bluuebunny/crossref_metadata_2025 huggingface dataset:
Sulindac metabolite and TNFa interaction in liver injury induced by sulindac and lipopolysaccharide in rats
Authors: Zou Wei , Beggs Kevin M , Sparkenbaugh Erica , Jones A. Daniel , Roth Robert A , Ganey Patricia E
Published: June 2021

Sulindac (SLD) is a nonsteroidal anti‐inflammatory drug (NSAID) that has been associated with a greater incidence of idiosyncratic hepatotoxicity in human patients than other NSAIDs. In previous studies, cotreatment of rats with SLD and a modestly inflammatory dose of lipopolysaccharide (LPS) led to liver injury, whereas neither SLD nor LPS alone caused liver damage. In studies presented here, the concentration of TNFα in plasma was significantly increased by LPS at 1 hr, and SLD enhanced this response. Etanercept, a soluble TNFα receptor, reduced SLD/LPS‐induced liver injury, suggesting a potential role for TNFα. SLD metabolites in plasma and liver were determined by LC/MS/MS. Cotreatment with LPS did not increase the concentrations of SLD or its metabolites, excluding the possibility that LPS contributed to liver injury through enhanced exposure to the SLD or its metabolites. The cytotoxicities of SLD and its sulfide and sulfone metabolites were compared in primary rat hepatocytes and HepG2 cells; SLD sulfide was more toxic in both types of cells than SLD or SLD sulfone. TNFα augmented the cytotoxicity of SLD sulfide in primary hepatocytes and HepG2 cells. These results suggest that TNFα enhances SLD sulfide‐induced hepatotoxicity, which might contribute to liver injury in SLD/LPS‐cotreated rats. (Supported by NIH GM075865 and ES004139.)
Evaluation of pain‐depressed behaviors in preclinical assays of pain and analgesia: Osteoarthritis‐depressed wheel running in rats
Authors: Cormier Jim , Dunbar Catherine , Benoit Laura , Morin Kathryn , Bilsky Edward J , Stevenson Glenn W
Published: June 2021

Pain stimulates some behaviors (e.g. withdrawal responses) and depresses other behaviors (feeding, locomotion). We are developing methods for testing candidate analgesics using measurements of pain‐depressed behaviors. Such assays may model important aspects of clinical pain and complement traditional assays that measure pain‐stimulated behaviors. We have previously shown that both feeding and open‐field locomotor activity can be reliably suppressed by a sub‐acute noxious stimulus (i.p. injection of acetic acid). The present study extended these findings by assessing the effects of a chronic pain manipulation (monosodium iodoacetate‐induced osteoarthritis) on wheel running in rats. Rats had 24hr voluntary access to running wheels for seven consecutive days. On day eight, rats were given intra‐articular MIA (.32‐3.2mg) into the left hind knee. Wheel running was monitored for 21 consecutive days following MIA administration. MIA produced a concentration‐dependent and prolonged decrease in running wheel activity. Parallel experiments demonstrated that MIA produced equi‐potent and concentration‐dependent tactile allodynia and shifts in hind‐limb weight‐bearing. Studies are currently underway to determine the efficacy and potency of morphine to (1) restore MIA‐depressed wheel running, (2) reverse MIA‐induced tactile allodynia, and (3) restore normal posture.

Funded by NIAMS/NIH grant R15 AR054975‐01 (GWS)
The PPAR‐gamma agonist, pioglitazone, reverses neuropathic pain behavior in the obese Zucker Diabetic Fatty Rat
Authors: Donahue Renee Rivas , Garrett Colleen , Taylor Bradley
Published: June 2021

The most widely used model of Type I diabetic neuropathic pain (DNP) involves the injection of streptozotocin (STZ). Here we tested the hypothesis that behavioral signs of Type II DNP develop in the Zucker Diabetic Fatty (ZDF) rat. To do this, we evaluated behavioral responses to noxious mechanical (paw pressure), noxious heat (Hargreaves test and 52.5ºC hot‐plate test), and non‐noxious cool (6ºC cold plate) stimuli, blood glucose levels, in both ZDF obese rats and their lean controls. Rats arrived at 12 wk of age and were tested once a week for 12 weeks. Obese animals arrived hyperglycemic. Paw pressure threshold did not differ between strains at any time. Obese but not lean ZDF rats developed cold allodynia and heat allodynia at approximately 15 wk of age, and this was maintained for the duration of the study. Both cold and heat allodynia, but not hyperglycemia, were significantly reduced 105 min after a single intra‐peritoneal injection of the PPAR‐gamma agonist, pioglitazone in obese ZDF rats. These results suggest that the ZDF rat represents a more robust model of Type II DNP than previously thought, and indicate that FDA‐approved thiazolinedione drugs rapidly reduce DNP via a mechanism that is independent of their anti‐diabetic properties. Supported by NS62306, DA18732, and DA19656 to BKT.
Effects of Acute Administration of Sigma Receptor Ligands on Mesolimbic Dopamine Neurotransmission in Rats
Authors: Tanda Gianluigi , Garcés‐Ramírez Linda , Green Jennifer L , Hiranita Takato , Katz Jonathan L
Published: June 2021

