bluuebunny/crossref_metadata_2025 huggingface dataset:
The Amyloid Precursor Protein of Alzheimer's Disease Localizes to Plaques on the Surfaces of Axoplasmic Organelles
Authors: DeGiorgis Joseph Alan , Scollins Brianne J , Walsh Rylie B
Published: June 2021

Alzheimer disease afflicts an estimated 26 million individuals worldwide and its incidence is predicted to reach 100 million by 2050. This debilitating neurodegenerative disease is characterized by loss of long‐term memory, language degeneration, and cognitive impairment. Mutations in the amyloid precursor protein (APP) lead to heritable forms of this disorder, however, little is known about the wildtype function of APP or how mutations in APP lead to AD pathogenesis. Here, we set out to study the distribution of APP at the ultrastructural level in extruded axoplasm of the squid giant axon as well as in reconstituted axoplasmic organelle/microtubule complexes. By immunogold electron microscopy using a C‐terminal APP antibody, gold particles distribute to plaques on the surfaces of 100 nm vesicles in axoplasm. Many of these organelles are bound to microtubules and APP often localizes to the organelle/microtubule interface. In reconstituted organelle/microtubule complexes, APP remains integral to the organelle membrane. These organelles bind to the microtubules in the absence of ATP and we have previously shown that they have Kinesin‐3 on their surfaces. These vesicles also move to the plus ends of microtubules indicating that APP is a cargo or maybe directly involved in the transport process. This work was funded by the Rhode Island IDeA Network of Biomedical Research Excellence (INBRE).
Endogenous and Psychostimulant Substrates but not Blockers Stimulate Dopamine Transporter Phosphorylation at a Proline‐Directed Site
Authors: ChallaSivaKanaka Sathyavathi , Foster James D , Vaughan Roxanne A
Published: June 2021

The dopamine transporter (DAT) clears extraneuronal dopamine (DA) and thus controls the spatio‐temporal dynamics of dopaminergic neurotransmission. DAT is the major target for psychostimulants such as cocaine (COC) and amphetamine (AMPH). DAT has a serine cluster on the distal N‐terminus that is phosphorylated in a PKC‐dependent manner and a proline‐directed site, Thr (T) 53, that is phosphorylated in vitro by the MAP kinases. COC and AMPH impact DAT regulatory properties including uptake activity and surface expression. Although, the mechanism of drug action on DAT is unclear, DAT phosphorylation is associated with altered surface expression and DAT activity. In this study, we examined the effect of DA and several psychostimulant drugs on the phosphorylation of DAT using a newly developed phospho‐specific antibody that detects pT53. Immunoblotting revealed that DAT substrates led to increased DAT T53 phosphorylation. This increase was transport or binding dependent as COC blocked the AMPH effect. In rat striatal synaptosomes METH‐stimulated DAT T53 phosphorylation was rapid, occurring within 60 sec. In contrast, uptake blockers did not influence T53 phosphorylation. Because phosphorylation at proline‐directed sites causes cis‐isomerization of the protein backbone, our findings indicate that translocation or binding of substrates induces major structural rearrangements of the membrane‐proximal N‐terminus. We hypothesize that substrate‐mediated phosphorylation‐dependent structural rearrangement resulting from prolyl cis‐isomerization could thus act as a mechanism affecting DAT function.

Supported by NIDA R01 DA 13147‐09
Sperm motility characteristics are regulated by changes in carboxy methylation and tyrosine phosphorylation of protein phosphatase PP2A
Authors: Dudiki Tejasvi , Kadunganattil Suraj , Ramdas Shandilya , Vijayaraghavan Srinivasan
Published: June 2021

The enzymes PP2A and PP1γ2 are the two major serine/threonine phosphatases in mammalian spermatozoa. PP2A is implicated in diverse cellular functions. In this study, we confirm the presence of PP2A in spermatozoa by western blot and show that it is carboxy methylated (Lys 309) and reciprocally tyrosine phosphorylated (Tyr 307). Mammalian spermatozoa acquire motility and their ability to fertilize eggs during their transit through the epididymis. PP2A of immotile sperm from late caput region of the epididymis is methylated with negligible tyrosine phosphorylation whereas PP2A from motile sperm of cauda epididymis is demethylated and tyrosine phosphorylated. Protein phosphatase assay shows that demethylation lowers catalytic activity of PP2A in motile caudal sperm compared to immotile caput sperm. Demethylation of PP2A occurred in the presence of nanomolar levels of okadaic acid or with increased intrasperm S‐adenosyl homocysteine levels. Increased PP2A demethylation converts caudal sperm motility into patterns resembling hyperactivation. These treatments however have no effect on immotile caput sperm. The enzymes PME‐1 and PPMT1 responsible for demethylation and methylation of PP2A respectively, along with SAH hydrolase are present in sperm. Thus PP2A regulated by methylation plays a role in modulating sperm motility characteristics essential for fertilization. (NIH‐HD38520)
Development of biosensor for serine/threonine kinases
Authors: Lee Jhao-hong , Yuan Chiun-Jye
Published: June 2021

Protein serine/threonine kinase has several important functions in cells, including regulation of glycogen, sugar and lipid metabolism. Many human diseases are found to be associated with the dysregulation of various protein serine/threonine kinases. There are several disadvantages of conventional methods for the mearurement of the activity of serine/threonine kinase, such as requirement of specific instruments, expensive, labor intensive and high risk of radioactive contamination. In this study, an impedance‐based biosensor was proposed for the detection of protein serine/threonine kinases was proposed and developed. Accordingly, the PKA (Protein kinase A) activity could be detected by seting the voltage at non‐redox potentials and record the impedance of electrode surface under different frequency. The peptide substrates of PKA (Protein kinase A) were coated on the electrode surface (working electrode with a diameter of 0.4 μm). After PKA phosphorylation at specific amino acid residue(s) of the substrate, the net impedance difference between non‐phosphorylated and phosphorylated peptide was determined. The results obtained in this study suggest that the activity of protein serine/threonine kinase can be detected by the developed electrochemical biosensor.

