bluuebunny/crossref_metadata_2025 huggingface dataset:
PROSTATIC HOX GENES: EXPRESSION DURING DEVELOPMENT AND IN TRAMP TUMOR CELLS
Authors: Wang Billy C , Keith Ashena L , Pira Charmaine U , Feenstra Jennifer M , Oberg Kerby C
Published: June 2021

Hox transcription factors play critical roles during development and reactivation may participate in tumorigenesis. For example, some prostate cancers overexpress androgen sensitive HOXC8, while others upregulate androgen insensitive HOXB13. A comprehensive Hox expression profile during prostate development has not been described. We generated a prostatic Hox expression profile in an effort to identify candidate Hox genes critical to prostate development and potentially tumorigenesis.

Mouse prostates at various stages of development [0, 2, 6, 13, and 24 weeks of age] were collected RNA isolated, cDNA generated and semi‐quantitative PCR performed. We also examined Hox expression in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) cell line. Hox genes with profiles of interest were subjected to quantitative PCR for confirmation.

Hoxa10 is expressed through out prostate development and in TRAMP cells implicating it as a marker of prostate tissue similar to its corresponding expression in the female endometrium. Hoxb3 and d8 are present during development, and in TRAMP cells, but not prostates of older males. Furthermore, Hoxd11 and b13 have robust expression in older males, but are absent in TRAMP cells. Our analysis has identified prostate marker genes, genes that appear to be developmentally regulated and genes that appear to be reactivated or specifically repressed in the TRAMP tumor cells.

Grant Funding Source

NIH, LLU Pathology Group
Evidence that neural crest precursors of spinal ganglia do not migrate in human embryos (DREM databases utilization # 1)
Authors: Gasser Raymond Frank
Published: June 2021

The objective was to determine if neural crest precursors of spinal ganglia (NC) actually move from one region to another (migrate) or do they attain their definite location as a result of changes in the size and shape (growth movements) of the neural tube (NT) and surrounding structures. Transverse (T) sections through the C‐4 level in 9 Carnegie human embryos were captured digitally at circa 29‐56 postfertilization days, stages (st) 11‐17, 20 and 23. Traditionally, the dorsal surface of the NT was used as the reference point for movement. In expanding masses structures not attached to the surface move away as the mass expands. The ideal reference point is the center of the mass (centroid). While no morphological centroid exists in embryos, the notochord (N) in T sections is more central than any other consistent structure and was used in the present study. NC could not be identified outside the NT at st 11 but are present dorsolaterally at st 12. The NT enlarges rapidly vertically and horizontally. At st 13 the spinal ganglion primordium is present dorsolateral to the NT. If NC cells migrated ventrally they would get closer to the horizontal plane thru the N but they do not. Instead, the primordium moves dorsolaterally in st 14‐17 and 20 and becomes progressively better defined. At st 23 the spinal ganglion is near its definitive location dorsolateral to N, caused by growth movements, not migration. Supported by NIH grant RO1 HD 37811.
Dyad Pedagogy: A New Education Paradigm for Allied Health Programs
Authors: Chaudhri Ali Aftab , Sherman Lloyd , Marquez Samuel
Published: June 2021

Allied health programs attract students from diverse educational backgrounds, though most have only been exposed to traditional lecture‐based modalities. While quantitative performances can be excellent in these programs, difficulties arise assessing if students can function in groups, a critical issue in the current interdepartmental health care model. The problem may be a consequence of the prevalence of lecture over non‐lecture class models, to which some have suggested re‐structuring course material into group projects. But would working in groups adversely affect learning required content? To explore this question, 21 sonography students in a gross anatomy class were randomly assigned into 9 dyads & 1 triad, and given weekly problem‐based assignments involving key anatomical concepts and oral projects. Dyads were assessed by pre‐ and post‐test comparisons, and qualitative performances on weekly assignments tracked over time. Early data showed improved post‐test results, increasingly sophisticated qualitative content in weekly assignments, and increased confidence in self‐expression. This suggests dyad pedagogy can be readily incorporated into anatomical instruction without any negative impact on learning critical content, and has the added value of encouraging students to learn from each other as well as helping them develop other workplace‐relevant skills.

Grant Funding Source

DO NOT HAVE FUNDING
Student laboratory presentations as a learning tool in anatomy education
Authors: Chollet Madeleine Blair , Garofalo Evan , Teaford Mark , DeLeon Valerie
Published: June 2021

Previous studies have shown that students who complete oral presentations in the anatomy laboratory believe they understand the material better and retain it longer; however, we have found no studies that empirically test such outcomes based on exam scores. The purpose of this study was to assess the effectiveness of oral lab presentations on exam performance. Specifically, we tested whether students (n = 132) performed better on exam questions on topics covered by their oral presentations than on other topics. Each student completed two graded[vbd1], 12‐minute lab presentations on two different assigned topics (e.g., thorax) during the course. Each student also took three examinations, each covering a third of the course material. Exam questions were characterized by type (memorization, pathway, analytical, other). A paired t‐test revealed that students performed better on topics covered by their presentations than on topics not covered by their presentations (p < 0.05). Two‐way repeated measures ANOVA (presentation‐by‐question type) also indicated a significant effect of question type on presentation‐related improvement in exam scores. For "pathway" questions, presenting students scored on average 5.6% higher on topics covered by their presentations than on topics not covered by their presentations (p < 0.05). These results demonstrate empirically that oral presentations are an effective learning tool.

