bluuebunny/crossref_metadata_2025 huggingface dataset:
A critical role of FADD in hematopoietic stem and progenitor cells
Authors: Zhang Jianke , Rosenberg Stephen
Published: June 2021

FADD is required for apoptotic signaling downstream of death receptors, through its association with caspase 8. FADD−/− mice die in utero. A lack of FADD in embryonic stem cells lead to severe defects in T and B cell development as shown by Rag‐1−/− blastocyst complementation assays, implicating FADD as a crucial mediator of lymphopoiesis. However, deletion of FADD following lymphoid lineage commitment did not lead to significant defects in T or B cell development. We reports the generation and analyses of organ‐specific as well as inducible FADD knockout mice in which FADD is deletion in multiple organs/cell types including the liver, hematopoietic stem/progenitor cells and lineage committed myeloid cells in adult animals. FADD deficiency resulted in diminished Fas‐induced apoptosis of hepatocytes, but had no significant impact on liver regeneration. Deletion of FADD specifically in monocytic precursors still allows generation of macrophages. However a lack of FADD leads to impaired maintenance of bone marrow hematopoietic progenitors in vivo. Furthermore, the ability of in vitro and in vivo differentiation into lymphoid, myeloid and erythroid cells were greatly decreased in FADD‐deficient bone marrow progenitor cells. Thus, while dispensable after lineage commitment, FADD plays a critical role in hematopoietic stem/progenitor cells. Studies were supported by NIH/intramural grants.
Reciprocal Relationship between Cell Death versus Heat Shock Proteins and Matrixmetalloproteinases in Normal and Cancer Cells
Authors: Philips Neena
Published: June 2021

Heat shock proteins are stress response factors that are collectively anti‐apoptotic and protein stabilization chaperones. They also have specific functions: HSP27 induces cell differentiation, HSP immunity, and HSP90 is associated with cancer progression. Matrixmetalloproteinases (MMPs) facilitate extracellular matrix remodeling and cancer metastasis. While there is a clear reciprocal relationship between cell death and apoptosis, the relationship between cell death and heat shock proteins is complex. In normal and cancer cells, increased cell viability is associated with increased expression of all heat shock proteins suggesting that heat shock proteins facilitate cell survival. However, in normal cells under oxidative stress or cancer cells treated with potential chemotherapeutics there is direct relationship between cell loss and HSPs, which may imply that the surviving cells are supported by the increased expression of HSPs. The relationship between cell loss and expression of MMPs in normal and cancer cells is also direct. Treatment options require combating of the reciprocal effect of cell toxicity versus stimulation of HSPs and MMPs in cancer cells.
Identification of proteins with altered expression in female Pregnancy Associated Plasma Protein‐A knockout mouse embryos
Authors: Rortvedt Ross , Lawrence Jim
Published: June 2021

Our research examines the insulin‐like growth factor (IGF) signaling network. Pregnancy Associated Plasma Protein‐A (PAPP‐A), cleaves the Insulin‐like growth factor binding protein – 4 ability to sequester IGFs. We have examined the effects of PAPP‐A on protein expression in 18 day post‐conception mice embryos in a wild type/PAPP‐A knockout model. We have used 2‐D gel electrophoresis and proteomics technologies to examine protein expression differences between the knockout and wild‐type embryos. Three female wild‐type/knock out pairs were examined. Three successful trials were run for each of the embryo samples resulting in significant, reproducible results for each. The images from the wild‐type and knock out samples were compared in order to identify protein expression differences. Comparison of the wild‐type and knock out gels resulted in the identification of 6 protein spots out of 3500 total spots which showed significant change in protein expression. We have identified the proteins found in each of these 6 spots.
KAP1 regulates TNF‐Induced NF‐kappaB transcriptional activity by influencing the interactions between p300 and NF‐kappaB
Authors: Togi Sumihito , Kamitani Shinya , Kawakami Shiho , Ikeda Osamu , Muromoto Ryuta , Sekine Yuichi , Nanbo Asuka , Matsuda Tadashi
Published: June 2021

KAP1 is thought to act mainly as a scaffold for protein complexes, which together silence transcription by triggering the formation of heterochromatin. Using siRNA‐mediated KAP1 knockdown, we found that endogenous KAP1 negatively regulated TNF‐induced IL‐6 production in HeLa cells. KAP1 is likely to modulate the binding of NF‐kappaB to the IL‐6 promoter because KAP1‐knockdown enhanced TNF‐induced NF‐kappaB‐luciferase activity, but not IkappaB degradation. Of importance, KAP1 associated with NF‐kappaB and inhibited the binding between NF‐kappaB and p300. Moreover, KAP1 negatively controls the acetylation of NF‐kappaB, which is critical for its nuclear retention. Taken together, KAP1 suppressed the acetylation of NF‐kappaB by interfering with the interactions between p300 and NF‐kappaB, resulting in reduced IL‐6 production after TNF‐stimulation.
Regulator of G protein Signaling 6 (RGS6) Modulates Doxorubicin‐induced DNA Damage Signaling by Regulating ATM Activity
Authors: Huang Jie , Chatterjee Tapan K , Mayuzumi Daisuke , Fisher Rory A
Published: June 2021

