bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Egfl7 During Vascular Development
Authors: Stuhlmann Heidi , Durrans Anna , Nichol Donna , Campagnolo Luisa
Published: June 2021

Epidermal growth factor domain‐like 7, Egfl7, encodes a 29kDa protein with an early and endothelial‐restricted expression profile. In vivo, Egfl7 mRNA is detected at mouse embryonic day 7.5. During development, expression is restricted to sites of blood island formation, the vascular endothelium, and endocardium. In adults, Egfl7 is down‐regulated except in the lung, pregnant uterus, and regenerating endothelium. We have used loss‐ and gain‐of‐function to determine the role of Egfl7 during vasculogenesis and angiogenesis. We are using a lentiviral siRNA‐mediated approach to establish Egfl7 knock‐down in ESC clones and transgenic mice. Embryoid bodies with Egfl7 knock‐down appear to have less organized vascular cords compared with controls. Tie2‐Egfl7 transgenic mice, with forced expression of the gene in endothelial cells, have been generated for gain‐of‐function analysis, and these display increased vessel lumen diameter, hemorrhaging, and other vascular abnormalities. We will report on analyses of these ESC and transgenic mice studies, and describe how they relate to the role of Egfl7 during embryogenesis. In addition, we will report on a second site of Egfl7 expression in embryonic germ cells (EGC) and embryonic stem cells (ESC), primordial stem cells, and during adult spermatogenesis.

Research Support: NIH HL082098, Tri‐Institutional Stem Cell Initiative (H.S.)
Animal models of arteriogenesis
Authors: Fernández Borja
Published: June 2021

Arteriogenesis is the process of growth of collateral arteries after the stenosis of a feeding artery. The mechanism varies among species. In dogs, occlusion of a coronary artery leads to the growth of muscular vessels, whereas in pigs only capillary vessels grow. Ligation of rabbit and mouse femoral arteries induce collateral artery growth in non‐ischemic areas. The vascular response is restricted to collateral segments and collateral ending branches remain quiescent. Collateral neo‐formation is a rare event. Leukocyte recruitment and vascular proliferation dominate the process in both species, but only rabbit collateral arteries develop a neo‐intima layer. Two experimental models illustrate the crucial roles of leukocytes and shear stress in arteriogenesis: Infusion of the leukocyte chemoattractant protein MCP‐1, and surgical creation of an arterio‐venous shunt, both after femoral ligation result in an exacerbated development of collateral arteries. In a hamster model of Bland‐White‐Garland syndrome (origin of the left coronary artery from the pulmonary artery), collateral arteries connecting the right and left coronary arteries appear spontaneously, creating a left‐to‐right shunt that allows perfusion of the left heart. This model supports the idea that shear stress regulates not only the growth, but also de formation of new collateral arteries during the postnatal life.
Notch signaling in early embryonic vascular differentiation
Authors: Vivian Jay , Copeland Jessica
Published: June 2021

The signaling cascades that direct the morphological differentiation of the vascular system during early embryogenesis are not well defined. Several signaling pathways, including the Notch and VEGF signaling, are critical for the formation of the vasculature in the mouse. To further understand the role of Notch signaling during endothelial differentiation, we are using a both loss‐of‐function and gain‐of‐function approaches in vivo. Embryos with activated Notch signaling in the vasculature display a variety of defects, and do survive not after E10.5. Most notably, the extraembryonic vasculature of the yolk sac displays remodeling differentiation defects, with few matured vessels. These phenotypes are distinct from tissue specific loss‐of‐function of Rbpj, a transcriptional regulator of Notch activity. Gene expression analysis indicated aberrant expression in a variety of genes in these models. In particular, VEGF and TGF‐beta family members show coordinate expression defects in the loss‐of‐function and gain‐of‐function models. This data suggests potential regulatory connections between Notch signaling and other signaling pathways during endothelial differentiation. Based on these findings, we are exploring other transgenic and embryo culture models to further understand the relevance of the of VEGF‐ and TGF‐beta‐related signaling during Notch‐mediated endothelial differentiation.
VelociGene coupled with VelocImmune yield Dll4 as a Tumor Angiogenesis Target
Authors: Gale Nicholas William
Published: June 2021

Clinically blocking VEGF can have efficacy in cancer and in vascular eye diseases. However, data suggests that in some cases tumor growth and angiogenesis can proceed in the face of potent VEGF blockade. To define new targets in the field of angiogenesis we exploited our mouse genetics approaches, VelociGene and VelociMouse. One new target, termed Delta‐like ligand 4 (Dll4), is induced by VEGF as a negative feedback regulator. Knockout or blockade of this feedback system during vascular development or in tumor settings results in uncontrolled excessive angiogenesis. Paradoxically, in tumors, this excessive angiogenesis results in suppression of tumor growth, apparently because the excess vasculature cannot support tumor perfusion. Dll4‐blockade is effective in several tumor models resistant to anti‐VEGF therapies and Dll4‐blockade can also be effectively combined with anti‐VEGF therapies. We have used our VelocImmune platform, a new mouse system for rapidly and efficiently generating fully human monoclonal antibodies against targets of interest, to generate high‐affinity fully human antibody therapeutic candidates that block Dll4.
Conformational entropy in molecular recognition by proteins
Authors: Wand A. Joshua
Published: June 2021

Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein‐protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally assess changes in residual protein entropy, a potentially important component of the change in the binding free energy. NMR spectroscopy has recently emerged as a powerful tool for characterizing the dynamics of proteins and has thereby gained access to their conformational entropy. We have previously shown that the dynamics of calmodulin varies significantly upon binding a variety of target domains. Surprisingly, the apparent change in the corresponding entropy is linearly related to the change in the overall binding entropy. We have completed a complimentary study of the dynamics of the bound calmodulin‐binding domains, which allows for the calibration of the dynamical “entropy meter.” The results indicate that changes in residual protein conformational entropy can indeed contribute significantly to the free energy of protein‐ligand association. This is not a generally held view and has significant implications for the maturation of high affinity interactions either by biological evolution or by human intervention.
Engineering Cellular Metabolism
Authors: Liao James C
Published: June 2021

Natural metabolic circuits are evolved for the organisms’ survival and fitness in natural environments. However, to harness organisms’ capability for a human application or to intervene a pathological process may require man‐made metabolic circuits that interact with existing intracellular networks. In the past few years, synthetic biological circuits have been studied to pave the way for applications useful to mankind. In this talk, we will demonstrate how cellular metabolic network can be reengineered to achieve a desired purpose. In particular, we will discuss general design approaches to engineer cellular metabolic networks. Guided by mathematical analyses, we have re‐wired the metabolic circuits for production of specific biochemicals; we have constructed a synthetic feedback control system for redirecting metabolic flux; and we have demonstrated a gene‐metabolic oscillator that mimics a key feature in circadian rhythm, namely the coupling between metabolism and gene expression oscillation.
Individual differences to cocaine activation and self‐administration in rats: role of the brain dopamine transporter
Authors: Zahniser Nancy R , Mandt Bruce H , Schenk Susan , Allen Richard M
Published: June 2021

Individuals differ in their responsiveness to psychostimulants like cocaine, which produce their activating effects largely by inhibiting the dopamine transporter (DAT). This differential responsiveness may impact subsequent drug abuse and addiction. Adult outbred Sprague‐Dawley rats also differ in their responsiveness to cocaine and can be classified as either low or high cocaine responders (LCRs or HCRs), based on the magnitude of each rat's locomotor activity resulting from its first exposure to low dose cocaine. Initially, cocaine inhibits striatal DAT activity only in HCRs; and LCR/HCR differences in rapid cocaine‐induced regulation of DATs parallel their behavioral differences. With repeated daily cocaine administration, however, cocaine begins to inhibit striatal DAT activity in LCRs. Also with repeated cocaine, LCRs more readily develop behaviors associated with enhanced drug responsiveness and reward than HCRs: viz. cocaine‐induced locomotor sensitization, conditioned place preference and greater motivation to self‐administer cocaine. Together, our findings suggest that the initial responsiveness of striatal DATs to cocaine helps to explain the variable locomotor activation to cocaine and may also contribute to the differential reinforcing effectiveness of cocaine in individuals. Supported by NIH grants DA004216, DA015050, DA023343.
Small RNAs as Mobile Signals in Development
Authors: Timmermans Marja C , Chitwood Dan , Nogueira Fabio , Madi Shahinez
Published: June 2021

Leaves of higher plants exhibit a varying degree of asymmetry along their dorsoventral axis. This asymmetry is specified via the polarized expression of class III homeodomain‐leucine zipper (hd‐zipIII) genes, which specify dorsal cell fate. Through detailed in situ hybridization analyses we were able to show that this polarized hd‐zipIII expression is set up by the graded expression pattern of a 21‐nucleotide microRNA, miR166. More recently, we have shown that this miR166 gradient is generated through the opposing activity of a different type of small regulatory RNA, the transacting siRNA tasiR‐ARF. A further twist to the story is added by the observation that tasiR‐ARF in turn is regulated by a third small RNA, miR390. Our observations identify a novel mechanism of pattern formation in which cell fates along a developmental axis are specified through the opposing activity of distinct small RNAs. tasiR‐ARF defines the dorsal side of the leaf by restricting the expression domain of miR166, which in turn delineates the ventral side by restricting expression of the hd‐zipIII transcription factors. Importantly, comparison of the expression patterns of small RNA precursor transcripts to those of the mature small RNA provides evidence that small regulatory RNAs can be mobile, and this offers the intriguing possibility that small RNAs may function as morphogen‐type signals in development.
GroEL‐GroES‐mediated protein folding
Authors: Horwich Arthur , Chapman Eli , Koculi Eda , Apetri Adrian , Fenton Wayne A. , Farr George , Horst Reto , Wüthrich Kurt
Published: June 2021

The chaperonin system assists the folding of a large variety of proteins by two actions: binding in the hydrophobic central cavity of an open ring and folding mediated in a subsequently GroES‐encapsulated hydrophilic chamber. We have been studying the GroEL system both in vivo and in vitro, and report on recent results. In one experiment in vitro, we have used hydrogen‐deuterium exchange and NMR spectroscopy to compare the folding trajectories of human DHFR while folding free in solution and inside a stable GroEL‐GroES complex formed by a single ring version of GroEL, SR1, and GroES. We observe that identical trajectories are taken, involving early formation of the central parallel beta‐sheet of DHFR followed by formation of flanking alpha helices. The recovery of native DHFR is only ~40% in the spontaneous reaction, however, where the remaining molecules are subject to aggregation, vs nearly 100% recovery of native DHFR in the GroEL‐GroES reaction. Thus the sequestered chaperonin cavity appears to forestall or, as evidenced by other experiments, can “correct” misfolded states to support productive folding.
Teaching Basic Immunology Using Flow Cytometry
Authors: Stoyan Tanja , Podshivalova Katie , Stevens Daniel , Eardley Diane , Sears Duane
Published: June 2021

