bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression analysis of Progranulin and TDP‐43 genes in FTLD‐U and FTLD‐MND
Authors: Mishra Manjari , Bigio Eileen H
Published: June 2021

Mutations in MAPT and Progranulin (PGRN) genes have been found in familial forms of FTLD‐U. MAPT mutations alter tau protein function or isoform ratios and PGRN mutations lead to haploinsufficiency. High PGRN expression was found in central nervous system diseases with microglial activation. PGRN is not present in the TDP‐43 or ubiquitinated inclusions of either familial or sporadic FTLD‐U. There are at least three possibilities regarding the interaction of PGRN and TDP‐43: neurodegeneration may result as a consequence of accumulation of TDP‐43, both may be the result of PGRN mutation, or TDP‐43 accumulation may be protective against neurodegeneration, but ineffective. Our gene expression microarray analysis of homogenates from superficial frontal cortex of post‐mortem frozen FTLD‐U brain showed altered expression of PGRN and TDP‐43 genes. Our array data showed TDP‐43 to be 1.5 fold increased in both FTLD‐MND and FTLD‐U while PGRN showed no gene expression differences between controls and FTLD‐MND. Only one case has a PGRN mutation and whether it is a pathogenic mutation is as yet unknown. Due to high stringency approaches in array analysis it is possible that some disease relevant information is lost. So we further investigated the array results using quantitative Real Time PCR. Preliminary data showed altered expression which will be discussed in detail. Increased PGRN from activated microglia may contribute to artificially normal PGRN.
Spinal Cord Malformation Containing a Papillary Neoplasm
Authors: Sharer Leroy R. , Heary Robert F. , Cho Eun‐Sook , Miller Douglas C.
Published: June 2021

A 36‐year‐old man had lower back pain for over one year. MRI revealed an intradural mass with a cystic component, inferior to the spinal cord at L2‐L3, with no bony abnormality of the spine. At surgery, the mass was entirely intradural, inferior to the spinal cord and intimately attached to nerve roots of the cauda equina. On histological reconstruction, it had dorsal columns with a midline sulcus, anterior horns containing anterior horn cells, dorsal roots, and a central canal, resembling a separate spinal cord, with occasional Rosenthal fibers. External to the CNS tissue were adipose cells, smooth muscle bundles and pacinian corpuscles. A cystic region contained a papillary, epithelial‐like neoplasm that was immunopositive for vimentin, EMA and cytokeratin and negative for GFAP, CEA, PLAP and AFP. Because of the resemblance of the solid tissue to the spinal cord, it was considered that this might be a variant of Type II split cord malformation (SCM) of Pang et al (Neurosurgery 31:451, 1992), with mesenchymal and renal cell components. SCM Type II's (diplomyelia) are generally lateral to each other, with two cords within the same dural compartment, unlike this case, in which the second cord was caudal to the first. Alternatively, this lesion might be a mature teratoma containing within it a papillary component. Post‐operatively the patient has no motor deficits.
Diagnostic Testing for Congenital Muscular Dystrophies in a Cohort of USA Patients
Authors: Moore Steven Allen , Lamande Shireen R. , Winder Thomas L.
Published: June 2021

The congenital muscular dystrophies (CMD) are a genetically and phenotypically heterogeneous group of neuromuscular and developmental diseases. Since the 1995 report that LAMA2 mutations are responsible for merosin‐deficient CMD, twelve additional CMD genes have now been identified. Diagnostic testing for CMD began at The University of Iowa in 1997 with an immunostaining panel of 4 antibodies that has now expanded to include 15 antibodies. Molecular genetic testing using direct DNA sequencing of the CMD genes responsible for dystroglycanopathies has been added since 2004. 147 muscle biopsies and 62 DNA samples have now been evaluated for patients with a clinical diagnosis of CMD; 33 cases had a biopsy and DNA. Immunostaining abnormalities were diagnostic in 63% of biopsies (53 dystroglycanopathy, 19 merosin‐deficient, 21 collagen VI‐deficient). Gene sequencing confirmed the diagnosis in 25 of the dystroglycanopathy patients across the clinical spectrum from WWS to MDC1C (3 POMT1, 5 POMT2, 1 POMGnT1, 7 FCMD, and 9 FKRP). Analysis of fibroblast culture collagen VI biosynthesis and cDNA has confirmed COL6 mutations in 6 patients. These data indicate that presumptive or definitive diagnoses can be made in ~70% of CMD patients. While the data suggest that dystroglycanopathies (as a group) are the most common form of CMD, a population‐based study will be needed to determine the true prevalence of CMD subtypes in the USA.
I‐Z‐I complex myopathy
Authors: Rhodes Roy H , Sharer Leroy R
Published: June 2021

A 6‐week‐old boy with mild dysmorphism, born with severe muscle weakness and requiring intermittent ventilatory support, had a muscle biopsy with unusual features. He died at 3 months of age. There was no family history of neuromuscular disease. His mother reportedly had hypothyroidism. Modified Gomori's trichrome stain of the muscle biopsy showed many round to elongated small bodies in many myofibers, interpreted as nemaline “rods.” Electron microscopy was performed and a few small nemaline bodies were found. Over half the cells had round or oval profiles of possibly mononuclear myoblasts largely or entirely filled with globoid dense bodies that had 11‐nm dense crossing filaments and thin actin‐like filaments that extended up to 1.5 μm from each side of the globoid bodies, corresponding to I‐Z‐I complexes of embryonic myoblasts and myotubes. Some myosin thick filaments were present. We found two previous cases with foci of I‐Z‐I–like complexes at polar “caps” and filling a few cells, but our case appears to be the only one with a significant number of cells filled with I‐Z‐I–like complexes as the major finding in a myopathy. All three patients are mildly dysmorphic males. A mutation affecting mononuclear myoblasts and early primary myotubes is suspected, although an acquired lesion cannot be ruled out. The findings may also have been altered by secondary events in this unique case, such as degeneration, regeneration, and stem‐cell activity.
Thymoma and Myasthenia Gravis in a patient with Sarcoidosis
Authors: KURUKUMBI MOHANKUMAR , WEIR ROGER , Kalyanam Janaki , Nasim Mansoor , Jayam‐Trouth Annapurni
Published: June 2021

Coexistence of thymoma, myasthenia gravis (MG) and sarcoidosis is very rare. To the best of our knowledge, thymoma, MG and sarcoidosis has not been reported together in the literature. We report an interesting case with coexisting thymoma, MG and sarcoidosis.

