bluuebunny/crossref_metadata_2025 huggingface dataset:
Mitochondrial dysfunction in endothelium impairs vasodilation in Zucker obese rats with insulin resistance
Authors: Katakam Prasad V.G. , Domoki Ferenc , Snipes James A , Busija Anna R , Busija David W
Published: June 2021

Mitochondria affect cerebrovascular tone by activation of mitochondrial ATP‐sensitive K+ channels (mitoKATP). Insulin resistance (IR) causes mitochondrial dysfunction, therefore, we evaluated the effects of diazoxide, a mitoKATP activator, on cerebral arteries of Zucker obese (ZO) rats and lean (ZL) controls. Diameter studies showed reduced vasodilation to diazoxide in ZO compared to ZL. Endothelial denudation reduced vasodilation to diazoxide in all arteries implicating endothelial mitoKATP. Inhibition of nitric oxide synthase (NOS) reduced vasodilation in intact arteries from ZL but not ZO, indicating diminished NO effects. Paradoxically, NOS inhibition in endothelium denuded arteries enhanced vasodilation in ZO implying the expression and uncoupling of NOS in vascular smooth muscle (VSM). Inhibition of cyclooxygenase (COX) enhanced vasodilation in intact arteries from ZL but not ZO. In contrast, endothelial denudation enhanced vasodilation in ZO but not ZL. Thus, ZO arteries exhibit enhanced production of both vasoconstrictor and vasodilator prostanoids. Fluorescence studies of rat cerebral microvascular endothelial cells showed enhanced NO production in response to diazoxide and also confirmed reduced NO generation in ZO arteries. Thus, endothelial mitochondrial dysfunction in ZO arteries results in the impairment of NOS and COX activity. Support: NIH HL077731, HL030260, HL 065380.
Heme Oxygenase Induced Endothelial BKCa Channel Activity Following Chronic Hypoxia
Authors: Riddle Melissa Anne , Paffett Michael L , Walker Benjimen R
Published: June 2021

Exposure to chronic hypoxia (CH) results in blunted myogenic and agonist‐induced tone in systemic arteries that persists upon return to normoxia. This impaired vasoconstrictor reactivity is reversed by either removal of the endothelium or by blockade of the CO producing enzyme heme oxygenase (HO). Interestingly, endothelial cell (EC) specific blockade of large conductance Ca2+‐activated K+ (BKCa) channels also restores vasoreactivity in arteries from CH rats. Patch clamp studies demonstrate EC BKCa activity following CH, but not in ECs from control rats, although channels are observed by immunofluorescence (IF) in cells from both groups. Since HO‐derived CO is an endogenous activator of BKCa channels, we hypothesized that HO and BKCa form a functional unit that results in enhanced channel activity following CH. In support of the hypothesis, the HO inhibitor zinc protoporphyrin IX (ZnPPIX) significantly decreased K+ currents in ECs from CH rats, but was without effect in EC from control rats. In addition, the HO substrate hemin increased K+ currents only in the CH group. HO‐2, but not HO‐1, was found to co‐localize with BKCa in cells from each group. These results suggest that although HO and BKCa channels are expressed in EC from each group, HO‐derived CO only stimulates channel activity following CH.
A Simple and Rapid Method for Collecting Cerebrospinal Fluid from Rat
Authors: Liang Xiaojuan , Zhao Tuocheng , Liu Zhengshang , Wang Yehong , Shao Qiming
Published: June 2021

The goal of this study was to establish a simple, reliable and efficient method for the collection of cerebrospinal fluid (CSF) from rat. Our new technique can easily obtain 150‐200 uL of blood free CSF from rat in less than 2 minutes.

Method

Under anesthesia, rat was flexed downward of the head at approximately 45 degree. A small incision was made in the skin. Overlying the occipital bone, the underlying tissue was prepared in such a way that the atlanto‐occipital membrane in between the occipital bone and the upper cervical vertebra was exposed. The remaining tissue above the atlanto‐occipital membrane was cut by scalp until the atlanto‐occipital membrane is clearly visible. A butterfly needle connected with 1 ml of syringe was used. When collecting CSF, the needle was directly punctured into the cisterna magna, and the non‐contaminated sample was drawn into the syringe until blood appears. We use hemostat to clamp the silicon tubing when the CSF becomes bloody. The tubing is cut with a pair of scissors from the clear part to avoid blood contamination. Then the clear CSF is transferred into eppendorf tubes through an insulin needle.

Results

This technique had a greater than 95% success of CSF collection. Non‐contaminated 100‐250 µl of CSF can routinely be obtained per rat.

In conclusion

This technique has proved to work well in experimental rat. The method is convenient, efficient and provides significant time saving.
Regulation of TRPC6 channel by hydrogen peroxide
Authors: Graham Sarabeth Marie , Ding Min , Sours‐Brothers Sherry , Ma Rong
Published: June 2021

The present study was performed to examine the effect of H2O2 on TRPC6 channel in HEK293t cells. In the TRPC6 expressing cells, H2O2 significantly stimulated Ca2+ entry in a dose‐dependent manner. This effect appeared through a distinct mechanism from DAG because the Ca2+ entry response and TRPC6 channel activity induced by a combination of OAG and H2O2 was significantly greater than the individual response of either OAG or H2O2. Inhibition of endogenous H2O2 by catalase substantially attenuated the OAG‐induced Ca2+ entry. NEM, a thiol‐specific oxidizing agent, were able to mimic the H2O2 response, and DTT, a thiol‐specific reducing agent, significantly inhibited the H2O2 response. DTNB, a cell membrane‐impermeable thiol oxidizing agent, did not affect TRPC6 channel activity when applied via the pipette solution in cell‐attached patches. Furthermore, the NEM‐induced TRPC6 activation was only observed in cell‐attached patches, but not in inside‐out patches. Taken together, these data indicate that H2O2 is an activator of TRPC6 channel. This H2O2 dependent response is through modification of thiol groups on intracellular proteins. Funding was provided by the American Diabetes Association.
Intracellular movement of p120‐catenin in endothelial cells and implications for transcriptional regulation
Authors: O'Donnell James John , Zhang Jihang , Forsyth Christopher B , Lum Hazel
Published: June 2021

