bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of a transcriptional repressor region of the human N‐cadherin gene
Authors: Marley Kate , Flitcroft Crysta , Workman Aspen , Madden Scott
Published: June 2021

Cadherins are calcium‐dependent transmembrane cell adhesion proteins. N‐cadherin can become misexpressed in epithelial tumors and is linked to increased motility and invasiveness. Loss of N‐cadherin expression in rat kidney cells is associated with degradation of kidney integrity and the N‐cadherin promoter becomes methylated in these cells. The objective of this study was to characterize a putative repressor region of the human N‐cadherin gene. N‐cadherin promoter luciferase reporter constructs were transiently transfected into HeLa cells and MDA‐MB‐435 cells, which both express N‐cadherin endogenously. Expression levels from reporters were highest from the +10bp to ‐462bp fragment and lowest from the +10bp to ‐1975bp fragment. Deletion reporters from ‐462bp to ‐1975bp are narrowing the region of focus for a relevant repressor element. A431 cells and BT‐20 cells do not express endogenous N‐cadherin and A431 also did not express any N‐cadherin reporters. However, BT‐20 cells had reporter expression similar to HeLa and MDA‐MB‐435 cells, suggesting there may be epigenetic modifications of the endogenous N‐cadherin promoter. Examination of the methylation status of the human N‐cadherin gene in BT‐20 cells in underway. This publication was made possible by the NIH Grant Number P20 RR16469 from the INBRE Program of the National Center for Research Resources.
A hydrophobic patch on proteinase 3, the target of autoantibodies in Wegener's granulomatosis, mediates membrane binding via NB1 receptors
Authors: Korkmaz Brice , Kuhl Angelika , Bayat Behnaz , Santoso Sentot , Jenne Dieter E.
Published: June 2021

Proteinase 3 (PR3), the target antigen of antineutrophil cytoplasmic antibodies which are found in patients with Wegener's granulomatosis, is a neutrophil serine protease localized within cytoplasmic granules. A small fraction of it is translocated to the plasma membrane of neutrophils after cytokine priming. Recently, the human neutrophil antigen NB1 was identified as a specific neutrophil cell surface receptor of PR3. We hypothesized that the unique hydrophobic cluster of PR3 that is not present in other PR3 homologs accounts for its binding to the NB1 receptor expressed on the cellular surface of NB1 expressing cells. Instead of generating and testing various artificial human PR3 mutants, we cloned and expressed the very closely related gibbon (Hylobates pileatus) PR3 homolog which did not bind to the human NB1 receptor. Moreover, a human‐gibbon hybrid constructed from the N‐ and C‐terminal half of the human and gibbon PR3, respectively, did also not interact with human NB1. The C‐terminal half of gibbon PR3 differs only by 9 residues from human PR3, among which four closely spaced hydrophobic residues are substituted in a non conservative manner (Phe166Leu, Trp218Arg, Gly219Ala, Leu223His). The NB1 bound PR3 was active and was rapidly cleared from the surface by alpha‐1‐protease inhibitor (a1‐PI).

Deutsche Forschungsgemeinschaft (SFB 571)
Investigation of amino acid residues involved in the sunflower thiolase reaction mechanism
Authors: Cadet Melissa , Gomez Iris , Dyer James H.
Published: June 2021

Acetoacetyl CoA thiolase is one of two thiolase activities found in sunflower cotyledons. This thiolase activity is involved in the oxidation of the short‐chain substrate acetoacetyl CoA to produce two acetyl CoAs. Crystal structures of thiolase from other organisms have identified conserved cysteines and a histidine present in the active site of the enzyme indicating that these residues probably play a key role in the reaction mechanism. The purpose of this research is to do site‐directed mutagenesis of the corresponding cysteine and histidine residues in the sunflower acetoacetyl CoA thiolase
Investigating the Details of the Catalytic Mechanism of Alkanesulfonate Monooxygenase
Authors: Robbins John M , Ellis Holly R
Published: June 2021

Elemental sulfur is essential for the synthesis and functionality of certain biological cofactors and proteins. When excess sulfate is not readily available, Escherichia coli expresses a series of proteins implicated in the uptake of sulfur from alternative environmental sources. Notably among these sulfate‐starvation induced proteins is the two‐component alkanesulfonate monooxygenase system composed of an NAD(P)H‐dependent FMN reductase (SsuE) and a FMNH2‐dependent monooxygenase (SsuD). The details of the catalytic mechanism governing the desulfonation reaction of SsuD are under active investigation.

Previous structural and kinetic studies have implicated an active site base serving a role in catalysis. Several possibilities for this active‐site base were identified, and mutagenesis was performed in order to assess the importance of each residue in catalysis. Kinetic Studies were performed to determine how these substitutions altered the kinetic parameters compared to wild type SsuD, and deuterium‐isotope studies were performed to evaluate further the mechanism of desulfonation. These results provided valuable insight into the overall catalytic mechanism of SsuD.
Structure and Function of Thimet Oligopeptidase
Authors: Wagner Jonathan , Rodgers David
Published: June 2021

Thimet Oligopeptidase is a neuropeptidase in the M3 family of zinc metallopeptidases. It is responsible for the degradation of many bioactive peptides. Its most well‐known substrate is neurotensin, which is active both in the central nervous system and the periphery, but TOP also cleaves a number of other small peptides. The most intriguing characteristic of TOP is its "fuzzy" substrate recognition that allows it to cleave oligopeptides with no obvious sequence similarity while maintaining a high degree of specificity for those sites. By crystallizing TOP in the presence of substrates we have found a unique binding site that interacts with the C‐terminal end of peptide substrates. We are currently investigating the energetics of peptide binding by calorimetry.
T‐cadherin, an Adiponectin Receptor in the Cardiovascular System
Authors: Denzel Martin Sebastian , Scimia Maria Cecilia , Ruiz‐Lozano Pilar , Shapiro Lawrence , Ranscht Barbara
Published: June 2021

