Gene/Protein Disease Symptom Drug Enzyme Compound
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1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
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PMID:The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. 439 82

Collagen-Chitosan (COL-CS) scaffolds supplemented with different concentrations (0.1-0.5%) of aloe vera (AV) were prepared and tested in vitro for their possible application in tissue engineering. After studying the microstructure and mechanical properties of all the composite preparations, a 0.2% AV blended COL-CS scaffold was chosen for further studies. Scaffolds were examined by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and thermogravimetry analysis (TGA) to understand the intermolecular interactions and their influence on the thermal property of the complex composite. Swelling property in phosphate buffered saline (pH 7.4) and in vitro biodegradability by collagenase digestion method were monitored to assess the stability of the scaffold in a physiological medium in a hydrated condition, and to assay its resistance against enzymatic forces. The scanning electron microscope (SEM) image of the scaffold samples showed porous architecture with gradual change in their morphology and reduced tensile properties with increasing aloe vera concentration. The FTIR spectrum revealed the overlap of the AV absorption peak with the absorption peak of COL-CS. The inclusion of AV to COL-CS increased the thermal stability as well as hydrophilicity of the scaffolds. Cell culture studies on the scaffold showed enhanced growth and proliferation of fibroblasts (3T3L1) without exhibiting any toxicity. Also, normal cell morphology and proliferation were observed by fluorescence microscopy and SEM. The rate of cell growth in the presence/absence of aloe vera in the scaffolds was in the order: COL-CS-AV > COL-CS > TCP (tissue culture polystyrene plate). These results suggested that the aloe vera gel-blended COL-CS scaffolds could be a promising candidate for tissue engineering applications.
ACS Appl Mater Interfaces 2013 Aug 14
PMID:Preparation and characterization of aloe vera blended collagen-chitosan composite scaffold for tissue engineering applications. 2383 42

External triggers such as pH or temperature can induce hydrogels to swell or shrink rapidly. Recently, these triggers have also been used to alter the three-dimensional (3-D) shapes of gels: for example, a flat gel sheet can be induced to fold into a tube. Self-folding gels are reminiscent of natural structures such as the Venus flytrap, which folds its leaves to entrap its prey. They are also of interest for applications in sensing or microrobotics. However, to advance the utility of self-folding gels, the range of triggers needs to be expanded beyond the conventional ones. Toward this end, we have designed a class of gels that change shape in response to very low concentrations of specific biomolecules. The gels are hybrids of three different constituents: (A) polyethylene glycol diacrylate (PEGDA); (B) gelatin methacrylate-co-polyethylene glycol dimethacrylate (GelMA-co-PEGDMA); and (C) N-isopropylacrylamide (NIPA). The thin-film hybrid is constructed as a bilayer or sandwich of two layers, with an A/B layer (alternating strips of A and B) sandwiched above a layer of gel C. Initially, when this hybrid gel is placed in water, the C layer is much more swollen than the A/B layer. Despite the swelling mismatch, the sheet remains flat because the A/B layer is very stiff. When collagenase enzyme is added to the water, it cleaves the gelatin chains in B, thus reducing the stiffness of the A/B layer. As a result, the swollen C layer is able to fold over the A/B layer, causing the sheet to transform into a specific shape. The typical transition is from flat sheet to closed hollow tube, and the time scale for this transition decreases with increasing enzyme concentration. Shape transitions are induced by enzyme levels as low as 0.75 U/mL. Interestingly, a shape transition is also induced by adding the lysate of murine fibroblast cells, which contains enzymes from the matrix metalloproteinase (MMP) family at levels around 0.1 U/mL (MMPs are similar to collagenase in their ability to cleave gelatin). We further show that transitions from flat sheets to other shapes such as helices and pancakes can be engineered by altering the design pattern of the gel. Additionally, we have made a rudimentary analog of the Venus flytrap, with two flat gels ("leaves") flanking a central folding gel ("hinge"). When enzyme is added, the hinge bends and brings the leaves together, trapping objects in the middle.
ACS Appl Mater Interfaces 2016 Jul 27
PMID:Enzyme-Triggered Folding of Hydrogels: Toward a Mimic of the Venus Flytrap. 2740 25