In endothelium, the adherens junction‐associated protein p120‐catenin (p120ctn) is known for cell‐cell adhesion, but a role in transcriptional regulation is unclear. We postulate that the junctional pool of p120ctn may redistribute to regulate transcription under conditions of loss of organized junctions. At subconfluent growth, immunofluorescent dectection of p120ctn in endothelial cells (EC) shows distribution primarily in the nucleus as confirmed with DAPI stain colocalization. In contrast, in fully confluent monolayers, p120ctn localizes at the plasma membrane with barely detectable expression in either cytoplasm or nucleus. The disruption of EC junctions with TNF (100 U/ml for 1 hr) causes a marked shift of p120ctn from the membrane to the nuclear and perinuclear regions. We observe that the EC express Kaiso, a transcription factor reported to be a binding partner with p120ctn. Transfection of minimal promoter reporter plasmid containing Kaiso binding elements (pKBS‐Luc) results in 30% repression of luciferase activity in EC. Finally, we show that siRNA‐mediated knockdown of p120ctn increased 2‐fold the KBS reporter activity, suggesting that p120ctn‐Kaiso represses gene transcription. Together, these data implicate p120ctn as a key player in regulating transcription in endothelial cells. The study is supported by NIH HL093715.
Pathological vascular remodeling in mice with disrupted circadian rhythm
Authors: Anea Ciprian Bogdan , Sullivan Jeniffer , Rudic Radu Daniel
Published: June 2021

The transcription factors Bmal1 and Clock are integral components of the molecular machinery that drive circadian rhythm. Herein, we undertook experiments to determine if mice with disruption (Bmal1‐KO) or mutation (Clockmut) of the circadian clock conditioned the adaptation of blood vessels to chronic disturbances. Implementing the common carotid artery ligation model of vascular remodeling, we found that Bmal1‐KO mice exhibit pathological remodeling, arterial dilatation, and thrombosis of the common carotid artery undergoing arterial ligation, identified through histomorphometric and high resolution ultrasound imaging techniques. The lumen diameter in the ligated left common carotid (LC) narrowed by 34±3.5% relative to contralateral (RC) while Bmal1‐KO were only able to adapt by a 5.2±11% reduction. In Clockmut mice, pronounced thickening occurred of the blood vessel wall after ligation, but only when the mice were housed under conditions of constant darkness (DD), not in standard light‐dark conditions, consistent with light‐cycle dependent worsening of circadian rhythms in these mice. The hyperplastic response to intraluminal wire injury of the femoral artery was also more pronounced in Bmal1‐KO and Clockmut mice relative to controls. These data suggest that the circadian clock may be an important contributing factor in the pathogenesis of vascular disease.
Erythrocytes (RBCs) of Humans and Rats with Type 2 Diabetes (DM2) Fail to Release ATP or Stimulate Dilation of Isolated Resistance Vessels in Response to Low Oxygen Tension
Authors: Sprague Randy , Hanson Madelyn , Achilleus David , Bowles Elizabeth , Stephenson Alan , Ellsworth Mary
Published: June 2021

Low oxygen tension‐induced ATP release from RBCs contributes to the matching of O2 supply with O2 demand in skeletal muscle. RBCs of humans with DM2 failed to release ATP in response to pharmacological activation of the pathway for low O2‐induced ATP release. We investigated the hypothesis that RBCs of humans and rats with DM2 (12 week Zucker Diabetic Fatty [ZDF] rats) would also fail to release ATP in response to low O2 and would not stimulate dilation of isolated arterioles exposed to reduced extraluminal O2. RBCs of healthy humans (n=10) and ZDF controls (n=4) released significantly more ATP (230±68 and 654±197%, respectively) when exposed to low O2 (19 ± 1 mm Hg) than did RBCs of both humans (n=6) and rats (n=3) with DM2 (56±29 and 64±4%, respectively) (P<0.05). To ascertain if there is a functional consequence of this failure to release ATP, we perfused isolated skeletal muscle arterioles with buffer containing RBCs and decreased the level of extraluminal O2. Vessels perfused with RBCs of healthy humans (n=3) increased their diameter by 20 ± 4% (P<0.01) when PO2 was decreased from 139 ± 5 to 16 ± 7 mm Hg. In contrast, when the buffer contained RBCs of humans with DM2, the increase was only 5.3% (n=2). These studies suggest that in DM2, ATP release from RBCs in response to reduced O2 is impaired which would diminish the role of these cells as regulators of microvascular perfusion. (ADA grant RA‐133, NIH grants HL‐64180 and HL‐89094)
Lycium barbarum Attenuates Increased Plasma Stress Hormone Levels Induced by a Short and Intense Exercise Challenge. A Randomized, Double‐blind, Placebo‐controlled Human Clinical Study
Authors: Amagase Harunobu , Nance Dwight M
Published: June 2021

