bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of cell proliferation by a novel POK family protein HKR3
Authors: Yoon Jae‐Hyeon , Kim Myung‐Hwa , Choi Won‐Il , Hur Man‐Wook
Published: June 2021

POK family proteins have been shown to play roles in cell proliferation, development, oncogenesis, and immunity. HKR3 is a ubiquitous transcription factor consisted of 688 amino acids and contains N‐terminus BTB/POZ‐domain and eleven Krüppel‐like zinc fingers. We investigated whether the HKR3 has a regulatory effect on cell cycle progression by regulating gene expression of p53 pathway (ARF‐HDM2‐p53‐p21CIP1). The HKR3 is a potent activator of the pathway and activates transcription of ARF, p53, and p21CIP1 genes. In particular, HKR3 potently activated transcription of tumor suppressor gene ARF. The activation of ARF by HKR3 involved the acetylation of histone H3 and H4 around the ARF promoter through interaction with transcription co‐activator p300. HKR3 induced cell cycle arrest and inhibited cell proliferation by decreasing the number of cells in S phase. Also, we found that mRNA expression of HKR3 is greatly increased when cells were treated with a DNA damage agent, etoposide. Our investigation suggests that HKR3 is one of the major regulators of the p53 pathway by acting at the level of transcription and may be a potential a tumor suppressor.
The NO trafficking between dinitrosyl‐iron complexes (DNIC) and E. coli transcriptional factors containing iron‐sulfur clusters
Authors: Yu Steve , Lo Fengchun , Hung Mu‐Cheng
Published: June 2021

SoxR protein is a transcriptional factor belonged to a member of the Mer family. The native form of the protein is presented as a dimeric structure and each monomer contains a redox‐active [2Fe‐2S] center. The iron‐sulfur cluster of SoxR is responsive to one‐electron oxidation by superoxide and resulted in subsequent expression of soxS gene for the rescue of oxidative stress such as the expression of superoxide dismutase or catalase. In addition to superoxide, SoxR is also sensitive to nitric oxide (NO) and its nitrosylated protein displayed paramagnetic features where its average g value is appeared at 2.03 in EPR. Despite the crystal structures of SoxR protein with and without the nucleic acids have already been unraveled [1], there is still not sufficient structural information to illustrate how the conformational changes mediated by redox chemistry or NO modulation [2] could lead with the transcriptional activation. In this study, we employed the x‐ray absorption spectroscopic methods to determine the structural insights of metal active sites in SoxR under the oxidized, reduced and nitrosylated conditions, respectively. We anticipate clarifying the major clues that control the corresponding gene regulations resulting from oxidative or nitrosative stress.

Financial Support: National Science Council (NSC 98‐2627‐M‐001‐004‐) and Academia Sinica in Taipei
Identification of a Consensus DNA Binding Sequence for nGdf5
Authors: Baldwin Michael C , Jensen J Daniel , Sperry Daniel K , Meinhart Christopher A , Bridgewater Laura C
Published: June 2021

Growth and differentiation factor 5 (Gdf5) is a member of the BMP family of secreted growth factors and is associated with joint formation and cartilage maintenance. Recently, however, a nuclear variant of Gdf5, called nGdf5, was discovered. It is produced by translation of the Gdf5 transcript from a downstream alternative start codon. A yeast one‐hybrid screen suggested that Gdf5 may interact with an enhancer element in the Col11a2 gene, suggesting the hypothesis that nGdf5 regulates transcription. CASTing (cyclic amplification and selection of targets) was performed to identify a consensus DNA binding sequence for nGdf5. CASTing involved several cycles of incubating nGdf5 with random oligonucleotides, imunoprecipitating the resulting DNA‐protein complexes, and then amplifying the selected DNA sequences by PCR. After four cycles of CASTing, the selected DNA probes were cloned, sequenced, and aligned. The sequence (T/G)GGTG(G/T)G was present in 15% of the selected and amplified probes, and another 58% contained 1‐ or 2‐bp mismatches to this consensus sequence. Electrophoretic mobility shift assays verified that nGdf5 binds specifically to (T/G)GGTG(G/T)G. These results suggest that the novel protein nGdf5 binds to DNA, either directly or in cooperation with a binding partner, and are consistent with the possibility that nGdf5 helps regulate transcription.
Mediator co‐activator function is controlled by activator‐induced structural shifts
Authors: Taatjes Dylan J , Ebmeier Christopher C , Bernecky Carrie
Published: June 2021

The human Mediator complex is a general factor that helps regulate expression of nearly all protein‐coding genes and thus lies at the heart of transcriptional regulation. Activator binding triggers major structural shifts within Mediator, suggesting a straightforward means to spatially and temporally regulate Mediator activity. Using mass spectrometry (MudPIT) and other techniques, we have compared the subunit composition of Mediator in three different structural states: bound to the activator SREBP‐1a, VP16, or an activator‐free state. As expected, consensus Mediator subunits were similarly represented in each sample. However, we identify a set of cofactors that interact specifically with activator‐bound but not activator‐free Mediator, suggesting activator binding triggers new Mediator‐cofactor interactions. Furthermore, MudPIT combined with biochemical assays reveal a non‐overlapping set of co‐regulatory factors associated with SREBP‐Mediator vs. VP16‐Mediator. These data define an expanded role for activators in regulating gene expression in humans and suggest that distinct structural shifts regulate Mediator function in gene‐specific ways.
Pirlindole analogs as probes of the active site of Monoamine Oxidase A
Authors: Ramsay Rona Ruth , Liu Gu , Westwood Nicholas J
Published: June 2021

Pirlindole analogs are antidepressants that selectively inhibit mitochondrial monoamine oxidase A. New analogs were synthesized to probe the interaction with the FAD prosthetic group. Previous work (Hynson, R.M.G., Wouters, J. and Ramsay, R.R. (2003) Biochem. Pharmacol. 65, 1867–74) showed that changing the position of the pirlindole nitrogen relative to the N5 of the isoalloxazine ring altered the intensity of the spectral shift at 500 nm. New derivatives masking the pirlindole nitrogen or changing it to C or O provide experimental support for the position of the inhibitor docked in the active site.

