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1.
Cell Death Dis ; 15(3): 232, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519456

RESUMO

Unlike the intense research effort devoted to exploring the significance of heparanase in cancer, very little attention was given to Hpa2, a close homolog of heparanase. Here, we explored the role of Hpa2 in breast cancer. Unexpectedly, we found that patients endowed with high levels of Hpa2 exhibited a higher incidence of tumor metastasis and survived less than patients with low levels of Hpa2. Immunohistochemical examination revealed that in normal breast tissue, Hpa2 localizes primarily in the cell nucleus. In striking contrast, in breast carcinoma, Hpa2 expression is not only decreased but also loses its nuclear localization and appears diffuse in the cell cytoplasm. Importantly, breast cancer patients in which nuclear localization of Hpa2 is retained exhibited reduced lymph-node metastasis, suggesting that nuclear localization of Hpa2 plays a protective role in breast cancer progression. To examine this possibility, we engineered a gene construct that directs Hpa2 to the cell nucleus (Hpa2-Nuc). Notably, overexpression of Hpa2 in breast carcinoma cells resulted in bigger tumors, whereas targeting Hpa2 to the cell nucleus attenuated tumor growth and tumor metastasis. RNAseq analysis was performed to reveal differentially expressed genes (DEG) in Hpa2-Nuc tumors vs. control. The analysis revealed, among others, decreased expression of genes associated with the hallmark of Kras, beta-catenin, and TNF-alpha (via NFkB) signaling. Our results imply that nuclear localization of Hpa2 prominently regulates gene transcription, resulting in attenuation of breast tumorigenesis. Thus, nuclear Hpa2 may be used as a predictive parameter in personalized medicine for breast cancer patients.


Assuntos
Neoplasias da Mama , Glucuronidase , Humanos , Feminino , Glucuronidase/genética , Glucuronidase/metabolismo , Neoplasias da Mama/genética , Transdução de Sinais , Núcleo Celular/metabolismo
2.
Appl Environ Microbiol ; 90(3): e0185123, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38426790

RESUMO

Symbiotic nitrogen fixation (SNF) by rhizobia is not only the main natural bionitrogen-source for organisms but also a green process leveraged to increase the fertility of soil for agricultural production. However, an insufficient understanding of the regulatory mechanism of SNF hinders its practical application. During SNF, nifA-fixA signaling is essential for the biosynthesis of nitrogenases and electron transfer chain proteins. In the present study, the TetR regulator NffT, whose mutation increased fixA expression, was discovered through a fixA-promoter-ß-glucuronidase fusion assay performed with Rhizobium johnstonii. Real-time quantitative PCR analysis showed that nffT deletion increased the expression of symbiotic genes including nifA and fixA in nifA-fixA signaling, and fixL, fixK, fnrN, and fixN9 in fixL-fixN signaling. nffT overexpression resulted in disordered nodules and reduced nitrogen-fixing efficiency. Electrophoretic mobility shift assays revealed that NffT directly regulated the transcription of RL0091-93, which encode an ATP-binding ABC transporter predicted to be involved in carbohydrate transport. Purified His-tagged NffT bound to a 68 bp DNA sequence located -32 to -99 bp upstream of RL0091-93 and NffT deletion significantly increased the expression of RL0091-93. nffT-promoter-ß-glucuronidase fusion assay indicated that nffT expression was regulated by the cobNTS genes and cobalamin. Mutations in cobNTS significantly decreased the expression of nffT, and cobalamin restored its expression. These results revealed that NffT affects nodule development and nitrogen-fixing reaction by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes and, thus, plays a pivotal regulatory role during symbiosis of R. johnstonii-Pisum sativum.IMPORTANCESymbiotic nitrogen fixation (SNF) by rhizobia is a green way to maintain soil fertility without causing environmental pollution or consuming chemical energy. A detailed understanding of the regulatory mechanism of this complex process is essential for promoting sustainable agriculture. In this study, we discovered the TetR-type regulator NffT, which suppressed the expression of fixA in Rhizobium johnstonii. Furthermore, NffT was confirmed to play pleiotropic roles in R. johnstonii-Pisum sativum symbiosis; specifically, it inhibited rhizobial growth, nodule differentiation, and nitrogen-fixing reactions. We revealed that NffT indirectly affected R. johnstonii-P. sativum symbiosis by participating in a complex regulatory network of symbiotic and carbohydrate metabolic genes. Furthermore, cobalamin, a chemical molecule, was reported for the first time to be involved in TetR-type protein transcription during symbiosis. Thus, NffT identification connects SNF regulation with genetic, metabolic, and chemical signals and provides new insights into the complex regulation of SNF, laying an experimental basis for the targeted construction of rhizobial strains with highly efficient nitrogen-fixing capacity.


