Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Induction of heat shock protein 70 (Hsp70) via sublethal stress protects neurons from subsequent lethal injuries. Here we show that specific and efficient intracellular transduction of Hsp70 can be achieved utilizing an 11 amino acid leading sequence from human immunodeficiency virus (TAT-Hsp70) in primary neuronal cultures. Western blot and immunohistochemistry demonstrated intracellular accumulation of Hsp70 in insoluble protein fractions and mitochondrial compartments. We then examined the effects of Hsp70 overexpression using TAT-Hsp70 in models of nitrosative and excitotoxic neuronal death in vitro. Neurons were pre-incubated with 300 nM TAT-Hsp 70 overnight, then exposed to either peroxynitrite (ONOO-) or glutamate. TAT-Hsp70 maintained cellular respiration, inhibited extracellular lactate dehydrogenase release, and/or reduced cell death assessed by flow cytometry vs. vehicle, wild-type Hsp70, and TAT-beta-galactosidase controls. Hsp70 transduction using a TAT fusion protein is an effective method to selectively increase Hsp70 in neurons and is sufficient to provide neuroprotection from nitrosative stress and excitotoxicity. Further study is needed to confirm whether TAT-Hsp70 is protective in in vivo models of brain injury.
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PMID:Selectively increasing inducible heat shock protein 70 via TAT-protein transduction protects neurons from nitrosative stress and excitotoxicity. 1599 87

Although the structure of an enzyme is often depicted as static, it is dynamic. Hence, a population of chemically identical enzymes has not one, but a distribution of structures at any moment in time. Does this have an effect on the activity of the enzyme? This article reviews experiments designed to test the hypothesis that this distribution of structures results in a distribution of enzyme activities. The experiments reviewed here use different enzymes, falvin adenine dinucleotide, beta-galactosidase, alkaline phosphatase, exonuclease I, lactate dehydrogenase I, alpha-chymotrypsin, the 20S proteasome, and horseradish peroxidase. All experiments come to the same conclusion, when measured individually, apparently identical enzymes show a distribution in rates of activity.
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PMID:Diversity in the activity of individual enzymes. 1637 26

The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l(-1)) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h(-1). Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h(-1). The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD+, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H2 removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H2 scavenging and acetate producing microorganisms.
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PMID:Metabolism of lactose by Clostridium thermolacticum growing in continuous culture. 1650 46

Adenoviral vectors are extensively used as gene-delivery vehicles in gene therapy. They are usually produced by HEK-293 cell (human embryonic kidney-293 cell) culture, which requires specially formulated serum-free medium, the cost of which is considerable or by supplementation with FBS (fetal bovine serum). The risk of infectious diseases such as BSE (bovine spongiform encephalopathy) and endogenous retrovirus derived from cattle is a serious concern. The present study reports the use of sericin protein derived from silkworm (Bombyx mori) as an effective supplement instead of FBS. Without FBS, HEK-293 cells significantly proliferated in the presence of 0.025-0.4% sericin, especially at 0.1%, but the effect was inferior to that of FBS. When a lower titre [MOI (multiplicity of infection) 0.03] of adenoviral vector pAxCAiLacZ was used as the inoculum, HEK-293 cells in the presence of 0.1% sericin produced a nearly 3-fold higher vector titre than culture in the presence of 5% (v/v) FBS. However, when a higher vector titre (MOI 3.7) was used as the inoculum, HEK-293 cells in the presence of sericin produced a slightly higher vector titre than in the presence of FBS, which might suggest that HEK-293 cells produce a maximum amount when a higher vector titre is used as the inoculum. These increases in vector production with sericin were confirmed by LacZ (beta-galactosidase reporter gene) activity assay. Supplementation with sericin decreased lactate dehydrogenase activity, an indicator of cell death, suggesting that sericin improved cell survival; hence, prolonging the culture period might be one of the reasons for increased vector production. On the basis of these results, sericin peptide seems to be a potent and effective alternative supplement for production of adenoviral vectors without such risks as BSE and retrovirus.
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PMID:Effect of the silk protein sericin on the production of adenovirus-based gene-therapy vectors. 1667 13