Sigma receptors (σRs) can modulate cocaine's behavioral effects, which are related to increased mesolimbic dopamine (DA) neurotransmission, and σR agonists can maintain responding in rats that self administer cocaine. We report here effects of σR ligands on DA transmission in rats implanted with vertical microdialysis probes in the nucleus accumbens shell. Cocaine (0.1‐1.0 mg/kg iv) or a non‐selective σ1/2 agonist (DTG: 1.0‐5.6 mg/kg iv) dose‐dependently increased DA levels to 275 and 150% of baseline, respectively. In contrast, the preferential σ1R agonist, PRE‐084 (0.32‐3.2 mg/kg iv), did not significantly modify DA levels. The preferential σ1R antagonist, BD 1063 (10‐30 mg/kg ip), administered alone transiently (<30 min) and dose‐dependently increased DA levels to a maximum of 150%, and later decreased DA levels to ~80% of baseline. The σ1/2 antagonist, BD 1008 (10 mg/kg ip) modestly decreased DA levels over the 2 hr following its administration. The effects of 5.6 mg/kg DTG were attenuated by BD 1008 (10 mg/kg ip), but not by BD 1063 (10‐30 mg/kg ip). In sum, the results show that acute administration of σR ligands can increase DA transmission in a brain area involved in the reinforcing effects of cocaine, and this effect appears to be mediated by the σ2R. Studies are in progress to understand if σR ligands can also modulate cocaine‐induced stimulation of DA transmission.
Effect of Acetyl‐L‐Carnitine (ALCAR) on the Striatal Outflow of Various Neurotransmitters Using Microdialysis and the Role of the GLT‐1/EAA‐2 Glutamate Transporter During Middle Cerebral Artery Occlusion (MCAO) in Rats
Authors: Jalal Fakhreya Yousuf , Böhlke Mark , Maher Timothy J.
Published: June 2021

We have shown that ALCAR is neuroprotective in rat models of ischemia. This study determined ALCAR's effect on extracellular fluid (ECF) neurotransmitters that are disrupted during ischemia. Using striatal microdialysis we measured ALCAR's effect on MCAO‐induced alterations in ECF dopamine (DA), serotonin (5HT), and their major metabolites 3,4‐dihydroxyphenylacetic acid (DOPAC), 4‐hydroxy‐3‐methoxyphenylacetic acid (HVA), and 5‐hydroxyindolacetic acid (5‐HIAA), as well as glutamate (GLU), GABA and taurine (TAU). ECF GLU was also monitored in the presence of dihydrokainate (DHK), a GLT‐1/EAA‐2 inhibitor before, and during permanent intraluminal MCAO. A marked decrease of DA, 5‐HT and their metabolites was observed during MCAO. The amino acids also greatly increased, likely reflecting massive neuronal cell death. Chronic pretreatment with ALCAR (400mg/kg/5days, i.p) attenuated the decrease in DA, DOPAC, HVA, 5‐HIAA (p<0.05), while the ECF level of 5‐HT was unaltered. ALCAR also attenuated the expected increase in GLU, GABA and TAU (p<0.01). In the presence of DHK, ECF GLU significantly increased in both control and ALCAR‐treated groups. The neuroprotection of ALCAR may partly result from restoration of the balance between excitatory and inhibitory neurotransmitters, especially via a reduction in GLU accumulation by modulation of GLT‐1 transporters.
Characterization of sodium‐dependent monocarboxylate transporter‐1 (SMCT1)‐mediated GHB transport in FRTL‐5 cells
Authors: Felmlee Melanie Ann , Cui Dapeng , Morris Marilyn E.
Published: June 2021

Purpose

To characterize γ‐hydroxybutyrate (GHB) uptake kinetics in Rat Thyroid Follicular (FRTL‐5) cells, which express the Na+‐dependent monocarboxylate (SMCT1) transporter.

Methods

SMCT1 expression and GHB uptake kinetics were analyzed in FRTL‐5 cells. Transport was evaluated with respect to Na+, pH, concentration and inhibitors.

Results

At pH 7.4, Na+‐dependent GHB uptake could be described with a simple Michaelis‐Menten equation (Km, 0.77 ± 0.41 mM; Vmax, 3.59 ± 2.0 nmol/mg/min) suggesting a single transporter‐mediated uptake process. GHB uptake was not altered at different pH values (5.5 ‐ 7.4) in the presence of Na+, but was significantly increased at low pH in the absence of Na+, suggesting an additional uptake pathway. Na+‐dependent GHB uptake was inhibited by MCT substrates (D‐lactate, L‐lactate, pyruvate and butyrate), NSAIDs (ibuprofen, ketoprofen and naproxen) and probenecid. IC50 values for L‐lactate, ibuprofen, ketoprofen and probenecid were 106.7, 31.6, 64.4 and 379 uM, respectively. All four inhibitors also inhibited GHB uptake in ratMCT1 transfected MDA/MB231 cells, suggesting they also inhibit proton‐dependent MCT1.

Conclusions

The present study characterized Na+‐dependent GHB transport in FRTL‐5 cells and furthers our understanding of the roles of Na+‐ and proton‐dependent MCTs in GHB disposition.

Support

NIH grant DA023223 and a fellowship for MAF from Pfizer Inc.
Organic cation transporter expression and function in human pulmonary epithelial cells
Authors: Ehrhardt Carsten , Tachon Gaelle , Endter Sibylle
Published: June 2021

In this study, the expression of members of the organic cation transporter (OCT) family (i.e., OCT 1, 2, 3 as well as OCTN1 and N2) in human bronchial (hBEpC, Calu‐3, 16HBE14o‐) and alveolar (A549) epithelial cells on mRNA and protein level was assessed. Experiments were carried out measuring the effect of time, temperature, pH, sodium concentration and various pharmacological agents on uptake of the cationic fluorophore, ASP+. Bidirectional transport studies of ASP+ across Transwell‐grown cell monolayers were also conducted. All cell types investigated were found positive for OCT1, OCTN1 and OCTN2 expression on mRNA level. OCT3 transcripts were positively identified in Calu‐3 monolayers. No cell type contained transcripts for OCT2. Protein levels were consistent with mRNA expression with the exception of OCT3. A saturable, temperature‐sensitive uptake of ASP+ was observed which was dependent on the pH. Salbutamol, formoterol, verapamil and amantadine resulted in a decrease in ASP+ uptake. In transport studies, 16HBE14o‐ monolayers exhibited net absorption of ASP+, whereas net secretion was found across Calu‐3 monolayers. OCTs are functionally expressed in epithelial cells of the alveolus and bronchi in vitro and might play important roles in the transport of endogenous compounds such as acetylcholine as well as xenobiotics.
Functional Interactions Between CYP1A2 and CYP2B4 Require Both Enzymes to Reside in the Same Phospholipid Vesicle
Authors: Reed James Robert , Backes Wayne L.
Published: June 2021