A 59 year old female patient with a history of sarcoidosis, was admitted to the hospital with a one‐day history of sudden onset of right sided partial ptosis and diplopia. Neurosarcoidosis with cranial nerve involvement was considered, but was ruled out by the clinical findings, and MG was confirmed by the positive tensilon test, electrophysiological findings and positive Acetylcholine receptor binding antibodies. On further evaluation, CT chest showed left anterior mediastinal mass and bilateral lymphadenopathy. Post surgical diagnosis confirmed the thymoma and sarcoidosis in the lymphnodes.

When two or more diseases of undetermined origin are found together, several interesting questions are raised. It is important to first confirm the diagnoses individually. Immunologic mechanisms triggering the occurrence of these diagnoses together, is difficult to address. Although the coexistence of thymoma, MG and sarcoidosis may be coincidental, it is noteworthy to report this case because of the multiple interesting features observed as well as the rarity of occurrence.
Diagnostic role of peripheral nerve biopsy in Churg‐Strauss Syndrome
Authors: Narendra Sonia , Fuller Christine
Published: June 2021

Churg‐Strauss syndrome (CSS) is an uncommon yet distinctive form of systemic necrotizing vasculitis. Though the diagnosis is often made on clinically grounds by the presence of adult‐onset asthma, peripheral eosinophilia, and vasculitis‐associated symptoms, tissue confirmation may be necessary in some cases. Herein we report a case of a 52 year‐old woman recently diagnosed with asthma who presented with acute worsening of pulmonary symptoms and new onset of mononeuritis multiplex. Her weakness and numbness began asymmetrically in her arms, progressing to involve both legs over one week. Laboratory findings included peripheral eosinophilia, elevated IgE and rheumatoid factor, and positive pANCA. Radioimaging studies revealed bilateral pulmonary infiltrates and mediastinal adenopathy. Nerve conduction studies documented diminished amplitude and conduction block in multiple tested nerves. Radial nerve biopsy revealed active axonal degeneration, vascular and perivascular eosinophil infiltrates, and rare granulomas. A diagnosis of CSS was established based on American College of Rheumatology criteria. She later developed ischemic bowel secondary to necrotizing vasculitis (established microscopically in resection specimen). In cases of CSS where peripheral nerve damage dominates the clinical picture, peripheral nerve biopsy may be critical in establishing an early diagnosis.
Caudate Nucleus Atrophy Associated with HIV Encephalitis
Authors: Newell Kathy L
Published: June 2021

Central nervous system infection with the human immunodeficiency virus (HIV) may be associated with neurological disease ranging from mild cognitive changes to dementia. Recent imaging studies have linked atrophy of the caudate nucleus with cognitive impairment in HIV infection. Here we report the neuropathological findings in a 43 year‐old man with HIV infection and altered mental status who died after an acute illness. General autopsy findings included cirrhosis of the liver, consistent with a history of hepatitis B and C infection, acute and chronic pancreatitis, mitral valvulopathy, and end‐stage kidney disease. Gross brain findings revealed mild frontal lobe atrophy, bilateral severe atrophy of the head and body of the caudate nucleus, and moderate diffuse ventricular enlargement. Microscopic sections identified numerous microglial nodules and p24 immunoreactive multinucleated giant cells throughout deep gray nuclei and white matter, consistent with HIV encephalitis (HIVE). Microscopic findings in the caudate nucleus revealed severe neuronal loss and astrocytosis, reminiscent of Huntington disease (HD) although the Vonsattel gradient of changes was not seen. Further pathological studies of the caudate nucleus in HIV infection may yield insights into the basis of cognitive deficits in HIVE and possibly other disorders of caudate atrophy associated with cognitive decline, such as HD.
The activity of γ‐glutamyltransferase and the histology of the liver of Hybro chickens treated with chromimum (VI) and vitamin C.
Authors: Halili Fetah Murtez , Gashi Agim Maliq , Bajraktari Ismet Dervish , Bislimi Bislim , Bislimi Kemajl , Fetahu Shukri , Sopa Ramadan , Muçolli Riza
Published: June 2021

In this study the effect of interaction of both vitamin C and chromium (VI) on the activity of γ‐glutamyltransferase (GT) on serum as well as histological structure of the liver of Hybro chicken was investigated. The activity of this enzyme is closely connected with liver and bilary channels’ disease. The chickens were divided into five groups (I–V) each consisting of 10 chickens, which were treated orally for six months with different concentration amounts of chromium (VI) and vitamin C. Our investigation shows that 6 months administration of two different concentration amounts of chromium (VI), resulted in non‐significant increase of γ‐GT activity and slight histopathological changes in liver, e.g. vacuolization of hepatocytes, infiltration of limphoplazmocytes around biliary channels and the central vein, proliferation of biliary ducts, desquamated cells and extravasated erythrocytes in some areas, etc. Histological findings in liver show that there is a slight difference between the chickens treated with chromium (VI) and vitamin C and of those of control group, whereas there is an increase of the activity of γ‐GT among the treated chickens compared to those of control group.
Sex bias in drug‐induced immune mediated hepatitis
Authors: Li Zhaoxia , Talor Monica Vladut , Rose Noel R. , Njoku Dolores B.
Published: June 2021