The flow cytometer is a mainstream instrument for analyzing cells being widely used in research and clinical laboratories. However, its use in the teaching laboratory has been limited due to instrument cost and extensive training required. In recent years, a new generation of flow cytometers has become available and these are easier to operate than older models and require less training. We have used such a flow cytometer (Guava Technologies, Inc) and find that it is an ideal instrument for teaching senior level undergraduate students basic techniques in immunology. Specifically, we have developed a quantitative exercise whereby students compare the distributions of CD4, CD8 and CD3 receptors expressed on mouse thymus and spleen cells. Students isolate cells from sacrificed animals, prepare cell suspensions, and stain cells with fluorochrome‐conjugated antibodies following standard protocols. They then acquire data on the flow cytometer, and analyze the data with user friendly, intuitive software. Besides learning the principles of flow cytometry and using the computer software, students learn to organize their scientific data in tables and graphs that allow them to draw scientific conclusions from their findings. We show that the new generation of user‐friendly flow cytometers make an excellent teaching tool for undergraduate laboratory instruction.
Human embryonic stem cells can differentiate into myocytes with structural and functional properties of cardiomyocytes
Authors: Yoo Hyun‐Tae , Yoo Young‐Mi , Kim Yong‐Jung , Lee Uhn
Published: June 2021

Adult cardiomyocytes have limited regenerative capacity and, therefore, significant loss of these cells may result in the development of progressive heart failure. Cardiomyocytes derived from human fetal cells could be useful in restoring heart function in cases of heart failure. We established primary cultures of cardiomyocytes obtained from human fetal cells and found that human fetal cardiomyocytes may constitute a suitable cell source for cellular cardiomyoplasty. These human cardiomyocyte cultures were found to form very large colonies with many flattened cells at the edges, while rhythmically contracting areas appeared at 1 month after plating. In the immunocytochemistry experiment, cells originating from heart cells stained positively with sarcomeric á‐actinin, troponin I, and desmin. Also, thirteen different lines of evidence confirm the cardiomyocytic nature of these cells. Our results demonstrated the presence of spontaneously contracting tissue within the developing cell mass (CM). Using IHC, the presence of cardiac specific proteins and their spatial organization were studied in dispersed cells forming the contracting CM. The establishment of this unique system may have an important impact on our understanding of human cardiac development and function, and can be a step forward in the development of human cardiomyocyte transplantation therapy.
Chondrogenic differentiation of MSC expanded under FCS‐free inflammatory condition
Authors: Felka Tino , Schäfer Richard , Schewe Bernhard , Kluba Torsten , Aicher Wilhelm K
Published: June 2021

Objectives:

Mesenchymal stromal cells (MSC) are a promising tool for tissue engineering of the intervertebral disc. The injured tissue is usually characterized by inflammatory processes. In this study we investigated the effect of IL‐1beta on the chondrogenic differentiation of MSC.

Methods:

Bone‐marrow‐derived MSC were cultured in FCS‐free, GMP‐compatible medium and characterized by flow cytometry. Chondrogenic differentiation of MSC was induced with and without IL‐1beta supplementation. After 28 days, micromasses were analyzed by immunostaining. In addition, the gene expression of several matrix proteins was investigated by quantitative PCR.

Results:

Micromasses differentiated under IL‐1beta supplementation are characterized by smaller dimensions, disruptions and weaker accumulation and expression of extracellular matrix proteins compared to micromasses differentiated without IL‐1beta supplementation.

Conclusion:

Inflammatory processes in the injured tissue could inhibit the chondrogenic differentiation of transplanted MSC. For cell therapy to be effective it will be necessary to develop strategies to ensure the steady expression of extracellular matrix proteins under inflammatory conditions.
A Composite Scaffold for the Engineering of Hollow Organs and Tissues
Authors: Eberli Daniel , Yoo James J. , Atala Anthony
Published: June 2021

Introduction

Several types of synthetic and naturally derived biomaterials have been used for augmenting hollow organs and tissues. However, each has desirable traits which were exclusive of the other. We fabricated a composite scaffold and tested its potential for the engineering of hollow organs in a bladder tissue model.

Materials and Methods

The composite scaffolds were configured to accommodate a large number of cells on one side and were designed to serve as a barrier on the other side. The scaffolds were fabricated by bonding a collagen matrix to PGA polymers with threaded collagen fiber stitches. Bladder cells were seeded on the composite scaffolds, and implanted in mice for up to 4 weeks and analyzed.

Results and Discussion

The composite scaffolds, consisting of an acellular matrix and PGA polymers, are tightly bonded, and maintained their structural properties. The bladder cells readily attached and proliferated on the scaffolds and formed bladder tissue structures in vivo. The biomechanical studies demonstrated that the tissues were readily elastic while maintaining their pre‐configured structures.