RGS proteins are well known negative regulators of heterotrimeric G protein signaling. We have shown previously that RGS6 sensitizes breast cancer cells to doxorubicin (Dox)‐induced DNA damage signaling (ATM and p53 phosphorylation), suggesting a possible role of RGS6 in tumor suppression. Unexpectedly, these actions of RGS6 were not dependent on its interaction with G proteins. Here we further reveal the mechanism by which RGS6 modulates DNA damage signaling. Expression of a dominant negative form of ATM completely blocked the ability of RGS6 to promote p53 phosphorylation in response to a submaximal dose of Dox in MCF7 cells. Thus, RGS6‐induced activation of p53 is dependent upon ATM activity. Moreover, ATM co‐immunoprecipitates with RGS6 under basal conditions, demonstrating that these two proteins form a complex without induction of DNA damage. RGS6 does not enhance phosphorylation of CHK2 in response to Dox suggesting that RGS6 interactions with ATM may modulate its activity on specific substrates. RGS6 knockdown in MCF7 cells by RNAi significantly reduces p53 activation in response to Dox, demonstrating that RGS6 is required, at least in part, for Dox‐induced DNA damage signaling. Our data indicate that RGS6 plays an important role in modulating chemosensitivity in cancer cells and identify RGS6 as a novel chemotherapeutic target. (NIH GM075033)
Immunohistochemical localization of transcription factors, Pax6 and Nurr1, in the adult echinoderm nervous system
Authors: García‐Rivera Enrique M. , Diaz‐Balzac Carlos A. , Lázaro‐Peña María I. , García‐Arrarás José E.
Published: June 2021

Studies of the echinoderm nervous system have been advanced recently with the unraveling of the sea urchin Strongylocentrotus purpuratus genome. The identification of gene sequences similar to those of other animals has made possible the study specific of genes and gene products in the echinoderm nervous system. Here we have focused on the transcription factors, Pax6 and Nurr1. Bioinformatic analysis of Pax6 revealed that its coding sequence and characteristic domains are conserved in the echinoderms. Similarly, sequence analyses of the echinoderm predicted protein similar to Nurr1, revealed that the C‐terminus, which contains the ligand‐binding domain of the nuclear receptor, is the most conserved segment, while the N‐terminus is poorly conserved. Immunohistochemistry experiments showed that anti‐Nurr1 and anti‐Pax6 labeled cells and fibers within the echinoderm nervous system. The neural specificity of these transcription factors was verified by co‐labeling with the neural‐specific marker RN1. Our results suggest that Pax6 may have a yet unknown function in the adult echinoderm and that Nurr1 is present in the deuterostomes, rather than just in vertebrates as was previously thought. Additionally, our findings underscore the importance of studying the role and distribution of transcription factors in the echinoderm nervous system as these are responsible for the development of the nervous system.
Cellular Distribution, Homo‐oligomerization and Functional Characterization of GnRH Receptors in Immortalized GnRH Neurons
Authors: Leung Po Ki , Hu Lian , Feng Hao , Krsmanovic Lazar Z , Catt Kevin J
Published: June 2021

Cellular distribution, receptor‐receptor interaction, and signaling pathways of the murine GnRH‐R tagged with Renilla luciferase (Rluc) and/or green fluorescent protein (GFP) were monitored in immortalized (GT1‐7) GnRH neurons. GFP‐tagged GnRH‐R was confined to a thin rim of cytosol at the plasma membrane, neuronal processes, and apparent synaptic junctions. GnRH stimulation caused redistribution of GnRH‐R‐GFP, with movement in close proximity to their bipolar extensions and increased intensity in synaptic connections. BRET2 assay revealed dose‐dependent, GnRH‐induced interaction between GFP‐tagged GnRH‐R and Rluc‐tagged Gαq, Gαs and Gαi. Firing of spontaneous action potentials (APs), calcium signaling and pulsatile GnRH secretion were comparable to those of intact GT1‐7 neurons. These findings indicate that specific subcellular localization, agonist‐induced redistribution, homologous oligomerization, and coupling to multiple G proteins of the GnRH receptor, modulate electrical activity, second messengers, and neurosecretion in hypothalamic GnRH neurons.
Interaction analysis of TcrX/Y two component system from Mycobacterium tuberculosis
Authors: Bhattacharya Monolekha , Biswas Ashis , Das Amit Kumar
Published: June 2021

TcrX/Y is one of the twelve two component system (TCS) present in Mycobacterium tuberculosis. The objective was to investigate the TcrX/Y interaction by in silico studies, pull down assay, radioactive phosphotransfer, surface plasmon resonance as well as crosstalk analysis of TcrY with TcrA ‐ a non‐cognate response regulator. Sequence alignment of TcrY with other histidine kinases revealed His256 as the residue responsible for autophosphorylation. Modeled structure of TcrX predicts that Asp59 may be involved in phosphotransfer with His256 of TcrY. Docking analysis pointed to the interaction of TcrY/His256 with TcrX/Asp54. TcrY/Y dimerisation was also observed by in silico and crosslinking studies. Autophosphorylation of TcrY has been observed followed by the phosphate transfer to TcrX. The phosphorylation process required divalent metal ions like Mg2+ or Ca2+ ions as evident from the radioactive phosphorylation studies. Interaction was not observed in TcrY/A suggesting the signal transduction process is specific in TcrX/Y system. TcrY hydrolyzes ATP and the Km value has been found to be 10 mM. TcrX/Y interaction has been determined by surface plasmon resonance and dissociation constant (KD) was evaluated to be 3.6 μM. We conclude from our results that TcrX/Y are part of the same signal transduction pathway without their involvement in crosstalk with non‐cognate counterpart.

