bluuebunny/crossref_metadata_2025 huggingface dataset:
Automated analysis of skeletal muscle fiber cross‐sectional area using CyteSeer image analysis program
Authors: Kostrominova Tatiana Y , Ingermanson Randall , Laris Casey A , Loy Patricia , McDonough Patrick M
Published: June 2021

The accurate quantification of skeletal muscle fiber cross‐sectional areas is desired in diverse areas of biomedical research. Majority of current studies employ time consuming analysis techniques with manual outlining of muscle fibers using Image J software. The goal of our study was to adapt CyteSeer, a recently developed automated image analysis program, for the quantification of skeletal muscle fiber cross‐sectional areas. Initial CyteSeer algorithm created masks located in the middle of the endomysium between adjacent muscle fibers. This caused the values of the CyteSeer analyzed muscle fiber cross‐sectional areas to be much larger (~25%) when compared with manual analysis of the same fibers. Subsequent modification of the CyteSeer algorithm allowed accurate quantification of skeletal muscle fiber cross‐sectional area. Automated analysis required only 5 seconds/image versus several hours/image needed for the manual analysis. Manual and automated analysis of muscle fiber cross‐sectional areas was performed side‐by‐side for samples from rats, mice, pigs, and monkeys. It showed very high degree of correlation with an average error of 3.2%. The results demonstrate that CyteSeer can be used for accurate automated quantification of skeletal muscle fiber cross‐sectional areas while greatly reducing time required for the analysis. Support: NIH R41AR55604 (PM).
Anatomy of the azygos lobe and vein in costoclavicular compression with decreased venous return in patients with thoracic outlet syndrome (TOS): MRI/MRA/MRV
Authors: Collins James D , Saxton Ernestina H , Ahn Samuel S , Gelabert Hugh , Carnes Alfred
Published: June 2021

The azygos lobe of the lung is a normal variant in 1% of anatomic specimens and 0.4% of chest radiographs. The right posterior cardinal vein, precursor of the azygos vein, invaginates the lung carrying pleural layers (azygos fissure) entrapping a portion of the right upper lobe. In thoracic outlet syndrome costoclavicular compression alters fascial planes, compressing the bicuspid valves in the draining veins of the supraclavicular fossa, neck and mediastinum, impeding venous return to the right heart and dilating veins proximal to the compression. The azygos vein dilates, particularly in compression of the brachiocephalic vein, providing collateral circulation from the abdomen, pelvis and thorax. Bilateral MRI/MRA/MRV displays the sites of costoclavicular compression of bicuspid valves within lymphatics, draining veins of the neck and supraclavicular fossae and the subclavian and axillary arteries with binding nerve roots (JNMA 1999; 91:333–341). Monitored multiplanar images with abduction external rotation and 2D Time of Flight MRA/MRV without contrast were acquired on a 1.5 Tesla GE Signa LX unit, 44 cm field of view, 512 × 256 matrix and saline water bags to enhance signal to noise ratio. We present two TOS patients with costoclavicular compression and impedance to venous return. One patient has an azygos lobe, the other a right aortic arch with anomalous course of the azygos vein.
Cloning of histone genes from Giardia lamblia for studies of post‐translational modification
Authors: Donkersloot John , Guerra Lupe , Lamphear Brenna , Best Aaron , Pikaart Michael
Published: June 2021

Covalent modifications of the N‐termini of core histone proteins are essential to gene transcription. One such modification is the monoubiquitylation of histone H2B, which has been shown in all eukaryotes tested so far. However, this process has not been examined in Giardia lamblia, a primitive eukaryotic parasite of the intestinal tract. Two techniques are being pursued to test for the monoubiquitylation of histone H2B: western blotting and liquid chromatography/mass spectroscopy (LC/MS). Discovering whether monoubiquitylated histone H2B occurs in G. lamblia will help to show when mechanisms of chromatin modifications arose in eukaryotes as well as provide important biological information about a dangerous parasite.
Binge alcohol inhibits hepatic class I and II histone deacetylases (HDACs): Potential mechanism for alcohol induced liver injury
Authors: Kirpich Irina , Gobejishvili Leila , Kharebava Giorgi , Zhang Jingwen , McClain Craig , Barve Shirish
Published: June 2021

Introduction

Binge, as well as chronic, alcohol consumption affects global histone acetylation and gene expression. It is becoming increasingly evident that these histone associated epigenetic modifications play a significant role in the development of hepatic pathology. The present work examined the effect of binge alcohol exposure on the expression of class I and II HDACs and their relevant contribution to histone acetylation changes.

Methods

C57/BL6 mice were gavaged 3 times (12 h intervals) with ethanol at a dose of 4.5 g/kg. Liver injury was evaluated by histopathology and plasma ALT activity. Hepatic HDAC gene expression was assessed by RT PCR.

Results

Mice binged with ethanol developed microvesicular liver steatosis with elevated serum ALT levels (49.07±5.61 U/L compared to 17.93±0.82 U/L in control group), and significantly increased liver triglycerides (48.02±3.83 vs. 19.90±3.48 mg/g liver). Notably, HDACs 1, 7, 9, 10, and 11 were down regulated while HDAC 3 was found to be up regulated. Suppression of both class I & II HDACs was associated with increased histone H3 acetylation.

