scholarly journals Glucose-6-phosphate dehydrogenase mRNA sequence abundance in primary cultures of rat hepatocytes. Effect of insulin and dexamethasone

1986 ◽  
Vol 237 (2) ◽  
pp. 617-619 ◽  
Author(s):  
R S Fritz ◽  
D J Stumpo ◽  
R F Kletzien
Keyword(s):  
Blot Hybridization ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Nutritional Regulation ◽  
Dot Blot ◽  
Hybridization Procedure ◽  
Hepatic Glucose ◽  
Glucose 6 Phosphate Dehydrogenase

Hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is subject to nutritional regulation. To assess the possible role of hormones in this regulation, the amounts of G6PDH mRNA were studied in primary cultures of rat hepatocytes treated with insulin and dexamethasone, alone or in combination. Relative concentrations of G6PDH mRNA were directly assessed by a dot-blot hybridization procedure with nick-translated cDNA probes. G6PDH sequence abundance increased when the cultures were treated with insulin or dexamethasone, but the G6PDH mRNA induced by dexamethasone was not expressed at the protein level as active enzyme. In cultures treated with insulin and dexamethasone in combination, enzyme activity and G6PDH sequence abundance were greater than those induced by insulin alone. Our results directly demonstrate that G6PDH mRNA amounts are modulated in liver by these two classes of hormones and can partially account for the dietary induction of the enzyme observed in vivo.

scholarly journals Ethanol modulation of the hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase activity in primary cultures of rat hepatocytes

1984 ◽  
Vol 217 (2) ◽  
pp. 543-549 ◽  
Author(s):  
D S Kelley ◽  
R F Kletzien
Keyword(s):  
Enzyme Activity ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Fold Increase ◽  
Defined Medium ◽  
Nutritional Regulation ◽  
G6pdh Activity ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Dose Dependent ◽  
Permissive Role

The hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Inoculation of hepatocytes from starved rats into primary cultures resulted in a 4-5-fold increase in G6PDH activity in 48 h in the absence of hormones. Parallel cultures treated simultaneously with glucocorticoids and insulin exhibited a 12-15-fold increase during the same time. Glucocorticoids by themselves did not elevate G6PDH activity, whereas insulin alone significantly stimulated enzyme activity. Thus the glucocorticoids acted in a ‘permissive’ role to amplify the insulin stimulation of G6PDH. Elevated concentrations of glucose in the culture medium increased enzyme activity in both the control cultures and those treated with hormones. Ethanol was found to potentiate G6PDH activity in cultures treated with glucocorticoids and insulin. The effect of ethanol was time- and dose-dependent. These results establish that insulin, glucocorticoids, glucose and ethanol interact in some undefined manner to regulate hepatic G6PDH activity.

scholarly journals Liver Protection from Apoptosis Requires Both Blockage of Initiator Caspase Activities and Inhibition of ASK1/JNK Pathway via GlutathioneS-Transferase Regulation

2002 ◽  
Vol 277 (51) ◽  
pp. 49220-49229 ◽  
Author(s):  
David Gilot ◽  
Pascal Loyer ◽  
Anne Corlu ◽  
Denise Glaise ◽  
Dominique Lagadic-Gossmann ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Primary Cultures ◽  
Radical Scavenging ◽  
Rat Hepatocytes ◽  
Jnk Pathway ◽  
Dual Inhibition ◽  
Kinase Pathway ◽  
High Expression Level

Hepatoprotection mediated by free radical scavenging molecules such as dimethyl sulfoxide (Me2SO) arose the question as to whether this effect involved one or several anti-apoptotic signals. Here, using primary cultures of rat hepatocytes andin vivothioacetamide-induced liver failure, we showed that Me2SO failed to prevent any cleavage of initiator caspase-8 and -9 but constantly inhibited procaspase-3 maturation and apoptosis execution, pointing to an efficient inhibition of cleaved initiator caspase activities. Evidence was recently provided that apoptosis might require both caspase and ASK1/JNK-p38 activities. We demonstrated that this kinase pathway was strongly inhibited in the presence of Me2SO whereas overexpression of ASK1 was able to restore caspase-3 activity and apoptosis. Interestingly, we also found that GST Μ1/2 and GST Α1/2 dropped under apoptotic conditions; furthermore transfection of GST Μ1, A1, or P1 to cells overexpressing ASK1, abolished caspase-3 activity and restored viability. This role of GSTs was further assessed by showing that their high expression level was tightly associated with inhibition of ASK1 activity in Me2SO-protected hepatocytes. Together, these results demonstrate that Me2SO-mediated hepatoprotection involves a dual inhibition of cleaved initiator caspase and ASK1/JNK-p38 activities. Furthermore, in highlighting the control of apoptosis by GSTs, these data provide new insights for analyzing the complex mechanisms of hepatoprotection.

