bluuebunny/crossref_metadata_2025 huggingface dataset:
Methionine kinetics and first pass disappearance in healthy adolescents
Authors: Hsu Jean W‐C , Castro Danny , Heird William C , Castillo Leticia
Published: June 2021

Methionine, an indispensable amino acid serves as precursor for important substrates. There are no data on methionine first‐pass disappearance (SU) and whole body kinetics in healthy children receiving methionine by the oral or intravenous route. We studied four healthy adolescents, (age 15.0±1.2 y; wt 51.9±6.3 kg) receiving a 2 day dietary period providing adequate energy and protein intakes. On day 3 the subjects received a dual, primed, 8h, continuous tracer infusion of L‐[13C]‐ and L‐[2H3]‐methyl methionine by the intravenous and enteral routes respectively, while consuming small isocaloric and isonitrogenous meals every 30 min. On the next day the route of tracer administration was reversed. HCY/Meth ratios were 0.89 and 0.66 respectively for the oral and IV tracers, while the SU of 13C‐ and 2H3‐methionine labels were 29±0.13% and 31±0.13 % respectively. Methionine transmethylation (TM), remethylation (RM) and transulfuration (TS) were 33±10, 14±5 and 18±6, and 23±3, 11±2 and 11±4 µmol/kg/h respectively, for the oral and IV tracer routes using HCY enrichment as precursor; and 29±19, 13±5 and 16±5 and 15±4, 8±2 and 7±4 µmol/kg/h respectively, for the oral and IV tracer routes when methionine enrichment was used. In this preliminary data, methionine SU in healthy adolescents was between 29‐31% and about 50% of methionine plasma flux was utilized for TM when the tracer was given by the oral route, while 22 and 28% were utilized for TS and RM respectively. Hence, the intestinal tract is a major site of whole body methionine TM. NIH DK‐62363, T32 HD‐007445, Ajinomoto 3‐ARP; USDA/ARS 25337387
Fast intestinal barrier recovery following ischemia/reperfusion damage in the human gut: myosin light chain mediated closure of the villus tip
Authors: Thuijls Geertje , Grootjans Joep , Derikx Joep PM , Heyer Frans , Matthijsen Robert , Heineman Erik , van Dam Ronald M , Dejong Kees HC , Buurman Wim A
Published: June 2021

In a recently developed human model of intestinal ischemia reperfusion (I‐IR) a rapid reversal of intestinal damage by shedding of injured enterocytes and villus tip closure was described. Here we investigated the putative involvement of myosin light chain kinase (MLCK) mediated actin contraction in villus tip closure.

During surgery, an isolated part of the jejunum scheduled to be removed was subjected to 30 min ischemia and subsequent reperfusion. Immunohistochemistry was used to study the localization of MLCK, phosphorylated‐MLC (pMLC), filamentous actin and claudin‐3. Protein levels of MLCK, pMLC and claudin‐3 were semi‐quantitatively analyzed using western blot. Intestinal claudin‐3 production was determined by qPCR.

After 30 min ischemia subepithelial spaces were noted as retractions of the basal membrane. A 30 min reperfusion period resulted in disintegration of epithelial lining at the villus tip, with epithelial cell damage and loss of claudin‐3. Both MLCK and pMLC increased following ischemia and were distributed around the disrupted villus tips after 30 min of reperfusion (MLCK; 918±430 versus 641±338 INT, P<0.01) (fig 1). After 60 min reperfusion complete barrier recovery was observed.

This study shows fast gut barrier recovery following I‐IR damage in the human gut after myosin light chain kinase mediated closure of the villus tip.

Figure 1

A) Before ischemia

B) After 30 min ischemia

C) After 30 min reperfusion

D) After 60 min reperfusion
Renal Proteomics in Rodent Meckel Gruber Syndrome
Authors: Gattone Vincent , Mason Stephen , Witzmann Frank , Ringham Heather , Lai Xian Yin
Published: June 2021

Wpk rats are a viable model for human Meckel Gruber syndrome (MKS), allowing a disease stage proteomic analysis of renal tissue. We hypothesize there are differentially expressed proteins associated with the inherited cystic trait and others associated with renal dysfunction in the later stage. These represent both mechanistic and diagnostic biomarkers for disease inheritance and progression. Wistar‐Wpk/Wpk rats are compared with intralitter normals at 10 days (early stage disease) and 21 days (late stage). Rats were anesthetized; kidneys flushed with 0.9% saline, and frozen in liquid nitrogen until all specimens were collected. Kidneys were homogenized in IPG (urea, diothiotreitol, and CHAPS) solution and 750 ug of protein was loaded for 2‐D electrophoresis. The stained gels were analyzed with Progenesis SameSpots software (Nonlinear Dynamics, Ltd.) which identified a total of 72 spots with a statistically different, normalized staining intensity in at least one group. We identified five distinctly interpretable patterns of protein expression which identify changes associated with cystic phenotype and disease progression/azotemia. Pattern 1 reflects proteins elevated while Pattern 2 reflects proteins decreased in cystic disease. Pattern 3 reflects proteins that are enhanced in cystic disease, but increase with the extent of the cysts. Patterns 4 are proteins that are increased in late stage cystic disease. Pattern 5 reflects proteins down regulated in response to the uremia. Selected spots/proteins which match these patterns are analyzed by LC‐MS/MS. The Wpk model of MKS presents an interesting study in differential protein expression. Characterization of protein changes may provide insight into the mechanisms of renal cyst development and lead to diagnostic biomarkers of disease progression.
Acute pulmonary eosinophilia by i.t. carbon nanotubes: a DAMP trigger
Authors: Herndon Betty , Halderman Charles , Quinn Tim , Nalvarte Elisabet
Published: June 2021