Conclusion

Inhibition of both class I & II HDACs and consequent changes in histone H3 acetylation and gene expression by binge alcohol consumption potentially plays a significant role in the development of hepatic steatosis and injury. This work was supported by NIH grants.
Manipulation of protein splicing side‐reactions to facilitate protein purification and expressed protein ligation
Authors: Nadelson Adam C. , York Daniel J. , Dorval Deirdre M. , Lewandowski Katherine T. , Connor Katherine R. , Reitter Julie N. , Mills Kenneth V.
Published: June 2021

The PolII intein from Pyrococcus abyssi can be modified by mutation to facilitate cleavage of either the flanking N‐extein or C‐extein in a temperature dependent fashion. Taking advantage of cleavage at the intein N‐terminus, we have created a fusion protein with a target protein at the N‐terminus, followed by the intein and a C‐terminal His tag. The fusion protein is bound to an affinity resin via its C‐terminus, and cleavage is induced such that the N‐terminal target protein is highly purified in one step. We have also designed a fusion protein that takes advantage of the C‐terminal cleavage side‐reaction. This fusion protein has an N‐terminal affinity domain, followed by the intein and the C‐terminal target protein. The fusion protein is bound to an affinity resin via its N‐terminus, and the C‐terminal segment is cleaved and purified. As this C‐terminal segment may have an N‐terminal Cys, the purified protein can serve as a nucleophile for expressed protein ligation.

This material is based upon work supported by the National Science Foundation under Grant No. 0320824 and CAREER grant No. 0447647.
Investigating the Chaperone Ability of Two sHsp Isoforms Based on Protection of a Model Substrate
Authors: Klein Roger D. , Pett Virginia , Bouchard Robert
Published: June 2021

ZmHsp17.0‐CII and ZmHsp17.8‐CII are Class II small heat shock proteins (sHsps) found in maize (corn). These highly conserved sHsps protect client proteins from irreversible aggregation in times of stress. Each sHsp was expressed as a fusion protein with a chitin‐binding domain on the C‐terminus that allows for chitin affinity column purification. The increase in light scattering of the model substrate citrate synthase (CS) after heat shock at 43 °C in solution was measured using a fluorescence spectrophotometer to detect protein aggregation. By comparing the light scattering curves of CS alone with a solution containing CS plus varying amounts of ZmHsp17.0‐CII and ZmHsp17.8‐CII, a clear, consistent reduction in aggregation was seen for all molar ratios of sHsp monomer: CS monomer of 1:1 and greater, with greater reduction at higher concentrations. In addition, more efficient substrate protection was seen in ZmHsp17.8‐CII than in ZmHsp17.0‐CII, which could indicate different substrate specificity or different chaperone capability of the two proteins. These results demonstrate that ZmHsp17.0‐CII and ZmHsp17.8‐CII are concentration‐dependant molecular chaperones.

This work was supported in part by a grant from The College of Wooster, and from the Howard Hughes Medical Institute through the Undergraduate Science Education Program.
Pre‐steady State Kinetic Analysis of Covalent Intermediates on the E. coli Pyruvate Dehydrogenase Complex
Authors: Balakrishnan Anand , Jordan Frank
Published: June 2021

Synthesis of [C2, C6′‐ 13C2]thiamin diphosphate (ThDP) enabled first detection by 1‐D 1H‐13C HSQC NMR of ThDP‐derived intermediates on the E1 component of the pyruvate dehydrogenase complex (PDHc) from E. coli, both in E1 and in the assembled complex. Using rapid chemical quench, pre‐steady state analyses were performed on the PDHc and on its E401K‐E1 and H407A‐E1 variants. The enamine intermediate (detected after acid quench as C2‐alphahydroxyethylThDP) forms at a rate of 80 s‐1 on both the E1 component and on PDHc, while the rates for both are > 100x slower for the E401K‐E1 and H407A‐E1 PDHc variants. Assembly to complex increases the rate of enamine formation for the E401K‐E1 variant ten‐fold. Formation of 2‐acetylThDP could be detected on the E1 component during its reaction with pyruvate and the artificial electron acceptor DCPIP. The rate of formation of this intermediate parallels that of the enamine, probably because protonation of the enamine on the enzyme is much slower than its oxidation. Apparently, the enamine is very well stabilized by the enzyme. On the E401K‐E1 variant 2‐acetylThDP could not be detected, in this case, protonation of the enamine being faster. The C2‐alphalactylThDP intermediate could not be detected under any of the conditions used, indicating that its formation slower than its decarboxylation. The method developed is applicable to all such complexes. Supported by NIH‐GM050380.
Altered binding of eukaryotic Elongation Factor 3 (eEF3) to the ribosomes: A key to understanding its functional requirement in yeast
Authors: Sasikumar Arjun Narayan , Kinzy Terri Goss
Published: June 2021

Translational elongation is a universally conserved process. In eukaryotes this involves two G‐proteins: eukaryotic Elongation Factor 1A (eEF1A), required for loading the cognate aminoacyl‐tRNA at the A‐site of the ribosome and eukaryotic Elongation Factor 2 (eEF2), essential for the translocation of the ribosome along the mRNA. Fungal translation is unique in its requirement for the ATPase eEF3. An essential protein with no known mammalian homologue, eEF3 is an ideal anti‐fungal drug target. eEF3‐ribosome binding is modulated by Stm1p, a non‐essential protein implicated in translation under nutrient stress. Our work shows that knocking out Stm1p adversely affects the ability of Cryptococcus neoformans eEF3 to complement the loss of WT eEF3 in Saccharomyces cerevisiae, reinforcing the functional relationship between the two. We also show that mutations in the chromodomain of eEF3 affect ribosome binding and cause semi‐dominant slow growth phenotypes. Studying the effects of altered ribosome binding of eEF3 will provide insights in its role in fungal translation. This work is supported by the National Institutes of Health.
An effective docking model for rapid screening of potential xanthine oxidase inhibitors
Authors: Stockert Amy , Fakunmoju Layomi , Mahfouz Tarek
Published: June 2021