The work was funded by Department of Biotechnology, Govt. of India.
Slow Onset Inhibitors of Bacterial Fatty Acid Biosynthesis: Residence Time, In Vivo Activity and In Vivo Imaging
Authors: Tonge Peter J , am Ende Christopher , Knudson Suan , Luckner Sylvia , Reddy B Gopal , England Kathleen , Liu Li , Borgaro Janine , Liu Nina , Pan Pan , Lu Hao , Li Huei‐Jiun , Fowler Joanna , Kisker Caroline , Slayden Richard A , Hooker Jacob
Published: June 2021

The goal of our structure‐based drug discovery effort is to develop slow onset enzyme inhibitors, based on the premise that the long residence time of the compounds on the target is critical for in vivo activity. Targets for drug resistant bacterial infections include the enoyl‐ACP reductase and β‐ketoacyl‐ACP synthase enzymes in the bacterial fatty acid biosynthesis pathway. We have developed slow onset diaryl‐ether inhibitors of the enoyl‐ACP reductases from Francisella tularensis and Mycobacterium tuberculosis and have shown that the in vivo antibacterial activity of the compounds in animal models of infection correlates with their residence time on the enzyme targets. We are exploring the mechanistic basis for slow onset inhibition and are using this information to develop inhibitors with longer residence times. We have also developed methods to introduce short‐lived isotopes into these compounds and are using the labeled molecules to identify the target(s) for the inhibitors in live cells and to image drug distribution in vivo.
TCF‐1 Regulation By A Wnt‐Kinase Network In Colon Cancer
Authors: Najdi Rani , Theisen Heidi , Atcha Fawzia , Edwards Robert , Marsh J. Lawrence , Waterman Marian L.
Published: June 2021

TCF‐1 and TCF‐4 are the major transducers of Wnt signaling in the intestine and the oncogenic drivers of colon cancer. However, knock‐out of the TCF1 gene leads to intestinal tumors while knockout of TCF‐4 suppresses growth and maintenance of crypt stem cells. These phenotypes suggest that TCF‐4 is Wnt‐promoting and TCF‐1 is a tumor suppressor. We show that a truncated dominant negative isoform of TCF‐1 (dnTCF‐1) predominates in the normal colon and is equally distributed between nuclear and cytoplasmic compartments in normal crypts. However, in primary human colon tumors, TCF‐1 is often cytoplasmic while TCF‐4 remains nuclear. This shift to the cytoplasm is regulated by a CaMKII/TAK1/NLK kinase cascade that triggers secretion of a Wnt ligand to selectively export TCF‐1 from the nucleus. In colon cancer, TCF‐1 expression is altered so that dnTCF‐1 is downregulated while to full‐length, Wnt‐promoting isoforms are upregulated. Our study shows that control of nuclear export is an important mechanism for regulating the balance of TCFs and that a Wnt/CaMKII kinase signal plays a key role in modulating Wnt activity in the human colon.

Supported by NIH
Physical and functional interactions between hnRNP K and PRMT family proteins
Authors: Huang Shih‐Ming , Hsieh Tsai‐Yuan , Liu Shu‐Ting
Published: June 2021

The mechanism underlying the protein‐protein interaction of hnRNP K and PRMT family proteins is unclear. We examined and confirmed the arginine methylation of hnRNP K protein by PRMT1, not CARM1, via their direct binding. We also studied hnRNP K protein complexes containing CARM1, as well as PRMT1, using co‐immunoprecipitation analysis. PRMT family proteins might be involved in the regulation of hnRNP K functions in nuclear receptor coactivator, transactivation, and p21 gene and protein expressions. In summary, the indirect binding ability of PRMT proteins and the arginine methylation status of hnRNP K protein are not necessary for the coactivator functions of these working complexes, suggesting that other factors determine their functional roles in some transcriptional systems. (Research support from National Science Council, Taipei, Taiwan, Republic of China, to Shih‐Ming Huang)
Regulation of the LEF1 gene's alternative promoter
Authors: Pate Kira T , Yokoyama Noriko N , Waterman Marian L
Published: June 2021

LEF1 is a DNA‐binding transcription factor and a downstream component of the Wnt signaling pathway. The LEF1 gene has multiple promoters and produces two distinct proteins: a full‐length, growth‐promoting form produced from promoter 1 (P1), LEF1, and a truncated, growth‐suppressing form produced from promoter 2 (P2), dnLEF1. In tissues where LEF1 is normally expressed, both promoters are active, producing both forms of LEF1. However, in colon cancer, LEF1 is aberrantly expressed, and only the full‐length, growth‐promoting protein is produced. P1 is a Wnt target, explaining its transcriptional upregulation in Wnt‐driven colon cancers. P2 is silent because it is actively repressed. A genomic region upstream of P2 that is necessary for its repression has been delimited, however, the protein components and exact mechanism of repression are unknown. In this study we describe one component of the repressor machinery, identified through site‐directed mutagenesis of the LEF1 core promoter 2. A YY1 binding site at the +25 location was identified, which, when mutated, greatly reduces repressor function. The binding of YY1 to the +25 site in vitro is shown by gel shift, while ChIP suggests in vivo binding of YY1 within a colon cancer cell line. The functional role of YY1 is confirmed through siRNA knockdown of YY1, which results in loss of repressor function. These findings suggest a pivotal role for YY1 in repression of P2.

Supported by NIH.
The Parkinson's disease associated LRRK2 exhibits weaker in vitro phosphorylation of 4E‐BP compared to autophosphorylation
Authors: Kumar Azad , Greggio Elisa , Beilina Alexandra , Kaganovich Alice , Chan Diane , Wolozin Ben , Cookson Mark
Published: June 2021

Mutations in the gene encoding leucine‐rich repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson's disease (PD). LRRK2 is a multi‐domain protein containing a central catalytic core, with GTPase and kinase domains, surrounded by a number of protein‐protein interaction domains. An important step forward in the understanding of the biology and the pathology of LRRK2 would be achieved by identification of its authentic physiological kinase substrates. In the present study we examined phosphorylation of 4E‐BP (eukaryotic initiation factor 4E (eIF4E)‐binding protein), a recently proposed substrate for LRRK2 and confirmed that LRRK2 is capable of phosphorylating 4E‐BP in vitro. The PD related LRRK2‐G2019S mutant was ~2 fold more active than wild type protein. Interestingly, LRRK2 autophosphorylation, which we have mapped to the GTPase domain, was stronger than 4E‐BP phosphorylation under conditions of molar excess of 4E‐BP to LRRK2. We also tested three other kinases (STK3, MAPK14/p38α and DAPK2) and found that MAPK14/p38α could efficiently phosphorylate 4E‐BP at the same site as LRRK2. Overall, our results suggest that 4E‐BP is a relatively poor direct substrate for LRRK2 compared to autophosphorylation. We suggest that increased 4E‐BP phosphorylation reported in some systems may be related to p38‐mediated cell stress rather than direct LRRK2 activity.