Tissue engineering aims to generate functional tissue constructs in which proper extracellular matrix (ECM) for cell survival and establishment of a vascular network are necessary. A modular approach via the assembly of modules mimicking the complex tissues' microarchitectural features and establishing a vascular network represents a promising strategy for fabricating larger and more complex tissue constructs. Herein, as a model for this modular tissue engineering, engineered bone-like constructs were developed by self-assembly of osteon-like modules and fast degradable gelatin microspheres. The collagen microspheres acting as osteon-like modules were developed by seeding human umbilical vein endothelial cells (HUVECs) onto collagen microspheres laden with human osteoblast-like cells (MG63) and collagenase. Both HUVECs and MG63 cells were well spatially patterned in the modules, and collagen as ECM well supported cell adhesion, spreading, and functional expression due to its native RGD domains and enzymatic degradation activity. The patterned modules facilitated both the cellular function expression of osteogenic MG63 cells and vasculogenic HUVECs; that is, the osteon-like units were successfully achieved. The assembly of the osteon-like modules and fast degradable gelatin microspheres promoted the vascularization, thus facilitating the osteogenic function expression. The study provides a highly efficient approach to engineering complex 3D tissues with micropatterned cell types and interconnected channels.
ACS Appl Mater Interfaces 2017 Feb 01
PMID:Vascularization in Engineered Tissue Construct by Assembly of Cellular Patterned Micromodules and Degradable Microspheres. 2807 50

Full-thickness skin equivalents are gathering increased interest as skin grafts for the treatment of large skin defects or chronic wounds or as nonanimal test platforms. However, their fibroblast-mediated contraction and poor mechanical stability lead to disadvantages toward their reproducibility and applicability in vitro and in vivo. To overcome these pitfalls, we aimed to chemically cross-link the dermal layer of a full-thickness skin model composed of a collagen type I hydrogel. Using a noncytotoxic four-arm succinimidyl glutarate polyethylene glycol (PEG-SG), cross-linking could be achieved in cell seeded collagen hydrogels. A concentration of 0.5 mg of PEG-SG/mg of collagen led to a viability comparable to non-cross-linked collagen hydrogels and no increased release of intracellular lactate dehydrogenase. Cross-linked collagen hydrogels were more mechanically stable and less prone to enzymatic degradation via collagenase when compared with non-cross-linked collagen hydrogels. Remarkably, during 21 days, cross-linked collagen hydrogels maintain their initial surface area, whereas standard dermal models contracted up to 50%. Finally, full-thickness skin equivalents were generated by seeding human epidermal keratinocytes on the surface of the equivalents and culturing these equivalents at an air-liquid interface. Immunohistochemical stainings of the cross-linked model revealed well-defined epidermal layers including an intact stratum corneum and a dermal part with homogeneously distributed human dermal fibroblasts. These results indicate that cross-linking of collagen with PEG-SG reduces contraction of collagen hydrogels and thus increases the applicability of these models as an additional tool for efficacy and safety assessment or a new generation of skin grafts.
ACS Appl Mater Interfaces 2017 Jun 21
PMID:Cross-linked Collagen Hydrogel Matrix Resisting Contraction To Facilitate Full-Thickness Skin Equivalents. 2855 35

Surgical blades are common medical tools. However, blades cannot distinguish between healthy and diseased tissue, thereby creating unnecessary damage, lengthening recovery, and increasing pain. We propose that surgical procedures can rely on natural tissue remodeling tools-enzymes, which are the same tools our body uses to repair itself. Through a combination of nanotechnology and a controllably activated proteolytic enzyme, we performed a targeted surgical task in the oral cavity. More specifically, we engineered nanoparticles that contain collagenase in a deactivated form. Once placed at the surgical site, collagenase was released at a therapeutic concentration and activated by calcium, its biological cofactor that is naturally present in the tissue. Enhanced periodontal remodeling was recorded due to enzymatic cleavage of the supracrestal collagen fibers that connect the teeth to the underlying bone. When positioned in their new orientation, natural tissue repair mechanisms supported soft and hard tissue recovery and reduced tooth relapse. Through the combination of nanotechnology and proteolytic enzymes, localized surgical procedures can now be less invasive.
ACS Nano 2018 02 27
PMID:Proteolytic Nanoparticles Replace a Surgical Blade by Controllably Remodeling the Oral Connective Tissue. 2936 50

The increasing emergence of antibiotic resistance necessitates the development of anti-infectives with novel modes of action. Targeting bacterial virulence is considered a promising approach to develop novel antibiotics with reduced selection pressure. The extracellular collagenase elastase (LasB) plays a pivotal role in the infection process of Pseudomonas aeruginosa and therefore represents an attractive antivirulence target. Mercaptoacetamide-based thiols have been reported to inhibit LasB as well as collagenases from clostridia and bacillus species. The present work provides an insight into the structure-activity relationship (SAR) of these fragment-like LasB inhibitors, demonstrating an inverse activity profile compared to similar inhibitors of clostridial collagenase H (ColH). An X-ray cocrystal structure is presented, revealing distinct binding of two compounds to the active site of LasB, which unexpectedly maintains an open conformation. We further demonstrate in vivo efficacy in a Galleria mellonella infection model and high selectivity of the LasB inhibitors toward human matrix metalloproteinases (MMPs).
ACS Infect Dis 2018 06 08
PMID:Binding Mode Characterization and Early in Vivo Evaluation of Fragment-Like Thiols as Inhibitors of the Virulence Factor LasB from Pseudomonas aeruginosa. 2948 68