T‐cadherin, an atypical GPI‐anchored cadherin is expressed on endothelial and muscle cells. It binds Adiponectin, a fat‐derived cytokine with proangiogenic and cardioprotective properties. Adiponectin's serum concentration is inversely related to fat mass. Adiponectin activates AMP dependent protein kinase (AMPK) but the detailed mechanism remains elusive. We report that T‐cadherin is required for the effects of Adiponectin in the vasculature and the heart in vivo. Adiponectin binding to the surface of muscle fibers and blood vessels is abolished in mice genetically deficient for T‐cadherin. T‐cadherin‐dependent binding of Adiponectin was confirmed by siRNA‐mediated depletion of T‐cadherin in C2C12 myotubes and with endothelial cells overexpressing T‐cadherin. In the hind limb ischemia angiogenesis model both T‐cadherin‐ and Adiponectin‐deficient mice displayed reduced angiogenesis after hypoxia as compared to the wild type. Cardiac hypertrophy after pressure overload in mice lacking T‐cadherin or Adiponectin was enhanced compared to the wild type. AMPK phosphorylation in the hypertrophic hearts was significantly reduced in both T‐cadherin‐ and Adiponectin‐deficient mice. Our work suggests a mechanistic link between T‐cadherin's and Adiponectin's functions. We propose a model in which T‐cadherin mediates Adiponectin's protective effects as a receptor or coreceptor.
Uncoupler induced graded mitochondrial depolarization and attenuated matrix calcium uptake are enhanced by complex V inhibition indicative of blocked ATP hydrolysis
Authors: Haumann Johan , Hartman Cory , Aldakkak Mohammed , Dash Ranjan K , Camara Amadou KS , Stowe David F
Published: June 2021

Mitochondrial (m) Ca2+ uptake occurs mainly via the Ca2+ uniporter and is highly dependent on the membrane potential (Δ Ψ m). We measured Ca2+ uptake at decreasing ΔΨ m in isolated mitochondria in the presence and absence of oligomycin (OMN) to test the effect of complex V reversal. [Ca2+]m, ΔΨm, pHm, and NADHm were measured spectrophotometrically using indo‐1, rhodamine 123, BCECF fluorescence and autofluorescence, respectively, in guinea pig heart isolated mitochondria. After energizing with pyruvic acid, 0, 10, 20, 30 or 100 µM dinitrophenol (DNP) was added to stepwise reduce Δ?m, after which 80 and 130 nM [Ca2+]e was added. OMN was added in some experiments to block complex V. The effect of DNP to decrease Δ?m, was exaggerated in the presence of OMN. Partial depolarization decreased the rate of matrix Ca2+ uptake, while uptake of Ca2+ resulted in further depolarization. With partial depolarization [Ca2+]m did not reach steady state but increased despite further depolarization. OMN reversed this increase and actually caused a decrease in [Ca2+]m with greater depolarization. pHm and NADHm were also lower with OMN. Our study demonstrates the importance of 1) electrical and chemical gradients for Ca2+ uptake, and 2) a likely effect of ATP hydrolysis at complex V to support ΔΨ m even during mild, graded uncoupling. Proton leaks may lead to reversal of ATP synthesis to maintain membrane potential. NIH, AHA, VA Merit grants.
Determination of malate‐aspartate shuttle activity in intact hearts using 13C NMR spectroscopy and modeling
Authors: Lu Ming , Saidel Gerald , Yu Xin
Published: June 2021

The malate‐aspartate (M‐A) shuttle, which transports cytosolic NADH into mitochondria, provides an important mechanism to regulate metabolic activity in these two compartments. However, direct measurement of M‐A shuttle flux has not been possible. In this study, we developed a novel approach that combines dynamic 13C NMR spectroscopy with biology detailed model of cardiac metabolism to directly quantify M‐A shuttle flux from experimental data. Dynamic 13C NMR spectra were acquired from isolated rat hearts perfused with either short chain (2.5mM [2‐13C]acetate) or long chain (0.32mM [U‐13C]palmitate) fatty acids (FA). A 3rd group of hearts were perfused with 2.5mM [2‐13C]acetate plus unlabeled glucose (5.0mM) and lactate (1.0mM) to induce high cytosolic redox state (HRX). TCA cycle flux (VTCA) and M‐A shuttle activity (VM‐A) were determined by least‐square fitting of a novel multi‐domain kinetic model of cardiac metabolism to dynamic 13C NMR spectra. Our results showed that, regardless of chain length, both VTCA and VM‐A were unchanged in FA only groups. At HRX, VTCA was unaltered compared to the other two groups, however, VM‐A was increased by 40% (P<0.05). Therefore, our proposed method provides the opportunity to directly quantify metabolic communication between subcellular compartments via M‐A shuttle under various pathophysiological conditions. This work was supported by R01HL073315 & R01HL086935.
Localizing compartmental gene expression in developing mouse prostate
Authors: Frank Sander , Chen Qian , Sikes Robert
Published: June 2021

The prostate gland is a significant source of male genitourinary tract morbidity. As developmental processes share several features in common with metastatic cancer, we hypothesize that a better understanding of genes involved in prostate morphogenesis may identify possible targets for therapeutic intervention. The prostate is derived from the urogenital sinus (UGS) which is composed of 4 subdomains: epithelium/mesenchyme (UGE/UGM) and dorsal/ventral halves (UGD/UGV). Each region develops into specific lobes of the prostate with unique properties. This study sought to validate prostate developmental cDNA arrays that showed about 530 UGE/UGM and 35 UGD/UGV differentiated genes. Male UGS were separated either into UGE/UGM or UGD/UGV. Total RNA was extracted and used to synthesize cDNA templates for each subdomain. Quantitative‐polymerase chain reaction (Q‐PCR) was performed to observe relative mRNA levels and confirmed localization for 10/12 primary and 6/10 secondary compartmental genes. Immunofluorescence (IF) localized 6 genes in different predicted compartments. With this research, we have established the basis for a map of regional gene expression in the murine UGS. This work identifies candidate genes for further study and functional analysis by other developmental laboratories. Funding: NIH DK63919 and P20RR016472. Individual support: UD science and engineering and Stetson awards.
Sphingosine kinase inhibition to sensitize hormone‐resistant prostate cancer cells to docetaxel
Authors: Pshezhetskiy Dmitry
Published: June 2021

