bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Dihydroquinonoid Formation in Substrate Specificity of Escherichia coli Dihydrofolate Synthetase‐ Folylpolyglutamate Synthetase.
Authors: Shearer Emily J , Stover Patrick J , Shane Barry
Published: June 2021

Escherichia coli dihydrofolate synthetase‐folylpolyglutamate synthetase (DHFS‐FPGS) (FolC) catalyzes the ATP‐dependent addition of glutamate to both dihydropteroate and pteroylglutamate substrates. The mechanism used by the enzyme to differentiate between folate and pteroate substrates was investigated using equilibrium binding and rapid‐reaction kinetic techniques, and structural modeling approaches. The absorbance spectrum of dihydropteroate displays a 40 nm red shift upon binding to DHFS‐FPGS· ATP, and the bound dihydropteroate exhibits increased fluorescence. Spectral and steady‐state kinetic data suggests that DHFS‐FPGS·ATP catalyzes the rapid formation of quinonoid dihydropteroate by removal of the N‐3 nitrogen proton and protonation of the C‐6 carbon of dihydropteroate. A similar spectral shift was observed for dihydrofolate when bound to enzyme in the presence of ATP or ADP. Rapid‐reaction kinetic studies demonstrate that the DHFS‐FPGS·ADP catalyzed formation of dihydrofolate quinonoid is associated with a conformational change in the enzyme. The results of this study suggest that the enzyme‐catalyzed formation of the quinonoid structure and the associated conformational change in the enzyme is required for effective DHFS activity but inhibitory for FPGS activity. These studies were supported by NIH R37DK58144 to PJS.
Evolutionary Origin of the Sulfilimine Chemical Bond in Basement Membranes
Authors: Fidler Aaron , Vanacore Roberto , Pedchenko Vadim , Bhave Gautam , Chetyrkin Sergei , Stothers Cody , McDonald Hayes , Rose Kristie , Aspirnauts the , Hudson Julie , Hudson Billy
Published: June 2021

Collagen IV is the primary protein constituent of basement membranes, conferring structural integrity to this specialized form of extracellular matrix and serving as a ligand for cell‐surface receptors. Collagen IV protomers, characterized by a triple‐helical arrangements of alpha‐chains, oligomerize forming complex networks. We recently discovered a novel sulfilimine bond (‐S=N‐ ) that covalently links methionine‐93 and hydroxylysine‐211 in the noncollagenous domains (NC1) between opposing protomers. In the present study, we sought to determine the distribution and origin of the sulfilimine bond in the animal kingdom. Representative species, spanning the major phyla Chordata, Hemichordata, Echinodermata, Mollusca, Arthropoda, Annelida, Nematoda, Ctenophora, and Cnidaria were investigated. The presence of the bond was detected by electrophoresis techniques and, in certain species, its presence was confirmed by mass spectrometry. Evidence for the bond was found for all phyla. Within Cnidaria, the bond was found in four species: Hydractinia, Ectopleura, Metridium, and Nematostella. In contrast, the bond was absent in Hydra, a result which is consistent with the absence of methionine‐93 and hydroxylysine‐211 in collagen IV genomic data of Hydra. The findings reveal that the sulfilimine bond arose at the divergence of sponge and Cnidaria, coinciding with the emergence of the basement membrane. The bond represents an essential innovation for strengthening the structural integrity of basement membranes for the genesis of tissues.
Probing the biological significance of c‐heme attachment in cytochrome c
Authors: Novick Bethany Rose , Sgheiza Valerie , Frink Melody , Kalmeta Breanna , Grimaldi Kyle , Bren Kara L. , Vacca‐Michel Lea
Published: June 2021

Heme is an iron‐containing porphyrin ring that some iron‐dependent bacteria are able to use as their iron source. c‐type heme is differentiated from other hemes (b, d, etc.) by covalent attachment of the heme to the protein chain via two thioether bonds to Cysteines. Covalent attachment of the heme group is energetically expensive, suggesting that there is a biological benefit to the attachment. One proposed advantage, supported by our studies, is that the covalent attachment stops iron‐dependent bacteria, such as Nontypable Haemophilus influenzae (NTHi), from stealing heme. In our study, we evaluated the role of c heme attachment by creating single‐ and double‐mutant cytochrome c proteins, which lacked one or both of the covalent attachments to the heme group, respectively. We then grew NTHi in the presence of wild‐type (two covalent heme attachments), single‐mutant (one covalent attachment) and double‐mutant (no covalent attachments) protein, as well as appropriate controls, to examine the effect of covalent heme attachment on NTHi growth. Results from our studies demonstrated that the covalent linkages do prevent NTHi from scavenging heme from wild‐type and single‐mutant cytochrome c, while non‐covalently attached heme (in double‐mutant cytochrome c) is readily scavenged and used as an iron source. This study was funded by the Rochester Institute of Technology.
Using biotinylation to determine the orientations of Pal in Escherichia coli
Authors: Shaw Juliana , Schmidt Rachel , Snyder Joy , Pichichero Michael , Michel Lea Vacca
Published: June 2021

Nontypable Haemophilus influenzae (NTHi) causes otitis media and other respiratory diseases in people of all ages, conferring the need for a vaccine against the bacteria. P6, a lipoprotein vaccine candidate for NTHi, was recently discovered to exhibit two populations: one facing out of the cell and one facing in towards the periplasm. We hypothesized that dual orientation is a novel, widespread phenomenon among bacterial lipoproteins. To corroborate our hypothesis, we performed a biotinylation experiment on Pal, homolog to P6, in Escherichia coli (E. coli). In the experiment, whole cells were incubated with a cell‐impermeable biotinylating reagent (NHS‐LC‐LC‐biotin) in order to label all surface exposed proteins. The labeled, outer proteins were separated from the unlabeled, internal proteins via streptavidin‐agarose beads. Standard protein detection and immunoblotting techniques confirmed that Pal exists in two orientations in E. coli, with the majority of Pal facing in toward the periplasmic space. Only one other protein (Lpp of E. coli) has been shown to exhibit dual orientations. The results of this experiment suggest that lipoprotein dual orientation may be more widespread than originally thought, which could have important implications in the field of bacterial lipoprotein biology. This study was funded by the Rochester Institute of Technology and NIH NIDCD RO1 08671 (to MEP).
Zhx2 and the balance between cardiovascular and hepatic health
Authors: Creasy Kate Townsend , Clinkenbeard Erica , Spear Brett T
Published: June 2021