Many autoimmune diseases are more prevalent in women than in men. Drug hapten‐induced immune‐mediated hepatitis (DIH) from halogenated volatile anesthetics or other drugs is also more prevalent in women than in men. However, precise mechanisms responsible for sex bias are unclear. We investigated sex bias in female and male BALB/c mice by immunizing with hepatic S100 proteins covalently linked to the trifluoroacetyl chloride (TFA) hapten formed from CYP2E1 metabolism of anesthetics. After three weeks we measured hepatitis, serum TFA and S100 antibodies, and assessed liver and spleen supernatant cytokines. We also measured splenic foxp3+CD4+CD25+ regulatory T cells two weeks following immunizations. We found that female mice developed more severe hepatitis and had higher anti‐TFA and −S100 antibodies than males (p < 0.01). Elevated pro‐inflammatory cytokines IL‐2, IL‐9, IL‐12p70, and IL‐17 were found in female livers (p < 0.01). Interestingly, male mice had higher levels of splenic IL‐10 (p < 0.05) and elevated splenic foxp3+CD25+CD4+ T cells at two weeks (p < 0.05). Sex bias in DIH is partially due to elevated anti‐inflammatory cytokine IL‐10 and induced regulatory T cells in males. Further investigations in these areas may uncover therapeutic agents to treat or prevent DIH.

Supported by AARDA, Mr. and Mrs. Joseph Scoby, The Gail I Zuckerman Foundation and NIHR21DK075828.
Decline in NRF2‐Regulated Antioxidant Pathway in Advanced COPD Patient Lungs Due to DJ‐1 Deficit
Authors: Malhotra Deepti , Thimmulappa Rajesh , Singh Anju , Acien‐Navas Ana , Elliot Mark , Hogg James , Tuder Rubin , Biswal Shyam
Published: June 2021

NRF2, a bZIP transcription factor regulates a battery of cytoprotective genes and protects mice from cigarette smoke‐induced emphysema by attenuating oxidative stress and inflammation. However, NRF2's role in human COPD is unknown. Here, we analyzed expression of NRF2 and its transcriptional targets in non‐emphysematous (NE) lungs, mild COPD lungs (GOLD 1‐2), and advanced COPD lungs (GOLD 3‐4). NRF2‐mediated transcriptional targets (NQO1, HO‐1 and GCLM) declined in advanced COPD, even though there was greater oxidative stress, as evident from increased lipid peroxidation and decreased GSH levels. NRF2 protein but not NRF2 mRNA was dramatically reduced in advanced COPD lungs. KEAP1 protein, the cytoplasmic inhibitor of NRF2, showed no significant difference between NE and COPD lungs. Conversely, DJ‐1, which stabilizes NRF2, showed a significant decrease in both mRNA and protein expression in advanced COPD lungs. Knockdown of DJ‐1 in human lung epithelial (Beas2B) cells and mouse lungs by siRNA significantly decreased Nrf2 protein expression and attenuated induction of antioxidants in response to cigarette smoke exposure. Taken together, in advanced COPD patient lungs, there is a significant decline in Nrf2 protein stability that results in decreased antioxidant protection and increased oxidative stress. Decline in the NRF2 pathway may play an important role in the pathological progression of COPD.
Cigarette smoke (cs) or degeneration of pulmonary C‐fibers (PCFs) in mice changes alveolar macrophages’ (AM's) NK1Rs and MMP‐12 responses to substance P (SP) in vitro
Authors: Xu Junyang , Xu Fadi
Published: June 2021

SP binds with AMs’ NK1Rs to increase MMP‐12, a key contributor to emphysema development in mice. Since SP is released from PCFs during CS, we asked whether pulmonary SP and AMs’ NK1Rs and MMP‐12 expression were affected by CS exposure or PCF degeneration, and these treatments altered the responses of AMs’ NK1Rs and MMP‐12 to SP in vitro. Female C3H/HeN mice were exposed to filtered air (FA) or CS for 16 weeks (Series I), or received vehicle or neonatal capsaicin treatment (NCT) to produce PCF degeneration (Series II). AMs and lungs were collected after euthanasia. SP and mRNA of NK1R and MMP‐12 were detected using enzyme immunoassay and real‐time PCR, respectively. CS significantly increased pulmonary SP by 31% associated with an elevation of NK1Rs and MMP‐12 in the lungs (89% and 247%) and AMs (119% and 294%), while NCT decreased pulmonary SP by 75% associated with reduction of NK1Rs and MMP‐12 in the lungs (64% and 48%) and AMs (65% and 55%). AMs from these mice were subsequently cultured with control medium and SP (10−8 M) for 24 h. SP profoundly upregulated AMs’ NK1Rs and MMP‐12 expression that were 76% and 52% higher in the AMs obtained from CS‐ than FA‐exposed mice. Surprisingly, these SP regulatory effects were almost eliminated after PCF degeneration. Our results suggest a CS‐ and PCF degeneration‐induced AMs’ plasticity in SP‐induced upregulation of NK1Rs and MMP‐12 expression (Supported by NIH 74183).
Scheduling process at DEA ‐ the example of cannabidiol
Authors: Ghozland Sandy G , Tolliver James M , Carr Susan M , Sannerud Christine A
Published: June 2021

DEA collects and reviews scientific, medical and other data on substances to determine their abuse potential and placement into the Controlled Substances Act (CSA). The scientific data reviewed includes in vitro pharmacology and pre‐clinical abuse liability studies. In particular, data on discriminative stimulus effects and rewarding properties is essential to determining whether a substance belongs in a class of drugs already scheduled, and to predict its abuse potential. The medical data reviewed includes laboratory‐controlled clinical studies. Databases and surveys on drug abuse patterns, illicit trafficking and seizures provide DEA with valuable information on the actual abuse of a drug and its consequences on health, public health and public safety.