Financial supported by NSC project NSC 100‐2311‐B‐009 ‐001
Trypanosoma cruzi regulates the extracellular matrix interactome network to promote cellular infection
Authors: Johnson Candice A , Nde Pius N , Cardenas Tatiana C , Pratap Siddharth , Lima Maria F , Villalta Fernando
Published: June 2021

The interactome networks of the extracellular matrix molecules operating in the early process of Trypanosoma cruzi infection of human cells are unknown. Here we show that T. cruzi regulates the laminin gama‐1 (LAMC1) and thrombospondin‐1 (THBS1) interactome networks in epithelial cells and that LAMC1 and THBS1 play fundamental roles in the initial infection process. Invasive T. cruzi up‐regulates the LAMC1 and THBS1 expression to rapidly recruit trypanosomes via galectin‐3 to the extracellular matrix facilitating cellular entry since silencing LAMC1 and THBS1 expression by stable RNAi dramatically reduces trypanosome recruitment and cellular entry. The global gene transcription regulated by T. cruzi, confirmed by RT‐PCR, functionally tested by RNAi, followed by gene and protein network construction elucidated the first critical extracellular matrix interactome sub‐networks of LAMC1 and THBS1 operating during the first step of T. cruzi infection. Thus, LAMC1 and THBS1 and their interactome sub‐networks play important roles in the pathogenesis of Chagas disease and may provide insights for the development of new strategies to block the first step of T. cruzi infection (supported by NIH grants AI AI080580 and AI007281)
Obligatory Structural Elements in Tobacco Etch Virus (TEV) RNA Required for Depurination by Pokeweed Antiviral Protein (PAP)
Authors: Domashevskiy Artem , Cheng Shu-Yuan
Published: June 2021

Pokeweed antiviral protein (PAP) from P. americana is a ribosome inactivating protein (RIP) and is an N‐glycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA, arresting protein synthesis at the translocation step. PAP is a potent antiviral agent against many plant, animal, and human viruses. PAP inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNA and depurinates them at multiple sites downstream of the cap structure (Hudak, K. A., et al. RNA (2002) 8: 1148–59). This mechanism does not clarify inhibitory effect of PAP on the replication of uncapped viruses, and thus the overall mechanism of PAP antiviral activity remains to be elucidated. In this work, fluorescence spectroscopy and HPLC techniques were used to quantitatively describe binding and enzymatic activities of PAP and its variants towards tobacco etch virus (TEV) RNAs, and thus understand the molecular basis behind PAP antiviral activity. The TEV RNA 5′‐leader is sufficient to confer cap‐independent translation, and contains an internal ribosome entry site (IRES) (Zeenko, V. and Gallie, D. R. J Biol Chem (2005) 280: 26813–24). We show that PAP possesses different binding and enzymatic domains within its structure, as well as that full 143‐nt TEV RNA leader sequence is required for PAP depurination of both m7GpppG‐capped and uncapped viral RNAs.
Functional validation of human pigmentation SNPs in zebrafish
Authors: Tsetskhladze Zurab , Canfield Victor A. , Copper Jean , Johnson Stephen L. , Kawakami Koichi , Cheng Keith C.
Published: June 2021

Pigmentation is a quantitative and polygenic trait with high diversity within and between human populations. Two coding single nucleotide polymorphisms (SNPs), A111T in SLC24A5 and L374F in SLC45A2 have been linked to normal pigmentation variation in European populations, and one SNP ‐ E272K in SLC45A2 has been potentially linked to the East Asian skin color. To directly test the effect of each of these polymorphisms on pigmentation, we used a direct functional assay based on mutant rescue in the zebrafish. For this assay, we first identified slc45a2 as the mutated gene in zebrafish albino mutants. Zebrafish mRNA containing mutant zebrafish allele did not rescue the mutant phenotype, while wild‐type slc45a2 mRNA did. In this study we describe how we were able to ascertain that A111T and L374F mutations appear to be hypomorphic mutations and that the E272K mutation is not. The method may also be applicable to the analysis of other polymorphisms linked to human phenotypes by genome‐wide association studies or DNA sequencing.

NIH funding: 5R01 AR052535
Isolation and Functional Studies of Eukaryotic Ribosomes using HaloTag Surface Display and Imaging Technology
Authors: Urh Marjeta , Daniels Danette , Mendez Jacqui
Published: June 2021