The goal of this study was to determine if the interactions between different P450 enzymes (CYP1A2 and CYP2B4) required both enzymes to be present in the same lipid vesicles, or if the proteins could interact when in different vesicles. When a single P450 and P450 reductase (CPR) were incorporated into separate vesicles, extremely slow reduction rates were observed, suggesting that the enzymes were anchored in the vesicles. Next, metabolism by systems were compared when two P450s (and the requisite CPR) were incorporated into separate vesicles (separate vesicle systems) and when both P450s and CPR were incorporated in the same vesicles (ternary system). In the ternary system, metabolism by CYP2B4 was significantly inhibited and that by CYP1A2 was stimulated by the presence of the alternate P450. In contrast, in the separate vesicles, the P450s were unable to interact and simple additive metabolism was observed. These results clearly demonstrate that the observed alterations in P450 metabolism require both P450s to be present in the same vesicles, and support a mechanism whereby these P450s form a physical complex in the membrane environment. (Supported by NIEHS R01‐004344).
Role of the Toll‐like receptor adaptor protein, TIRAP in regulation of gene expression of hepatic drug metabolizing enzymes
Authors: Ghose Romi , Guo Tao
Published: June 2021

Background

Altered expression of drug metabolizing enzymes (DMEs) in inflammation can affect the metabolism and toxicity of drugs. We have recently shown that the Toll‐like receptor (TLR) 2 ligand, lipoteichoic acid (LTA) can regulate the gene expression of drug metabolizing enzymes (DMEs). However, the role of the TLR2 adaptor protein, TIRAP in regulating hepatic DME genes is not known. We hypothesize that TIRAP mediates TLR2‐dependent regulation of hepatic DME gene expression.

Methods

TIRAP+/+ and TIRAP−/− mice were i.p. injected with LTA, (6mg/kg) or saline. Livers were harvested and RNA and proteins were analyzed by real‐time PCR and western blotting, respectively.

Results

Cyp2b10, Cyp2a4 and Sultn were significantly reduced by LTA in TIRAP+/+ mice (~70‐80% reduction), but not in TIRAP−/− mice. Gene expression of the DMEs is regulated by members of the nuclear receptor superfamily (PXR, CAR and RXRα). RNA levels of PXR and CAR was significantly reduced by LTA treatment in TIRAP+/+, but not in TIRAP−/− mice. Interestingly, RNA levels of the pro‐inflammatory cytokines, IL‐1β, IL6 and TNFα were induced by LTA in both the TIRAP+/+ and TIRAP−/− mice.

Conclusions

TIRAP is involved in regulating hepatic DME gene expression by LTA, but is not involved in regulating LTA‐mediated induction of pro‐inflammatory cytokines in the liver.
Dynamic DNA and histone methylation influences the ontogeny of xenobiotic metabolizing genes during postnatal mouse liver maturation
Authors: Hart Steven N , Li Ye , Cui Yue , Klaassen Curtis , Zhong Xiao‐bo
Published: June 2021

After birth, liver transitions from hematopoiesis to meet new metabolic demands, including xenobiotic (drug) processing. During this maturation, expression of many xenobiotic processing genes increases in distinct patterns. Since the epigenetic code is implicated in regulating gene expression and cellular differentiation during development, we hypothesized that these mechanisms may also be involved in the ontogenic expression of xenobiotic processing genes. In the current study, ChIP‐on‐chip assays representing 14% of the mouse genome were used to define the levels and distributions of DNA methylation (meDNA), histone 3 lysine 4 dimethylation (H3K4me2), and histone 3 lysine 27 trimethylation (H3K27me3) during postnatal liver development. In addition to the epigenetic data, gene expression microarrays defined ontogenic expression patterns. meDNA was not observed in the examined xenobiotic processing genes. Gradual increases in H3K4me2 from prenatal to adult ages were observed in several xenobiotic processing genes (e.g. Cyp3a11 and Cyp2d22). Higher levels of H3K27me3 in prenatal liver coincided with the suppression of gene expression of some xenobiotic processing genes at that age (e.g. Cyp3a16). In conclusion, these data suggest that the epigenetic code is involved in the functional transition of the postnatal liver. This work was supported by National Institute of Health Grant 5P20 RR021940.
Expression of Estrogenicity Genes in a Lineage Cell Culture Model of Human Breast Cancer Progression
Authors: Fu Jiaqi , Weise Amy , Runge‐Morris Melissa
Published: June 2021

The mRNA levels of estrogen receptors (ERα and ERβ), cytosolic sulfotransferases (SULT1E1, SULT1A1, SULT2A1 and SULT2B1), steroid sulfatase (STS), aromatase (CYP19), 17β‐hydroxysteroid dehydrogenases (17βHSD1 and 2), CYP1B1 and catechol‐O‐methyltransferase (COMT) were examined in MCF7 and in MCF10A‐derived cell lines representing a lineage cell culture model for breast cancer progression. Relative to MCF7, MCF10A‐derived cells expressed low levels of ERα but comparable levels of ERβ. In benign parental MCF10A breast epithelial cells, SULT1E1 mRNA was induced in confluent relative to subconfluent cell cultures. Proestrogenic 17βHSD1 mRNA was expressed in all of the MCF10A lineage cell lines while 17βHSD2 mRNA was most abundant in MCF10A cells. CYP1B1, SULT1A1 and SULT2B1 were expressed in MCF7 cells, while STS and COMT were widely detected across cell lines. Treatment with the potent histone deacetylase inhibitor trichostatin A (TSA) induced SULT1E1, SULT2B1 and CYP19 mRNA, but suppressed CYP1B1, STS, COMT, 17βHSD1 and 17βHSD2 mRNA in MCF10A lineage cell lines. In MCF7 cells, TSA treatment suppressed ERα, CYP1B1, STS, COMT, SULT1A1, and SULT2B1 but induced ERβ, CYP19 and SULT2A1 mRNA expression. The results indicate that relative to ER‐positive MCF7 cells, key determinants of breast estrogen metabolism are differentially regulated during neoplastic progression. Supported by ES05823 and ES06636.
Renal microcirculatory dysfunction in sepsis‐induced acute kidney injury (AKI) in mice
Authors: Wang Zhen , Mayeux Philip R
Published: June 2021