Cannabidiol (CBD) is one of many cannabinoids present in marijuana, and as such is in schedule I of the CSA. DEA is currently conducting a scientific review of CBD to elucidate its pharmacology and abuse liability and to identify gaps in the published literature.
Discriminative‐stimulus effects of the novel CB1 agonist AM 4054 in squirel monkeys
Authors: Delatte Marcus Sherode , Thakur Ganesh , Connolly Jennifer , Zakarian Ani , Makriyannis Alexandros , Bergman Jack
Published: June 2021

Discriminative‐stimulus (Sd) properties of cannabinergic (CB) ligands other than the CB‐1 partial agonist Δ9–THC have not been studied in nonhuman primates. The present studies were conducted to systematically examine the Sd effects of the novel CB‐1 full agonist AM 4054 in squirrel monkeys (n=4) trained under a two‐lever procedure to discriminate i.m. injections of saline from 0.01 mg/kg AM 4054. Results show that the AM 4054 Sd was acquired with varying speed among monkeys. Following acquisition, ≤ 5% and ≥ 97% drug‐lever responding occurred following injection with, respectively, saline and AM 4054. Under test conditions, AM 4054 produced dose‐related (0.001–0.01 mg/kg) increases in drug‐lever responding, with little or no effect on response rate and >90% substitution following 0.0056 mg/kg AM 4054. The Sd effects of 0.01 mg/kg AM 4054 were fully evident 1 hr after injection and dissipated gradually over 12 hr. Dose‐related substitution for AM 4054 also occurred with the CB‐1 agonists Δ9–THC (0.03–3 mg/kg) and methanandamide (0.3–10 mg/kg). In antagonism experiments, SR 141716A dose‐dependently reduced the Sd effects of 0.01 mg/kg AM 4054, with full attenuation after 1.0 mg/kg of the CB‐1 antagonist. Together, these data demonstrate that the discriminative stimulus effects of AM 4054 are CB‐1‐mediated and overlap those of the CB1 agonists Δ9–THC and AM 356 (supported by NIH/NIDA DA19205 and DA7252).
In vivo characterization of 6ß‐naltrexamine, a putative opioid neutral antagonist
Authors: Pyzocha Natasha , Mercer Hannah , Raymond Tyler , Lowery John , Bilsky Edward
Published: June 2021

Opioid analgesics remain a mainstay for the treatment of acute and chronic pain. The use of these agents is associated with significant side effects including addiction and gastrointestinal (GI) dysfunction. Our laboratory has previously reported on the in vivo pharmacology of 6ß‐naltrexol, an active metabolite of naltrexone and a putative neutral antagonist. The current studies assessed the in vivo pharmacology of a related compound, 6ß‐naltrexamine, which also acts as a neutral antagonist in opioid‐exposed cell systems. I.v. administration of 6ß‐naltrexamine, in male ICR mice, resulted in a time related blockade of the antinociceptive effects of i.v. hydrocodone, ID50 value (95% CI) of 2.03 (1.68–2.46) mg/kg. The antagonist had a peak effect at 60 minutes and duration of action of approximately 90 minutes in the 55°C tail‐flick test. 6ß‐naltrexamine also dose‐dependently reversed hydrocodone‐induced GI inhibition with an ID50 value (95% CI) of 4.14 (2.23–7.71) mg/kg. These results indicated that 6ß‐naltrexamine is a relatively potent opioid antagonist that exhibits a CNS/PNS potency ratio similar to naltrexone and naloxone. We are currently assessing the efficacy of 6ß‐naltrexamine in animal models of acute and chronic dependence.
Comparison of the development of tolerance following chronic in‐vivo exposure to opioid (morphine) versus cannabinoid receptor agonists (WIN‐55,212‐2)
Authors: Maguma Hercules T , Taylor David A
Published: June 2021

Few studies have determined if the heterologous tolerance that develops in the guinea pig longitudinal muscle‐myenteric plexus (LM/MP) following in‐vivo opioid treatment extends to cannabinoids; nor, have studies compared the nature of tolerance that develops after chronic in‐vivo exposure to opioids with that after cannabinoids. To induce tolerance, morphine was injected twice daily for 7 days and WIN‐55,212‐2 once daily for 5 days. The LM/MP was used to assess the degree and character of tolerance by comparing the ability of opioid, adenosine and cannabinoid agonists to inhibit neurogenic contractions. Animals exposed to morphine showed tolerance to all inhibitory agonists (ratios of IC50 values of 4.8 [DAMGO], 3.6 [2‐chloroadenosine] and 4.9 [WIN‐55,212‐2]). In contrast, WIN‐55,212‐2 exposure resulted in reduced maximum responses and subsensitivity to WIN‐55,212‐2 only (IC50 ratio of 10.5) with no change in sensitivity all other agonists including nicotine. Thus, the heterologous tolerance in the LM/MP after in‐vivo opioid treatment extends to cannabinoid agonists and may involve non‐receptor coupled changes. In contrast, tolerance that develops after in‐vivo WIN‐55,212‐2 exposure is associated with reduced sensitivity and maximum responses specific to cannabinoid agonists that suggests tolerance following chronic in‐vivo cannabinoid exposure may involve receptor‐dependent modifications.
Effects of opioid ligands on the relative reinforcing strength of food and saline in monkeys trained under concurrent FR30 schedules of food and heroin availability
Authors: Paronis Carol A , Bergman Jack
Published: June 2021