The understanding of ribosome complex assembly, structure, and function has exploded in recent years by achievements in the areas of single molecule studies, electron microscopy, and X‐ray crystallography. To facilitate further biochemical and cellular characterization, we have developed a protein capture and labeling technology, HaloTag, and applied it for functional studies of ribosomes, including efficient isolation and covalent attachment to surfaces as well as fluorescent labeling to visualize in vivo localization. Using mass spectrometry analysis we show successful capture intact 80S ribosomes directly from a mammalian cellular lysates. A notable advantage for analysis with mass spectrometry is the feature that HaloTag remains bound to the resin during the elution step. Therefore the sample is not plagued with the capture molecule, allowing for a more efficient analysis of the sample and detection of dilute and small proteins. Further experiments were done to show that the isolated ribosomes were bound to mRNAs and retained activity in vitro for elongation and complete protein synthesis. Correlative pulse‐chase cellular imaging experiments were done to follow old and newly assembled ribosomal populations after starvation. The multiple capabilities of this technology demonstrate the ability to fully characterize ribosomal assembly, interactions, function, and cellular localization.
iTRAQ based quantitative proteomics analysis reveals that G6PD deficiency affects aflatoxin B1 metabolsim in A549 cells
Authors: Lin Shin-Ru , Wu Chih-Ching , Wu Yi-Hsuan , Cheng Mei-Ling , Chiu Daniel Tsun-Yee
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD), a metabolic enzyme in the pentose phosphate pathway, plays major roles in the regeneration of NADPH and maintenance of cellular redox status. In the present studies, we explored the effects of G6PD knockdown on global proteome change. Toward this end, we used G6PD‐knockdown A549 cells as the cell model to investigate the effects of altered redox status on proteome change by applying iTRAQ‐based LC‐MS/MS. In the iTRAQ analysis, 19 dysregulated proteins (5 proteins down‐regulated; 14 proteins up‐regulated) involved in metabolic pathway, protein folding and cellular component movement were identified in G6PD‐knockdown A549 cells compared to control ones. The data of RT‐PCR reveals that 3 down‐regulated genes; 8 up‐regulated genes are dysregulated at transcriptional level. Among them epoxide hydrolase 1 (EPHX1) was down‐regulated in G6PD‐knockdown cells which have been reported to be involved in regulating aflatoxin B1 (AFB1) metabolism. Indeed, increased susceptibility toward AFB1‐induced cell death were observed to accompany with decreased induction of EPHX1 in G6PD‐knockdown A549 cells compared to control ones. Take together, these data implicate that imbalanced redox status plays a role in regulating cellular detoxification of AFB1.
Rab38, Varp and VAMP7 interactions define a biased trafficking pathway in lung alveolar type II cell
Authors: Zhang Linghui , DeBolt Kristine M , Fukuda Mitsunori , Fisher Aron B , Huang Shaohui
Published: June 2021

We have reported that Rab38, a rat Hermansky‐Pudlak Syndrome (HPS) protein, targets to ~30 % lamellar bodies (LBs) in alveolar type II (ATII) pneumocyte; importantly, Rab38 reexpression in cultured ATII cells isolated from the Rab38‐null Fawn‐hooded hypertension (FHH) rat lungs rescues the giant LB pathology of HPS (Zhang et al., 2011, AJP Lung Cell. Mol. Physiol.). In this study, we demonstrate a biased trafficking pathway mediated by Rab38 interaction with its downstream effecter Varp, which preferentially recruits VAMP7‐positive vesicles to Rab38‐labeled LBs. When expressed alone in cultured FHH ATII cells, mStrawberry (mStr) tagged Varp was predominantly found in cytosol. In contrast, when coexpressed with EGFP‐Rab38, mStr‐Varp was dramatically recruited to Rab38‐positive LB surfaces. EGFP‐VAMP7 labeled both LB limiting membranes and vesicular compartments in cultured ATII cells; coexpressing EGFP‐VAMP7 and str‐Varp similarly localized the latter molecule to VAMP7‐positive LB and vesicle surfaces. These results indicate that Varp can mediate vesicle tethering/fusion between VAMP7‐positive transporting vesicles and Rab38‐labeled LBs. Consistently, reexpressing Rab38 in FHH ATII cells preferentially recruits VAMP7‐positive vesicles to Rab38‐labeled LBs. Future studies will identify cargo(s) carried by the VAMP7‐positive vesicles, and demonstrate the vesicle tethering/fusion process occurring on LB surface in live ATII cells. Supported by NIH 5T32HL007748 and 5P01HL19737.
BIG2, an ARF guanine nucleotide‐exchange protein, regulates cell migration via effects integrin β1 cycling and actin cytoskeleton remodeling
Authors: LI CHUN-CHUN , Shen Xiaoyan , Aponte Angel , Shen Rong-Fong , Billings Eric M , Moss Joel , Vaughan Martha
Published: June 2021

Brefeldin A‐inhibited guanine nucleotide‐exchange protein (BIG)2 activates ADP‐ribosylation factors (ARFs), a family of ~20‐kDa GTPase proteins involved in regulation of vesicular trafficking, by accelerating the replacement of ARF‐bound GDP with GTP. Mechanisms of additional BIG2 function(s) are less clear. We identified an involvement of BIG2 in integrin β1 action during cell migration using small interfering RNA (siRNA) and Difference Gel Electrophoresis (DIGE) analysis. Amounts of Arp2, Arp3, and cofilin, proteins known to act downstream of integrin β1 in cell movement, were increased in the cytosol fraction after BIG2 depletion. Treatment of HeLa cells with BIG2 siRNA, but not control siRNAs, resulted in perinuclear accumulation of integrin β1 and delayed return of internalized integrin β1 to the cell surface. At the same time, motility of BIG2‐depleted cells was decreased, and formation of the actin‐based membrane protrusions at leading edges of migrating cells was blocked, as was recruitment of Arp2, Arp3, and cofilin to the leading edge. Taken together, these data reveal a novel mechanism through which BIG2 may serve to coordinate cytoskeleton and membrane traffic, in cell migration, via integrin β1 action.
Role of SLC10A1 SNPs in regulating cytochrome P450 expression
Authors: Varechtchouk Olga , Chaudry Amarjit , Schuetz Erin
Published: June 2021