Assuntos
Rhizobium , Rhizobium/genética , Rhizobium/metabolismo , Fixação de Nitrogênio/genética , Glucuronidase/metabolismo , Carboidratos , Nitrogênio/metabolismo , Solo , Vitamina B 12/metabolismo , Simbiose/genética
3.
Int J Biol Macromol ; 264(Pt 1): 130145, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38382789

RESUMO

Mycophenolate mofetil (MMF) is a viable therapeutic option against various immune disorders as a chemotherapeutic agent. Nevertheless, its application has been undermined by the gastrotoxic metabolites (mycophenolic acid glucuronide, MPAG) produced by microbiome-associated ß-glucuronidase (ßGUS). Therefore, controlling microbiota-produced ßGUS underlines the potential strategy to improve MMF efficacy by overcoming the dosage limitation. In this study, the octyl gallate (OG) was identified with promising inhibitory activity on hydrolysis of PNPG in our high throughput screening based on a chemical collection of approximately 2000 natural products. Furthermore, OG was also found to inhibit a broad spectrum of BGUSs, including mini-Loop1, Loop 2, mini-Loop 2, and mini-Loop1,2. The further in vivo experiments demonstrated that administration of 20 mg/kg OG resulted in predominant reduction in the activity of BGUSs while displayed no impact on the overall fecal microbiome in mice. Furthermore, in the MMF-induced colitis model, the administration of OG at a dosage of 20 mg/kg effectively mitigated the gastrointestinal toxicity, and systematically reverted the colitis phenotypes. These findings indicate that the OG holds promising clinical potential for the prevention of MMF-induced gastrointestinal toxicity by inhibition of BGUSs and could be developed as a combinatorial therapy with MFF for better clinical outcomes.


Assuntos
Colite , Ácido Gálico/análogos & derivados , Microbioma Gastrointestinal , Camundongos , Animais , Ácido Micofenólico/farmacologia , Ácido Micofenólico/uso terapêutico , Imunossupressores/uso terapêutico , Glucuronidase/metabolismo , Bactérias/metabolismo , Colite/tratamento farmacológico
4.
Nat Commun ; 15(1): 1564, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38378682

RESUMO

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.


Assuntos
Encefalomielite Autoimune Experimental , Linfócitos T Reguladores , Camundongos , Animais , Humanos , Interleucina-2/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo , Matriz Extracelular/metabolismo , Heparitina Sulfato/metabolismo
5.
Front Endocrinol (Lausanne) ; 15: 1310466, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38352710