Previous studies in monkeys trained to “choose” between injections of heroin or delivery of food pellets revealed that buprenorphine increased the relative reinforcing strength of low, but not high, doses of heroin while antagonizing some of the direct effects of heroin. The present studies extend these findings by evaluating the effects of mu‐opioid full and partial agonists in rhesus monkeys responding under concurrent FR30 schedules (30 responses on one lever results in food delivery, 30 responses on the alternative lever results in i.v. injection of saline or heroin). Under this schedule, the distribution of behavior is related to available i.v. dose: when saline, 0.003 and 0.01 mg/kg/inj heroin are available 4, 37, and 88% of responses, respectively, occur on the injection lever. Opioid ligands were studied by administering doses 10 min before the start of the session. Preliminary results indicate that, like morphine, codeine (0.3–3.0 mg/kg) butorphanol (0.003–0.03 mg/kg) and nalbuphine (0.03–0.3 mg/kg) all dose‐dependently increase responding on the injection lever during sessions of saline availability. These results suggest that the effects of opioid ligands on the relative reinforcing strength of saline or food do not depend on the efficacy of the pretreatment drug. Ongoing studies are evaluating the effects of these drugs on the relative reinforcing strength of heroin. (Supported by NIH Grant DA15723)
Effects of strain on MDMA (“ecstasy”)‐induced locomotor hyperactivity and sensitization in mice
Authors: Ansah Twum Ampofo , Ferguson Marcus , Nayyar Tultul
Published: June 2021

MDMA (3,4‐Methylenedioxymethamphetamine; “ecstasy”) is a popular illicit recreational amphetamine congener that possess both stimulant and hallucinogenic properties. It acts by releasing dopamine (DA) and serotonin (5‐HT) via the reuptake transporters. Whereas the locomotor activating effects of amphetamine primarily depend on the DA system, 5‐HT system appears to be essential in mediating MDMA effects. Selective 5‐HT reuptake inhibitors, which inhibit the binding of MDMA to the 5‐HT transporter, blocks hyperactivity stimulated by MDMA and the locomotor response to MDMA is attenuated in knockout mice lacking the 5‐HT1B receptor. It has been reported that DA transporter (DAT) expression is 50% higher in DBA/2 mice than C57BL/6 mice. An altered expression of DAT (the main target of psychostimulants) could influence psychostimulant response. The goals of the current studies were to determine possible strain differences in sensitivity to MDMA and to evaluate the behavioral response to repeated MDMA treatment. Baseline locomotor activity was significantly higher in C57BL/6 mice than in DBA/2 mice. MDMA produced dose‐dependent increase in locomotor activity in C57BL/6 mice but not in DBA/2 mice. In C57BL/6 mice, repeated treatment with MDMA produced behavioral sensitization, believed to model the motivational aspects of psychostimulant addiction. In addition, female mice of either strain exhibited greater sensitivity to MDMA than male mice. The results of these studies will facilitate efforts to elucidate how genetics may impact vulnerability to drug addiction. Supported by NIH grants, DA020677, NS041071 and ES014156.
The drug self‐administration assay measures the pharmacodynamic and pharmacokinetic potencies of receptor antagonists
Authors: Norman Andrew B. , Norman Mantana K. , Buesing William R. , Tabet Michael R. , Tsibulsky Vladimir L.
Published: June 2021

According to pharmacological theory, competitive antagonists increase the concentration of agonist required to produce a defined magnitude of response. In the drug self‐administration paradigm, the maximum concentration of drug that induces lever pressing represents the satiety threshold, an equieffective drug concentration. In rats trained to self‐administer cocaine or apomorphine, SCH23390 and (‐)eticlopride at doses between 5 and 30 nmol/kg i.v. produced acceleration of self‐administration with a concomitant increase in the satiety thresholds, which rapidly increased and then gradually decreased over time. The magnitude of the maximal increase in the satiety thresholds was proportional to the dose of antagonist. A plot of the agonist dose ratio as a function of the antagonist dose according to the method of Schild should represent the dose of antagonist required to occupy 50% of the receptors underlying the satiety response (Kdose), a measure of antagonist pharmacodynamic potency. Furthermore, the absorption and elimination t1/2s for the antagonists, calculated from pharmacokinetic modeling of the time courses of the antagonist effects, were approximately 5–7 min and 40–50 min, respectively. Both rate constants for each antagonist were independent of the agonist and of the dose of antagonist. Self‐administration is regulated according to pharmacological principles. Supported by PHS grant DA018538.
Assessment of Reinforcing Effects of Benztropine Analogues and Their Effects on Cocaine Self‐Administration: Comparisons with Monoamine Uptake Inhibitors
Authors: Hiranita Takato , Newman Amy H , Katz Jonathan L
Published: June 2021

Reinforcing effects of monoamine uptake inhibitors (cocaine, nisoxetine, citalopram, WIN 35428, methylphenidate (MPD), AHN 1055 (4′4″diF‐benztropine), AHN 2005 (N‐allyl 4′4″diF‐BZT), and JHW 007 (N‐butyl 4′4″diF‐BZT) were assessed, along with their effects on cocaine self administration. Rats self administered cocaine (.03–1 mg/kg/injection) or saline on a fixed ratio 5 response schedule. The cocaine dose effect curve was bitonic, with maximum responding maintained at 0.3 mg/kg/inj, iv, and low rates maintained by saline. WIN 35428, MPD, and AHN 1055 maintained responding above saline levels, with cocaine equipotent to MPD, 10‐fold < WIN 35428, and more effective than AHN 1055. None of the other drugs maintained responding. AHN 2005 and JHW 007 administered before sessions (10–100 mg/kg po) produced dose related decreases in cocaine self administration, shifting the dose effect curve down, without effects on responding maintained by food reinforcement. Increasing doses of AHN 1055 (10–100 mg/kg po) shifted the cocaine self administration dose effect curve up, to the left and down. MPD (3–30 mg/kg po) dose dependently shifted the cocaine self administration dose effect curve leftward, whereas both nisoxetine and citalopram produced relatively small shifts down that did not depend on dose. The effects suggest that the BZT analogues may have utility as medications for cocaine abuse. Support by NIDA IRP.
Effects of acute stress and environmental enrichment on cocaine self‐administration in socially housed monkeys
Authors: Nader Michael A. , Campbell Jennifer E. , Garrett Nicholas R. , Icenhower Michelle R. , Czoty Paul W.
Published: June 2021