Background

The Zinc fingers and homeoboxes 2(Zhx2) gene has been identified as being responsible for the mouse Hyplip2 trait characterized by hyperlipidemia and atherosclerosis. This finding is supported by a recent human GWAS study identifying ZHX2 as an important risk factor for carotid intima media thickness, a measure of subclinical atherosclerosis. Mice with mutated Zhx2 incur less cardiovascular damage from a high‐fat diet than wildtype mice.

Objective

To explore the role of Zhx2 on lipid metabolism and homeostasis, as well as its impact on cardiovascular and hepatic health.

Methods

Wild‐type and Zhx2‐mutant mice were fed either a control or high‐fat (15%) diet for 8 weeks. Body weight was measured and serum was collected during this period, and tissues were collected post‐sacrifice. Quantitative RT‐PCR analysis was performed to assess gene expression.

Results

Mice with low Zhx2 levels have elevated serum ALT levels, increased hepatic AFP mRNA, and elevated liver pathology and lipid accumulation. Additionally, Lpl, Elovl3, and Cyp8B1, genes involved in cholesterol/lipid metabolism, were identified as Zhx2 targets.

Conclusions

These data indicate that reduced Zhx2 levels may be cardioprotective but at the same time lead to increased liver damage in mice on a high‐fat diet. Further studies will investigate the role of Zhx2 in human cardiovascular and liver disease.

This research is supported by PHS Grants DK074816 and RR021954 from the NIH.
Preliminary Investigation of The Nutritional Qualities, Phytochemical Properties and Antioxidant Activities of Aqueous Extract of Mistletoe Leaves (Tapinanthus Bangwensis) Grown on Orange Trees.
Authors: Omojufehinsi Moyosore , Nwoke Stella C , Imaga Ngozi A , Magbagdeola Abiola O , Ebuehi Albert O. , Okafor Uzoma A
Published: June 2021

Mistletoe (Tapinanthus bangwensis) plant has been used traditionally in Nigeria and other parts of Africa for the treatment and management of ailments such as diabetics, female reproductive disorders, arthritis, rheumatism, hypertension, asthma, epilepsy and diabetes. In view of the traditional usage of Mistletoe, this has no detailed scientific data; we investigated the nutritional potentials of aqueous crude extract of Tapinanthus bangwensis (Mistletoe) and determination of in vitro antioxidant activity, qualitative and quantitative physiochemical properties.

The proximate analysis revealed the crude protein to be 6.8 ± 0.4%, crude fat (9.65 ± 0.3%), total carbohydrate (12.72 ± 0.1%), moisture (67.2 ± 0.1%), ash (3.5 ± 0.1%), crude fibre (0.13±0.2%) and the energy value is 164.94KJ/mole. The mineral evaluation in mg/100g dry weight showed Potassium (147.3±0.01) >; Calcium (90.70±0.025) >; Phosphorus (62.9±0.025) >; Sodium (54.6±0.03) >; Magnesium (43.05±0.02) >; Manganese (34.00±0.01) >; Zinc (24.90±0.015) and Iodine (2.05±0.01). Further tests revealed that heavy metals such as lead, chromium and cadmium were not detected. The qualitative phytochemical analysis revealed the presence of tannins, steroidal glycosides, phenols, saponin, terpenoids and flavonoids while the quantitative (in mg/100g) phytochemical analysis revealed tannin (108.65±0.04 ) >;phenol (66.7±0.01)>; flavonoid (44.58±0.03)>; Cynogenic glycoside (24.53±0.02)>; saponin (2.40±0.02). Reducing sugar was revealed to be 33.07±0.05.
Multiple cycles of acetyl‐CoA enolization catalyzed by citrate synthase (CS) and ATP‐citrate lyase (ACL)
Authors: Li Qingling , Anderson Vernon E. , Brunengraber Henri , Zhang Guo‐Fang
Published: June 2021

Enolization of acetyl‐CoA on CS had been identified in Eggerer¡s experiments with [3H]acetyl‐CoA (Biochem. Z. 343: 111, 1965). To test whether CS catalyzes sequential cycles of acetyl‐CoA enolization, we incubated [2H3, 13C2]acetyl‐CoA with pig heart CS + oxaloacetate (OAA) (added as either a bolus, a bolus of OAA/malate, or malate + NAD + malate dehydrogenase). We measured the mass isotopomer distribution (MID) of acetyl‐CoA and citrate. The productions of M4 + M3 + M2 acetyl‐CoA, and of M4 + M3 + M2 citrate revealed up to 3 cycles of acetyl‐CoA enolization. This was confirmed when condensation of unlabeled acetyl‐CoA + OAA was conducted in 50% D2O. Acetyl‐CoA was up to M3 labeled and citrate was M1 labeled. Thus, up to 3 cycles of acetyl‐CoA enolization were catalyzed by CS.