It has recently been shown that docetaxel chemotherapy is effective in prolonging life in patients with prostate cancer. We have investigated potential ways of increasing the effectiveness of chemotherapy in this disease. We have previously reported that sphingosine kinase‐1 (SK1) inhibition is a key step in docetaxel‐induced apoptosis in the PC‐3 prostate cancer cell line (Pchejetski et al, Cancer Res 2005) and that pharmacological SK1 inhibition is chemosensitizing in the docetaxel‐resistant prostate cancer LNCaP cell line (Pchejetski et al, Mol Cancer Ther, 2008). In this current study we have addressed the mechanism of docetaxel‐induced apoptosis of PC‐3 cells and identified SK1‐dependent and ‐independent components. We have shown that SK1 inhibition by docetaxel is a two‐step process involving an initial loss of enzyme activity followed by a decrease in SK1 gene expression. We have demonstrated that both pharmacological and siRNA‐mediated SK1 inhibition leads to a four‐fold decrease in the docetaxel dose required to cause prostate cancer cell apoptosis.

Overall we have identified a mechanism of docetaxel‐induced SK1‐mediated prostate cancer cell apoptosis. This work points to potential ways of increasing the effectiveness of chemotherapy for prostate cancer by SK1 inhibition.
Pank1 plays an important role in coenzyme A homeostasis during fasting
Authors: Leonardi Roberta , Wang Jina , Miller Karen , Rock Charles O. , Jackowski Suzanne
Published: June 2021

Coenzyme A (CoA) is an essential cofactor synthesized in five steps from pantothenate, and pantothenate kinase (PanK) catalyzes the first and most regulated step. The Pank1 gene encodes two of the four PanK isoforms, PanK1α and 1β. Deletion of exon 3 in the mouse Pank1 gene resulted in loss of expression of both isoforms. The liver of the Pank1 knockout animals contained 40% less total CoA, and the knockout mice showed a blunted response to a pyruvate challenge, indicating impairment in gluconeogenesis. Fasting increases CoA levels to support β‐oxidation and gluconeogenesis. Following a 48h fast, the CoA levels increased by 70% (from 122 ± 4 to 207 ± 23 nmoles/mg of tissue) in livers from wild type and by 50% (from 71 ± 7 to 107 ± 11 nmole/mg of tissue) in livers from knockout mice. Concomitantly, the knockout livers accumulated five times more triglycerides than controls, and the knockout liver homogenates exhibited a decreased rate of palmitic acid oxidation. These results indicate that the reduction in CoA decreases the efficiency of fatty acid β‐oxidation, and that in addition to contributing a large portion of the cofactor pool, Pank1 plays an important role in responding to the increased demand for CoA during fasting. We also observed that the Pank1 knockout males are hyperphagic and 15% heavier than controls, which correlated with a 50% lower malonyl‐CoA in the hypothalamus. Supported by NIH GM062896, CA21765, and ALSAC.
Pseudomonas aeruginosa motility requires condensing enzyme FabF1
Authors: Zhu Kun , Frank Matthew W , Rock Charles O , Zhang Yong‐Mei
Published: June 2021

Bacterial motility along growth surfaces is classified into swarming, swimming and twitching, and all three modes of translocation are important in virulence. Previous study on genome‐wide screen of motility defective mutant of the opportunistic pathogen Pseudomonas aeruginosa has identified fabF1 as the only gene required for all three types of motility. FabF1 is an elongation condensing enzyme homolog with predicted function in fatty acid biosynthesis. Analysis of the membrane fatty acid composition showed that the fabF1 mutant produced significantly reduced level of cis‐vaccinate comparing to the wild type strain. In addition, the fabF1 mutant exhibited increased sensitivity to antibiotics, indicating an increased permeability to small molecules through the mutant membrane probably due to the altered membrane fatty acid composition. Transmission electron microscopy analysis showed that the fabF1 mutant possessed intact flagella which is required for swarming and swimming motilities, demonstrating the assembly of flagella was not affected by the decreased level of cis‐vaccinate. Supplement of unsaturated fatty acids such as oleate and cis‐vaccinate in the growth medium did not reverse the fabF1 mutant phenotype. In contrast, overexpression of fabF1 on a plasmid restored the cis‐vaccinate level and rescued the motility defect of the fabF1 mutant suggesting a direct relationship between the cis‐vaccinate level with the motility phenotype in P. aeruginosa. (Supported by GM34496)
The Phospholipid Transfer Protein Sec14 is Required for Trafficking from Endosomes and Regulates Distinct trans‐Golgi Export Pathways
Authors: Curwin Amy J , McMaster Christopher R
Published: June 2021

Vesicle fission, transport, and fusion are affected by membrane lipid composition. A protein known to regulate both lipid metabolism and vesicular transport is the phosphatidylcholine/phosphatidylinositol transfer protein Sec14 of Saccharomyces cerevisiae. We determined that decreasing Sec14 function affected secretion via the endosome dependent invertase route as well as the endosome independent route followed by Bgl2. Trafficking of a series of other secreted proteins including Hsp150, Cts1, Scw4, Scw10, Exg1, Cis3, and Ygp1 still occurred. Upon diminution of Sec14 function the v‐SNARE Snc1 accumulated in endosomes and the trans‐Golgi but was competent to traffic from the Golgi to the plasma membrane. Sec14 was required for trafficking of Ste3 and the lipophilic dye FM4‐64 from the plasma membrane to the vacuole at the level of endosomes. The results from a synthetic genetic array screen for genes whose inactivation impaired growth of cells with a temperature sensitive SEC14 allele, sec14ts, were consistent with Sec14 regulating endosome function. We go on to determine that lipid ligand occupancy by Sec14 can differentially regulate its functions. (Supported by CIHR and the Canada Research Chairs Fund)
Differential enterocyte partitioning of fatty acids in intestinal fatty acid‐binding protein‐null mice (IFABP‐/‐)
Authors: Lagakos William , Storch Judith , Zhou Yin Xiu , Russnak Tim , Agellon Lou
Published: June 2021