RESUMO

Introduction: Due to the relatively long life span of rodent models, in order to expediate the identification of novel therapeutics of age related diseases, mouse models of accelerated aging have been developed. In this study we examined skeletal changes in the male and female Klotho mutant (kl/kl) mice and in male and female chronically aged mice to determine whether the accelerated aging bone phenotype of the kl/kl mouse reflects changes in skeletal architecture that occur with chronological aging. Methods: 2, 6 and 20-23 month old C57BL/6 mice were obtained from the National Institute of Aging aged rodent colony and wildtype and kl/kl mice were generated as previously described by M. Kuro-o. Microcomputed tomography analysis was performed ex vivo to examine trabecular and cortical parameters from the proximal metaphyseal and mid-diaphyseal areas, respectively. Serum calcium and phosphate were analyzed using a colorimetric assay. The expression of duodenal Trpv6, which codes for TRPV6, a vitamin D regulated epithelial calcium channel whose expression reflects intestinal calcium absorptive efficiency, was analyzed by quantitative real-time PCR. Results and discussion: Trabecular bone volume (BV/TV) and trabecular number decreased continuously with age in males and females. In contrast to aging mice, an increase in trabecular bone volume and trabecular number was observed in both male and female kl/kl mice. Cortical thickness decreased with advancing age and also decreased in male and female kl/kl mice. Serum calcium and phosphate levels were significantly increased in kl/kl mice but did not change with age. Aging resulted in a decline in Trpv6 expression. In the kl/kl mice duodenal Trpv6 was significantly increased. Our findings reflect differences in bone architecture as well as differences in calcium and phosphate homeostasis and expression of Trpv6 between the kl/kl mutant mouse model of accelerated aging and chronological aging. Although the Klotho deficient mouse has provided a new understanding of the regulation of mineral homeostasis and bone metabolism, our findings suggest that changes in bone architecture in the kl/kl mouse reflect in part systemic disturbances that differ from pathophysiological changes that occur with age including dysregulation of calcium homeostasis that contributes to age related bone loss.


Assuntos
Cálcio , Glucuronidase , Animais , Feminino , Masculino , Camundongos , Envelhecimento/genética , Glucuronidase/genética , Glucuronidase/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Fosfatos , Microtomografia por Raio-X
6.
Biotechnol Lett ; 46(2): 223-233, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38310624

RESUMO

Bilirubin, a key active ingredient of bezoars with extensive clinical applications in China, is produced through a chemical process. However, this method suffers from inefficiency and adverse environmental impacts. To address this challenge, we present a novel and efficient approach for bilirubin production via whole-cell transformation. In this study, we employed Corynebacterium glutamicum ATCC13032 to express a ß-glucuronidase (StGUS), an enzyme from Staphylococcus sp. RLH1 that effectively hydrolyzes conjugated bilirubin to bilirubin. Following the optimization of the biotransformation conditions, a remarkable conversion rate of 79.7% in the generation of bilirubin was obtained at temperate 40 °C, pH 7.0, 1 mM Mg2+ and 6 mM antioxidant NaHSO3 after 12 h. These findings hold significant potential for establishing an industrially viable platform for large-scale bilirubin production.


Assuntos
Bilirrubina , Corynebacterium glutamicum , Glucuronidase/genética , Glucuronidase/metabolismo , Corynebacterium glutamicum/metabolismo , Staphylococcus , China
7.
Gut Microbes ; 16(1): 2310277, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38332701

RESUMO

Up to 40% of transplant recipients treated long-term with tacrolimus (TAC) develop post-transplant diabetes mellitus (PTDM). TAC is an important risk factor for PTDM, but is also essential for immunosuppression after transplantation. Long-term TAC treatment alters the gut microbiome, but the mechanisms of TAC-induced gut microbiota in the pathogenesis of PTDM are poorly characterized. Here, we showed that vancomycin, an inhibitor of bacterial beta-glucuronidase (GUS), prevents TAC-induced glucose disorder and insulin resistance in mice. Metagenomics shows that GUS-producing bacteria are predominant and flourish in the TAC-induced hyperglycemia mouse model, with upregulation of intestinal GUS activity. Targeted metabolomics analysis revealed that in the presence of high GUS activity, the hydrolysis of bile acid (BAs)-glucuronic conjugates is increased and most BAs are overproduced in the serum and liver, which, in turn, activates the ileal farnesoid X receptor (FXR) and suppresses GLP-1 secretion by L-cells. The GUS inhibitor vancomycin significantly eliminated GUS-producing bacteria and inhibited bacterial GUS activity and BAs levels, thereby enhancing L-cell GLP-1 secretion and preventing hyperglycemia. Our results propose a novel clinical strategy for inhibiting the bacterial GUS enzyme to prevent hyperglycemia without requiring withdrawal of TAC treatment. This strategy exerted its effect through the ileal bile acid-FXR-GLP-1 pathway.


