bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating the Triceps Surae in Chronic Inflammatory Demyelinating Polyneuropathy Patients using Magnetic Resonance Imaging
Authors: Fanous Jacob , Gilmore Kevin J. , Kimpinski Kurt , Rice Charles L.
Published: June 2021

Introduction

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an acquired autoimmune disease, primarily characterized by peripheral nerve demyelination. Patients present with sensory and motor deficits, including but not limited to symmetrical diffused muscle weakness. Studies to date have mainly focused on the neuropathic aspects of CIDP and its involvement in paresis. Impairments in skeletal muscle quality and quantity may be a consequence of motor nerve deficits, but these aspects have not been investigated comprehensively. Thus, the purpose of this study was to use magnetic resonance imaging (MRI) to explore anatomical and functional differences in the triceps surae muscle complex in a group with CIDP with a healthy control group.

Methods

To date, five patients with CIDP and six healthy control subjects were matched on anthropomorphic characteristics. Both groups underwent isometric plantar flexion strength measurements on a custom dynamometer. On separate days, MRI (T1 and T2) of the leg musculature was acquired via serial axial plane scans in a 3.0‐Tesla magnet. All MRI scans were analyzed using OsiriX imaging processing software. Total muscle volume was computed using the T1 weighted anatomical images with a 3D FLASH sequence: (0.9mm slice thickness with slice separation of 1mm ranging from 280 to 400 slices). T2 weighted relaxation times were determined from images with a spin‐echo sequence (5.0mm slice thickness; 16 echoes between 13.2–211.2ms). Both total volume (T1) and relaxation times (T2) were calculated separately for the three components of the triceps surae: soleus, medial gastrocnemius (MG), and lateral gastrocnemius (LG).

Results

CIDP patients had ~34% less plantar flexion strength compared with controls. CIDP patients had ~14%, ~28% and ~38% smaller muscle total volumes in the soleus, MG and LG, respectively, compared to controls. When strength was normalized to total triceps surae volume it was ~26% lower in CIDP. T2 relaxation times were significantly longer in CIDP with the soleus, MG and LG showing ~37%, ~31% and ~29% longer relaxation times, respectively.

Conclusion

CIDP patients were significantly weaker compared to control subjects. When strength was normalized to total triceps surae volume the difference remained indicating CIDP patients have lower intrinsic muscle contractile quality. This is further reinforced by the significantly longer T2 relaxation times, which likely reflects an increase in intramuscular fat infiltration in the CIDP group. The results indicate that muscle quantity and quality are affected by alterations in axonal function due to CIDP.

Support or Funding Information

Supported by NSERC

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Muscle‐intensive and High‐Impact Exercises Differentially Influence Whole Bone 3D Morphology in Young Outbred Male Mice
Authors: Smolinsky Amanda N. , Aldridge Kristina , Middleton Kevin M.
Published: June 2021

Skeletal morphology is predominantly dictated by the genetic background and the habitual loading environment of individual bones. Mechanical loads placed on bones are derived from intracorporeal contractions of muscles and from extracorporeal ground reaction forces generated by bearing weight and by substrate reaction forces during locomotion. We investigated the relative effects of these two sources of load on the growing skeleton using an outbred mouse model system. The 3D shape of the femur was compared across groups of mice following discrete exercise regimens designed to emphasize specific loading environments. Hsd:ICR (CD‐1) mice (N=80; 20 per group) were randomly assigned to one of four treatment groups at four weeks old: swimming which emphasizes muscle forces, dropping which emphasizes impact forces, wheel running which results in a combination of muscle and impact forces, and non‐exercised controls which were free to move about their cages. Durations of swimming, running, and impact loading were matched so that mice received approximately the same intensity. After 20 days mice were euthanized and right hind limbs were micro‐CT scanned (~20 μm3 voxel size). Femoral shape was defined by digitizing 23 landmarks on each scan. To adjust for size, each individual femur was scaled to the geometric mean of all possible linear distances among landmarks. Group differences were analyzed using Euclidean Distance Matrix Analysis (EDMA), and differences >5% were considered substantial. In all three activity groups, we find decreased robusticity of the femoral diaphysis and increased distal condylar breadth compared to non‐exercised controls. Additionally, each group displays unique femoral morphologies when compared to controls. The swimming treatment group shows increased length of the femoral neck. The wheel running treatment group shows a narrower femoral neck with a larger femoral head. The impact loading group shows a lengthened and reoriented distal diaphysis. Overall, the running and swimming groups show similar patterns of morphology that diverge from the non‐exercised mice, suggesting muscle forces differentially affect the loading environment during running in mice compared to impact forces. Further, these results indicate the regional sensitivity of femoral morphology to muscle‐ and impact‐derived loads, and emphasize how activity patterns during adolescence can lead to distinct morphologies in the limb skeleton.

Support or Funding Information

Research support: University of Missouri School of Medicine

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Morphological Variants of The Pes Anserinus of Tibia in South Indian Population
Authors: Chettiar Ganesh Kumar , Murlimanju B.V.
Published: June 2021

Introduction

The tendons inserting at the antero medial surface of the tibia are called ‘pes anserinus' because of the resemblance to the ‘goose foot’. The tendons radiate during the insertion and it has been reported that the variations exist in the tendons. In this context, the objective of the present study was to study the morphological variations of the pes anserinus of the tibia in South Indian population.

