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1.
bioRxiv ; 2023 Oct 24.
Article in English | MEDLINE | ID: mdl-37693559

ABSTRACT

The translational repressor Nanos (Nos) regulates a single target, maternal hunchback (hb) mRNA, to govern abdominal segmentation in the early Drosophila embryo. Nos is recruited specifically to sites in the 3'-UTR of hb mRNA in collaboration with the sequence-specific RNA-binding protein Pumilio (Pum); on its own, Nos has no binding specificity. Nos is expressed at other stages of development, but very few mRNA targets that might mediate its action at these stages have been described. Nor has it been clear whether Nos is targeted to other mRNAs in concert with Pum or via other mechanisms. In this report, we identify mRNAs targeted by Nos via two approaches. In the first method, we identify mRNAs depleted upon expression of a chimera bearing Nos fused to the nonsense mediated decay (NMD) factor Upf1. We find that, in addition to hb, Upf1-Nos depletes ~2600 mRNAs from the maternal transcriptome in early embryos. Virtually all of these appear to be targeted in a canonical, hb-like manner in concert with Pum. In a second, more conventional approach, we identify mRNAs that are stabilized during the maternal zygotic transition (MZT) in embryos from nos- females. Most (86%) of the 1185 mRNAs regulated by Nos are also targeted by Upf1-Nos, validating use of the chimera. Approximately 60% of mRNAs targeted by Upf1-Nos are not stabilized in the absence of Nos. However, Upf1-Nos mRNA targets are hypo-adenylated and inefficiently translated at the ovary-embryo transition, whether or not they suffer Nos-dependent degradation in the embryo. We suggest that the late ovarian burst of Nos represses a large fraction of the maternal transcriptome, priming it for later degradation by other factors during the MZT in the embryo.

2.
Oral Dis ; 27(2): 378-387, 2021 Mar.
Article in English | MEDLINE | ID: mdl-32609943

ABSTRACT

OBJECTIVES: To identify factors which influence the intraoral prevalence of human herpes viruses (HHVs) using mucosal swabs, saliva samples and qPCR analysis. METHODOLOGY: In this cross-sectional observational study, matched saliva and oral swabs were collected from a total of 115 subjects: 70 immunocompetent subjects with no mucosal abnormalities, 22 with mucosal abnormalities and 23 therapeutically immunocompromised individuals. Extracted DNA was analysed by multiplex qPCR for detection and quantification of HHVs 1-6. RESULTS: At least one human herpes virus was detected in 77.1% of immunocompetent individuals with no mucosal abnormalities, with EBV the most commonly detected at 61.4%. HHV-6 was detected in 17.1%, HSV-1 in 4.3% and CMV in 1.1%. Detection was higher in saliva than in oral swabs. There was no detection of HSV-2 or VZV. Neither presence of oral mucosal abnormality nor therapeutic immunocompromise was related to increased detection of human herpes virus. CONCLUSION: Commensal detection rates of EBV are high, and caution in clinical correlation of positive detection is warranted. Commensal CMV rates are low, and detection is likely to be clinically relevant. This study presents a comprehensive commensal detection rate of HHVs 1-6 by qPCR in saliva and swabs.


Subject(s)
Herpesviridae Infections , Viruses , Cross-Sectional Studies , DNA, Viral , Herpesviridae Infections/diagnosis , Humans , Saliva
3.
J Thorac Cardiovasc Surg ; 148(6): 3139-45, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25224551