Assuntos
Diabetes Mellitus , Microbioma Gastrointestinal , Hiperglicemia , Camundongos , Animais , Tacrolimo/farmacologia , Tacrolimo/uso terapêutico , Vancomicina/farmacologia , Imunossupressores/uso terapêutico , Hiperglicemia/induzido quimicamente , Hiperglicemia/tratamento farmacológico , Bactérias/genética , Bactérias/metabolismo , Glucuronidase/metabolismo , Glucuronidase/farmacologia , Ácidos e Sais Biliares/farmacologia , Peptídeo 1 Semelhante ao Glucagon
8.
Cells ; 13(3)2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38334603

RESUMO

Heparanase (Hpa1) is expressed by tumor cells and cells of the tumor microenvironment and functions to remodel the extracellular matrix (ECM) and regulate the bioavailability of ECM-bound factors that support tumor growth. Heparanase expression is upregulated in human carcinomas, sarcomas, and hematological malignancies, correlating with increased tumor metastasis, vascular density, and shorter postoperative survival of cancer patients, and encouraging the development of heparanase inhibitors as anti-cancer drugs. Among these are heparin/HS mimetics, the only heparanase-inhibiting compounds that are being evaluated in clinical trials. We have synthesized dicarboxylated oxy-heparins (DCoxHs) containing three carboxylate groups per split residue (DC-Hep). The resulting lead compound (termed XII) was upscaled, characterized, and examined for its effectiveness in tumor models. Potent anti-tumorigenic effects were obtained in models of pancreatic carcinoma, breast cancer, mesothelioma, and myeloma, yielding tumor growth inhibition (TGI) values ranging from 21 to 70% and extending the survival time of the mice. Of particular significance was the inhibition of spontaneous metastasis in an orthotopic model of breast carcinoma following resection of the primary tumor. It appears that apart from inhibition of heparanase enzymatic activity, compound XII reduces the levels of heparanase protein and inhibits its cellular uptake and activation. Heparanase-dependent and -independent effects of XII are being investigated. Collectively, our pre-clinical studies with compound XII strongly justify its examination in cancer patients.


Assuntos
Antineoplásicos , Neoplasias da Mama , Humanos , Animais , Camundongos , Feminino , Heparina/farmacologia , Heparina/química , Glucuronidase/metabolismo , Antineoplásicos/uso terapêutico , Carcinogênese , Neoplasias da Mama/tratamento farmacológico , Microambiente Tumoral
9.
Int J Mol Sci ; 25(3)2024 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-38339199

RESUMO

Multiple cis-acting elements are present in promoter sequences that play critical regulatory roles in gene transcription and expression. In this study, we isolated the cotton FDH (Fiddlehead) gene promoter (pGhFDH) using a real-time reverse transcription-PCR (qRT-PCR) expression analysis and performed a cis-acting elements prediction analysis. The plant expression vector pGhFDH::GUS was constructed using the Gateway approach and was used for the genetic transformation of Arabidopsis and upland cotton plants to obtain transgenic lines. Histochemical staining and a ß-glucuronidase (GUS) activity assay showed that the GUS protein was detected in the roots, stems, leaves, inflorescences, and pods of transgenic Arabidopsis thaliana lines. Notably, high GUS activity was observed in different tissues. In the transgenic lines, high GUS activity was detected in different tissues such as leaves, stalks, buds, petals, androecium, endosperm, and fibers, where the pGhFDH-driven GUS expression levels were 3-10-fold higher compared to those under the CaMV 35S promoter at 10-30 days post-anthesis (DPA) during fiber development. The results indicate that pGhFDH can be used as an endogenous constitutive promoter to drive the expression of target genes in various cotton tissues to facilitate functional genomic studies and accelerate cotton molecular breeding.