We examined the effects of orally consumed Lycium barbarum, as a standardized juice (GoChi), in healthy adults (18‐60 years old). Subjects consumed 120 ml/day of GoChi or placebo for 30 days. Subjects were given a short and intense exercise challenge on a cyclergometer at 70% of age‐adjusted maximum heart rate both pre‐ and post‐treatment. Changes in plasma levels of stress hormones (dehydroepiandrosterone (DHEA) and cortisol) and lactic acid were assessed pre‐ and post‐treatment, and before and immediately after the exercise challenge. Hormone levels were measured by ELISA and analyzed with ANOVA. DHEA, cortisol and lactic acid levels were all significantly increased in the placebo group (N = 19) after exercise for both pre‐ and post‐treatment challenges (by 26‐57%, 28‐125%, and 140‐170%, respectively). Whereas a significant increase in levels of DHEA and cortisol after the initial exercise challenge was seen at pre‐treatment in the GoChi group (N = 20), at post‐treatment, the exercise‐induced increase of DHEA and cortisol was significantly attenuated. Lactic acid level was not changed by GoChi or placebo. These results suggest that consumption of GoChi may increase adaptability to a physical stressor, but has no detectable effect on lactate levels. Based on this and previous studies, GoChi may improve physical activities, in part, by modifying adrenal steroid regulation.
Quantitative PCR validation of hormones and growth and immune factors from gene array of rat cecal cells fed resistant starch and control diets
Authors: Keenan Michael J , Hegsted Maren , Martin Roy J , Raggio Anne M , McCutcheon Kathleen L , Newman Susan S , Brown Ian L , Birkett Anne , Tulley Richard T , Skaf Jihad , Zhou Jun
Published: June 2021

Previously we reported the gene array results on cecal cells from rats fed three diets: resistant starch (RS, 3.2 kcal/g), standard control (CC, 3.6 kcal/g), and energy balanced control (EC, 3.2 kcal/g). We now can report the validation assay performed by quantitative PCR (n=8) for 15 hormones, and growth and immune factors with the greatest fold changes and levels of significance from the gene array. The 18S ribosomal RNA gene was used as the endogenous control. Three t‐tests, RS‐EC, RS‐CC, and CC‐EC, were performed with results for RS‐EC presented. Fourteen of 15 factors found different by the gene array were validated. Large increases (>2) in fold change and highly significant (p<0.0001) differences for RS were: 1) hormones ‐ adrenomedullin, glucagon, neurotensin and peptide YY; 2) growth factors ‐ amphiregulin, bone morphogenetic protein 2, epiregulin, and vascular endothelial growth factor alpha; and 3) immune factor ‐ interleukin 1 alpha. Results demonstrate dietary RS is associated with increased gene expression of hormones and factors that may positively impact the health of the gut and body of the animals. Funding sources were National Starch and LSU AgCenter.
Microglial activation during HSV‐1 infection is increased by vitamin E deficiency
Authors: Sheridan Patricia Ann , Beck Melinda A
Published: June 2021

Previous studies from our laboratory have shown that dietary a‐tocopherol (VE) is critical to regulating the cytokine and chemokine response in the brain to herpes simplex virus (HSV) infection. Microglial cells have been identified as a source of pro‐inflammatory cytokine production during HSV‐1 infection in both humans and mice. During HSV‐1 infection, microglia from BALB/c mice produce a vigorous, but not protective response to HSV‐1. In VE deficient (VE‐D) mice, the pro‐inflammatory response was even more robust than in the VE adequate (VE‐A) mice. In this study, we determined that VE‐D mice have increased proliferation and activation of microglia and increased numbers of infiltrating macrophage following HSV‐1 infection compared to VE‐A mice. Administration of U‐83836E, a lazaroid compound that is a potent inhibitor of membrane lipid peroxidation with anti‐oxidant properties similar to VE, alters the response of microglia in both VE‐A and VE‐D mice infected with HSV‐1. These data suggest that oxidative stress during HSV‐1 infection alters microglial responses and that VE deficiency acts synergistically to amplify this response.

Support: P30ES10126 and DK56350.
STRUCTURE‐FUNCTION ANALYSIS OF TRANSFROMED PHYTOCHEMICAL GLYCOSIDES
Authors: Ji Geun Eog
Published: June 2021

Glycosides are important food components which can confer beneficial functions for human health. Transformation, especially hydrolysis, of the sugar moieties tends to improve the bioavailability of the administered glycosides in vivo. Selected strains of Bifidobacterium possessed potent ginsenoside‐transforming activities. Final major products such as compound K, Rh2, and Rh1 were differentially obtained depending on the microorganisms. When compared between various ginsenosides, compound K (CK) showed anti‐tumor activities and Rh1 showed strong anti‐allergic effect. Bifidobacterium sp. Int‐57 efficiently hydrolyzed genistin to genistein and daidzin to daidzein, respectively, in Puerariae radix (PR) and soybean. Transformation of the platycodin saponins from Platycodon grandiflorum A. DC reduced the cytotoxicity on V79‐4 (Chinese hamster lung fibroblasts) and the hemolytic toxicity, while the sensory scores responsible for the pungency, bitterness and after‐tastes were improved. Additionally, transformation of gastrodin from Gastrodia elata Blume to 4‐hydroxybenzyl alcohol, furostanol diosgenin saponins from Trignoella foenum‐graecum to spirostanol forms, and baicalin and wogonoside from Scutellariae radix to baicalein and wogonin, respectively, were carried out by selected strains of Bifidobacterium and Lactobacillus. In conclusion, proper transformation may improve the functionality and the sensory value and reduce the cyto‐toxicity of the functional glycosides present in various functional food raw materials.
Effect of functional materials producing microbial strains isolated from Kimchi on antiobesity and inflammatory cytokines in 3T3‐L1 preadipocytes
Authors: Park Jeong‐Eun , Moon Youn ‐Joeng , Cha Youn‐Soo
Published: June 2021