Carbon‐based nanoparticles have promising applications in nanomedicine, electronics and drug delivery. However, lung toxicity studies in rodents continue to reflect tissue effects. Our in vivo studies with single wall carbon nanotubes (SWCNT) administered intratracheally in pulmonary surfactant (50 mg in 50 microliters i.t.) produced a rapid pulmonary eosinophilia and airway mucin upregulation within minutes. Nanotube clusters produced pulmonary tissue granulomas within 1 week, at which time mucin‐laden macrophages, some with nanoparticle fibers, traveled to the mesothelial space and the draining lymph nodes. The responses of the SWCNT‐treated rat airway and mesothelium was of interest given the known response of human mesothelium to fibers such as asbestos. Toxicity of SWCNT on Met‐5A human mesothelial cells was investigated in vitro. Viability was evaluated in the presence and absence of surfactant with increasing doses of SWCNT. Cell toxicity, evaluated by WST‐1 assay showed TC50 of 63 μg/mL without and 70 μg/mL with 20% surfactant in the culture medium, a nonsignificant difference. Transmission electron microscopy on mesothelial cells treated with SWCNT showed intracellular nanotube clusters indenting the nuclear membrane with projecting fibers. These data suggested that the SWCNT‐induced rapid eosinophilia might be triggered by such damage, through cellular release of endogenous signals. Our early efforts with nuclear protein HMGB1 in tissue suggests that the SWCNT are responsible for a "DAMP‐type" trigger of eosinophilia in vivo in the rat model. TEM efforts with pleural fluid and nodes continue to trace the path of the SWCNT in the pulmonary‐ mesothelial interface.
Changes in infrared spectra in a liquid polydimethylsiloxane (Dow Corning MDX‐4‐4011) after passive contact exposure to certain aqueous media
Authors: Shanklin Douglas Radford
Published: June 2021

The repetitive molecule dimethylsiloxane has asymmetry with methyl groups on one side and alternating silicon‐oxygen on the other. This arrangement enhances chemical attack on the siloxane bonds. Passive layered exposure at room temperature of MDX‐4‐4011 to normal saline (0.9% NaCl w/v), a potassium equivalent (0.9% KCl w/v), and Betadine solution (Purdue Frederick Co., Norwalk, CT, povidone‐iodine, 10%) for 12 hours and 13 and 66 days yielded FTIR changes in energy level (transmission/absorbance) and wave number specificity in the siloxane signature zone, and both. These were complex and varied between solutions. The effects of 0.9% NaCl in the signature range 1062 ± 40 λwere:

Transmission after exposure to 0.9% KCl was 4% after 12 hours, 0% after 13 days, and 0.5% after 66 days; after 10% Betadine it was zero after both 12 hours and 12 days, and 1% after 66 days. Changes in wave number were similar to those after saline. Decreased transmission is equated to molecular disorganization and silanolization of polydimethysiloxane. These passive effects of water and ions are indicative of first stage siloxane decomposition in a bioenergetic environment. Internal funding only.
Enteric neural crest cell migration
Authors: Anderson Richard
Published: June 2021

The formation of a functional enteric nervous system requires the coordination of a complex web of interactions, facilitating the migration, proliferation and differentiation of precursor cells within the gastrointestinal tract. During development, the enteric nervous system is derived from neural crest cells that emigrate from the post‐otic hindbrain ("vagal" level) into and along the developing gut. In mice, vagal neural crest cells enter the developing foregut around E9.5 and migrate rostrocaudally in chains to colonise the entire length of the gut by E14.5. The migration of enteric neural crest cells into and along the gastrointestinal tract is a critical process. Failure of neural crest cells to colonize the entire length of the gastrointestinal tract results in a region of the gut that lacks a functional enteric nervous system. In humans, this condition is called Hirschsprung's disease. Our research is focused on understanding the cellular and molecular mechanisms that influence enteric neural crest cell migration into and along the developing gut. We have shown that the migration of enteric neural crest cells is influenced by multiple factors, including: (i) factors that are secreted by the gut mesenchyme, which regulate the entry of neural crest cells into the gut and promote neural crest cell migration along the gut; and (ii) neural crest cell‐cell interactions, as we have shown that cell‐cell interactions are required for the chain migration of neural crest cells and that changes that altered the equilibrium of these interactions disrupt migration. Together, these studies highlight that enteric neural crest cell migration is a complex process, requiring the integration of multiple factors.
Calreticulin‐Knockdown Suppresses Cell Proliferation, Migration and Adhesion in Human Bladder Cancer Cells
Authors: Lu Yi‐Chien , Chang Cheng‐Chi , Lee Hsinyu
Published: June 2021

Bladder cancer is a common urothelial cancer. Through proteomic approaches, calreticulin (CRT) has been identified as a urinary marker for bladder cancer. Calreticulin is a multifunctional Ca2+‐binding molecular chaperone in endoplasmic reticulum (ER) and regulates various cellular functions such as Ca2+ homeostasis, gene expression, and cell adhesion. In the present study, J82 bladder cancer cells stably transfected with RNAi of CRT were generated (J82 CRT‐RNAi) to investigate the physiological effects of CRT in bladder tumor. The results showed that knockdown of CRT expression suppressed cell proliferation, migration and adhesion. We further demonstrated that the mRNA expression and protein secretion levels of vascular endothelial growth factor‐A (VEGF‐A), an important angiogenic factor, were also decreased in these cells. In addition, we also observed that compared to control vector transfected cells, tumors derived from J82 CRT‐RNAi cells were significantly smaller in nude mice. These results suggested that CRT may play an important role in bladder tumor formation.
A novel role for p12/CDK2AP1 cell cycle regulator in inhibiting cancer cell growth
Authors: Zolochevska Olga , Figueiredo Marxa L
Published: June 2021