Assuntos
Arabidopsis , Gossypium , Gossypium/genética , Gossypium/metabolismo , Regiões Promotoras Genéticas , Plantas/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Glucuronidase/genética , Glucuronidase/metabolismo
10.
Mol Genet Metab ; 141(3): 108145, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38301529

RESUMO

Mucopolysaccharidosis type VII (MPS VII) is an ultra-rare, life-threatening, progressive disease caused by genetic mutations that affect lysosomal storage/function. MPS VII has an estimated prevalence of <1:1,000,000 and accounts for <3% of all MPS diagnoses. Given the rarity of MPS VII, comprehensive information on the disease is limited and we present a review of the current understanding. In MPS VII, intracellular glycosaminoglycans accumulate due to a deficiency in the lysosomal enzyme that is responsible for their degradation, ß-glucuronidase, which is encoded by the GUSB gene. MPS VII has a heterogeneous presentation. Features can manifest across multiple systems and can vary in severity, age of onset and progression. The single most distinguishing clinical feature of MPS VII is non-immune hydrops fetalis (NIHF), which presents during pregnancy. MPS VII usually presents within one month of life and become more prominent at 3 to 4 years of age; key features are skeletal deformities, hepatosplenomegaly, coarse facies, and cognitive impairment, although phenotypic variation is a hallmark. Current treatments include hematopoietic stem cell transplantation and enzyme replacement therapy with vestronidase alfa. Care should be individualized for each patient. Development of consensus guidelines for MPS VII management and treatment is needed, as consolidation of expert knowledge and experience (for example, through the MPS VII Disease Monitoring Program) may provide a significant positive impact to patients.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Mucopolissacaridose VII , Gravidez , Feminino , Humanos , Mucopolissacaridose VII/diagnóstico , Mucopolissacaridose VII/genética , Mucopolissacaridose VII/terapia , Glucuronidase/metabolismo , Hepatomegalia , Esplenomegalia , Glicosaminoglicanos , Doenças Raras/tratamento farmacológico
12.
Oncogene ; 43(5): 354-362, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38040805

RESUMO

Klotho, a 1012 amino acid transmembrane protein, is a potent tumor suppressor in different cancer types. Klotho is composed of two internal repeats KL1 and KL2, and the tumor suppressor activity is primarily attributed to the KL1 domain. Despite its significant role in regulating various cancer-related pathways, the precise mechanism underlying its tumor suppressor activity remains unresolved. In this study, we aimed to identify the sequence responsible for the tumor suppressor function of Klotho and gain insights into its mechanism of action. To accomplish this, we generated expression vectors of truncated KL1 at the C and N-terminal regions and evaluated their ability to inhibit the colony formation of several cancer cell lines. Our findings demonstrated that truncated KL1 1-340 (KL340) effectively inhibited colony formation similar to KL1, while truncated KL1 1-320 (KL320) lost this activity. Furthermore, this correlated with the inhibitory effect of KL1 and KL340 on the Wnt/ß-catenin pathway, whereas KL320 had no effect. Transcriptomic analysis of MCF-7 cells expressing the constructs revealed enriched pathways associated with tumor suppressor activity in KL1 and KL340. Interestingly, the α-fold predictor tool highlighted distinct differences in the α and ß sheets of the TIM barrel fold of the truncated Klotho constructs, adding to our understanding of their structural variations. In summary, this study identified the 340 N-terminal amino acids as the sequence that possesses Klotho's tumor suppressor activity and reveals a critical role in the 320-340 sequence for this function. It also provides a foundation for the development of Klotho-based therapeutic approaches for cancer treatment.