This research was supported by the Intramural Research Program of the NIH, National Institute on Aging
Modification of a 3‐d deuterated‐retinol dilution (DRD) equation for estimation of vitamin A (VA) stores in humans
Authors: Duca Frank Anthony , Green Michael H
Published: June 2021

Isotope dilution analysis has been successfully used to estimate vitamin A stores in humans via the Furr‐Olson DRD equation: estimated total body stores = F × dose × {S ×a × [(1/D:H) −1]} (Am J Clin Nutr 1989;49:713). The method requires a blood sample 21 d after isotope administration and involves several assumptions (factors F, S and a). Using VA tracer data from rat and human studies, we refined a modified DRD equation (J Nutr 2008; 138:971) that uses a 3‐d sample, a time that is more feasible in the field. We found that the VA specific activity ratio in plasma vs. stores (factor S in DRD equation) varies with VA status in both rats and humans. Also, the amount of tracer irreversibly lost between dose administration and blood sampling (factor a) increases as status decreases. In contrast, retention (factor F) increases as status increases. Applying these findings to the original DRD equation, we developed a new equation in which S, F, and a vary with a subject's 3‐d plasma VA deuterium:hydrogen (D:H) ratio. For data from humans of varying age, gender and ethnicity, VA stores predicted by the new equation agree better with estimates from the original 21‐d Furr‐Olson equation than do values obtained using other published 3‐d DRD equations. Thus the new equation provides an improved tool to determine VA stores in humans.

Funded by the College of Health and Human Development, Penn State

Grant Funding Source: The College of Health and Human Development
Common metabolites and reducing/chelating agents do not mobilize iron from intracellular ferritin but can (like lysosomal extracts) dissolve ferritin iron mineral
Authors: La Alice , Patel Sonali , Patel Arpitaben , Nguyen Trinity , Sauble Eric N. , Linder Maria C.
Published: June 2021

Although in several cell types release of storage Fe involves movement of ferritin (Ft) to and degradation in lysosomes (Lys), we wondered whether common Fe binding metabolites and reducing agents might also play a role, and also contribute to solubilization of the Fe mineral within Ft. HepG2 cells, loaded overnight with 59Fe so radioactivity was almost exclusively with Ft, were washed and incubated with and without mM concentrations of ascorbate, citrate and glutathione for 1–12h. Loss of 59Fe from cellular Ft was measured by rocket immunoelectrophoresis and phosphorimaging. No changes in Ft 59Fe contents were observed. The same was the case when cells were treated with agents that increased or reduced glutathione. To begin to understand how Lys juices might dissolve the Fe in Ft once the protein “shell” had been degraded, Fe mineral cores in Ft were purified after boiling to dissolve the protein. Portions of the cores were incubated with the same metabolites and reducing agents, at pH 5 or 7, and amounts of Fe in the supernatants were measured at various times thereafter. All 3 reagents efficiently solubilized Ft Fe, as did extracts from purified Lys. These findings support previous conclusions that Ft Fe is only released in conjunction with its degradation in Lys, and identifies substances that could be in Lys that would be able to dissolve the Fe exposed by degradation of the Ft shell. Supported in part by USPHS Grant HD 46949.
Hepatic iron loading increases rat ZIP8 expression
Authors: Wang Chia‐Yu , Nam Hyeyoung , Jenkitkasemwong Supak , Duarte Stephanie
Published: June 2021

Zip8 is a member of the ZIP (Zrt‐, Irt‐like Protein) family of metal‐ion transporters. Zip8 amino acid sequence shares approximately 60% homology to Zip14, which can transport both zinc and iron. Similar to Zip14, overexpression of rat Zip8 in HEK293T cells significantly enhances the uptake of iron. In the present study, Western blot analysis revealed ~3‐fold higher levels of Zip8 in iron‐loaded rat liver compared to iron‐normal controls (mean non‐heme iron concentrations of 1813 vs. 30 μg/g liver, respectively, n=6/group). Higher Zip8 protein levels were not associated with elevated Zip8 transcript abundance, suggesting post‐transcriptional regulation. In the H4IIE rat hepatocyte cell line, we further demonstrated that Zip8 is glycosylated and detectable at the cell surface. These data suggest that Zip8 may contribute to hepatic iron transport, particularly during states of iron overload. Supported by NIH.

Grant Funding Source: NIH
Inhibitory action of green tea on non‐heme iron absorption is not reversed by ascorbic acid in intestinal cells
Authors: Kim Eun‐Young , Han Okhee
Published: June 2021

There is persuasive epidemiological evidence that regular consumption of green tea promotes human health. Because the polyphenolic compounds in green tea have a wide range of effects in vivo and vitro, including chelation of iron, it is prudent to test whether the regular intake of green tea impair the utilization of dietary iron. The influence of green tea extract (GTE) on intestinal iron absorption was initially examined in Caco‐2 intestinal cells. Addition of GTE significantly (P < 0.001) decreased transepithelial iron transport in a dose ‐ dependent manner although apical iron uptake was significantly (P < 0.001) increased by GTE. Since ascorbic acid is known to promote non‐heme iron absorption, the possible counteractive effect of ascorbic acid on the inhibitory action of GTE in iron absorption was tested. The addition of ascorbic acid to the GTE treated cells did not significantly change the inhibitory action of GTE on non‐heme iron absorption. Our data suggest that GTE inhibits non‐heme iron absorption by reducing basolateral iron transport rather than by decreasing apical iron import. Furthermore, the inhibitory effects of GTE on non‐heme iron absorption can not be reversed by ascorbic acid.
How does iron deficiency affect endurance training in female collegiate rowers?
Authors: DellaValle Diane M. , Haas Jere D.
Published: June 2021