Methods

The present study included 53 formalin fixed human lower limbs. The tendons inserting into the antero medial surface of the tibia were carefully dissected. The morphological variants were observed and the pictures were taken. The variants were tabulated and analysed.

Results

It was observed that, 48 lower limbs (90.6%) had convergent type of the pes anserinus in which the gracilis tendon was in between the semitendinosus and semimembranosus tendons and all joined together. The semitendinosus was giving an extra slip, which was attaching to the medial condyle of tibia in 7 cases (13.2%). The pes anserinus was inserting into the fascia cruris in 8 specimens (15.1%). The remaining 5 lower limbs (9.4%) were having variant pattern. Semimembranosus muscle was taking in the formation of pes anserinus in 3 lower limbs (5.7%). The gracilis and sartorius were absent in one lower limb each (1.9%).

Conclusion

The present study has provided the morphological knowledge about the variations of pes anserinus of the tibia in South Indian population. We believe that this information is enlightening to the orthopaedic and plastic surgeons.

Support or Funding Information

NIL

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
MYOFASCIAL PLANTAR PAIN AND THE RELATIONSHIP BETWEEN THE INNERVATION OF ABDUCTOR HALLUCIS MUSCLE. NEW PHYSIOPATHOLOGICAL APPROACH
Authors: Wada Juliano , Akamatsu Flávia Emi , Itezerote Ana Maria , Hojaij Flávio , Andrade Mauro , Jacomo Alfredo Luiz
Published: June 2021

The plantar myofascial pain syndrome is a common cause of chronic pain and dysfunction of the abductor hallucis muscle has been reported as a possible factor implicated in its physiopathology. We decided to investigate the anatomical correspondence of the muscle innervations to the clinical evaluation of patients with chronic foot pain.

Methods

We dissected 30 feet from fixed cadavers (ADG) and identified the entry points of nerve branches into each of four quadrants determined for the abductor hallucis muscle belly. The clinical group (OPG) consisted of 30 patients evaluated by digital algometer (Force Ten FDX, Wagner Instruments, Greenwich, CT) and the pression which triggered painful sensation in each of the quadrants of the muscle were obtained. We analyzed anatomical data with Poisson distribution and logarithm link function followed by Bonferroni multiple comparisons. For the patient's group, the same methods for normal distribution were employed.

Results

Branches of the medial plantar nerve were predominantly found in the posterolateral quadrant (Q1) and there was a match to the area of the muscle in which lower pressures were necessary to produce pain

Conclusion

Anatomical distribution of the branching of the medial plantar nerve into the abductor hallucis muscle is topographically similar to the muscle areas where patients with chronic foot pain referred higher sensitivity to external pressure. These findings suggest that the nervous supply to the muscle may be implicated in the physiopathology of the myofascial plantar pain.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LOW LEVEL LASER THERAPY INCREASES MESENCHYMAL CELL PROLIFERATION AND COLLAGEN II AFTER EXPERIMENTAL INJURY
Authors: Akamatsu Flávia Emi , Teodoro Walcy R. , Itezerote Ana Maria , Saleh Samir , Martinez Carlos Augusto Real , Ribeiro Marcelo Lima , Pereira José Aires , Hojaij Flávio , Andrade Mauro , Jacomo Alfredo Luiz
Published: June 2021

BACKGROUND

Tendon is a mechanosensitive tissue which transmits muscle derived forces to bones. Photobiomodulation (PBM) also known as low‐level laser (or light) therapy (LLLT), has been used in therapeutic approaches in tendon lesions but uncertainties about its mechanisms of action have prevented its widespread use so far. We investigated the response of LLLT therapy in experimental lesions of the calcaneous tendon in rats.

METHODS AND MATERIAL

Thirty adult male Wistar rats weighing 250 to 300 g were surgically submitted to bilateral partial transverse section of the calcaneous tendon. The right tendon was treated with LLLT whereas the left tendon served as control. On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each which were treated with LLLT (Konf, Aculas – HB 750), 780 nm and 80 mW for 20 seconds, three times/week for 7, 14 and 28 days. The Sca‐1 was examined by immunohistochemistry and histomorphometry and COLA1, COLA2 and COLA3 gene expression by qRT‐PCR.

RESULTS

COLA2 gene expression was higher in LLLT treated rats as compared to the control group. Interestingly, the histomorphometric analysis coincided with increased number of mesenchymal cells, characterized by Sca‐1 expression in the lesion region (p<0.001).