Cytochrome's P450 (CYPs) are a group of enzymes that have a major role in metabolizing drugs, and their variable expression can lead to pharmacokinetic and pharmacodynamic differences within the population. Genetic variants in CYPs have been extensively investigated, but they only explain a part of the interindividual variation in hepatic CYP expression. A recent study has proposed SLC10A1, a bile acid uptake carrier, to be a novel regulator of CYP expression. Hence, we hypothesized that functional SNPs (single nucleotide polymorphisms) in SLC10A1 may have an effect on CYP expression. Towards this end we resequenced all exons, promoter, and 3′‐UTR of SLC10A1 in 98 human livers (43 over‐ and 55 under‐expressing) to look for functional SNPs. We found a total of eight SNPs. An exon 1 synonymous SNP (rs4646285) occurred with a higher frequency in livers overexpressing SLC10A1, whereas a 3′‐UTR SNP (rs45593332) occurred with a higher frequency in the livers underexpressing SLC10A1. The genotypes of rs4646285 correlated with total mRNA of SLC10A1 as well as the downstream target CYP3A4 (p=0.02). An allelic expression imbalance assay demonstrated samples having imbalanced SLC10A1 expression, although none of the SLC10A1 SNPs explained the cause of this imbalance. In conclusion, an exonic SLC10A1 SNP led to increased SLC10A1 mRNA expression and increased CYP3A4 expression and may help explain some of the variation in the pharmacokinetics of CYP3A4 substrate drugs. This work was funded by the Pediatric Oncology Education (POE) summer program at St. Jude Children's Hospital (O.V.).
Conditional Deletion of Cytochrome P450 Reductase in Mouse Bone Results in Antley‐Bixler Syndrome‐Like Craniofacial and Bone Mass Defects
Authors: Panda Satya Prakash , Guntur Anyonya R. , Kar Rekha , Tang Kejian , Masters Betties Sue
Published: June 2021

NADPH‐cytochrome P450 oxido‐reductase (CYPOR) is the obligate electron donor for cytochromes P450, dehydrocholesterol reductase, heme oxygenase, and squalene monooxygenase. Genetic screening of patients had revealed the presence of mutations in CYPOR leading to severe developmental defects including sexual ambiguities and various bone defects such as craniosynostosis and mid‐face hypoplasia, originally described by Antley and Bixler (1975). To probe the possible roles played by CYPOR during bone development, we have generated a conditional knockout (CKO) mice using cross‐bred Cprlox/lox and Dermo1 Cre mice. Since the Dermo1 Cre recombinase is active from 9.5 dpc, CYPOR is deleted in both chondrocytes and osteoblasts in the CKO mice. Here we report the first mouse model showing craniofacial abnormalities upon deletion of CYPOR in bone. Differential staining of CKO mice skulls show premature fusion of spheno‐occipital synchondroses and anterior intraoccipital synchondroses. Teeth malocclusion was noticed in the adult CKO mice. Micro CT analysis of the tibia revealed that deletion of CYPOR leads to a reduction in the bone mineral density compared in age‐ and sex‐matched littermate controls, with cortical bone thickness remaining unaffected. These results recapitulate the human CYPOR deficiency phenotype and offer an approach to study the phenotypic sequelae of CYPOR mutations. Supported by GM081568 to BSM
Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)
Authors: Polusani Srikanth Reddy , Kar Rekha , Riquelme Manuel , Masters Bettie Sue , Panda Satya Prakash
Published: June 2021

Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron‐transferring enzyme, which provides electrons to various redox partners, such as cytochromes P450, heme oxygenase, cytochrome b5 and squalene monooxygenase. Human patients with severe forms of a CYPOR mutation exhibit bone defects such as cranio‐ and humero‐radial synostoses and long bone fractures, identified as Antley‐Bixler‐like Syndrome. CYPOR knock‐down in multiple osteoblast cell lines decreased the expression of Connexin 43 (Cx43), which is known to play a critical role in bone formation, modeling, and remodeling as well as decreased Gap Junction Intercellular Communication and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock‐down cells was due to transcriptional repression. Primary osteoblasts isolated from calvariae from bone‐specific Por gene knock‐down mice recapitulated the findings in the cell lines. The possibility of up‐regulating Cx43 by vitamin D3 and retinoic acid, which are CYPOR metabolites is being pursued. Furthermore, the role of CYPOR in cell migration is being investigated as Cx43 is implicated in this process. Our study provides new insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR‐mediated bone defects. Support: GM081568 to BSM and. RK and MAR by EY012085 and AQ‐1507 to J X Jiang
Effect of propionylcarnitine on mitochondrial energy metabolism in elderly rat heart
Authors: Kerner Janos , Virmani Ashraf , Koverech Aleardo , Hoppel Charles
Published: June 2021

Background and objective

Propionyl‐L‐carnitine (PLC) has been shown to have an anaplerotic effect in rat heart. The goal of the present study was to test if PLC attenuates the age‐associated decreased myocardial carnitine content and improves mitochondrial oxidative phosphorylation.

Approach and methods

To achieve our objective we isolated subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) from hearts of 24 month old elderly Fisher 344 rats injected intraperitoneally with PLC or saline. We determined (a) the myocardial carnitine and acylcarnitine contents; (b) the oxidative capacity of isolated mitochondria with non‐lipid and lipid substrates; and (c) the activity of electron transport chain complexes.

Results

(a) Exogenous PLC is readily taken up and metabolized by the heart as indicated by the increased carnitine and acetylcarnitine content. (b) PLC has no effect on oxidative phosphorylation as assessed by unaltered oxygen consumption with isolated mitochondria. (c) PLC significantly increased the cytochrome oxidase (COX) activity in both mitochondrial populations as reflected by the increased first order rate constant.

Conclusion

The data demonstrate that exogenous PLC corrects the age‐associated loss of myocardial carnitine and selectively and significantly increases COX activity.

Supported in part by POI AG15885 and a gift from RostaQuo S.p.A..
Cholecalciferol increases 7‐dehydrocholesterol reductase activity in adult human epidermal keratinocytes
Authors: Zou Ling , Porter Todd D
Published: June 2021

7‐Dehydrocholesterol reductase (DHCR7) catalyzes the final step in cholesterol synthesis by reducing the 7–8 double bond of 7‐dehydrocholesterol. In skin cells 7‐dehydrocholesterol also can be converted to cholecalciferol (vitamin D3) by ultraviolet radiation, providing a second pathway for 7‐dehydrocholesterol metabolism in these cells. We hypothesized that keratinocytes might regulate cholecalciferol levels by modulating the activity of DHCR7, a hypothesis supported by evidence that DHCR7 is a phosphoprotein, and is activated by the addition of ATP to liver microsomes.