Conclusions

We demonstrate that a composite scaffold can be fabricated with two completely different polymer systems for the engineering of hollow organs.
Interplay of angiotensin II and angiotensin 1–7 in the regulations of matrix metalloproteinases of human cardiocytes
Authors: Pan Chun‐Hsu , Wen Cheng‐Hao , Lin Chih‐Sheng
Published: June 2021

Angiotenisn 1–7 (Ang 1–7) is considered to play an opposing role to angiotensin II (Ang II). However, the modulation of MMPs and TIMPs by Ang 1–7 is largely unclear in cardiocytes, and the counteractive effects of Ang 1–7 on Ang II‐mediated MMPs and TIMPs expression need to be identified. We examined the mRNA expression of MMPs and TIMPs in human cardiac fibroblasts (HCF) and cardiac myocytes (HCM) stimulated by Ang II or Ang 1–7, and analyzed the antagonistic effects of Ang 1–7 to Ang II. Ang II decreased transcript expression of MMP‐1, MMP‐2, TIMP‐1, TIMP‐2 and TIMP‐3, but downregulated MMP‐9 expression in the HCF cells. Transcript expression of MMP‐9 and TIMP‐2 were downregulated by Ang 1–7 in the cells. In the HCM cells, Ang II induced MMP‐1 and MMP‐9 expression, but all of examined MMPs and TIMPs, except MMP‐9, were markedly decreased by Ang 1–7. Ang 1–7 could counteract the effects of Ang II‐mediated regulations on MMP‐1, MMP‐9 and TIMP‐1 in the HCF cells. The regulations of all examined MMPs by Ang II were reversed to basal expression by Ang 1–7 in the HCM cells. Opposite regulation in the examined MMPs and TIMPs expression was found between Ang II and Ang 1–7, and the antagonistic effects of Ang 1–7 to Ang II were also identified. These results might raise a potential role of Ang 1–7 to attenuate Ang II‐induced ECM remodeling in heart. This work was supported by National Science Council (NSC 95‐2313‐B‐009‐002‐MY3), Taiwan.
RGS2 is a negative regulator of Gq/11‐mediated signaling and cell proliferation in adult rat ventricular fibroblasts
Authors: Zhang Peng , King Michelle , Ayrapetov Marina , Mende Ulrike
Published: June 2021

Cardiac fibroblasts (CF) contribute to cardiac remodeling by assuming an “activated” myofibroblast (MyoFb) phenotype. Many G protein‐coupled receptors are involved in CF activation. G proteins are controlled by Regulators of G protein Signaling (RGS). RGS expression and function in CF and MyoFb are unknown. We set out to identify which RGS are expressed in isolated adult rat ventricular CF (passage P0‐P2) and to begin to delineate their function. Morphologic changes and expression of MyoFb markers (e.g., α‐smooth muscle actin) indicated gradual conversion from CF (P0) to MyoFb (P2). We detected mRNA of 14 RGS by RT‐PCR that were differentially regulated during CF to MyoFb conversion. Only RGS2 mRNA was markedly up‐regulated in response to short‐term stimulation of Gq/11‐coupled Angiotensin II (Ang II) and Gs‐coupled β‐adrenergic receptors. Adenoviral RGS2 expression blunted Ang II‐induced phospholipase C β activation by 65±2% (P0), 66±2% (P1) and 78±3% (P2) (n=3, P<0.05) and CF proliferation (BrdU incorporation) by 37±5% (n=10, P<0.05). RGS2 had no effect on Gs‐coupled cAMP accumulation. Thus, while RGS2 is selectively up‐regulated in response to Gq/11 and Gs activation, it negatively regulates only Gq/11‐mediated signaling and proliferation in both CF and MyoFb. These data point to RGS2 as an important regulator of CF and MyoFb signaling and function.

Supported by the NIH and American Heart Association.
Axonal degeneration as a self‐destructive defense mechanism against neurotropic virus infection
Authors: Tsunoda Ikuo , Tanaka Tomoko , Hasanovic Faris , Fujinami Robert S.
Published: June 2021

C57BL/WldS (Wallerian degeneration slow mutant) mice are protected from axonal (Wallerian) degeneration by overexpression of a fusion protein (WldS). We tested whether delay of axonal degeneration in WldS mice could be beneficial in mice infected with GDVII and DA strains of Theiler's murine encephalomyelitis virus (TMEV), which spread in the central nervous system (CNS) using axonal transport. We infected WldS mice and their parent strain, C57BL/6 (B6) mice, with virulent GDVII virus. One week after GDVII virus infection, 97% of B6 mice died, while 36% of WldS mice survived. LD50 in B6 and WldS mice were 1.1 and 23 plaque forming units, respectively. WldS mice had neuronal death (apoptosis) and marked loss of MAP‐2 immunoreactivity, suggesting that apoptosis and dendritic pathology cannot be prevented by the WldS gene. In contrast, infection with the less virulent DA virus resulted in limb paralysis only in WldS mice with larger numbers of virus‐infected cells in the CNS. Although prolonged survival of axons in WldS mice may be beneficial during GDVII virus infection, it could favor virus spread during DA virus infection. This suggests that axonal degeneration in B6 mice might be a beneficial mechanism that limits the spread of some neurotropic viruses. Supported by the University of Utah (Funding Incentive Seed Grant to IT) and the NIH (NS34497 to RSF).
Transforming Growth Factor‐β stimulates smooth muscle differentiation in epicardial cells lacking the Type III Receptor
Authors: Barnett Joey V. , Compton Leigh A. , Love Joey D. , Austin Anita F.
Published: June 2021