Diets rich in natural antioxidants are associated with reduced risk of heart diseases. This study was aimed to evaluate the preventive role of naringin on cardiac troponin T (cTnT), lactate dehydrogenase (LDH)-isoenzyme, cardiac marker enzymes, electrocardiographic (ECG)-patterns and lysosomal enzymes in isoproterenol (ISO)-induced myocardial infarction (MI) in male Wistar rats. Rats subcutaneously injected with ISO (85mg/kg) at an interval of 24h for 2 days showed a significant increase in the levels of cTnT, intensity of the bands of LDH-isoenzyme (LDH1 and LDH2) and the activities of cardiac marker enzymes such as creatine kinase-MB (CK-MB), creatine kinase (CK), LDH, aspartate transaminase (AST) and alanine transaminase (ALT) in serum with subsequent decrease in the activities of CK, LDH, AST and ALT in the heart and alterations in ECG-patterns. The activities of lysosomal enzymes (beta-glucuronidase, beta-N-acetyl glucosaminidase, beta-galactosidase, cathepsin-B and cathepsin-D) were increased significantly in serum and the heart of ISO-induced rats, but the activities of beta-glucuronidase and cathepsin-D were decreased significantly in the lysosomal fraction of the heart. Pretreatment with naringin (10, 20 or 40mg/kg) daily for a period of 56 days positively altered the levels of cTnT, intensity of the bands of the LDH1 and LDH2-isoenzyme and the activities of cardiac marker enzymes, ECG-patterns and lysosomal hydrolases in ISO-induced rats. Thus, naringin possess cardioprotective effect in ISO-induced MI in rats.
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PMID:Preventive effect of naringin on cardiac markers, electrocardiographic patterns and lysosomal hydrolases in normal and isoproterenol-induced myocardial infarction in Wistar rats. 1718 15

The present study investigated cerebrospinal fluid (CSF) biomarkers for estimating degeneration of the central nervous system (CNS) in experimental dogs with GM1 gangliosidosis and preliminarily evaluated the efficacy of long-term glucocorticoid therapy for GM1 gangliosidosis using the biomarkers identified here. GM1 gangliosidosis, a lysosomal storage disease that affects the brain and multiple systemic organs, is due to an autosomal recessively inherited deficiency of acid beta-galactosidase activity. Pathogenesis of GM1 gangliosidosis may include neuronal apoptosis and abnormal axoplasmic transport and inflammatory response, which are perhaps consequent to massive neuronal storage of GM1 ganglioside. In the present study, we assessed some possible CSF biomarkers, such as GM1 ganglioside, aspartate aminotransferase (AST), lactate dehydrogenase (LDH), neuron-specific enolase (NSE) and myelin basic protein (MBP). Periodic studies demonstrated that GM1 ganglioside concentration, activities of AST and LDH, and concentrations of NSE and MBP in CSF were significantly higher in dogs with GM1 gangliosidosis than those in control dogs, and their changes were well related with the months of age and clinical course. In conclusion, GM1 ganglioside, AST, LDH, NSE and MBP could be utilized as CSF biomarkers showing CNS degeneration in dogs with GM1 gangliosidosis to evaluate the efficacy of novel therapies proposed for this disease. In addition, we preliminarily treated an affected dog with long-term oral administration of prednisolone and evaluated the efficacy of this therapeutic trial using CSF biomarkers determined in the present study. However, this treatment did not change either the clinical course or the CSF biomarkers of the affected dog, suggesting that glucocorticoid therapy would not be effective for treating GM1 gangliosidosis.
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PMID:Cerebrospinal fluid biomarkers showing neurodegeneration in dogs with GM1 gangliosidosis: possible use for assessment of a therapeutic regimen. 1719 62

Genetic studies of Streptococcus mutans have benefited greatly from the numerous techniques that have been successfully adapted for use in this organism. One notable exception is the lack of a negative selection system that can be employed for the easy isolation of markerless in-frame deletions. In this study, we report the development of a galK/galactose-based negative selection system in S. mutans for this purpose. This system consists of a recipient strain (IFD140) that contains a deletion in the galKTE operon and a suicide vector (pIFD-Sm) that carries the S. mutans galK open reading frame fused to the constitutive lactate dehydrogenase (ldh) promoter. Using this system we created a markerless in-frame deletion in the beta-galactosidase (lacG) gene within the S. mutans lactose operon. After vector integration, plasmid excision after counterselection appeared to have occurred in 100% of the galactose-resistant colonies and resulted in in-frame deletions in 50% of the screened isolates. Based on the ratio of galactose-resistant cells to total cells, we determined that plasmid excision occurred at a frequency of approximately 1/3000 cells. Furthermore, the simplicity of this system should make it adaptable for use in numerous other gram-positive and gram-negative organisms.
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PMID:Construction of a counterselection-based in-frame deletion system for genetic studies of Streptococcus mutans. 1731 32