Understanding the sequence of events leading to sepsis‐induced renal injury, especially at the early stages of sepsis, is critical for developing new therapy to reduce mortality that is currently greater than 50%. The cecal ligation and puncture (CLP) model of sepsis was used to study the development of AKI in male 40‐week C57BL/6 mice. Intravital video microscopy (IVVM) was used to monitor and quantitate changes in the peritubular microenvironment in sham surgery mice and at 2, 4, or 6 h following CLP. IVVM revealed a dramatic fall in peritubular capillary perfusion as early as 2 h post CLP. The percentages of cortical peritubular capillaries with continuous flow dropped from 85 ± 4% to 66 ± 6% (P < .05, n = 6) and continued to worsen through 6 h. We also observed that cellular levels of NAD(P)H, an indicator of cellular redox stress measured by IVVM, were elevated following CLP but lagged behind the fall in capillary perfusion. These data show that a compromised renal cortical microcirculation is a very early event in the development of sepsis. The data also indicate a critical relationship between peritubular capillary dysfunction and the development of tubular stress and renal injury during sepsis. This study suggests that therapeutic approaches targeting the peritubular capillary that improve the renal microcirculation could reduce sepsis‐induced AKI. Supported by AHA Grant In Aid #850227Z.
Role of renin‐angiotensin system and prostaglandins upon renal hypertrophy development during early diabetes mellitus
Authors: Lopez Pedro , Vazquez Beatriz , Ibarra Maximiliano , Rangel Josseline , Anguiano Liliana
Published: June 2021

Diabetic nephropathy is the first cause of end‐stage renal disease. The exact pathogenesis has not been described. Hypertrophy has been mentioned as one of the most earlier changes induced by diabetes mellitus (DM). Angiotensin II (Ang II) and prostaglandins (PG) have been involved in hypertrophic processes. The aim of this work was to study if Ang II and PG participate in early diabetes hypertrophy development. We induced DM with streptozotocin (65 mg/Kg) in Wistar rats. Four treatment groups were formed: Control; Celecoxib (Cel), a COX‐2‐specific inhibitor, 20 mg/Kg; Losartan (Los), an AT1 receptor antagonist, 40 mg/Kg, and Captopril (Cap), an ACE inhibitor, 20 mg/Kg. Treatments were given during one week after DM induction. Proteinuria, creatinine clearance, kidney weight, protein/DNA ratio and cellular area were measured. Cortex COX‐2 expression was determined using immunoblot. Cel did not modify kidney weight. Cap but not Los diminished kidney weight. All treatments improved proteinuria and none modified creatinine clearance. Cel, but neither Cap nor Los, decreased protein/DNA ratio and cellular area. Cel and Cap reverted COX‐2 increase in renal cortex, but Los augmented COX‐2 expression. Our results suggest that PG participate in developing of DM‐induced hypetrophy, while Ang II by itself does not, but probably another peptide in this system could be involved. PAPIIT IN210307 SIP 20080066 CONACYT 46217
Monitoring the anti‐cancer effects and chemosensitizing abilities of novel cyclotides from Psychotria leptothyrsa
Authors: Gerlach Samantha Lee , Göransson Ulf , Mondal Debasis
Published: June 2021

Cyclotides are plant proteins with exceptional structural stability, due to their cysteine knot motif and cyclic peptide backbone. Cycloviolacin O2 (CyO2), a cyclotide from Viola odorata (Violaceae family) has anti‐tumor effects and causes cell death by membrane permeabilization. Despite their history of use, little is known about cyclotide distribution in the Rubiaceae family. We observed that Psychotria leptothyrsa, a member of the Rubiaceae family, expresses six novel cyclotides, psyle A ‐ psyle F. Their structures were elucidated by RP‐HPLC, LC‐MS and MS/MS sequencing. In the breast cancer line, MCF‐7 and its drug resistant sub‐line MCF‐7/ADR, we monitored the cytotoxic effects of psyle cyclotides, by using MTT assays. Cytotoxicities were also measured in presence or absence of Doxorubicin (5 ‐ 0.1 µM). Psyle E showed a potent cytotoxic effect (IC50 < 1µM) comparable to CyO2. Furthermore, co‐exposure to CyO2 or psyle E significantly enhanced doxorubicin induced toxicity in MCF‐7/ADR cells. We used a Sytox Green pore formation assay to determine membrane permeabilization. Cy02 showed the highest pore formation ability, followed by psyle C and psyle A, which correlates with their chemosensitizing abilities. We document several novel cyclotides with potent anti‐cancer effects and demonstrate that cyclotides may be promising chemosensitizing agents against drug resistant cancers. Supported by LCRC.
A novel designer natriuretic peptide integrating human C‐type natriuretic peptide, atrial natriuretic peptide, and B‐type natriuretic peptide
Authors: Lee Candace Y.W. , Burnett John C.
Published: June 2021

Background

The 17‐amino‐acid (AA) ring of C‐type natriuretic peptide (CNP) is essential for activation of NP receptor (NPR)‐B and the 2nd messenger cGMP. CNP induces minimal hypotension as NPR‐B is more abundantly found in veins vs arteries, but possesses limited renal properties. We designed a novel peptide fusing the CNP ring with AAs from ANP and BNP to test our hypothesis that this unique peptide, CAB‐NP, would exert enhanced renal actions without inducing excessive hypotension.

Methods

CAB‐NP was synthesized (SLRRSSCFGLKLDRIGSMSGLGCKVLRRH) and infused (21 pmol/kg/min iv) in 5 anesthetized dogs. GFR was assessed by inulin clearance. Mean±SE, P<.05*, <.01 †, BL vs 30 min infusion.