This study investigated the inhibitory effect of lactic acid bacteria isolated from Kimchi (Korea traditional fermented food) (KLAB) treatment on cellular lipid accumulation and inflammatory cytokines in preadipocyte 3T3L‐1 cell lines. 3T3‐L1 cells were treated with GLAB significantly reduced lipid accumulation, as well as GPDH activity. And mRNA levels of PPAR? and C/EBPα, the master adipogenic transcription factors, are regulated by KLAB. Moreover, mRNA levels of PPAR? target genes (fat‐specific marker genes), such as aP2 are down‐regulated by GLAB. And, verify the role of KLAB on inflammatory cytokines in preadipocyte. White aipocyte tissue is now recognized as an important endocrine organ which secretes various signal factors and protein termed adipockine. In this study, we investigated the effects of KLAB on TNF‐α ‐induced atherogenic changes of the adipokines in 3T3‐L1 cells. The results suggested that treatment of lactic acid from Kimchi might play a role in the prevention of fat accumulation and improve obesity‐induced cardiovascular disease, particularly atherosclerosis, by attenuating the TNF‐α ‐induced changes of adipokines. Furthermore, animal research suggests the possible action of lactic acid from Kimchi as a facilitator of lipid profiles. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (NO. R01‐2008‐000‐20979‐0))
Effect of vitamin D supplementation on calcium absorption and retention in adolescent girls
Authors: Park Clara Yongjoo , Hill Kathleen M , Elble Ann E , Martin Berdine R , Peacock Munro , McCabe George P , Weaver Connie M
Published: June 2021

In adolescents, cross‐sectional studies show either no or negative relationship between vitamin D status and calcium absorption. Through a crossover‐design metabolic balance study, we investigated the effect of vitamin D supplementation on calcium absorption and retention in adolescent girls (mean serum 25(OH)D: 19.83 ng/ml). Sixteen girls age 11‐14 yrs consumed a controlled diet (providing 400 IU vitamin D and 1136 mg Ca/d) for two 3‐week metabolic balance periods separated by a 1‐week washout period. Sunlight exposure was minimized by sunscreen with a sun protection factor = 15. After the first metabolic balance period, subjects received 1000 IU vitamin D3/d supplementation for 4 weeks. Calcium absorption was measured each metabolic balance period using a stable calcium isotope method and indirectly through a 5‐hr PTH suppression profile. All urine and fecal samples were collected and analyzed to measure calcium retention. Statistical analysis was done by repeated measures ANOVA. Vitamin D supplementation resulted in a mean increase in serum 25(OH)D of 4.65 ng/ml (p=0.0007), but it had no effect on serum fasting 1,25(OH)2D (p=0.72), calcium absorption (p=0.33), PTH suppression (p=0.92), or calcium retention (p=0.10). Vitamin D supplementation at 1000 IU/d did not affect calcium homeostasis in pubertal girls with vitamin D status levels considered suboptimal in adults.

Grant Funding Source

Delavau, LLC
Age‐dependent liver repopulation by transplanted hepatocytes
Authors: Stock Peggy , Ebensing Sabine , Hempel Madlen , Bielohuby Maximilian , Bidlingmaier Martin , Christ Bruno
Published: June 2021

The low liver repopulation efficacy of transplanted hepatocytes is probably due to their poor proliferative capacity linked to the age of the liver donors. The age‐dependent proliferative potential of wild type F344 rat hepatocytes was investigated after transplantation into old and young CD26‐deficient F344 rat host livers. Repopulation rates were determined by flow cytometry and assay of CD26 enzyme activity.

Hepatocytes isolated from either young or old donor rats formed small and rare clusters of donor‐derived hepatocytes in senescent host livers. However, both hepatocytes from juvenile and from senescent donor livers transplanted into juvenile host livers developed cell clusters significantly larger and more frequently as compared with senescent host livers. Repopulation of juvenile host livers by old or young hepatocytes amounted to 20 % as compared 2 % in senescent hosts. No functional difference between transplanted juvenile and senescent hepatocytes was observed in the host liver tissue in terms of glycogen storage. As compared to senescent host animals, serum levels of IGF1 were significantly higher in juvenile rats. Thus, in rats, the age of the recipient liver seems to be critical for the efficient repopulation by transplanted hepatocytes involving regulation along the growth hormone‐IGF1‐axis.
Pecking at the origin of vertebrate diversity: insights from the beak of the finch
Authors: Abzhanov Arkhat
Published: June 2021

The faces of vertebrates are often readily recognizable as they display a number of species‐specific characteristics. It is likely that this stunning diversity of cranial morphology in vertebrates was generated by alterations in craniofacial development. We are employing a combination of genetic, genomic, molecular, bioinformatic, 2‐D and 3‐D imaging and modeling approaches to understand evolution of craniofacial structures, such as highly adaptive beak morphologies in such species as Darwin's Finches (a classic example of species multiplication and diversification caused by natural selection) and their relatives, the African Seedcrackers Pyrenestes ostrinus (textbook example of adaptive polymorphism), and other avian and reptilian species. The major goal of these studies is to use both novel approaches on well‐studied evolutionary systems to address some of the long‐standing questions in animal development and evolution.
Multi‐color FISH assays for characterization of marker chromosomes and epigenetic changes
Authors: Liehr Thomas , Weise Anja , Mrasek Kristin , Ewers Elisabeth , Mkrtchyan Hasmik , Kosyakova Nadezda
Published: June 2021