IFABP is expressed at high levels in the mammalian small intestine (SI) and binds the major product of dietary triacylglycerol (TG) digestion, fatty acids (FA). The precise role of IFABP in processing these diet‐derived lipids is unknown. IFABP‐null mice grow normally but have a modest increase in food intake. We investigated the acute metabolism of FA in fasted WT and IFABP−/− SI mucosa in vivo. Two min after intraduodenal administration of [14C]‐oleate, mucosal [14C] was recovered primarily in TG, with no difference between WT and IFABP−/−. However, recovery of [14C] in the phospholipid (PL) fraction was greater in IFABP−/− mice (p<0.01), resulting in a reduced TG/PL ratio. No changes were found in the expression of LFABP or lipid synthetic genes, suggesting the results may be due to a non‐transcriptional, trafficking function of IFABP. Appearance of TG and [14C] in the plasma after an oral gavage of [14C]‐oleate in olive oil was unaffected by IFABP‐ablation, confirming that dietary PL is not a major source for chylomicron PL. Food deprivation increased [14C]‐oleate oxidation in WT and IFABP−/− mucosa similarly, and the expression of genes involved in mitochondrial and peroxisomal FA oxidation were also unchanged. Overall, given the absence of transcriptional changes, the effects of IFABP−/− on metabolism suggest it may be involved in FA trafficking into complex lipids. Source of research support: NIHDK38389 (JS)
The influence of phospholipid metabolism and regulatory networks on the subcellular localization of phosphatidic acid in Saccharomyces cerevisiae
Authors: Chang Yu‐Fang , Henry Susan A.
Published: June 2021

Phosphatidic acid (PA) is a signaling molecule that plays an important role in mediating yeast phospholipid biosynthesis. In cells grown in the absence of inositol, the transcriptional repressor Opi1p is localized to the endoplasmic reticulum (ER) through interactions with Scs2p via its FFAT motif (two phenylalanines in an acidic tract) and the binding with PA. Under these conditions, the UASINO‐containing phospholipid biosynthetic genes are expressed at the maximum level through activation via the Ino2p‐Ino4p activation complex. When inositol is added to the medium, the increased synthesis of phosphatidylinositol causes a rapid depletion of PA in the ER. Opi1p then translocates to the nucleus and represses the transcription of target genes (Loewen et al. 2004. Science 304, 1644 ‐ 1647). Here we present studies utilizing Opi1p reporter constructs to monitor the change in PA metabolism. Strains carrying a single genomic copy of a wild type OPI1 or a mutant OPI1FFAT fused to GFP driven by the native OPI1 promoter were constructed. These functional constructs were introduced into strains contain mutations known to perturb PA metabolism or Opi1p localization including sec14ts, scs2Δ, single mutations or in combination with pct1Δ. INO1 expression was examined to compare with Opi1p localization results and to monitor Opi1p repressor activity under changing metabolic conditions.
Fetal lipid metabolism is regulated by the Liver X Receptor (LXR) in mice
Authors: Plosch Torsten , van Straten Esther M.E. , Huijkman Nicolette C.A. , Baller Julius F.W. , Kuipers Folkert
Published: June 2021

The Liver X Receptors LXRa/ß are vitally important in regulating cholesterol and fatty acid metabolism in the adult. Aim of this study was to elucidate the role of LXR in the fetal mouse liver. Pregnant C57BL/6 mice or Lxra+/− mice crossed heterozygously were fed a diet containing the synthetic LXR agonist T0901317 from E10.5 until delivery. In fetuses and dams, gene expression and lipid levels were monitored. In the mouse fetus, hepatic expression of LXR target genes (e.g., Srebp1c, Fas and Abcg5) is low during gestation and increases after birth. Lxra itself is expressed at ~40 % of adult levels, whereas the expression of Lxrb is 2.5‐3 times higher than in adult control liver. Upon administration of T0901317, LXR target genes are activated in the fetal liver: expression of the cholesterol transporter Abcg5 is 45 times induced. Expression of Srebp1c and Fas is increased 2.0‐ to 5.3‐fold and 1.7‐ to 2.0‐fold, respectively. Consequently, triglyceride concentrations in fetal liver and plasma are raised up to 2.7‐ and 1.5‐fold. Cholesterol levels in plasma are doubled upon treatment, whereas hepatic cholesterol concentrations are unchanged. In Lxra−/− fetuses, these changes are reduced but not absent.

Conclusion

Lxra and Lxrß are functionally active in fetal mouse liver when stimulated with the synthetic agonist T0901317. Similar to adult mice, cholesterol homeostasis and de novo lipogenesis can be influenced experimentally by administration of this agonist.