Prostate cancer is the second most prevalent type diagnosed in men in the western world, and since early‐stage cellular growth is dependent on androgens, androgen deprivation has been used as the main therapy intervention. However, following initial responsiveness, tumors relapse to an androgen‐independent state that ultimately leads to mortality, suggesting that alternatives are needed to enhance therapy efficacy. Since antiandrogens promote cell cycle arrest in part by decreasing CDK2 activity, we hypothesized that this effect could be enhanced by a novel CDK2‐associating molecule, p12/CDK2AP1. We assessed the effects of antiandrogen bicalutamide (cdx), p12, or both on cell cycle; both cdx and p12 induced cell cycle arrests, with a G1 arrest predominant for cdx‐treated, and either G1 or S‐G2 arrests for p12‐expressing cells. Interestingly, combination of cdx/p12 greatly augments the G1 arrest. Apoptosis was most prevalent with cdx/p12 and in the Lapc4 line. CCK8 assay revealed that the cdx/p12 combination was the most effective in reducing viability (up to 60%), colony formation ability, and cellular invasion (by up to 73%). Finally, although the effects of cdx or p12 varied among androgen‐dependent and ‐independent cell lines, the cdx/p12 combination was effective in reducing growth in all lines examined, representing a promising and novel combination for prostate cancer treatment.
Application of FISH Technique in Invasive Breast Cancer Diagnosis
Authors: Salameh Yasmeen M. , Al‐Kahlout Basema I , Bargal Hala W. , Afifi Nahla M.
Published: June 2021

Breast cancer is the leading cause of cancer death among women worldwide. The aim of this study is to improve the prognosis of Breast Cancer in women living in State of Qatar by accurate diagnosis. In the study 70 Breast Cancer patients (year 2000 ‐ 2006) were included. It was found that the mean diagnostic age of breast cancer patients were (46.9 ± 10.4), the majority (48.4 %) of patients was obese (P‐value 0.006) and all of them were positive for estrogens (E) and progesterone (P) receptors. Invasive Ductal Carcinoma (IDC) was the most common type found (93.3 %).

Immunohistochemical investigations revealed 30% of samples were negative for the expression of (E) & (P) Receptors. On the other hand HER‐2/neu testing by Immunohistochemistry showed that 22.9 % of the subjects were diagnosed as negative for the HER‐2/neu protein and 76.1 % subjects with positive HER2/neu receptor (54.9 % +2 expressing, 22.2 % +3 or higher)

By FISH application to the selected cases, of the total Border line area of IHC tested for HER2 (+2 expression), we have found 60% of them were actually positive for the gene amplification, while 24% were found to be negative. Unfortunately 16% of the +2 expressing cases couldn't be determined for HER2 amplification due excessive denaturation. As FISH is more accurate than IHC in diagnosis of HER‐2 positive cases it's recommended to use FISH in diagnosis to make better prognosis and further training to master FISH technique to avoid any technical error. This study was funded by QU & UREP.
T(14;18) TRANSLOCATION: A POTENTIAL MARKER OF HIGH‐GRADE TRANSFORMATION IN HCV‐ASSOCIATED MALT LYMPHOMAS
Authors: Libra Massimo , Malaponte Grazia , Bruni Bibiana , Skarmoutsou Evangelia , Vella Nadia , Mazzarino Clorinda , Stivala Franca
Published: June 2021

MALT lymphomas account for approximately 8% of non‐Hodgkin's lymphomas (NHLs) and occur mostly in the gastrointestinal tract, often as a result of chronic stimulation of B‐cells triggered by autoimmune processes or persistent infection, such as Helicobacter pylori or Hepatitis C Virus (HCV) infection.

Among the several genetic abnormalities involved in MALT development, we have recently demonstrated that t(14;18) translocation in MALT lymphomas clusters among HCV‐infected patients. It has been shown that t(14;18) translocation is also detected in diffuse large B‐cell lymphoma (DLBCL) originated from low‐grade B‐cell lymphoma. MALT lymphomas may undergo histological transformation to high‐grade lymphoma. Therefore, we hypothesized that t(14;18) may play a role in the MALT lymphoma progression. To this aim we analyzed t(14;18) translocation in tumor DNA samples obtained from metachronous biopsy specimens from patients with MALT lymphoma that developed DLBCL in the presence and absence of HCV infection. The results of the present study confirm that t(14;18) translocation clusters among HCV‐associated lymphomas and show for the first time that the condition of Bcl‐2‐translocated cases in MALT lymphomas renders them prone to tumour progression in high‐grade.
CHMP1A expression and function in colon tumors
Authors: Wolf B. Christopher , Li Jing , Belogortseva Natalia , Park Maiyon
Published: June 2021

CHMP1A (Charged multivesicular protein 1A) is a member of the ESCRT‐III family that functions in endosome mediated vesicle trafficking and in gene silencing. However, its function in colon tumors has not been reported. Our preliminary data indicates that CHMP1A might function in tumor development since CHMP1A knockout mice exhibited abnormalities in colon tissue. CHMP1A expression and localization was studied in normal and tumor cells and it is inconclusive at this time. However, CHMP1A mRNA is slightly reduced in colon tumors compared to corresponding normal tissues in a human cancer array. CHMP1A showed diffuse expression including the nucleus of the colon tumor compared to normal tissues which showed no nuclear expression. In addition, knockdown of CHMP1A in normal and tumor cells caused an increase in growth compared to the control cells. Collectively, these results are in agreement with our preliminary data indicating CHMP1A is a tumor suppressor in colon. Supported by NIH Grant 5P20RR016477 to the West Virginia IDeA Network for Biomedical Research Excellence
Regeneration advantage in beta‐catenin overexpressing transgenic mice after partial hepatectomy
Authors: Nejak‐Bowen Kari D , Monga Satdarshan S
Published: June 2021