Multicolor fluorescence in situ hybridization (mFISH) assays are essential for a precise description of chromosomal rearrangements. Routine application on human chromosomes started in 1996 with the simultaneous use of all 24 human whole chromosome painting probes in multiplex‐FISH (M‐FISH) and spectral karyotyping (SKY), even though the principle of mFISH was already described in 1989. Numerous approaches for chromosomal differentiation based on mFISH assays were reported recently. Predominantly, these have been established to characterize marker chromosomes, but also in many other fields mFISH is applied nowadays (for overview see the mFISH homepage http://www.med.uni‐jena.de/fish/mFISH/mFISHlit.htm). Here we review the recent developments and applications of mFISH including variants of M‐FISH, FISH‐banding techniques and applications in 2‐D and 3‐D‐FISH. Also a recently invented possibility to do sort of 'microsatellite analyses' in situ on human chromosomes called parental origin determination FISH (pod‐FISH) is presented. Overall, mFISH approaches are and will be essential scientific and diagnostic tools irrespective of being called death due to recent chip‐ and array‐technology developments. Supported in parts by the Stiftung Leukämie, Stefan‐Morsch‐Stiftung, DAAD 415‐br‐probal/po‐D/07/09624 and D07/00070 and the DFG (LI 820/11‐1, LI 820/13‐1, LI 820/17‐1).
Embryonic Stem Cell‐Derived Hematopoietic Cells: Are they Immunocompetent?
Authors: Zavazava Nicholas , Tabayoyong William , Kim Eun‐Mi , Bonde Sabrina
Published: June 2021

Bone marrow cells are well established as a source of hematopoietic cells. Alternatively, embryonic stem (ES) cells have emerged as an exciting new source of hematopoietic progenitor cells (HPCs). However, deriving immunocompetent hematopoietic cells from ES cells remains a huge challenge. The efficiency of deriving hematopoietic cells is less than 10 % and their engraftment short‐lived. In contrast, HOXB4‐transduced ES cells more readily formed CD45+ HPCs that permanently engrafted in immunodeficient mice. Our preliminary studies show that natural killer cells target ES‐cell‐derived H60‐expressing c‐kit and Sca‐1 progenitor cells, limiting their long‐term engraftment in immunocompetent mice. As expected, deletion of NK cells allowed long‐term engraftment of the HPCs. In addition, we tested whether these newly formed HPCs can establish immunity to immunodeficient mice. Surprisingly, although HOXB4 provided self‐renewal properties to the HPCs, it obliterated their ability to form lymphocytes. Our current studies are focused on identifying the transcription factors regulating lymphopoiesis and on how HOXB4 regulates these factors. More interestingly, our newly generated human inducible pluripotent stem cells appear to be very similar to blastocyst‐derived ES cells.
Single‐molecule analysis of transcription
Authors: Ebright Richard H. , Weiss Shimon , Chakraborty Anirban , Wang Dongye , Korlann You , Kapanidis Achillefs , Margeat Emmanuel
Published: June 2021

We are using single‐molecule fluorescence resonance energy transfer to define fundamental aspects of transcription initiation, elongation, and termination.

In published work, we have shown that initial transcription proceeds through a "scrunching'' mechanism, in which RNA polymerase (RNAP) remains fixed on promoter DNA and pulls downstream DNA past its active center. We have shown further that putative alternative mechanisms for RNAP‐active‐center translocation in initial transcription, involving "transient excursions" of RNAP or "inchworming" of RNAP, do not occur. The results support a model in which a stressed intermediate, with DNA‐unwinding stress and DNA‐compaction stress, is formed during initial transcription, and in which accumulated stress is used to drive breakage of RNAP‐promoter interactions during promoter escape.

In unpublished work, we are assessing opening and closing of the RNAP active‐center‐cleft, movements of modules of sigma relative to RNAP in transcription initiation, movements of modules of the RNAP active center in transcription elongation, and movements of RNAP relative to DNA in transcription termination. In further unpublished work, carried out in support of these studies, we have developed reagents and procedures that permit incorporation of a fluorescent probe at any position of interest within a transcription complex.
Crystallographic studies of ribosomal frameshifting
Authors: Selmer Maria , Chen Yang
Published: June 2021

A recent 2.8 Åcrystal structure of the 70S ribosome from Thermus thermophilus shows many details of how the ribosome interacts with its mRNA and tRNA substrates (1). During translation, specific tRNA‐mRNA combinations can cause frameshifting, deviations from the normal three‐base reading frame. The aim of this work is to gain structural and functional understanding of why these deviations occur and how the ribosome in the normal case prevents them from occurring.

Mutated frameshift suppressor tRNAs containing eight‐base anticodon loops can cause a +1 frameshift through decoding of a four‐base codon. The crystal structures of such ASLs (anticodon stem‐loops) bound to their cognate four‐base mRNA codons in the context of the ribosomal A site showed that three bases of the extended anticodon covered four bases of the mRNA (2).

When the tRNA balance is altered, tRNA3Ser, that normally decodes AGU and AGC, can cause ‐1 frameshifting at a GCA alanine codon. Nitrocellulose filter binding studies indicate that this occurs through a doublet decoding mechanism.

The current status of our crystallographic studies of frameshift‐related ribosomal complexes will be presented.