Assuntos
Perfilação da Expressão Gênica , Glucuronidase , Humanos , Glucuronidase/genética , Glucuronidase/metabolismo , Células MCF-7 , Hormônios
13.
Chem Biodivers ; 21(1): e202301255, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37997005

RESUMO

Klotho is a human protein with versatile functions associated with longevity and well-being. α-Klotho (α-KL) deficiency in the circulatory system is associated with reduced life expectancy with numerous disorders such as chronic kidney disease, atherosclerosis, infertility, skin atrophy, emphysema, and osteoporosis. The antagonistic effects of Klotho protein against intractable cancers have also been well documented over the past two decades. In addition, recent findings have also illuminated the importance of soluble Klotho during cognitive development, oxidative stress, cellular apoptosis, and neurodegenerative disorders. The low-cost and sustainable production of alpha Klotho protein is extremely important for its widespread use against different diseases. Here, we report heterologous, functional, and extracellular production of mouse α-KL (mα-KL) protein in model microalga Chlamydomonas reinhardtii. The secretion of mα-KL into the extracellular environment facilitated downstream processes and warranted low-cost purification in high-titer. Furthermore, the anticarcinogenic efficiency of recombinant mα-KL was examined and validated on Rattus norvegicus AR42J pancreas tumors. Microalgae-based photosynthetic, low-cost, and scalable production of mα-KL could be used to develop a variety of cosmetics, pharmaceuticals, and wellness products, all aimed at serving health and well-being.


Assuntos
Chlamydomonas reinhardtii , Microalgas , Camundongos , Humanos , Ratos , Animais , Glucuronidase/metabolismo , Chlamydomonas reinhardtii/metabolismo , Microalgas/metabolismo , Estresse Oxidativo
14.
Biochem Biophys Res Commun ; 693: 149357, 2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38091839

RESUMO

Klotho is well known as a gene with antiaging properties. It has membrane and soluble forms, providing a unique system that controls various metabolic processes essential to health and disease. Klotho deficiency has been revealed to be associated with various aging-related disorders. Based on its various known and unknown protective properties, upregulating the Klotho gene may be a possible therapeutic and/or preventive approach in aging-related complications. Some agents, such as hormonal compounds, renin-angiotensin system inhibitors, antioxidants, peroxisome proliferator-activated receptor gamma (PPAR-γ) agonists, statins, vitamin D receptor agonists, antioxidants, anti-inflammatory agents, mammalian target of rapamycin (mTOR) signaling inhibitors, and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) inhibitors, can possibly lead to the upregulation and elevation of Klotho levels. Demethylation and deacetylation of the Klotho gene can also be considered other possible Klotho-enhancement methods. Some emerging techniques, such as RNA modifications, gene therapy, gene editing, and exosome therapy, probably have the potential to be applied for increasing Klotho. In the present study, these current and emerging Klotho-enhancement strategies and their underlying mechanisms were comprehensively reviewed, which could highlight some potential avenues for future research.


Assuntos
Glucuronidase , Transdução de Sinais , Glucuronidase/genética , Glucuronidase/metabolismo , Antioxidantes , Regulação para Cima
15.
Methods Mol Biol ; 2722: 227-239, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-37897610

RESUMO

To study the gene regulatory mechanisms modulating development is essential to visualize gene expression patterns at cellular resolution. However, this kind of analysis has been limited as a consequence of the plant tissues' opacity. In the last years, ClearSee has been increasingly used to obtain high-quality imaging of plant tissue anatomy combined with the visualization of gene expression patterns. ClearSee is established as a major tissue clearing technique due to its simplicity and versatility.In this chapter, we outline an easy-to-follow ClearSee protocol to analyze gene expression of reporters using either ß-glucuronidase (GUS) or fluorescent protein (FP) tags, compatible with different dyes to stain cell walls. We detail materials, equipment, solutions, and procedures to easily implement ClearSee for the study of vascular development in Arabidopsis thaliana, but the protocol can be easily adapted to a variety of plant tissues in a wide range of plant species.


