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1.
J Biol Chem ; 297(4): 101219, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34560100

RESUMO

Polyamines are fundamental molecules of life, and their deep evolutionary history is reflected in extensive biosynthetic diversification. The polyamines putrescine, agmatine, and cadaverine are produced by pyridoxal 5'-phosphate-dependent L-ornithine, L-arginine, and L-lysine decarboxylases (ODC, ADC, LDC), respectively, from both the alanine racemase (AR) and aspartate aminotransferase (AAT) folds. Two homologous forms of AAT-fold decarboxylase are present in bacteria: an ancestral form and a derived, acid-inducible extended form containing an N-terminal fusion to the receiver-like domain of a bacterial response regulator. Only ADC was known from the ancestral form and limited to the Firmicutes phylum, whereas extended forms of ADC, ODC, and LDC are present in Proteobacteria and Firmicutes. Here, we report the discovery of ancestral form ODC, LDC, and bifunctional O/LDC and extend the phylogenetic diversity of functionally characterized ancestral ADC, ODC, and LDC to include phyla Fusobacteria, Caldiserica, Nitrospirae, and Euryarchaeota. Using purified recombinant enzymes, we show that these ancestral forms have a nascent ability to decarboxylate kinetically less preferred amino acid substrates with low efficiency, and that product inhibition primarily affects preferred substrates. We also note a correlation between the presence of ancestral ODC and ornithine/arginine auxotrophy and link this with a known symbiotic dependence on exogenous ornithine produced by species using the arginine deiminase system. Finally, we show that ADC, ODC, and LDC activities emerged independently, in parallel, in the homologous AAT-fold ancestral and extended forms. The emergence of the same ODC, ADC, and LDC activities in the nonhomologous AR-fold suggests that polyamine biosynthesis may be inevitable.


Assuntos
Proteínas Arqueais , Bactérias , Proteínas de Bactérias , Poliaminas Biogênicas , Carboxiliases , Euryarchaeota , Evolução Molecular , Ornitina Descarboxilase , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Bactérias/enzimologia , Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Poliaminas Biogênicas/biossíntese , Poliaminas Biogênicas/química , Carboxiliases/química , Carboxiliases/genética , Carboxiliases/metabolismo , Euryarchaeota/enzimologia , Euryarchaeota/genética , Ornitina Descarboxilase/química , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
2.
J Med Chem ; 64(15): 10951-10966, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34260245

RESUMO

Influenza viruses cause approximately half a million deaths every year worldwide. Vaccines are available but partially effective, and the number of antiviral medications is limited. Thus, it is crucial to develop therapeutic strategies to counteract this major pathogen. Influenza viruses enter the host cell via their hemagglutinin (HA) proteins. The HA subtypes of influenza A virus are phylogenetically classified into groups 1 and 2. Here, we identified an inhibitor of the HA protein, a tertiary aryl sulfonamide, that prevents influenza virus entry and replication. This compound shows potent antiviral activity against diverse H1N1, H5N1, and H3N2 influenza viruses encoding HA proteins from both groups 1 and 2. Synthesis of derivatives of this aryl sulfonamide identified moieties important for antiviral activity. This compound may be considered as a lead for drug development with the intent to be used alone or in combination with other influenza A virus antivirals to enhance pan-subtype efficacy.


Assuntos
Antivirais/farmacologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Vírus da Influenza A/efeitos dos fármacos , Sulfonamidas/farmacologia , Antivirais/síntese química , Antivirais/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Relação Estrutura-Atividade , Sulfonamidas/síntese química , Sulfonamidas/química , Internalização do Vírus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
3.
PLoS Pathog ; 16(4): e1008407, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32240278