Supported by the Dutch Heart Foundation (2004T048).
Increased TGFb signaling in type I diabetic mice is associated with parasympathetic dysfunction of the heart
Authors: Georgescu Serban , Naggar Jack C , Welzig Michael C , Zhang Yali , Park Ho‐Jin , Wooten Eric C , Aronovitz Mark J , Karas Richard H , Huggins Gordon S , Galper Jonas B
Published: June 2021

Diabetic Autonomic Neuropathy (DAN), a severe complication of diabetes, is associated with parasympathetic dysfunction, severe arrhythmias and sudden death. We had shown in cardiac myocytes that TGFb attenuates the negative chronotropic response to Carbamylcholine and decreases the expression of M2 muscarinic receptor and Gai2 (components of the cardiac parasympathetic response). Increased TGFb signaling has been implicated in the end organ damage mechanisms in patients and in type I diabetic animal models. Our hypothesis: the decreased response of the diabetic heart to parasympathetic stimulation might be due to increased TGFb signaling. Here we present a new animal model for DAN. Compared to WT, Akita type I diabetic mice showed a decreased Carbamylcholine‐induced bradycardia and a faster recovery to 80% of initial heart rate. Compared to WT, the diabetic heart showed significant increases in mRNA expression of TGFbRII and PAI‐1 (a target of TGFb signaling). Western blots using hearts lysates showed increased phospho‐Smad2/3 and TGFbRII levels in Akita mice. Together with our previous data that TGFb stimulation attenuates the parasympathetic response in cardiac myocytes, these in vivo results suggest that in type I diabetic mice elevated TGFb signaling is associated with parasympathetic dysfunction of the heart, and that TGFb may be involved in the arrhythmia mechanisms in the diabetic heart.
Mst3 Plays an Important Role in Hypoxia‐induced placental Apoptosis in Preeclampsia
Authors: Yuan Chiun‐Jye , Wu Hung‐Yi , Lin Chia‐Ying
Published: June 2021

Preeclampsia, a disorder unique to humans, affects 5‐10% of all pregnancies, and remains a leading cause of fetal and maternal morbidity and mortality. A key histopathologic correlation is shallow invasion and aberrant remodeling of maternal spiral arteries, leading to decreased uteroplacental perfusion and hypoxia. In preeclampic human placenta, hypoxia is thought to be the main cause to elevate oxidative stress and induced trophoblast cell death. Recently, mammalian Ste20‐like protein kinase 3 (Mst3) was found to be elevated in human preeclampic placenta that exhibit extensive sign of apoptosis, suggesting the participation of Mst3 in preeclampsia. Hypoxia is postulated to induce the expression of Mst3 and the apoptosis of human placenta during preeclampsia. The role of Mst3 in hypoxia‐induced apoptosis was demonstrated in both human placental explant culture and the human trophoblast cell line 3A‐sub‐E. Further studies show that, in human trophoblast, hypoxia may induce the expression of Mst3 via oxidative stress and the activation of JNK. The activated Mst3 then triggers apoptosis of human trophoblasts by both caspase‐dependent and independent pathways. (The work was supported by National Science Council, ROC (Taiwan) under Contract No. NSC‐97‐2311‐B‐009‐001‐MY3)
Intracellular TIAF1 self‐association induces apoptosis
Authors: Chang Nan‐Shan
Published: June 2021

TIAF1 is a small TGF‐beta1‐induced factor, which supports fibroblast growth, T helper cell activation and effect on allograft organ rejection, and probably induces regulator TREG cell differentiation. However, overexpressed TIAF1 induces apoptosis in monocytic and epithelial cells and non‐fibroblasts. Here, we demonstrate that TIAF1 self‐association leads to apoptosis. When cells were overexpressed with CFP‐TIAF1 and YFP‐TIAF1, TIAF1 underwent self‐association and the cells died. Also, TGF‐beta enhances the self‐association for causing apoptosis in TGF‐beta‐sensitive normal epithelial cells. Smad4, an adaptor in the TGF‐beta signaling, binds TIAF1 and blocks the apoptosis. Transiently overexpressed TIAF1 sequesters endogenous Smad proteins in the cytoplasm, and blocks the activation of promoter element driven by Smad proteins. Small interfering RNA‐targeting TIAF1 augments the apoptotic function of overexpressed Smad4. Together, self‐association of TIAF1 induces apoptosis, and that Smad4 interrupts the event.
Induction of apoptosis by Solenopsin B: evalutaion by DNA microarray and RT2 Profiler™ PCR array analysis.
Authors: Sample Robert Cressten , Sullivan Donna C , Kelley Kashonda , Rockhold Robin
Published: June 2021

The effect of synthetic fire ant venom alkaloid Solenopsin B (Sol B) on the human monocytic cell line U937 was examined to determine its ability to induce apoptosis and efficacy as a therapeutic agent. Sol B treated cells displayed a >50% reduction in viability along with DNA laddering, a hallmark of apoptosis. The apoptosis mechanism was further examined using DNA microarrays and quantitative RT‐PCR (qRT‐PCR). U937 cells were incubated in the presence of Sol B and total cellular RNA isolated. cDNA was synthesized, labeled with cy3/cy5 and hybridized to microarrays or used in qRT‐PCR RT2 Profiler(tm) PCR array analysis. Microarray analysis revealed that 661 genes and 620 ESTs were up regulated >1.5 fold, including several apoptosis and cell cycle genes. PCR arrays representing functional gene groupings of 90 apoptosis and 92 cell cycle genes showed up regulation of >90% and >70% respectively. Genes examined using both microarray and qRT‐PCR showed correlations of ~93% for apoptosis and ~79% for cell cycle genes. Further, transmission electron microscopy revealed Sol B treatment resulted in loss of cell membrane integrity further verifying the ability of Sol B to induce apoptosis. Collectively these findings indicate that Sol B induced programmed cell death in human cells by triggering the apoptotic pathway.
A Novel Mechanism for Acute Hyperglycemia Induced Apoptosis In Renal Epithelial Cells
Authors: Wisdom Erika , Thekkumkara Thomas
Published: June 2021