The goal of this study is to determine whether iron deficiency (ID) impairs the ability of rowers to increase their training quality (TQ) and performance in response to iron (Fe) supplementation. Female rowers (N=186) were screened for ID to assess ferritin (sFer), transferrin receptor, red blood cell indices, & alpha‐glycoprotein at the beginning of a training season. Across all schools, 27% of rowers had impaired Fe status (sFer <20 ug/L), and 10% were identified as anemic (Hgb<12.0 g/dL). During the first week of training (BL), rowers across a range of Fe status (N=59) had body composition measured, & completed both a VO2max test and a 4K time trial (TT) at a work rate (WR) of 85% of their VO2max on the rowing ergometer. Blood lactate was measured during testing. Continuing subjects (N=47) were then randomized to receive either 100 mg/d FeSO4 or a placebo for 8‐weeks (during their training season), and the 4K TT at the BL WR was repeated at 8‐weeks. During the trial, physical activity (PA) and measures of TQ were recorded daily, and accelerometry was used bi‐weekly to assess PA during and outside of training. The following hypotheses will be tested: 1) Fe supplementation of ID rowers will improve Fe status, and TQ; and 2) as TQ improves, endurance performance will improve. Funded by grants from the American Dietetic Association Foundation, Cornell University's Division of Nutritional Sciences, and the N.S. Meinig Professorship.
Zinc Deficiency Induces Apoptosis but Zinc Induces Necrosis in Rat Hepatoma Cells
Authors: Sankavaram Kavitha , Chong Leelyn , Bruno Richard S , Freake Hedley C
Published: June 2021

Altered concentrations of intracellular zinc influence cellular homeostasis and are believed to modulate apoptosis. We reported earlier that both zinc supplementation and DTPA (diethylenetriaminepentaacetic acid) induced zinc deficiency generate reactive oxygen species (ROS) and decrease cell viability in rat hepatoma (H4IIE) cells. In this study, we examined the effects of altered zinc availability on mode of cell death, signaling pathways and associated morphological changes in these cells. Fluorescence microscopy of cells with Hoechst 33342 and PI indicated that DTPA induced apoptosis but zinc induced both necrosis and apoptosis. Cells were also stained with both PI and FTTC‐labeled annexin V and then analyzed by flow cytometry. The results confirmed that DTPA induced apoptosis (20%) whereas zinc induced both necrosis (50%) and apoptosis (10%). Zinc induced necrosis prior to apoptosis and at a greater rate. Features of apoptosis, such as chromatin condensation, nuclear fragmentation, elevated levels of p53, p21, and Fas/Fas ligand mRNA accompanied DTPA and zinc induced cell death. Decreased expression of Bcl2 and elevated expression of Bax were observed with DTPA but not with zinc treatment. These results indicate that zinc deficiency leads to mitochondrial dependent apoptosis, modulating ROS and apoptosis proteins in H4IIE cells. However, elevated zinc leads predominantly to cell necrosis.

Grant Funding Source: UConn Research Foundation
Breast Milk Zinc Transfer to Appropriate‐ for‐Gestational‐Age (AGA) and Small‐for‐Gestational‐Age (SGA) Bangladeshi Infants: Studies of Milk Volume, Using Deuterium‐To‐Mother, and Milk Zinc Concentrations
Authors: Islam M Munirul , Brown Kenneth H
Published: June 2021

Objective

To measure breast milk zinc transfer by mothers of AGA and SGA infants.

Methods

We studied breast milk transfer (using the deuterium “dose‐to‐the‐mother” technique) and milk zinc concentration of 73 mother‐infant pairs (33 AGA, 40 SGA) at 4, 12 and 24 wks post‐partum. In each round, 30 g deuterium was given orally to the mother following baseline blood and saliva collection from mothers and infants. Saliva samples were then obtained for deuterium analysis on days 1, 2, 3, 4, 13, and 14 from infants and days 1, 2, 13 and 14 from mothers. Breast milk zinc concentration was measured in milk collected at baseline and day 4 each round.

Results

Breast milk intake increased significantly with infant age (643 ± 190; 756 ± 150; 804 ± 150 g/d at 4, 12 and 24 weeks, respectively; p=0.000), and differed by birth weight (BW) groups only at 4 weeks post‐partum (AGA = 714 ± 193 vs SGA = 579 ± 164 g/d; p=0.005). Milk zinc concentration decreased significantly from 2.46 mg/L at 4 weeks to 1.02 mg/L at 24 weeks (p=0.000), but did not differ by BW group at any time point. Zinc transfer through breast milk declined significantly with infant age (1.60 ± 0.62; 1.08 ± 0.41; 0.81 ± 0.32 mg/d at 4, 12 and 24 weeks respectively; p=0.000), but did not differ by BW group.