RESUMO

Influenza A viruses are human pathogens with limited therapeutic options. Therefore, it is crucial to devise strategies for the identification of new classes of antiviral medications. The influenza A virus genome is constituted of 8 RNA segments. Two of these viral RNAs are transcribed into mRNAs that are alternatively spliced. The M1 mRNA encodes the M1 protein but is also alternatively spliced to yield the M2 mRNA during infection. M1 to M2 mRNA splicing occurs at nuclear speckles, and M1 and M2 mRNAs are exported to the cytoplasm for translation. M1 and M2 proteins are critical for viral trafficking, assembly, and budding. Here we show that gene knockout of the cellular protein NS1-BP, a constituent of the M mRNA speckle-export pathway and a binding partner of the virulence factor NS1 protein, inhibits M mRNA nuclear export without altering bulk cellular mRNA export, providing an avenue to preferentially target influenza virus. We performed a high-content, image-based chemical screen using single-molecule RNA-FISH to label viral M mRNAs followed by multistep quantitative approaches to assess cellular mRNA and cell toxicity. We identified inhibitors of viral mRNA biogenesis and nuclear export that exhibited no significant activity towards bulk cellular mRNA at non-cytotoxic concentrations. Among the hits is a small molecule that preferentially inhibits nuclear export of a subset of viral and cellular mRNAs without altering bulk cellular mRNA export. These findings underscore specific nuclear export requirements for viral mRNAs and phenocopy down-regulation of the mRNA export factor UAP56. This RNA export inhibitor impaired replication of diverse influenza A virus strains at non-toxic concentrations. Thus, this screening strategy yielded compounds that alone or in combination may serve as leads to new ways of treating influenza virus infection and are novel tools for studying viral RNA trafficking in the nucleus.


Assuntos
Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Antivirais/farmacologia , Núcleo Celular/virologia , Vírus da Influenza A/metabolismo , Influenza Humana/virologia , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Avaliação Pré-Clínica de Medicamentos , Humanos , Vírus da Influenza A/genética , RNA Mensageiro/genética , RNA Viral/genética , Replicação Viral/efeitos dos fármacos
4.
Biochem J ; 476(18): 2579-2594, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31467246

RESUMO

The only known function of S-adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that 'eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.


Assuntos
Adenosilmetionina Descarboxilase/metabolismo , Putrescina/metabolismo , Ralstonia pickettii/enzimologia , Shewanella/enzimologia , Espermidina/metabolismo , Adenosilmetionina Descarboxilase/química , Adenosilmetionina Descarboxilase/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Putrescina/química , Ralstonia pickettii/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Shewanella/genética , Espermidina/química
5.
Mol Microbiol ; 111(1): 159-175, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30281855

RESUMO

Polyamines such as spermidine and spermine are primordial polycations that are ubiquitously present in the three domains of life. We have found that Gram-positive bacteria Staphylococcus aureus and Enterococcus faecalis have lost either all or most polyamine biosynthetic genes, respectively, and are devoid of any polyamine when grown in polyamine-free media. In contrast to bacteria such as Pseudomonas aeruginosa, Campylobacter jejuni and Agrobacterium tumefaciens, which absolutely require polyamines for growth, S. aureus and E. faecalis grow normally over multiple subcultures in the absence of polyamines. Furthermore, S. aureus and E. faecalis form biofilms normally without polyamines, and exogenous polyamines do not stimulate growth or biofilm formation. High levels of external polyamines, including norspermidine, eventually inhibit biofilm formation through inhibition of planktonic growth. We show that spermidine/spermine N-acetyltransferase (SSAT) homologues encoded by S. aureus USA300 and E. faecalis acetylate spermidine, spermine and norspermidine, that spermine is the more preferred substrate, and that E. faecalis SSAT is almost as efficient as human SSAT with spermine as substrate. The polyamine auxotrophy, polyamine-independent growth and biofilm formation, and presence of functional polyamine N-acetyltransferases in S. aureus and E. faecalis represent a new paradigm for bacterial polyamine biology.


Assuntos
Acetiltransferases/metabolismo , Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/enzimologia , Enterococcus faecalis/crescimento & desenvolvimento , Espermidina/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/crescimento & desenvolvimento , Acetilação , Processamento de Proteína Pós-Traducional , Espermidina/análogos & derivados , Espermina/metabolismo
6.
Carbohydr Polym ; 186: 243-251, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29455984

RESUMO

For targeted delivery with nanoparticles (NPs) as drug carriers, it is imperative that the NPs are internalized into the targeted cell. Surface properties of NPs influence their interactions with cells. We examined the responses of retinal pigment epithelial cells, NIH 3T3 fibroblast cells, and Chinese hamster ovary cells to gold nanoparticles (Au NPs) in their nascent form as well as coated with end-thiolated hyaluronate (HS-HA). The grafting density of HS-HA on Au NPs was calculated based on total organic carbon measurements and thermal gravimetric analysis. We imaged the intracellular NPs by 3D confocal microscopy. We quantified viability and generation of reactive oxygen species (ROS) of the cells to Au NPs and monitored cell-surface attachment via electrical cell-substrate impedance sensing. The results confirmed that receptors on cell surfaces, for HA, are critical in internalizing HS-HA-Au NPs, and HA may mitigate ROS pathways known to lead to cell death. The 50- and 100-nm HS-HA-Au NPs were able to enter the cells; however, their nascent forms could not. This study shows that the delivery of larger Au NPs is enhanced with HS-HA coating and illustrates the potential of HA-coated NPs as a drug delivery agent for inflamed, proliferating, and cancer cells that express CD44 receptors.