Supported by INTAS 99‐00433
The Use of Short lived Isotopes for Exploring Drug Binding Partners of Isoniazid in Mycobacterium Tuberculosis
Authors: Borgaro Janine , Liu Li , Hooker Jacob , Tonge Peter
Published: June 2021

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), is a serious global health threat. The mycobacterial cell wall is composed of mycolic acids, which rely on the synthesis of long chain fatty acids by the fatty acid biosynthesis pathway (FASII) which is a target for some of the common anti‐TB drugs such as isoniazid. Following activation of isoniazid, it forms a covalent adduct with NAD(H) which inhibits InhA, the FASII enoyl reductase enzyme, in vitro. While it is clear that the adduct inhibits InhA, there is considerable evidence that the mode of action of isoniazid is complex and that it interacts with other proteins in the cell. In order to answer some of the questions concerning the protein targets, we are incorporating a short lived radioisotope into isoniazid. Bacteria will then be exposed to the labeled drug and fractionated under non‐denaturing conditions using methods such as size exclusion chromatography and isoelectric focusing with in‐line radiation detection to identify fractions containing protein‐drug complexes.
High‐Throughput Yeast Screen to Identify Vacuolar H+‐ATPase subunit a specific inhibitors
Authors: Hinton Ayana Makeda , Forgac Michael
Published: June 2021

Vacuolar H+‐ATPases (V‐ATPases) are multi‐subunit proteins critical for acidification of intracellular compartments. V‐ATPases are important for biological processes such as bone resorption and disease related processes including metastasis. V‐ATPase localization is determined by the subunit a isoform. There are four a isoforms in humans, a1‐a4. V‐ATPases at the plasma membrane of osteoclasts have a3 and those at the plasma membrane of certain renal cells have a4. Genetic defects in a3 and a4 lead to osteopetrosis and renal tubular acidosis, respectively. We would like to exploit the specificity of a isoform function to develop isoform specific inhibitors that can be used as therapeutics for diseases. A high‐throughput screen will be developed in which each of the human a isoforms will be expressed in a yeast strain lacking the endogenous a subunit genes. These yeast strains will be grown in the presence of compounds from small molecule libraries. Isoform‐specific inhibitors will be identified by their ability to selectively block growth at neutral pH in yeast strains expressing the appropriate a isoforms. Thus far our experiments indicate that expression of the human a subunit alone does not complement the loss of the yeast a subunits. Thus, to develop the inhibitor screen we must determine the minimal number of mammalian V0 subunits necessary to have a functional V‐ATPase containing the human a subunit.
One‐Dimensional Inter‐ligand Nuclear Overhauser Effect – fragment based drug discovery targeting M.tuberculosis KasA
Authors: Kapilashrami Kanishk , Machutta Carl , Picart Francis , Bommineni Gopal Reddy , Tonge Peter
Published: June 2021

β‐keto‐acyl synthase A (KasA) catalyzes the decarboxylative Claisen condensation of Malonyl AcpM with the growing acyl chain in the Fatty acid biosynthesis pathway (FAS II) in Mycobacterium tuberculosis. Thiolactomycin (TLM), a natural broad spectrum antibiotic, is a competitive inhibitor of KasA, and binds to the Malonyl AcpM binding site. However, TLM has a poor Kd with KasA because of lack of appropriate contacts. This study is based on the hypothesis that the pathethine chain of AcpM should be bound spatially adjacent to TLM in the active site. We use 2D NOESY‐NMR and subsequently an enhanced 1D version of the same technique to understand the relative orientation of the two ligands in the bound state. This version uses pulse field gradients and “excitation sculpting” to attain a higher sensitivity and cleaner, easier to interpret spectra. Understanding the relative orientation helped us design molecules with potentially higher affinity to KasA.
Endothelial Nitric Oxide Synthase Gene Polymorphisms (894G> T and −786T> C) and Risk of Coronary Artery Disease
Authors: Alokail Majed Saleh , Alkharfy Khalid M. , Al‐Daghri Nasser M. , Al‐Attas Omar S. , Draz Hossam M. , Hussain Tajamul S. , Sabico Shaun L. , Bamakhramah Ahmed A.
Published: June 2021

Endothelial nitric oxide synthase gene polymorphisms, either independently or through gene environmental interactions, are associated with cardiovascular diseases in multiple ethnic populations. However, no information is available with regard to such associations in Saudi population despite a high incidence of cardiovascular abnormalities. Here we studied the associations of 894G> T and −786T> C polymorphisms of endothelial nitric oxide synthase gene with coronary artery disease in Saudi population. Variants 894G> T and −786T> C were studied in 142 coronary artery disease patients and 145 normal controls by PCR‐ restriction fragment length polymorphism analysis and allele specific PCR respectively. Carriers of GT and TT genotypes of 894G> T polymorphism were significantly high (p< 0.0001) in patients (47.2%, and 7%, respectively), than in controls (27.6% and 4.8%, respectively). Likewise, carriers for TC and CC genotypes of −786T> C polymorphism were significantly high (p< 0.001) in patients (50% and 32%, respectively) than in controls (34.5% and 22.5%, respectively). Genotype distributions of 894G> T and −786T> C polymorphisms in the disease and control populations matched with those found in Caucasian populations. This study, for the first time, suggests an independent association of 894G> T and −786T> C polymorphisms of endothelial nitric oxide synthase gene with coronary artery disease in Saudi population.
Expression and purification of active human recombinant cathepsin K in E. coli
Authors: Tha Soe , McConnell Matthew , Berryman Laura , Chmielowski Jennifer , Bordowitz Michael , Liang Kenneth , Lafollette Jessica , Wen Lisa , McConnell Rose
Published: June 2021