Results

CAB‐NP increased plasma cGMP (3.5±0.6 to 10.9±1 † pmol/mL), urinary cGMP excretion (980±96 to 4587±825* pmol/min), net renal cGMP generation (823±88 to 4091±824* pmol/min), urine flow (0.2±.03 to 1.2±.4* mL/min) and Na+ excretion (19±7 to 191±55* †Eq/min). Proximal (85±3 to 68±1 † %) and distal (98±1 to 91±2 † %) FRNa were reduced; GFR was preserved (46±4 to 45±3 mL/min). MAP mildly decreased (121±4 to 111±5* mmHg). PCWP (6±1 to 4±1 † mmHg) and RAP (3±1 to 2±1* mmHg) were reduced. Plasma renin activity (11±1 to 2±0.3 † ng/mL/hr) and aldosterone (15±4 to 7±2* ng/dL) were suppressed.

Conclusions

CAB‐NP exerts favorable in vivo actions without excessively lowering BP. The therapeutic potential of CAB‐NP warrants further studies.

Supported by the NIH, HFSA, and ASCPT.
Survey of "Off‐ Label" Drug Use in Pediatric Department of a Teaching Hospital ‐ A Prospective Study
Authors: Patel Kamlesh Prahladbhai , Panchal Dhaval J. , Patel Varsha J. , Bhavsar Rekha H.
Published: June 2021

Objective

: To detect extent of off label drug use in pediatric outpatients and to correlate different variables of patients.

Method

: The data of 400 patients were collected prospectively in a proforma during one year study period. Anatomical Therapeutic Chemical (ATC) Classification was used for the prescribed drugs. The drugs were ascribed off label status in different categories using standard references. The data were analyzed with Epi Info 2002 software. The χ2 test was used and value of p<0.05 was considered statistically significant.

Result

: Out of total 1744 drug prescriptions, 39.73% were prescribed in off label manner and 63% patients received at least one or more off label drug prescription. Use in child of inappropriate age, administration in sub therapeutic doses and in high doses were responsible for off label use of drugs. Antiemetics, antianemics, nasal preparations, vitamins and drugs for obstructive airway disease were commonly prescribed in off label manner. Infectious diseases, neonates and infants were the risk factors for being prescribed a drug in off label manner.

Conclusion

Off label use of drugs is highly prevalent in pediatric practice. Greater emphasis on pediatric prescribing in Pharmacology curriculum, continuous medical education programmes for practitioners and availability of updated National Formulary may help in improvement.

Research support ‐ none.
Effect of Lepidium meyenii (Maca) on female fertility in rats
Authors: Pino‐Figueroa Alejandro J , Maher Timothy J.
Published: June 2021

Maca is a native South American plant with many purported beneficial health effects. The aim of this study was to determine the effect of methanol and pentane extracts from Lepidium meyenii (Maca) on fertility in female rats. Inefficient female breeders were divided into 3 groups (N=6/group) and treated with vehicle (control), crude extract (30 mg/Kg), or pentane extract (3 mg/Kg) 21 days prior to mating. Males were then placed in the cages during a 12 day mating period, and exchanged among groups at the midpoint of the mating period. The female rats were then separated and caged individually. Pregnancy occurrence was monitored and daily body weight determined until the day of delivery. At delivery the number of pubs was recorded and thereafter weights and mortality of pubs during 6 days was registered. The total number of pubs delivered from the treated animals averaged 79 per group compared with 56 from the control group. The rate of pregnancy was 100% for both treatment groups compared with 67% for the control group. The mortality of pubs at postnatal day (PND) 6 was 10.7% for the control group and 7.6% and 2.5%, for the treated groups, respectively. The weights of the pubs at birth and the rate of increase in body weight through PND6 were not significantly different between the groups. Additional studies are needed to further explore the fertility‐enhancing properties of the constituents of the Maca plant.
Synthesis of Some New Benzothiazol‐2‐yl‐Benzylidene‐Imines and their Antibacterial Activity
Authors: Hoti Ramiz , Vehapi Idriz , Bicaj Muhamet , Kalaj Vuksan , Mulliqi‐Osmani Gjyle
Published: June 2021

Nowel benzothiazol‐2‐yl‐benzylidene imines are synthesized by condensation of benzaldehydes and corresponding 2‐aminobenzothiazoles. Condensation of benzaldehyde 1a and 2‐aminobenzothiazoles 2(a,b) gave benzothiazol‐2‐yl‐benzylidene imines 3(a,b). By condensation of 3‐nitrobenzaldehyde 1b and 2‐aminobenzothiazoles 3(a,b) , corresponding benzothiazol‐yl‐3‐nitrobenzylidene imines 3(c, d) were synthesized. The antibacterial activity of products 4(a‐d) were investigated and results were submitted for their activities against E. coli, Clebsiella and S. aureus. Compound 3d showed light bactericide activity against S. aureus. Emphatic activity against E. coli exhibited compounds 3d and 3c , whereas 3d and 3b exhibited the strongest activity against Clebsiella. In general there was a direct positive correlation between increasing concentration of the compound and antibacterial activity.
Intracavernosal pressure responses to sodium nitrite
Authors: Matt Christopher Jude , Casey David B , Badejo Adeleke M , Hodnett Christopher , Dhaliwal Jasdeep S , Fokin Alex , Murthy Subramanyam N , Kadowitz Philip J
Published: June 2021