Grant Funding Source

None
Understanding Anatomy Through the Art of Sculpting
Authors: Svigals Philip Zachary , Márquez Samuel
Published: June 2021

Understanding anatomical structures in the context of their topographical position provides a basis for mastery of our discipline. However, the art of sculpture offers the opportunity for the health professional to improve his/her perception while greatly enhancing their understanding of anatomy. As a professional sculptor and medical student, one of the authors (PZS) was asked by Director of anatomy at SUNY Downstate Medical School (SM) to create a life size model of a heart with coronary vessels. An interactive version of the sculpture was posted online allowing students to rotate the model 360 degrees while multiple copies of the piece were distributed throughout the labs. Using images from Grant's and Netter Atlases PZS transformed the 2D into 3D structures by evaluating the contours, volume and proportion; improving the right brain's visual‐spatial perception and the left brain's praxis. Through molding the clay into anatomically correct structures by viewing the piece from every angle, spatial relationships were registered, understood and committed to memory. Practice analyzing the form trains the eye to see in greater detail and to better synthesize a sense of the whole. These skills allow a physician to readily differentiate the abnormal from the normal and make accurate decisions in a surgical or clinical setting. Common goals of surgeons and artists lie in the careful evaluation of their patients/subjects in all dimensions and visualizing the finished product in order to properly plan and execute a surgical procedure or sculpture. As the plastic surgeon places the scalpel to tissue to reconstruct an absent ear or deformed nose, so too must the sculptor meet similar concerns about size, shape, position and proportion as they place the chisel to stone. A sculpture workshop to help teach anatomy and develop valuable tactile and observational skills for the physician is being planned for the fourth year curriculum.

Grant Funding Source

n/a
Global re‐organization of replication domains during embryonic stem cell differentiation
Authors: Gilbert David Michael , Hiratani Ichiro , Hiratani Mari , Ryba Tyrone , Takebayashi Shin‐Ichiro
Published: June 2021

We constructed high‐resolution replication timing profiles in mouse and human embryonic stem cells (mESCs and hESCs) before and after differentiation to multiple lineages. We demonstrate that domains of coordinate replication ("replication domains") delineate distinct units of chromosomes with cell‐type specific boundaries. During differentiation, smaller discordantly replicating domains consolidate into larger coordinately replicated units. Moreover, induced pluripotent stem (iPS) re‐acquire the smaller replication domains of ESCs. When mESCs were followed through intermediate stages of differentiation, early to late (EtoL) replication changes preceded‐while late to early (LtoE) changes followed‐loss of pluripotency as measured by loss of Oct4 expression. However, changes in radial sub‐nuclear positioning, whether toward (EtoL) or away (LtoE) from the periphery, as well as replication domain consolidation, all took place during loss of pluripotency. We conclude that replication profiles can identify chromosome segments that undergo higher‐order organizational changes during major cell fate transitions. In particular, smaller replication domains and a higher density of timing transition regions define novel characteristics of the pluripotent state. Supported by NIH grants GM083337 and GM085354.
Water Release upon DNA Binding by E. coli and T. aquaticus Pol I DNA Polymerases
Authors: Baker John T. , Deredge Daniel J. , Datta Kausiki , LiCata Vince J.
Published: June 2021

Klentaq (T. aquaticus) and Klenow (E. coli) are a thermophilic‐mesophilic pair of Type I DNA polymerases with similar morphologies. The thermodynamic linkage between binding affinity and the osmotic pressure induced by non‐interacting osmolytes can be used to calculate the number of water molecules bound or released during a macromolecular process such as DNA binding. Approximately 600 water molecules are released when Klentaq polymerase binds to either primed‐template or blunt‐end DNA. In contrast, only approximately 500 waters are released when Klenow binds primed‐template DNA, however, the necessity of measuring Klenow DNA binding at significantly higher salt concentrations may be the source of this difference. For both proteins, the measured water release is significantly higher than that predicted from the burial of surface area upon complex formation calculated from structural data (approximately 300 waters). Steric displacement of non‐surface associated water within the DNA binding cleft of the proteins may account for this difference, and indicates that these binding reactions will be more enhanced at intracellular osmotic pressures than would be predicted from surface area calculations.
Topoisomerase I from Pseudomonas aeruginosa can act as a drug target for fluoroquinolones
Authors: Jain Teesta , Grove Anne
Published: June 2021

Despite the recognition of Pseudomonas aeruginosa as the most prevalent opportunistic human pathogen causing life‐threatening conditions in immunocompromised patients with neutropenic cancer and bone marrow transplant, little has been accomplished to resolve the infection due to the intrinsic resistance to a wide variety of antimicrobial agents and active efflux pumps in P. aeruginosa. P. aeruginosa encodes a topoisomerase (PAT) with sequence similarity to the eukaryotic type IB topoisomerase from vaccinia virus (VT). The human topoisomerases are effective molecular targets for many antitumor drugs. The VT is also a model system to study the much larger human topoisomerase. Binding the duplex DNA, PAT forms a covalent protein DNA complex where the active site tyrosine attacks a phosphodiester bond on one of the DNA strands. Residues in the active site are conserved, notably Tyr292 which is predicted to form the covalent bond to the DNA, and substitution of the Tyr292 renders the enzyme inactive. Fluoroquinolones are antibacterial agents in clinical use as inhibitors of type II topoisomerases. Sparfloxacin, a synthetic fluoroquinolone, inhibits PAT at high concentrations, but other drugs belonging to this family do not. We therefore hypothesize that PAT could act as a structurally optimized target for fluoroquinolones that target the covalent complex between the DNA and the PAT.
Isolation and Characterization of Histone Deacetylase Kinases from Failing Human Heart
Authors: Huynh Khai Quang , Hood David B , Mckinsey Timothy A
Published: June 2021