Transforming growth factor (TGF) β receptor III (TGFβR3) binds all 3 TGFβ ligands and inhibin with high affinity but lacks the serine/threonine kinase domain found in the type I and type II receptors (TGFβR1, TGFβR2). TGFβR3 facilitates signaling via TGFβR1/TGFβR2 but also has been suggested to play a unique and nonredundant role in TGFβ signaling. Coronary vessel development is impaired in Tgfbr3 null mice with few vessels formed and mice die at embryonic day (E) 14.5. In null mice, vessels throughout the yolk sac and embryo form and recruit smooth muscle in a pattern indistinguishable from heterozygous or wild‐type littermates. Null mice form 2 coronary ostia and appear to initiate smooth muscle recruitment by E14. To directly address the role of Tgfbr3 in smooth muscle differentiation in epicardial cells we generated immortalized cell lines from wildtype, heterozygous null, and null animals at E11.5 and E13.5. Cells from all genotypes grew as tightly packed epithelium and expressed Zonula Occludens‐1 (ZO‐1). The addition of 250 pM TGFβ1 or TGFβ2 demonstrated a loss of epithelial morphology as measured by the loss of ZO‐1. TGFβ1 or TGFβ2 induced the smooth muscle marker SM22α in all genotypes. Therefore, TGFβR3 is not required in epicardial cells for smooth muscle differentiation and the defect in coronary vessel development seen in Tgfbr3 null mice is likely not due to defects in smooth muscle recruitment.
Kinetics of presynaptic filament assembly in the presence of SSB and mediator proteins
Authors: Morrical Scott Walker , Liu Jie , Berger Christopher
Published: June 2021

Enzymes of the RecA/Rad51 family catalyze DNA strand exchange reactions that are important for homologous recombination and DSB repair. RecA/Rad51 recombinases are activated by their assembly into presynaptic filaments on ssDNA, a process that is regulated by ssDNA‐binding (SSB) and mediator proteins. Mediator proteins stimulate strand exchange by accelerating the rate‐limiting displacement of SSB from ssDNA by the incoming recombinase. The use of mediators is a highly conserved strategy in recombination, but the precise mechanism of mediator activity is unknown. Here we present a detailed kinetic analysis of presynaptic filament assembly using the well‐defined bacteriophage T4 recombination system (UvsX recombinase, Gp32 SSB, and UvsY mediator). We demonstrate that the ATP‐dependent assembly of UvsX presynaptic filaments on Gp32‐covered ssDNA is limited by a salt‐sensitive nucleation step in the absence of mediator. Mediator protein UvsY selectively enhances filament nucleation, allowing UvsX assembly and Gp32 displacement to occur at higher salt concentrations. UvsY promotes filament nucleation by selectively stabilizing the pre‐nucleation complex that forms prior to a slow isomerization step. These and other results provide a rigorous kinetic model for presynaptic filament assembly involving all three major protein components—recombinase, SSB, and mediator, on native ssDNA.
Notch regulation of telomerase induction in pulmonary fibrosis
Authors: Hu Biao , Liu Tianju , Wu Zhe , Phan Sem H.
Published: June 2021

Injury in diverse tissues, including the lung, is accompanied by induction of telomerase activity which is primarily due to induced TERT gene expression. Because Notch signaling pathway plays important roles in development and cell differentiation, its role in regulation of TERT gene expression is examined. Lung injury and fibrosis in mice was induced by endotracheal injection of bleomycin. Analysis of lung tissues revealed that bleomycin‐induced fibrosis caused significant reduction in Notch protein (western blot) and mRNA (real time qPCR) levels. Fibroblasts from fibrotic lungs expressed high levels of TERT, which was suppressed by ectopic overexpression of the intracellular domain of Notch1 (NIC). Conversely, Notch signaling deficiency in cells from FX (GDP‐4‐keto‐6‐deoxymannose 3,5‐epimerase‐4‐reductase) knockout mice, or inhibition of Notch signaling by γ‐secretase inhibitor, resulted in significant induction of telomerase activity. Moreover gel shift assays indicated that the Notch signal mediator, CBF1, could bind to the TERT promoter. Thus Notch signaling may be important in suppression of TERT gene expression in normal adult somatic cells, and that its induction in tissue injury may be due at least in part to relief from this repression. Supported by NIH grants HL28737, HL31963, HL 52285 and HL77297, and an Award to SHP from the Sandler Program in Asthma Research.
The genes coding for the Lucina pectinata oxygen reactive binding hemoglobins have different structures
Authors: Rivera Linda Edith , López‐Garriga Juan , Cadilla Carmen L.
Published: June 2021

Lucina pectinata is a mollusk that houses intracellular symbiotic bacteria and produces three types of hemoglobin. HbI is sulfide‐reactive whereas HbII and HbIII are oxygen‐reactive proteins. Their gene structures were examined to understand their evolutionary origin. The L. pectinata HbII and HbI genes contain two introns, interrupting the coding region at position B12.2 and G7.0. The HbIII gene has three introns, including one located 60 bases before the start codon, and two other introns at conserved positions. The 5′UTR and coding regions of HbII and HbIII are identical in length but their 3′UTRs are of different size. The HbII 3′UTR is relatively long when compared with the other globins reported in the literature, including L. pectinata HbI and HbIII. Alternate polyadenylation sites were detected in HbIII and HbII. The L. pectinata Hb genes may be under different cellular controls that direct them to exert their particular functions, which requires characterization, analysis and functional studies of the regulatory elements of the cognate genes.
RAG2 PHD finger couples histone H3 lysine 4 trimethylation with V(D)J recombination
Authors: Matthews Adam Goon Wai , Kuo Alex J , Ramon‐Maiques Santiago , Han Sunmi , Champagne Karen S , Kutateladze Tatiana G , Yang Wei , Gozani Or , Oettinger Marjorie A
Published: June 2021