We hypothesized that ACL could also catalyze enolization of acetyl‐CoA. We incubated [2,2,4,4‐2H4]citrate + Mg++, CoA, ATP and rat liver ACL. The production of M2 + M1 + M acetyl‐ CoA, and the progressive de‐labeling of acetyl‐CoA demonstrated multiple cycles of enolization of acetyl‐CoA on ACL. This was confirmed when cleavage of unlabeled citrate was conducted in 50% D2O, by the accumulation of M1 + M2 + M3 acetyl‐CoA. Thus, multiple cycles of acetyl‐CoA enolization were catalyzed by ACL.(Supported by the NIDDK Metabolomic Initiative grant R33DK070291).
Evaluation of the bioavailability of vitamin D2 in mushrooms in healthy adults
Authors: Bogusz Jaimee , Pagonis Garyfallia , Holick Michael F
Published: June 2021

Mushrooms can produce vitamin D2 when exposed to sunlight or ultraviolet radiation. The goal of this research was to determine if ingesting dried white button mushroom extract (Agaricus bisporus) containing D2 (Monterey Mushrooms, Inc., CA) was as effective at increasing and maintaining vitamin D status as supplemental D3 and D2. Thirty healthy adults (6 male, 19 female, mean age 35.2 years) were randomized to take capsules containing 2000 IU D2, 2000 IU D3, or 2000 IU mushroom D2 once a day for three months during the winter. Twenty‐five subjects completed the study. Serum 25‐hydroxyvitamin D [25(OH)D] was measured once a week for 12 weeks. Baseline serum 25(OH)D levels were not significantly different; 17.1 ± 1.2, 19.4 ± 2.3, and 20.9 ± 2.3 ng/mL for the supplemental D3 and D2, and mushroom D2 groups respectively. Serum 25(OH)D levels gradually increased and plateaued at 7 weeks and were maintained for the following 5 weeks. After supplementation for 12 weeks, serum 25(OH)D levels were not statistically significantly different in all three groups: 34.4 ± 1.2, 29.2 ± 2.0, and 31.1 ± 2.4 ng/mL for D3, D2, and mushroom D2 respectively. These results demonstrate ingestion of mushrooms containing D2 is as effective at increasing and maintaining serum 25(OH)D levels as supplemental D2 and D3. Supported by the Mushroom Council.
EFFECT OF TAPINANTHUS BANGWENSIS LEAF EXTRACT, CLOMIPHENE CITRATE AND FOLIC ACID ON REPRODUCTIVE HORMONES IN FEMALE SPRAGUE DAWLEY RATS.
Authors: Nwoke Stella C , Magbagbeola Abiola O. , Ebuehi Albert O. , Omojufehinsi Moyosore , Gbotolorun Stella , Okafor Uzoma A , Ani Anthony , Ajayi Godwin O.
Published: June 2021

Folkloric claims has implicated the plant Tapinanthus bangwensis, indiginous to Nigeria as a possible adjuvant in the treatment of infertility in women. This study seeks to scientifically validate this claim. Hence, the effect of administration of aqueous extract of Tapinanthus bangwensis leaves for 52 days on reproductive hormones of female rats was investigated. 30 female sprague‐dawley rats were grouped into 5 groups of 6 rats each. Group 1 (control), 2,3,4 and 5 received 1ml‐distilled water, 250mg/kg and 500mg/kg, 0.714mg/kg clomiphene citrate and 11.43mg/kg folic acid respectively. Hormonal analysis using Eliza kits indicated increase In the serum FSH level (5.63± 0.1), LH (37.96± 0.13), progesterone (0.13± 0.20) and Estradiol (20.06±0.15) for grp 2, when compared with the control (4.64± 0.22), FSH,(2.12± 0.15) LH, (0.05±0.20) Progesterone, and (11.62±0.13) Estradiol respectively. Also reduction in the serum prolactin level 122.46±0.10 was observed in group 2, when compared to it's control 179.63±0.14. Again administration of the extract in group 3 produced a significant increase in FSH 26.74± 0.012, LH 41.74±0.01, Progesterone 1.61± 0.02 and Estradiol 30.70±0.02 at P<0.05, compared to control, and a significant reduction in Prolactin 98.46±0.02. Group 4 showed FSH (5.19±0.012), LH (83.18±0.014), Progesterone (0.09±0.021), Estradiol (18.50±0.025) and Prolactin (181.80±0.011), while FSH (5.45±0.023), LH (3.49±0.04), Progesterone (0.28±0.025), Estradiol (19.10±0.03) and. Prolactin (225.16±0.031) was for group 5. We conclude, that the hormonal effect of plant extract compares more favourably to folic acid and clomiphene citrate that are known fertility enhancers.
Crosslinking mass spectrometry with novel aryl‐dialdehyde reagents reveal extended conformational dynamics of RNA polymerase II
Authors: Trnka Michael J , Burlingame A. L.
Published: June 2021

Chemical crosslinking mass spectrometry is a means of probing the structures of protein machines in their native states. Bifunctional reagents covalently label stable complexes, reflecting their topology. Identification of the modified sites generates distance restraints, which together with crystallography of individual subunits and cryoEM visualization of entire complexes, can be used to model large three‐dimensional structures. Furthermore, solid‐state protein structures are conformationally constrained but crosslinking can reveal the extent of their dynamics. Here, we probe RNA polymerase II (pol II) with aryl‐dialdehyde (ADA) crosslinking reagents. ADA reagents preserve positive charge at the site of crosslinking, which facilitates the enrichment of crosslinked peptides by cation exchange chromatography relative to conventional reagents. Furthermore, these highly charged precursors are well suited to analysis by electron transfer dissociation (ETD) mass spectrometry, which provides more comprehensive product ion coverage than collisional dissociation and facilitates identification of crosslinked peptides in complex searches. The data indicate that the pol II clamp domain is more dynamic than indicated by crystallography.