Histone deacetylase 5 (HDAC5) represses pathological cardiac gene expression through interactions with pro‐hypertrophic transcription factors. In response to stress signals, HDAC5 is phosphorylated and exported to the cytoplasm, resulting in derepression of downstream target genes that promote pathological cardiac remodeling. Overexpression and in vitro studies have identified multiple putative HDAC5 kinases, most of which are members of the Ca2+/calmodulin‐dependent protein kinase (CaMK) superfamily (e.g., protein kinase D (PKD), microtubule associated kinase (MARK) and CaMK). We employed an unbiased approach to identify endogenous HDAC5 kinases from human heart. A six‐step biochemical purification followed by mass spectrometry uncovered three HDAC5 kinases in the myocardium of patients with end‐stage heart failure: PKD1, PKD2 and MARK3. Kinetic studies with recombinant enzymes and HDAC5 substrate in vitro confirmed the capacity of multiple members of the CaMK superfamily to phosphorylate HDAC5, including checkpoint kinase (CHK) 1/2, salt inducible kinase (SIK) 1, mitogen‐ and stress‐activated kinase (MSK) 1/2, death‐associated protein kinase (DAPK) 1, testis‐specific serine/threonine kinase (TSSK) 1 and PIM‐1. However, only PKDs exhibited low Km values and high catalytic activity (kcat/Km) toward HDAC5, and co‐immunoprecipitation studies revealed selective physical association of HDAC5 with PKD. These findings support a role for PKD isoforms in the control of pathological cardiac remodeling through their neutralizing effects on HDAC5.
The tumor suppressor Ikaros engages the VPAC1 promoter in activated HuT 78 cells
Authors: Benton Keith D , Hermann Rebecca J , Dorsam Sheri , Van der Steen Travis , Nelson Ashley , Dovat Sinisa , Dorsam Glenn Paul
Published: June 2021

DNA sequence specific recruitment of transcription factors to gene promoters is essential for normal gene expression. Total Ikaros (IK) protein and its phosphorylation pattern change during activation of lymphocytes, altering its subnuclear localization and DNA binding. IK has been shown to decrease expression of an anti‐proliferative, G‐protein coupled receptor, vasoactive intestinal peptide receptor 1 (VPAC1) when ectopically overexpressed in NIH‐3T3 cells. The VPAC1 promoter contains 24 putative binding sites for IK, including four high affinity motifs. VPAC1 mRNA levels are dependent on the activation status of CD4 T cells, as TCR signaling downregulates VPAC1 expression. IK is hypothesized to engage the VPAC1 promoter upon activation and downregulate expression. Binding of IK to the VPAC1 promoter was queried using chromatin immunoprecipitation (ChIP). IK was enriched at the VPAC1 promoter in an activation (PMA/ionomycin) dependent manner in HuT 78 cells. Surprisingly, HuT 78 activation did not decrease VPAC1 mRNA. Future studies will overexpress DNA binding, non DNA binding, and alanine/aspartate mutant IK isoforms to determine their effect on IK binding. Engagement of the master regulator or lymphopoesis, IK, at the neuropeptide receptor VPAC1 promoter strengthens the neuroimmunomodulation connection at the molecular level.
Daphnoretin, a novel protein kinase C activator, induces growth arrest irreversibly on Human Hepatoma HepG2 Cells
Authors: Huang Yi John , Chen Hua‐Chien , Chou Chen‐Kung
Published: June 2021

Daphnoretin, a dicoumarin type natural product originally isolated from plant Wikstroemia indica C. A. Mey has been shown as a protein kinase C (PKC) activator with novel structure. Previous studies demonstrated that daphnoretin treatment inhibited the expression of hepatitis B virus surface antigen (HBsAg) in human hepatoma Hep3B cells. In this study, we found that daphnoretin inhibited cell growth and induced a G1‐phase cell cycle arrest in human hepatoma HepG2 cells. Treatment of HepG2 cells with daphnoretin significantly up‐regulated the expression of p21cip1 and decreased the kinase activity but not the protein levels of cdk2/cyclin E complex in HepG2 cells. Unlike the reversible effect of phobol ester, a well known protein kinase C activator, 48 hour‐treatment of daphnoretin caused an irreversible growth arrest in HepG2 cells associated with persistent accumulation of p21 in HepG2 cells. The transcriptome and miRNA expression profile analysis indicates that daphnoretin may coordinately regulate a set of growth related genes at transcriptional and miRNA mediated translational level in cultured HepG2 cells.
Expression and regulation of epithelial sodium channels (ENaC) by cGMP/PKG in bone and osteoblasts
Authors: Li Qingnan , Huang Jian , Nie Hongguang , Mao Sufeng , Zhao Runzhen , Han Dongyun , Yang Guozhu , Lu Li , Chen Jun , Ji Honglong
Published: June 2021