Assuntos
Ouro/química , Ácido Hialurônico/química , Nanopartículas Metálicas/química , Receptores de Hialuronatos/química , Microscopia Confocal , Espécies Reativas de Oxigênio/metabolismo
7.
Antiviral Res ; 150: 193-201, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29294299

RESUMO

Ebola virus (EBOV) is an enveloped negative-sense, single-stranded RNA virus of the filovirus family that causes severe disease in humans. Approved therapies for EBOV disease are lacking. EBOV RNA synthesis is carried out by a virus-encoded complex with RNA-dependent RNA polymerase activity that is required for viral propagation. This complex and its activities are therefore potential antiviral targets. To identify potential lead inhibitors of EBOV RNA synthesis, a library of small molecule compounds was screened against a previously established assay of EBOV RNA synthesis, the EBOV minigenome assay (MGA), in 384 well microplate format. The screen identified 56 hits that inhibited EBOV MGA activity by more than 70% while exhibiting less than 20% cell cytotoxicity. Inhibitory chemical scaffolds included angelicin derivatives, derivatives of the antiviral compound GSK983 and benzoquinolines. Structure-activity relationship (SAR) studies of the benzoquinoline scaffold produced ∼50 analogs and led to identification of an optimized compound, SW456, with a submicromolar IC50 in the EBOV MGA and antiviral activity against infectious EBOV in cell culture. The compound was also active against a MGA for another deadly filovirus, Marburg virus. It also exhibited antiviral activity towards a negative-sense RNA virus from the rhabdovirus family, vesicular stomatitis virus, and a positive-sense RNA virus, Zika virus. Overall, these data demonstrate the potential of the EBOV MGA to identify anti-EBOV compounds and identifies the benzoquinoline series as a broad-spectrum antiviral lead.


Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Quinolinas/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/química , Relação Dose-Resposta a Droga , Descoberta de Drogas , Avaliação Pré-Clínica de Medicamentos , Ebolavirus/genética , Humanos , Quinolinas/química , Bibliotecas de Moléculas Pequenas
8.
Langmuir ; 33(31): 7660-7668, 2017 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-28658954

RESUMO

Presbyopia, the inability to focus at arm's length, and cataracts that cloud vision are associated primarily with changes in the mechanical and optical properties of the lens. The optical properties, particularly the refractive index, of the human lens originate from the cytoplasm of the lens fiber, which contains a highly concentrated solution (∼40%) of globular proteins referred to as α, ß, and γ crystallins, of which ß is the most abundant. In this study, we focus on the synthesis and characterization of a ß-crystallin biomimetic in an effort to understand and develop treatments for presbyopia and cataract. Polyacrylamide was used as a protein analogue. The side chains were endowed with aromatic and acidic functionality. Acrylic acid was incorporated into the copolymer and cross-linked with diamines to form nanoparticles. The composition and cross-linking condition of the biomimetic copolymers were optimized to match the hydrodynamic radius (Rh), refractive index, size, density, and intrinsic and dynamic viscosities with those of ßhigh lens crystallins. The refractive indices and densities of the nanoparticles' dispersion at different concentrations matched that of ßhigh lens crystallins, and the viscosity of the nanoparticles approached that of ßhigh lens crystallins. The biocompatibility findings for primary porcine retinal pigment epithelial (ppRPE) cells and porcine lens epithelial (pLE) cells showed both cell types tolerated up to 30 mg/mL of nanoparticles. These materials have the potential for use as replacements for the crystallins in developing an accommodating intraocular lens nanocomposite hydrogel that closely replicates the natural autofocusing ability of the original.