Previously, our lab demonstrated an essential role for β‐catenin in liver regeneration. In order to elucidate the effect of β‐catenin overexpression on regeneration, we utilized recently generated transgenic (TG). Using the partial hepatectomy (PHx) model of liver regeneration, we found a dramatic increase in cell proliferation in TG mice as compared to WT at 40H after PHx, before the onset of proliferation in WT FVB mice that occurs at 72H. Coincident with the earlier onset of proliferation, β‐catenin translocates to the nucleus at 40H post‐PHx in TGs, whereas this translocation does not occur until 72H in WT. Analysis of cyclin D1 expression by Western blotting showed that levels of this cell‐cycle regulator are several‐fold higher in TG than in WT at 40H. The differences in liver weight to body weight ratios between WTs and TGs at all time points after PHx are unremarkable. However, restoration of gross lobular architecture is noted in TG livers harvested 1M after PHx, while WT livers from the same time point show restored mass but not normal lobulation. To determine the mechanism of β‐catenin activation during liver regeneration, we utilized immunoprecipitation studies and found that β‐catenin dissociates from Met earlier in TG mice than in WTs after PHx. This was confirmed by increase in β‐catenin phosphorylation (and thus activation) 40H after PHx in TG mice as compared to T0, while in WT mice, β‐catenin phosphorylation does not increase until 72H tyrosine‐654, suggesting that Met/β ‐catenin interactions play a key role during the initiation of liver regeneration in TG mice. In conclusion, β‐catenin overexpression causes an upregulation of downstream targets such as cyclin D1, which gives these mice a regenerative advantage.
Toxicity caused by Chelidonium majus in primary human, canine, monkey, and rat hepatocytes
Authors: Rudolf Hendrik , Ullrich Anett , Ludwig Kaja , Runge Dieter
Published: June 2021

Herbal extracts of Chelidonium majus have been widely used in European countries and in Chinese herbal medicines for different applications, based on their anti‐viral, anti‐inflammatory and anti‐microbial properties and their use against various liver disorders including cancer. Recently, several cases of liver disease have been related to C. majus intake. Therefore, we have used primary hepatocyte cultures of human, rat, canine and monkey to elucidate the potential hepatotoxicity of C. majus.

Water‐soluble extracts were prepared and incubated with primary hepatocytes. Cellular viability and functions were analyzed by means of lactate dehydrogenase (LDH) and urea release and via the MTT assay. The amounts of different alkaloids present in C. majus extracts were determined by HPLC analysis.

The liquid extract showed a significant concentration‐dependent toxicity in human hepatocytes and at concentrations of 7.5 mg/ml for animal hepatocytes. In addition, C. majus lead to a decrease in urea release. The decline in urea production varied between the different species with regard to cellular sensitivity and to its maximal extent.

The data suggest, that human hepatocytes seemed to be more sensitive to C. majus extracts than canine, rat and monkey hepatocytes, with rat hepatocytes being the most insensitive cells. In addition, these results demonstrate the necessity to use human hepatocytes in order to evaluate possible toxicological effects of complex plant extracts in humans.
Hyperbaric oxygen (HBO2) induces a two‐phase antinociceptive response of unusually long duration in mice
Authors: Chung Eunhee , Zelinski Lisa M , Ohgami Yusuke , Shirachi Donald Y , Quock Raymond M
Published: June 2021

HBO2 is approved by the FDA for limited clinical indications but not for chronic pain. Earlier we showed that HBO2 treatment produces an antinociceptive effect mediated by nitric oxide (NO) and opioid mechanisms [Zylstra et al., FASEB J 22, 711.16 & 711.17, 2008]. In this study, we assessed the antinociceptive effect of multiple HBO2 treatments. Male NIH Swiss mice (20‐30 g) were exposed to HBO2 at 2.5 ATA for 4×60‐min then returned to their home cages for different time intervals before testing with the acetic acid‐induced abdominal constriction test. Results revealed a robust antinociception of at least 6 hr after the last HBO2 treatment. This response was followed a day later by an emerging and equally robust late‐phase antinociceptive effect that surprisingly lasted for two weeks following the last HBO2 treatment. The early phase was antagonized by pretreatment with naltrexone (NTX) and the late phase was antagonized by the selective 5‐HT1A antagonist WAY‐100635 but not NTX. Continuous treatment with NTX or the NO synthase‐inhibitor L‐NAME during the 4 days of HBO2 treatment also reduced the late‐phase response. Based on these findings, we suggest that the early‐phase antinociception involves an opioid pathway, while the longer‐acting late‐phase antinociception involves an NO‐opioid‐5‐HT1A pathway. (Supported in part by NIH Grant GM‐77153, the College of Pharmacy and the Chico Hyperbaric Center.)
Differential involvement of 5‐hydroxytryptamine1A (5‐HT1A) receptors in the antinociceptive effects of hyperbaric oxygen (HBO2) and nitrous oxide (N2O) in mice
Authors: Winkle Christina K , Rich Roxanne M , Chung Eunhee , Shirachi Donald Y , Quock Raymond M
Published: June 2021

Earlier we reported that a 60‐min exposure to HBO2 induced an antinociceptive response that lasted for up to 90 min following exposure [Zylstra et al., FASEB J 22:711.17, 2008]. Recent studies have reported a sustained antinociceptive effect of a selective, high‐efficacy 5‐HT1A receptor agonist in rodents [Deseure et al., Eur J Pharmacol 568:134, 2007]. To determine whether supraspinal or spinal 5‐HT1A receptors might be involved in HBO2‐ or N2O‐induced antinociception, male NIH Swiss mice (20‐30 g) were exposed to HBO2 (2.5 ATA, 60 min) then removed to room air for 90 min or N2O acutely (11 min). Mice were pretreated with intracerebroventricular (i.c.v.) or intrathecal (i.t.) selective 5HT1A antagonist WAY‐100635 prior to antinociceptive testing using the abdominal constriction test. Results show that both i.c.v. and i.t. pretreatments with WAY‐100635 produced comparable and dose‐related antagonism of the antinociceptive response to HBO2. On the other hand, i.c.v. and i.t. pretreatment with WAY‐100635 had no effect on N2O‐induced antinociception. These results suggest that both supraspinal and spinal 5HT1A receptors are involved in the 90‐min antinociceptive response to a 60‐min exposure to HBO2. However, there does not appear to be a role for 5HT1A receptors in the antinociceptive response to N2O. (Supported in part by NIH Grant GM‐77153, the College of Pharmacy and the Chico Hyperbaric Center.)
Exposure to nitrous oxide (N2O) increases levels of nitric oxide (NO) metabolites and β‐endorphin (β‐EP) in ventricular cisternally (VC)‐perfused rats
Authors: Zelinski Lisa M , Ohgami Yusuke , Quock Raymond M
Published: June 2021