To test this hypothesis, DHCR7 activity was measured in lysates of rat hepatoma cells (McA‐RH7777), immortalized human keratinocytes (HaCaT), and adult human epidermal keratinocytes (HEKa) after treatment of the cells with cholecalciferol or calcitriol for 3 h. Cholecalciferol had no effect on DHCR7 activity in hepatoma cells or HaCaT cells, but increased DHCR7 activity in HEKa cells by 3‐fold at 1 μM and 4‐fold at 10 μM. Calcitriol (100 nM), the active form of vitamin D, had no effect on DHCR7 activity in HaCaT cells at 3 or 24 h, but decreased DHCR7 activity in HEKa cells after 3 h by 40%.

These findings suggest that cholecalciferol regulates its own levels in keratinocytes by increasing the activity of DHCR7, thereby decreasing the levels of the cholecalciferol precursor, 7‐dehydrocholesterol.
Characterization of a lysophospholipid acyltransferase in Candida albicans
Authors: Oelkers Peter M. , Ayyash Mariam , Alghami Amal , Haidar - Ahmad Ibrahim , Khadr Lara
Published: June 2021

We have begun to characterize the role of the lysophospholipid acyltransferase, 1881, in Candida albicans. 1881 is homologous to the LPT1 gene in S. cerevisiae. A cDNA copy of the 1881 gene has been generated and the four CUG codons changed to UCG so to allow maintenance of the primary structure upon expression and translation in a lpt1 S. cerevisiae strain. In parallel, a homozygous, C. albicans strain has been generated which has both 1881 alleles removed by homologous recombination. This strategy of over and abrogated expression will allow us to address the question, what is the substrate specificity of 1881 with respect to degree of unsaturation of acyl‐CoA species? We will also address the questions, does removal of 1881 from the genome affect C. albicans a) virulence in C. elegans or CaCo2 cell based assays? b) membrane composition? c) hyphal growth? d) sensitivity to azoles and other antifungals? and e) viability in hypoxia?
Type II pneumocyte lamellar bodies contain components of the Niemann‐Pick type C (NPC) pathway
Authors: Roszell Blair R. , Tao Jian-Qin , Yu Kevin J. , Huang Shaohui , Bates Sandra R.
Published: June 2021

Type II cell lamellar bodies, the primary storage organelle for lung surfactant, contain about 10% cholesterol. Cellular cholesterol trafficking has been shown to be regulated by the NPC pathway. A form of alveolar proteinosis characterized by high cholesterol to phospholipid ratio has been reported in an NPC2 patient, which suggested the NPC pathway played a role in regulating surfactant cholesterol in type II cells. Our objective was to define the localization and function of components of the NPC pathway in alveolar type II cells. Immunocytochemical analysis for NPC1, NPC2, and lysosomal acid lipase (LAL) was performed in isolated primary rat type II cells. The ATP‐binding cassette transporter (ABCA3) was used to identify the limiting membrane of lamellar bodies. 3D projections of a single lamellar body acquired by confocal microscopy confirmed NPC1 expression along the entirety of the limiting membrane. NPC1 was localized in alternating adjacent domains with ABCA3. NPC2 and LAL were located within the lumen of the organelle. Upon pharmacological inhibition of the NPC pathway with U18666A compound, cholesterol was found to accumulate within lamellar bodies. These results suggest that the NPC pathway is important for regulating lamellar body cholesterol. [HL‐19737 and NRSA T32‐HL‐07748‐17]
Comparative Lipidomic Analysis of the Symbiosis Between Aiptasia pallida and Symbiodinium
Authors: Schmeitzel John L , Klein Joshua , Smith Marc , Schwarz Jodi , Garrett Teresa A
Published: June 2021

Our research focuses on identifying lipids involved in the symbiosis between the anemone Aiptasia pallida and its photosynthetic dinoflagellate Symbiodinium. Liquid chromatography quadrupole time‐of‐flight mass spectrometry (MS) was used to analyze lipid extracts of aposymbiotic and holosymbiotic A. pallida and Symbiodinium. By comparing the intensity of each lipid (as defined by RT and m/z) between aposymbionts and holosymbionts, we observed lipidomic changes that occur when A. pallida and Symbiodinium enter symbiosis. The lipidome of A. pallida displays numerous changes as a result of symbiosis formation; [M‐H] − ions at m/z 765.4, 1033.5, and 1061.6 displayed >900‐fold increase in intensity. To begin to characterize these lipids, we partially purified via normal phase HPLC the lipid at m/z 765.4. Using collision induced dissociation MS and chemical modifications of functional groups we hypothesize that the lipid at m/z 765.4 is a diacyl‐glycerosulfolipid, with a head group attached to the sulfate with the formula C6H9O4. This work will lay the foundation for biochemical classification of all of the lipids in this systeme, and determination of what role they play in the symbiosis. This work was funded by a Cottrell College Science Award to J. Schwarz and E. Eberhardt and an ASBMB Undergraduate Affiliate Network Summer Research Award to J. Schmeitzel.
Transcriptome Analysis of Chlorella protothecoides to Identify Novel Pro‐Lipid Genes for Biofuel Production
Authors: Davidson Janeth O , Overton Jackson , Waikel Rebekah
Published: June 2021