Conclusions

Milk zinc concentrations and total milk zinc transfer of Bangladeshi mothers are consistent with published data, and do not differ for mothers of AGA and SGA infants.
Sub‐cellular zinc distribution: a novel role for lysosomal‐mediated cell death in malignant breast tumor cells
Authors: Lopez Veronica , Foolad Farnaz , Kelleher Shannon L.
Published: June 2021

Numerous studies implicate abnormally high zinc (Zn) levels in the etiology of breast cancer. ZnT2 plays role in Zn sequestration, protecting cells from the cytotoxic effects of excess Zn. We previously determined that ZnT2 is over‐expressed in malignant breast tumor cells. Intriguingly, ZnT2‐attenuation resulted in caspase‐independent apoptosis. Emerging evidence suggests that lysosomal rupture releases catabolic enzymes and activates or potentiates apoptosis. Numerous cytotoxic conditions, such as reactive oxygen species (ROS) generation induce permeabilization of lysosomal membranes. Thus, the primary objective of this study was to determine if ZnT2‐attentuation was associated with lysosome‐mediated apoptosis in tumor cells. We determined that ZnT2‐attenuated cells had significantly higher cytoplasmic Zn level compared with ZnT2‐expressing cells. Curiously, ZnT2‐attenuation significantly increased ROS generation, suggesting that re‐distribution of Zn resulted in lysosomal swelling. Taken together, these results illustrated that ZnT2‐attenuation increased ROS potentially revealing lysosomal membrane instability as an apoptotic signal in malignant breast tumor cells. Our data provide novel insight suggesting that changes in the Zn microenvironment regulate lysosome‐mediated cell death. Supported by DOD‐BCRP to SLK.
Effect of cranberry juice intake on human gut microbial community and urinary metabolites in a randomized, placebo‐controlled intervention
Authors: Khoo Christina , Hullar Meredith AJ , Li Fei , Stepaniants Karina , Levy Lisa , Roderick Robin , Lampe Johanna
Published: June 2021

Cranberry polyphenols support health by reducing pathogenic bacteria adhesion and growth. This randomized, double‐blind, cross‐over pilot study compared effects of consuming a light 27% cranberry juice beverage(LCB) vs. a placebo beverage (LPB) on fecal microbial community and urine metabolites. Healthy subjects(n=5), 20–40 y, received 480 ml/d LPB or LCB in two 6‐wk intervention periods, with a 1 mth washout. Fecal samples were collected at baseline, 3 and 6 wk and 24h urine at baseline and 6 wk. Microbial community structure, measured by terminal restriction fragment length polymorphism and quantitative PCR (QPCR) of the 16S rRNA gene, differed significantly (MRBP; p=0.036) between treatments at 6 wks. Bifidobacteria(as % of total Eubacteria) significantly increased when subjects received the LCB (F= 6.19, p<0.015)with no significant effect on Bacterodetes, Lactobacillus sp., Enterobacteria, or C. coccoides/E.rectale groups (by qPCR and adjusted for period effect). LCB intake significantly changed 49 urine metabolites, notably of nucleotide metabolism, and potential intake biomarkers, including protocatechuic acid‐3‐glucoside (Metabolon, Inc, NC) to help guide cranberry health research. The results suggest that cranberry juice intake may modulate human gut microbial community without decreasing percentages of some beneficial commensal bacteria. Supported by Ocean Spray Cranberries, Inc.
Inhibitory Effects of Poly‐gamma‐glutamate on Abdominal Fat Adipogenesis in Dietary Induced Obese Rats
Authors: Chang Moon‐Jeong , Kim Boram , Park Jee‐Ho , Kim Sun‐Hee
Published: June 2021

High molecular weight polymer, poly–γ‐glutamate(γ‐PGA) that produced by Bacillus Subtilis subsp. Chungkookjang isolated from traditional Korean fermented sticky soybean products is a multi‐anionic‐ γ‐polypeptide. The purpose of the present study was to test the biological efficacy of γ‐PGA on weight control and abdominal fat adipogenesis in dietary induced obese rats. Sixty male Sprague‐Dawley rats were fed a normal fat diet(10%) or high fat diet(45%) for 4 weeks and subsequently fed a normal fat diet (NN), a normal fat diet containing 0.1% γ‐PGA (NNP), a high fat diet(HH), a high fat diet containing 0.1% γ‐ PGA (HHP), a normal fat diet (HN), or a normal fat diet containing 0.1% γ‐PGA(HNP) for 4 weeks. γ‐ PGA prevented the high fat diet induced elevations in body weight, the weights of liver and visceral adipose tissues (epididymal and perirenal). γ‐PGA also significantly decreased C/EBP β mRNA expression after they had been increased by a high fat diet. γ‐PGA significantly increased hormone sensitive lipase mRNA level and decreased aP2 mRNA expression. In conclusion, γ‐PGA prevents and improves obesity. These effects appear to stem from the action of γ‐PGA in altering mRNAs expression that control adipogenesis, or increasing hormone sensitive lipolysis in white adipocyte.

This study was supported by 2009 Seoul B&RD program.
Sensory evaluation and effectiveness of natural drink for lowering blood cholesterol concentrations
Authors: Han Chan‐Kyu , Kim Sung‐Soo , Cho Kyung‐Dong , Park Ju‐Hun , Lee Bog‐Hieu , Lee Kee Seung , Nam Jong‐Hyun
Published: June 2021

The Koreans with the average serum cholesterol value ranged from 200~ 240 mg/dL are quite high, and thus it is a serious medical problem in the society. In this study, natural drink mostly made with medicinal herbs such as Albizzia julibrissin Durazz, Cassia tora L. seed, Maximowiczia chinensis, Morus alba L., and Glycyrrhiza uralensis was developed as anticholesterolemic drink. Sensory evaluation and anticholesterolemic effects of natural herbal drinks were tested. Sensory evaluation was performed by 146 panels (male 79, female 67) on the strength of color, flavor, taste, preference and overall acceptance based on 9 point‐hedonic scale. Anticholesterolemic effect of natural herbal drink was examined for serum lipid fractions by 15 adults at 0, 4, 8 and 12 week after use of one can (140 ml/can) a day. Sensory test results indicated that 6.0 pt was attained for the color, 5.3 pt., 4.1 pt., 5.7 pt. for flavor of Korean herbal medicine, astringent taste, and sweet taste, respectively. Overall acceptance(5.6 pt.) was not so high. However, the blood levels of total lipid, and triglyceride were decreased since after 8 weeks of the consumption and those of total cholesterol and LDL‐cholesterol also decreased significantly. From the findings, our natural herbal drink might be a good functional drink having anticholesterolemic effect. However, the natural herbal drink needs to be improved in the taste, flavor, and overall acceptance.
100% Tart cherry juice reduces plasma triglycerides and CVD risk in overweight and obese subjects
Authors: Martin Keith R , Bopp Jennifer , Neupane Srijana , Vega‐Lopez Sonia
Published: June 2021