Assuntos
Nanopartículas , Animais , Biomimética , Cristalinas , Humanos , Cristalino , Suínos , beta-Cristalinas
9.
J Biomed Mater Res B Appl Biomater ; 105(5): 977-988, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-26873608

RESUMO

The natural vitreous is a biological hydrogel consisting primarily of a collagen and anionic hyaluronate. It is surgically removed in many ocular diseases and replaced with fluids, gases, or silicone oils. We have been interested in developing synthetic hydrogels as vitreous substitutes. In this study, we combined the stiffness and hydrophobicity of polymethacrylamide (PMAM) and the anionic nature of polymethacrylate (PMAA) to make copolymers that would mimic the natural vitreous. We used bis-methacryloyl cystamine (BMAC) to introduce thiol groups for reversible crosslink. The Mn of copolymers ranged from ∼100 k to ∼200 k Da (polydisperisty index of 1.47-2.63) and their composition as determined by titration, 1 H NMR and disulfide test were close to the feed ratio. The reactivities of monomers were as follows: MAM > MAA ∼ BMAC. Copolymers with higher MAA contents gelled faster, swelled more, and had higher storage modulus (1.5 to 100 Pa) comparable to that of the natural vitreous. We evaluated the biocompatibility of copolymers by electric cell-substrate impedance sensing (ECIS) using human retinal pigment epithelial cells, primary porcine retinal pigmented epithelial cells, human microvascular endothelial cells adult dermis, and a fibroblast line 3T3. The biocompatibility decreases as the content of BMAC increases. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 977-988, 2017.


Assuntos
Células Epiteliais/metabolismo , Hidrogéis , Teste de Materiais , Ácidos Polimetacrílicos , Epitélio Pigmentado da Retina/metabolismo , Corpo Vítreo , Animais , Células Epiteliais/citologia , Humanos , Hidrogéis/síntese química , Hidrogéis/química , Hidrogéis/farmacologia , Camundongos , Células NIH 3T3 , Ácidos Polimetacrílicos/síntese química , Ácidos Polimetacrílicos/química , Ácidos Polimetacrílicos/farmacologia , Epitélio Pigmentado da Retina/citologia , Suínos
10.
Biomacromolecules ; 17(12): 4064-4074, 2016 12 12.
Artigo em Inglês | MEDLINE | ID: mdl-27936721

RESUMO

Sulfonate-containing hydrogels are of particular interest because of their tunable mechanical and swelling properties, as well as their biological effects. Polysulfonate copolymers were synthesized by reacting 2-acrylamido-2-methylpropanesulfonic acid (AMPS), acrylamide (AM), and acrylic acid (AA). We found that the incorporation rate of sulfonate-containing monomer and the molecular weight of the copolymer were significantly enhanced by increasing the ionic strength of the solution. We introduced thiol groups by modifying the pendant carboxylates or copolymerizing along with a disulfide-containing monomer. The thiol-containing copolymers were reacted with a 4-arm acrylamide-terminated poly(ethylene glycol) via a thiol-ene click reaction, which was mediated by a photoinitiator, a redox initiator, or a base-catalyzed Michael-Addition. We were able to tailor the storage modulus (33-1800 Pa) and swelling capacity (1-91 wt %) of the hydrogel by varying the concentration of the copolymers. We determined that the injectable sulfonate-containing hydrogels were biocompatible up to 20 mg/mL, as observed by an electric cell-substrate impedance sensing (ECIS) technique, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using three different cell lines: human retinal pigment epithelial cells (ARPE-19), fibroblasts (NIH 3T3), and Chinese hamster ovary cells (CHO).


Assuntos
Materiais Biocompatíveis/química , Fibroblastos/citologia , Hidrogéis/química , Polímeros/química , Epitélio Pigmentado da Retina/citologia , Compostos de Sulfidrila/química , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Impedância Elétrica , Humanos , Camundongos , Polimerização , Reologia , Engenharia Tecidual
11.
Acta Biomater ; 43: 327-337, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27481290