Till recently nitrite and nitrate were considered as inert biological end products of nitric oxide (NO) metabolism. However, studies in experimental animals and human subjects have shown that nitrite is reduced back to NO with significant vasodilator activity. The vadsoilatory effect of sodium nitrite in the corpus cavernosum (erectile function) has not yet been determined, and therefore was investigated. The erectile responses were evaluated by measuring intracavernosal pressure (ICP) with a 27 gauge needle placed in the crura. Intracavernosal (IC) injections of sodium nitrite (10 ‐ 100 µmol/kg) increased ICP in a dose‐dependent manner. The increases in pressure ranged from 5 to 50 mm Hg and were similar to responses elicited by IC injection of SNP. Maximum increases in ICP in response to sodium nitrite and SNP were similar to increases in pressure observed upon electrical stimulation of the corporal nerve. The IC injections of acetylcholine induced smaller increases in ICP than sodium nitrite or SNP. The IC injections of sodium nitrate however did not have any significant vasodilator effect. The results indicate that nitrite is converted to NO causing vasodilation and increases in cavernosal blood flow and erection in the rat. Additionally, it suggests that nitrite reductase activity is present in the corpora cavernousum of the rat and is consistent with the hypothesis that nitrite represents a storage form of NO.
Repeated acute and accumulative effects of Cordymax‐6 on sexual function enhancement in orchiectomized rats
Authors: Tan Ningzhi , Gao Ling , Zhao Chunsheng , Yang Jieying , Zhu Jia‐Shi
Published: June 2021

Cordyceps sinensis (Cs) is widely used in anti‐fatigue and sexual function enhancement as a traditional Chinese medicine (JACM 4:289 & 429, 1998). We reported the improvement of the sexual enhancement activity of CM6 (a super CM product) through PDE5 inhibition and central pathways (FASEB J 22:1137.5, 2008). In this study, we investigated the repeated effects of CM6 on acute sexual enhancement in rats. Male SD rats underwent bilateral orchiectomy and recovered for 2 wks. They then received oral CM6 (0.6, 1.2 or 1.8g/kg) or testosterone propionate (2mg/kg) for 3 days repeatedly with a washout interval of 3 wks. Erection latency (EL) was assessed on Days 1 & 3 of the treatments. During the first 3‐day treatment, EL was shortened in orchiectomized rats received CM6 at 1.8g/kg on Day 1 (p=0.003), and at 1.2 and 1.8g/kg on Day 3 (p=0.022, p=0.0028), compared to controls. In the second treatment period after a 3‐wk washout, EL was shortened in all 3 dosage groups on day 1 (p<0.0005) and day 3 (p<0.013). We demonstrate in this study the dose‐dependent, EL‐shortening activity of CM6 in bilateral orchiectomized rats during repeated treatments in acute and 3‐day accumulative experimental settings, confirming the sexual enhancement function of CM6.
Studies with Skeletal Muscle Troponin I (sTnI), a New Biomarker of Skeletal Muscle Injury
Authors: Knapton Alan , Morales Azorides , Robins Terry , Herman Eugene
Published: June 2021

Available biomarkers of myotoxicity such as creatine kinase (CK), are of limited value. Recently, new assays (Pathway Diagnostics) have been developed which can detect the slow and fast isoforms of skeletal muscle troponin I (sTnI). This study evaluated the use of sTnI as a biomarker to detect alterations induced by a known skeletal muscle toxicant (2, 3, 5, 6 tetramethyl p‐phenylenediamine,TMP). Adult male and female Wistar(W) and Sprague Dawley rats(SD)(5 rats/group) were dosed with 25, 50 or 75 µmole/kg (sc) daily for 2 days or 200 mg/kg cyclophosphamide (CP)(1day) (ip). Tissues were collected 24 hrs(TMP) or 48 hrs (CP) after dosing. Rats given CP had normal muscle morphology and no detectable sTnI. Female rats were more sensitive to TMP (mortality vs males). TMP‐induced patchy muscle necrosis with inflammation. Increases in fast sTnI were noted in male (2/5) and female (3/5) SD rats treated with 25 µmole/kg TMP (male avg 31 ng/ml vs female avg 65 ng/ml). At 50 µmole/kg all animals had increased levels of fast sTnI. The levels of fast sTnI were significantly higher in female SD than male SD rats at the 50 µmole/kg dose (avg 2354 vs 347 ng/ml). Low levels of slow sTnI were detected in both female (25 and 50 µmole/kg) and male (75 µmole/kg) SD rats. CK levels were increased over baseline only at the highest tolerated doses. Thus, sTnI appears to be a sensitive biomarker that could be used to detect chemically‐induced myotoxicity.
Traditional Chinese medicine ″Anti‐cancer agent″ inhibits tumor growth in Sarcoma 180 mice via enhancing immune function
Authors: Huang Yue‐yan , Xia Man‐li
Published: June 2021

The present study was to explore the anti‐tumor effect of "Anti‐cancer agent", a traditional Chinese herbal compound, on transplanted tumor Sarcoma 180 (S180) in mice and its underlying mechanism. The tumor‐inhibited experiment was carried out in vivo by rules of anti‐tumor therapeutics screening. The mice, which had been transplanted with S180, were given different doses (4, 8, 16 g/kg/d) of "Anti‐cancer agent" by gastric perfusion for 7 days, while cyclophosphamide (CTX) was given in the positive control group. The growth of tumor S180, weight of immune organ, body weight, tumor pathological changes were measured. We found that the growth of tumor S180 in mice was markedly inhibited by the "Anti‐cancer agent" in a dose‐dependent manner. The inhibitory rates of high, medium, and low dose "Anti‐cancer agent" for tumor S180 in mice were 35.36%, 51.95% and 44.45%, respectively. The "Anti‐cancer agent" significantly inhibited the atrophy of thymus and spleen in mice induced by tumor S180, and had no harmful effect on body weight and general health status in mice. The results indicate that the "Anti‐cancer agent" inhibited the growth of tumor in S180 mice, which may relate to reducing the atrophy of immune organ induced by S180.
Angiotensin II‐induced microvascular responses in mouse brain
Authors: Vital Shantel A , Russell Janice M , Granger D. Neil
Published: June 2021