Transcription is known to be regulated by chromatin‐associated proteins containing protein domains that specifically recognize distinct covalent post‐translational histone modifications. However, it has been unclear whether similar mechanisms are involved in mammalian DNA recombination. Here, we show that RAG2 – an essential component of the eukaryotic RAG1/2 V(D)J recombinase, that mediates antigen receptor gene assembly in developing lymphocytes – contains a plant homeodomain (PHD) finger that specifically recognizes histone H3 trimethylated at lysine 4 (H3K4me3). A high‐resolution crystal structure of the RAG2PHD‐H3K4me3 complex reveals the molecular basis of H3K4me3‐recognition by RAG2. Mutations that abrogate RAG2's recognition of H3K4me3 severely impair V(D)J recombination in vivo. Reducing the level of H3K4me3 similarly leads to a decrease in V(D)J recombination in vivo. Notably, a conserved tryptophan residue (W453) that constitutes a key structural component of the K4me3‐binding surface and is essential for RAG2's recognition of H3K4me3 is mutated in patients with immunodeficiency syndromes. Together our results identify a novel function for histone methylation in mammalian DNA recombination. In addition, we provide the first evidence suggesting that disrupting the read‐out of histone modifications can cause an inherited human disease.
Roles of renal microRNA‐216a (miR‐216a) in TGF‐beta signaling and diabetic nephropathy
Authors: Kato Mitsuo , Wang Lin , Wang Mei , Yuan Hang , Lanting Linda , Putta Sumanth , Todorov Ivan , Zhang Jane , Rossi John J , Natarajan Rama
Published: June 2021

Diabetic Nephropathy (DN) is a major microvascular complication of diabetes. Key features include accumulation of extracellular matrix (ECM) proteins such as collagen type1‐alpha 2 (Col1a2). Transforming growth factor‐beta 1 (TGF‐b), a key regulator of ECM genes, is increased in renal glomerular mesangial cells (MC) in DN. We recently found that miR‐192 levels were significantly enhanced in diabetic glomeruli, as well as TGF‐b‐treated MC. miR‐192 could target E‐box repressor Zeb2 and mediate TGF‐b‐induced Col1a2 expression. We now note that miR‐216a levels were also significantly enhanced in diabetic glomeruli and TGF‐b‐treated MC. miR‐216a could target YB‐1, an RNA‐binding protein (located in processing bodies), which regulates mRNA stability and translation. YB‐1 levels were reduced in diabetic glomeruli and TGF‐b‐treated MC. YB‐1‐TSC‐22 mRNA interaction was reduced by TGF‐b and also by miR‐216a or YB‐1 shRNA, while miR‐216a inhibitor increased this interaction. TSC‐22 protein levels were enhanced by TGF‐b due to reduced YB‐1. Since TSC‐22 is known to augment TGF‐b signaling via Smad3/4, these data suggest that TGF‐b induced miR‐216a and TSC‐22 translation due to reduced YB‐1 can enhance Col1a2 expression. These results uncover novel roles for renal miRs in controlling TGF‐b‐induced Col1a2. miRs could be novel targets for diabetic vascular diseases. Supported by NIH (NIDDK) and JDRF.
Polyubiquitination determinants on Ubc1, an APC‐dependent E2
Authors: Brenni Monica Carolina Rodrigo , Morgan David O.
Published: June 2021

The Anaphase‐Promoting Complex (APC) is an E3 ubiquitin ligase that assembles polyubiquitin chains on substrates important for cell cycle progression. In budding yeast, the APC collaborates with two E2 ubiquitin‐conjugating enzymes, Ubc4 and Ubc1. We have demonstrated that Ubc4 and Ubc1 have very different enzymatic behaviors: APC reactions with Ubc4 result in rapid monoubiquitination of multiple nonspecific lysines on the substrate, whereas APC reactions with Ubc1 result in the assembly of polyubiquitin chains that are linked specifically through lysine 48 (K48) of ubiquitin. We are now mapping the surfaces on Ubc1 that are responsible for its K48 linkage specificity. We focused in two clusters of amino acids that are found on the protein surface near the catalytic cysteine of Ubc1 but are not present in Ubc4. Changing these amino acids in Ubc1 to those in Ubc4 resulted in a specific defect in K48‐linked ubiquitination activity. Nonspecific Ubc1 activity toward other lysines was not affected. We therefore speculate that these amino acid clusters in Ubc1 help guide K48 of the attacking ubiquitin toward the catalytic cysteine. We now hope to change the residues in this region of Ubc4 to those found in Ubc1, to test if these amino acids are sufficient to confer K48 specificity on Ubc4.
Plasminogen activator inhibition in Alzheimer disease brain
Authors: Fabbro Shay , Seeds Nicholas
Published: June 2021

Amyloid beta (Aβ) plaques are a pathological hallmark of Alzheimer's disease (AD). Several proteases are known to cleave Aβ including plasmin, the product of tissue plasminogen activator (tPA) cleavage of the pro‐enzyme plasminogen. Although several in vitro studies have demonstrated a clear link between the PA/plasmin system and AD, there has been little analysis of the PA/plasmin system in the brains of AD patients. In this study, we utilized zymography and immunocapture assays to determine the tPA activity in frontal cortex tissue of AD patients. The amount of active tPA is significantly reduced (~50%) in brain homogenates of AD patients compared to age‐matched controls. Interestingly, western blot analysis showed that both tPA and plasminogen protein levels were similar between the AD and control brain tissues; suggesting that tPA activity was inhibited in the AD brain. An analysis of endogenous PA inhibitors showed that while plasminogen activator inhibitor‐1 levels were unchanged, neuroserpin levels were significantly elevated (1.6‐fold) in brains of AD patients. Immunohistochemical studies demonstrate that both tPA and neuroserpin protein are associated with Aβ plaques in the AD brain tissue, providing further support for our hypothesis. Thus, neuroserpin inhibition of tPA activity explains the lower plasmin levels and possibly the Aβ accumulation seen in AD brain tissue. Neuroserpin may be a target for AD therapy.
Refolding of ybeA: a native knotted protein
Authors: Yeh Yi‐Chen , Chen Tzu‐Hsuan , Chu Hsueh‐Liang , Tsai Chuan‐Mei , Chang Chia‐Ching
Published: June 2021