Accumulating epidemiological evidence shows that diets rich in fruits, e.g., cherries, and vegetables can significantly reduce CVD risk. In this randomized, placebo‐controlled crossover study, we recruited 10 participants (38.1 ± 12.5 y; 8 females, 2 males) with BMI>25.0 (32.2 ± 4.6; 5 obese, 5 overweight) to consume 8 fl oz. daily of either 100% tart cherry juice or an alternate placebo beverage, for 4 weeks each with a 2 week intervening washout period. Fasting blood samples were collected at the beginning and end of each arm for measurement of biomarkers of dyslipidemia and glycemia. Total cholesterol (C) (188.1 ± 21.0 mg/dl) was not different between treatments (p>0.05) but plasma triglycerides (TG), TG/HDL‐C, and VLDL were significantly reduced 10%, 17% and 15%, respectively (p<0.05) after tart cherry juice consumption. LDL‐C and HDL‐C were not different between treatments. Baseline fasting glucose and insulin levels were 100.0 ± 10.3 mg/dl and 12.8 ± 5.8 uIU/ml, respectively. HOMA, QUICKI, and McAuley indices of insulin resistance were not significantly different at the end of both phases. Baseline hsCRP was 7.0 ± 5.2 mg/l indicating increased CVD risk in these participants. Consumption of tart cherry juice or placebo did not result in significant differences in hsCRP. Collectively, the data suggest that 100% tart cherry juice may reduce the risk of CVD by reducing plasma TG.
Green tea extract (GTE) attenuates adipose inflammation by restoring glutathione redox status in high fat diet‐induced nonalcoholic fatty liver disease (NAFLD)
Authors: Park Hea Jin , Chung Min‐Yu , Koo Sung I , Bruno Richard S
Published: June 2021

Adipose and hepatic inflammation contributes to NAFLD. We hypothesized that the anti‐inflammatory activities of GTE would decrease inflammation, restore glutathione redox status and attenuate hepatic injury in NAFLD. Wistar rats (16‐wk old, n=63) were fed a low‐fat (LF) diet containing no GTE or a high‐fat (HF) diet containing 0, 1, or 2% GTE for 8 wks. HF controls had greater (p<0.05) adipose and hepatic monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNF‐α) compared to LF controls. GTE decreased MCP‐1 by 25–64% and TNF‐α by 40–42% in adipose and liver. GTE increased adipose and hepatic glutathione (GSH) by 19–43% that was decreased by HF feeding. HF controls had decreased adipose glutathione disulfide (GSSG) that was normalized by GTE to levels of LF controls. GTE lowered serum alanine aminotransferase (ALT) by 39% relative to HF controls. Adipose TNF‐α and hepatic TNF‐α and MCP‐1 were correlated to ALT. Adipose TNF‐α and GSH/GSSG ratio were positively correlated whereas hepatic MCP‐1 and GSH were inversely correlated suggesting that tissue inflammation alters thiol redox status. ALT and adipose total GSH were inversely correlated suggesting that higher adipose thiol levels are associated with the amelioration of hepatic injury. Thus, GTE decreased HF‐induced inflammation and hepatic injury in NAFLD by altering tissue GSH redox status. Supported by the USDA‐NRI (2007‐02303) to RSB.
Quercetin from Onion Peel Extract Improves the Features of Metabolic Syndrome in Male Smokers
Authors: Lee Kyunghea , Lee Hye jin , Kim Myeong ok , Cha Young jun , Park Eunju , Shin Min jeoung
Published: June 2021

Objective

This study tested the effects of supplemental quercetin derived from onion peel extract on the components of metabolic syndrome in healthy male smokers.

Methods

Randomized assigned subjects were instructed to take either a placebo (n=43) or 4 quercetin capsules per day for a total of 100 mg of quercetin (n=49). Blood pressure and blood lipids were measured at baseline and after 10 weeks of intervention.

Results

Quercetin supplementation significantly reduced serum total cholesterol (p<0.05) and LDL‐cholesterol (p<0.01), whereas these effects were not shown in the placebo group. In addition, there were significant increases in serum HDL‐cholesterol both in the placebo (p<0.005) and quercetin supplementated group (p<0.001); however, changes in HDL‐cholesterol were significantly greater in subjects receiving supplemental quercetin compared to the placebo. Both systolic (p<0.05) and diastolic blood pressure (p<0.01) was significantly reduced in the quercetin supplemented group. Glucose levels were significantly decreased after 10 weeks of quercetin supplementation (p<0.05).