Funding was provided by the Biomedical Technology Research Centers program of the NIH National Institute of General Medical Sciences NIH NIGMS 8P41GM103481
Human melanoma metabolic network analysis with combined 13C NMR/bioreactor technique: testing the Warburg effect
Authors: Shestov Alexander , Mancuso Anthony , Locasale Jason W , Glickson Jerry D
Published: June 2021

The bioreactor techniques are becoming an important tool to study cancer cell metabolism(1). The goal of the present work was to develop a 13C metabolic “bonded cumomer” modeling approach adapted for fitting bioreactor data obtained with 13C glucose as substrate and to calculate the extent of the Warburg effect and how much energy comes from aerobic glycolysis vs oxidative phosphorylation.

Results

Figure 1 shows experimental time courses for labeled glutamate obtained during [1,6‐13C2] glucose perfusion of melanoma cells cultured in a CST bioreactor. Using extracted fluxes we estimated that 54% of the energy coming from oxidative phosphorylation (from which 44% are from glucose) and 46% from aerobic glycolysis and the extent of the Warburg effect (aerobic glycolysis) was 87%.

Conclusions

Under aerobic conditions glycolysis provides nearly as much ATP as oxidative phosphorylation. The model is validated by excellent agreement between model predicted and experimentally measured values of CMRO2 and cytosolic glutamate pool size. Dynamic high‐resolution MR spectra is very sensitive to changing/adding biochemical pathways and flux values, and bonded cumomer modeling allows one to check precisely the feasibility of assumed general bionetworks and particular metabolic pathways.

This work was supported by NIH grants 2U24‐CA083105 and 5R01CA129544–02
Signal amplification is critical for the induction of unfolded protein response‐mediated apoptosis
Authors: Banerjee Dipak K , Banerjee Aditi , Martinez Juan A , Longas Maria O , Baksi Krishna , Kotapadi Ashok , Prasad Kilari PVRK , Santiago Jesus
Published: June 2021

Angiognesis (proliferation and differentiation of endothelial cells) is essential for normal growth and development as well as for solid tumor progression and metastasis. Our objective is to understand the dynamic relationship between protein N‐glycosylation and angiogenesis. Using a protein N‐glycosylation inhibitor we establish that the anti‐angiogenic action of tunicamycin is mediated by ER stress‐coupled unfolded protein response (upr). The cells are incapable of forming colonies and exhibited nuclear fragmentation. To evaluate upr‐mediated programmed cell death is the consequence of a “cellular tsunami”, genome and proteome were analyzed. There is differential expression of 577 genes, and correlation with protein folding dynamics by Raman Spectroscopy exhibited decreased in area at 1672 cm−1 by 41.85% at 3 h and 55.39% at 12 h; at 1684 cm−1 by 20.63% at 3 h and 40.08% at 12 h; and also at 1994 cm−1 by 33.33% at 3 h and 32.92% at 12 h, respectively. The ER resident protein, mannosylphospho dolichol synthase (DPMS) is unable to remain phosphorylated, and loses catalytic activity irrespective of mRNA and protein expression. Thus, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form undergo further degradation. Supported in part by grants from Susan G. Komen for the Cure BCTR0600582 (DKB) and NIH/NIMHD 8G12MD007583 (KB)
Computational Model for Monitoring of the Membrane Potential with the Fluorescent Probe DiSC3(5)
Authors: Lemeshko Victor V , Alvarez Jose A.
Published: June 2021

The optical properties of cationic dicarbocyanide dyes have been shown to depend on the transmembrane electrical potential of liposomes or living cells. In this work, we developed the most complete computational model allowing optimization of experimental conditions to monitor the membrane potential of membrane vesicles composed of neutral and charged phospholipids at fixed ionic strength of incubation media in a desirable voltage range. The model allows predicting potential‐dependent changes of the fluorescent properties of DiSC3(5) on concentration of the dye and on the lipid composition of the liposomes of certain sizes. It also takes into account the values of the surface potentials of the inner and outer leaflets of the lipid bilayer, as well as their changes due to the DiSC3(5) incorporation into the lipid phase. The probe dimerization in the aqueous and lipid phases, and the possibilities of the voltage‐dependent transitions of the dimers between two leaflets of the lipid bilayer and between the aqueous and lipid phases were also included in the model, in addition to the monomer transitions. The model was adjusted to several literature data obtained with DiSC3(5) probe as an indicator of the membrane potential in experiments with liposomes and biomembrane vesicles. Colciencias (Colombia) research grants #111840820380 and #111852128625.
The Molecular Mechanism of Inhibition of Newly Identified Inhibitors of Human MDR1 P‐Glycoprotein
Authors: Vogel Pia D. , Brewer Fran K. , Follit Courtney A. , Rossi Isolina R. , Wise John G.
Published: June 2021

The ABC‐transporter superfamily of proteins includes members like the ABCB1 P‐glycoprotein (Pgp) that cause pronounced problems in cancer chemotherapies and antiviral treatments. While these normally protective proteins are used in many tissues to prevent the accumulation of toxins, in chemotherapies their over‐expression, and the fact that they can transport many different compounds across the plasma membrane out of the cell, causes clinically intractable multidrug resistance phenomena. In previous studies, we used targeted molecular dynamic simulations to elucidate important conformations in the catalytic transport cycle of Pgp (Biochemistry 2012 51:5125–41) and then applied these structures to massively parallel in silico drug docking routines. This approach has allowed us to identify several novel compounds that inhibit Pgp by targeting the nucleotide binding domains of the transporter. In earlier studies, we pioneered the use of spin labeled ATP analogs and electron spin resonance spectroscopy to identify and quantitate nucleotide binding to Pgp under equilibrium binding conditions (Biochemistry 2005 44:14010–14019). In the present study, we have investigated the mechanism of inhibition of a number of newly identified, novel inhibitors of Pgp by applying routine biochemical techniques as well as our novel ESR‐based biophysical techniques. Supported by NIH 1R15GM094771–01A1 to PDV and JGW.
Development of Antagonists for the Protease Activated Receptor‐2
Authors: Hoffman Justin , Flynn Andrea , Tillu Dipti V , Sherwood Cara L , Zhang Zhenyu , Patek Renata , Vagner Josef , Price Theodore J , Boitano Scott
Published: June 2021