Cathepsin K, a lysosomal cysteine protease, is abundantly expressed in osteoclasts and involved in bone remodeling and resorption. It has been reported that inhibitors of cathepsin K show great potential in the treatment of osteoporosis. The objective of this research is to prepare functional recombinant cathepsin K enzyme which will be used in the development of new cathepsin K inhibitors. Human procathepsin K cDNA has been amplified by PCR and successfully subcloned into pET15b expression vector. Overexpression of the recombinant His‐tagged fusion protein in BL21(DE3)pLysS host cells was found mainly in inclusion bodies. The inclusion bodies were solubilized by 6 M guanidine hydrochloride and the recombinant procathepsin K protein purified using Ni‐NTA affinity column. The purified protein was refolded by dilution followed by dialysis. The procathepsin K protein has been autoactivated and its proteolytic activity assayed by monitoring the increase in absorbance at 405 nm due to hydrolysis of chromogenic substrate, Z‐Phe‐Arg‐pNA. The availability of the functional cathepsin K enzyme will permit a drug discovery program for development of newer effective therapy for the treatment of osteoporosis in the near future.

Supported by National Cancer Institute at NIH (Grant No. 3R15CA086933‐04 and 3R15CA086933‐04A2S1)
An unopened knot protein: YbeA
Authors: Chang Yu‐Juan , Yeh Yi‐Chen , Ho Yen‐Peng , Chang Chia‐Ching
Published: June 2021

YbeA is one of the methyltransferase which contains deep embedded trefoil knot in its backbone structure. It is challenge to observe the knot transitions during the protein folding process. In this study, the conformation rigidity of YbeA folding intermediates were analyzed by trypsin digested matrix‐assisted laser desorption/ionization time of flight (MALDI‐TOF) mass spectrometry technique. We find that the knotted domain is the most stable region in the entirety structure of YbeA, both in unfolded and folded state. By using fluorescence resonance energy transfer (FRET) measurement, we find that the knot structure remains intact both in unfolded and refolded states. Therefore, we summarized that knot conformation is formed during its translational process and this unique structure becomes an unopened knot. This unique knot structure may facilitate cell to against stress of environment change.
Expression and purification of the Trypanosoma brucei VDAC in Escherichia coli
Authors: Lawrence Stacey Alicia , Sharma Shvetank , Singha Ujjal K , Duncan Melanie , Hamilton VaNae N , Chaudhuri Minu
Published: June 2021

The Voltage Dependent Anion‐Selective Channel (VDAC), a transmembrane â‐barrel protein on the mitochondrial outer membrane, is highly conserved among all eukaryotes. VDAC is the most abundant protein on the outer membrane and is responsible for regulating the transport of metabolites that are necessary for respiration and energy production by mitochondria. Trypanosoma brucei is a hemoflagellated protozoan, which is the causative agent of African Trypanosomiasis. T. brucei belongs to the group of earliest eukaryotes and possesses VDAC, which is similar in structure with bacterial outer membrane porin Omp85. The objective of this study is to determine whether the recombinant T. brucei VDAC (rTbVDAC) can function as a selective anion channel when reconstituted in an artificial membrane. To obtain the purified recombinant protein, we cloned the ORF for TbVDAC in a bacterial expression vector, pQE30. The protein was expressed in E. coli after induction with IPTG (1 mM). The expression conditions were optimized for maximum production. The rTbVDAC was solubilized under native conditions and subjected to purification by Nickel‐agarose chromatography. Currently, we are optimizing the purification process to obtain a sufficient quantity of purified protein that will be used for subsequent electrophysiological studies to determine to what extent this recombinant protein can function as VDAC.
Genetic Polymorphism of lipoprotein lipase, Beta 3 Adrenergic Receptor and Plasma Cell Membrane Glycoprotein and The Susceptibility to Dyslipidemia
Authors: El Din Hemimi Neveen Salah , Tawfik Lamis M , Elhannan Sahar Elsaid
Published: June 2021

Background

Dyslipidemia is one of the most common disorders in the modern society to the extent that it is the major determinant of the degree of excessive morbidity and mortality.

Aim

Identification of subject at risk of dyslipidemia in order to carry more effective interventions to prevent the development or progression of the associated complication such as cardiovascular diseases.

Material and Methods

The study group consisted of 202 individual with different lipid profiles. Individuals were genotyped for LPL Hind III, LPL S447X, β 3 ADR and PC1 by using PCR‐RFLP. Statistical analysis was done by using SPSS program.

Results

Carriers of H+/H+ genotype were at significant high risk of developing dyslipidemia manifested by high triglycerides, high LDL and low HDL (OR=1.9, 2.3 and 2.0) respectively. SS genotype was associated with significant increase risk of total cholesterol (OR= 2.7). C allele of β 3 ADR was a risk factor for low level HDL (OR= 2.3). Carriers of Q allele of PC1 were at significant risk of developing atherogenic phenotype manifested by low level HDL and high level LDL (OR=2.4 and 5.1) respectively. Furthermore combined analysis revealed that carrier of two or more of the previous haplotypes were at the highest risk of developing dyslipidemia.

Conclusion

H+, S, C and Q alleles were risk factors of developing atherogenic lipid profile.