ABSTRACT

OBJECTIVES: Measurements of stroke volume variation for volume management in mechanically ventilated patients are influenced by various factors, such as tidal volume, respiratory rate, and chest/lung compliance. However, research regarding the effect of positive end-expiratory pressure on stroke volume variation is limited. METHODS: Patients were divided into responder and nonresponder groups according to the prediction of fluid response by the passive leg raising test and hemodynamic parameters, including stroke volume variation, measured in all patients at the following ventilator settings: (1) conventional ventilation (C), tidal volume 10 mL · kg(-1) with positive end-expiratory pressure settings of 0 (C0), 5 (C5), and 10 cmH2O (C10) and (2) lung protective ventilation (P), tidal volume 6 mL · kg(-1) with positive end-expiratory pressure settings of 0 (P0), 5 (P5), and 10 cmH2O (P10). RESULTS: Regardless of ventilator setting, stroke volume variation in the responder group had an increasing trend as increased positive end-expiratory pressure level and was significantly higher than in the nonresponder group at each positive end-expiratory pressure level. The area under the curve was (1) 0.899 at C0, 0.942 at C5, and 0.985 at C10; and (2) 0.901 at P0, 0.932 at P5, and 0.947 at P10. Optimal threshold values given by receiver operating characteristic curve analysis were (1) 13.5%, 13.5%, and 14.5%; and (2) 13.5%, 13.5%, and 14.5%, respectively. CONCLUSIONS: The threshold value of stroke volume variation in predicting fluid responsiveness may change when positive end-expiratory pressure 10 cmH2O is applied. This must be considered when stroke volume variation is used to detect the fluid responsiveness to prevent volume overload in this mechanical ventilation setting.


Subject(s)
Aortic Valve Insufficiency/surgery , Aortic Valve Stenosis/surgery , Cardiac Surgical Procedures , Monitoring, Physiologic/methods , Positive-Pressure Respiration , Stroke Volume , Adult , Aortic Valve Insufficiency/diagnosis , Aortic Valve Insufficiency/physiopathology , Aortic Valve Stenosis/diagnosis , Aortic Valve Stenosis/physiopathology , Area Under Curve , Cardiac Surgical Procedures/adverse effects , Female , Fluid Therapy , Humans , Intensive Care Units , Male , Middle Aged , Positive-Pressure Respiration/adverse effects , Predictive Value of Tests , Prospective Studies , ROC Curve , Time Factors , Treatment Outcome
4.
PLoS One ; 7(4): e34016, 2012.
Article in English | MEDLINE | ID: mdl-22514614

ABSTRACT

In Drosophila melanogaster, specification of wing vein cells and sensory organ precursor (SOP) cells, which later give rise to a bristle, requires EGFR signaling. Here, we show that Pumilio (Pum), an RNA-binding translational repressor, negatively regulates EGFR signaling in wing vein and bristle development. We observed that loss of Pum function yielded extra wing veins and additional bristles. Conversely, overexpression of Pum eliminated wing veins and bristles. Heterozygotes for Pum produced no phenotype on their own, but greatly enhanced phenotypes caused by the enhancement of EGFR signaling. Conversely, over-expression of Pum suppressed the effects of ectopic EGFR signaling. Components of the EGFR signaling pathway are encoded by mRNAs that have Nanos Response Element (NRE)-like sequences in their 3'UTRs; NREs are known to bind Pum to confer regulation in other mRNAs. We show that these NRE-like sequences bind Pum and confer repression on a luciferase reporter in heterologous cells. Taken together, our evidence suggests that Pum functions as a negative regulator of EGFR signaling by directly targeting components of the pathway in Drosophila.


Subject(s)
Drosophila Proteins/metabolism , Drosophila melanogaster/metabolism , ErbB Receptors/metabolism , Mitogen-Activated Protein Kinases/metabolism , RNA-Binding Proteins/metabolism , Receptors, Invertebrate Peptide/metabolism , Animals , Drosophila Proteins/genetics , ErbB Receptors/genetics , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Mitogen-Activated Protein Kinases/genetics , Protein Binding , RNA-Binding Proteins/genetics , Receptors, Invertebrate Peptide/genetics , Signal Transduction/genetics , Signal Transduction/physiology
5.
Korean J Anesthesiol ; 62(2): 113-8, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22379564