Efforts to improve the efficacy of algae for biodiesel are centered on increasing synthesis of triacylgycerol (TAG), which can be chemically converted to a useable fuel source. Our approach to enhance TAG production utilizes environmental manipulation of algae to increase the expression of pro‐lipid genes. To identify novel pro‐lipid genes, we employed comparative transcriptome analysis. A cost‐benefit analysis of lipid production in several algal strains under typical lipid producing conditions (low nitrogen) led to the selection of Chlorella protothecoides. The genome of Chlorella protothecoides, like many others, has not yet been sequenced. Therefore, next generation sequencing was employed to obtain a transcriptome assembly and a differential transcriptome analysis of algae grown in growth conditions (non‐lipid producing) compared to algae grown in low nitrogen conditions (lipid producing). Differential transcriptome analysis yielded both homologs to known and novel genes up‐regulated during lipid production. The assembled mRNA sequences for pro‐lipid genes were used to design primer sets for TaqMan PCR, which was then used to confirm preferential expression of these genes in lipid producing conditions. Expression analysis of pro‐lipid genes, which were confirmed via relative RT‐PCR, is currently being used as a rapid screening panel to determine optimal lipid producing conditions (nutrients, light, pH). It is anticipated that these findings will aid in the large‐scale production of algae derived biofuels.
Phosphorylation of phosphatidate phosphatase on Ser10 by protein kinase A regulates triacylglycerol synthesis in Saccharomyces cerevisiae
Authors: Su Wen-Min , Casciano Jessica , Carman George M
Published: June 2021

The Saccharomyces cerevisiae PAH1‐encoded phosphatidate (PA) phosphatase catalyzes the penultimate step in the synthesis of triacylglycerol (TAG). It also provides the diacylglycerol used for the synthesis of phospholipids via the Kennedy pathway. PA phosphatase was a substrate for protein kinase A (PKA). Phosphorylation inhibited PA phosphatase activity with a 43% decrease in the specificity constant (kcat/Km) for the enzyme. Site direct mutagenesis followed by phosphopeptide mapping analyses indicated Ser10, Ser677, Ser773, Ser774, and Ser788 were the target sites for PKA phosphorylation. Of the five sites, the phosphorylation Ser10 had the greatest effect on PA phosphatase regulation. The phosphorylation‐deficient mutant S10A, which exhibited a 50% decrease in phosphorylation, abolished the phosphorylation‐mediated inhibition of PA phosphatase activity. Yeast cells expressing the phosphorylation‐mimic S10D mutation exhibited a 37% decrease in TAG, whereas cells expressing the phosphorylation‐deficient S10A mutation exhibited a 38% increase in TAG. The alterations in TAG synthesis were accompanied by corresponding changes in the synthesis of phospholipids. Supported by NIH grant GM 50679.
Alteration of sphingolipid metabolism during 4‐HPR induced cell death in the ARPE‐19 human retinal pigment epithelial cell line
Authors: Duncan Todd , Samuel William , Kutty R. Krishnan , Redmond T. Michael
Published: June 2021

A close association between the production of ceramide and the onset of programmed cell death has been well established. Ceramide generation can occur by de novo synthesis, sphingomyelin hydrolysis, or via acylation of sphingosine (salvage pathway). Ceramide acts as a second messenger in activating the apoptotic cascade. N‐(4‐hydroxyphenyl)‐retinamide (4‐HPR), a retinoic acid derivative, is known to induce cell death in many cell types including the human retinal pigment epithelial cell line ARPE‐19. Although the end result, cell death, is well established, the cellular and molecular mechanism(s) by which 4‐HPR causes cell death in ARPE‐19 are not well defined. Therefore, we investigated whether 4‐HPR can induce the production of ceramide in ARPE‐19 cells, which in turn can lead to apoptosis. Our results show, that 4‐HPR treatment of ARPE‐19 cells for 24‐h lead to the accumulation of dihydroceramide, as well as bioactive sphinganine (10‐ and 20‐fold increase, respectively). However, ceramide levels in 4‐HPR treated ARPE‐19 cells were similar to controls. It is possible that this alteration in sphingolipid metabolism, that is accumulation of sphinganine and dihydroceramide, caused by 4‐HPR contributes to cell death in ARPE‐19 cells. Support: Intramural Research Program of the National Eye Institute, National Institutes of Health.
Differential gene expression in capillary endothelial cells after inducing unfolded protein response with a protein N‐glycosylation inhibitor
Authors: Baksi Krishna , Banerjee Aditi , Banerjee Dipak K
Published: June 2021

Angiogenesis is a hallmark of breast tumor growth and progression. Our objective has been to understand the dynamic relationship between asparagine‐linked (N‐linked) protein glycosylation and angiogenesis. We have observed cell cycle arrest in G1 and induction of apoptosis by ER stress‐mediated unfolded protein response following transcriptional and translational attenuation in tunicamycin treated capillary endothelial cells. The effect is irreversible under tumor microenvironment. cDNA microarray analysis identified 133 and 464 genes sharing only 10 genes in 3h and 32h tunicamycin treated cells, respectively. 72.2% and 77.6% of these are upregulated whereas 27.8% and 22.41% are down regulated. Among these 45 genes encode for transcription (75% and 20% of which are upregulated) whereas 15 genes encode for cell cycle (40% and 20% of which are upregulated) at 3h and 32h, respectively. Only 10 genes encode for translation, 80% of which are upregulated at 32h. Interestingly, only one gene is upregulated at 32h for protein folding, DNA inhibition and angiogenesis. Real‐time PCR validated and confirmed the expression profile of a number of these genes. To our knowledge, it is the first detailed study assessing the significance of a genome‐wide expression profiling for a glycotherapeutic treating breast cancer. Supported by NIH/NCRR/RCMI G12‐RR03035 (KB) and Susan G. Komen for the Cure BCTR056206 (DKB) grants.
Towards a Well Defined Meningitis Vaccine: Chemoenzymatic Synthesis of Meningococcal Glycoconjugate Vaccine Candidates
Authors: McCarthy Pumtiwitt , Saksena Rina , Peterson Dwight , Lee Che-Hung , An Yanming , Vionnet Justine , Cipollo John , Vann Willie
Published: June 2021

