Effects of glycine betaine and glycerophosphocholine on thermal stability of ribonuclease

1998 ◽  
Vol 274 (4) ◽  
pp. F762-F765 ◽  
Author(s):  
Maurice B. Burg ◽  
Eugenia M. Peters
Keyword(s):  
Thermal Stability ◽  
Tissue Culture ◽  
Glycine Betaine ◽  
Metabolic Cost ◽  
Rnase A ◽  
Renal Cells ◽  
And Function ◽  
Thermal Stability Of

Urea in renal medullas is sufficiently high to perturb macromolecules, yet the cells survive and function. The counteracting osmolytes hypothesis holds that methylamines, such as glycine betaine (betaine) and glycerophosphocholine (GPC) in renal medullas, stabilize macromolecules and oppose the effects of urea. Although betaine counteracts effects of urea on macromolecules in vitro and protects renal cells from urea in tissue culture, renal cells accumulate GPC rather than betaine in response to high urea both in vivo and in tissue culture. A proposed explanation is that GPC counteracts urea more effectively than betaine. However, we previously found GPC slightly less effective than betaine in counteracting inhibition of pyruvate kinase activity by urea. To test another macromolecule, we now compare GPC and betaine in counteracting reduction of the thermal stability of RNase A by urea. We find that urea decreases the thermal transition temperature and that betaine and GPC increase it, counteracting urea approximately equally. Therefore, the preference for GPC in response to high urea presumably has some other basis, such as a lower metabolic cost of GPC accumulation.

Survival and Function of Bioengineered Cardiac Grafts

Circulation ◽  
1999 ◽  
Vol 100 (suppl_2) ◽  
Author(s):  
Ren-Ke Li ◽  
Zhi-Qiang Jia ◽  
Richard D. Weisel ◽  
Donald A. G. Mickle ◽  
Angel Choi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Subcutaneous Tissue ◽  
Scar Tissue ◽  
Myocardial Scar ◽  
Fetal Rat ◽  
Myocardial Scar Tissue ◽  
And Function ◽  
Cardiac Graft

Introduction —Patients with congenital heart disease frequently require graft material for repair of cardiac defects. However, currently available grafts lack growth potential and are noncontractile and thrombogenic. We have developed a viable cardiac graft that contracts spontaneously in tissue culture by seeding cells derived from fetal rat ventricular muscle into a biodegradable material. We report our investigations of the in vitro and in vivo survival and function of this bioengineered cardiac graft. Methods and Results —A cardiomyocyte-enriched cell inoculum derived from fetal rat ventricular muscle was seeded into a piece of Gelfoam (Upjohn, Ontario, Canada), a biodegradable gelatin mesh, to form the graft. For in vitro studies, growth patterns of the cells within the graft were evaluated by constructing growth curves and by histologic examination; in in vivo studies, the graft was cultured for 7 days and then implanted either into the subcutaneous tissue of adult rat legs or onto myocardial scar tissue in a cryoinjured rat heart. Five weeks later, the graft was studied histologically. The inoculated cells attached to the gelatin mesh and grew in 3 dimensions in tissue culture, forming a beating cardiac graft. In both the subcutaneous tissue and the myocardial scar, blood vessels grew into the graft from the surrounding tissue. The graft implanted into the subcutaneous tissue contracted regularly and spontaneously. When implanted onto myocardial scar tissue, the cells within the graft survived and formed junctions with the recipient heart cells. Conclusions —Fetal rat ventricular cells can grow 3-dimensionally in a gelatin mesh. The cells in the graft formed cardiac tissue and survived and contracted spontaneously both in tissue culture and after subcutaneous implantation. Future versions of this bioengineered cardiac graft may eventually be used to repair cardiac defects.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

scholarly journals Extracellular vesicles as mediators and markers of acute organ injury: current concepts

2021 ◽  
Author(s):  
Birte Weber ◽  
Niklas Franz ◽  
Ingo Marzi ◽  
Dirk Henrich ◽  
Liudmila Leppik
Keyword(s):  
Extracellular Vesicles ◽  
Inflammatory Diseases ◽  
Organ Injury ◽  
Isolation And Characterization ◽  
Communication Processes ◽  
Incidence And Mortality ◽  
New Biomarkers ◽  
And Function

AbstractDue to the continued high incidence and mortality rate worldwide, there is a need to develop new strategies for the quick, precise, and valuable recognition of presenting injury pattern in traumatized and poly-traumatized patients. Extracellular vesicles (EVs) have been shown to facilitate intercellular communication processes between cells in close proximity as well as distant cells in healthy and disease organisms. miRNAs and proteins transferred by EVs play biological roles in maintaining normal organ structure and function under physiological conditions. In pathological conditions, EVs change the miRNAs and protein cargo composition, mediating or suppressing the injury consequences. Therefore, incorporating EVs with their unique protein and miRNAs signature into the list of promising new biomarkers is a logical next step. In this review, we discuss the general characteristics and technical aspects of EVs isolation and characterization. We discuss results of recent in vitro, in vivo, and patients study describing the role of EVs in different inflammatory diseases and traumatic organ injuries. miRNAs and protein signature of EVs found in patients with acute organ injury are also debated.