In Vitro Culture of Human Thyroid Cells: Preliminary Findings on the Role of the Pathological Condition of the Donors

1997 ◽  
Vol 25 (2) ◽  
pp. 153-160
Author(s):  
Francesca Mattioli ◽  
Marianna Angiola ◽  
Laura Fazzuoli ◽  
Francesco Razzetta ◽  
Antonietta Martelli
Keyword(s):  
Pathological Condition ◽  
Primary Cultures ◽  
S Phase ◽  
Human Thyroid ◽  
Thyroid Cells ◽  
Toxicological Studies ◽  
Predictive Indicator

Although primary cultures of human thyroid cells are used for endocrinological and toxicological studies, until now no attention has been paid toward verifying whether the hormonal conditions to which the gland was exposed in vivo prior to surgery could influence in vitro responses. Our findings suggest that the hormonal situation in vivo cannot be used as a predictive indicator of triiodothyronine and thyroxine release and/or S-phase frequency in vitro, either with or without the addition of bovine thyrotropin.

Role of Nitric Oxide in Down-Regulation of CYP2B1 Protein, but Not RNA, in Primary Cultures of Rat Hepatocytes

2001 ◽  
Vol 60 (1) ◽  
pp. 209-216 ◽  
Author(s):  
Luc Ferrari ◽  
Ning Peng ◽  
James R. Halpert ◽  
Edward T. Morgan
Keyword(s):  
Nitric Oxide ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Down Regulation

scholarly journals Attenuation of late simian virus 40 mRNA synthesis is enhanced by the agnoprotein and is temporally regulated in isolated nuclear systems.

1985 ◽  
Vol 5 (6) ◽  
pp. 1327-1334 ◽  
Author(s):  
N Hay ◽  
Y Aloni
Keyword(s):  
Gel Electrophoresis ◽  
Blot Hybridization ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Wild Type Virus ◽  
Insertion Mutant ◽  
Dot Blot ◽  
Nuclear Systems

Studies were performed to verify the physiological significance of attenuation in the life cycle of simian virus 40 and the role of agnoprotein in this process. For these purposes, nuclei were isolated at various times after infection and incubated in vitro in the presence of [alpha-32P]UTP under the standard conditions which lead to attenuation. Attenuation was evident by the production of a 94-nucleotide attenuator RNA, revealed by gel electrophoresis. In parallel, the synthesis of agnoprotein was studied at various times after infection by labeling the cells for 3 h with [14C]arginine, lysing them, and analyzing the labeled proteins by gel electrophoresis. Both attenuation and the synthesis of agnoprotein were predominant towards the end of the infectious cycle. At earlier times, there was almost no attenuation and no synthesis of agnoprotein. Moreover, there was almost no attenuation even at the latest times after infection in nuclei isolated from cells infected with simian virus 40 deletion mutants that do not synthesize agnoprotein. Finally, analysis by dot blot hybridization showed higher amounts of cytoplasmic viral RNA in cells infected with an agnoprotein gene insertion mutant, delta 79, that does not produce agnoprotein, compared with cells infected with wild-type virus. The present studies indicate that attenuation is temporally regulated and suggest that agnoprotein enhances attenuation in isolated nuclei and that may also enhance it in vivo.

scholarly journals Alpha 1-antichymotrypsin contributes to stem cell characteristics and enhances tumorigenicity of Glioblastoma

2020 ◽  
Author(s):  
Montserrat Lara-Velazquez ◽  
Natanael Zarco ◽  
Anna Carrano ◽  
Jordan Phillipps ◽  
Emily S Norton ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Migration ◽  
Brain Tumor ◽  
Primary Cultures ◽  
Cellular Migration ◽  
Tumor Initiating Cells ◽  
The Impact