ABSTRACT

BACKGROUND: The effects of dexmedetomidine on the propofol-sparing effect and intraoperative hemodynamics during remifentanil-based propofol-supplemented anesthesia have not been well investigated. METHODS: Twenty patients undergoing breast surgery were randomly allocated to receive dexmedetomidine (group DEX) or placebo (group C). In the DEX group, dexmedetomidine was loaded (1 µg/kg) before anesthesia induction and was infused (0.6 µg/kg/h) during surgery. Anesthesia was induced with a target-controlled infusion (TCI) of propofol (effect site concentration, Ce; 3 µg/ml) and remifentanil (plasma concentration, Cp, 10 ng/ml). The Ce of TCI-propofol was adjusted to a bispectral index of 45-55, and Cp of TCI-remifentanil was fixed at 10 ng/ml in both groups. Mean arterial blood pressure (MAP) and heart rate (HR) were recorded at baseline (T-control), after the loading of study drugs (T-loading), 3 min after anesthesia induction (T-induction), tracheal intubation (T-trachea), incision (T-incision), 30 min after incision (T-incision30), and at tracheal extubation (T-extubation). MAP% and HR% (MAP and HR vs. T-control) were determined and the propofol infusion rate was calculated. RESULTS: The propofol infusion rate was significantly lower in the DEX group than in group C (63.9 ± 16.2 vs. 96.4 ± 10.0 µg/kg/min, respectively; P < 0.001). The changes in MAP% at T-induction, T-trachea and T-incision in group DEX (-10.0 ± 3.9%, -9.4 ± 4.6% and -11.2 ± 6.3%, respectively) were significantly less than those in group C (-27.6 ± 13.9%, -21.7 ± 17.1%, and -25.1 ± 14.1%; P < 0.05, respectively). CONCLUSIONS: Dexmedetomidine reduced the propofol requirement for remifentanil-based anesthesia while producing more stable intraoperative hemodynamics.

6.
Osong Public Health Res Perspect ; 3(4): 213-21, 2012 Dec.
Article in English | MEDLINE | ID: mdl-24159517

ABSTRACT

OBJECTIVES: In this study, we demonstrated that TBEV-infected ticks have been distributed in the ROK, combined with our previous results. These results suggest that TBEV may exist in the ROK, and H. longicornis, H. flava, and I. nipponensis may be potential vectors of TBEV. In addition, these results emphasize the need for further epidemiological research of TBEV. METHODS: We examined for the presence of RNA of TBEV by reverse transcriptase-nested polymerase chain reaction (RT-nested PCR) using ixodid ticks captured in 25 localities of 10 provinces. Ticks were collected by the flagging and dragging method or using sentinel BG traps at forests, grass thickets, and grassland. A total of 13,053 ticks belonging to two genera and four species were collected and pooled (1292 pools), according to collection site, species of tick, and developmental stage. RESULTS: Among 1292 pools, the envelope (E) protein gene of TBEV was detected using RT-nested PCR in 10 pools (3 pools of the 1,331 adult ticks and 7 pools of the 11,169 nymph ticks) collected from Gangwon-do province, Jeonrabuk-do province, and Jeju Island. The minimum infection rates for TBEV of Haemaphysalis longicornis, Haemaphysalis flava, and Ixodes nipponensis were 0.06%, 0.17%, and 2.38%, respectively. Phylogenetic analysis based on the partial E protein gene was performed to identify relationships between the TBEV strains. This showed that 10 Korean strains clustered with the Western subtype. CONCLUSION: In this study, we investigated the prevalence of tick-borne encephalitis virus (TBEV) in ixodid ticks from various regions of the Republic of Korea (ROK) during 2011-2012 to identify whether TBEV is circulating and to determine the endemic regions of TBEV.

7.
Anesth Analg ; 112(5): 1076-81, 2011 May.
Article in English | MEDLINE | ID: mdl-21127282

ABSTRACT

BACKGROUND: Although sedation is often performed during spinal anesthesia, the details of intraoperative dreaming have not been reported. We designed this prospective study to compare 2 different IV sedation protocols (propofol and midazolam infusion) with respect to dreaming during sedation. METHODS: Two hundred twenty adult patients were randomly assigned to 2 groups and received IV infusion of propofol or midazolam for deep sedation during spinal anesthesia. Patients were interviewed on emergence and 30 minutes later to determine the incidence, content, and nature of their dreams. Postoperatively, patient satisfaction with the sedation was also evaluated. RESULTS: Two hundred fifteen patients (108 and 107 in the propofol and midazolam groups, respectively) were included in the final analysis. The proportion of dreamers was 39.8% (43/108) in the propofol group and 12.1% (13/107) in the midazolam group (odds ratio=4.78; 95% confidence interval: 2.38 to 9.60). Dreams of the patients receiving propofol were more memorable and visually vivid than were those of the patients receiving midazolam infusion. The majority of dreams (36 of 56 dreamers, 64.3%) were simple, pleasant ruminations about everyday life. A similarly high level of satisfaction with the sedation was observed in both groups. CONCLUSIONS: In cases of spinal anesthesia with deep sedation, dreaming was almost 5 times more common in patients receiving propofol infusion than in those receiving midazolam, although this did not influence satisfaction with the sedation. Thus, one does not need to consider intraoperative dreaming when choosing propofol or midazolam as a sedative drug in patients undergoing spinal anesthesia.