1. Selmer, M et al., Science 313(5795):1935‐1942 (2006)

2. Dunham, C.M. et al., RNA 13(6):817‐23 (2007)
Approaches to assigning protein function in E. coli
Authors: Armstrong Richard N.
Published: June 2021

Understanding the functional genomics of organisms has emerged as a fundamental challenge of biochemistry. Almost 40% of the genes in one of the best‐understood organisms in the biosphere, Escherichia coli, still have no experimentally verified function. In this work the challenge of, and criteria for, assigning protein function in the context of the glutathione (GSH) transferase paralogues encoded in the E. coli genome will be discussed. The E. coli genome harbors eight genes encoding canonical GSH transferase homologues including YliJ, YncG, Gst YfcF, YfcG, YghU, SspA and YibF. Only one of these gene products (SspA) has a well‐defined function. The structures and possible functions of GSH transferase homologues, YfcG, YghU and YghU, will be discussed with respect to GSH and glutathionylspermidine homeostasis in E. coli. Supported by NIH Grant GM030910.
Mechanism of intrinsic hydrolysis in Ras and the effect of Raf on oncogenic Ras mutants
Authors: Mattos Carla , Buhrman Greg , Kumar Senthil , Wink Glenna
Published: June 2021

The Ras GTPase and its effector Raf are key mediators of the Raf/MEK/ERK signal transduction pathway, which plays a central role in cell growth and malignant transformation. While it is well accepted that signaling through the Ras/Raf interaction is critical in the development of many cancers, most studies to date have focused on the properties of the individual components of this complex. Thus, the biochemical properties of Ras and its oncogenic mutants have been well characterized in vitro in the absence of Raf and numerous structures of wild type and oncogenic Ras mutants have been used to study the possible mechanisms through which Ras becomes defective in its ability to hydrolyze GTP. We show that in the context of the Ras/Raf‐RBD‐CRD complex the highly oncogenic mutant RasQ61L is unable to hydrolyze GTP and that the high transformation efficiency in RasQ61L is correlated with an anti‐catalytic conformation of the active site in this and other highly transforming mutants. We propose a new mechanism through which intrinsic hydrolysis occurs in Ras and explain the structural features that render the RasQ61L mutant a severely impaired enzyme in the presence of Raf. We also present crystal structures supporting a role for Gln61 in our proposed mechanism and show how the G12V mutant disrupts this structure while in the non‐transforming G12P mutant it is well tolerated.
Dietary Hydroxypropylmethylcellulose Reduced Weight Gain and Insulin Resistance in Diet‐Induced Obese Mice
Authors: Kim Hyunsook , Bartley Glenn E. , Young Scott A , Anderson Kerr
Published: June 2021

Consumption of a diet high in soluble dietary fiber has been suggested to induce anti‐obesity effects and improve insulin resistance. To investigate effect of hydroxypropyl methylcellulose (HPMC), a highly viscous soluble dietary fiber, C57BL/6J mice were fed a high‐fat diet for five weeks until they became obese. Then obese C57BL/6J mice were fed either 6 % HPMC or 6 % microcrystalline cellulose (MCC) for another five weeks. Body weight gain was significantly lower in HPMC fed mice compared with MCC. Adipose tissue and liver weight were significantly decreased by dietary HPMC. HPMC significantly reduced plasma LDL‐cholesterol levels resulting in a significantly increased HDL/LDL ratio. Genes related to lipid metabolism in liver and adipose tissues were modulated after HPMC. HPMC feeding markedly reduced area under curve for 2‐hr insulin response, indicating enhanced insulin sensitivity. 2‐hr glucose response tended to be reduced after HPMC compared with MCC. These data suggest that HPMC feeding induces beneficial effects on amelioration of body weight gain and plasma lipid profiles induced high‐fat diets, thus resulting in improved insulin sensitivity. Therefore, dietary application of HPMC is recommendable for the management of insulin resistance and obesity, thus reducing incidences of metabolic syndrome.
Iron and zinc deficiencies affect zinc kinetic parameters in rats
Authors: Yokoi Katsuhiko , Konomi Aki
Published: June 2021

Co‐occurrence of zinc (Zn) and iron (Fe) deficiencies is common. The epidemiological studies suggested dietary origin of the co‐occurrence. The aim of this study was to test whether Fe deficiency affects Zn kinetics using rats and the stable Zn tracer. Forty 4‐wk‐old male SD rats were assigned into 4 dietary groups of 10: Zn deficiency (ZD), Fe deficiency (FD), Zn/Fe deficiency (ZFD), and Control (AIN‐93G). After rats were fed experimental diets for 4 weeks, baseline blood was collected from the jugular vein and the intravenous 67‐Zn tracer was administered via the femoral vein. Then, blood samples were collected 2‐128 min after the intravenous dose. The plasma 67‐Zn/68‐Zn isotope ratios were measured using ICP‐MS, and Zn kinetic parameters were calculated using the two‐compartment model. The Zn kinetic parameters of FD were not different from those of Control. The rats in ZD had the significantly smaller central and peripheral Zn pool sizes, and the significantly smaller Zn transfer rate between the two compartments and Zn loss from the system. The rats in ZFD had the significantly smaller Zn transfer rate and Zn loss, whereas the Zn pool sizes were not different. In conclusion, simple Zn deficiency induces changes in Zn kinetics. Although simple Fe deficiency does not affect Zn kinetics, it modulates Zn deficiency‐induced changes in Zn kinetics.
The construction, analysis and legacy of the radiation chimaera
Authors: Brynmor Thomas David
Published: June 2021

By August 1945 Jacobson and his associates in Chicago had observed that following the ingestion of 89Sr the murine spleen exerted a beneficial effect, which they attributed to a 'radiation protection factor' that could not be isolated from the spleen but which could be transferred to irradiated recipients in splenic implants or in suspensions of spleen cells. In 1952 Congdon, Lorenz and Uphoff at Bethesda successfully used bone marrow cells to protect mice against the lethal effects of whole body irradiation and recognized the possibility that protection could be due to the transplantation of blood cell precursors. This possibility was endorsed by the investigations of Barnes and Loutit in Harwell, which revealed that the radiation protection factor was antigenic and that it was preserved under conditions which were known to sustain living cells but not under conditions which were adequate for the preservation of unstable chemical compounds. Within four years the use of markers proved that donor bone marrow cells repopulate the medullary cavities of irradiated mice. The radiation chimaera had been constructed and the stage was set for three decades of research and development during which the transplantation of blood cell precursors was established as a routine treatment for impaired haematopoiesis, a unique model for the study of stem cell populations was analysed and the generation of a rich legacy was commenced.
Use of Case Presentations and Peer‐Teaching to Enhance the learning of Cell and Tissue Biology in an Integrated Medical Curriculum
Authors: Pettepher Cathleen C , Bader David , Nanney Lillian B , Petrusa Emil
Published: June 2021