Assuntos
Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Ureia/metabolismo , Xilitol/metabolismo , Plantas/genética , Expressão Gênica , Glucuronidase/genética , Glucuronidase/metabolismo , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética
16.
Nat Rev Cardiol ; 21(1): 11-24, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37443358

RESUMO

Cardiovascular disease is the leading cause of death in patients with chronic kidney disease (CKD). As CKD progresses, CKD-specific risk factors, such as disordered mineral homeostasis, amplify traditional cardiovascular risk factors. Fibroblast growth factor 23 (FGF23) regulates mineral homeostasis by activating complexes of FGF receptors and transmembrane klotho co-receptors. A soluble form of klotho also acts as a 'portable' FGF23 co-receptor in tissues that do not express klotho. In progressive CKD, rising circulating FGF23 levels in combination with decreasing kidney expression of klotho results in klotho-independent effects of FGF23 on the heart that promote left ventricular hypertrophy, heart failure, atrial fibrillation and death. Emerging data suggest that soluble klotho might mitigate some of these effects via several candidate mechanisms. More research is needed to investigate FGF23 excess and klotho deficiency in specific cardiovascular complications of CKD, but the pathophysiological primacy of FGF23 excess versus klotho deficiency might never be precisely resolved, given the entangled feedback loops that they share. Therefore, randomized trials should prioritize clinical practicality over scientific certainty by targeting disordered mineral homeostasis holistically in an effort to improve cardiovascular outcomes in patients with CKD.


Assuntos
Doenças Cardiovasculares , Insuficiência Renal Crônica , Humanos , Glucuronidase/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Rim , Insuficiência Renal Crônica/complicações , Minerais/metabolismo
17.
Anal Chim Acta ; 1285: 342007, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38057056

RESUMO

BACKGROUND: The identification and quantification of viable Escherichia coli (E. coli) are important in multiple fields including the development of antimicrobial materials, water quality, food safety and infections diagnosis. However, the standard culture-based methods of viable E. coli detection suffer from long detection times (24 h) and complex operation, leaving the unmet requirement for fast assessing the efficiency of antimicrobial materials, early alerting the contamination of water and food, and immediately treatment of infections. RESULTS: We present a digital ß-d-glucuronidase (GUS) assay in a self-priming polydimethylsiloxane (PDMS) microfluidic chip for rapid E. coli identification and quantification. The GUS expression in viable bacteria was investigated to develop a fast GUS assay at the single-cell level. Single E. coli were stochastically discretized in picoliter chambers and identified by specific GUS activity. The digital GUS assay enabled identifying E. coli within 3 h and quantifying within 4 h for different E. coli subtypes. The specificity of our method was confirmed by using blended bacteria including E. coli, Bacillus, Shigella and Vibrio. We utilized digital GUS assay to enumerate viable E. coli after incubated with antibacterial materials for assessing the antibacterial efficiency. Moreover, the degassed chip can realize automatic sample distribution without external instruments. SIGNIFICANCE: The results demonstrated the functionality and practicability of digital GUS assay for single E. coli identification and quantification. With air-tight packaging, the developed chip has the potential for on-site E. coli analysis and could be deployed for diagnosis of E. coli infections, antimicrobial susceptibility testing, and warning the fecal pollution of water. Digital GUS assay provides a paradigm, examining the activity of metabolic enzyme, for detecting the viable bacteria other than E. coli.


Assuntos
Escherichia coli , Qualidade da Água , Escherichia coli/metabolismo , Microfluídica , Antibacterianos/farmacologia , Glucuronidase/metabolismo
18.
J Am Chem Soc ; 146(1): 125-133, 2024 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-38118176

RESUMO

Siastatin B is a potent and effective iminosugar inhibitor of three diverse glycosidase classes, namely, sialidases, ß-d-glucuronidases, and N-acetyl-glucosaminidases. The mode of inhibition of glucuronidases, in contrast to sialidases, has long been enigmatic as siastatin B appears too bulky and incorrectly substituted to be accommodated within a ß-d-glucuronidase active site pocket. Herein, we show through crystallographic analysis of protein-inhibitor complexes that siastatin B generates both a hemiaminal and a 3-geminal diol iminosugar (3-GDI) that are, rather than the parent compound, directly responsible for enzyme inhibition. The hemiaminal product is the first observation of a natural product that belongs to the noeuromycin class of inhibitors. Additionally, the 3-GDI represents a new and potent class of the iminosugar glycosidase inhibitor. To substantiate our findings, we synthesized both the gluco- and galacto-configured 3-GDIs and characterized their binding both structurally and kinetically to exo-ß-d-glucuronidases and the anticancer target human heparanase. This revealed submicromolar inhibition of exo-ß-d-glucuronidases and an unprecedented binding mode by this new class of inhibitor. Our results reveal the mechanism by which siastatin B acts as a broad-spectrum glycosidase inhibitor, identify a new class of glycosidase inhibitor, and suggest new functionalities that can be incorporated into future generations of glycosidase inhibitors.