Protease activated receptor‐2 (PAR2) is a G‐Protein Coupled Receptor with a significant role in many diseases including allergic asthma and chronic pain. PAR2 is activated by protease cleavage to reveal tethered ligand sequences that bind the receptor to initiate intracellular signaling. Peptide/peptidomimetic ligands based on the primary trypsin cleavage site have been explored as PAR2 agonists/antagonists. We describe a PAR2 peptidomimetic (C391) derived from the major trypsin cleavage site sequence (SLIGRL) that blocks both PAR2 peptidomimetic and protease induced signaling in vitro and thermal hyperalgesia in vivo. We also describe the first antagonists developed from the primary Kallikrein14 cleavage site sequence (SSKGRS). Kallikrein14 derived peptidomimetic antagonist potency was increased through palmitoylation (Pam) with polyethylene glycol spacers (PEGs) to construct synthetic tethered ligands. We demonstrate that: 1) these ligands bind to PAR2; 2) do not directly stimulate cell signaling pathways (e.g., Ca2+, MAPK); 3) do not limit trypsin‐like protease activity; and 4) inhibit trypsin‐like protease and peptidomimetic‐induced PAR2 signaling. These peptidomimetics represent a novel set of high potency PAR2 antagonists.
Disabeled‐2 (Dab‐2) modulates platelet‐cancer cell interactions through its sulfatide binding domain
Authors: Andreano Kaitlyn Jo , Fu Xiangping , Capelluto Daniel , Finkielstein Carla
Published: June 2021

Platelets’ role in cancer progression and metastasis has largely been attributed to platelet‐mediated enhancement of tumor cell survival, extravasation (exiting form capillaries and entering organs), and angiogenesis. Correlations exist between the ability of some tumor cells to aggregate platelets in vitro and their metastatic potential in vivo that it is manifested as a hypercoagulable state found in most cancer patients. We have identified a tumor suppressor molecule (Dab‐2) that it is released upon platelet activation and that modulates the extent of blood clotting. Our results show Dab‐2 is released from alpha‐granules in the platelet to the membrane surface where specifically binds to the integrin receptor, thus inhibiting platelet aggregation. Binding of Dab‐2 to integrins is modulated by sulfatides, a form of glycosphingolipid that accumulate in platelet membrane and effectively compete for Dab‐2 levels. Interestingly, we found that the N‐terminal region of Dab‐2 specifically binds to sulfatides through two conserved sulfatide‐binding sites. Upon activation, sulfatides protect Dab‐2 from thrombin cleavage, facilitate its internalization and modulate the surface expression of P‐selectin, a coagulation protein needed for stabilization of platelet aggregates. P‐selectin also mediates both tumor cell adhesion to vascular endothelial cells and the interaction between activated platelets and cancer cells (emboli) during metastasis. Interestingly, our data show that Dab‐2/sulfatide recognition influences the stability of platelet aggregates heterotypically with cancer cells through sulfatide binding and, indirectly, by controlling P‐selectin levels. Thus, we hypothesize that manipulation of Dab‐2 function in response of platelet activation will impact emboli formation offering an alternative route for therapeutic strategies aimed to control metastatic processes.
Induction of hepatic glutathione synthesis via alterations in sulfur amino acid metabolism in mice treated with silymarin acutely
Authors: Kwon Do Young , Ahn Chul Won , Choi Yoo Jin , Kim Young Chul
Published: June 2021

It has been known that silymarin exhibits protective effects against chemical‐induced liver injury, but the underlying mechanism of its beneficial action remains unclear. We examined the silymarin‐induced changes in the metabolism of sulfur‐containing amino acids in association with its effects on antioxidant capacity of liver. Male mice were treated with silymarin (100 or 200 mg/kg, po) every 12 hr for a total of 3 doses. Hepatic methionine level was increased, but the activity and protein expression of methionine adenosyltransferase were decreased dose‐dependently in liver of the mice treated with silymarin. S‐Adenosylmethionine or homocysteine was not altered whereas cystathionine, cysteine and glutathione (GSH) were increased significantly. Betaine‐homocysteine methyltransferase and cysteine dioxygenase were down‐regulated, but cystathionine β‐synthase was induced. γ‐Glutamylcysteine synthetase was unchanged by silymarin treatment. Oxygen radical scavenging capacity of liver cytosol against peroxyl radical and peroxynitrite was increased, and hepatic lipid peroxidation was diminished in the silymarin‐treated mice. It is concluded that silymarin enhances hepatic GSH biosynthesis by elevating cysteine availability via increment in cysteine synthesis and inhibition of its catabolism to taurine, which may subsequently contribute to the antioxidant defense of liver.
ApaH diadenosine polyphosphatase from Legionella pneumophila
Authors: Madaio Michael Paul , Patron Alejandra Rizo , Thomson Joshua G. , Baltus Andrew , Edelstein Martha , Zhuang David , Edelstein Paul , O'Handley Suzanne
Published: June 2021