Exposure to N2O has been shown to cause neuronal release of β‐EP from rat hypothalamic cells (Zuniga et al., Brain Res. 420:66, 1987). The antinociceptive of N2O in rodents is antagonized by inhibition of NO production (McDonald et al., JPET 269: 601, 1994). The present study was carried out to determine whether NO activity was increased concomitantly with the release of β‐EP in VC‐perfused rats. Male Sprague Dawley rats, 200‐250 g body weight, were anesthetized with urethane and mounted in a stereotaxic headholder. An inflow cannula was inserted into the lateral cerebral ventricle and an outflow cannula was inserted into the cisternum magnum. Artificial cerebrospinal fluid (aCSF) was infused into the lateral ventricle; perfusate fractions were collected from the cisternum and analyzed for NO metabolites and β‐EP content. Exposure to 70% N2O increased perfusate levels of nitrite and nitrate, compared to fractions collected from rats under room air. Exposure to 70% N2O also increased the concentration of β‐EP, compared to levels in fractions collected under room air. These findings indicate a functional link between increased NO activity and neuronal release of β‐EP in response to N2O and support our hypothesis that N2O may stimulate an NO‐dependent neuronal release of β‐EP. (Supported in part by State of Washington Initiative Measure No. 171 and the SURF Program in Pharmacology and Toxicology.)
In vivo characterization of the antinociceptive effects of UFP‐512 and BG‐156: Selective Delta Opioid Receptor Ligands
Authors: Lopez Alexa , Balboni Gianfranco , Martinez Heather , Giuvelis Denise , Negus Steve , Salvadori Severo , Bilsky Edward J.
Published: June 2021

Delta opioid receptor (DOR) agonists have demonstrated efficacy in several preclinical models of pain. In addition, several groups have demonstrated novel antidepressant and anxiolytic effects for this class of compounds. Modification of the N‐terminal tyrosine by 2',6′‐dimethyl‐L‐tyrosine (Dmt) can have significant effects on opioid peptide affinity, selectivity, and efficacy at the DOR. The studies presented here assess the in vivo activity of two structurally related peptides that possess DOR‐selectivity (UFP‐512 and BG‐156). Both compounds produced dose‐related antinociception in the mouse 55°C tail‐flick assay (i.c.v. A50 values of 8.0 and 5.4 nmol, respectively). I.v. administration of these compounds produced limited antinociception (<20% MPE). In contrast, systemic administration of UFP‐512 produced potent, dose‐related antinociception in the mouse acetic acid writhing assay. In Rhesus monkeys, the intramuscular administration of UFP‐512 dose‐dependently reversed capsaicin‐induced thermal hyperalgesia, with a 10 mg/kg dose producing ~50% reversal. The data indicates that these peptides produce antinociception and antihyperalgesia in both rodents and monkeys, and that systemic injections of the compound may act primarily at peripheral DORs. Additional studies are being conducted to confirm receptor selectivity and site(s) of action in vivo.
Assessment of the efficacy and side effect profile of morphine and MMP2200, a mixed delta/mu agonist
Authors: Giuvelis Denise , De Felice Milena , Lowery John , Polt Robin , Porreca Frank , Bilsky Edward
Published: June 2021

We have previously reported on the chemistry and preclinical pharmacology of a series of glycosylated enkephalin analogs. These compounds exhibit high affinity/efficacy at delta and mu opioid receptors (DOR and MOR). The current studies present a more detailed characterization of the pharmacology of MMP2200, a lead glycopeptide that incorporates the disaccharide lactose into the peptide structure. MMP2200 produced dose‐related antinociception in the mouse 55°C tail‐flick assay following a variety of routes of administration. The compound was also effective at reversing thermal hyperalgesia (CFA model) and tactile allodynia (plantar incision) in rats. At equi‐antinociceptive doses MMP2200 produced significantly less stimulation of locomotor activity compared to morphine in mice. MMP2200 produced dose‐related inhibition of gastrointestinal transit and respiration similar to that produced by morphine. In summary, MMP2200 acts as a mixed delta/mu agonist in vivo. This profile may account in part for the potent antinociceptive effects seen in several different rodent models of pain, as well as the decreased propensity to produce locomotor stimulation compared to morphine. For other measures (inhibition of GI transit and respiratory depression), the compound may exert too strong an effect on the MOR to differentiate it from morphine.
Discriminative stimulus effects of DOM in rhesus monkeys: interactions between 5‐HT1A and 5‐HT2A receptor agonists
Authors: Li Jun‐Xu , Rice Kenner C. , Koek Wouter , France Charles P.
Published: June 2021