Subject(s)
Anesthesia, Spinal , Deep Sedation , Dreams/drug effects , Hypnotics and Sedatives/administration & dosage , Midazolam/administration & dosage , Propofol/administration & dosage , Adult , Anesthesia, Spinal/adverse effects , Chi-Square Distribution , Deep Sedation/adverse effects , Female , Humans , Hypnotics and Sedatives/adverse effects , Infusions, Intravenous , Male , Midazolam/adverse effects , Middle Aged , Odds Ratio , Patient Satisfaction , Patient Selection , Propofol/adverse effects , Prospective Studies , Republic of Korea , Surveys and Questionnaires , Time Factors
8.
Endocr J ; 57(10): 863-71, 2010.
Article in English | MEDLINE | ID: mdl-20724799

ABSTRACT

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) reduces ovulation rate in rats. The present study was to investigate whether TCDD alters the progression of cell cycle, and thus resulting in the blockade of ovulation in gonadotropin-primed, immature rats. The ovulation rate and ovarian weight were reduced in intact rats given TCDD (32 µg/kg BW in corn oil) by gavage one day before pregnant mare's serum gonadotropin (PMSG; 5 IU/rat) injection. Flow cytometry demonstrated that the percentage of granulosa cells in S-phase was increased at 24 h following PMSG treatment, but declined at 8 h following hCG treatment in corn oil-treated rats. Interestingly, the number of S-phase cells in TCDD-treated rats was reduced 24 and 48 h following PMSG treatment. TCDD, however, increased the percentage of cells in G2/M-phase at 24 h following PMSG treatment. TCDD inhibited the mRNA levels of Cdk2 at 0 h and 24 h, and cyclin D2 at 24 h and 48 h following PMSG treatment. Protein levels of aryl hydrocarbon receptor in granulosa cells were elevated in TCDD-treated rats at 12 h and 24 h following PMSG treatment. The present study indicates that TCDD reduces S-phase cells and inhibits levels of Cdk2 and cyclin D2 at 24 h following PMSG treatment, implying the ovulation-inhibiting action of TCDD may be exerted through the attenuation of cell cycle progression via AhR-mediated cascade.


Subject(s)
Cell Cycle/drug effects , Endocrine Disruptors/pharmacology , Granulosa Cells/drug effects , Growth Inhibitors/pharmacology , Ovulation Inhibition/drug effects , Polychlorinated Dibenzodioxins/pharmacology , Animals , Cyclin D2/genetics , Cyclin D2/metabolism , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Female , Gene Expression Regulation/drug effects , Granulosa Cells/metabolism , Organ Size/drug effects , Ovary/drug effects , Ovary/growth & development , Ovulation Induction , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Aryl Hydrocarbon/metabolism , Reproductive Control Agents/pharmacology , Time Factors
9.
Cell Biol Int ; 33(9): 957-63, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19524685

ABSTRACT

Phellodendri Cortex (PC) is a traditional herbal medicine, widely used in Korea and China. The effects of the methanol extract of Phellodendri Cortex (PC extract) on 1-methyl-4-phenylpyridinium (MPP(+))-induced neuronal apoptosis in PC-12 cells have been investigated. MPP(+)-induced apoptosis in PC-12 cells was accompanied by an increased bax/bcl-2 ratio, release of cytochrome c to the cytosol and activation of caspase-3. PC extract inhibited the downregulation of bcl-2 and the upregulation of bax, as well as the release of mitochondrial cytochrome c into the cytosol. In addition, PC extract attenuated caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP). These results suggest that the PC extract has protective effects against MPP(+)-induced neuronal apoptosis in PC-12 cells.