CONCLUSION

LLLT effectively interferes in tendon tissue repair after injury by stimulating mesenchymal cells proliferation and synthesis of collagen type II, which could provide structural support to the interstitial tissues during the healing process of the calcaneous tendon. Further studies are needed to confirm LLLT role in tendon healing.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Probing the role of distal residues in DinB and Pol Kappa in the extension step of DNA damage bypass
Authors: Stern Hannah R. , Mills Caitlyn L. , Ondrechen Mary Jo , Beuning Penny
Published: June 2021

DNA is constantly subjected to damage from endogenous and exogenous sources. Replicative DNA polymerases are typically unable to replicate damaged DNA, but specialized DNA polymerases in the Y family possess this ability. Escherichia coli has two Y family polymerases that are specialized to bypass damage when copying DNA in a process called translesion synthesis (TLS). DinB is one of these polymerases and is involved in bypassing deoxyguanosine adducts at the N2 position. Humans have four Y family polymerases, including DNA polymerase kappa. E. coli DinB and human pol kappa both bypass minor groove adducts and are inhibited by major groove adducts. However, pol kappa is more efficient in copying past DNA damage in the extension step of translesion synthesis. In order to probe the importance of particular residues in the extension step of TLS, the computational tool POOL was utilized. This method identified active site residues and residues previously observed to be important for activity. POOL also predicted more distant residues that do not have direct contact with substrates that may have catalytic importance, but the residues are in different regions of DinB and pol kappa. To study the contribution of these distal residues on the extension step of TLS, DinB and pol kappa variants with mutations at the predicted distal positions were constructed and are being assayed for bypass of damage. We have identified variants with a range of activity on undamaged and damaged DNA; in particular several mutations in the DinB little finger domain severely reduce activity.

Support or Funding Information

Support from NSF‐MCB‐1517290, American Cancer Society RSG‐12‐161‐01‐DMC, and the PhRMA Foundation (predoctoral fellowship in informatics awarded to CLM)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Wavelength of light impacts circadian regulation in Rdl mutant Drosophila melanogaster
Authors: Walters Benjamin Patricia , MacDonald Stephanie Patricia , Rogers Ryan Patricia
Published: June 2021

Drosophila melanogaster are a useful human analog for circadian rhythm research. They have a short life cycle, similar genetic loci to humans with respect to spatial and temporal gene expression and easily traceable mutant phenotypes. This study analyzes the impact of specific photon wavelength on circadian rhythm in Drosophila melanogaster. In flies, Resistant to Dieldrin (Rdl) encodes a subunit of the GABA receptor, which is upregulated by the circadian oscillator, WAKE. This interaction induces RDL localization to the plasma membrane to modulate sleep; however, the specific function of RDL and its downstream interactions to facilitate sleep/wake switching, are yet to be characterized. The Drosophila Activity Monitor TriKinetics System and a custom‐built light controlling apparatus were used to characterize the impact of varied light wavelengths on locomotion in control, RdlMD‐RR mutant flies and flies overexpressing Rdl in a subset of circadian neurons. Preliminary data suggests RdlMD‐RR flies show less activity during daylight hours, and severely diminished locomotion in darkness compared to controls; thus, it is the intent to characterize the specific wavelength associated with induction of this phenotype. These data will provide information related to sleep latency and quality of sleep as different wavelengths of light would amplify or diminish sleep onset and duration.

Support or Funding Information

This work was supported by funds from the Department of Sciences at Wentworth Institute of Technology.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Direct pharmacologic regulation of the ETS transcription factor PU.1
Authors: Lee Sangchoon , Poon Gregory M. K.
Published: June 2021

The ETS‐family transcription factor PU.1 is essential for the self‐renewal of hematopoietic stem cells (HSC) as well as their differentiation to various lineage‐committed hematopoietic progenitors. Specific to the bone marrow niche, PU.1 regulates the expression of several key cytokines (e.g. CXCR4) and their receptors (e.g. GM‐CSF receptor, FLT3) involved in HSC mobilization. Extensive studies have shown that incomplete loss of PU.1 activity, to ~20% wildtype levels, leads to lineage arrest and aggressive leukemic disease. Conversely, lentiviral overexpression of PU.1 resolves the differentiation block in leukemic blasts, and suggests the activation of PU.1 as a potential therapeutic approach. To this end, we have identified by phage display technology a panel of selective PU.1‐binding oligopeptides. Several of these peptides promote PU.1 binding to cognate DNA sites in a dose‐dependent manner and confer resistance to PU.1 against inhibition by netropsin. To determine the essential residues responsible for their activity, we screened a targeted library of truncated hit peptides and found that as few as four residues were sufficient to enhance site‐specific DNA binding by PU.1. These peptide sequences represent leads for developing direct‐acting PU.1‐specific modulators with respect to target DNA binding and interactions with major binding partners. The resulting low‐MW reagents could significantly expand the pharmacology of transcription factors and help overcome barriers in hematopoietic research and therapy that are currently imposed by genetic approaches.