Rational drug design involves the examination of enzyme mechanism and active site structure in order to develop a compound which is predicted to alter the enzyme function. Docking is a method which predicts the preferred orientation for ligand binding. Docking results can be extrapolated to find inhibitors for target proteins. We use Glide (Grid‐base Ligand Docking with Energetics) to generate ligand poses to search for possible locations of ligand molecules in the active‐site region. We selected xanthine oxidase as our model based on the largely understood kinetic mechanism and available crystal structure. We used computer calculations to investigate the different binding modes or docking energies for each potential compound. We downloaded available protein structures (xanthine oxidase from cow) and 1jr0 (xanthine dehydrogenase from Rhodobacter capsulatus) from the protein databank website. We examined potential active site modifications based on the available mechanistic information including: ionization states, cofactor bonds, and molybdenum bond order. Mutant active sites were similarly modified. The docking model was further defined by assigning grids to limit the active site region as well as the ligand binding region. Docked ligands were limited to those with available kinetic characterization in order to provide comparison following completion of computational analysis. Correlation of docking energies to available kinetic parameters identifies this model as an effective tool for the screening of potential xanthine oxidase inhibitors.
Short‐term feeding effects of origanum syriacum crude extract on blood constituents in rats
Authors: Al‐Qirim Tariq , Shahwan Moyad , Bader Ammar
Published: June 2021

This study was carried out to investigate the effect of the whole crude extract of Origanum syriacum on different biochemical parameters in blood using animal model. The aerial parts of Origanum syriacum were extracted with petroleum ether, chloroform, methanol and water, all these extracts were combined to prepare the whole extract then administrated orally in two doses 100mg/kg and 400mg/kg. The results obtained indicate that the extract showed a significant decreasing effect on low density lipoprotein (LDL‐cholesterol), triglycerides and glucose in both doses, while a non significant increase of urea and high density lipoprotein (HDL‐cholesterol) was observed as compared to normal saline treated controls. This study showed that the whole extract of Origanum syriacum possess a potential pharmacological activity in lowering the major lipid profile parameters in which we have reported for the first time.

This work is sponsored by Al‐Zaytoonah University of Jordan.
A Novel Control Point in Cholesterol Biosynthesis
Authors: Gill Saloni , Stevenson Julian , Kristiana Ika , Brown Andrew J.
Published: June 2021

Targeting the cholesterol synthesis pathway has proved invaluable for the treatment of heart disease, as exemplified by the success of the statins. This class of drugs targets 3‐hydroxy‐3‐methyl‐glutaryl‐coenzyme A reductase (HMGCR), which is billed as the ‘rate‐limiting’ enzyme. Perhaps due to the success of the statins, relatively little is known about the other enzymes involved in cholesterol synthesis, including squalene monooxygenase (SM). SM carries out the first oxygenation step on squalene, before cyclization into the basic steroid structure. Our aim was to investigate the regulation of SM using Chinese hamster ovary (CHO) cells as a model cell system. Surprisingly, cholesterol treatment caused the accumulation of squalene, suggesting that SM may serve as a ‘rate‐limiting’ enzyme beyond HMGCR. This squalene accumulation was also seen in mutant CHO cells that have sterol‐independent transcription, implying that cholesterol regulates SM post‐transcriptionally. Indeed, we found that SM protein was degraded within hours of cholesterol treatment. Proteasomal inhibition blocked SM degradation and reversed the squalene accumulation observed with cholesterol treatment, indicating that the cholesterol‐induced degradation of SM is a flux controlling step in cholesterol synthesis. Therefore, we have identified a significant new control point in the feedback regulation of cholesterol synthesis.
Effects of fatty acids on the growth of beta‐cells and on albumin‐mediated cholesterol effflux from endothelial cells
Authors: Tuei Vivian C. , Ha Ji‐Sook , Bhagavan Nadhipuram V. , Ha Chung‐Eun
Published: June 2021

There is mounting evidence that free fatty acids (FFAs) plays a role in the pathogenesis of diabetes and cardiovascular diseases. However, the effects of human serum albumin (HSA)/FFA complexes on ß‐cells as well as on HSA‐mediated cholesterol efflux are not well known. Thus, we exposed the insulinoma ß‐cells, HIT‐TI5 to different HSA/FFAs complexes for 24hrs followed by assessment of insulin secretion and cell viability. Also, a human endothelial cell line, EA.hy926 was used as the model system to study cholesterol efflux. In the presence of HSA, palmitate and stearate induced significant cell death at 0.1mM or higher, which was not significantly inhibited by the two apoptosis inhibitors, cyclosporin A and ZVAD‐FMK. In contrast, myristate, palmitoleate, oleate, and elaidate bound to HSA showed minimal effects on cell viability. Additionally, insulin secretion was significantly reduced by HSA/oleate complexes. We also found differential effects on cell viability by HSA mutants/FFAs complexes. For efflux studies, HSA complexed to oleate or palmitate at 1:5.3 molar ratios showed significant reduction in cholesterol efflux but not with arachidonate. In summary, this study showed that FFA‐induced changes of cellular functions are dependent upon the types and concentrations of FFAs which might be modified by HSA variants.