The amiloride‐sensitive epithelial sodium channels (ENaC) are essential for maintaining sodium homeostasis, transducting mechanical stimuli, and sensing acidosis. The expression of ENaC in bone and the correlation of Na content with bone disease have been reported. We thus studied the distribution and regulation of ENaC by cGMP/PKG signal in normal and cGK knockout mouse bone and primary cultured osteoblasts. Our immunostaining and RT‐PCR results showed that three ENaC subunits, namely ,α, β and γ were expressed in osteoblasts of normal mice. Amiloride‐sensitive Na currents were detected in primary osteoblasts clamped in whole‐cell mode. Furthermore, we found that 8‐pCPT‐cGMP (0.2mM, 24hr) stimulated ENaC channel activity in oocytes (P<0.05, n=9). On the other hand, regulation of ENaC expression by cGMP was examined by comparing the protein expression levels of ENaC in normal and cGKII knockout mice. The protein expression levels for ENaC, as measured by densitometry of Western blot strikingly reduced in cGKII knockout mice which are dwarfs (P<0.05, n=3). Similar observations were seen in immunohistochemical images. Our results suggest that regulation of ENaC channel expression and function by cGMP/cGKII signal pathway may be a potential target for developing pharmaceutical intervention for metabolic bone diseases. Supported by HL87017 and National Natural Science Foundation of China 30740085.
Acetylation of Nrf2 regulates its transcriptional activity and nucleo‐cytoplasmic localization
Authors: Garduno LaKisha , Theodore Melanie , Kawai Yumiko , Arinze Ifeanyi J.
Published: June 2021

Nrf2 (nuclear factor erythroid factor 2‐related factor 2) induces transcription of a variety of genes by binding to antioxidant response element(s) (ARE) on target gene promoters. Nrf2 is activated by separation from its inhibitor protein Keap1 in the cell cytoplasm through covalent modifications on both Keap1 and Nrf2. However, covalent modification(s) downstream of this activation are not as well‐delineated. Using K562 and HepG2 cells, we have explored the role of acetylation in the biological action of Nrf2. Acetylation of Nrf2 was confirmed by co‐immunoprecipitation assay, using anti‐acetyl‐lysine antibody. Nrf2‐induced transcription from an ARE‐driven reporter gene construct (Gαi2‐luc) was increased by an expression plasmid for the acetyltransferase CBP (cAMP response element‐binding protein‐binding protein). This effect was blocked by the adenovirus protein E1A, which inhibits the action of CBP/p300 proteins. Treatment of cells with resveratrol, a potent activator of SIRT1, a class III NAD+‐dependent histone/protein deacetylase, inhibited Nrf2‐induced transcription. Resveratrol induced cytoplasmic localization of GFP‐Nrf2; CBP induced essentially nuclear localization, but E1A reversed this localization to the cytoplasmic compartment, suggesting that acetylation/deacetylation alters the nucleo‐cytoplasmic localization of Nrf2. Supported by NIH grant #S06‐GM08037.
PECTINASE PRODUCTION BY UV‐2DG RESISTANT MUTANT OF Aspergillus niger WITH A CATABOLITE REPRESSION RESISTANCE ACTIVITY
Authors: Uzoma okafor Anthony , Ihuaku Okochi Veronica , Joy Okpuzor , Adedotun Adekunle A , Albert Ebuehi O , Blessing Onyegeme‐Okerenta M
Published: June 2021

Strain improvement of microfungi for hyper production of industrial enzymes is important in the biodegradation of agrowastes to valuable product. Wild strain of Aspergillus niger previously isolated from agrowastes in Lagos Nigeria, was screened for pectinase production in various agro wastes. This strain was further subjected to UV‐mutagenesis; through the use of a selective culture medium containing 2 deoxy D glucose (2dg) it was possible to isolate hyper productive mutant strains (resistant to 2dg). Pectinase production by the mutant was monitored for 72h in both SSF and SmF and the effect of catabolite repression was also studied using glucose and sucrose. The wild strain of Aspergillus niger produced high level of pectinase in all the agrowaste studied with the highest pectinase activity in wheat bran medium (348.54 IU/mg protein).Three 2dg UV mutant were isolated, the best 2dg UV mutants (2dg UV A.n 2) showed 465% and 230% higher pectinase activity than the parent strain in SSF and SmF respectively. This mutant is also noted to be resistance to catabolite repression in both glucose and sucrose containing medium. The result indicated that UV mutation and 2 dg were effective mutagenic and selectant for strain improvement of Aspergillus niger for enhanced pectinase production.
Purification and Characterization of the Rhizobium leguminosarum lipid A Oxidase, LpxQ
Authors: Ingram Brian O'Neal , Raetz Christian R. H.
Published: June 2021

The lipid A component of lipopolysaccharides from the plant endosymbionts Rhizobium leguminosarum contains an unusual proximal sugar unit consisting of a 2‐amino‐2‐deoxy‐gluconate moiety in place of glucosamine. This modification results from the actions of the enzyme LpxQ, which generates the 2‐amino‐2‐deoxy‐gluconate unit from a glucosamine‐containing precursor. LpxQ is localized to the outer membrane and is dependent on molecular oxygen for activity. LpxQ was shown to be active when expressed heterologously in E. coli. Lipid A was isolated from E. coli expressing LpxE and LpxQ and was shown to be oxidized by ESI‐mass spectrometry. A new substrate and assay were developed to monitor lipid A oxidation in vitro. A purification scheme was developed. Purified LpxQ was shown to be dependent on a divalent cation and not stimulated by FAD or PQQ. Hydrogen peroxide was found to be a by‐product of lipid A oxidation. The mechanism and cofactors involved in the oxidation reaction are under investigation. This work is supported by NIH Grant R37‐GM‐51796 to C. R. H. R.
Zn‐finger domain of carboxyltransferase plays a role in nucleic acid binding and enzymatic activity
Authors: Meades Glen , Waldrop Grover
Published: June 2021