scholarly journals Modelling the Human Placental Interface In Vitro—A Review

Micromachines ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 884
Author(s):  
Marta Cherubini ◽  
Scott Erickson ◽  
Kristina Haase
Keyword(s):  
Drug Testing ◽  
Cell Types ◽  
In Vitro Models ◽  
Placental Development ◽  
Scientific Challenge ◽  
Induced Pluripotent ◽  
And Function ◽  
And Control

Acting as the primary link between mother and fetus, the placenta is involved in regulating nutrient, oxygen, and waste exchange; thus, healthy placental development is crucial for a successful pregnancy. In line with the increasing demands of the fetus, the placenta evolves throughout pregnancy, making it a particularly difficult organ to study. Research into placental development and dysfunction poses a unique scientific challenge due to ethical constraints and the differences in morphology and function that exist between species. Recently, there have been increased efforts towards generating in vitro models of the human placenta. Advancements in the differentiation of human induced pluripotent stem cells (hiPSCs), microfluidics, and bioprinting have each contributed to the development of new models, which can be designed to closely match physiological in vivo conditions. By including relevant placental cell types and control over the microenvironment, these new in vitro models promise to reveal clues to the pathogenesis of placental dysfunction and facilitate drug testing across the maternal–fetal interface. In this minireview, we aim to highlight current in vitro placental models and their applications in the study of disease and discuss future avenues for these in vitro models.

scholarly journals Therapeutic Application of Brain-Specific Angiogenesis Inhibitor 1 for Cancer Therapy

Cancers ◽  
2021 ◽  
Vol 13 (14) ◽  
pp. 3562
Author(s):  
Mitra Nair ◽  
Chelsea Bolyard ◽  
Tae Jin Lee ◽  
Balveen Kaur ◽  
Ji Young Yoo
Keyword(s):  
Angiogenesis Inhibitor ◽  
Suppressor Gene ◽  
Chemotherapeutic Agents ◽  
Tumor Models ◽  
In Vivo Models ◽  
Herpes Simplex Viruses ◽  
Multiple Tumor ◽  
And Function

Brain-specific angiogenesis inhibitor 1 (BAI1/ADGRB1) is an adhesion G protein-coupled receptor that has been found to play key roles in phagocytosis, inflammation, synaptogenesis, the inhibition of angiogenesis, and myoblast fusion. As the name suggests, it is primarily expressed in the brain, with a high expression in the normal adult and developing brain. Additionally, its expression is reduced in brain cancers, such as glioblastoma (GBM) and peripheral cancers, suggesting that BAI1 is a tumor suppressor gene. Several investigators have demonstrated that the restoration of BAI1 expression in cancer cells results in reduced tumor growth and angiogenesis. Its expression has also been shown to be inversely correlated with tumor progression, neovascularization, and peri-tumoral brain edema. One method of restoring BAI1 expression is by using oncolytic virus (OV) therapy, a strategy which has been tested in various tumor models. Oncolytic herpes simplex viruses engineered to express the secreted fragment of BAI1, called Vasculostatin (Vstat120), have shown potent anti-tumor and anti-angiogenic effects in multiple tumor models. Combining Vstat120-expressing oHSVs with other chemotherapeutic agents has also shown to increase the overall anti-tumor efficacy in both in vitro and in vivo models. In the current review, we describe the structure and function of BAI1 and summarize its application in the context of cancer treatment.

scholarly journals Necrostatin-1 Supplementation to Islet Tissue Culture Enhances the In-Vitro Development and Graft Function of Young Porcine Islets

2021 ◽  
Vol 22 (16) ◽  
pp. 8367
Author(s):  
Hien Lau ◽  
Shiri Li ◽  
Nicole Corrales ◽  
Samuel Rodriguez ◽  
Mohammadreza Mohammadi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Oxygen Consumption ◽  
Insulin Secretion ◽  
Oxygen Consumption Rate ◽  
Consumption Rate ◽  
In Vitro Development ◽  
Porcine Islets ◽  
Vitro Development