Source of fund: Ain Shams University.
Hepatic Adipose Triglyceride Lipase (ATGL) mediates hepatic triglyceride turnover, fatty acid channeling and PPAR‐alpha activity
Authors: Ong Kuok Teong , Mashek Mara , Mashek Douglas
Published: June 2021

Adipose triglyceride lipase (ATGL) catalyzes the first step in triglyceride (TAG) hydrolysis. Because most studies have focused on ATGL in adipose tissue, its role and function in other tissues is relatively unknown. To elucidate the metabolic functions of hepatic ATGL, we employed gain and loss‐of‐function studies in primary hepatocyte cultures and adenovirus‐mediated knockdown in vivo. ATGL overexpression in primary hepatocyte cultures exhibited resistance to fatty acid‐induced steatosis, increased catabolism of hydrolyzed fatty acids and increased PPAR‐α activity. Treament of hepatocytes with siRNA against ATGL resulted in decreased rates of TAG hydrolysis and increased TAG accumulation along with suppressed fatty acid oxidation and PPAR‐α activity. However, the large changes in TAG turnover and fatty acid oxidation did not affect media TAG suggesting that rates of VLDL synthesis were unaltered. Interestingly, ATGL shRNA‐treated mice exhibited 150% more hepatic TAG and increased liver weight. Serum TAG levels of the mice did not change in response to ATGL knockdown while the expression of PPAR‐α and its target genes decreased, thus supporting the previous data from in vitro studies. Taken together, these data show that ATGL is a major TAG lipase in the liver that acts, perhaps in part through PPAR‐α, to channel hydrolyzed fatty acids towards oxidative pathways as opposed to VLDL secretory pathways.
Identification of cyclin‐dependent kinase phosphorylation sites in yeast phosphatidate phosphatase
Authors: Choi Hyeon‐Son , Morgan Jeanelle M , Su Wen‐Min , Han Gil‐Soo , Carman George M
Published: June 2021

The PAH1‐encoded phosphatidate (PA) phosphatase (PAP) plays a major role in controlling the cellular content of PA in the yeast Saccharomyces cerevisiae. The enzyme is phosphorylated in vivo on multiple residues, and one of the protein kinases involved is cyclin‐dependent kinase (CDK). In vitro, CDK phosphorylates purified recombinant PAP on both serine and threonine residues. An analysis of purified truncated forms of PAP revealed that the sites of CDK phosphorylation were at the C‐terminal end of the enzyme. A site‐directed mutagenesis study showed that Ser602 and Thr723 were the major sites of phosphorylation. In vitro, the S723A mutation caused a 50 % reduction in the CDK phosphorylation of PAP, whereas the S602A mutation caused the complete loss of the CDK phosphorylation of the enzyme. This indicated that phosphorylation of Ser602 was required for the phosphorylation of Thr723. Yeast strains that express S602A, T723A, and S602A T723A mutant enzymes have been constructed to examine the effects of CDK phosphorylation on PAP function in lipid metabolism. Supported by NIH grant GM‐50679.
Hypertonicity induces cytidylyltransferaseα redistribution in nucleoplasmic lamin A/C speckles in MDCK cells
Authors: Favale Nicolás Octavio , Sterin‐Speziale Norma Beatriz
Published: June 2021

Papillary collecting ducts works in high renal interstitial osmolarity, and increase their phosphatidylcholine (PtdCho) synthesis that occurs by Kennedy pathway. CCTα is the regulatory enzyme which is an amphitropic enzyme, but in most cell lines it has been localized predominantly in the nucleus. We have demonstrated that hypertonicity induces lamin A/C distribution to nucleoplasm speckle. In the present work we examined the nuclear distribution of CCTα in MDCK cells. We evaluated the relationship between the lamin A/C speckles and intranuclear CCTα. Confocal microscopy showed that under hypertonic conditions CCTα colocalize with speckles and colocalization involves protein interaction. ClNa‐TritonX‐100 extraction and immunoprecipitation assays were performed and demonstrated that CCTα interacts with Lamin A/C. Silencing of Lamin A/C causes decreases in lamin A/C speckles number and CCTα redistribution to soluble nuclear matrix. Results herein demonstrate that hypertonicity induces CCTα redistribution from soluble nuclear matrix to nuclear speckles, and this distribution dependent on lamin A/C scaffold. We propose that under hypertonic condition CCTα interact with lamin A/C‐speckles thus establishing two different intranuclear enzyme pools. This work was supported by the University of Buenos Aires (UBACyT B076), CONICET (PIP 5557) and ANPCyT (PICT 33154) grants.
The role of proline in the membrane re‐entrant helix of caveolin‐1
Authors: Aoki Satoko , Thomas Annick , Decaffmeyer Marc , Brasseur Robert , Epand Richard M
Published: June 2021

Caveolin‐1 (Cav1) has a segment of hydrophobic amino acids comprising approximately residues 103 to 122. We have performed an in silico analysis of the conformational preference of this segment of Cav1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro110 residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the FLAG‐tagged P110A mutant of Cav1 in HEK 293 cells. We find that this mutation causes a large reduction of endocytosis of caveolae. We also evaluate the topology of the proteins with confocal immunofluorescence microscopy. The cells expressing Cav1 show that the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG‐tagged P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. Thus, the single Pro residue in the membrane‐inserting segment of Cav1 plays an important role in both the membrane topology of the protein as well as its function in the endocytosis of caveolin. (Supported by Canadian Post‐Doctoral Research Fellowships 2008–09 and by the Natural Sciences and Engineering Research Council of Canada, grant 9848)
Exploring the Dynamics of the Proteasome Interaction Network by Quantitative Proteomics
Authors: Huang Lan
Published: June 2021