Previously, we reported that acute (10‐20 min) exposure of proximal tubule epithelial cells to 25 mM high glucose (HG) induces a time‐dependent dual effect, involving an early (12h) proliferation and a late (48h) apoptosis. In this study, conditioned medium from cells treated with HG (acute) significantly inhibited high glucose‐induced [3H]thymidine incorporation into DNA in fresh cells at 12h. Treatment with conditioned medium increased p53 expression that was inhibited by ROS inhibitor NAC. Under similar conditions, we observed increased chromatin condensation and DNA fragmentation. However, in HG (acute) treated cells replenishment with fresh normal glucose medium at 6h induced only cell proliferation further, confirming the role of secretory factor(s). Consistent with these results, we observed decreased ROS production, DNA fragmentation, and chromatin condensation. Although additional studies are needed to identify HG mediated inhibitory factor(s), our study demonstrates for the first time that a single HG exposure for 20 min alone is sufficient to induce cellular factor(s) capable of eliciting ROS mediated cellular apoptosis leading to epithelial cell dysfunction. Supported by NIH Grant DK072140
Cytosolic expression of dimerization defective mutants of mature Smac potentiate induction of apoptosis without interacting with inhibitor of apoptosis proteins (IAPs)
Authors: Burke Stephen P. , Smith Jeffrey B.
Published: June 2021

Mature Smac, a homodimeric mitochondrial protein lacking the first 55 amino acids, exits mitochondria and promotes apoptosis by directly antagonizing IAPs. We used the ubiquitin fusion method to express mutant and wild type mature Smac in the cytosol of the 911 line of human embryonic retinoblasts. Mutations V78E, V81D, and F88D in the dimeric interface of Smac each prevented homodimerization, as determined by pull down of transiently expressed mutant Smac‐V5 by transiently expressed mutant Smac‐GST. Each of the Smac mutants also failed to heterodimerize with wild type Smac. We induced apoptosis in 911 cells that stably expressed cytosolic Smac by a 24 h treatment with zIEALal, a peptidyl aldehyde inhibitor of the proteasome. Apoptosis was measured by FACS analysis with annexin V PE and 7‐AAD. Each of the monomeric Smac mutants potentiated the induction of apoptosis by zIEALal at least as well as wild type Smac. Caspase activation in 911 cells that stably expressed cytosolic Smac was demonstrated by western analysis of cleaved PARP after a 10 h zIEALal treatment. Cells that expressed the F88D‐Smac mutant accumulated significantly more cleaved PARP than those that expressed wild type Smac. Interestingly, stable expression of wild type Smac in the cytosol of 911 cells markedly decreased its ability to interact with IAPs, which may be due to an as yet unidentified post‐translational modification. Following transient expression in 911 cells, monomeric F88D‐Smac failed to co‐immunoprecipitate with any of the 6 IAPs tested (cIAP1, cIAP2, XIAP, Survivin, Livin, or Apollon). These findings suggest that mature Smac can exert a proapoptotic action that is independent of a direct interaction with IAPs. Supported by grant GM60383 from NIH.
EXPLORING DOUBLE‐STRANDED RNA AS A POSSIBLE CHEMOTHERAPEUTIC AGENT
Authors: Schramm Danielle N. , Lepine Sandrine , Bell Jessica K.
Published: June 2021

Pattern recognition receptors (PRRs) function in the innate immune system to alert the host to pathogenic components. Double‐stranded RNA (dsRNA), viral genomic material or a viral replication intermediate, trigger the PRRs toll‐like receptor 3 (TLR3), retinoic acid‐inducible gene I (RIG‐I), melanoma differentiation‐associated gene 5 (MDA5), and dsRNA‐dependent protein kinase receptor (PKR). Previous studies have reported that dsRNA stimulation induced TLR3‐dependent apoptosis.

Objectives

Examine 786‐O, OVCAR‐3, OVCA‐420, SKOV‐3, DOV‐13, CAOV‐3 cells for dsRNA receptor expression and determine dsRNA‐induced response.

Methods

PRR expression was confirmed by RT‐PCR. Expression levels were determined via qPCR. Apoptotic response to dsRNA stimulation was measured by Hoechst staining 48h post‐stimulation.

Results

786‐O, SKOV‐3 and DOV‐13 show no response to dsRNA. RT‐PCR analysis showed that cell lines contained mRNA for all dsRNA receptors. qPCR showed a significant increase in PRR expression in those cell lines that underwent dsRNA‐induced apoptosis.

Conclusions

Four dsRNA receptors are present in our cell lines. dsRNA receptor expression does not correlate with dsRNA induced apoptosis, but increased dsRNA receptor expression suggests receptor levels or altered signaling pathways may reduce sensitivity to dsRNA. Funding provided by National Cancer Institute & Jimmy V Foundation.
Rapamycin enhances G protein signaling in the budding yeast Saccharomyces Cerevisiae
Authors: Zhu Ming , Wang Yuqi
Published: June 2021

Heterotrimeric G proteins are common signal transducers that are important for many cellular processes. Mechanisms that ensure accurate magnitude and specificity of G protein signaling are under intensive investigation. Here we demonstrate that in Saccharomyces cerevisiae, the activity of heterotrimeric G protein (Gpa1/Ste4/Ste18) that is required for mediating mating responses is augumented by the treatment of rapamycin, a drug that specifically inhibits TOR, i.e., the target of rapamycin. Both basal and mating pheromone‐induced activation of a mitogen‐activate protein kinase Kss1 are enhanced by rapamycin treatment. Genetic analysis indicates that the rapamycin effects are dependent on the existence of Ste4, Ste5, Ste11, Ste7 but not Ste2. Furthermore, the effect is abolished by overexpression of Gpa1, the alpha subunit of the G protein. Together, these data indicate that rapamycin directly or indirectly impact the interaction between Gpa1 and Ste4, and suggest that TOR might play an important role in regulating the magnitude of G protein signaling. Studies that are designed to investigate how Gpa1 and Ste4 interaction is impacted by rapamycin and TOR are underway.