Previous research has shown that social rank can influence the reinforcing effects of cocaine in monkeys (Morgan et al. 2002; Czoty et al. 2005). It has been hypothesized that dominant and subordinate monkeys represent a continuum from environmental enrichment to socially‐derived stress. Whereas social rank reflects a chronic condition, the effects of additional enrichment or stress on cocaine self‐administration (SA) has not been examined. The goal of these studies was to determine if sensitivity to cocaine reinforcement could be altered by acute presentation of environmental enrichment or stressors. Adult male dominant and subordinate cynomolgus monkeys self‐administered cocaine (0.003–0.1 mg/kg) under a concurrent schedule with 1‐g food pellets as the alternative; dose‐response curves were determined in single sessions. When SA was stable, one of two manipulations was performed prior to SA sessions: either the monkey was placed into a larger enclosure for 2 days or he was exposed to a toy snake for 10 min. Acute enrichment shifted the cocaine dose‐response curves to the right for all monkeys, while the acute stressor shifted the cocaine curves to the left only in subordinate monkeys. These results indicate that the model is sensitive to acute manipulations and that environmental enrichment impacts all monkeys, while the effects of stress are more prominent in subordinate monkeys. DA10584.
Blockage of the Proposed Precipitating Stage for Parkinson's Disease by Antioxidants: A Potential Preventative Measure for PD
Authors: Muthian Gladson , King Jennifer , Mackey Veronica , Prasad Kedar , Charlton Clivel
Published: June 2021

Parkinson's disease (PD) is due to the degeneration of dopamine (DA) neurons and loss of nigrostriatal (NS) tyrosine hydroxylase (TH) and DA. In another study we show that fetal exposure to toxins makes the NS DA neurons vulnerable and that later challenges cause the vulnerable neurons to succumb, indicative of a sensitization stage and a precipitating stage for idiopathic PD. In this study we determine whether the precipitating stage can be blocked by treating with antioxidants, as a potential preventative measure for PD. Adult mice were treated every other day for 27 days with 25 or 50 μg/animal of curcumin (CU) or with 25 or 50 mg/kg BW of an antioxidant cocktail (AO). The goal was to know if CU or AO can block the TH depleting effects of MPTP, so at the 20th day treatment point a 7 day 30 mg/kg/day of MPTP was included. Western blot for TH, brain histology and motor activities were determined. The treatments with CU and AO block, in a dose‐dependent manner, the TH‐depleting effects and the hypokinesia of MPTP. MPTP depletes TH by about 60 and 80% in the striatum and midbrain. In the striatum and midbrain CU substantially and partially blocks the effects of MPTP and AO blocks the effects of MPTP and may enhance TH expression. Both CU and AO reduce the incidence of death and hypokinesia caused by MPTP. The results demonstrate that pre‐symptoms therapeutic interventions may be effective in blocking or delaying the on‐set of PD. Supported by NIH RO1NS041674 and NIH R21NS049623
Early environmental exposure to methanol as a sensitization factor for Parkinson's disease: a closer look into the etiology of PD
Authors: Mackey Veronica Renee , Charlton Clivel G.
Published: June 2021

Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of substantia nigra (SN) dopaminergic neurons that project to the striatum. The etiology of PD is unknown; there is the possibility of early exposure to toxicants creating a vulnerability stage for PD to occur later in life. Here, we investigate whether methanol (MeOH) exposure in early life causes sensitization of the basal ganglia to challenges, such as 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) that precipitates the symptoms of PD. C57/bl6 mice were exposed to sub‐chronic levels of MeOH in utero. At 12 wks of age, the offspring were treated with MPTP for seven days. Tyrosine hydroxylase (TH) protein was analyzed in the midbrain and striatum. MPTP increases the expression of TH in the midbrain and reduces it in the striatum, suggesting that the increase of TH in the midbrain may be due to impairment of its transport from cell bodies in the SN to the terminals in the striatum. Lastly, the fetal MeOH sensitized the mice to MPTP reducing effects, suggesting that fetal exposure to MeOH may be a pre‐disposing factor for PD.

This project was supported, in part by NIH Grant 1R21 NS 049623‐01A1
Endothelium‐derived hyperpolarising factor (EDHF) underlies sex‐differences in the pressure‐induced myogenic response
Authors: Chan Melissa , Villar Inmaculada , Scotland Ramona , Ahluwalia Amrita
Published: June 2021

Sex‐differences exist in the predominant endothelial vasodilator that moderates vascular tone in resistance arteries i.e. nitric oxide (NO) and prostacyclin (PGI2) in male arteries and EDHF in female murine resistance arteries (Scotland et al [2005] Circulation, 111, 796). These differences are thought to underlie the protection of females from hypertension. A major determinant of blood pressure is the myogenic response i.e. the constriction response to rises in intraluminal pressure. We investigated whether sex‐differences in the magnitude of the myogenic response, relate to differences in endothelial vasodilator activity. Using in vitro perfusion myography we demonstrated that the myogenic response was greater in arteries from male than female C57BL/6 mice and enhanced in arteries of ovariectomised than sham‐operated mice (p<0.05, n=15). Inhibition of NO using L‐NAME [300μM] enhanced myogenic responses in all arteries (p<0.01), but block of EDHF activity, using TRAM‐34 [10μM] and apamin [50nM], enhanced responses in arteries of female sham‐operated but not ovariectomised mice (p>0.05). Block of PGI2 synthesis, using indomethacin [5μM], had no significant effect. Thus, an oestrogen‐mediated suppression of myogenic responses in female mice is associated with enhanced release of EDHF; an effect that is likely to contribute to the relative protection of females from hypertension.
Botanical Drug Product Development in the U.S.A.: Importance of Genotoxicity Testing
Authors: Wu Kuei‐Meng , Ghantous Hanan , Birnkrant Debra B.
Published: June 2021

