin contributes to evolutionary diversity of metabolic pathways that have relevance to understanding human diabetes.
Signaling Networks in Chemotaxis and Cytokinesis
Authors: Devreotes Peter N. , Borleis Jane , Cai Huaqing , Franca‐Koh Jonathan , Swaney Kristen , Kamimura Yoichiro , Long Yu , Rahdar Meghdad , Tang Michelle , Willard Stacey
Published: June 2021

The mechanisms of sensing shallow gradients of extracellular signals is remarkably similar in Dictyostelium amoebae and mammalian leukocytes. An extensive series of studies have indicated that the upstream components and reactions in the signaling pathway are quite uniform while downstream responses such as PIP3 accumulation and actin polymerization are sharply localized towards the high side of the gradient.

We have recently shown that activation of TorC2 and PKB occurs at the leading edge of chemotaxing cells and plays a critical role in directed cell migration. Within seconds of stimulation of chemotactically sensitive cells, two PKB homologs, PKBA and PKBR1, transiently phosphorylate at least 10 proteins. The enzymes are activated by phosphorylation of their hydrophobic motifs (HMs) through TorC2. Cells deficient in both PKBs or TorC2 lack most of the phosphorylated substrates and are specifically impaired in directional sensing. Activation of PKBA requires PIP3 while activation of PKBR1 is independent of PIP3. This redundant circuit explains previous observations on the role of PIP3 in chemotaxis. In cells lacking PTEN, high levels of PIP3 overactivate PKBA and impair chemotaxis by triggering many extraneous pseudopodia. In the absence of PIP3, chemotaxis can occur almost normally because PKBR1 is activated by TorC2 independently of PIP3.