Conclusions

The results will be discussed that daily supplementation of onion peel extract improved the components of metabolic syndrome including blood lipid profiles, glucose and blood pressure, suggesting quercetin's beneficial role in onion peel extracts as a preventive measure against cardiovascular risks.
Chemopreventive Effect in HepG2 cells exposed to L. Reuteri with Antioxidant Supplements
Authors: Green Kevin M. , Ibrahim Salam , Holmes‐McNary Minnie
Published: June 2021

There is the limited treatment for hepatocellular carcinoma (HCC) which is one of the most common cancers. Preventive control has been strongly emphasized for HCC. Researchers have begun to consider complementary and alternative medicines (CAM) as a means to treat and reduce the risk of gastrointestinal diseases. The probiotic bacteria Lactobacillus reuteri (L. reuteri) is shown to exert beneficial effects on the host by decreasing side effects of illnesses such as diarrhea, lactose intolerance, colorectal and liver cancer. Antioxidants are bioactive nutrients found in fruits and vegetables that can prevent or slow oxidative damage. Antioxidants act as “free radical scavengers“ to prevent free radical damage. Both probiotic and antioxidants are shown to have a beneficial inhibitor response in promoting apoptosis. To date, there is no research study on HCC with the probiotic L. Reuteri. Therefore, we hypothesize that probiotic Lactobacillus reuteri (L. reuteri) supplemented with selected antioxidants will enhance cell death in hepatocellular carcinoma. In this human‐derived HEPG2 cell culture study L. reuteri with our selected antioxidants demonstrates the effectiveness in altering the cancerous morphology and demonstrates a synergistic chemopreventive effect.

Grant Funding Source: NSF Opt‐ED, Biology
Changes in Biomarkers of SH Rats Fed Novel and Traditional Sources of Vegetables
Authors: Powe Michelle , Stevens Amy M , Dawkins Norma L , Pace Ralphenia D
Published: June 2021

Purslane, sweet potato greens and collard greens have been known to decrease the complications of hypertension due to their high antioxidant capacity. The purpose of this study was to determine if the consumption of these greens decreased blood pressure, weight, and injury to the glomeruli of the kidney. Five week old Spontaneously Hypertensive (SHR) rats (n=44) were separated equally into four diet groups: control (AIN‐76A diet), purslane, sweet potato greens, and collard greens each added at 4%. SHR were fed their respective diets for six weeks. Blood pressure measurements and weight were recorded weekly and twice per week, respectively.

Animals were euthanized and the kidneys were removed and placed in 10 % formalin. Tissues were fixed on two slides; stained with hematoxylin and eosin stain (H&E) and the other stained with Periodic acid‐Schiff (PASH).

Minimal variation in food intake was observed among the treatment groups, however, a significant reduction (P < 0.05) in systolic blood pressure in animals fed purslane and sweet potato greens were observed. Preliminary results in kidney glomeruli are inconclusive.

The results from these studies indicate that these novel and traditional vegetables show significant promise in significantly reducing risk factors associated with heart disease and other inflammatory chronic diseases.

Grant Funding Source: USDA/AFRI
Ingestion of Wheat Germ in Healthy Subjects Does Not Acutely Elevate Plasma Wheat Germ Agglutinin Concentrations
Authors: Kuzma Jessica N , Cordain Loren
Published: June 2021

Dietary fiber content (15 g/day) of the typical U.S. diet is considerably lower than recommended values (25–30 g), hence the public is encouraged to increase consumption of whole grains, fruits, and vegetables. Whole grains are a rich source of the lectin wheat germ agglutinin (WGA), which has been poorly studied in humans but has elicited adverse health effects in laboratory animals. It is unclear if it can breach the gut barrier, enter circulation, and influence physiology. Apart from animal studies, characterization of lectin behavior in humans is limited to tomato lectin (TL) and peanut lectin (PNA) feeding studies; both of which were able to detect lectin in systemic circulation within hours of ingestion of tomato juice and peanuts, respectively.

The purpose of this study was to determine if WGA enters the circulation of healthy subjects following ingestion of wheat germ. Fourteen normal males and females between 17 and 40 yrs completed preliminary screening and reported to the lab after an overnight fast. Following baseline blood measurements, subjects consumed 50 g wheat germ within a 60 min interval. Blood samples were obtained at 30, 60, 90, 120, 240 min, and at 24 hrs post ingestion. Venous plasma concentrations of WGA were determined via WGA‐specific Enzyme‐Linked Immunosorbent Assay (ELISA) at the six time intervals, including baseline.

According to the protocol utilized, WGA was not detected in venous plasma samples from any of the subjects following consumption of 50 g of wheat germ. Further research will be required to determine if WGA enters the bloodstream and binds formed elements such as erythrocytes, platelets, and leukocytes in addition to other tissues and organs. This research was supported by the department of Health and Exercise Science, Colorado State University.
Effects of 20% fat and 1% cholesterol diet‐induced obesity on gene expression profiles of T cells
Authors: Han Sung Nim , Kim Min Soo
Published: June 2021

Obesity accompanies various metabolic and immunological changes. Higher incidence and severity of infectious illnesses and a poor antibody response in obese persons support the dysregulation of immune functions with obesity. We studied the gene expression profile of T cells to understand the molecular changes associated with obesity in T cells. C57BL mice were fed diets containing 5% fat (Control) or 20% fat and 1% cholesterol (HFD) by weight for 12 wks. Samples were collected at 4 and 12 wks after being on diets. T cells were purified from splenocytes by negative selection and cultured with media or plate‐bound anti‐CD3 and soluble anti‐CD28 for 2 hours. Gene expression profile was assessed using Illumina microarray analysis containing 45281 probes. Genes showing more than 2 fold changes and p values less than 0.05 after Benhamini‐Hochberg multiple‐testing correction were selected. Proliferative responses of T cells were determined at 72 hrs after stimulation. Proliferative response of T cells were similar between control and HFD groups at 4 wks, but, 20% lower in HFD group at 12 wks. While 37 and 43 genes showed significant upregulation in 4wk control and HFD groups upon stimulation and 27 genes showed significant upregulation in 12wk control group, none of the genes from 12wk HFD group showed significant upregulation with stimulation. Genes failed to show significant upregulated expression in 12wk HFD group include IL2, Gfi1, Bcl2, and CD69. These findings suggest that obesity is associated with an impairment of T cell activation.

Supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009‐0063409).