Abstract Background Glioblastomas (GBMs) are the most common primary brains tumors in adults with almost 100% recurrence rate. Patients with lateral ventricle proximal GBMs (LV-GBMs) exhibit worse survival compared to distal locations for reasons that remain unknown. One potential explanation is the proximity of these tumors to the cerebrospinal fluid (CSF) and its contained chemical cues that can regulate cellular migration and differentiation. We therefore investigated the role of CSF on GBM gene expression and the role of a CSF-induced gene, SERPINA3, in GBM malignancy in vitro and in vivo. Methods We utilized patient-derived CSF and primary cultures of GBM brain tumor initiating cells (BTICs). We determined the impact of SERPINA3 expression in glioma patients using TCGA database. SERPINA3 expression changes were evaluated at both the mRNA and protein levels. The effects of knockdown (KD) and overexpression (OE) of SERPINA3 on cell behavior were evaluated by transwell assay (for cell migration), and alamar blue and Ki67 (for viability and proliferation respectively). Stem cell characteristics on KD cells were evaluated by differentiation and colony formation experiments. Tumor growth was studied by intracranial and flank injections. Results GBM CSF induced a significant increase in BTIC migration accompanied by upregulation of the SERPINA3 gene. In patient samples and TCGA data we observed SERPINA3 to correlate directly with brain tumor grade and indirectly with GBM patient survival. Silencing of SERPINA3 induced a decrease in cell proliferation, migration, invasion, and stem cell characteristics, while SERPINA3 overexpression increased cell migration. In vivo, mice orthotopically-injected with SERPINA3 KD BTICs showed increased survival. Conclusions SERPINA3 plays a key role in GBM malignancy and its inhibition results in a better outcome using GBM preclinical models.

Characterization of Marine Temperate Phage-Host Systems Isolated from Mamala Bay, Oahu, Hawaii

1998 ◽  
Vol 64 (2) ◽  
pp. 535-542 ◽  
Author(s):  
Sunny C. Jiang ◽  
Christina A. Kellogg ◽  
John H. Paul
Keyword(s):  
Water Samples ◽  
Blot Hybridization ◽  
Temperate Phage ◽  
Dot Blot ◽  
Dot Blot Hybridization ◽  
Double Stranded Dna ◽  
The Family ◽  
Marine Viruses

ABSTRACT To understand the ecological and genetic role of viruses in the marine environment, it is critical to know the infectivity of viruses and the types of interactions that occur between marine viruses and their hosts. We isolated four marine phages from turbid plaques by using four indigenous bacterial hosts obtained from concentrated water samples from Mamala Bay, Oahu, Hawaii. Two of the rod-shaped bacterial hosts were identified as Sphingomonas paucimobilis andFlavobacterium sp. All of the phage isolates were tailed phages and contained double-stranded DNA. Two of the phage isolates had morphologies typical of the family Siphoviridae, while the other two belonged to the families Myoviridae andPodoviridae. The head diameters of these viruses ranged from 47 to 70.7 nm, and the tail lengths ranged from 12 to 146 nm. The burst sizes ranged from 7.8 to 240 phage/bacterial cell, and the genome sizes, as determined by restriction digestion, ranged from 36 to 112 kb. The members of the Siphoviridae, T-φHSIC, and T-φD0, and the member of the Myoviridae, T-φD1B, were found to form lysogenic associations with their bacterial hosts, which were isolated from the same water samples. Hybridization of phage T-φHSIC probe with lysogenic host genomic DNA was observed in dot blot hybridization experiments, indicating that prophage T-φHSIC was integrated within the host genome. These phage-host systems are available for use in studies of marine lysogeny and transduction.

scholarly journals Epidemiology of enteric adenovirus infection in prospectively monitored Argentine families

1992 ◽  
Vol 109 (3) ◽  
pp. 539-546 ◽  
Author(s):  
A. S. Mistchenko ◽  
K. H. Huberman ◽  
J. A. Gomez ◽  
S. Grinstein
Keyword(s):  
Buenos Aires ◽  
Blot Hybridization ◽  
Dot Blot ◽  
Severe Diarrhoea ◽  
Natural Conditions ◽  
Dot Blot Hybridization ◽  
Faecal Samples ◽  
Enteric Adenovirus ◽  
Enteric Adenoviruses

SUMMARYTo examine the role of enteric adenoviruses (EAV) in an urban area of Buenos Aires (Argentina), we prospectively studied faecal samples from 49 families of newborns. These were monitored weekly for diarrhoea for 2 years.A total of 180 samples from cases of diarrhoea and 766 samples obtained during diarrhoea-free periods were studied by dot-blot hybridization with an EAV-specific DNA probe. EAV were found in 6/180 (3·3%) cases of diarrhoea and 6/766 (0·8%) asymptomatic samples (P < 0·015). Incidence of EAV was 3·9 cases per 100 person-years in children < 60 months old. EAV-related diarrhoeas were slight and of short duration. In addition, 129 faeces from hospital out-patients, 1–30 months old, were also studied. EAV was identified in 7/129 cases (5·4%). These cases were 9·5 ±3·5 months old and the diarrhoea was mild or severe, of 3±1·5 days of duration.We suggest that EAV are low-risk causes of diarrhoea under natural conditions, although a few children may develop more severe diarrhoea. The diagnosis of EAV needs to be considered in these patients.

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