Most cellular processes in a cell are mediated by large protein assemblies or multi‐protein complexes, not by individual proteins. The protein complexes resemble molecular machines with highly ordered dynamic structures that assemble, store and transduce biological information. Systematic identification and characterization of these macromolecular machines will provide the essential knowledge base and set the stage for linking proteome dynamics and architecture to cellular functions. The 26S proteasome is a macromolecular machine existing in all eukaryotes and is responsible for ubiquitin/ATP dependent protein degradation in both cytosol and nucleus. It consists of two subcomplexes, the 20S core particle (CP) and the 19S regulatory complex (RP). The 20S CP is responsible for various catalytic activities, while the 19S RP is involved in several biochemical functions including recognition and unfolding of polyubiquitinated substrates, assisting in opening the gate of the 20S chamber and subsequently translocating unfolded substrates into the 20S for degradation. Selective degradation in cells is tightly controlled and plays an important role for regulating cell cycle progression, signal transduction and maintaining genome stability, etc. Disruption of normal proteolytic destruction pathways can lead to a wide range of human disease, including cancer and neurodegenerative disorders. Despite intensive research, the unknown still exceeds what are currently known on ubiquitin/proteasome dependent degradation. In order to understand the regulation of the 26S proteasome function, we have developed an integrated proteomic approaches to comprehensively characterize the 26S proteasome complexes. In addition, the QTAX strategy, quantitative identification of tandem‐affinity purified cross‐linked (x) protein complexes was developed to capture protein interaction of all natures in living cells, which has been successfully applied to map the dynamic interaction network of the 26S proteasome under various physiological conditions. Coupled with protein interaction network analysis, validity of the identified proteasome interactions have been confirmed and the biological significance of the identified interactions has been suggested. The methodologies presented here can be applied for the study of other protein complexes and their interaction networks.
The Role of Integrins in the Regulation of Vascular Hypertrophy in a Profilin 1 Transgenic Mouse Model
Authors: Abouelnaga Zeinb Ahmed , Hassona Mohamed Darwish Hessein , Abdulrahman Basant , Elnakish Mohammad , Hassanain Hamdy
Published: June 2021

Increased actin polymerization and stress fiber formation generate mechanical forces that play a role in the modulation of cellular morphology and function. Mechanical stress has a direct effect on the extracellular matrix (ECM) and integrins. To study the effect of stress fiber formation on integrin‐associated activation of hypertrophic signaling in vascular smooth muscle cells (VSMCs), we have overexpressed the human profilin 1 cDNA in vascular smooth muscle cells of FVB/N mice. Western blotting showed a significant increase in á1, â1 and á7 integrin subunits in the aorta of profilin 1 mice compared to control mice (278.9%, 354.7% and 265.7 % respectively, p<0.05). There was significant activation of phospho‐ERK1/2 and phospho‐JNK compared to controls (512% and 371% respectively, p<0.05). In addition, there is a significant increase in â‐pix and Rac 1 (356.4% and 430.6% respectively, p < 0.05). Our results additionally demonstrated an increase in superoxide production in the aortas of profilin1 mice (400%, p<0.05). In conclusion, increased stress fiber formation in VSMCs activates á1, â1 and á7 integrins that trigger the activation of hypertrophic signaling and modulate changes in oxidative stress.
Superoxide enhances cell proliferation in pulmonary artery adventitial fibroblasts from chronically hypoxic calves and augments expression of the redox‐sensitive transcription factor, Egr‐1
Authors: Martinez Maylyn , Hartney Tanya , Frid Maria , Stenmark Kurt , Nozik‐Grayck Eva
Published: June 2021

Superoxide (O2−) has been implicated in chronic hypoxia‐induced pulmonary hypertension (CHPH) and pulmonary vascular remodeling. In a calf model of CHPH, the pulmonary artery adventitial fibroblast (PAAF) proliferates in vivo and in vitro and contributes to vascular remodeling. Hypoxia‐induced proliferation of PAAF is mediated in part by a redox‐sensitive transcription factor, early growth response‐1 (Egr‐1). We hypothesized that ROS are responsible for the proliferation observed in PAAF from the chronically hypoxic (HA) calf through Egr‐1. We cultured PAAF isolated from HA neonatal calves in the presence or absence of the antioxidant, superoxide dismutase (SOD, 200 U/ml) for up to 6 days and measured changes in cell counts by hemocytometer and MTT assay. We exposed cells to xanthine and xanthine oxidase (8 mU/mL) to generate O2−, and measured Egr‐1 mRNA expression by real‐time PCR. We found that SOD decreased proliferation in PAAF from HA calves. Treatment of HA PAAF with xanthine/xanthine oxidase increased Egr‐1 mRNA expression by 2 fold (p<0.05 by unpaired t‐test). These data provide evidence that O2− contributes to the proliferation of PAAF from HA calves and support the hypothesis that O2− may regulate cell proliferation by upregulating the transcription factor, Egr‐1.
Marinobufagenin is an upstream modulator of Gadd45a stress signaling in cytotrophoblast cells
Authors: Uddin Mohammad Nasir , Horvat Darijana , DeMorrow Sharon , Agunanne Enoch , Puschett Jules B
Published: June 2021

Objective

Marinobufagenin (MBG), a cardiotonic steroid, is increased in preeclampsia (PE). Gadd45a is involved in the pathophysiological changes observed in PE. Cytotrophoblast (CTB) cells play an important role in placental development.

Study aim

to elucidate the role of MBG in Gadd45a signaling in CTB cells.