Many drugs acting on serotonin (5‐HT) systems, including 5‐HT releasers and reuptake inhibitors, indirectly activate multiple 5‐HT receptor subtypes; however, the impact of the interactions between these receptors is not fully understood. In rodents, activity at 5‐HT1A receptors enhances actions at 5‐HT2A receptors, although the generality of this finding is unknown. This study examined interactions between 5‐HT receptor agonists in rhesus monkeys discriminating between saline and 0.32 mg/kg of the 5‐HT2A receptor agonist DOM. The selective 5‐HT1A receptor agonists 8‐OH‐DPAT and F13714 shifted the DOM dose response curve to the right and this effect was reversed by the 5‐HT1A receptor antagonist WAY100635. The non‐selective 5‐HT1A receptor agonist buspirone had no effect while the 5‐HT2A receptor agonists 2C‐T‐7 and dipropyltryptamine shifted the DOM dose response curve to the left. Thus, agonism at 5‐HT1A receptors attenuated the discriminative stimulus effects of a drug acting at 5‐HT2A receptors. That this interaction in monkeys may be opposite to what has been observed in rodents underscores the need to examine these interactions under a broader range of conditions and in multiple species. CPF is supported by a Senior Scientist Award (DA17918).
Effects of cholecystokinin and oxytocin in rats trained to discriminate 22 from 2 hours food deprivation
Authors: Jewett David , Edwards Kayla , Dunbar Andrew , Filtz Catherine , Novinska Noah , Sime Lindsey , Johnson Kelsey , Callahan Jamie , Levine Allen
Published: June 2021

Cholecystokinin and oxytocin have previously been shown to decrease food intake in rats. We tested the effects of cholecystokinin and oxytocin, in rats trained to discriminate 22 hr food deprivation from 2 hr food deprivation in a two‐lever, operant choice task. After rats acquired the discrimination, subjects were food restricted for 22 hr and administered cholecystokinin(1‐10 µg/kg,i.p.) or oxytocin (0.01‐3.2 mg/kg, i.p.) before generalization tests. Food intake was measured for 1 hr after the generalization test. Cholecystokinin dose‐dependently decreased the discriminative stimulus effects of 22 hr deprivation. Oxytocin did not affect significantly affect the discriminative effects of deprivation, but significantly reduced response rate and food intake. The findings support the notion that CCK affects feeding stimulated by energy needs, whereas oxytocin affects feeding via other mechanisms.
Effects of Chronic Exposure to the CB1 Agonist AM 411 in Squirrel Monkeys
Authors: Desai Rajeev I , Wood Lindsey , Thakur Ganesh , Makriyannis Alexandros , Bergman Jack
Published: June 2021

Cannabinoid CB1 agonists produce tolerance that may be associated with dependence and increased sensitivity to the behavioral effects of the CB1 antagonist SR141716A, perhaps indicative of antagonist‐precipitated withdrawal. In the present studies, the effects of the long‐acting CB1 agonist AM 411 and the CB1 antagonist SR141716A on operant responding were examined in squirrel monkeys before and during chronic AM 411 treatment (1.0 mg/kg/day, im). Prechronically, AM 411 (0.01‐1 mg/kg) produced dose‐related decreases in responding; the highest dose decreased behavior to approximately 20% of control values. In contrast, SR 141716A (1‐10 mg/kg) had little effect on behavior; the highest dose decreased responding only to >80% of control values. Re‐determination of drug effects during the chronic regimen revealed a >10‐fold rightward shift in the dose‐response function for AM 411, indicative of tolerance. On the other hand, SR 141716A (0.32‐3.2 mg/kg) produced dose‐related decreases in behavior; the highest dose decreased responding to <50% of control values. These data are consistent with the view that chronic CB1 receptor activation produces tolerance to the effects of CB1 agonists and enhances sensitivity to the behaviorally disruptive effects of CB1 antagonists. The increased sensitivity to the effects of SR 141716A may be indicative of antagonist‐precipitated withdrawal (supported by DA19205).
Roscovitine Prevents Dopamine Mediated Degeneration of Nigral Neurons with Reduced Vesicular Monoamine Transporter2 Expression.
Authors: Shepherd Kennie Raviie , Wang Min Zheng , Miller Gary W
Published: June 2021

The vesicular monoamine transporter 2 (VMAT2) is an abundant protein in the nigrostriatal system, which plays a significant role in the maintenance and homeostasis of the dopaminergic system. For example, VMAT2 is responsible for the packaging of dopamine (DA), preventing its degradation and subsequent production of ROS. In the present study, the addition of physiological concentrations of DA (10µM) to primary nigral neurons isolated from VMAT2 low expresser mice have significant production of ROS, caspase‐3 activation, and loss of tyrosine hydroxylase (TH) positive neurons and processes. In addition, 80% of DA neurons demonstrated increased expression of the cell cycle inhibitor p27 in the cytoplasm, and an increase in Brdu labeling. The neurotoxic effects of DA were prevented by the pre‐treatment of 1mM ascorbate, pargyline (10µM), and nomifensine (20µM), indicative of ROS production via DA metabolism as a mediator of the neurotoxic effects. Furthermore, roscovitine (10‐25µM), a cyclin‐dependent kinase that triggers and coordinates the transition between the G2/M phases of the cell division cycle, prevented loss of DA neurons. Taken together, the results suggest that vulnerable DA neurons undergo cell death via re‐entry into the cell cycle; cell cycle inhibitors may represent potential therapeutics for the treatment of neurodegenerative diseases.
Characterization of As(GS)3 and [(GS)2AsSe]‐ transport by the human multidrug resistance protein 2 (MRP2/ABCC2)
Authors: Carew Michael Wayne , Leslie Elaine MacKay
Published: June 2021