[Funding: Hawaii EXPORT Centre, HCF, and an award from the American Heart Association].
Lipidomic analysis of carrageenan‐induced hyperalgesia reveals bilateral eicosanoid production in the spinal cord
Authors: Dumlao Darren Sabio , Buczynski Mathew , Svensson Camilla , Fitzsimmons Bethany , Wirkus Jennifer , Jacobsen Faith , Hua Xiao‐Ying , Yaksh Tony , Dennis Edward
Published: June 2021

Using a high throughput mass spectrometric lipidomics approach, we determined the contribution of bioactive lipids to carrageenan‐induced hyperalgesia. Quantitative analysis of CSF and spinal cord tissue for 171 different eicosanoids, ethanolamides and fatty acids revealed 102 distinct lipid species. There was a bilateral time‐dependent increase of 12‐lipoxygenase (12‐LOX) metabolites at 2 hours, cyclooxygenase (COX) metabolites at 8 hours, and anandamide between 12 and 24 hours in spinal cord. Systemic treatment with morphine, the COX inhibitor ketorolac, or the LOX inhibitor NDGA were used to investigate the correlation between spinal bioactive lipid levels and hyperalgesia. The inhibitors reduced allodynia but affected lipid profiles differentially. Morphine did not alter the lipid profile, while ketorolac and NDGA caused a global reduction of many bioactive lipid mediators. These findings suggest that COX and 12‐LOX play an important role in the induction of carrageenan mediated hyperalgesia potentially via cross talk between their pathways. This work was supported by GM64611 and GM069338 (E.A.D.).
JAM‐A regulates the progression of Non‐alcoholic Fatty Liver Disease (NAFLD)
Authors: Kissig Megan Elizabeth , Naik Ulhas P.
Published: June 2021

Non‐alcoholic fatty liver disease (NAFLD) is characterized by an abnormal amount of fat accumulation in the liver, specifically more than 5% fat by weight. Little is known about how the fat accumulates in the liver, but it has been found that intestinal permeability due to leaky tight junctions may be a contributing factor. Our lab studies junctional adhesion molecule‐A (JAM‐A), a protein located at the tight junctions of epithelial and endothelial cells. Through its ability to homodimerize at the apical part of the lateral membrane, JAM‐A helps regulate the permeability and stability of the junction.

This study's aim was to find what effect JAM‐A has on the development of NAFLD. To analyze this relationship, groups of Jam‐A (+/+) and Jam‐A (−/−) mice were put on either a high‐fat or low‐fat diet for 20 weeks. During this time the mice were weighed every two weeks and blood samples were taken every four weeks. At the end of the 20 weeks, the mice were sacrificed and the livers and fat pads were removed.

We found that the variation in weight between the Jam‐A (−/−) groups was greater than that between the Jam‐A (+/+) groups. Also, the average area of the adipocytes in the high‐fat Jam‐A (−/−) group was greater than the average area in the high‐fat Jam‐A (+/+) group. Since the liver sends fat to tissues in the form of cholesterol, we compared the levels in the plasma. The high‐fat Jam‐A (−/−) mice had significantly higher LDL‐cholesterol and total cholesterol levels in the plasma than the other groups as well. After examining the stained sections of the livers, it was found that the livers of the high‐fat Jam‐A (−/−) mice showed more fat droplet accumulation than the high‐fat Jam‐A (+/+) mice. This suggests that ultimately the absence of JAM‐A from the tight junctions promotes the development of NAFLD. This project was funded by the Arnold and Mabel Beckman Foundation.
Inhibition of Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9) secretion by prodomain overexpression or pefabloc treatment as measured by a luciferase assay
Authors: De Vries Luc , Robichon Céline , Cathala Claudie , Perez Michel , Junquero Didier , Cussac Didier
Published: June 2021

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protease that acts as a regulator of circulating plasma cholesterol levels by enhancing the endocytosis of the low density lipoprotein receptor (LDL‐R) at the cell surface of hepatocytes. In this study, we show that overexpression of PCSK9's prodomain reduces the secretion of its mature form, as measured by a newly developed PCSK9‐luciferase assay. Similarly, treatment of cells with the non‐specific permeable serine protease inhibitor pefabloc decreases the secretion of mature PCSK9. We show by overexpression and immunoprecipitation that the prodomain is interchangeable on the catalytic domain of PCSK9. Medium from PCSK9‐transfected CHO cells overexpressing the prodomain or treated by pefabloc leads to enhanced surface LDL‐R expression in hepatic cells in contrast to medium from PCSK9‐transfected CHO cells only, demonstrating the functional consequences of both modes of inhibition.
Single tryptophan mutants of apolipophorin III: binding interaction to lipopolysaccharides
Authors: Martinon Daisy , Weers Paul
Published: June 2021

Apolipoproteins have potential to neutralize lipopolysaccharide (LPS) toxicity. To gain insight in this binding interaction, apolipophorin III (apoLp‐III) from the Greater wax moth, Galleria mellonella, was used as a model. Single tryptophan mutants of apoLp‐III were engineered to determine binding interaction with the three components of LPS (Lipid A, core and O‐antigen polysaccharides). Five single‐trytophan mutants (F20W, L80W, L119W, I138W and F145W) were designed and produced in a bacterial expression system. Based on circular dichroism analysis, the secondary structure of the mutant proteins was similar to that of the wild‐type protein. The protein stability, measured as the resistance to guanidine‐HCl induced denaturation, was slightly decreased. This indicates that the overall a‐helical fold was maintained in the mutant proteins. Tryptophan fluorescence spectroscopy was used to analyze the LPS binding interaction. Binding to LPS resulted in an increase in tryptophan fluorescence emission intensity for I138W and F145W‐apoLp‐III, a decrease for F20W and L119W‐apoLp‐III, and no significant change was observed for L80W‐apoLp‐III. Thus the tryptophan residues relocate into distinct environments, indicating that the apoLp‐III helices bind to different parts of LPS.
The role of fibronectin in microfibril assembly and homeostasis
Authors: Sabatier Laetitia , Fagotto‐Kaufmann Christine , Chen Daliang , Annis Douglas S. , Mosher Deane F. , Reinhardt Dieter P.
Published: June 2021