Methods

CTB cells were treated with various concentrations of MBG (0, 0.1, 1, 10 or 100 nM). Gadd45a expression and p38 phosphorylation were measured by immunoblotting. The degree of cell cycle arrest was determined by FACS analysis. sFlt‐1 and 8‐isoprostane were measured by EIA. Gadd45a expression was inhibited in CTB cells via siRNA‐mediated knock down.

Results

Gadd45a expression and p38 phosphorylation were significantly upregulated in the 1, 10 and 100 nM MBG‐treated cells compared to vehicle‐treated cells. 0.1 nM MBG had no effect. There was a significantly higher percentage (80%) of cells in the G0/G1 phase in the cells treated with ≥1.0 nM MBG compared to basal and to 0.1 nM treated cells (55%). The application of MBG ≥1 nM significantly increased the secretion of sFlt‐1 and 8‐isoprostane. The RNAi‐mediated inhibition of Gadd45a attenuated MBG‐induced CTB cell stress signaling.

Conclusions

MBG caused upregulation of p38 phosphorylation and cell cycle arrest, and increased the secretion of sFlt‐1 and 8‐isoprostane. These alterations occurred as a result of Gadd45a stress signaling.
A Novel Mono‐carbonyl Analogue of Curcumin Induces Apoptosis by Activating ER Stress in Non‐Small Lung Cancer Cells
Authors: Xiao Jian , Liang Guang , Huang Yi , Li Xiaokun , Studer Elaine , Zhou Huiping
Published: June 2021

Non‐small lung cancer (NSCLC) is one of the most common cancers worldwide. Curcumin, a well‐known food additive and constituent of traditional medicine, has been used to treat several different cancers including NSCLC in clinical trials. However, its poor bioavailability and pharmacokinetic profiles due to its instability under physiological conditions have limited its application in anti‐cancer therapies. In this study, we synthesized a novel mono‐carbonyl analogue of curcumin, (1E,4E)‐1,5‐bis (2‐methoxyphenyl) penta‐1,4‐dien‐3‐one (B63), and examined its proapoptotic effect in NSCLC cells.

Methods

H460 or H358 cells were treated with different amounts of curcumin and B63 for various time periods. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry. The activation of ER stress was determined by Western blot analysis. The ER Ca2+ content was measured using the fluorescent Ca2+ indicator fura 2/AM. Lentiviral shRNA was used to knock down the ER stress responsive gene CHOP.

Results

B63 had better bioavailability and more potent proapoptotic activity than curcumin. B63, not curcumin, significantly induced depletion of ER Ca2+ store and activation of the unfolded protein response (UPR). Downregulation of CHOP expression inhibited B63‐induced apoptosis.

Conclusion

Activation of the UPR represents a key molecular mechanism underlying B63‐induced apoptosis in NSCLC cells.
p19INK4d function linked to DNA repair relies on sequential phosphorylation
Authors: Marazita Mariela C. , Ogara M. Florencia , Sonzogni Silvina , Pignataro Omar P. , Cánepa Eduardo T.
Published: June 2021

p19INK4d(p19), a member of INK4 cell cycle inhibitors, plays a role in DNA repair and becomes phosphorylated upon DNA damage. We aimed to evaluate first the involvement of predicted p19 phosphorylation sites in its DNA repair activity, second to demonstrate the phosphorylation event in those sites, and last to establish the subcellular localization of the phosphorylation process. p19 mutants were made replacing Ser or Thr by Ala at S13, S66, S76, T89, T141. Wi‐38 cells were transfected with the mutants and cell cycle arrest ability by 3H‐timidine incorporation, DNA repair capacity by UDS and apoptosis by caspase‐3 activity were assayed. Although none of the mutated sites are involved in cell cycle arrest capacity, S76 and T141 are necessary for p19 DNA repair function. Mutation of S76 and T141 to glutamic acid to mimic phosphorylation leads to the recovery of the function. To evaluate in vivo phosphorylation of these mutants we performed metabolic labeling with 32P‐orthophosphate. While S76A entirely supressed phosphorylation, T141A didn't completely abrogated. The mutant S76E retrieves the phosphorylation potential. p19 translocates into the nucleus after genotoxic treatment. We found that p19 phosphorylation begins in the citoplasm. These results show p19 is sequentially phosphorylated starting in the cytoplasm and being necessary for exerting an effective DNA repair function.

Research supported by ANPCyT.
Identification of regulators of the Wnt signaling pathway using a genetic screen in Drosophila melanogaster
Authors: Jack Jessica , Engelhardt Nicole , Kennell Jennifer A.
Published: June 2021

The Wnt signaling pathway is crucial because it controls the transcription of genes that are necessary for animal development; however, regulation of this pathway is not fully understood. Therefore, we conducted a genetic screen with the goal of identifying new genes that regulate the Wnt pathway in the fruit fly, Drosophila melanogaster. We generated transgenic fly lines to randomly mis‐express genes in the eye; these lines were then screened for their ability to negatively regulate the Wnt pathway. We initially found 78 potential Wnt regulators; however, a secondary screen was necessary because the primary screen potentially yielded negative regulators of either Wnt signaling or apoptosis. To specifically select for Wnt regulators, we crossed the potential candidates with lines that promoted apoptosis in the eyes. Wnt regulators were then identified by their inability to prevent cell death. We performed inverse PCR to determine the identity of mis‐expressed genes that tested positive as Wnt regulators in both screens. In the future, we plan to investigate the potential role of these genes in the regulation of Wnt signaling.
Groucho‐mediated long‐range transcriptional repression: roles for discontinuous spreading and an HDAC‐dependent increase in nucleosome density
Authors: Courey Albert J , Winkler Clint J. , Ponce Alberto , White Kevin , Negre Nicolas
Published: June 2021