The importance of learning normal histology is often lost on students who find studying slides via microscopes or virtual images boring. It is not until they begin studying how disease processes present in pathology that they discover the real need for knowing normal microstructure and function of cells, tissues and organs. The authors were interested in finding ways to enhance both excitement for the discipline and the desire to learn normal histology. The focus of this study was to determine if utilizing clinical case presentations (ie;, burn patient) in the histology laboratory would be an effective means of teaching the relevant normal histology of each organ system. Case presentations were put together in the format of a brief introduction to the patient history containing all pertinent information followed by a series of detailed questions specifically addressing a particular cell, tissue (ex: gland) or structural component of the organ system that might be affected (blood vessels, sweat glands, melanocytes, etc). The students then worked in teams to answer the various questions and determine what structures normally present in the organ system being discussed were damaged or injured and not functioning properly. These responses were then reported and explained to the remainder of the class at the end of the laboratory period. The students enthusiastically shared their responses with their classmates with additional details provided by attending faculty if incomplete or incorrect. The students have since requested that clinical cases be presented on a regular basis to enhance the learning environment in the histology laboratory.
The Role of Insulin‐like Signaling for the Central and Peripheral Regulation of Nutrient Homeostasis and Life Span
Authors: White Morris F.
Published: June 2021

It is incontrovertible that insulin‐like signaling is critical for development and growth of metazoans: Animals cannot survive without insulin‐like signaling, as it regulates nutrient storage and utilization throughout life. Regardless, the life‐span of lower metazoans‐C. elegans and Drosophila‐benefits from reduced insulin secretion or attenuation of the insulin‐like signaling cascades. One of the most important effects of insulin in higher animals and humans is to maintain blood glucose concentration within a narrow physiologic range. While glucose homeostasis is important, insulin‐like signaling can have detrimental effects upon life span. In higher animals and humans, reduced insulin secretion or resistance to the action of insulin causes glucose intolerance that can progress to life‐threatening diabetes. During insulin resistance, compensatory hyperinsulinemia is essential to prevent the fatal effects of hyperglycemia owing to insulin resistance. However, increased circulating insulin might have damaging effects upon peripheral tissues and the brain that can promote age‐related disease and reduce life span. By manipulating the expression of the insulin receptor substrates (Irs1, Irs2 and Irs4) in mice, we reveal the role of insulin‐like signaling upon peripheral and central nutrient homeostasis, and suggest that the brain is a site where reduced insulin‐like signaling can have a consistent effect to extend mammalian lifespan‐just as reduced insulin‐like signaling extends the life span of worms and flies.
What a difference a day makes: carotenoid assessment using 24 hour recalls
Authors: Arab Lenore , Jardack Patricia , Verba Jennifer M , Craft Neal
Published: June 2021

Due to infrequent and seasonal consumption of specific foods rich in carotenoids, and intra individual variability in fruit and vegetable consumption it has been assumed that 24 hour recalls are inadequate for measuring carotenoid consumption in individuals. We examined the increase in correlations with each additional day of intake in the Energetics study, an NIH funded validation DietDay, of a Web based, self administered 24 hour recall among African Americans and Caucasians. 90% of the subjects conducted 6 or more repeat 24 hour recalls online on non‐adjacent days. Intakes from supplements were included. Due to the expected right tailed distribution, plasma and dietary intake values were log normalized. For alpha and beta carotene and lycopene, adjustment for LDL cholesterol levels proved beneficial. Comparisons of Pearsons' correlations for the means of 2,3,4,5 and 6 days of recall started at 0.30 and rose to 0.5 at 4 days for beta cryptoxanthin. Across the carotenoids, the improvement with additional days was greatest from 2 to 4 days and continued in general to increase with additional days. In conclusion‐ a few days of 24 hour recalls provide stable estimates for most carotenoid levels as measured against plasma levels. The largest correlations in this population were seen with beta cryptoxanthin and the combined lutein and zeaxanthin measures. Supported by NIH R01CA105048.
Effect of a pecan meal on postprandial plasma concentrations of hydro‐ and lipophilic oxygen radical absorbance capacity (ORAC) in human volunteers
Authors: Hudthagosol Chatrapa , Hasso‐Haddad Ella , Rajaram Sujatha , Oda Keiji , Sabaté Joan
Published: June 2021

Objective

Pecans (Carya illinoinensis) are rich in phenolic substances which may contribute to the body's antioxidant defenses. The objective of this study was to determine the effect of pecans on postprandial antioxidant responses.

Methods

Test meals containing 100g whole pecans, 100g ground pecans, or an energy‐equivalent amount of refined olive oil as control were administered in a randomized crossover design to 16 healthy volunteers with a 1‐week washout. Plasma was sampled at baseline and at intervals up to 24 hours after consumption of the test meal. Oxygen radical absorbance capacity (ORAC) assays were carried out using the procedure described by Prior et al. (2003). Mixed model analyses of data were performed using SAS version 9.2.