Proteases are involved in almost every important cellular activity, from embryonic morphogenesis to apoptosis. To study protease activity in situ, hydrogels provide a synthetic mimic of the extracellular matrix (ECM) and have utility as a platform to study activity, such as those related to cell migration, in three-dimensions. While 3-dimensional visualization of protease activity could prove quite useful to elucidate the proteolytic interaction at the interface between cells and their surrounding environment, there has been no versatile tool to visualize local proteolytic activity in real time. Here, micron-sized gels were synthesized by inverse suspension polymerization using thiolene photo-click chemistry. The size distribution was selected to avoid cellular uptake and to lower cytotoxicity, while simultaneously allowing the integration of peptide-based FRET sensors of local cell activity. Proteolytic activity of collagenase was detected within an hour via changes in fluorescence of embedded microgels; incubation of microgel sensors with A375 melanoma cells showed upregulated MMP activity in the presence of soluble fibronectins in media. The microgel sensors were readily incorporated into both gelatin and poly(ethylene glycol) (PEG) hydrogels and used to successfully detect spatiotemporal proteolytic activity of A375 melanoma cells. Finally, a tumor model was constructed from a hydrogel microwell array that was used to aggregate A375 melanoma cells, and local variations in proteolytic activity were monitored as a function of distance from the cell aggregate center.
ACS biomaterials science & engineering 2018
PMID:Synthesis of microgel sensors for spatial and temporal monitoring of protease activity. 2952 70

The abundant tumor extracellular matrix (ECM) could result in insufficient tumor retention and ineffective intratumor penetration of therapeutic agents as well as an acidic and hypoxic tumor microenvironment (TME), leading to unsatisfactory therapeutic outcomes for many types of therapies. Therefore, developing strategies to modulate the TME by selectively degrading the condensed ECM may be helpful to improve existing cancer therapies. Herein, collagenase (CLG)-encapsulated nanoscale coordination polymers (NCPs) are synthesized based on Mn2+ and an acid-sensitive benzoic-imine organic linker and then modified by polyethylene glycol (PEG). Upon intravenous (iv) injection, these CLG@NCP-PEG nanoparticles show efficient accumulation within the tumor, in which CLG would be released because of the collapse of NCP structures within the acidic TME. The released CLG enzyme could then specifically degrade collagens, the major component of ECM, leading to a loosened ECM structure, enhanced tumor perfusion, and relieved hypoxia. As a result, the second wave of nanoparticles, chlorin e6 (Ce6)-loaded liposomes (liposome@Ce6), would exhibit enhanced retention and penetration within the tumor. Such phenomena together with relieved tumor hypoxia could then lead to greatly enhanced photodynamic therapeutic effect of liposome@Ce6 for mice pretreated with CLG@NCP-PEG. Our work thus presents a unique strategy for TME modulation using pH-responsive NCPs as smart enzyme carriers.
ACS Appl Mater Interfaces 2018 Dec 19
PMID:Collagenase-Encapsulated pH-Responsive Nanoscale Coordination Polymers for Tumor Microenvironment Modulation and Enhanced Photodynamic Nanomedicine. 3046 76

Cellular machineries guide the bottom-up pathways toward crystal superstructures based on the transport of inorganic precursors and their precise integration with organic frameworks. The biosynthesis of mesocrystalline spines entails concerted interactions between biomolecules and inorganic precursors; however, the bioinorganic interactions and interfaces that regulate material form and growth as well as the selective emergence of structural complexity in the form of nanostructured crystals are not clear. By investigating mineral nucleation under the regulation of recombinant proteins, we show that SpSM50, a matrix protein of the sea urchin spine, stabilizes mineral precursors via vesicle-confinement, a function conferred by a low-complexity, disordered region. Site-specific proteolysis of this domain by a collagenase initiates phase transformation of the confined mineral phase. The residual C-type lectin domain molds the fluidic mineral precursor into hierarchical mesocrystals identical to structural crystal modules constituting the biogenic mineral. Thus, the regulatory functions of proteolytic enzymes can guide biomacromolecular domain constitutions and interfaces, in turn determining inorganic phase transformations toward hybrid materials as well as integrating organic and inorganic components across hierarchical length scales. Bearing striking resemblance to biogenic mineralization, these hybrid materials recruit bioinorganic interactions which elegantly intertwine nucleation and crystallization phenomena with biomolecular structural dynamics, hence elucidating a long-sought key of how nature can orchestrate complex biomineralization processes.
ACS Cent Sci 2019 Feb 27
PMID:On Biomineralization: Enzymes Switch on Mesocrystal Assembly. 3083 24


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