Support or Funding Information

Supported by NIH HL129063.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of Sp1 and Sp3 Occupation of the Distal Manganese‐Superoxide Dismutase Promoter in the Presence of HIV‐1 Tat
Authors: Manes Terrin , Cota‐Gomez Adela
Published: June 2021

The introduction of antiretroviral therapies (ART) is largely responsible for the development of chronic HIV infection, but long‐term exposure to viral proteins can contribute to oxidative stress and chronic inflammation which could promote the development of HIV‐Associated co‐morbidities such as pulmonary arterial hypertension (HA‐PAH). Previously, we have reported that the HIV transcriptional regulator, Tat, can induce oxidative responses by depleting cellular glutathione and repressing the mitochondrial manganese‐superoxide dismutase (MnSOD, sod2); there are Tat‐regulated elements within the proximal region of the human sod2 promoter, designated Tat‐sensitive Sp‐responsive elements (TSS). Tat alters the pattern of binding by the Sp1 and Sp3 transcription factors on the sod2 TSS, resulting in transcriptional repression. Recently, we observed evidence of Tat‐regulated regions containing predicted Sp‐binding sequences within more distal sections of the sod2 promoter, which we have termed the DTSS (Distal Tat‐sensitive Sp‐responsive elements). The previous discoveries that Tat regulates the proximal promoter by changing the pattern of Sp binding prompted us to investigate if Tat influences transcription from the distal elements via modulation of Sp1 and Sp3 ratios. In this study, we transiently transfected luciferase‐reporter plasmids containing varying regions of the sod2 promoter in the presence or absence of a Tat‐expressing plasmid into human pulmonary artery endothelial cells (HPAEC). To further investigate the DTSS and the occupancy of Sp1 and Sp3, we collected Sp1 and Sp3‐bound DNA via Chromatin Immunoprecipitation (ChIP). Our data shows that under basal conditions certain regions of this promoter are highly expressed while others are repressed by an already existing biological ratio of Sp1 and Sp3. However, in the presence of Tat, all of these regions are repressed. ChIP analysis shows that Tat changes the amounts of Sp1 and Sp3 bound at the predicted Sp‐binding clusters. With this data we conclude that Tat represses sod2 by altering the ratio of Sp1 and Sp3 at both proximal and distal regions thereby extending the known regulatory sequences of the sod2 gene that are responsive to HIV Tat.

Support or Funding Information

National Institutes of Health (NIH) 1RO1HL125050‐01

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Iron Metabolism Receptors, TfR1 and TfR2, Bind and Stabilize Transferrin Through Non‐Conserved Interactions
Authors: Kleven Mark Daniel , Enns Caroline
Published: June 2021

Hereditary hemochromatosis, a disease marked by chronic iron overload from insufficient expression of the hormone hepcidin, is one of the most common genetic diseases in people of Northern European ancestry. One form of HH (Type III) results from mutations in the transferrin receptor‐2 (TfR2). How TfR2 functions to promote hepcidin expression remains unclear, though it appears to be an important part of a mechanism to modulate hepcidin expression based on systemic iron levels. This responsiveness may be achieved through TfR2's competition with its homolog, TfR1, for their ligand, the iron transport protein, transferrin (Tf). TfR1 is expected to be the preferred receptor for diferric, or holo‐, Tf and that TfR2 will bind holo‐Tf when Tf‐iron saturation levels are elevated. To better understand the nature of these protein‐protein interactions, a surface plasmon resonance binding study with the full‐length receptors was conducted for the first time. In agreement with previous studies, holo‐Tf binds to TfR1 significantly stronger than TfR2. However, the binding constant for Tf‐TfR2 is still far below that of physiological holo‐Tf levels, inconsistent with the hypothetical model and suggests that other factors mediate the interaction. Apo‐Tf only weakly binds TfR2 at serum pH, thus is not be able to effectively compete with holo‐Tf under these conditions. Like TfR1, TfR2 is able to bind apo‐Tf with higher affinity at the lower pH of endosomes, suggesting that it could function to take up iron, where stabilization of the apo‐Tf‐TfR complex is key. Tf‐binding Tf‐binding to a TfR2 chimera containing the TfR1‐helical domain indicates that the differences in the helical domain account for differences in on‐rate of Tf, but non‐conserved inter‐receptor interactions are necessary stabilize the Tf‐TfR1/TfR2 chimera complex. One possible site of stabilization, the apical arm junction, is not important for TfR1‐Tf complex stabilization. The junction is critical for the TfR2‐Tf interaction, however; highlighting one non‐conserved stabilization mechanism. Our results highlight the differences in Tf interactions with the two TfRs.

Support or Funding Information

NIH T‐32 Training Grant GMMBI0231C; NIH R01DK072166

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
LIMD2 is an intracellular activator of Integrin Linked Kinase(ILK) activity and GSK‐3/Akt/beta‐catenin signaling
Authors: Dedhar Shoukat , Awrey Shannon
Published: June 2021

Integrin‐Linked Kinase (ILK) is an intracellular, focal adhesion, scaffold protein that couples beta 1‐integrins and growth factors to actin cytoskeletal organization and signal transduction via GSK‐3 beta and Akt. ILK has an atypical kinase catalytic domain, and has been considered to be a pseudokinase.

Here, we demonstrate that a small LIM domain only protein, LIMD2, which is highly overexpressed in several types of cancers, and which interacts directly with ILK within the kinase domain ( Peng, H et al, Cancer Res, 2014), forms a complex with ILK in cancer cells and stimulates ILK kinase activity. LIMD2 overexpression results in the stimulation of the phosphorylation of GSK‐3 beta on Ser‐9, as well as of Akt on Ser‐473, in an ILK‐dependent manner. Furthermore we demonstrate a striking LIMD2 induced stimulation of phosphorylation of beta‐catenin on Ser‐552, shown previously to be a target of Akt, leading to 14.3.3‐dependent stablization of beta‐catenin and stimulation of Lef/TCF transcriptional activity that results in tumor cell invasion (Fang et al. J Biol Chem, 2007).