The Drosophila Groucho (Gro) protein is the founding member of a family of metazoan transcriptional corepressors with diverse roles in development. Gro directs long‐range repression, which contrasts with the short‐range repression directed by the corepressor CtBP. However, the mechanistic difference between these two types of repression is not clear. To determine the structure of domains of Gro‐mediated repression, we examined the genome‐wide distribution of Gro‐binding sites in early embryos and wing discs by ChIP‐chip. Our findings suggest that communication between the sites to which repressors recruit Gro and promoters may involve a combination of linear Gro spreading and DNA looping. This, in turn, leads to an increase in nucleosome density. The increase in nucleosome density appears to require histone deacetylase activity, as the wing discs of flies raised in media containing HDAC inhibitors show dramatically increased levels of histone H3 and histone H4 acetylation coincident with reduced repression and decreased histone density at Gro target genes.
Epigenetic marks of Insulin‐related transcription factor network by DNA methylation
Authors: Ito Shuhei , Sato Shinya , Yoshioka Tetsuya , Ohgane Jun , Tanaka Satoshi , Yagi Shintaro , Shiota Kunio
Published: June 2021

Numerous data of transcription factors for Insulin gene expression has been accumulated and used to characterize the Insulin‐producing cells induced from pluripotent stem cells. DNA methylation is a pivotal mark of epigenetic regulation of genes. There are numerous tissue‐dependent differentially methylated regions (T‐DMRs). DNA methylation profiles, which are consisting of methylated and unmethylated T‐DMRs, are unique to the cell types. Here, we analyzed DNA methylation profile of insulinoma MIN6 cell line by genome‐wide DNA methylation analysis method for T‐DMR analysis (D‐REAM). D‐REAM analysis revealed that MIN6 cells have unique DNA methylation profile distinctive from that of insulin non‐expressing cells including embryonic stem cells. Several transcription factors which are involved in Insulin 2 gene expression had T‐DMRs uniquely hypomethylated in MIN6 cells. Interestingly, main part of the Insulin transcription factor network, which involves transcription factors regulating other transcription factors, are under the epigenetic regulation by DNA methylation. Thus, Insulin‐expressing cells had a distinctive DNA methylation profile from others. DNA methylation profile is powerful to characterize the cells for regenerative medicine and the T‐DMR information will provide critical information for evaluating the Insulin‐producing cells.

This work was supported by NEDO and NIBIO.
Ring finger protein 14 regulates beta‐catenin/TCF‐mediated transcription
Authors: Wu Beibei , Piloto Sarah , Schilling Thomas F , Waterman Marian L
Published: June 2021

Beta‐catenin‐dependent Wnt signaling controls many fundamental events during development and homeostasis. Beta‐catenin and transcription factors of the T cell factor/lymphoid enhancer factor (TCF/LEF) family interact with many protein factors and cooperatively regulate gene transcription. We have discovered ring finger protein 14 (RNF14) as a positive regulator of Wnt signaling in both mammalian cells and zebrafish. RNF14 interacts with TCF1 and can be recruited to response elements in Wnt target genes. We have also demonstrated that RNF14 is required for efficient activation of Wnt signaling and that it helps stabilize beta‐catenin recruitment to DNA. RNF14 has been reported as a co‐activator of androgen receptor (AR)‐mediated transcription; however we have found that RNF14 participates in the Wnt pathway independent of AR signaling. Thus our findings suggest a novel role of RNF14 in enhancing beta‐catenin/TCF‐mediated transcription.
ABL‐N induces caspase‐dependent apoptosis in human breast cancer cells via activation of c‐Jun‐NH2‐terminal kinase
Authors: Liu Bin , Han Mei , Wang Jun‐jie , Sun Rong‐hua , Wen Jin‐kun
Published: June 2021

Acetylbritannilactone (ABL), derived from Chinese herb Inula Britannica L, has shown promising abilities as a preventing agent against cancers but exhibits poor effects. Structural modification of ABL resulted in the identification of a derivative 5‐(5‐(ethylperoxy)pentan‐2‐yl)‐6‐methyl‐3‐methylene‐2‐oxo‐2,3,3 a,4,7,7a‐hexahydrobenzofuran‐4‐yl 2‐(6‐methoxynaphthalen‐2‐yl)propanoate (ABL‐N) that manifested improved efficacy and specificity against cancer cells. Here, we reported that ABL‐N induced the activation of caspase‐3 in MDA‐MB‐231 and MDA‐MB‐468 human breast cancer cell lines, as evidenced by the cleavage of endogenous substrate Poly (ADP‐ribose) polymerase (PARP). The activation of caspase‐3 preceded ABL‐N‐induced chromatin condensation and nucleosome fragmentation, the typical apoptotic morphologies. Pretreatment of cells with pan‐caspase inhibitor z‐VAD‐fmk or caspase‐3‐specific inhibitor z‐DEVD‐fmk inhibited ABL‐N‐induced apoptosis. Treatment of cells with ABL‐N also resulted in an increase in the expression of pro‐apoptotic members (Bax and Bad) with a concomitant decrease in Bcl‐2. Furthermore, c‐Jun‐NH2‐terminal kinase (JNK) and p38 MAP kinase (p38) were activated in the inductionof apoptosis and JNK‐specific inhibitor SP600125 attenuated ABL‐N‐mediated apoptosisand c‐jun phosphorylation. However, the p38‐specificinhibitor SB203580 had no effect upon these processes. Moreover, neither of the caspase inhibitors prevented ABL‐N‐induced JNK activation, while JNK specific inhibitor, at least partially inhibited ABL‐N‐induced apoptosis, indicating that JNK is upstream of caspases in ABL‐N‐initiated apoptosis. Taken together, this study showed for the first time that ABL‐N induced apoptosis in human breast cancer cell through caspase‐3 and JNK signaling pathway.
The effect of retinol supply over cytokines and blood chemistry in healthy adult subjects
Authors: Perez Valentina Guzman , Corredor Carlos , Monterrey Pedro , Cortes Yadira , Carreño Alejandra , Alvarez Lorena , Valencia Lina
Published: June 2021