YbeA is a hypothetic methyltransferases (MTases) enzyme. X‐ray crystallography study indicated that the backbone of this protein form a deep trefoil knot. It is interesting to reveal the mechanism of knot transition during its folding process. In this study we have cloned, expressed and folded this protein from inclusion body of E. coli. The conformational analysis of its unfolded, folding intermediates and native form, by spectrophotometers, and dynamic light scattering indicated that YbeA is a native knot protein. There is no knot transition during the folding process. Meanwhile, this deep knot protein makes itself thermal stable in high temperature environment (above 125 °C) which was analyzed by differential scanning calorimeter. Therefore, this knot structure may be used in thermal stable enzyme engineering.
Refolding Circulary Permutated GFP From Inclusion Bodies; Accessing the Chromophore of Green Fluorescent Protein
Authors: Kim TaeSoo , Melief Edward , Tonge Peter J
Published: June 2021

The Green Fluorescent Protein(GFP) which is derived from the jellyfish Aequorea Victoria has become an invaluable tool in molecular and cellular biology research due to its ability to form a chromophore in vitro. Its amino acid components, Ser65, Tyr66, and Gly67 undergo post‐translational, autocatalytic reaction to create a five membered ring chromophore. This mature P‐hydroxybenzylidene imidazolinone chromophore is formed in exposure to molecular oxygen via an intramolecular reaction. Even though several possible mechanisms have been suggested for chromophore biosynthesis, none have been proven definitive. The chemical ligation of unnatural amino acids onto the chromophore residues will halt the chromophore formation without affecting protein refolding process. Because the chromophore is located in the center of the beta‐barrel protein, making any subtle changes to the chromophore is difficult to accomplish. Therefore, a permutated variant of GFP has been constructed where N and C termini of the protein are linked and a new N terminus closer to S65, T66, G67 is created. The truncation of N terminus results in GFP where unnatural amino acids can be ligated in place of the residues that comprise the chromophore. Subsequent refolding of this protein will help eluciadate the mechanisms of chromophore formation. The focus of current studies is to find the optimal renaturing condition for this variant of GFP from the inclusion bodies, before the chromophore has a chance to form.
Isoform Specific Regulatory Site for PKA: AGC‐Specific aH‐aI Loop
Authors: yang jie , Kenndy Eileen , Wu Jian , Deal Michael , Brown Simon , Pennypacker Juniper , Ghosh Gourisankar
Published: June 2021

In an initial study of ATP binding site in the catalytic subunit of PKA, a distantly coupled mutation pair of F327A/K285P was identified through a yeast genetic screen. It was found that while the wild‐type mammalian C‐subunit can sustain growth in a yeast strain devoid of PKA‐expressing tpk genes, the F327A mutant, a C‐terminal residue that constitutes part of the ATP binding site, failed to do so. Screening for suppressors by means of restored cell survival in the tpk‐null strain identified several suppressor mutations, one of which was K285 to Pro. K285 lies in an AGC‐specific insert between the aH‐aI helices of the large lobe of the kinase domain. In this study, we confirmed an important role of F327 in stabilizing ATP binding. We also showed that the second site mutation of K285P did not restore PKA activity; rather, through allosteric connection with F327A, the double mutant exhibited decreased inhibition by the inhibitory regulatory subunit of PKA. Thus a low yet constant PKA activity was maintained which appeared to be sufficient for cell viability.

Further characterization of the mutants unexpectedly showed that K285 constitutes an interacting site that is critical for the RII‐, but not the RI‐ subunit of PKA; this isoform‐specific interaction was not previously appreciated. This study highlighted a unique feature of the K285 site in the AGC specific insert, which may have considerable relevance to other AGC kinases.
Generation and characterization of mBAP‐9, a new in vitro brown adipogenesis model
Authors: Wu Yu , Kim Ji‐Young , Smas Cynthia M.
Published: June 2021

We have established mBAP‐9, a new brown preadipocyte cell line, from interscapular BAT of C57BL/6 mice. mBAP‐9 preadipocytes undergo spontaneous adipogenesis which is enhanced by treatment with a combination of dexamethasone, 3‐methyl‐1‐isobutylxanthine, indomethacin, insulin and triiodotyronine. mBAP‐9 adipogenesis is accompanied by formation of multilocular lipid droplets, induction of Ucp1 and PPARα transcripts, and expression of PGC‐1α . Using a candidate gene approach based on in silico analysis of the GNF Symatlas database, we identified 10 new adipocyte differentiation‐dependent genes that exhibit marked upregulation during mBAP‐9 adipogenesis. Three of these genes, carboxymethylene butenolidase‐like (Cmbl), a new adipose tissue α/β hydrolase (Atabh), and Tmem182 were further characterized. Cmbl, Atabh and Tmem182 are induced 24‐fold, 13‐fold, and 45‐fold, respectively, during in vitro adipogenesis of mBAP‐9 preadipocytes and evidence differential regulation by β‐adrenergic agonists in mBAP‐9 adipocytes. Transcripts for Cmbl, Atabh and Tmem182 are also upregulated during in vitro white adipogenesis of 3T3‐L1 cells, are expressed in BAT and in white adipose tissue (WAT) in vivo, and evidence dysregulation in obese adipose tissue(s). We conclude that the mBAP‐9 preadipocyte cell line we have developed is a novel and useful tool for detailed molecular studies of adipogenesis.
A Central Role for CD36 in Mediating Oxidized LDL and Fatty Acid Induced Insulin Resistance
Authors: Kennedy David J , Kuchibhotla Sai , Vanegas Difernando , Nimako George , Febbraio Maria , Silverstein Roy L
Published: June 2021

Elevated levels of oxidized LDL (oxLDL) and fatty acids (FA) are highly associated with insulin resistance. Clinical and experimental evidence also demonstrate increased expression of scavenger receptor CD36 in insulin resistant states. As oxLDL and FA are CD36 ligands, we tested the hypothesis that they influence insulin resistance in a CD36 dependant manner.