Since LIMD2 upregulation in tumor cells is associated with increased cell invasion and metastasis, we examined the effect of inhibiting LIMD2 induced ILK activity on breast, thyroid and pancreatic cell invasion through collagen and matrigel. As expected, overexpression of LIMD2 induced tumor cell invasion, and this invasion was blocked by ILK siRNA or by a specific pharmacological inhibitor of ILK, QLT‐0267. Inhibition of ILK in these cells also resulted in dramatic suppression of Akt Ser‐473 and beta‐catenin Ser‐552 phosphorylation as well as of LEF‐TCF transcrptional activity.

In summary, we describe a new paradigm for the intracellular activation of low activity protein kinases through protein‐protein interactions. Specifically we identify a new signaling axis through the activation of ILK by LIMD2, which results in the stimulation of a GSK‐3/Akt/Beta‐catenin pathway important in tumor cell invasion.

Support or Funding Information

Supported by funds from the Cancer Research Society, Quebec, Canada

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Anti‐metastatic activity of capsaicin and natural capsaicin‐like compounds in human lung adenocarcinoma
Authors: Richbart Stephen D. , Nolan Nicholas A. , Akers Austin T. , Colclough Kate W. , Hurley John D. , Williams Cathryn D. , Rollyson William D. , Brown Kathleen C. , Denning Krista L. , Brown Linda G. , Egleton Richard D. , Dasgupta Piyali
Published: June 2021

Lung adenocarcinoma (LAC) accounts for 65% of all non‐small cell lung cancer (NSCLC) cases. A substantial proportion of LAC present with extrapulmonary and distant metastasis at the time of their diagnosis. One of the earliest events of the metastatic process is the invasion of malignant cells through the surrounding extracellular basement membrane into the blood and lymph. The long‐term goal of our laboratory is to identify nutrition‐based agents which will suppress the growth and progression of human LACs. Capsaicin is the pungent ingredient of chili peppers. Published reports have revealed that capsaicin inhibits the invasion and metastasis of several types of human cancers including melanoma, prostate cancer, and cholangiosarcoma. However, the clinical application of capsaicin as an anti‐cancer drug is limited by its unpleasant side‐effect profile. This led us to compare the anti‐metastatic activity of capsaicin with natural non‐pungent capsaicin‐like compounds, namely capsiate and capsiconiate. The structure and bioactivity of capsaicin closely resembles capsiate. There are no published reports involving the biological activity of capsiconiate. We measured the anti‐invasive activity of these compounds by two independent invasion assays, namely the Boyden chamber assay and spherical invasion assay. We found that capsaicin and capsiate displayed anti‐invasive activity in three human LAC cell lines. In contrast, capsiconiate did not suppress the invasion of any LAC cell lines. Furthermore, we tested the anti‐metastatic activity of capsaicin in a syngeneic mouse model of metastasis. We observed that the daily dietary administration of capsaicin in AIN‐76A diet (with 5% lipid level) robustly decreased the area metastatic foci (in the lung) relative to vehicle‐treated mice. The results of our studies may foster the development of novel anti‐metastatic therapies for human LAC.

Support or Funding Information

Funding for our study was supported by an NIH R15‐AREA Grant (2R15CA161491‐02) and a NASA Undergraduate Fellowship to NAN

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Investigation of metabolic profiling of leelamine in vitro and in vivo by LC‐MS/MS
Authors: Shrestha Riya , Lee Taeho , Lee Sangkyu
Published: June 2021

Leelamine is a diterpene compound found in the bark of pine trees that is generating considerable interest owing to its potent anticancer properties. The present study aimed to investigate the metabolic profile of leelamine in human liver microsomes (HLMs) and mice using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). We found that leelamine undergoes only Phase I metabolism, which generates one metabolite that is mono‐hydroxylated at the C9 carbon of the octahydrophenanthrene ring (M1) both in vitro and in vivo. The structure and metabolic pathway of M1 were determined from the MSn fragmentation obtained by collision‐induced dissociation using LC‐MS/MS in HLMs. Cytochrome p450 (CYP) 2D6 was found to be the dominant CYP enzyme involved in the biotransformation of leelamine to its hydroxylated metabolite while CYP2C19, CYP1A1, and CYP3A4 contributed to some extent. Moreover, further investigation of the in vivo metabolism of leelamine in mice using urinary and fecal analysis detected only one metabolite, M1, in the urine but none in the feces. In conclusion, leelamine was metabolized to mono‐hydroxyl metabolite by CYP2D6 and mainly excreted in the urine.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Milk plus Carbohydrate Supplementation during Interval Walking Training Enhanced the Improvement of Blood Glucose and Blood Pressure Regulations in Older People
Authors: UCHIDA KOJI , MASUKI SHIZUE , MORIKAWA MAYUKO , FURIHATA MAYUKA , MANABE KAZUMASA , OGAWA YU , KATAOKA YUFUKO , AIDA TAKAMICHI , NAKANO SAKURA , NOSE HIROSHI
Published: June 2021

Aims

We examined whether the milk + carbohydrate (CHO) supplementation during interval walking training improved the symptoms of lifestyle‐related diseases with enhanced methylation of the pro‐inflammatory gene, NFKB2.