Akt is expected to be an effective target for the treatment of ischemia-reperfusion injury (I/R) due to its anti-apoptotic properties and its ability to activate the endothelial nitric oxide synthase (eNOS) enzyme. Therefore, this study was aimed to determine the efficacy of an active mutant of Akt (myr-Akt) to decrease I/R injury in a model of orthotopic liver transplantation in pigs. In addition, we analyzed the contribution of nitric oxide in the Akt-mediated effects by using an eNOS mutant (S1179DeNOS) that mimics the phosphorylation promoted by Akt in the eNOS sequence. Donors were treated with adenoviruses codifying for myr-Akt, S1179DeNOS or beta-galactosidase 24 h before liver harvesting. Then, liver grafts were orthotopically transplanted into their corresponding recipients. Levels of transaminases and lactate dehydrogenase (LDH) increased in all recipients after 24 h of transplant. However, transaminases and LDH levels were significantly lower in the myr-Akt group compared with vehicle. The percentage of apoptotic cells and the amount of activated-caspase 3 protein were also markedly reduced in myr-Akt-treated grafts after 4 days of liver transplant compared with vehicle and S1179DeNOS groups. In conclusion, myr-Akt gene therapy effectively exerts cytoprotection against hepatic I/R injury regardless of the Akt-dependent eNOS activation.
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PMID:Gene transduction of an active mutant of akt exerts cytoprotection and reduces graft injury after liver transplantation. 1739 Nov 22

The present study was designed to evaluate the possible beneficial effect of lipoic acid in preventing the renal damage induced by cyclosporine A in rats. Male albino rats of Wistar strain were divided into four groups and treated as follows. Two groups received cyclosporine A by oral gavage (25 mg/kg/body weight) for 21 days to induce nephrotoxicity, one of which simultaneously received lipoic acid treatment (20 mg/kg body weight) for 21 days. A vehicle (olive oil) and a lipoic acid drug control were also included. Cyclosporine A induced renal damage was evident from the decreased activities of tissue marker enzymes (alkaline phosphatase, acid phosphatase, lactate dehydrogenase, aspartate transaminase and alanine transaminase) and decreased activities of ATPases (Na+, K+-ATPase, Ca2+-ATPase and Mg2+ ATPase). An apparent increase in the levels of serum constituents (urea, uric acid and creatinine) and urinary marker enzymes (N-acetyl-beta-D-glucosaminidase, beta-glucosidase, beta-galactosidase, cathepsin-D and gamma-glutamyl transpeptidase) along with significant decline in creatinine clearance were seen in the cyclosporine treated rats, which was reversed upon treatment with lipoic acid. Ultrastructural observations were also in agreement with the above abnormal changes. Lipoic acid effectively reverted these abnormal biochemical changes and minimized the morphological lesions in renal tissue. Hence, this study clearly exemplifies that lipoic acid might be an ideal choice against cyclosporine A induced cellular abnormalities.
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PMID:Therapeutic efficacy of DL-alpha-lipoic acid on cyclosporine A induced renal alterations. 1761 14

Macroautophagic activity is most directly and precisely measured by a cargo sequestration assay. Long-lived, cytosolic proteins that are degraded exclusively by the autophagic-lysosomal pathway, such as lactate dehydrogenase (LDH) are suitable as endogenous sequestration probes. Autophagic sequestration is measured as transfer of the protein from the soluble (cytosolic) to the sedimentable (organelle-containing) cell fraction, using leupeptin or other proteinase inhibitors to block inactivation and degradation of the protein inside autophagic vacuoles. A convenient separation method is electrodisruption of the cells, followed by sedimentation of the organelle fraction through a Nycodenz density cushion. A promising variant of the cargo assay is to use a protein probe that is processed by the autophagic-lysosomal pathway so as to generate an intravacuolar fragment. Because there is no cytosolic background, subcellular fractionation is unnecessary, allowing the use of the autophagic fragment assay to measure autophagic activity in whole cells. In hepatocytes, a small fragment, p10(BHMT), made by autophagic processing of the enzyme betaine:homocysteine methyltransferase, thus accumulates in an autophagy-dependent manner in the presence of leupeptin. Autophagic sequestration can also be measured by using exogenous cargo probes, such as radiolabeled di- and trisaccharides, which can be loaded into the cytosol of hepatocytes by reversible electrodisruption or mechanical stress. Raffinose is the preferable probe for measurement of autophagic activity, whereas sucrose (which can be hydrolyzed in amphisomes and lysosomes by added endocytosed invertase) and lactose (which is hydrolyzed in lysosomes by the endogenous beta-galactosidase) are useful for dissection of the various steps in the autophagic-lysosomal pathway and for studying autophagic-endocytic interactions. Furthermore, the intralysosomal hydrolysis of autophagocytosed lactose can be measured in whole cells (as formation of the hydrolysis product, galactose), thus providing a background-free assay (autophagic lactolysis) of the overall autophagic-lysosomal pathway.
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PMID:Sequestration assays for mammalian autophagy. 1920 Aug 76


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