Assuntos
Inibidores Enzimáticos , Glucuronidase , Piperidinas , Humanos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Glucuronidase/metabolismo , Glicosídeo Hidrolases/metabolismo
19.
PLoS One ; 18(11): e0294791, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38015969

RESUMO

Both mTOR and α-klotho play a role in the pathophysiology of renal disease, influence mineral metabolism and participate in the aging process. The influence of mTOR inhibition by rapamycin on renal α-klotho expression is unknown. Rats with normal (controls) and reduced (Nx) renal function were treated with rapamycin, 1.3 mg/kg/day, for 22 days. The experiments were conducted with rats fed 0.6% P diet (NP) and 0.2% P diet (LP). Treatment with rapamycin promoted phosphaturia in control and Nx rats fed NP and LP. A decrease in FGF23 was identified in controls after treatment with rapamycin. In rats fed NP, rapamycin decreased mRNA α-klotho/GADPH ratio both in controls, 0.6±0.1 vs 1.1±0.1, p = 0.001, and Nx, 0.3±0.1 vs 0.7±0.1, p = 0.01. At the protein level, a significant reduction in α-klotho was evidenced after treatment with rapamycin both by Western Blot: 0.6±0.1 vs 1.0±0.1, p = 0.01, in controls, 0.7±0.1 vs 1.1±0.1, p = 0.02, in Nx; and by immunohistochemistry staining. Renal α-klotho was inversely correlated with urinary P excretion (r = -0.525, p = 0.0002). The decrease in α-klotho after treatment with rapamycin was also observed in rats fed LP. In conclusion, rapamycin increases phosphaturia and down-regulates α-klotho expression in rats with normal and decreased renal function. These effects can be observed in animals ingesting normal and low P diet.


Assuntos
Hipofosfatemia Familiar , Sirolimo , Feminino , Ratos , Animais , Sirolimo/farmacologia , Glucuronidase/metabolismo , Proteínas Klotho , Rim/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Hipofosfatemia Familiar/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo
20.
Int J Mol Sci ; 24(22)2023 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-38003224

RESUMO

Hepatocellular adenomas are benign endothelial tumors of the liver, mostly associated with female individual users of estrogen-containing medications. However, the precise factors underlying the selective development of hepatic adenomas in certain females remain elusive. Additionally, the conventional profile of individuals prone to hepatic adenoma is changing. Notably, male patients exhibit a higher risk of malignant progression of hepatocellular adenomas, and there are instances where hepatic adenomas have no identifiable cause. In this paper, we theorize the role of the human gastrointestinal microbiota, specifically, of bacterial species producing ß-glucuronidase enzymes, in the development of hepatic adenomas through the estrogen recycling pathway. Furthermore, we aim to address some of the existing gaps in our knowledge of pathophysiological pathways which are not yet subject to research or need to be studied further. As microbial ß-glucuronidases proteins recycle estrogen and facilitate the conversion of inactive estrogen into its active form, this process results in elevated levels of unbound plasmatic estrogen, leading to extended exposure to estrogen. We suggest that an imbalance in the estrobolome could contribute to sex hormone disease evolution and, consequently, to the advancement of hepatocellular adenomas, which are estrogen related.


Assuntos
Adenoma de Células Hepáticas , Carcinoma Hepatocelular , Microbioma Gastrointestinal , Neoplasias Hepáticas , Humanos , Masculino , Feminino , Adenoma de Células Hepáticas/metabolismo , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/patologia , Glucuronidase/metabolismo , Estrogênios/metabolismo
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