RESUMO

UNLABELLED: The vitreous humor of the eye is a biological hydrogel principally composed of collagen fibers interspersed with hyaluronic acid. Certain pathological conditions necessitate its removal and replacement. Current substitutes, like silicone oils and perfluorocarbons, are not biomimetic and have known complications. In this study, we have developed an in situ forming two-component biomimetic hydrogel with tunable mechanical and osmotic properties. The components are gellan, an analogue of collagen, and poly(methacrylamide-co-methacrylate), an analogue of hyaluronic acid; both endowed with thiol side groups. We used response surface methodology to consider seventeen possible hydrogels to determine how each component affects the optical, mechanical, sol-gel transition temperature and swelling properties. The optical and physical properties of the hydrogels were similar to vitreous. The shear storage moduli ranged from 3 to 358Pa at 1Hz and sol-gel transition temperatures from 35.5 to 43°C. The hydrogel had the ability to remain swollen without degradation for four weeks in vitro. Three hydrogels were tested for biocompatibility on primary porcine retinal pigment epithelial cells, human retinal pigment epithelial cells, and fibroblast (3T3/NIH) cells, by electric cell-substrate impedance sensing system. The two-component hydrogels allowed for the tuning and optimizing of mechanical, swelling, and transition temperature to obtain three biocompatible hydrogels with properties similar to the vitreous. Future studies include testing of the optimized hydrogels in animal models for use as a long-term substitute, whose preliminary results are mentioned. STATEMENT OF SIGNIFICANCE: Although hydrogels are researched as long-term vitreous substitute, none have advanced sufficiently to reach clinical application. Our work focuses on the development of a novel two component in situ forming hydrogel that bio-mimic the natural vitreous. Our thiol-containing copolymers can be injected as an aqueous solution into the vitreous cavity wherein, at physiological temperature, the rigid component will instantaneously form a physical gel imbedding the random coil copolymer. Upon subsequent oxidation, the two components will form disulfide cross-links and a stable reversible hydrogel capable of providing osmotic pressure to reattach the retina. It may be left in the eye permanently or easily removed by injection of a simple reducing agent to cleave the disulfide bonds, rather than surgery. This contribution is significant because it is expected to provide patients with a much better quality of life by improving surgical outcomes, creating much less post-operative burden, and reducing the need for secondary surgeries.


Assuntos
Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacologia , Fenômenos Mecânicos , Corpo Vítreo/efeitos dos fármacos , Animais , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Módulo de Elasticidade , Humanos , Camundongos , Células NIH 3T3 , Transição de Fase , Polímeros/química , Reologia , Sus scrofa , Tomografia de Coerência Óptica , Temperatura de Transição
12.
Carbohydr Polym ; 132: 472-80, 2015 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-26256372

RESUMO

Hyaluronan (HA) and its derivatives have been extensively researched for many biomedical applications. To precisely tailor the property of HA by derivatizing it to a pre-determined extent is challenging, yet critical. In this paper, we used 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and N-methylmorpholine (NMM) to derivatize HA via a triazine-based coupling reaction. Using a fractional factorial (FF) design, we observed that water content in the solvent, and molar ratios of CDMT and NaHCO3 to the carboxylate were the significant factors controlling the derivatization. We investigated how the effect of each factor changes as reaction conditions change. Moreover, by altering the amount of CDMT and NaHCO3, we developed a cubic regression model for precise control of the extent of derivatization using a response surface methodology (RSM) with a D-optimal design. No spurious peaks were detected by (1)H NMR spectrum and only 10% decrease of molecular weight of the derivatized HA was determined by GPC. The HA with 6% modification was relatively biocompatible up to 15 mg/mL.


Assuntos
Ácido Hialurônico/química , Triazinas/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Ácido Hialurônico/toxicidade , Espectroscopia de Ressonância Magnética , Morfolinas/química , Compostos de Sulfidrila/química , Água/química
13.
J Biomed Mater Res A ; 103(7): 2300-8, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25369214

RESUMO

Hyaluronan (HA) is a naturally occurring glycosaminoglycan widely researched for its use as a biomaterial in tissue engineering, drug delivery, angiogenesis, and ophthalmic surgeries. The mechanical properties of this biomaterial can be altered to a required extent by chemically modifying the pendant reactive groups. However, derivatizing these polymers to a predetermined extent has been the Achilles heel for this process. In this study, we have investigated the factors controlling the derivatization of the carboxyl moieties of HA with amine containing thiol, cystamine dihydrochloride (Cys), via carbodiimide crosslinking chemistry. We used fractional factorial design to screen and identify the significant factor(s) affecting the reaction, and response surface methodology (RSM) to develop a model equation for predicting the degree of thiolation of HA. Also, we analyzed the reaction mechanism for potential side reactions. We observed that N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) (mole ratio with repeat unit of HA) is the significant factor controlling the degree of amidation. The quadratic equations developed from RSM predict the formulation for a desired degree of amidation of HA and percentage of potential side product. Hence, derivatizing HA to a predetermined extent with minimal side product can be achieved using the statistical design of experiments.