Although angiotensin II (AngII)‐induced hypertension increases the risk for ischemic stroke, the mechanisms linking AngII to this cardiovascular disease remain poorly understood. We determined whether AngII (delivered over a 2 wk period with an Alzet pump) promotes leukocyte (LECA)‐ and platelet (PECA)‐endothelial cell adhesion in cerebral venules and/or alters blood brain barrier (BBB) permeability in C57Bl/6 (WT), AT1‐R−/− mice, and AT1‐R−/−‐>WT bone marrow chimeras (BMC). Intravital video microscopy was used to monitor blood cell adhesion from a cranial window. AngII infusion induced dose‐dependent (400‐2000 ng/kg/min) increases in mean arterial pressure (MAP), LECA, and PECA. AT1‐R−/−, AT1‐R−/−‐>WT BMC, and WT mice treated with Losartan (25 mg/kg/day) all exhibited no changes in MAP, LECA and PECA in response to AngII. BBB permeability, measured by Evans blue extravasation, was increased in response to AngII in WT and AT1‐R−/−‐>WT BMC mice, but not in AT1‐R−/− mice and WT mice treated with Losartan. These findings implicate AT1‐receptors (AT1‐R) on blood cells in the inflammatory and pro‐thrombogenic phenotype induced during AngII‐induced hypertension. The accompanying BBB dysfunction appears to involve AT1‐R on endothelial cells, rather than blood cells. (Supported by HL26441)
Oxidative stress mediates homocysteine proatherosclerotic effects on human endothelial cells through a MAP kinase pathway
Authors: Alkhoury Kenan , Parkin Susan Mary , Graham Anne Margaret
Published: June 2021

Homocysteine (Hcy) is implicated in inducing cellular oxidative stress (OXS) which mediates endothelial cell (EC) damage via unknown mechanisms. We have previously shown Hcy induces activation of MAPK JNK and increases EC:leukocyte interactions. However, the JNK activator has yet to be identified. OXS is known to cause activation of the JNK MAP kinase pathway. Objective: to study the effects of antioxidants on Hcy EC damage.

Human umbilical vein EC (HUVEC) were cultured for 5‐9 days in medium containing 1mM DL‐Hcy alone, with 5mM N‐acetylcysteine (NAC) or 100µM vitamin C. and JNK kinase assays conducted on cell lysates Reactive oxygen species (ROS) generated in HUVEC were also detected after treatment, by confocal microscopy. Confluent cells after treatment were subjected to flowing neutrophils (1x106 cells/ml) at 1.1dynes and tethered, rolled, fixed and transmigrated neutrophils on HUVEC counted.

DL‐Hcy caused activation of JNK (1.5fold) and increased ROS accumulation. NAC reversed these effects. Furthermore, DL‐Hcy significantly increased HUVEC:neutrophil interactions. However, the presence of either NAC or Vitamin C reversed these Hcy mediated effects. These results show that OXS mediates Hcy effects through a MAPK kinase dependent pathway. Antioxidants block Hcy stimulated JNK activation, so preventing HUVEC: neutrophil interactions under flow conditions.

Funding: HR UK, ORSAS
Multiscale modeling of the lymphatic system relates lymphangion properties to edema formation and resolution
Authors: Quick Christopher M , Stewart Randolph H , Venugopal Arun M , Dongaonkar Ranjeet M , Moore James E , Laine Glen A
Published: June 2021

The central challenge of lymphatic research is to relate what is known about contractile lymphangions to the formation and resolution of edema. Because lymphatic system function emerges from the complex interaction of lymphangions that act both like blood vessels and cardiac ventricles, we have developed two complementary modeling approaches. First, we developed a lymphangion model that merges classical hemodynamics with the time‐varying elastance model of ventricles. By theoretically connecting lymphangions, we discovered that lymphangion structure optimizes stroke work, and lymphatic networks optimize lymph flow. Second, we developed an organ‐level compartment model that incorporates microvascular water and protein flux, interstitial storage capacity, and lymphatic drainage. This approach revealed that interstitial volume is a simple function of microvascular permeability, interstitial compliance, and the effective lymphatic resistance, RL. Merging these two modeling approaches provides a critical link between local lymphangion properties and global lymphatic function characterized by RL. Multiscale modeling thus provides a strategy to translate basic lymphatic biology to clinical edema research.
Lymphatic pacemaking
Authors: Imtiaz Mohammad Shariq , von der Weid Pierre‐Yves , Crowe Melissa , van Helden Dirk
Published: June 2021

Lymphatic vessels display rhythmic constriction‐dilation cycle which is known as vasomotion. The rhythmic constrictions propel fluid through the lymphatic chambers which are connected by valves. Our previous study has shown that lymphatic smooth muscle pacemaking occurs through a mechanism that involves Ca2+ release from intracellular Ca2+ stores through inositol 1,4,5‐trisphosphate (IP3) receptor, and depolarization through L‐type Ca2+ channels. The aims of this study were to extend our study on single lymphatic chambers to understand lymph propulsion in chains of valve connected lymphatic chambers.

Our study shows that phase locking of Ca2+ oscillations can occur across many lymphatic chambers and result in phase waves which manifest as slowly propagating waves of contractions within and across chambers. Our study also indicates that electrical coupling between chambers across valves is relatively poor. Numerical simulations indicate that chambers could partially interact through mechanical coupling between chambers.
Increased arachidonic acid‐induced thromboxane generation impairs skeletal muscle arteriolar dilation with genetic dyslipidemia
Authors: Goodwill Adam G. , Stapleton Phoebe A. , James Milinda E. , d'Audiffret Alexandre C. , Frisbee Jefferson C.
Published: June 2021

This study determined if arachidonic acid (AA)‐induced skeletal muscle arteriolar dilation is altered with hypercholesterolemia in ApoE and LDLR gene deletion mice fed normal diet compared to control C57/Bl/6J (C57) mice, and potential contributing mechanisms underlying demonstrated differences. Gracilis muscle arterioles were isolated, with mechanical responses assessed following challenge with AA under control conditions and after elements of AA metabolism pathways were inhibited. Conduit arteries from each strain were used to assess AA‐induced production of PGI2 and TxA2. Arterioles from ApoE and LDLR exhibited a blunted dilation to AA versus C57. While responses were cyclooxygenase‐dependent in all strains, inhibition of thromboxane synthase or blockade of PGH2/TxA2 receptors improved dilation in ApoE and LDLR only. AA‐induced generation of PGI2 was comparable across strains, although TxA2 generation was increased in ApoE and LDLR. Arteriolar reactivity to PGI2 and TxA2 was comparable across strains. Treatment with TEMPOL improved dilation and reduced TxA2 production with AA in ApoE and LDLR. These results suggest that AA‐induced arteriolar dilation is constrained in ApoE and LDLR via an increased production of TxA2. While partially due to elevated oxidant stress, additional mechanisms contribute which are independent of acute alterations in oxidant tone. (NIH R01 DK64668, AHA EIA 0740129N)
Isoproterenol‐ and iloprost‐induced ATP release is enhanced by selective phosphodiesterase inhibition in human erythrocytes (RBCs)
Authors: Adderley Shaquria , DuFaux Eileen , Sridharan Meera , Hanson Madelyn , Bowles Elizabeth , Stephenson Alan , Sprague Randy
Published: June 2021