The Nudix diadenosine polyphosphatase, NudA, is a virulence factor in Legionella pneumophila (LP), the causative agent of Legionnaires’ disease. The nonNudix diadenosine polyphosphatase, ApaH, is a virulence factor in E. coli and acts additively with the E. coli NudA for virulence. An LP apaH knockout mutant had no apparent phenotype; however, a double apaH/nudA knockout mutant could not be created, indicating that this dual mutation is lethal, and that apaH is important. We subcloned apaH, but ApaH was insoluble, so we subcloned apaH using the maltose binding protein (MBP) as a solubility tag. We obtained a soluble fusion protein with diadenosine polyphosphatase activity. Neither a MBP nor His tag could be used for purification indicating that the protein may not be folded properly, and thus we are in the process of subcloning the apaH, once again, to put the MBP solubility tag at the c‐terminal end instead of the n‐terminal end where it was originally placed. Once the ApaH diadenosine polyphosphatase has been purified, it will be characterized further enzymatically. This research has been supported by an NIH AREA grant and the Merck/AAAS Undergraduate Science Research Program.
Isolation of potential new SPase inhibitors producing strains by soft agar overlay assay method.
Authors: Ahmed Sibtain , Smith Peter A , Roberts Tucker C , Jensen Paul R
Published: June 2021

The arylomycins are a class of compounds that target bacterial signal peptidases (SPases). In an effort to discover new compounds in this class and other small molecule SPase inhibitors, a rapid screening method was developed and applied to bacteria cultured from marine sediment samples. The assay consisted of a soft agar overlay using an engineered E. coli strain MG1655 ΔlepB::kan + pTetBHR2‐LepB designated as CUA‐604. Sixty marine sediment samples from Fiji and 200 from the Channel Islands were processed for the cultivation of marine bacteria. The primary isolation plates were overlaid with strain CUA‐604 and following 24–48 h, colonies showing zones of inhibition were removed and purified using standard microbiological methods. Six strains from Fiji and six from the Channel Islands were confirmed as inhibiting the growth of CUA‐604. It was determined that pH and iron limitation were not the cause of inhibition. The most active strain was identified as a Bacillus sp. To isolate the active compound, multiple overlay plates were prepared and the agar in the zones of inhibition removed and extracted with a variety of organic solvents. The crude butanol, methanol, and acetone extracts were active at a concentration of 10 mg/mL. The results of the bioassay‐guided isolation of the active constituents will be presented.
Diadenosine phosphatases of the Nudix hydrolase superfamily
Authors: DiDonato Tessa , Williford Kelsey , Thomson Joshua , Ramos Julian , Sheibley Daniel , Glick Sarah , O'Handley Suzanne
Published: June 2021

The diadenosine polyphosphatases are a family of enzymes within the Nudix hydrolase superfamily. The diadenosine polyphosphatases from Legionella pneumophilia and Bartonella bacilliformis have been found to be important in the pathogen's ability to invade its host cells. It is of interest to know whether these enzymes act in the same way in M. tuberculosis and M. leprae. If they are all found to be involved in invasiveness and thus in virulence, then these enzymes could be novel antibiotic targets. Orf176 (NudH) is a diadenosine polyphosphatase in E. coli; phenotype work is currently underway with a NudH− knockout mutant. We are also subcloning in preparation for complementation studies using the M. tuberculosis and M. leprae diadenosine polyphosphatases incorporated into the NudH− knockout mutant. Both overexpression (even from leaky expression) and knocking the gene out result in either a lag phase and/or slower growth as compared to the wild‐type, thus we are still investigating a phenotype that we can use for complementation studies. This research has been supported by an NIH AREA grant and a RIT honors summer research fellowship.
The molecular mechanism of histone deacetylase inhibitor (HDACi) PCI‐24781 mediating anti‐tumor activity in neuroblastoma cells
Authors: Zhan Qing Lei , Ngai Sai Ming
Published: June 2021

Neuroblastoma accounts for 7–10% of all childhood cancers, and it is the second most common solid tumor diagnosed during infancy. The survival rate among children with high‐risk neuroblastoma is less than 40%, despite intensive multi‐modality treatment, highlighting the urgent needs for new treatment strategies. Epigenetic changes are known to be important for tumorigenesis and a number of drugs targeting epigenetic regulation have been developed for anticancer therapies. Among those drugs, the most effective ones are histone deacetylase inhibitors (HDACi). Numerous HDACi are currently in phase I/II clinical trials for various tumor types. However, little is known about the role of PCI‐24781, a novel HDACi in nueroblastoma. In the present study, we investigated the underlying mechanisms that mediate anti‐tumor activity of PCI‐24781 in neuroblastoma cell line SK‐N‐DZ. PCI‐24781 treatment inhibited tumor growth at very low doses in SK‐N‐DZ, while normal cell line HS‐68 shown a relatively resistance to treatment. PCI‐24781 caused the accumulation of acetylated histone H3 and cell cycle arrest in G2/M phase in SK‐N‐ DZ. Treatment of SK‐N‐DZ with PCI‐24781 also induced apoptosis signaling pathways via up‐regulating some genes associated with apoptosis, including death receptor DR4, caspase3, p21 and p53. Furthermore, the 2D electrophoresis‐based proteomic analysis revealed different protein expression profile in SK‐N‐DZ cells treated with PCI‐24781 for 24 h and 36 h. The identified proteins involved in many biological processes, including energy metabolism, DNA repair, transcription regulation, cell cycle and proliferation. Immunoblotting analysis further confirmed that PCI‐24781 could increase the expression of prohibitin, hHR23a and RuvB2.