Immunization efforts against meningitis have been effective at reducing incidence of the disease worldwide. Glycoconjugate vaccines are the most commonly used form of meningococcal vaccine. They are composed of capsular polysaccharides attached to a carrier protein. The chemical methods used to make these vaccines leads to heterogeneous mixtures. Well defined vaccines are needed to elucidate the relationship between vaccine structure and generated immune response. In this work we describe our efforts to produce these types of structures using chemoenzymatic synthesis. Chemically synthesized lactosides, containing azido groups at the aglycon were used as substrates for recombinant sialyltransferase enzymes from Camplyobacter jejuni and Neisseria meningitidis serogroup C. Optimization of this method led to glycans of varying chain lengths. These azido‐containing oligosialic acids, which mimic polysaccharides found in the capsule of Neisseria, were then conjugated to alkyne‐modified Hc fragment of the tetanus toxoid protein using the “click” chemistry method of 1,3‐cycloaddition. The resulting glycoconjugates were shown to be more immunogenic in mice than unconjugated oligosialic acids.
Development of a small anti‐cancer molecule targeting both the intrinsic and extrinsic pathways of apoptosis
Authors: Titorenko Vladimir , Goldberg Alexander , Beach Adam
Published: June 2021

Our chemical genetic screen for small molecules that increase lifespan in yeast cellular models of aging by targeting mitochondria‐controlled apoptosis identified lithocholic acid (LCA) as one of such molecules. Aging is one of the major risk factors of cancer, an age‐related disease whose onset can be delayed and incidence reduced by certain pharmacological antiaging interventions. We therefore sought to examine if LCA exhibits an anti‐tumor effect in cultured human cancer cells by activating certain anti‐cancer processes that may play an essential role in cellular aging. We found that LCA, at concentrations that are not cytotoxic to primary cultures of human neurons, kills the neuroblastoma (NB) cell lines BE(2)‐m17, SK‐n‐SH, SK‐n‐ MCIXC and Lan‐1. In the case of BE(2)‐m17, SK‐n‐SH and SK‐n‐ MCIXC cells, the LCA anti‐tumor effect is due to apoptotic cell death. In contrast, the LCA‐triggered death of Lan‐1 cells is not caused by apoptosis. Our findings imply that LCA kills BE(2)‐m17 and SK‐n‐MCIXC cell lines by triggering not only the intrinsic (mitochondrial) apoptotic cell death pathway driven by mitochondrial outer membrane permeabilization and initiator caspase‐9 activation, but also the extrinsic (death receptor) pathway of apoptosis involving activation of the initiator caspase‐ 8. Our data suggest a molecular mechanism underlying a potent and selective anti‐tumor effect of LCA in cultured human NB cells. Importantly, we found that a similar mechanism underlies a broad anti‐tumor effect of LCA in cultured mammalian cancer cells derived from different tissues and organisms. Supported by NSERC of Canada.
Effect of xanthopterin and isoxanthopterin on nitric oxide production by a RAW264.7 cell line
Authors: Metzger Robert P , Abadi Parisa , Mascuch Samantha , Gerwick Lena , de Peyster Ann
Published: June 2021

The pteridines xanthopterin (2‐amino‐4,6(3H,5H)‐pteridinedione) and isoxanthopterin (2‐amino‐4,7(3H,8H)‐pteridinedione) are products of the degradation of tetrahydrobiopterin, a coenzyme used by aryl amino acid hydroxylases, glyceryl ether monooxidase and nitric oxide synthases. These pteridines have cytotoxic effects on a number of cell lines including human mammary carcinoma MCF‐7 cell lines (Lord et al 2005, Cancer Lett. 222, 119). We wished to test the possibility that the pteridines could affect the activity of a nitric acid synthase. Accordingly, we performed experiments in which xanthopterin and isoxanthopterin were tested for their effect on nitric oxide (NO) production by a RAW264.7 mouse macrophage cell line with the resultant nitrite detected by the Griess assay. Both pteridines showed a dose‐dependent reduction of nitric oxide production with respect to controls. Whether this lowering of nitric oxide concentrations is due to inhibition of the inducible nitric oxide synthase will have to be examined. Furthermore, this finding may be significant since nitric oxide affects apoptosis (Brüne 2003, Cell Death and Differentiation 10, 864) and because of the well‐recognized association between chronic inflammation, cell proliferation, cancer and other diseases (Korhonen et al 2005 Current Drug Targets – Inflammation and Allergy 4, 471).
Cytotoxicity and active component in bitter melon (Momordica charantia) seed extracts
Authors: Chipps Elizabeth , Jayini Renuka , Malkawi Ahmed M , Mottaleb Mohammad Abdul , Islam Rafiq
Published: June 2021