Results

Least square means±SE for the 5‐hour area under the curve (AUC) after the whole pecan, ground pecan and control meals were 80.0±3, 77.2±3.0, 72.8±8 mmol·h/L trolox equivalents for hydrophilic; and 415±16, 412±16, 374±16 mmol·h/L trolox equivalents for lipophilic components, respectively. Both hydrophilic and lipophilic ORAC values increased about 10% (p<0.001) following the whole pecan test meals.

Conclusion

Although modest, the increases in plasma antioxidant capacity following pecan consumption may be physiologically important and further study is needed.

Funding: National Pecan Shellers Association
Pecan‐enriched meal inhibits postprandial LDL oxidation in healthy subjects
Authors: Mccarthy Katie , Gaban‐Chong Natasha , Hudthagosol Chatrapa , Oda Keiji , Sabaté Joan , Hasso‐Haddad Ella
Published: June 2021

Objective

Pecans (Carya illinoinensis) are a source of tocopherols and phenolics which may confer health benefits. The objective of this study was to test the effect of pecans on postprandial concentrations of circulating phenolic components and oxidized‐LDL (ox‐LDL) responses.

Methods

Plasma levels of ox‐LDL and polyphenols were measured in 16 healthy subjects after consumption of test meals with either 100g of whole pecans, 100g of ground pecans or an energy‐equivalent amount of refined olive oil as control in a randomized cross‐over design at 1‐week intervals. Phenolic concentrations were determined by the Folin‐Ciocalteau reagent and oxidized‐LDL by ELISA (Alpco Diagnostics, Salem, NH).

Results

The change in log(ox‐LDL) over 24 hours was examined using a mixed model statistical approach with repeated measures over time. A significant decrease in log ox‐LDL was found at 2 hours (‐29.6%, p=0.04) and 3 hours (‐33.3%, p<0.02); however, levels at 1, 5, 8 or 24 hours were not different from baseline. A 21% increase (p<0.05) in phenolic 5‐hour AUC was observed after the ground pecan compared to control meal (LSMƒpSE 8.25ƒp0.9 vs 6.91ƒp0.9 mmol·h/L gallic equivalents, respectively).

Conclusion

Pecan consumption may increase postprandial circulating phenolic compounds and protect LDL cholesterol from oxidation.

Funding

National Pecan Shellers Association
Effects of an antioxidant spice mixture on formation of lipid oxidation products during hamburger cooking and malondialdehyde levels following ingestion by human volunteers
Authors: Henning Susanne M , Zhang Yanjun , Li Zhaoping , Bowerman Susan , Zerlin Alona , Li Luyi , Lee Ru‐Po , Heber David
Published: June 2021

Oxidized lipids may be formed during the cooking of fatty meats and absorbed into the body where they may contribute to the cellular processes of atherosclerosis and carcinogenesis. Herbs and spices contain a wide array of phytochemicals with strong antioxidant capacity in vitro. In order to determine whether a mix of antioxidant spices including oregano, rosemary, paprika, cumin, ginger, and garlic could reduce the formation of oxidized lipids during cooking or digestion of hamburgers, ten subjects consumed a single hamburger prepared with spice mix or salt alone. A crossover design was used and the malondialdehyde (MDA) measured by HPLC after reaction with thiobarbituric acid in the burgers and in 6‐hour urine collected from subjects after burger consumption. The spice mix significantly reduced formation of oxidized lipids during cooking by 70 % from 1.8+0.2 to 0.5+0.02 μmol MDA per 250g meat. In urine collected for 6 h after ingestion of the hamburger, urinary MDA was decreased by 49 % from 13.7+7 to 7.0+3 μmol/g creatinine (p<0.01) by comparison to the burger made with salt but no spices. In summary the use of spice and herbs in cooking of high fat foods decreased postprandial circulating lipid oxidation products and may reduce their formation or absorption in humans. These finding suggest a benefits for antioxidant spices in reducing the risk of processes involving oxidized lipids including CVD and carcinogenesis
Acyl‐CoA Synthetase‐isoform 1 (ACSL1) is essential for FA oxidation in adipose tissue: lessons from adipose‐specific knockout of ACSL1
Authors: Ellis Jessica Mary , Wu Pei‐Chi , Li Lei , Coleman Rosalind Ann
Published: June 2021

Long chain acyl‐CoA synthetase (ACSL) catalyses the conversion of fatty acids into their acyl‐CoA derivatives. ACSL1, one of five ACSL isoforms, is highly expressed in white and brown adipose tissue where it was thought to be important for triacylglycerol formation. To investigate the role of ACSL1 in adipose tissue we created an adipose‐specific knockout of ACSL1, the ACSL1A‐/− mouse. Although we expected these mice to have a reduced ability to store fatty acids as triacylglycerol in adipose, compared to control mice (ACSL1flox/flox) when fed a low‐fat diet, subcutaneous and visceral fat depot mass was 30% higher in ACSL1A‐/− mice, and when fed a diet containing 45% kcal fat, weight gain was similar. In mature adipocytes isolated from ACSL1A‐/− mice, the rate of fatty acid oxidation was 50% lower, suggesting a role for ACSL1 in FA oxidation rather than TAG synthesis. ACSL1A‐/− mice were cold intolerant with body temperature dropping below 30°C within 2‐5 h in a 4°C environment. ACSL1A‐/− mice had reduced oxygen consumption in response to adrenergic stimuli but had intact cold‐stimulated transcriptional expression of PGC‐1α and UCP1 in brown adipose tissue, showing that adrenergic signaling was intact. The reduced FA oxidation in white adipocytes, together with the cold intolerance of the ACSL1A‐/− mice, strongly indicates that ACSL1 activates fatty acids destined for oxidation.

Grant Funding Source

AHA‐Pre‐doctoral Fellowship Mid‐Atlantic Affiliate