Arsenic (As) is a human carcinogen and toxicant, despite this, the key pathways in As metabolism and excretion are not fully understood. Multidrug resistance protein 2 (MRP2/ABCC2) is expressed at the canalicular surface of hepatocytes and can transport glutathione (GSH or GS‐) conjugates, including As(GS)3. Studies show an increased detoxification of As when administered with the micronutrient selenium (Se). One proposed mechanism for this is through the formation of [(GS)2AsSe]−, which has been isolated from rabbit bile. The purpose of the current study was to investigate the ability of MRP2 to transport As(GS)3 and [(GS)2AsSe]−. Transport activity was measured using membrane vesicles prepared from MRP2 transfected HEK293 cells. As(GS)3 (0.1 μM) transport by MRP2 was maximal at 5 min (7.7 pmol mg−1). The apparent Km and Vmax of As(GS)3 for MRP2 were 4.2 ± 0.9 μM and 134 ± 12 pmol mg−1 min−1, respectively. Transport of [(GS)2AsSe]− (0.1 and 1 μM) showed an ~ 3‐fold increase over As(GS)3. These data support a role for MRP2 in transporting [(GS)2AsSe]−, potentially contributing to As detoxification. Funding was provided by the Alberta Cancer Board, AHFMR and CIHR.
Inhibition of fulminant hepatitis by liquiritigenin, a licorice flavonoid, as a consequence of the induction of hepatic transporters and phase II enzymes
Authors: Kim Young Woo , Kim Sang Chan , Kim Sang Geon
Published: June 2021

Liquiritigenin (LQ), an active component in licorice, has an inhibitory effect on lipopolysaccharide (LPS)‐induced iNOS expression. This study investigated the effects of LQ on the expression of hepatic transporters and phase II enzymes, and on galactosamine/LPS‐induced fulminant hepatitis. Immunoblot and RT‐PCR analyses showed that the protein and mRNA levels of MRP2 and BSEP were increased in the liver of rats treated with LQ. Induction of hepatic transporters was verified by their increased localizations in canalicular membrane. In addition, LQ treatment enhanced the expression levels of major hepatic phase II enzymes. LQ treatments attenuated galactosamine/LPS‐induced hepatitis in rats, as supported by decreases in the plasma ALT, liver necrosis and plasma TNF‐alpha. Consistent with these, intravenous administration of LQ elevated bile flow rate and biliary excretion of bile acid, GSH and bilirubin. These results demonstrate that LQ has the ability to induce transporters and phase II enzymes in the liver, which may be associated with a hepatoprotective and choleretic effect. Our findings may provide insight into understanding the action of LQ and the therapeutic use of licorice for liver disease.
A Cytochrome P450‐derived Epoxygenated Metabolite of Anandamide is a Potent, Cannabinoid Receptor 2 Selective Agonist
Authors: Snider Natasha Tasheva , Nast James A , Hollenberg Paul F
Published: June 2021

Oxidation of the endocannabinoid anandamide by cytochrome P450 (P450) enzymes has the potential to affect signaling pathways within the endocannabinoid system and pharmacological responses to novel drug candidates targeting this system. We previously reported that the human cytochrome P450s 4F2, 3A4 and 2D6 are high‐affinity, high‐turnover anandamide oxygenases in vitro, forming the novel metabolites hydroxyeicosatetraenoic acid ethanolamides (HETE‐EAs) and epoxyeicosatrienoic acid ethanolamides (EET‐EAs). The objective of this study was to investigate the possible biological significance of this metabolic pathway. We report that the anandamide epoxide 5,6‐EET‐EA is a potent and selective cannabinoid receptor 2 (CB2) agonist. The Ki values for binding of 5,6‐EET‐EA to membranes from Chinese hamster ovary (CHO) cells recombinantly expressing either the human CB1 of the CB2 receptor were 11.4 μM and 8.9 nM, respectively. In addition, 5,6‐EET‐EA inhibited the forskolin‐stimulated cyclic AMP accumulation in CHO cells stably expressing the CB2 receptor (IC50 = 9.8 ± 1.3 nM). Within the central nervous system the CB2 receptor is expressed on activated microglia and it is considered a possible target for the treatment of neuroinflammation. BV‐2 microglial cells stimulated with low doses of interferon‐gamma exhibited increased capacity for converting anandamide to 5,6‐EET‐EA which correlated with increased protein expression of microglial P450s 4F13 and 3A1. Finally, we demonstrate that 5,6‐EET‐EA is more stable than anandamide in brain homogenate and it is primarily metabolized by epoxide hydrolase. Combined, our results suggest that epoxidation of anandamide by P450s to form 5,6‐EET‐EA represents an endocannabinoid bioactivation pathway in context of immune cell function.
Comparative characterization of UDP‐glucuronic acid (UDP‐GlcUA) binding‐site directed inhibitors with human UGT2B7 and 1A10.
Authors: Bratton Stacie Moseley , Laakkonen Liisa , Patana Anne‐Sisko , Mazur Anna , Miller Grover P. , Finel Moshe , Radominska‐Pandya Anna
Published: June 2021

Recent crystallography, homology modeling, and mutagenesis studies of human UGTs have demonstrated that UGT2B7 and 1A10 possess a number of distinctive UDP‐glucuronic acid contact amino acids. We have investigated the specificity of these co‐factor binding sites further using 8 bi‐dentate inhibitors containing a lipophilic and a uridine moiety. Ki and IC50 values were calculated for PP55B and inhibition types were determined. Models of the UGT1A10 UDP‐GlcUA binding site were built using the crystal structure of the UGT2B7 C‐terminal domain as a template and using sequence homology to available structures of plant glucosyltransferases with the GT‐B folding motif. We have detected potential differences in the tertiary structure/function of the C‐terminal domain of these two isoforms and subsequently docked PP55B at the UDP‐GlcUA binding site in order to model inhibitor complexes for each enzyme, including its possible location with respect to the N‐terminal domain. Establishing the roles of individual UGT isoforms is important for UGT‐based drug discovery. The goal of theses studies is aimed at the identification of potent, selective UGT inhibitors, which could help to define these roles and provide the basis for the design of therapeutic agents. NIH‐DK60109, GM075893 (AR‐P)
Lysophosphatidic acid (LPA) receptor roles in LPA and serum stimulation of lung fibroblast proliferation
Authors: Leuschen Bridget A. , Schulte Nancy A. , Toews Myron L.
Published: June 2021