Assuntos
Carbodi-Imidas/química , Ácido Hialurônico/química , Compostos de Sulfidrila/química , Amidas/química , Propriedades de Superfície
14.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 22(2): 329-32, 2014 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-24763000

RESUMO

This study was aimed to explore the role of full-automatic blood analyzer Sysmex XE-2100 in early screening and diagnosing the hematologic malignancies. A total of 288 samples of the patients with hematologic malignancies was examined. Then, the scatter plots, alarm information and blood smears were analyzed. The results indicated that 76% of these samples showed abnormal scatter plots. CMML and AML-M3 patients had their own characteristic scatter plots, while others hadnt's. The coincident rate of CMML and AML-M3 determined by scatter plots with practical diseases was 100%; the coincident rate of ALL determined by scatter plots with practical disease was 67%. The coincident rate of alarm information of blast cells was 92.5%, the coincident rate of immature granulocytes was 77.1%, the coincident rate of nucleated red blood cells was 33.3%, the coincident rate of atypical lymphocytes was 31.3%. It is concluded that the abnormal scatter plots and alarm information are very important for finding the patients with hematologic malignancies and determining the disease type. The alarm information has high reliability for blast cells and immature granulocytes, but has only mirror value for nucleated red blood cells and atypical lymphocytes.


Assuntos
Neoplasias Hematológicas/sangue , Testes Hematológicos/instrumentação , Testes Hematológicos/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Contagem de Leucócitos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Adulto Jovem
15.
J Am Chem Soc ; 133(5): 1428-37, 2011 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21210688

RESUMO

Degeneration of the hippocampus is associated with Alzheimer's disease and occurs very early in the progression of the disease. Current options for treating the cognitive symptoms associated with Alzheimer's are inadequate, giving urgency to the search for novel therapeutic strategies. Pharmacologic agents that safely enhance hippocampal neurogenesis may provide new therapeutic approaches. We discovered the first synthetic molecule, named P7C3, which protects newborn neurons from apoptotic cell death, and thus promotes neurogenesis in mice and rats in the subgranular zone of the hippocampal dentate gyrus, the site of normal neurogenesis in adult mammals. We describe the results of a medicinal chemistry campaign to optimize the potency, toxicity profile, and stability of P7C3. Systematic variation of nearly every position of the lead compound revealed elements conducive toward increases in activity and regions subject to modification. We have discovered compounds that are orally available, nontoxic, stable in mice, rats, and cell culture, and capable of penetrating the blood-brain barrier. The most potent compounds are active at nanomolar concentrations. Finally, we have identified derivatives that may facilitate mode-of-action studies through affinity chromatography or photo-cross-linking.


Assuntos
Carbazóis/química , Carbazóis/farmacologia , Descoberta de Drogas/métodos , Neurogênese/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Animais , Carbazóis/uso terapêutico , Carbazóis/toxicidade , Relação Dose-Resposta a Droga , Estabilidade de Medicamentos , Células HeLa , Humanos , Masculino , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Fármacos Neuroprotetores/toxicidade , Relação Estrutura-Atividade
16.
Inorg Chem ; 47(14): 6115-7, 2008 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-18553910

RESUMO

Two 2D Mn (II) complexes, [Mn3(TzDC)2(phen)3] x 2 H2O (1; H3TzDC = 1,2,3-triazole-4,5-dicarboxylic acid, phen = 1,10-phenanthroline) and [Mn3(TzDC)2(bipy)3] x 4 H2O (2; bipy = 2,2'-bipyridine), were synthesized by hydrothermal reactions and characterized magnetically, and complex 1 was the first example of the chiral complex with a Kagomé lattice connectivity obtained through spontaneous resolution.

17.
J Biomol NMR ; 34(2): 117-27, 2006 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-16518698

RESUMO

Acireductone dioxygenase (ARD) from Klebsiella ATCC 8724 is a metalloenzyme that is capable of catalyzing different reactions with the same substrates (acireductone and O2) depending upon the metal bound in the active site. A model for the solution structure of the paramagnetic Ni2+-containing ARD has been refined using residual dipolar couplings (RDCs) measured in two media. Additional dihedral restraints based on chemical shift (TALOS) were included in the refinement, and backbone structure in the vicinity of the active site was modeled from a crystallographic structure of the mouse homolog of ARD. The incorporation of residual dipolar couplings into the structural refinement alters the relative orientations of several structural features significantly, and improves local secondary structure determination. Comparisons between the solution structures obtained with and without RDCs are made, and structural similarities and differences between mouse and bacterial enzymes are described. Finally, the biological significance of these differences is considered.


Assuntos
Proteínas de Bactérias/química , Dioxigenases/química , Klebsiella/enzimologia , Modelos Químicos , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
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