This work is supported by American Heart Association grants 302970.
Inhibitors of p97/Cdc48 AAA ATPase as Potential Anti‐Cancer Therapeutics
Authors: Chou Tsui‐Fen , Brown Steven , Rosen Hugh , Deshaies Raymond J.
Published: June 2021

p97/Cdc48 is an important AAA ATPase and has been implicated in mediating turnover of proteins involved in tumorgenesis. We set out to develop small‐molecule inhibitors of p97 to use as tools to study its function and to evaluate whether it is a viable anti‐cancer target. We carried out a high throughput screen with a library of 15,000 compounds to identify molecules that inhibit p97's ATPase activity. A primary screen using 10 uM concentration identified 32 compounds exhibiting more than 35% inhibition. A dose response curve of 10 concentrations confirmed 9 compounds with IC50 less than 20 uM. To evaluate whether an active‐site cysteine is critical for the observed activity, we performed dose response curves of 9 inhibitors against C522A‐p97. One of the 9 compounds inhibited wild type p97 with 0.3 uM IC50 whereas the IC50 is 100‐fold higher toward C522A‐p97 (hereafter named as 15K1). Several cell‐based assays demonstrated that 15K1 is likely to target p97 in cells. After incubation with 15K1, accumulation of poly‐ubiquitinated proteins was observed in HeLa and RPMI‐8226 cancer cells, which recapitulated the effect of p97 depletion. Moreover, 15K1 caused accumulation of two unstable GFP fusion proteins, TCR‐GFP and UbG76V‐GFP, whose degradation depends on p97. Furthermore, the SV40‐transformed MRC5 cells were 3.4‐fold more sensitive to the growth inhibitory effects of 15K1 than the MRC5 wild type cells.
Optimization of expression and purification of acetoacetyl CoA thiolase from sunflower cotyledon
Authors: Gomez Iris , Schiedel Anke , Oeljeklaus Silke , Dyer James
Published: June 2021

Sunflower (Helianthus annus L) cotyledons have two thiolase activities which have been identified previously. Thiolase I (acetoacetyl CoA thiolase) has specificity for the four‐carbon acetoacetyl CoA, resulting in the production of two Acetyl CoA molecules. Thiolase II (oxoacyl CoA thiolase) is active towards short, medium and long chain acyl CoAs. The purpose of this research was to optimize the expression and purification of a Thiolase I from an E. coli expression system. The sunflower thiolase was cloned into the pBAD‐HisB expression vector, and this construct was transformed into BL‐21 E. coli which were induced for production of the Thiolase I with 0.2% arabinose. The soluble Thiolase 1 was purified using Ni‐NTA agarose affinity chromatography with a single band appearing on a Coomassie Blue stained SDS‐PAGE in the fraction eluted from the column with 100 mM imidazole.
Mismatches between the conceptual level of tests and faculty beliefs
Authors: Costa Manuel Joao , Gonçalves Leonor , Ludovico Paula , Oliveira Mário , Rodrigues Fernando , Sousa João Carlos
Published: June 2021

Student learning of biochemical concepts can be promoted by the conceptual quality of items in written assessments. We present a "faculty centered" development prototype aimed at the improvement of written assessments.

Summary of work

A pool was created with all the written items presented in 2007‐2008 to freshmen and sophomores of a 6 year medical degree ‐ included were disciplines such as Biochemistry, Molecular Biology, Cell Biology, etc. Items were ranked by faculty (n=6) of different disciplines (including biochemistry faculty), according to a simple scale (1 to 5), based on: i. the presence of a context; ii. the type of cognitive process required to solve the item; iii. presence/absence of irrelevant difficulties.

Summary of results

There was considerable inter‐rater variability on the classifications which were, in general, below initial expectations. Faculty regarded very highly the learning potential of the experience and converged on changes to improve item quality for 2008‐2009.

Conclusions

The conceptual level of items in written knowledge tests may be lower than faculty believe.

Take‐home messages

The prototype can be generalized to other institutions. Analysis of future new items and further empirical evidence are needed to corroborate the prototype's power for the improvement of the conceptual level of Biochemistry and Molecular Biology tests.
Snack selection influences satiety responses in adult women
Authors: Furchner‐Evanson Allison , Petrisko Yumi , Howarth Leslie S. , Nemoseck Tricia , Kern Mark
Published: June 2021

Some research suggests that snacking can promote a healthy body weight while other studies suggest the opposite; therefore, identification of satiating snack foods may prove to be valuable for weight control. The effect of different snack foods on satiety and plasma glucose and hormone responses was assessed. Nineteen fasted adult women consumed two 238 kcal snacks (dried plums or low‐fat cookies), 238 kcal white bread, or water on separate days. Subjects completed satiety index (SI) scales prior to snack consumption and again every 15 minutes for 2 hours following initiation of intake. Blood samples were collected at baseline and 15, 30, 45, 60, 90, and 120 minutes following intake. At the end of the 120‐minute test period, subjects were presented with a meal to be consumed until satisfied. Satiety index was higher (p=0.05) for dried plums versus low‐fat cookies. There was no difference in post‐snack consumption between the dried plums and low‐fat cookie trials. Dried plums elicited lower (p=0.05) plasma glucose and insulin area under the curve than low‐fat cookies and tended (p=0.056) to promote a greater plasma ghrelin area over the curve. These results suggest that consuming dried plums as a snack suppresses appetite relative to a commercially available low‐fat cookie perhaps by producing lower glucose and/or appetite‐regulating hormone concentrations.

Supported by the CA Dried Plum Board.
Measurement of de novo lipogenesis in humans during lactation
Authors: Mohammad Mahmoud A , Sunehag Agneta L , Haymond Morey W
Published: June 2021

Background

Milk fat is composed of 98% triglycerides (TG) and provides infants with ~50 % of their calories. Mammary gland de novo lipogenesis contributes significantly to milk fat in animals. However, there are no data regarding this process in humans.

Hypotheses

Incorporation of 13C carbons from [U‐13C]glucose into fatty acids (FAs) and glycerol in TG will be, higher in milk TG than in plasma, and higher during H‐CHO than H‐F diet.