These studies will enable a better understanding of molecular mechanisms underlying PCI‐24781‐mediated anti‐tumor effects in neuroblastoma cells SK‐N‐DZ.
Differential Protein Analysis of Developing Mouse Cochlea using Label‐Free Quantitative Mass Spectrometry
Authors: Darville Lancia , Sokolowski Bernd
Published: June 2021

The sensory epithelium of the inner ear contains the receptor or hair cells responsible for hearing. To obtain insights into the proteome of these epithelial cells during development, we quantitatively compared proteins expressed in postnatal day (P) 14 versus P30 mice using label free shotgun proteomics with mass spectrometry. Proteins from the cochlear sensory epithelia were extracted, enzymatically digested, separated by strong cation exchange (SCX), and analyzed using nano LC‐MS/MS on an Orbitrap. Proteins were quantified and compared for P14 and P30 cochleae using spectral counts calculated by MaxQuant. Results show that proteins unique to the P14 and P30 proteome number 368 and 255, respectively, with 746 proteins common to both. An initial comparison of up‐regulated proteins, as defined by at least a 10‐fold increase in spectral count, revealed an increase in 69 and 52 proteins, respectively, on P14 and P30. Analyses, using gene ontology annotations, showed that oxidoreductase proteins had the greatest decrease in expression on P30 relative to P14. In comparison, cytoskeletal and transfer proteins were up‐regulated on P30 compared to P14. The use of label free quantitation to investigate the cochlear proteome revealed protein identifications that may have biological significance to hearing maturation.

Supported by NIDCD grant DC004295 to BS.
Development and Testing of a Systematic Approach for Computational Enzyme Function Determination
Authors: Dodge Gregory , Arroyo E. Nicole , Bernstein Herbert , Craig Paul
Published: June 2021

With the success of the Structural Genomics Initiative and other programs, the number of uncharacterized but crystallized proteins is on the rise. To date, there are over 3500 structures in the Protein Data Bank tagged as unknown function. The use of computational tools to speed up the analysis of these structures has been widely adopted, with varying measures of success. ProMOL, a plugin for PyMOL, was developed in a collaboration between RIT and Dowling College. ProMOL functions by searching for conserved active site “motif templates” in a queried protein structure. A combination of ProMOL, NCBI's BLAST algorithm, and University of Helsinki's DALI alignment server were used to attempt to assign functions to 4 previously uncharacterized proteins: PDBIDs 3DS8, 3FLE, 3LP5, and 2L57. The results from the computational analysis were then confirmed in vitro via functional assays. The computational approach successfully assigned the function of 3DS8, 3FLE (both lipases), and 2L57 (a glutaredoxin). It appears that a combination of BLAST alignment, DALI alignment, and ProMOL alignment is sufficient to computationally predict the function of uncharacterized proteins. This approach may be suitable for classroom settings as well, due to the low cost and ease of accessibility of these tools. The research is supported by 2R15GM078077–02 and 3R15GM078077–02S1.
Thermodynamics of cellulose solvation in novel solvent mixtures
Authors: Das Ritankar , Chu Jhih‐Wei
Published: June 2021

Biomass contains abundant amounts of cellulose as crystalline microfibrils. A limiting step to using cellulose as an alternative energy source, however, is the hydrolysis of the biomass and subsequent transformation into fuels. Cellulose is insoluble in most solvents including organic solvents and water, but it is soluble in some ionic liquids like BMIM‐Cl. This project aims to find alternative solvents that are less expensive and are more environmentally benign than the ionic liquids.

All‐atom molecular dynamics simulations were performed on dissociated glucan chains separated by multiple (4–5) solvation shells, in the presence of several novel solvents and solvent mixtures. The solubility of the chains in each solvent was indicated by contacts calculations after the equilibration of the molecular dynamics. It was discovered that pyridine and imidazole acted as the best solvents because their aromatic electronic structure was able to effectively disrupt the inter‐sheet interactions among the glucan chains in the axial direction, and because perturbation of the solvent interactions in the presence of glucan chains was minimal.
Regulation of Prostaglandin E2 signaling by cysteinyl leukotrienes in human mast cells
Authors: Kondeti Vinay , Duah Ernest , Emory Ashley , Thodeti Charles K , Paruchuri Sailaja
Published: June 2021

Cysteinyl leukotrienes (cys‐LTs), LTC4, LTD4 and LTE4, are critical mediators of airway hyper‐responsiveness and asthma. We have previously shown that LTD4 regulates mast cell (MCs) functions through the activation of G‐protein‐coupled receptors, CysLT1R and CysLT2R. The role of Prostaglandin E2 (PGE2), the other arachidonic acid metabolite in asthma is controversial as it act as both pro‐ and anti‐inflammatory, depending on the receptor through which it exerts its effect. Although cysLTs were shown to induce PGD2 generation in mast cells, it is not known whether there is cross‐talk between cysLTs and PGE2. Here, we found that PGE2 by itself does not activate calcium and MIP‐1β in mast cells, but cys‐LT priming induced robust PGE2‐dependent calcium flux and MIP‐1β generation. Further, using immunoprecipitation assays, we found that PGE2 receptor EP1R interacts with CysLT1R and this interaction was enhanced upon LTD4 stimulation. Importantly, we found that LTD4 and PGE2 synergistically increased the production of pro‐inflammatory cytokine, MIP1β in mast cells which was substantially inhibited by blocking protein kinase C (PKC)s. Taken together, our results suggest that cysLTs augment inflammation by activating PGE2 signaling through the priming of PGE2 receptors. This unravels a novel phenomenon by which inflammatory mediators generated from the same precursor can cooperate to enhance inflammation.
Sphingosine Kinase 1 is regulated by PPARα in response to free fatty acids and mediates skeletal muscle IL‐6 production and signaling in diet‐induced obesity
Authors: Ross Jessica Shavonne , Hu Wei , Rosen Bess , Snider Ashley J , Obeid Lina M , Cowart L. Ashley
Published: June 2021