Genotoxicity testing (GT) is an important part of preclinical safety assessment of new drugs and is required prior to Phase I/II clinical trials. GT is designed to detect genetic damage such as gene mutations and chromosomal aberration, which may be reflected in teratogenic and tumorigenic potential of the drug. Botanical new drugs in the U.S. are entitled to a waiver for preclinical Pharm/Tox studies, including GT, in support of an initial clinical trial under IND, contingent on previous human experience. Recently, ethical concerns have been raised over conducting Phase I/II clinical trials of new drug with positive GT findings in healthy volunteers. Although the relevance of this issue depends on drug's indication, duration of treatment, and specific findings related to GT, the regulatory view toward it has been to not expose patients to genotoxic compounds unnecessarily in clinical trials. This philosophy may impact on herbal supplement marketing and botanical drug development, in that GT data are often lacking while consumers are exposed to the herbal supplement, or healthy volunteers are tested in an initial Phase I/II clinical trial on the botanical drug. Granted that GT is comparably cost‐efficient (relative to animal toxicity studies), has high statistical power, and is highly reproducible, botanical drug sponsors should be encouraged to prioritize obtaining this information in their product development.
Characterization of a Sphingosine‐1‐phosphate Receptor Antagonist
Authors: Kennedy Perry Courtland , Zhu Ran , Macdonald Timothy L. , Lynch Kevin R.
Published: June 2021

Sphingosine 1‐phosphate (S1P) is an extracellular lipid mediator that acts via a set of G‐protein‐coupled receptors (S1P1 – S1P5) to influence angiogenesis, immune cell trafficking, cell migration, proliferation and survival. The S1P receptor agonist prodrug, FTY720, is an immunomodulator that is in late stage clinical trials for multiple sclerosis. In the course of making conformationally‐constrained FTY720 analogs, we discovered multiple S1P receptor type‐selective compounds including antagonists such as VPC03090‐P. This compound is a pro‐drug that is converted to the active compound, VPC03090‐P, by sphingosine kinase 2. The parent compound is orally available and long lived; the ratio of active to parent drug in mouse plasma at 24 hours post dosing is 20:1. VPC03090‐P behaves as a competitive antagonist at the S1P1, S1P3 and S1P5 receptors, shifting agonist concentration‐response curves in both GTP[γ‐35S] binding and calcium mobilization assays by one log order or more when present at a concentration of 1 μM. Studies of the effect of this compound on lymphocyte trafficking and capillary permeability are ongoing. We anticipate that this unique tool will help elucidate the complex nature of S1P signaling. This research is supported by NIH grants R01 GM067958 and T32 GM007055.
Acute drug‐induced inactivation of the h5‐HT7 receptor: a pharmacological profile
Authors: Knight Jessica , Smith Carol , Toohey Nicole , Teitler Milt
Published: June 2021

The h5‐HT7R is one of 13 5‐HT receptors expressed in humans. It is involved in the control of cognition, emotion, and cardiovascular tone. Recently we reported that a 30 min exposure to nM levels of the antipsychotic drug risperidone results in an inactivation of the h5‐HT7R‐mediated stimulation of cAMP production (Smith et al, Mol. Pharmacol., 2006). To further investigate this unusual effect of this highly prescribed drug we have been testing other drugs for similar inactivating properties. 9‐OH‐risperidone, the active metabolite of risperidone, and methiothepin, a potent 5‐HT receptor antagonist, also potently inactivate the receptor. Preliminary studies indicate amoxapine, ritanserin, metergoline, lisuride, bromocryptine, SB‐269970 also produce this effect: clozapine, TFMPP and tenilapine did not produce the inactivating effects. It appears that a majority of h5‐HT7 receptor antagonists produce this unusual and dramatic effect. h5‐HT7‐mediated MAPK phosphorylation is also inactivated by risperidone, 9‐OH‐risperidone, and methiothepin, consistent with data indicating an irreversible or pseudo‐irreversible interaction is occurring between the drugs and the receptor. Further studies are underway to investigate the mechanism and in vivo relevance of these observations.

Supported by MH56650 (M.T.)
The pharmacological evaluation of novel and high‐affinity melatonin receptor ligands
Authors: Witt‐Enderby Paula Ann , Julius Justin , Attia Mohamed I , Zlotos Darius P
Published: June 2021

Melatonin exerts its multiple pharmacological actions via activation of two G protein‐coupled receptors, MT1 and MT2. The exploration of the physiological role of the distinct receptor subtypes requires the use of highly selective ligands (both agonists and antagonists). The compounds being presented serve as MT2 selective ligands. The introduction of the bulky lipophilic substituent at the position corresponding with the C‐2 position of melatonin provides MT2 selectivity potentially through an interaction with the NH group of the His‐208. Competitive binding assays were performed with 2‐[125I]‐iodomelatonin in Chinese hamster ovary cells stably transfected with the human MT1 and MT2 melatonin receptors. Competition analysis showed that large electron rich aromatic ring structures enhance binding and MT2 receptor selectivity. With the creation of highly selective, high affinity ligands, the binding pockets of each receptor subtype can be better understood and further the development of novel melatonergic drugs.
Palmitoylation of the TPβ isoform of the human thromboxane A2 receptor: Implications for receptor trafficking
Authors: Kinsella B. Therese , Reid Helen M.
Published: June 2021