A prospective and experimental assessment of vitamin A supplementation effect on the levels of cytokines, total cholesterol (CT), LDL cholesterol, triglycerides (TG) and glucose (G) in 59 healthy adult subjects was performed. A single vitamin A capsule per week (retinol palmitate: 15.152 retinol equivalents [ER]/capsule) was supplied to each individual during one month (4 capsules in total). Cytokines interleukin 2, 4, 6, 10, TNF‐α, IFN‐γ, CT, HDL, LDL, TG and G were evaluated in blood at 0 day (before supplementation) and 28 day (after supplementation). The subjects registered by themselves the food intake every day during the following (28 days). At 28 day no change was observed in cytokines levels, suggesting that cytokines are not affected by vitamin A supplementation. An increase in CT, LDL y TG: 3,39%; 6,78% y 6,78 % respectively was observed (p<0,005), the diet had no effect on this increase. In addition, there was a reduction of 10,17% in HDL levels. As a conclusion, vitamin A supplement had a positive effect on CT, LDL, TG and G levels.
Regulation of CMO1 and CMO2 expression during mouse embryonic development
Authors: Wassef Lesley , Kuhn Elizabeth , Kim Youn‐Kyung , Quadro Loredana
Published: June 2021

β‐carotene‐15, 15′‐oxygenase (CMO1) and β‐carotene‐9,10′‐oxygenase (CMO2) are the only enzymes known to generate vitamin A from β‐carotene. Both are expressed in mouse and human embryonic and extraembryonic tissues, suggesting a role for β‐carotene as a localized source of vitamin A for developing tissues. Studies indicate that β‐carotene cleavage activity is regulated by nutrient availability in adult tissues. Here we investigate whether nutrient availability may regulate the expression of CMO1 and CMO2 during mouse embryogenesis.

Wildtype and CMO1−/− pregnant females were administered β‐carotene by IP injection at 13.5 dpc and sacrificed one day later to collect maternal serum and liver, embryos, yolk sac and placenta. This procedure leads to detectable amounts of β‐carotene in these tissues. Real‐time PCR analysis demonstrated that, in maternal liver, CMO1 expression is down‐regulated while CMO2 is up‐regulated with increasing amounts of β‐carotene. Furthermore, HPLC analysis revealed that administration of β‐carotene also altered maternal serum retinol levels, suggesting the existence of highly regulated mechanisms to ensure that adequate amount of vitamin A are available to the embryo. Experiments are ongoing to analyze the changes in CMO1 and CMO2 expression in embryonic tissues in response to maternal β‐carotene availability and vitamin A status.

Grant Funding Source: NIH 5R01HD57493.
Moderate Energy Restriction Does Not Cause a Deterioration in the Iron Status Due to an Iron‐Deficient Diet, While Exercise Ameliorates It
Authors: Asai Tsuyoshi , Okumura Yuka , Kondo Emi , Matsuo Tatsuhiro , Okamura Koji
Published: June 2021

To evaluate the effect of energy restriction and exercise on the iron status in rats fed a moderately iron‐deficient diet, 30 male rats (4 weeks old) were given a moderately iron‐deficient diet (13mg Fe/kg diet) for 3 weeks. Half of the rats were fed ad libitum (A), while the other half were restricted to only 70% of the diet consumed by A (D). The rats of groups A and D were also divided into subgroups, a climbing exercise group (E) (5 min×6 sets/d, 3 d/w) and a sedentary group (S). The body weight and weights of the liver, spleen, heart, kidney, flexor hallucis longus (FHL) were less (P<0.05) in D than A, and only FHL was affected by exercise (E>S, P<0.05). The iron content (μ g/g) in the liver and kidney was higher in D than A (P<0.05), while the content in the spleen also tended to be higher in D than A, whereas the iron content in the whole tissue tended to be higher in the spleen in A than D, while the other tissues showed no differences. Exercise increased the iron content in the kidney whereas no effect of exercise was observed in the other tissues. The plasma iron concentration did not differ between A and D. Total iron binding capacity (TIBC) was lower (P<0.05) in D than A. Hemoglobin (Hb) was higher (P<0.05) and transferrin saturation (TfS) tended to be higher in D than A. Plasma iron, Hb and TfS were higher (P<0.05) and TIBC was lower (P<0.05) in E than S. We conclude that moderate energy restriction does not appear to deteriorate the iron status induced by an iron deficient diet, while exercise appear to ameliorate the iron status.

Grant Funding Source: Osaka Univ of Health and Sport sci
Using zebrafish as a model organism to understand the impact of maternal zinc status on embryonic zinc homeostasis during development
Authors: Ho Emily , Hardin Karin , Wong Carmen P , Hobson Brad , Traber Maret G , Tanguay Robert L
Published: June 2021

Zinc is a micronutrient important in several biological processes including growth and development. Previous studies suggest that zinc deficiency during pregnancy causes adverse effects in the offspring that can persist even in the presence of adequate postnatal zinc. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we developed a model using zebrafish to examine the impact of maternal zinc deficiency during development. A purified zebrafish diet was developed and tested for zinc deficiency. After 12wks, adult fish were spawned and the embryos obtained collected to examine the developmental consequences of zinc deficiency. In the zinc deficient fish, abnormalities such as pericardial edema, yolk sac edema, pigmentation, lower response to stimuli and deformed body axis were observed. We also saw decreased embryonic zinc levels and differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.