Subjects

7 healthy lactating women were studied in a crossover, randomized design during isocaloric, isonitrogenous, H‐F (30%CHO, 55%fat) and H‐CHO (60%CHO, 25%fat) diets. Following feeding for 7 days on each diet, women were continuously infused with [U‐13C]glucose for 5h following a 12h fast and during 7h of feeding. Incorporation of 13C from [U‐13C]glucose into FAs and glycerol was measured in both plasma and milk using GC‐MS.

Results

Incorporation of 13C from [U‐13C]glucose into milk FAs increased (p<0.05) with the increased chain length of the FAs from C2 to C12 then, declined in C14 and was not detected in FAs longer than C16. During meal absorption, enrichment of 13C was ~3 fold higher in milk FA and ~7 fold higher in milk glycerol compared to plasma FAs and glycerol. During fasting, 25% and 6% of medium chain FAs (MCFAs) (C6‐C12) were derived from glucose following H‐CHO and H‐F diets, respectively. However, during feeding, de novo lipogenesis from glucose was 60% and 20% of MCFAs during both H‐CHO and H‐F diet, respectively.

Conclusions

The human mammary gland is capable of de novo lipogenesis, mainly MCFAs, which is increased during a H‐CHO diet. RO1DK 55478, HD 37857, MO1‐RR‐00188‐34.
Contribution of complementary feeding (CF) to nutrient intake of low‐income rural infants, aged 6 to 12 mo, in Guatemala City: Principal sources of macro‐ and micronutrients
Authors: Hernandez Liza Mariana , Campos Raquel , Enneman Anke , Solomons Noel W.
Published: June 2021

Objective

To rank the food and beverage elements of the infants' diets in terms of the principal contributors to each macro‐ and micronutrient of interest.

Methods

3 non‐consecutive 24‐h recalls were collected from mothers of 64 infants, aged 6‐12 mo, in the rural village Santo Domingo Xenacoj. We modeled Maternal Milk (MM) intake based on total energy recommendations. The macro‐ and micronutrient contribution from each of the food and beverage items reported were ranked in relation to the total reported intakes among the infants.

Results

The top five sources of protein were: Incaparina; MM; white rolls; pasta; and tortilla, accounting for 52.5% of the total. MM was the leading sources of fat, vit C, ribo, and nia, and figured among the top 10 sources of all macro‐ and micronutrients with the exception of CHO, Fe and Zn. Table sugar is fortified with Vit A in Guatemala and that item was the first source of the vitamin, accounting for 24.1% of its total consumption; MM was second, providing 21.6%. Overall, 21%of CF items were fortified. 5% of CF could be classified as special "baby foods," with the remainder being family food.

Conclusions

The contribution of MM is indispensable in contributing toward an adequate macro‐ and micronutrient intake of infants in the second semester of life.

Funded in part by Sight & Life and Wyeth Nutrition
The association between carotid plaque and cardiovascular disease risk factors in Korean patients with type 2 DM
Authors: Park JiEun , Hwang Ji‐Yun , Choi YoungJu , Huh KapBum , Kim WhaYoung
Published: June 2021

The presence of carotid plaque is a cardiovascular disease risk factor and the prevalence of cardiovascular disease has been increasing in type 2 DM patients with carotid plaque. This study was performed to investigate the association between the presence of carotid plaque and cardiovascular disease risk factors in Korean patients with type 2 DM. The subjects were 696 Korean patients with type 2 DM. Anthropometric and hematological variables, presence of carotid plaque, carotid intima‐media thickness (IMT), and dietary intake were assessed. In male subjects, there was no difference between individuals with and without carotid plaque in hematological variables and nutrient intakes. In females, however, serum adiponectin concentration was lower (p=0.02) and carbohydrate intake was higher (p=0.05) in subjects with carotid plaque. The association between SNP 276 genotype and adiponectin concentration was not observed. In males, current smokers were more prevalent in subjects with carotid plaque than without carotid plaque (p=0.029). Carotid IMT was related to the incidence of metabolic syndrome in both genders. In conclusion, carotid plaque was related to behavioral factors such as smoking in male subjects, whereas it was related to adiponectin concentration and carbohydrate intake in female subjects. (This work was supported by the second stage of BK21 project and by Seoul R&BD Program, No. 10526.)
Dietary patterns and metabolic syndrome in Korean type 2 DM patients
Authors: Lee Yoon Jung , Yeo Eun‐Sil , Kim Wha Young , Park Jong Suk , Yang Eun Ju
Published: June 2021

The metabolic syndrome(MetSyn) is a worldwide health problem. The role of diet for the risk of MetSyn remains uncertain. This study examined the association between dietary patterns and the risk of MetSyn in Korean type 2 DM patients (>20y old, n=515). Factor analysis was used to identify dietary patterns from food frequency questionnaire data. Three major dietary patterns were identified (Eigenvalues>1.25): Traditional Korean, Convenience/pork, and Meat/fish. The Traditional Korean dietary pattern was characterized by high intakes of vegetables, mushrooms, seaweed, legumes, tofu, fruit, Kimchi, anchovy, and fish. The Convenience/pork dietary pattern was characterized by high intakes of pork, canned fish, eggs, noodles, bread, and beverage. The Meat/fish dietary pattern was characterized by high intakes of beef, chicken, fish, seafood, and noodles. The Traditional Korean pattern was not associated with the risk of MetSyn in type 2 DM patients. Both Convenience/pork and Meat/fish patterns were negatively associated with the risk of MetSyn in type 2 DM patients. In particular, Convenience/pork and Meat/fish patterns were negatively associated with HDL‐cholesterol. The prevalence of MetSyn in Korean type 2 DM patients may be related to low intakes of protein and fat. (This work was supported by the second stage of BK21 project and by Seoul R&BD Program, No.10526)