Methods

Twenty nine subjects (~70 yr) having performed interval walking training for >6 months but with >130 mmHg of systolic pressure and >110 mg/dl of blood glucose participated in the study. After the baseline measurements of peak aerobic capacity (VO2peak) by graded walking test, blood constituents, blood glucose by continuous method (iPro2R), and carotid arterial compliance with Doppler ultrasound (Vivid7R) and non‐invasive arterial pressure measurement (FinometerR), we randomly divided subjects into two groups: MILK (6 men, 9 women) and CNT (6 men, 8 women) consuming either milk (10 g protein and 9.5 g CHO) + CHO (8.8 g) or CHO (7 g) alone during 5‐month interval walking training, respectively. After the training, we measured the same variables as before.

Results

In MILK, VO2peak and carotid arterial compliance increased and mean blood glucose decreased with a reduced fluctuation after standardized meal intake during the day (all, P<0.05); however, these changes were minimal in CNT (all, P>0.06). This was accompanied by enhanced methylation of NFKB2 in MILK than CNT (P=0.045).

Conclusions

Milk + CHO supplementation during interval walking training enhanced the improvement of lifestyle‐related diseases with enhanced NFKB2 methylation.

Support or Funding Information

This study was supported by a grant from the Japan Society for the Promotion of Science (15H01830).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Sphingosine ‐1‐Phosphate on Expression of MAPK and Akt Signaling Pathways in Hypoxic Human Extravillous Trophoblasts
Authors: Swan Kenneth F. , Pridjian Gabriella , Swayze Thomas , Gagen Brennan R. , Intapad Suttira
Published: June 2021

Preeclampsia (PE), is a serious pregnancy disorder characterized by placental ischemia, hypertension, and proteinuria. A successful pregnancy requires the migration and invasion of extravillous trophoblasts (EVTs), from the villous tips of the placenta into the maternal decidua and subsequently the spiral arteries. Failure of EVTs to adequately migrate and invade into the spiral arteries leads to insufficient blood flow to the developing placenta commonly found in PE. Sphingosine‐1‐phosphate (S1P) functions as a key regulator of cellular homeostasis in angiogenesis, inflammation, and endothelium permeability. S1P acts on its receptor to control cell migration through the PI3‐Akt and p38 MAPK signaling pathways. We have previously reported that expression of placenta ceramidase, sphingosine kinases 1, and sphingosine kinases 2 were altered in reduced uterine perfusion (RUP) mouse model. In the present study, we tested the hypothesis that serine 473 phosphorylated Akt (pAkt) and phosphorylated p38 MAPK expressions are decreased in hypoxic condition and treatment with S1PR 1 agonist and antagonist alter these expression levels.

Methods

The human extravillous trophoblasts (EVTs) cell line SGHPL‐4 was cultured in either normoxia 21% oxygen or hypoxia 2% oxygen conditions for 24 hours. The cultures were treated for one hour with vehicle control or with S1PR1 receptor agonists (SEW2871, 1uM) or S1PR antagonist (W146, 18 nM).

Results

Both pAkt and p38 MAPK were significantly decreased in hypoxia EVTs compared to normoxia EVTs (p38 MAPK: 50% and pAkt: 25% decrease). SEW2871 increased p38 MAPK in hypoxia EVTs to a level similar to normaxia EVTs. Inhibition of S1PR1 by W146 decreased p38 MAPK in both hypoxia and normoxia. SEW2871 normalized the differences of pAkt expression between normoxia and hypoxia EVTs. While W146 did not change the pAkt in normoxia, it significantly increased pAkt in hypoxia EVTs cells. This data suggests that activation of S1P receptor type 1 increases the expression of angiogenic pMAPK in hypoxic SGHPL‐4 trophoblast cells.

Support or Funding Information

Dr. Intapad was supported by funding from the AHA 16SDG27770041, ASN Kidney Research Career Development grant, and Start‐up funds from Tulane University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Hydrogen Sulfide (H2S) on Homocysteine Mediated Glutamate Excitotoxicity, Endoplasmic Reticulum Stress and Pyroptosis in Retina
Authors: George Akash K. , Singh Mahavir , Homme Rubens P. , Majumder Avisek , Tyagi Suresh
Published: June 2021