Acetyl‐CoA carboxylase catalyzes the first, committed step in fatty acid synthesis, converting acetyl‐CoA to malonyl‐CoA. In contrast to the single polypeptide of eukaryotes, the bacterial form of the enzyme is a multi protein complex: a biotin carboxylase, a carrier protein, and a carboxyltransferase (CT). The crystal structures of the E. coli and S. aureus CT component reveal a small, independent, four cysteine Zn binding domain that lacks a complement in the eukaryotic homologue. Zn fingers are common features of proteins that bind nucleic acids. Through electrophoretic mobility shift assays, CT was found to bind DNA in a cooperative, non‐specific manner. Enzymatic analysis of single cysteine to alanine mutants reveals no change in Km for both substrates malonyl‐CoA and biocytin, and 0.7 to 12.7% of wildtype Vmax. No activity is observed for multi‐site mutants. DNA binding is similarly affected, with the single mutants having a 10‐fold increase in Kd; and multi‐site mutants losing all DNA binding ability. This shows that the Zn domain is required for catalysis as well as for DNA binding. What distinguishes CT from other DNA binding enzymes is that enzymatic function and DNA binding are inextricably linked through the Zn domain. The unique Zn domain, adjacent to the active site of CT, makes for a unique target for the development of novel antibiotics capable of highly specific binding and presumably marginal toxicity.
Structure and function of prolyl peptidases
Authors: Chiu Thang Kien , Li Min , Davies David R.
Published: June 2021

Prolyl peptidases are serine proteases that are involved in a large variety of biological functions, including Type II diabetes, cancer, and neurological function, and have been implicated in Lou Gehrig's disease, Chagas' disease and Celiac Sprue. There are ten members in this enzyme family, which all recognize and cleave proteins and small peptides at proline residues, but differ from each other in the position of the proline residue that they recognize: aminoacyl‐, di‐, tri‐, and endo‐ prolylpeptidase (i.e., APP, DPP, TPP, and PEP) recognize and cleave the first, second, third, and internal proline residue from the N‐terminus, respectively. Previous studies have provided indirect evidence for an involvement of a conformational change in enzyme catalysis. We now directly demonstrate from the structures of prolyl endopeptidase that the apoenzyme exists in different conformational states, addition of substrate induces a conformational change from an opened to a closed state, and catalysis occurs through an induced‐fit mechanism. The significance of this finding for conformationally‐targeted drug design efforts against this family of enzyme with highly degenerate substrate recognition will be discussed.
Ferritin‐specific diiron ligands control Fe/O2 chemistry in iron mineralization; diiron substrate vs diiron oxygenases
Authors: Theil Elizabeth C , Tosha Takehiko , Hasan Mohammad R.
Published: June 2021

Catalytic coupling of Fe(II) and O2 at diiron sites in ferritin initiates iron mineralization. A diferric peroxo (DFP) intermediate is common to catalysis in ferritins and in diiron cofactor oxygenases. Two of six residues in the ferritin Fe2 site of the catalytic center differ from cofactor Fe2 sites. Conserved Gln137 and variable Asp/Ser/Ala140 are substituted to Glu and His in diiron oxygenases, respectively. Here, we report how mutation of ferritin‐specific diiron ligands alters ferroxidation activity. The substitution of Gln137 for Glu showed no detectable DFP but normal ferroxidation rates, indicating reaction mechanism alterations. Asp140 natural variants, Ser/Ala140 mutants, varied the formation/decay kinetics of DFP, but not mechanism. His140 ferritin formed no DFP and ferroxidation rates decreased 40‐fold. Protein structures (X‐ray crystallography with H‐L Ng and T Alber, LBNL) indicate that His140 can block Fe/O2/H2O access to diiron catalytic centers from the protein cavity. These new observations indicate that DFP formation depends on Fe2 site residues that use His to yield higher valent iron intermediates in the context of related diiron oxygenases, contrasting with His inhibition in the ferritin context where Asp/Ser/Ala are required for turnover with release of diferric oxo/hydroxo mineral precursors from the catalytic site. Part support; DK20251 (ECT, MRH and TT) and the JSPS (TT).
A unique conformation of the inhibitory ε subunit in a crystal structure of the Escherichia coli F1‐ATPase
Authors: Duncan Thomas M , Cingolani Gino
Published: June 2021

We report a structure at 3.3 Åresolution for E. coli F1‐ATPase (EcF1, depleted ofδ). It was solved by combining molecular replacement and 4‐fold non‐crystallographic symmetry averaging. The sequences of α3β3γε subunits were unambiguously interpreted using selenomethionine‐labeled protein. The general architecture of the α3β3γ 'core' is like that of bovine mitochondrial F1 (MF1), but with greater asymmetry between pairs of αβ subunits. The conformation of ε (δin MF1) is the most striking feature. Unlike δ in MF1,ε's C‐terminal domain (CTD) adopts a highly extended state: helix‐1 extends up to pack on one side ofγ's 'foot' domain, the following loop packs below the 'DELSEED' motif of subunit βDP, and helix‐2 inserts into the central cavity of EcF1 to form an antiparallel coiled‐coil with part ofγ's N‐terminal helix. This interaction blocks specific contacts between γ and the CTDs of αDP/βDP subunits, which are shifted away fromγ's central rotary shaft. Remaining C‐terminal residues of ε are non‐helical and extend across the central cavity to contact βTP near the inner surface of its catalytic nucleotide‐binding site. Intrusion ofε's CTD into the central cavity is likely to block subunit motions involved in rotary catalytic coupling in F1 and in the intact ATP synthase. This structure sheds light on an inhibitory mechanism that is unique to bacterial and chloroplast ATP synthases. Support: NIH, 1R01GM083088‐01A2.
Acyl‐enzyme complex of the class D β‐lactamase OXA‐1 and doripenem
Authors: Schneider Kyle Douglas , Bonomo Robert , Leonard David , Powers Rachel
Published: June 2021