Bitter melon (Momordica charantia) has been used as traditional medicine for centuries. In many cases, whole extracts were used and the active component(s) responsible for the given activity is unknown. In this study, HEK293T and HCT116 cells treated with bitter melon seed extracts (BMSE), especially water (BMSE‐W) and 50% ethanol extracts, showed marked cytotoxicity. The cytotoxic activity in BMSE‐W was gathered in the unbound fractions when applied to various matrices, unaffected by digestion with trypsin and proteinase K, and eluted in total volume in size‐exclusion HPLC, suggesting small, organic nature of the active component(s). GC‐MS analysis of the active HPLC fractions identified methoxy‐phenyl oxime (MPO) and 2‐hydroxy‐2‐methyl‐ 2‐cyclopenten‐1‐one (2‐HMCP) as major components. Acetophenone oxime (AO), a structural homolog of MPO, exhibited similar cytotoxicity, but not 2‐hydroxy‐3‐methyl‐2‐ cyclopenten‐1‐one (3‐HMCP), a structural homolog of 2‐HMCP. Further studies showed acetophenone and methyl benzoate had no effect, indicating the oxime (C=N‐OH) functional group is essential. Increased PARP and actin cleavage, and chromatin condensation was seen in BMSE‐W and AO treated cells suggesting apoptosis as a potential cause for the cytotoxicity. In summary, this report identifies an oxime in BMSE as a cytotoxic factor and potential anticancer agent. Supported by NIH.
AKIP1 regulation of PKA and NF‐κB in the heart
Authors: Sastri Mira , Chang Philip , Fridolfsson Heidi , Paneerselvam Mathivadhani , Ng Daniel , Taylor Susan , Patel Hemal
Published: June 2021

Ischemic preconditioning, an endogenous protective mechanism to salvage ischemic myocardium, was described nearly 25 years ago; however, there has been little to no clinical translation. Further, there has been a critical lack of knowledge to understand the molecular mechanisms that bring about the protection. Nuclear factor‐kappa B (NF‐κB) and protein kinase A (PKA) are key regulators of gene transcription in the heart and have contradictory roles in cell death and survival. Activation of NF‐κB and PKA has been shown to be protective as well as promote cardiac cell death in the setting of ischemia or oxidant stress. The answer as to why these two important regulators of gene transcription and cardiac physiology produce such a dichotomous response upon stress induction may provide insights into how protective signaling in the heart is integrated. We have discovered a novel scaffolding protein known as A‐kinase interacting protein 1 (AKIP1) that is expressed at low levels in the heart and is induced by stress. Our data shows that AKIP1 binds to and regulates nuclear localization of PKA catalytic subunit (PKAc) and increases nuclear PKAc activity. Further, AKIP1 interacts with and enhances NF‐κB nuclear localization in a PKAc dependent manner where disruption of AKIP1 binding to PKAc enhances nuclear NF‐κB. In cardiomyocytes and in the heart, oxidant/ischemic stress caused an increase of AKIP1 levels. Further, our data suggests that overexpression of AKIP1 was protective to the heart. Therefore, we infer that AKIP1 may be a key molecular regulator/scaffold that assembles PKAc and NF‐κB signaling complexes to alter the physiologic response of the heart in the basal and stressed states. Understanding the dynamics and physiologic implications of the interaction of PKAc and NF‐κB with AKIP1 may provide a novel therapeutic control point for limiting cardiac injury associated with ischemic stress.
Tissue transglutaminase response in myelogenous leukemia cells following the addition of 8‐bromoadenosine 3′,5′‐cyclic monophosphate
Authors: Miller Logan T. , Birckbichler Paul J.
Published: June 2021

In this study K562 cells were utilized to evaluate the morphological and also chemical changes preceding the introduction of various chemicals into the growth media. The K562 cells, a myelogenous leukemia cell line, are differentiation‐incompetent, programmed cell death‐competent cancer cells. The chemical change that was of interest was the expression of tissue transglutaminase (tTg); tTg covalently modifies proteins by the formation between an ε‐amino group of a lysine residue and a γ‐carboxamide group of a glutamine residue. The expression of tTg is a widely used marker for apoptosis. One chemical that was of particular importance was 8‐Bromoadenosine 3′, 5′‐cyclic monophosphate (8‐Br‐cAMP). The chemical induced morphological changes as determined by May Grunwald‐Giemsa stained cytospin preparations. The intracellular concentration of cAMP was evaluated using a microtiter plate assay. This study also analyzed the apoptotic response the K562 cells underwent. Apoptosis is a biochemical process that can occur in multi‐cellular organisms; it has characteristics which include blebbing, apoptotic bodies, and DNA fragmentation. Particularly, in relation to this study blebbing and DNA fragmentation were common occurrences with the K562 cells. tTg expression was compared to the formation of stable apoptotic bodies. Support for the project was provided by SRU as well as an RFP Student Research Grant.
Apoptotic signaling of the Amblyomin‐X involves endoplasmic reticulum stress, cell cycle regulation and survival pathways
Authors: Chudzinski-Tavassi Ana Marisa , Morais Kátia Luciano Pereira , Souza Jean Gabriel , Simons Simone Michaela , Berra Carolina Maria , Lameu Claudiana
Published: June 2021

The Amblyomin‐X, a Kunitz‐type inhibitor obtained in a recombinant protein form, promotes apoptosis in murine renal adenocarcinoma (RENCA), decreased proteasomal activity and increased pool of poly‐ubiquitinylated proteins in some tumor cell lines, suggesting an endoplasmic reticulum (ER)‐stress, in addition to D1 cyclin expression decrease. However, the mechanistic effects of this protein are still unclear.

Objective

Evaluate the involvement of ER‐stress, cell cycle regulation and/or survival in tumor cells treated or not with Amblyomin‐X.

Methods

The genic expression was perfomed by ABI 7500 Real Time PCR System (Life Technologies) using forward and reverse specific genes primers. The intracellular calcium mobilization was measured by confocal microscope and microfluorometry.

Results

RENCA cells treated with Amblyomin‐X showed significant differences in genes related to ER‐stress and cell cycle regulation, as observed for caspase‐12, which showed increased expression and for mTOR, which, in turn, showed reduction in its expression. Moreover, Amblyomin‐X did not show instantly and transiently intracellular calcium mobilization, but showed increase in this intracellular ion concentration after 5h of protein treatment.

Conclusion

The results suggest that ER‐stress, cell cycle regulation and survival pathway proteins are involved in this process pro‐apoptotic.

Supported by FAPESP