3T3‐L1 adipocytes treated with oxLDL or with FA showed time and dose dependent increases in CD36 expression, activated Jun kinase (JNK), induced inhibitory IRS‐1 serine phosphorylation, increased adipocyte lypolysis and decreased glucose uptake. Pre‐incubation with either a CD36 monoclonal antibody or JNK kinase inhibitor attenuated these changes.

Next, ApoE null mice with or without a CD36 null mutation (CD36+ or CD36−) were fed a high fat diet for 12 weeks and examined for physiologic and biochemical markers of insulin resistance. These mice have increased oxLDL and FA levels and their isolated lipoproteins are CD36 ligands, inducing CD36‐dependent foam cell formation and atherosclerosis. Compared with CD36+ mice, CD36− mice had improved glucose clearance, decreased JNK activation and IRS‐1 serine phosphorylation in adipose tissue, increased AKT activation in both liver and muscle, as well as increased levels of adiponectin in adipose, muscle and liver.

These data suggest a central role for CD36 in mediating oxLDL and FA induced insulin resistance.
TIAF1 physically heterodimerizes with WOX1 in the mitochondria: a role in apoptosis
Authors: Chang Nan‐Shan
Published: June 2021

Overexpressed TIAF1 (TGF‐beta1‐induced antiapoptotic factor) promotes the growth of fibroblasts, but induces apoptosis in monocytic and non‐fibroblast cells. Functional property of TIAF1 in regulating cell growth and death is largely unknown. Here, we show that knockdown of endogenous TIAF1 by siRNA prevented cell growth inhibition and apoptosis by staurosporine, tumor necrosis factor, UV light, and TGF‐beta1. We have shown that overexpressed tumor suppressor WOX1 (WWOX or FOR) induced cell death at the mitochondrial level. Interestingly, WOX1 induced‐growth suppression and death of fibroblasts and prostate cancer cells was blocked in TIAF1‐knockdown cells. Analysis by fluorescence resonance energy transfer (FRET) and co‐immunoprecipitation revealed that TIAF1 physically heterodimerizes with WOX1 to its C‐terminus (designated D3 region). WOX2, an isoform of WOX1, possesses a truncated C‐terminus and did not bind TIAF1. Time‐lapse microscopy revealed that UV light induced an initial increased in the heterodimerization, followed by dissociation between WOX1 and TIAF1. Overexpressed WOX1 and TIAF1 caused apoptosis synergistically, suggesting their role in programmed cell death via the mitochondrial pathway.
Origin and Evolution of One of the Most Ancient Rossmann Folds
Authors: Duax William , Huether Robert , Pletnev Vladimir , Umland Timothy , Connare Sanjay , Weeks Charles
Published: June 2021

The β‐ketoacyl [acyl carrier protein] reductase (β‐k‐ACPR) enzymes, a 690‐member subset of the SCOR short‐chain oxidoreductase family, are essential to fatty acid synthesis in bacteria and plants. There is one member of the family in every bacterial and archeal genome sequenced to date. We have discovered that (1) the most primitive member of the family was an NADP reductase, (2) that NADP binding was originally contingent upon a Ser or Thr residue in a specific turn, (3) that a specific dimer assembly is stabilized by the stacking of aromatic groups and (4) that a previously undetected GGMXM sequence at the C‐terminus, conserved in all species of proteobacteria, stabilizes the functionally required tetramer by multiple hydrogen bonding and aromatic ring stacking crosslinking the four monomers together. Our analysis indicates that the primordial members of the β‐k‐ACPR family probably arose in the GC‐rich α‐proteobacteria and that they are distinguished by the presence of multiple open reading frames (MORFs), an extreme codon bias in their DNA and an amino acid bias in their protein composition. This research was supported by NIH grant No. DK26546.
Control of local actin assembly during exocytosis by compartment mixing
Authors: Bement William
Published: June 2021

Localized actin assembly is an essential feature of many fundamental cell processes, but how it is controlled is poorly understood. Here we have investigated the trigger for assembly of actin “coats” around exocytosing secretory granules in Xenopus eggs. We show that the trigger is fusion of the secretory granule with the plasma membrane, and the resultant mixing of the membrane contents of these two compartments. Specifically, diacylyglcerol from the plasma membrane incorporates into the membrane of the exocytosing secretory granules after fusion with the plasma membrane, and directs sustained recruitment of protein kinase C beta which, in turn, promotes local activation of Cdc42 which then directs actin assembly. The secretory granule membrane contributes Cdc42, which is localized (in the inactive, GDP‐bound form) to the secretory granule surface prior to exocytosis. Thus, actin assembly is limited to the surface of exocytosing secretory granules by virtue of compartment mixing. This mechanism provides a broadly useful means for cells to precisely limit actin assembly at the plasma membrane and other areas where membrane fusion occurs.