The class D β‐lactamase OXA‐1 belongs to the oxacillinase family of lactamase enzymes, OXA‐1 efficiently hydrolyzes penicillin β‐lactams, yet is relatively ineffective at turning over carbapenems. Alternatively, other class D β‐lactamases, such as OXA‐24, are proficient carbapenemase enzymes, but at the expense of their penicillinase activity. To explore this difference in substrate specificity, the structure of OXA‐1 in complex with the clinical carbapenem, doripenem, was determined to 1.4 Åresolution. Initial difference electron density maps contoured at 3σ suggest the presence of the carbapenem in an acyl‐enzyme complex with the catalytic serine (Ser70). Refined data and further studies are expected to yield valuable insight into the cause of the substrate specificity of the oxacillinases in comparison to the carbapenemases.
How does Bacillus thuringiensis PI‐Phospholipase C bind to mixed component vesicles? Insights from mass spectrometry H‐D exchange
Authors: Shi Xiaomeng , Roberts Mary F. , Gershenson Anne , Meklemburg Robert , Wintrode Patrick L.
Published: June 2021

It is difficult to determine the orientation and conformation of peripheral membrane proteins when docked to target membranes. Often, flexible regions of the proteins provide the major contacts with the membranes. B. thuringiensis PI‐PLC has two discrete binding sites for phospholipids ‐ the active site binding PI (or substrate competitors such as phosphatidylglycerol (PG)), and an activator site specific for phosphatidylcholine (PC). We have performed H/D exchange mass spectrometry (HDXMS) experiments on PI‐PLC in solution or when tightly bound to 1:1 and 1:9 PG/PC small unilamellar vesicles. In the absence of vesicles, most regions of the protein exchange rapidly. However, with SUVs, the regions around helix B and helix C show reduced exchange, consistent with protection by the membrane. These results agree with mutagenesis studies identifying residues on those helices as important for membrane binding. Other significant changes include reduced exchange in β‐strand E and dramatically increased exchange in helix E. The H/D exchange rates were also determined for a constructed covalent dimer (disulfide‐linked W242C), thought to mimic the membrane‐induced dimeric structure but in the absence of membranes, and a mutant protein, W47A/W242A, with very low affinity for vesicles that exists as a dimer in solution. Results provide insight into self‐association of PI‐PLC monomers. Clearly, for peripheral membrane protein dynamics, HDXMS can provide information on structural changes not accessible by other methods (Supported by N.I.H. GM60418).
The Intermitochondrial Localization of Human Pantothenate Kinase 2
Authors: Garcia Matthew , Jackowski Suzanne
Published: June 2021

Pantothenate kinase (PanK) catalyzes the first step in Coenzyme A (CoA) biosynthesis. Three genes express the four catalytically active isoforms of mammalian PanK; PanK1α1β 2, and 3. PanK activity is feedback inhibited by CoA, with each isoform exhibiting a distinct sensitivity to the CoA thioester pool. Mutations in the human PanK 2 gene result in a progressive and fatal neurodegenerative disease, called PKAN. Our lab and others have shown that human PanK2 is associated with mitochondria. The localization of PanK2 was investigated using a human neuroblastoma cell, SHSY. Selective permeabilization of mitochondrial compartments with increasing concentrations of digitonin, combined with Proteinase K digestion of exposed proteins, revealed that PanK2 was located in the intermitochondrial space. PanK enzyme activity in isolated mitochondrial fractions was cryptic, and restoration of full activity accompanied detergent solubilization of the mitochondrial membrane. Long‐chain acylcarnitine has been shown to activate PanK2 in vitro. Current experiments are investigating the impact of acylcarnitine on the metabolic activity of overexpressed PanK2 in SHSY cells. The localization of PanK2 places it in close proximity to the site of acylcarnitine formation, thereby enabling its rapid activation. Supported by GM62896, CA21765, and ALSAC.
Increased Expression of Hepatic Scavenger Receptor B 1 and Apo A‐I in Streptozotocin‐induced Diabetic Rats
Authors: Niesen Melissa , Ness Gene C.
Published: June 2021

Elevated serum cholesterol levels and an increase in cardiovascular events are frequently seen in diabetes. We used microarray and RT‐PCR analysis to investigate the expression of key hepatic genes in the male Sprague‐Dawley rat, a predominantly HDL animal, in which diabetes was induced by injection of 65 mg/kg of streptozotocin. As expected, HMG‐CoA reductase, glucokinase, fatty acid synthase, acetyl CoA carboxylase, stearoyl CoA desaturase, pyruvate kinase and thyroid hormone responsive protein expression was decreased. On the other hand, expression of serine dehydratase, carnitine palmitoyl transferase, carnitine acetyltransferase, insulin‐like growth factor binding protein‐1, INSIG‐1 and 2, PPAR gamma coactivator 1 alpha, and ABCC‐3 was increased in the diabetic rats. The expression of scavenger receptor B 1 and Apo A‐I were increased about 3‐fold in the streptozotocin‐induced diabetic animals. This may be a consequence of the severe insulin deficiency which promotes robust lipolysis. These genes are critical to reverse cholesterol transport. Consistent with these findings, a decrease in hepatic scavenger receptor B 1 has been reported in an animal model of Type 2 diabetes with increased insulin levels. (This investigation was supported by NIH grant DK075414).
NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a potential step in cholesterol egress from lysosomes.*
Authors: Infante Rodney , Brown Michael , Goldstein Joseph
Published: June 2021