Fibrillins are members of a protein family that constitutes the backbone of extracellular microfibrils. Assembly of fibrillins into microfibrils is often compromised in pathological situations such as the Marfan syndrome and other fibrillinopathies. We have used a cellular model system to analyze assembly of fibrillin‐1 in dependence of fibronectin. Fibrillin‐1 assembly was strictly dependent on the presence of fibronectin fibers. Immunofluorescence and electron microscopy experiments showed that fibrillin‐1 and fibronectin colocalized to the same fiber system at early stages of the assembly of both proteins. Using a fibronectin assembly inhibiting peptide (FUD), we observed that homeostasis of fibrillin‐1 fibers is dependent on the constant availability of fibronectin fibers. Solid phase binding assays demonstrated that fibronectin interacted with the C‐terminal region of all fibrillins. We also demonstrated that multimerization of the C‐terminal half of fibrillins is required for interaction with fibronectin. Further interaction mapping studies revealed that fibrillins require the complete collagen/gelatin site in fibronectin. FUD inhibits the fibrillin‐1 interaction to fibronectin likely through conformational changes in the gelatin binding domain of fibronectin. In summary, our results show that fibrillin network formation and homeostasis requires fibronectin.
Asparagine Linked Oligosaccharides Present on a Non‐Consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies
Authors: Valliere‐Douglass John F. , Eakin Catherine M. , Kodama Paul , Mujacic Mirna , Brady Lowell J. , Wang Wes , Wallace Alison , Yan Boxu , Reddy Pranhitha , Treuheit Michael J. , Balland Alain
Published: June 2021

We report that N‐linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NXS/T where X is not proline. Characterization of a recombinant human IgG2 antibody indicated that an Asn residue in the CH1 sequence TVSWN162SGAL was glycosylated. This highly atypical modification has also been observed in IgG1 antibodies derived from human donors. Site directed mutagenesis of the CH1 domain sequence in a recombinant‐human IgG1 antibody resulted in an increase in non‐consensus glycosylation to 3.15%, a greater than four fold increase over the level observed in the wild‐type, by changing the ‐1 and +1 amino acids relative to the Asn residue at position 162. Following these results, we developed enrichment techniques to isolate molecules with non‐consensus N‐glycans based on selective endo‐ and exoglycosidase digestion followed by lectin chromatography. By applying our enrichment techniques, we subsequently discovered additional occurrences of non‐consensus glycosylation that are present on amino acid sequences not previously described. Modeling of these residues from crystal structures provided valuable information of structural characteristics shared by these sites. These new findings shed light on the sequence requirements necessary for N‐glycosylation when there is not a Ser or Thr residue present in the +2 position with respect to the glycosylated Asn. We believe that further understanding of the phenomenon of non‐consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery.
Kinetics of yeast dissociation from lectin beads: I. alpha methyl mannose
Authors: Zem Gregory C. , Dreyfuss J , Allen J , Kawashima R , Daco E , Kanda Y , Yaghoobian J , Danialian S , Givens W , Baghoomian A , Zograbyan N , Haroutounian S , Bimanand N , Silani S , Khorrami H , Shirazi M Hashemieh , Keihani K , Tafreshi G , Jahanbakhsh A , Solati A , Balazadeh H , Phan H , Papazian L , Moayer A , Oppenheimer S. B.
Published: June 2021

Here we examine the kinetics of alpha methyl mannose induced dissociation of yeast (Saccharomyces cerevisiae) that have been previously allowed to bind to concanavalin A (Con A) derivatized agarose beads, a model for eluting glycans from Con A beads in purification protocols and for releasing pathogens from human cells. This study tests the statistical reliability of this model using one glycan concentration with hundreds of replicate experiments. Future dose‐response studies will precisely identify the reagents that are most effective at the lowest concentrations. In 963 trials, yeast release from the Con A beads was measured by counting yeast remaining on single yeast bound beads in the presence and absence of 1.5M alpha methyl mannose over a 60 min time course. Two‐ sample t tests showed significant differences in the amount of yeast remaining bound in the sugar versus control (no sugar) samples after 20 min (p less than 0.0001), 40 min (p less than 0.0001) and 60 min (p less than 0.0001), where the sugar caused substantially more dissociation than in its absence. Repeated measures‐ANOVA indicated a significant effect of the glycan treatment on the number of bound yeast (F= 624, p less than 0.0001), as well as a significant interaction between the treatment and time elapsed (F= 339, p less than 0.0001). This is therefore a reliable, simple, precise and inexpensive model for dissociation kinetic studies. Two other studies in these proceeding using different reagents support this conclusion (Supported by NIH NIGMS SCORE (S0648680), RISE, MARC, the Joseph Drown Foundation and the Sidney Stern Memorial Trust).
Matrigel„§ invasion and migration of capillary endothelial cells are blocked by Tunicamycin a glycosylation inhibitor
Authors: Banerjee Aditi , Duran Elizabeth , Baksi Krishna , Banerjee Dipak K
Published: June 2021