Previously, we reported that the β agonist, isoproternol (ISO), and the PGI2 analog, iloprost (ILO), stimulate increases in cAMP and ATP release from RBCs and that increases in cAMP are a requisite for this ATP release. Accumulation of cAMP depends on a balance between its synthesis and hydrolysis by phosphodiesterases (PDEs). Recently, we reported that PDE3B is present in human RBC membranes and that PDE3 inhibitors potentiate ILO‐induced increases in cAMP. In contrast, PDEs 2 and 4 regulate ISO‐induced increases in cAMP in human RBCs. Here we hypothesized that, based upon their effects on cAMP accumulation, PDE3 inhibitors will augment ILO‐induced ATP release while inhibitors of PDEs 2 and 4 will augment ISO‐induced ATP release from human RBCs. Pretreatment of RBCs with the PDE3 inhibitor, cilostazol (10 μM), augmented ILO (10 nM)‐induced ATP release (n=9, P<0.05). On the contrary, pretreatment of RBCs with either the selective PDE2 inhibitor, EHNA (30µM), or the PDE4 inhibitor, rolipram (20 μM), alone had no effect on ISO (1µM)‐induced ATP release. However, the combination of EHNA and rolipram potentiated the ISO‐induced ATP release (n=11, P<0.05). These results provide strong support for the hypothesis that increases in cAMP and ATP release produced by ILO and ISO are regulated by different PDEs that are specific to each signaling pathway. Supported by NIH grants HL‐64180 and HL‐89094 and ADA grant RA‐133.
Investigating transient and prolonged VEGF signaling through regulation of intracellular calcium
Authors: Noren David Paul , Levchenko Andre , Popel Aleksander
Published: June 2021

Vascular endothelial growth factor (VEGF) is a key regulator of vascular structure and function and has a profound influence on both angiogenesis and vascular permeability. While several signaling pathways have been shown to transmit information from VEGF receptors, recent attention has highlighted a role for intracellular calcium in regulating EC proliferation, migration, and the production of vasoactive factors like nitric oxide. Here we present a mathematical model describing the regulation of intracellular calcium via VEGF. The model is able to reproduce several of the interesting dynamics normally associated with VEGF stimulation of calcium release. These include both the initial rapid transient increases in intracellular calcium and a biphasic response leading to more prolonged and increased calcium levels. Results from the model were validated through in vitro experiments and provide insight into how VEGF can regulate both immediate and extended signaling to endothelial cells. This work was supported by NIH grants R01 HL79653 and R33 HL87351.
Ornithine decarboxylase (ODC) regulates the proliferative phenotype of rat pulmonary microvascular endothelial progenitor cells
Authors: Olson Jack W. , Koloteva Anna , Parra Glenda , Alvarez Diego F.
Published: June 2021

Nucleosome Assembly Protein1 Like 1 (NAP1‐L1) enforces the progenitor phenotype of a population of rat pulmonary microvascular endothelial cells (PMVECs). These lung resident progenitor cells are characterized by their high proliferative potential (HPP), self‐renewal capability and vasculogenic behavior. PMVECs also are populated with non‐progenitor cells distinguished by their minimal NAP1‐L1 protein content, low proliferative potential (LPP) and inability to self‐renew and undergo vasculogenesis. Since polyamines are essential for cell survival and proliferation, we began to test whether ODC, the rate‐limiting enzyme in polyamine synthesis, regulates the NAP1‐L1 engendered phenotype of HPPs. LPPs have very low ODC activity compared to HPPs. Heterologous NAP1‐L1 expression switched LPPs to a HPP phenotype ‐ cells with high proliferative potential and ODC activity. DFMO (alpha‐difluoromethylornithine), a highly specific irreversible inhibitor of ODC, reduced the proliferation of HPPs by about 80%. These data support the concept that the HPP status of rat PMVEC progenitor cells requires NAP1‐L1‐increased ODC activity.
Development of an interactive, FLASH‐animated model for teaching regulation of glomerular filtration
Authors: Gookin Jody , Lit Ari , Bonin Alex , Groch Chris
Published: June 2021

Teaching renal physiology in a manner that promotes an integrated concept of dynamic function is a challenge. The glomerular filtration rate (GFR) is largely controlled by changes in vascular resistance of the afferent and efferent arterioles. The aim of this teaching project was to develop an interactive model capable of animating 1) the effect of afferent and efferent arteriolar resistance on GFR and 2) the autoregulatory mechanisms by which the kidney controls GFR in response to changes in systemic blood pressure.

In the program, afferent and efferent arteriolar resistance are controlled by moving a slider to either constrict or relax positions. For each position the effect on renal blood flow, glomerular hydrostatic pressure, GFR, filtration fraction, and tubular flow are animated and categorically summarized. A "show notes" feature provides a written explanation of the observed physiology. Systemic blood pressure is controlled by moving a slider to the 180, 80 or <60 mmHg positions. Each intervention results in animation of an uncompensated response, followed by increasing levels of autoregulatory responses which are selected by the student.

Use of this model may translate into improved problem‐solving skills compared to students receiving traditional instruction in glomerular physiology.

Funded by a Teaching Innovation Grant from North Carolina State University College of Veterinary Medicine.