High levels of circulating homocysteine (Hcy) in the blood i.e. hyperhomocysteinemia (HHcy) have been linked to several visual disorders and are associated with impairment of retinal functions. The precise molecular mechanisms of how HHcy affects the retinal biology are unknown. The main objective of this study was to investigate the effect of HHcy on retinal glutamatergic dysfunction, endoplasmic reticulum (ER) stress and inflammation and whether H2S; a potent anti‐oxidant, anti‐inflammatory, vasoactive gas can mitigate the Hcy effects. We used HHcy mouse model CBS+/− and the wild type (WT) treated with H2S donor GYY4137 (133 μM/kg‐1/d‐1). The animal groups were designated as follows: Group‐I: WT control + No treatment; Group‐II: CBS+/− +No treatment, Group‐II: CBS+/− + GYY4137 and Group‐II: WT control + GYY4137. Novel Object Recognition Test (NORT) and Light Dark Box Test (LDBT) were employed to check the visual functions. The bovine serum albumin (BSA) labeled fluorescein isothiocyanate (FITC‐BSA) was used to study the vascular permeability in the retinas of these mice from all the four groups. Western blotting and qPCR were carried out to analyze the protein and mRNA expression levels in the retinas from all the groups. Our NORT and LDBT data showed a significant impairment of the visual functions in the CBS+/− mice as compared to the WT control mice. We also found a significant amount of leakage in the blood vessels in the retina of the CBS+/− mice in comparison to the WT control mice and that was mitigated by GYY4137 treatment. We noticed oxidative stress, ER‐stress, Glutamate excitotoxicity and inflammation were prominent in the tissue samples collected from the CBS mice in comparison to WT controls. Interestingly, we found that GYY4137 treatment could mitigate the glutamatergic dysfunction, ER stress, neurovascular dysfunction and the pyroptosis along with improvement in the visual functions in the CBS+/− mice treated with H2S.

Support or Funding Information

NIH Heart, Lung & Blood Institute (HL‐74815), Institute of Neurological Disorders & Stroke (NS‐084823).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Subcellular Ca2+ Puffs Mediated By Different Inositol Trisphosphate Receptor Isoforms
Authors: Lock Jeffrey T. , Alzayady Kamil J. , Yule David I. , Parker Ian
Published: June 2021

The second messenger inositol trisphosphate (IP3) evokes Ca2+ liberation through IP3 receptor/channels (IP3Rs) in the ER membrane to generate a hierarchy of cytosolic signals, including local Ca2+ puffs that arise from concerted openings of clustered IP3Rs, and global Ca2+ waves that propagate through the cell. Mammalian cells express three isoforms of IP3Rs that are encoded by different genes, have ~ 60–80% sequence homology and are reported to display differing functional characteristics. Imaging Ca2+ puffs with single‐channel resolution provides important information on the localization and properties of IP3Rs in intact cells. However, interpretation has been complicated because studies have been limited to native cell types that typically express varying proportions of two or three IP3R isoforms, with additional complexity arising from hetero‐multimerization within the tetrameric IP3R. Here, we applied high‐resolution TIRF microscopy to image Ca2+ puffs in HEK‐293 cell lines in which IP3R isoforms were knocked‐out in pairs by CRISPR/Cas9 technology to generate cells expressing exclusively IP3R types 1, 2 or 3. Puffs were evoked by photoreleased i‐IP3 in all three cell lines, indicating that IP3Rs of each isoform are able to assemble into the clusters that underlie these local Ca2+ signals. Moreover, Ca2+ puffs mediated by all three isoforms displayed largely similar mean amplitudes, temporal characteristics and spatial extents, except that type 3 IP3R‐mediated puffs showed appreciably faster kinetics. A quantal analysis of fluorescence signals revealed the single‐channel Ca2+ flux to be equivalent between isoforms, further suggesting that clusters of different IP3R isoforms contain similar numbers of active channels. Finally, mapping the distances between puff centroid localizations suggested that clusters of type 1 IP3Rs are more dispersed than clusters of type 2 or 3 IP3Rs. Overall, our results indicate that, although there is substantial functional redundancy among the IP3R isoforms, each exhibit distinct properties and spatial arrangements that likely shape the spatial‐temporal patterning of cellular Ca2+ signals observed in native cells.

Support or Funding Information

This work was supported by NIH grants R01 DE019245 (D.I.Y) and R37 GM048071 (I.P)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of mucus layer on the transcellular absorption of lipophilic drugs in rat small intestine
Authors: KISHIMOTO HISANAO , MIYAZAKI KAORI , SHIRASAKA YOSHIYUKI , INOUE KATSUHISA
Published: June 2021

Purpose

The mucus layer, which is adjacent to the intestinal wall, is considered to be a physiological barrier to intestinal drug absorption, since a drug must diffuse through this lipophilic layer prior to permeation across the intestinal epithelial cells. Thus, mucus layer permeation is suggested to be a rate‐limiting step in the intestinal absorption of lipophilic drugs. The major glycoprotein components of the mucus layer are mucins, which presumably play a pharmaceutical role in intestinal drug absorption, however, there have been no reports that describe the contribution of mucins to intestinal drug absorption at the molecular level. In this study, we examined the involvement of the mucus layer in the intestinal permeability of various lipophilic drugs in rat small intestine. Furthermore, we evaluated the regional distributions of mucins (MUCs) in rat gastrointestinal tract and explored the relationship between the effect of the mucus layer on passive transcellular transport and the expression pattern of mucins.

Methods

Drug permeation experiments were conducted using the in vitro sac method. The amount of mucus glycoproteins liberated from the intestinal wall were determined by the alcian blue method. The mRNA and protein expression levels of mucins were evaluated by quantitative real‐time RT‐PCR (qPCR) and Western blotting, respectively.