Subject(s)
Apoptosis/drug effects , Neurons/drug effects , Neuroprotective Agents/pharmacology , Phellodendron/chemistry , Poly(ADP-ribose) Polymerases/metabolism , 1-Methyl-4-phenylpyridinium/toxicity , Animals , Apoptosis/physiology , Caspase 3/metabolism , Caspase Inhibitors , Cell Nucleus Shape/drug effects , Cell Nucleus Shape/physiology , Cell Survival/drug effects , Cell Survival/physiology , Cytochromes c/metabolism , Methanol/chemistry , Neurons/metabolism , Neuroprotective Agents/chemistry , Plant Extracts/chemistry , Plant Extracts/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , RNA, Messenger/drug effects , RNA, Messenger/metabolism , Rats , bcl-2-Associated X Protein/agonists , bcl-2-Associated X Protein/metabolism
10.
Gene ; 361: 133-9, 2005 Nov 21.
Article in English | MEDLINE | ID: mdl-16203113

ABSTRACT

DJ-1 is linked to an early-onset autosomal recessive Parkinson's disease (PD) characterized primarily by selective loss of dopaminergic (DA) neurons, which results in motor disturbances. However, our understanding on how mutations in DJ-1 are related to PD is unclear. Here, we isolated the DJ-1 orthologue, DJ-1beta, in Drosophila and characterized its expression and loss-of-function mutants. We observed its strongest expression in the adult stage of development and ubiquitous expression in the larval brain. Our homozygous mutants showed severe defects in locomotor ability without loss of DA neurons, consistent with the previous mice DJ-1 mutant studies ([Goldberg, M.S., Pisani, A., Haburcak, M., Vortherms, T.A., Kitada, T., Costa, C., Tong, Y., Martella, G., Tscherter, A., Martins, A., et al., 2005. Nigrostriatal dopaminergic deficits and hypokinesia caused by inactivation of the familial Parkinsonism-linked gene DJ-1. Neuron 45, 489-496.]; [Kim, R.H., Smith, P.D., Aleyasin, H., Hayley, S., Mount, M.P., Pownall, S., Wakeham, A., You-Ten, A.J., Kalia, S.K., Horne, P., Westaway, D., Lozano, A.M., Anisman, H., Park, D.S., Mak, T.W., 2005. Hypersensitivity of DJ-1-deficient mice to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and oxidative stress. Proc. Natl. Acad. Sci. USA 102, 5215-5220.]; [Chen, L., Cagniard, B., Mathews, T., Jones, S., Koh, H.C., Ding, Y., Carvey, P.M., Ling, Z., Kang, U.J., Zhuang, X., 2005. Age-dependent motor deficits and dopaminergic dysfunction in DJ-1 null mice. J. Biol. Chem. 280, 21418-21426.]). The locomotor activity of DJ-1beta mutants was further decreased by paraquat-induced oxidative stress. Moreover, we found that Drosophila DJ-1 is prominently localized in mitochondria, suggesting that DJ-1 functions as a protector against oxidative stress in mitochondria.


Subject(s)
Drosophila Proteins/genetics , Drosophila/physiology , Gait Disorders, Neurologic/physiopathology , Mutation/genetics , Nerve Tissue Proteins/genetics , Oxidative Stress/physiology , Amino Acid Sequence , Animals , Behavior, Animal/physiology , Blotting, Northern , Blotting, Western , Brain/metabolism , Brain/pathology , COS Cells , Chlorocebus aethiops , Disease Models, Animal , Dopamine/metabolism , Drosophila/genetics , Drosophila Proteins/metabolism , Gait Disorders, Neurologic/genetics , Gene Expression Profiling , Humans , Microscopy, Confocal , Mitochondria/metabolism , Mitochondria/pathology , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Neurons/pathology , Parkinson Disease/physiopathology , Protein Deglycase DJ-1 , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Transfection
11.
Mol Biotechnol ; 27(1): 1-6, 2004 May.
Article in English | MEDLINE | ID: mdl-15122042