Angiogenesis is a hall mark of breast tumor growth, progression and metastasis, and requires a critical balance between pro‐ and anti‐angiogenic factors. Our laboratory has been studying the key issue(s) of protein N‐glycosylation to regulate angiogenesis and the capillary invasion. We have observed that (i) eFactor VIIIC (a Mr 270 kDa asparagine‐linked glycoprotein) expression precedes capillary endothelial cell proliferation; and (ii) the presence of anti‐Factor VIIIC monoclonal antibody makes cells less invasive. The role of eFactor VIIIC N‐glycans on tissue invasion was then tested in cells treated with tunicamycin. Considerable inhibition of Matrigel™ invasion raised a question of matrix metalloproteinases (MMPs) and analyzed after treating cells with tunicamycin (1μg/ml). The expression of MMP‐9, MMP‐2 and TIMP‐2 was quantified by western blotting. MMP‐9 and MMP‐2 were down‐regulated but TIMP‐2 was upregulated. This was supported by QRT‐PCR and gelatin zymography. To verify the effect in vivo we have used Matrigel™ implants in nude mice and observed reduced number of blood vessels following tunicamycin treatment. Quantitative assessment of paraffin sections of the plugs histologically or immunohistochemically for CD34 and CD144 confirmed reduced microvessel density as well. Cell migration was also inhibited. We conclude that tunicamycin targets tissue invasion and cell migration, and eliminate breast tumor growth. Supported by NIH U54‐CA096297 and Komen for the Cure BCTR06582 (DKB), and NIH/NCRR/RCMI G12‐RR03035 (KB).
Age related decrease in post‐synaptic signaling components and caveolin‐1 in membrane/lipid rafts and synaptosomal membrane fractions in the CNS
Authors: Bonds Jacqueline , Patel Hemal H. , Roth David M. , Peart Jason , Patel Piyush M. , Head Brian P.
Published: June 2021

Several synaptic resident proteins involved in neurotrophic signaling are compartmentalized to membrane/lipid (M/L) rafts. Their expression and localization to discrete microdomains is dependent on cholesterol binding and scaffolding protein caveolin (Cav). Consequently, Cav may serve to organize synaptosomal associated proteins within the M/L rafts and thus be a key mediator of protective neuronal signaling. We investigated the effect of aging on M/L raft protein composition. Sucrose density fractionation was performed on brains from three age groups of C57BL/6 mice: young (Yg), mature (Mat) and aged (Ag). The majority of Cav‐1 was detected in buoyant fractions (BFs) 4 and 5 of Yg brains. However, Cav‐1 was reduced in the BFs of Mt and Ag brains. IPs of Mt and Ag BFs showed a reduction in the co‐IP between Cav‐1 and PSD‐95 as well as almost no detection of NR2A, NR2B and AMPAR. Isolation of synaptosomal membranes from Yg Cav‐1 KO mice showed a reduction in PSD‐95 and NR subtypes compared to wild type. These findings demonstrate that cav expression and its ability to properly organize signaling complexes may be a key determinant of age related changes in synaptic and neuroprotective signaling.
Uncovering Novel Mechanisms for Limiting the Accumulation of Isoaspartyl‐Damaged Proteins
Authors: Patananan Alexander Nikolich
Published: June 2021

Although most organisms require protein L‐isoaspartyl O‐methyltransferase (PIMT) for the repair of isoaspartyl‐damaged proteins, recent studies have shown that Saccharomyces cerevisiae lacks any enzyme resembling PIMT. Using high‐performance liquid chromatography, matrix‐assisted laser desportion/ionization mass spectrometry, and a biochemical radiolabeling approach, the levels of isoaspartyl protein damage in S. cerevisiae were determined to be similar to other organisms with PIMT. Addition of 10 μM MG‐132 and/or 5 mM 3‐methyladenine proteasome and autophagy inhibitors, respectively, indicated that autophagy and the proteasomal degradation pathways both were important in the removal of isoaspartyl‐damaged proteins in specific environments and growth phases. Interestingly, combining various synthetic peptides with S. cerevisiae extract lysed without protease inhibitors resulted in the favorable cleavage of the isoaspartyl‐containing synthetic peptide KASAisoDLAKY, suggestive of an isoaspartyl‐specific protease. Understanding which proteins are most susceptible to damage, and the proteolytic pathways that prevent their accumulation, may aid in the development of therapeutic interventions that enhance repair pathways and thus the human healthspan. This research was supported by the UCLA Cellular and Molecular Training Grant.
GAMT, a p53‐Inducible Modulator of Apoptosis, Is Critical for the Adaptive Response to Nutrient Stress
Authors: Chu Kiki , Ide Takao , Brown‐Endres Lauren , Lee Sam W
Published: June 2021

The p53 tumor suppressor protein has a well‐established role in cell fate decision‐making processes through many mechanisms. However, recent discoveries indicate that p53 has much broader cellular functions that are non‐tumor‐suppressive roles, such as regulating glucose metabolism and mitochondrial respiration. Here, we identify GAMT (guanidinoacetate methyltransferase), an enzyme involved in creatine synthesis, as a novel p53 target gene and a key downstream effector of adaptive response to nutrient stress. We show that GAMT is not only involved in p53‐dependent apoptosis in response to genotoxic stress but is important for apoptosis induced by glucose deprivation. Additionally, p53→GAMT up‐regulates fatty acid oxidation (FAO) induced by glucose starvation, utilizing this pathway as an alternate ATP‐generating energy source. These results highlight that p53‐dependent regulation of GAMT allows cells to maintain energy levels sufficient to undergo apoptosis or survival under conditions of nutrient stress. p53→GAMT pathway represents a new link between cellular stress responses and processes of creatine synthesis and FAO, demonstrating a further role of p53 in cellular metabolism.
Salidroside inhibits clinorotation‐induced apoptosis in pulmonary microvascular endothelial cells by activating PI3k/Akt pathway and inactivating of caspase 3
Authors: Kang Chunyan , Yuan Ming , Zhou Lin , Zhang Ye , Liu Changting
Published: June 2021