Results and Discussion

The membrane permeability of the lipophilic drugs in the rat gastrointestinal tract was increased by pretreatment with dithiothreitol (DTT), which is commonly used as a mucus remover, and the alternation was remarkable in the upper part of the small intestine. Furthermore, the enhancing effects of DTT tended to be pronounced on the permeability of drugs with higher log D values in the case of neutral drugs with non‐dissociable groups. These results indicated that the effect of the mucus layer on intestinal permeability depends on the log D values and the physiological charge of the compound. On the other hand, the mRNA expression analysis of MUC genes revealed that the intestinal expression of MUC5AC is considerably higher in the duodenum, and those of MUC1, MUC2, and MUC3A are gradually increased toward the lower intestine in rats. Furthermore, Western blotting showed abundant MUC5AC protein in the mucus solution liberated from jejunum, but not from ileum and colon by the treatment with DTT. Therefore, these results suggest that rMUC5AC is involved in the absorption of lipophilic drugs in the upper part of rat small intestine.

Conclusions

MUC5AC could be involved in the barrier function to limit the transcellular permeation of hydrophobic drugs in the upper part of the gastrointestinal tract.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of spontaneous DNA damage and single‐stranded DNA in generation of enlarged G2 phase cells in rad52 mutants of Saccharomyces cerevisiae
Authors: England Corbin J. , Weis Monica F. , Lewis L. Kevin
Published: June 2021

The DNA of living creatures is continuously being damaged by endogenous and exogenous sources and different DNA repair pathways exist to mend the different types of damage. Double‐stranded breaks (DSBs) in the DNA of eukaryotes can be repaired by either homologous recombination (HR) or nonhomologous end‐joining (NHEJ). The initial steps of HR in the budding yeast S. cerevisiae lead to formation of long ssDNA tails at the broken ends. These tails arise through short resection by the Mrx and Sae2 proteins, followed by longer resection by Exo1 and Sgs1‐Dna2 and possibly other enzymes. The tails are subsequently coated with the single‐stranded DNA binding protein complex Rpa, which then recruits other proteins to the site such as DNA damage checkpoint sensor proteins and RAD52 group strand exchange proteins.

After exposure to DNA damaging agents, yeast cell cultures exhibit an increase in G2 phase cells due to activation of the dominant DNA damage cell cycle checkpoint. rad52 haploid and diploid mutants grown in normal liquid cell culture display an increase in large‐budded cells. DAPI staining indicated that over 90% of these cells were in G2 phase. Creation of double mutant strains with RAD52 and any of seven different DNA damage checkpoint genes co‐inactivated resulted in loss of the high G2 cell phenotype, suggesting constitutive activation of DNA damage checkpoints in the mutants. rad52 cells were also found to have other unusual characteristics, including (a) larger average sizes than wildtype cells, especially among large‐budded cells, (b) faster cell sedimentation rates, and (c) strongly enhanced light scattering in aqueous suspensions. The role played by formation of long 3′ ssDNA tails at DSBs during HR has been investigated using rad52 double mutants incapable of forming long tails. DNA damage checkpoint protein and single‐stranded binding protein fusions with GFP are also being employed to analyze differences in foci formation within WT and rad52 cells.

Support or Funding Information

National Institutes of Health

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Using Hydroxyproline to Measure Collagen in Teeth With and Without Whitening Strips
Authors: Meyer Seann , Fiorica Antonino , Keenan Kelly
Published: June 2021

Billions of dollars are spent in the United States every year on tooth whitening. Whether it is done in a dentist's office or with over the counter products, the active ingredient is often hydrogen peroxide which has long been known to damage proteins. The outermost part of the tooth is the enamel and contains several proteins while dentin, the layer underneath, contains 90 to 95% collagen. This protein contains the modified amino acid hydroxyproline and the level of this amino acid has been used to measure the amount of collagen. Hydroxyproline can be detected using a colorimetric test: the original test required use of perchloric acid but the method was modified with use of hydrochloric acid replacing the perchloric acid. Teeth were purchased, placed in artificial saliva and treated with whitening strips following the manufacturer's instructions while other teeth did not receive the strips. Afterwards, the teeth were dialyzed against 10% EDTA, pH 9.0 for 2 weeks to remove calcium and then dialyzed against water to wash the teeth. They were then placed in liquid nitrogen and hammered into shards which were turned into a powder using a blender. The powder was suspended in an acetic acid solution to make the collagen fraction(COL). This fraction was treated with pepsin for four hours, neutralized with saturated Tris solution and centrifuged for 1 hour. The supernatant was the pepsin soluble fraction (PSC). Both COL and PSC were then hydrolyzed by adding 6 M HCl overnight at 95 °C. The resulting hydrolyzed samples were neutralized with 10 M sodium hydroxide and tested for hydroxproline. Results indicated a significant drop in hydroxproline content after one treatment and an even greater loss after three treatments. The drop in the level of hydroxyproline following treatment with whitening strips does suggest a loss of collagen from the dentin due to the whitening strips.

Support or Funding Information

Authors express gratitude to Research and Professional Development Grant from Stockton University.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.