ABSTRACT

Identifying cellular substrates repertoire of individual proteases will facilitate our understanding of their physiological and pathological roles. In this article, we employed a yeast-based screening method to isolate CED-3 substrates. This method uses a transcription factor anchored to the plasma membrane by fusion to a library of cellular protein sequences. When a fusion protein is cleaved by CED-3, the transcription factor is released from the plasma membrane and enters the nucleus where it turns on the expression of reporter genes. We identified seven candidate clones by screening a genomic library using this method. Of these seven clones, two were cleaved by purified CED-3 in vitro. Therefore, the method described here may be generally used for genomewide screening to isolate potential substrates of specific proteases.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caspases/metabolism , Cysteine Endopeptidases/metabolism , Molecular Biology/methods , Yeasts/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Caenorhabditis elegans Proteins/analysis , Caenorhabditis elegans Proteins/isolation & purification , Caspases/analysis , Caspases/isolation & purification , Cysteine Endopeptidases/analysis , Cysteine Endopeptidases/isolation & purification , Gene Library , Receptors, Mating Factor , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Substrate Specificity , Transcription Factors/genetics , Transcription Factors/metabolism , Yeasts/metabolism
12.
Anal Biochem ; 323(1): 7-11, 2003 Dec 01.
Article in English | MEDLINE | ID: mdl-14622952

ABSTRACT

The cerebral deposition of amyloid beta-peptide (Abeta) is a major factor in the etiology of Alzheimer's disease. beta-Secretase (BACE) initiates the generation of Abeta by cleaving the amyloid precursor protein at the beta-site and is therefore a prime target for therapeutic intervention. Here we report a cell-based method suitable for monitoring BACE activity and the efficacy of protease inhibitors. A fusion protein containing the amino-terminal transmembrane domain of Golgi alpha-mannosidase II, a Drosophila Golgi integral membrane protein, linked to human alkaline phosphatase (AP) by a short beta-site sequence, was expressed in Drosophila S2 cells. While the uncleaved fusion protein was retained in the Golgi apparatus, cleavage of the beta-site by BACE resulted in the release of AP to the culture medium, where it was easily detected and quantified. Three peptidomimetic inhibitors (LB83190, LB83192, LB83202) were tested for their efficacy with this cell-based assay. While LB83190 and LB83192 effectively blocked BACE activity, LB83202, a carboxylated derivative of LB83192, did not. This is consistent with the inability of LB83202 to permeate the cell membrane. The present cell-based assay could provide a convenient tool for high-throughput screening of substances that can interfere with BACE in living cells.


Subject(s)
Drug Monitoring/methods , Endopeptidases/analysis , Protease Inhibitors/pharmacology , Alkaline Phosphatase/analysis , Alzheimer Disease/drug therapy , Amyloid Precursor Protein Secretases , Animals , Aspartic Acid Endopeptidases , Cell Line , Drosophila , Endopeptidases/drug effects , Endopeptidases/metabolism , Humans , Mannosidases , Recombinant Fusion Proteins/metabolism , Transfection
13.
Biochem Biophys Res Commun ; 296(2): 419-24, 2002 Aug 16.
Article in English | MEDLINE | ID: mdl-12163035

ABSTRACT

We report here a genetic assay suitable for detecting site-specific proteolysis in secretory pathways. The yeast enzyme invertase is linked to the truncated lumenal region of the yeast Golgi membrane protein STE13 via a protease substrate domain in a Saccharomyces cerevisiae strain lacking invertase. When the substrate is cleaved by a specific protease, the invertase moiety is released into the periplasmic space where it degrades sucrose to glucose and fructose. Therefore, site-specific proteolysis can be detected by monitoring the growth of yeast cells on selective media containing sucrose as the sole carbon source. We confirmed the validity of this assay with yeast Kex2 and human TMPRSS2 proteases. Our data suggest that this in vivo assay is an efficient method for the determination of substrate specificity and mutational analysis of secreted or membrane proteases.


Subject(s)
Biological Assay/methods , Glycoside Hydrolases/metabolism , Proprotein Convertases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/metabolism , Genetic Vectors , Glycoside Hydrolases/genetics , Humans , Mutation , Reproducibility of Results , Saccharomyces cerevisiae/genetics , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Substrate Specificity , Subtilisins/genetics , Subtilisins/metabolism , beta-Fructofuranosidase
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