The trafficking of lipoprotein‐derived cholesterol from lysosomes is dependent on the polytopic membrane‐bound Niemann‐Pick C1 (NPC1) and the soluble Niemann‐Pick C2 lysosomal proteins. The requirement for these proteins in the transport of cholesterol out of the lysosomes remains unclear. Previously, we demonstrated the ability of the N‐terminal luminal domain of NPC1, hereinafter called NPC1(NTD), to bind cholesterol with nM affinity. We and others also showed the ability of NPC2 to bind cholesterol with similar affinity. Recently, we established an in vitro assay detecting the movement of [3H]cholesterol between these two proteins and model membranes. NPC2 readily transfers or receives [3H]cholesterol to and from phosphatidylcholine liposomes, relative to NPC1(NTD). The bidirectional transport of [3H]cholesterol between NPC1(NTD) and liposomes is accelerated over a 100 fold in the presence of NPC2. The naturally occurring mutant (Pro120Ser) form of NPC2 did not demonstrate significant cholesterol binding and had a reduction in the facilitation of cholesterol transport between NPC1(NTD) and liposomes. These studies begin to define the dual requirement of both proteins in the egress of lipoprotein‐derived cholesterol out of the lysosomes, thus explaining why mutations in either protein cause a similar clinical phenotype.

*Infante, RE, et al. (2008) NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci USA 105:15287‐15292.
Optimizing polymerase chain reaction conditions for the human perlecan (HSPG2) promoter
Authors: Richards Matthew Thomas , Rohe Benjamin , Farach‐Carson Mary C
Published: June 2021

Perlecan, also called HSPG2, is a heparan sulfate proteoglycan predominantly located in basement membranes and the matrix surrounding endothelial, mesenchymal and stromal cells. The reactive stroma surrounding prostate cancer cell lines produces high levels of the protein which may play a role in delivery of growth and angiogenic factors, aiding survival and growth of metastatic tumors. The overall goal of this project was to study the promoter in order to understand the up‐regulation of perlecan in the tumor reactive stroma which occurs via transcriptional increases in perlecan biosynthesis. The sequence for the human HSPG2 promoter was found using public databases and compared to a published human sequence (Iozzo et al., 1997) and a mouse promoter sequence found in online databases. Several transcription factor binding sites of interest were identified for further study including NFkB, CREB, Smad3, Elk‐1, c‐Jun and TCF/LEF‐1. Currently we are working to build a promoter‐reporter construct by isolating the promoter region using polymerase chain reaction (PCR) amplification. Our current strategy seeks to optimize the PCR conditions with different sets of primers. The next step will be creating a fluorescence protein reporter construct of the full promoter sequence in order to test the effects of the identified pathways. This research was funded by the HHMI program and NIH/NCI P01 CA098912.
CaM Kinase Kinase Control of Prostate Cancer Cell Survival
Authors: Hart Brendon , Schmitt John M.
Published: June 2021

AKT has been implicated in promoting cell survival within certain cells. Current research has shown that hormones that increase the concentration of intracellular calcium can activate AKT that in turn leads to cancer cell survival. Interestingly, LNCaP cells express the M3‐subtype of GPCR's that may couple to increases in intracellular calcium and activation of the Ca2+/Calmodulin‐dependent Protein Kinases (CaM Ks). Specifically CaM Kinase Kinase (CaM KK) phosphorylates its direct substrates CaM Kinase I, CaM Kinase IV, and AKT. AKT promotes cell survival through phosphorylation of its target BAD that prevents caspase activation. Our goals were to examine the mechanism of carbachol activation of AKT and BAD in LNCaP prostate cancer cells and evaluate whether CaM Ks may be mediating carbachol's activation of AKT and cell survival. The results suggest that both AKT and BAD were phosphorylated in response to a five‐minute stimulation with carbachol in LNCaP cells. AKT and BAD phosphorylation were blocked by the selective CaM KK inhibitor, STO‐609, suggesting the involvement of CaM KK in the pathway. In addition, BAD phosphorylation was also blocked by treating cells with the AKT inhibitor, AKT‐X. Finally, our results suggest that carbachol treatment of LNCaP cells promoted cell survival through CaM KK, AKT, and the anti‐apoptotic protein, BAD.
TRAF6 and motor neuron loss
Authors: Butler Carrie Lynn , Dennis John S. , Kane Michael J. , Citron Bruce A.
Published: June 2021

TNF Receptor Associated Factor Six (TRAF6) activates transcription factor pathways including NF‐kB and c‐Jun. These pathways play roles in cell survival. Amyotrophic lateral sclerosis (ALS) and spinal cord injury are two examples of motor neuron disorders. We sought to identify factors that comprised a common dysregulation theme under motor neuron loss conditions. We analyzed gene expression changes in motor neurons mechanically injured in culture and in vivo traumatic impact injury rat spinal cords with Affymetrix gene arrays and found TRAF6 mRNA to be upregulated. These results were extended to neurodegenerative motor neuron loss, examining TRAF6 mRNA levels in human ALS spinal cords and in the wobbler mouse model of motor neuron disease. The gene expression changes, determined by qRT‐PCR, indicated that TRAF6 was upregulated in human ALS and in the mouse model spinal cords. We have generated cDNA clones of human and mouse TRAF6 to facilitate cause and effect experiments to better understand the relationships between TRAF6 and neuronal survival. Although our protein interactions studies will better define the function of TRAF6 in motor neuron loss, the elevation in TRAF6 in motor neurons programmed for cell death suggests that TRAF6 plays a role in motor neuron loss in spinal cord tissues. This study was supported by the Office of Research and Development, Medical Research Service, Department of Veterans Affairs, the Bay Pines Foundation, and the Alzheimer's Association.
Role of LFA‐1 in the transmission and pathogenesis of human immunodeficiency virus type 1 (HIV‐1)
Authors: Walker Tiffany Nicole , Hildreth James EK
Published: June 2021