We have previously demonstrated that clinorotation induced apoptosis in microvascular endothelial cells, but the underlying mechanisms are still not clear. In this study, we test the hypothesis that clinorotation could induce apoptosis in human pulmonary microvascular endothelial cells (HPMVEC) by inhibiting the PI3K/Akt pathway and activating caspase‐3. TUNEL assay and flow cytometric analysis indicated that 72h clinorotation significantly induced apoptosis in HPMVEC. Western blot results showed that clinorotation inhibited the PI3K protein expression and phosphorylation level of Akt. Caspase‐3 activity assay showed that clinorotation increased the activity of caspase‐3.Salidroside, one of main active ingredients extracted from the root of Rhodiola rosea, inhibited the apoptosis and attenuated the decrease of PI3K protein expression and phosphorylation level of Akt. Furthermore, salidroside also antagonized the activation of caspase‐3. Our results suggest that PI3K/Akt pathway and caspase‐3 involve in the apoptosis of HPMVEC after clinorotation, this effect can be reversed by salidroside, which may have a potential application in astronauts during spaceflight.(Funded by PHRP 06Z048)
Inflammatory Cytokines Induce Oxidative Stress And Apoptosis In PC12 Cells
Authors: Habib Nawaf , Goswami Gauri , Mungre Sue
Published: June 2021

Inflammation causes oxidative stress, which plays a critical role in neurodegenerative diseases such as multiple sclerosis and diabetic neuropathy. We have shown that PC12 cells exposed to metals such as manganese and cobalt (which causes oxidative stress) underwent apoptotic cell death. Treatment with reducing agents such as glutathione reversed the effect, suggesting oxidative stress processes were involved. Caspase activity was induced, indicating that cell death was apoptotic. It is known that inflammation contributes to progression of the disease. We investigated whether inflammatory cytokines contribute to cell death by activating oxidative pathways. PC12 cells treated with interferon gamma showed 60% loss in viability. Inhibitors of caspase‐3, −8 and −9 activity prevented cell death. Inducible nitric oxide synthase gene transcription was upregulated within 6 hours of treatment with IFN gamma as well as TNF/LPS treatment. Increase in the level for nitrite was observed using Griess reagent. and might be selectively exposed to iNOS‐mediated oxidative damage as a consequence of elevated TNFα levels. Our results suggest that oxidative stress induces cell death involving iNOS and caspase induction.
Cloning and expression of a putative type II metacaspase from Schizophyllum commune
Authors: Betancourt Dillon , Fox Kristin M.
Published: June 2021

Metacaspases are cysteine proteases similar in sequence to caspases. In Saccharomyces cerevisiae they undergo caspase‐like processing and have proteolytic activity. Experiments in several organisms have shown that metacaspases function similarly to caspases and are involved in apoptosis, although some work suggests that they are non‐apoptotic proteases. Type I metacaspases have a proline rich domain at the N‐terminus and Type II metacapases contain an linker region between the p20 and p10 subunits, but lack the proline rich domain. Five metacaspase genes are present in the genome of S. commune and one of them, scp1‐a type I, has been cloned and expressed in E.coli. In this report a type II metacaspase from S. commune, scp3, was successfully cloned into pQE‐80. Due to a mis‐predicted intron/exon boundary in the genome there is a deletion of a 74bp sequence, 640bp after the start codon in the cloned open reading frame. Next, scp3‐pQE‐80 was successfully expressed in E. coli strain M15[pREP4] and purified using Ni+ affinity chromatography. Research supported by Merck/AAAS.
Developmental regulation of caspases during death and regeneration events in an invertebrate chordate
Authors: Robison Leah Danielle , Lauzon Robert
Published: June 2021

Programmed cell death (PCD) by apoptosis is an essential process in the development and maintenance of multicellular animals. We study the colonial marine urochordate Botryllus schlosseri which undergoes synchronized apoptosis throughout its entire adult generation every week. During a colony's death phase, the old adult cells are engulfed and recycled by circulating phagocytes simultaneously as asexually‐derived buds reach functional maturity. Thus, Botryllus is a unique model organism to study the cyclical regulation and function of cysteinyl aspartases called “caspases” during organismal apoptosis. It is well documented that caspases ‐8 and ‐9 play a role in apoptosis initiation, while caspases ‐3 and ‐7 are used in its execution. We have investigated whether caspase expression is developmentally regulated during asexual development in Botryllus. An expressed sequence tag (EST) sequencing project has uncovered several candidate sequences of the apoptotic death program, including several caspase orthologs. Using standard, semi‐quantitative PCR, we have previously demonstrated that initiator and executioner caspases are developmentally regulated in Botryllus, and exhibit maximal expression at the onset of a colony's death phase. However, as these experiments made use of genotypically distinct colonies for comparison, the effects of genetic background on caspase expression could not be excluded. In the current study, we sought to investigate the regulation of caspase expression using clonal replicates and quantitative polymerase chain reaction (Q‐PCR). Experiments are currently underway to determine whether caspase expression is associated with specific death and regeneration events in the lifecycle of Botryllus.