Pre-weaned porcine islets (PPIs) represent an unlimited source for islet transplantation but are functionally immature. We previously showed that necrostatin-1 (Nec-1) immediately after islet isolation enhanced the in vitro development of PPIs. Here, we examined the impact of Nec-1 on the in vivo function of PPIs after transplantation in diabetic mice. PPIs were isolated from pancreata of 8–15-day-old, pre-weaned pigs and cultured in media alone, or supplemented with Nec-1 (100 µM) on day 0 or on day 3 of culture (n = 5 for each group). On day 7, islet recovery, viability, oxygen consumption rate, insulin content, cellular composition, insulin secretion capacity, and transplant outcomes were evaluated. While islet viability and oxygen consumption rate remained high throughout 7-day tissue culture, Nec-1 supplementation on day 3 significantly improved islet recovery, insulin content, endocrine composition, GLUT2 expression, differentiation potential, proliferation capacity of endocrine cells, and insulin secretion. Adding Nec-1 on day 3 of tissue culture enhanced the islet recovery, proportion of delta cells, beta-cell differentiation and proliferation, and stimulation index. In vivo, this leads to shorter times to normoglycemia, better glycemic control, and higher circulating insulin. Our findings identify the novel time-dependent effects of Nec-1 supplementation on porcine islet quantity and quality prior to transplantation.

scholarly journals Alcohol-and-HIV-Induced Lysosomal Dysfunction Regulates Extracellular Vesicles Secretion in Vitro and in Liver-Humanized Mice

Biology ◽  
2021 ◽  
Vol 10 (1) ◽  
pp. 29
Author(s):  
Raghubendra Singh Dagur ◽  
Moses New-Aaron ◽  
Murali Ganesan ◽  
Weimin Wang ◽  
Svetlana Romanova ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Extracellular Vesicles ◽  
Treated Group ◽  
Human Hepatocytes ◽  
In Vivo Studies ◽  
People Living With Hiv ◽  
Humanized Mouse Model ◽  
And Function

Background: Alcohol abuse is common in people living with HIV-1 and dramaticallyenhances the severity of HIV-induced liver damage by inducing oxidative stress and lysosomaldysfunction in the liver cells. We hypothesize that the increased release of extracellular vesicles(EVs) in hepatocytes and liver humanized mouse model is linked to lysosome dysfunction. Methods:The study was performed on primary human hepatocytes and human hepatoma RLWXP-GFP (Huh7.5 cells stably transfected with CYP2E1 and XPack-GFP) cells and validated on ethanol-fed liverhumanizedfumarylacetoacetate hydrolase (Fah)-/-, Rag2-/-, common cytokine receptor gamma chainknockout (FRG-KO) mice. Cells and mice were infected with HIV-1ADA virus. Results: We observedan increase in the secretion of EVs associated with a decrease in lysosomal activity and expressionof lysosomal-associated membrane protein 1. Next-generation RNA sequencing of primary humanhepatocytes revealed 63 differentially expressed genes, with 13 downregulated and 50 upregulatedgenes in the alcohol–HIV-treated group. Upstream regulator analysis of differentially expressedgenes through Ingenuity Pathway Analysis identified transcriptional regulators affecting downstreamgenes associated with increased oxidative stress, lysosomal associated disease, and function andEVs biogenesis. Our in vitro findings were corroborated by in vivo studies on human hepatocytetransplantedhumanized mice, indicating that intensive EVs’ generation by human hepatocytes andtheir secretion to serum was associated with increased oxidative stress and reduction in lysosomalactivities triggered by HIV infection and ethanol diet. Conclusion: HIV-and-ethanol-metabolisminducedEVs release is tightly controlled by lysosome status in hepatocytes and participates in thedevelopment of double-insult-induced liver injury.

scholarly journals Phosphorylation-dependent interaction of the synaptic vesicle proteins cysteine string protein and synaptotagmin I

2002 ◽  
Vol 364 (2) ◽  
pp. 343-347 ◽  
Author(s):  
Gareth J.O. EVANS ◽  
Alan MORGAN
Keyword(s):  
Rat Brain ◽  
Direct Interaction ◽  
Secretory Vesicle ◽  
Brain Membrane ◽  
Synaptotagmin I ◽  
Phosphorylated Form ◽  
Order Of Magnitude ◽  
And Function

The secretory vesicle cysteine string proteins (CSPs) are members of the DnaJ family of chaperones, and function at late stages of Ca2+-regulated exocytosis by an unknown mechanism. To determine novel binding partners of CSPs, we employed a pull-down strategy from purified rat brain membrane or cytosolic proteins using recombinant hexahistidine-tagged (His6-)CSP. Western blotting of the CSP-binding proteins identified synaptotagmin I to be a putative binding partner. Furthermore, pull-down assays using cAMP-dependent protein kinase (PKA)-phosphorylated CSP recovered significantly less synaptotagmin. Complexes containing CSP and synaptotagmin were immunoprecipitated from rat brain membranes, further suggesting that these proteins interact in vivo. Binding assays in vitro using recombinant proteins confirmed a direct interaction between the two proteins and demonstrated that the PKA-phosphorylated form of CSP binds synaptotagmin with approximately an order of magnitude lower affinity than the non-phosphorylated form. Genetic studies have implicated each of these proteins in the Ca2+-dependency of exocytosis and, since CSP does not bind Ca2+, this novel interaction might explain the Ca2+-dependent actions of CSP.

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