Palmitoylation is a prevalent feature of G protein coupled receptors (GPCRs). We investigated whether the TPα and TPβ isoforms of the human thromboxane (TX)A2 receptor are palmitoylated and the functional consequences thereof. Consistent with three cysteines within its unique C‐tail, TPβ, but not TPα, is palmitoylated at Cys347 and, to a lesser extent, at Cys373,377. Whilst impairment of palmitoylation did not affect ligand affinity, [Ca2+]i mobilization by TPβC347S, but not TPβC373S or TPβC373,377S was significantly reduced relative to TPβWT suggesting that palmitoylation at Cys347 is required for Gq/phospholipase Cβ coupling. As TPβ, but not TPα, undergoes agonist‐induced and tonic‐ internalization, we investigated whether palmitoylation affects TPβ internalization. It was found that agonist‐ and tonic‐internalization by TPβC373S, TPβC373,377S and TPβC347,373,377S was significantly reduced compared to TPβWT. Whilst TPβC347S underwent reduced agonist‐ internalization, it underwent tonic‐ internalization to a similar extent as TPβ. The deficiency in agonist‐induced internalization by TPβC347S was overcome by over‐expression of β‐arrestins. Collectively, data herein suggest that whilst palmitoylation of TPβ at Cys373,377 is critical for agonist‐ and tonic‐ internalization, palmitoylation at Cys347 may determine which pathway is followed. *This work was supported by The Wellcome Trust.
Interactions of the Dopamine D2 Receptor with the Calcium‐Binding Protein S100B
Authors: Neve Kim A , Liu Yong
Published: June 2021

S100B is a calcium‐binding protein with numerous regulatory activities in the mammalian brain. Using a bacterial two‐hybrid assay, we have identified a novel interaction between S100B and the third cytoplasmic loop of the dopamine D2 receptor (D2‐IC3). The binding of S100B to D2‐IC3 was confirmed using a polyHis pull‐down assay. The binding of heterologous S100B to full‐length D2 receptor in HEK293 cells, and of endogenous S100B to endogenous D2 receptor in the rat neostriatum, was demonstrated by co‐precipitation. A putative S100B binding motif is near the N‐terminus of D2‐IC3. Although S100B is considered a glial protein, S100B expression was detected in microtubule‐associated protein‐2 (MAP2) expressing neurons in neostriatal cultures. Expression of S100B in HEK293 cells also stably expressing the D2 receptor significantly increased D2 receptor stimulation of extracellular signal‐regulated kinases (ERKs), while causing little change in NGF‐stimulated activation of ERKs, and also potentiated D2 receptor inhibition of cyclic AMP accumulation. Taken together, these fundings suggest that the interaction of S100B with the D2 dopamine receptor enhances D2 receptor signaling. (MH045372 and VA Merit Review)
Identification and characterization of CIP98 as a novel regulator of mGluR6
Authors: Tian Liantian , Kammermeier Paul J. , Cismowski Mary J.
Published: June 2021

Group III metabotropic glutamate receptors (mGluRs) –mGluR4, 6, 7 and 8 – couple exclusively to Gαi/o proteins. mGluR6 has a unique role as the primary postsynaptic neurotransmitter receptor in retinal ON bipolar cells, which receive glutamatergic input directly from photoreceptor cells. Activated mGluR6 initiates a signaling cascade through Gαo that results in inhibition of a cation channel and hyperpolarization of ON bipolar cells. To identify potential regulators of mGluR6 signaling, yeast two‐hybrid screens were developed using intracellular regions of the receptor as baits against a rat retinal library. CIP98 (CASK interacting protein, ‘whirlin’) was identified from these screens. CIP98 has 3 PDZ domains and a proline‐rich domain, but little is known about CIP98 function. We verified expression of CIP98 in ON bipolar cells using single cell RT‐PCR. Using a reconstitution system in sympathetic neurons from the rat superior cervical ganglion (SCG), where we have shown mGluR6 modulation of N‐type calcium channels, we found a dose‐dependent shift in the mGluR6 response to agonist when co‐expressed with a CIP98 truncation mutant. Endogenous CIP98 expression in cultured SCG neurons and hippocampal neurons was confirmed, and knock‐down of endogenous CIP98 with siRNA was used to further examine the impact of CIP98 on mGluR6 function. Together, our data suggest that CIP98 directly regulates mGluR6 signaling.
Characterisation of the adenosine A1 receptor in Saccharomyces cerevisiae
Authors: Stewart Greg , Sexton Patrick , Scammells Peter , Dowell Simon , Christopoulos Arthur
Published: June 2021

Different ligands for G protein‐coupled receptors can induce unique receptor conformations, leading to differing profiles of G protein coupling. Given that S. cerevisiae expression systems are useful tools for studying the coupling of GPCRs to individual G protein pathways, experiments were undertaken to investigate whether different pharmacological profiles can be detected in this system for ligands of the adenosine A1 receptor (A1R), and to what extent the findings correlated to a mammalian (CHO) expression system. The human A1R was introduced into individual strains of S. cerevisiae, each expressing different chimeras from yeast and mammalian Gα subunits, followed by pharmacological characterisation. We found that, in the yeast, the A1R only couples to Gαi/o family proteins when stimulated with the full agonist, R‐PIA. The pA2 values generated from analysis of the interaction between the A1R antagonist, DPCPX, and R‐PIA correlated with that observed in mammalian cell assays. A novel partial agonist at the A1R, VCP‐189, was also tested. VCP‐189 appeared to selectively activate Gαi subtypes, but no response was seen with Gαo or Gαz strains. In agreement with the S. cerevisiae assay, the response to R‐PIA and VCP‐189 in the mammalian assays is pertussis toxin sensitive. Further studies investigating Gαi/o subtype selectivity in mammalian cells are currently underway.