Source of research support: Union College Internal Education Fund.
Advanced Glycation End Products activate MMP‐9 secretion in macrophage cells, the involvement of MAP Kinase and NF‐kappaB signaling pathways
Authors: Banker Greg , Zhang Fan , Kent K. Craig , Liu Bo
Published: June 2021

Matrix metalloproteinases (MMPs) are integral to pathogenesis of vascular diseases including abdominal aortic aneurysms (AAAs). We have previously shown that advanced glycation end products (AGEs) and their receptor RAGE are critical to AAA formation, in part through a novel function of AGEs, i.e. to induce MMP‐9 expression. The purpose of this study is to investigate the signal transduction mechanisms underlying AGE's regulation of MMP‐9. Using the RAW 264.7 macrophage cell line, we found that AGE (300 μg) activates multiple signaling pathways. The induction of all three MAP Kinases peaked between 30–60 min while the induction of NF‐κB peaked after 4 h, suggesting NFκB may be downstream of the MAP Kinases in the regulation of MMP‐9. Inhibition of ERK activity with PD098059 or UO126 (10–20 μM) and NFκB with PAO (5–20 μM) dose dependently decreased the amount of MMP‐9 induced by AGE. Inhibition of JNK with SP600125 or BI7803, however, further stimulated MMP‐9 expression induced by AGE. Similarly, inhibition of JNK potentiated the induction of MMP‐9 by TNF‐α. Taken together, our data suggest that AGE stimulates MMP‐9 expression through a mechanism involving ERK and NFκB. JNK MAP kinase seems to play a negative role in AGE signaling in macrophages. We believe that further exploration of the mechanism of AGE regulated MMP‐9 will further our understanding of the signaling network that may contribute to the pathogenesis of AAA.
Glycogen‐Synthase Kinase‐3beta/beta‐Catenin Axis Promotes Myocardial Angiogenesis: Role of VEGF
Authors: Thirunavukkarasu Mahesh , Maulik Nilanjana
Published: June 2021

The GSK‐3β/β‐catenin axis is one of most interesting pathways involved in the molecular mechanism of angiogenesis. Recently we have shown the critical role of GSK‐3β/β‐catenin in ischemic preconditioning (IP) mediated angiogenesis. We have observed for the first time that IP induces the β‐catenin driven expression of vascular endothelial growth factor (VEGF), Bcl‐2, survivin in chronic rat myocardial infarction (MI) model increasing the vessel density improving myocardial function. β‐catenin is found to be a critical mediator during angiogenesis & it is phosphorylated by GSK‐3β. During IP induction of the PI‐3 kinase pathway leads to phosphorylation of GSK‐3β, leading to the release, accumulation of β‐catenin in cytosol & the subsequent translocation of β‐catenin into the nucleus drives the expression of its target genes (VEGF, Bcl‐2, survivin) through activation of the T‐cell transcription factors/lymphoid‐ enhancer binding factor transcription factors. These astounding results paved the way for the induction of angiogenesis in the ischemic heart by administration of β‐catenin after an MI which depicted cardioprotection as was observed with IP. The efficacy of β‐catenin overexpression in the induction of collateral vessel formation remodeling was further ascertained by Adeno –shRNA‐β‐catenin to silence the gene expression of β‐catenin thereby abolishing the IP mediated cardioprotection.
Functional roles of the AMP‐activated protein kinase in Huntington's disease
Authors: Chern Yijuang , Ju Tz‐Chuen
Published: June 2021

Huntington's disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats in Huntingtin (Htt) gene. Accumulated evidence from different laboratories suggests the involvement of energy metabolism, excitotoxicity, and oxidative stress in HD pathogenesis. The common pathways linking Htt mutation with energy deficiency in HD remain largely unknown. We reported herein that a metabolic sensor (AMP‐activated protein kinase, AMPK) was overactivated in striatal cell lines (ST14A; STHdhQ7, STHdhQ109), as well as brains of a transgenic mouse model of HD (R6/2) and human HD patients. AMPK is a downstream mediator of many different types of stress. It composes a catalytic (α) and two regulatory subunits (β and γ), and can be activated by CaMKK and LKB1 via phosphorylation at the threonine residue 172 of AMPKα. In addition to suppressing metabolic enzymes (e.g., acetyl‐CoA carboxylase) and reducing ATP consumption upon stress, AMPK also phosphorylates a wide variety of proteins with different functions. We demonstrated in the present study that an agonist (CGS21680) of the A2A adenosine receptor (A2A receptor) not only produced beneficial effects on several major symptoms of HD in R6/2 mice, it also normalized the overactivated AMPK via a cAMP/protein kinase A‐ dependent pathway. Our findings shed new light on the metabolic aspects as well as the protective effects of cAMP‐elevating reagents in HD.
Heat shock protein 90 inhibitors exhibit anti‐inflammatory response and differential cytoprotection pattern from oxidative stress‐ induced injury.
Authors: Kacimi Rachid , Yenari Midori
Published: June 2021

Geldanamycin have emerged as promising therapeutic agents in cancer. Accordingly, we tested the role of GA, 17AAG and radicicol in LPS‐ activated microglia. BV2 cells were treated with LPS with or without GA analogs. 24hr later, media and cell extracts were assayedfor NO and iNOS protein; in parallel injury was examined by viability assays. Pretreatment with GA and its analogs markedly prevent iNOS/NO accumulation elicited by LPS. Conversely, GA and its analogs prevented LPS ‐induced subsequent BV2 death. In addition, treatment of astrocytes with GA for 24hr induces increase of HSP70i expression. Moreover, we found that GA protect from oxidative stress induced by H2O2 in glial cells. To examine the mechanisms of delayed protection in LPS induced injury, cells were incubated with GA and analogs for 1 day, thereafter, cells were subjected to LPS for another 24hr. GA and to lesser extend 17AAG but not radicicol showed a partial blunting in pro‐inflammatory induction of iNOS/NO accumulation and display protection in the delayed window from LPS ‐induced BV2 cell death. Our data together, showed that GA and its analogs exhibit dual protection in brain resident cells: acutely by blocking the signaling that drive inflammatory response elicited by endotoxin or TLR3 agonist, and a delayed protection against oxidative stress. These data support usefulness of the HSP90 targeting to protect from neuro‐inflammatory disease.