Both LPA and serum stimulate lung fibroblast proliferation and contraction, important components of wound healing and of the pathologic airway remodeling in fibrotic lung diseases. LPA and its LPA1 receptor have been identified as critical mediators of pulmonary fibrosis. We previously showed that the LPA1 agonist NAEPA and the LPA3 agonist OMPT stimulated collagen gel contraction by HFL‐1 human lung fibroblasts and that the LPA1+3 antagonist Ki16425 blocked contractions to both LPA and serum (FASEB J 22, A728.9, 2008). The present studies tested these same agents on HFL‐1 proliferation. HFL‐1 cells were serum‐starved, stimulated for 24 hr with various agents, and [3H]thymidine incorporation was assessed. Stimulation by LPA was mimicked by both NAEPA and OMPT and was completely blocked by Ki16425. Thus LPA1 and/or LPA3 receptors mediate LPA stimulation of HFL‐1 proliferation. In contrast, Ki16425 inhibited stimulation by serum by only about 40%. Though LPA is the major serum component for fibroblast contraction, it makes a smaller but significant contribution to serum stimulation of fibroblast proliferation. Supported by an ASPET SURF Award to B.A.L.
A new in vitro model of sepsis‐induced renal epithelial cell injury
Authors: Pathak Elina , Mayeux Philip R
Published: June 2021

Previous studies by our laboratory have shown that sepsis produces a rapid inflammatory response in the kidney that leads to tubular injury. There are no in vitro models that adequately replicate the tubular microenvironment during sepsis. To address this need, we have established a new model of sepsis‐induced renal epithelial injury using serum from septic mice and a mouse renal epithelial cell line. mIMCD‐3 cells (epithelial cells from mouse inner medullary collecting duct) were treated with media containing 5% serum collected from mice at 4h that have undergone sham surgery (Sham) or cecal ligation and puncture (CLP) to induce sepsis. We chose 4h CLP serum because cytokines are elevated at this time. Real‐time PCR analysis showed a time‐dependent induction in iNOS mRNA in cells treated with CLP compared to Sham (1.4 ± 0.5 fold at 6h and 5.3 ± 0.7 fold at 18h; P < .05 compared to Sham). This was accompanied by a two‐fold increase in nitric oxide generation at 6h as measured by total nitrate/nitrite levels (P < .05 compared to Sham). We also measured lactate dehydrogenase release (LDH) to assess toxicity and found a two‐fold increase in LDH at 18h in CLP‐treated cells (P < .05 compared to Sham). These data suggest that exposure of tubular epithelial cells to serum from septic mice is a unique, highly relevant in vitro model with which to study the mechanism of sepsis‐induced kidney injury. Supported by NIH DK075991.
THRX‐198321: A bifunctional muscarinic receptor antagonist and beta2‐adrenoceptor agonist which binds in a bimodal multivalent fashion to orthosteric and allosteric sites at both receptors
Authors: Steinfeld Tod , Hughes Adam , Klein Uwe , Smith Jacqueline A. M. , Mammen Mathai
Published: June 2021

Muscarinic receptor antagonists and β2‐adrenoceptor (β2‐AR) agonists are among the preferred therapies for COPD and asthma. Using a multivalent design strategy, we have identified THRX‐198321, a bimodal bifunctional multivalent ligand functioning as a muscarinic receptor antagonist (Ca2+ flux pKI = 9.9) and β2‐AR agonist (cAMP accumulation pEC50 = 9.4) activities. In radioligand binding studies, the apparent affinities of THRX‐198321 for muscarinic receptors (pKi = 9.7) and β2‐AR (pKi = 8.8) were several orders of magnitude greater than those of its component muscarinic and β2 fragments. In kinetic studies, THRX‐198321 retarded the dissociation of radiolabeled orthosteric ligands from muscarinic receptors and β2‐AR in a manner consistent with allosteric modulation. These data revealed the existence of a novel allosteric site on the β2‐AR. Additionally, the muscarinic pharmacophore fragment of THRX‐198321 retarded the dissociation of labeled β2‐AR orthosteric ligands from β2‐AR, while the β2‐AR pharmacophore fragment of THRX‐198321 retarded the dissociation of labeled muscarinic receptor orthosteric ligands from muscarinic M2 and M3 receptors. In summary, THRX‐198321 is a potent bimodal bifunctional multivalent ligand at muscarinic and β2‐AR that binds to the orthosteric and allosteric sites on each receptor. This class of ligand may offer clinical benefit for patients with respiratory disease.
Alpha‐2 Adrenergic Receptor Homology Models using Rhodopsin or Beta‐2 Adrenergic Receptor Templates
Authors: Montgomery Megan D , Bylund David B
Published: June 2021

Alpha‐2 adrenergic receptor agonists are used as adjuncts to general anesthetics and in the pharmacological treatment of many disorders, including hypertension and attention‐deficit hyperactivity disorder. Clinically available alpha‐2 agonists are not subtype‐selective and their use is limited by adverse effects resulting from actions at non‐targeted subtypes. To develop subtype‐selective ligands for alpha‐2A, ‐2B, or ‐2C receptors, more binding site structural information is needed; however, the only high‐resolution crystal structures of GPCRs are of bovine rhodopsin and modified human beta‐2 and turkey beta‐1 adrenergic receptors. Our goal was to develop homology models of the alpha‐2 receptor subtypes, using either the rhodopsin or beta‐2 receptor crystal structure as a template, that are capable of identifying characteristics important in ligand binding. Alpha‐2A and ‐2C amino acid sequences were each aligned with each template sequence and homology models were created using Sybyl7.1. Fifteen alpha‐2 agonists, including catecholamines and imidazoline derivatives, were docked into the models using FlexiDock. A comparison of calculated binding energies with experimental binding affinities resulted in poor correlations in models of alpha‐2A (r2 = 0.396 and 0.606 for rhodopsin‐ and beta‐2‐based, respectively) and alpha‐2C (r2 = 0.349 and 0.338).

Supported by a gift from Procter and Gamble.