Plasma fatty acids increase in obesity and diabetes; subsequent lipid overload to cells and tissues mediates numerous pathological processes. This is due in part to aberrant synthesis of bioactive lipids. We previously demonstrated that palmitate (PAL) increased sphingosine‐1‐phosphate through regulation of sphingosine kinase 1 (SphK1); however, potential functions and in vivo relevance of this have not been addressed. Data demonstrate that PAL mediated SphK1 induction through PPARα. Moreover, PAL treatment induced IL‐6 in a SphK1‐dependent manner, which was attenuated by inhibition of sphingosine‐1‐phosphate receptor 3 (S1PR3). Diet‐induced obesity in mice demonstrated a requirement for SphK1 for IL‐6 expression in muscle and plasma. Moreover, muscle IL‐6 signaling was activated in wild type but not SphK1‐/‐ mice. These data indicate that PPARα regulates SphK1 expression in fatty acid oversupply and links PAL to muscle IL‐6 production. Moreover, the activation of muscle IL‐6 signaling in diet‐induced obesity suggests a potential role for SphK1 in obesity‐associated pathology. This work was supported by a VA Merit award and a COBRE award (NIH P20 RR017677) to LAC and a GAANN fellowship in Lipidomics and Systems Biology to JSR.
The pro‐cysteine drug, L‐2‐Oxothiazolidine‐4‐carboxylic acid (OTC) is a novel inducer of nSMase2 mRNA and protein expression in the liver
Authors: Deevska Gergana , Karakashian Claudia , Sunkara Manjula , Peppers Benjamin , Morris Andrew , Nikolova‐Karakashian Mariana
Published: June 2021

Neutral sphingomyelinase 2 (nSMase2) is a signaling enzyme that mediates hepatic response to IL‐1β. It has been suggested that nSMase2 is activated by oxidative stress and during aging leading to hyperresponsiveness to inflammation. These changes in nSMase2 activity are not caused by increased protein, but are due to aging‐associated decline in the levels of hepatic GSH, a known nSMase2 inhibitor. In this study we sought to investigate the effect of a cysteine pro‐drug, L‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) on nMSase2 activity in vivo in young and aged mice (5 and 23 months old). OTC has been shown to replenish hepatic GSH and to improve the overall morbidity in elderly. C57Bl6 mice were fed three different diets for 4 weeks: a standard chow (Std), sulfur amino acid deficient control (SAA Def) and supplemented with 0.5% OTC diet (OTC). As anticipated, aging was associated with significant increases in hepatic nSMase activity. Mass‐Spec analyses showed significant accumulation of C16 and C24:1 ceramides in the aging liver. OTC diet lead to increase in hepatic GSH and abolished any aging‐associated differences. Surprisingly, the mice fed the OTC diet had significantly elevated nSMase activity than mice fed the other two diets. These changes were accompanied by corresponding increases in nSMase2 mRNA and protein levels suggesting that OTC has GSH‐independent effect. The effects were seemingly specific for nSMase2 and liver. Direct addition of OTC to HepG2 cells recapitulated the effects observed in vivo and lead to elevated nSMase2 mRNA and protein expression. Together, these studies provide evidence for a specific regulation of nSMase2 mRNA transcription and protein expression by a drug involved in the control of cellular red‐ox homeostasis.

Support:R01AG19223,R01AG026711to MNK, AHA11POST7650060 to GD.
Characterization and immunolocalization of inositol phosphorylceramide in Leishmania (Viannia) braziliensis
Authors: Takahashi Helio Kiyoshi , Castro Erica Valadares , Toledo Marcos Sergio , Mortara Renato Arruda , Straus Anita Hilda
Published: June 2021

Leishmania ssp is a protozoan parasite responsible for cutaneous, mucocutaneous and visceral disease. Leishmania expresses a class of sphingolipids, inositol phosphorylceramides (IPC). Lipids of Leishmania (Viannia) braziliensis promastigotes were extracted and analyzed by electrospray ionization‐mass spectrometry by positive and negative ion modes to detect parental ions of inositol and sphingosines. IPC species were identified at m/z 752 (d32:0), m/z 778 (d34:1), m/z 780 (d34:0), m/z 806 (d36:1), m/z 808 (d36:0). IPC of L. braziliensis displays a prominent ion at m/z 778.4, and its structure determined as d20:1/14:0, differing from IPC (d16:1/18:0) isolated from L. major and L. amazonensis, thus suggesting expression of sphingosine synthases with different specificities, as serine steaoryltransferase for L. braziliensis, and serine myristoyltransferase for L. major and L. amazonensis. These results show that L. braziliensis expresses IPC with a longer sphingosine compared to other Leishmania species. Using a specific monoclonal antibody to IPC and confocal microscopy it was demonstrated that IPC is present in the inner leaflet membrane and may be involved in intracellular signaling. The absence of IPC in mammals and the presence of an unusual sphingosine (d20:1) suggest that L. braziliensis IPC may represent a potential target for new therapeutic drugs. Supported by FAPESP, CNPq, CAPES.
Mechanisms of sterol regulatory element binding protein‐1 activation by membrane phospholipids
Authors: Smulan Lorissa Joy , Walker Amy K.
Published: June 2021

The sterol regulatory element‐binding protein (SREBP‐1a, ‐1c, and 2) family of transcription factors control genes involved in cholesterol, fatty acid, and triacylglycerol biosynthesis. Misregulation of these genes can contribute to development of the metabolic syndrome. Although cholesterol regulation of SREBP's has been well characterized, regulation by nutritional cues is less defined. Previously, we identified an additional level of SREBP‐1 regulation whereby proteolytic maturation is controlled by levels of intracellular phosphatidylcholine (PC). Importantly, we have found that ablation of PC biosynthesis results in increased protein expression of nuclear, transcriptionally active SREBP‐1 in multiple metabolically active cells, including human hepatoma (HepG2) and mouse macrophage cells. Inhibition of PC biosynthesis may alter the lipid composition of intracellular membrane compartments leading to SREBP‐1 activation, and further experiments will elucidate the mechanisms involved in low‐PC based activation of SREBP‐1. All together, nutritional or genetic factors which block PC biosynthesis could inappropriately activate SREBP‐1, leading to increased risk for development of diseases associated with the metabolic syndrome. This work is funded by the NIH (R01 DK084352).