HIV‐1infection induces formation of a virological synapse wherein CD4, chemokine receptors, and cell adhesion moleculessuch as the integrin LFA‐1 [RNSB1]form localized domains on the cell surface. Previous studies have shown that LFA‐1 on the surface of HIV‐1 particlesnot only retains its cellular function but also enhances virus attachment to susceptible cells by binding its counterreceptor ICAM‐1. Findings from our laboratory revealed that monoclonal antibodies (Mab) specific for LFA‐1 blocked HIV‐1 mediated syncytia formationand enhanced neutralization by antibodies from AIDS patients and by gp120 Mabs. The objective of this study is to determine if LFA‐1/ICAM‐1 interaction plays a limiting role in apoptosis, a process linked to both HIV‐1 infection and integrin biology such that disruption of this interaction leads to increasedapoptosis in HIV‐1‐infected cells. Using the TUNEL assay, we observed increased cell death in HIV‐1‐infected primary T cells treated with anti‐LFA‐1 Mab[RNSB2], as well as increased caspase 8 activation. HIV‐1‐infected cells treated with anti‐LFA‐1 Mab also released less virus compared to HIV‐1‐infected cells alone. These results suggests that anti‐LFA‐1 Mab blocks HIV‐1 infection by inducing apoptosis and that LFA‐1‐mediated signaling may be necessary to prevent HIV‐1‐induced apoptosis, thereby contributing to the spread of infection.

Supported by NIH (R01HD040772)
Overcoming the challenges of online science education via a holistic approach
Authors: Ortiz Phillip A. , Mawn Mary V.
Published: June 2021

The faculty and instructional design team of Empire State College's Center for Distance Learning have developed a number of innovative online natural science courses. These include both upper and lower level coursework, for majors and non‐majors, and across the spectrum of SMT. Starting less than ten years ago from a distance learning curriculum that was strictly print‐based, we have developed and/or refined a number of pedagogical approaches to maximize student learning by recognizing that a successful online course is one in which three key aspects are met: (1) course content is presented in a pedagogically sound way, (2) instructors are well versed in the appropriate pedagogy and instructional technology, and (3) a community of learners amongst the students is established. During this presentation, we will discuss how these key aspects were addressed in online courses such as Genetics and Evolution: One Long Argument. Similar to the legs of a stool, we will also discuss how these elements are interrelated and critical to the effective design and delivery of online science coursework.
Evaluation of itunes U Podcasts on Undergraduate Biology Education
Authors: Smith Christopher J , Beliz Tania , Mach Jean
Published: June 2021

Recent changes in undergraduate science education include online or hybrid courses and electronic material such as textbooks and supplemental media. Electronically enhanced courses are attractive to educators as a way to bolster course material and provide students with more and varied learning options, approaches fitting to the era of internet information access. This study was undertaken to evaluate one such electronic supplement to undergraduate‐level science courses. Audio podcasts of biology class lectures were posted on the College of San Mateo's iTunes University website, provided by the Apple Corporation. Surveys were administered to students of three consecutive semesters to evaluate the use, effectiveness and outcome, as determined by course grade, of the podcasts. The results showed that the majority of students accessed the recorded lectures and those that used them the most felt that doing so improved their performance in the class. Data indicated that students most at risk of poor academic performance used the podcasts most frequently. This study indicates that electronic media supplementation enhanced science student performance. Further studies are underway in order to more effectively implement and use electronic supports and supplements to contribute to student success.
The t10c12 conjugated linoleic acid (CLA) isomer prevents the development of obesity‐related hypertension by increasing plasma adiponectin and activating endothelial nitric oxide synthase (eNOS) in fa/fa Zucker rats
Authors: DeClercq Vanessa , Taylor Carla G. , Zahradka Peter
Published: June 2021

Obesity‐related hypertension may be due to increased activation of the adipose tissue renin‐angiotensin system (RAS) and/or reduced eNOS activity. CLA, a mixture of positional and geometric isomers of linoleic acid, has been shown to reduce blood pressure (BP) in obese insulin‐resistant rats. We investigated whether CLA isomers target RAS or eNOS by exploring expression of molecules related to BP regulation in obesity. Fa/fa Zucker rats were randomly assigned to 1 of 4 groups: (1) control (2) c9t11‐CLA (3) t10c12‐CLA (4) captopril. Systolic BP significantly increased in untreated obese rats during the 8‐week study. This increase of systolic BP was attenuated in the t10c12‐CLA isomer or captopril groups. Angiotensinogen, eNOS and adiponectin expression were examined by Western blot analysis and plasma adiponectin by ELISA. The t10c12‐CLA isomer increased plasma adiponectin levels however, no differences were seen in protein expression in white adipose tissue. Feeding the t10c12‐CLA isomer significantly increased phospho‐eNOS expression in white adipose tissue and the aorta. No changes were observed in angiotensinogen expression in white adipose tissue or aorta. In conclusion, the t10c12‐CLA isomer attenuates the development of obesity‐related hypertension, at least in part, by elevating circulating adiponectin and activating eNOS.

Grant Funding Source

NSERC