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1.
L. Zavalova S. Lukyanov I. Baskova E. Snezhkov S. Akopov S. Berezhnoy E. Bogdanova E. Barsova E. D. Sverdlov 《Molecular & general genetics : MGG》1996,253(1-2):20-25
We previously detected in salivary gland secretions of the medicinal leech (Hirudo medicinalis) a novel enzymatic activity, endo-ɛ(γ-Glu)-Lys isopeptidase, which cleaves isopeptide bonds formed by transglutaminase (Factor
XIIIa) between glutamine γ-carboxamide and the ɛ-amino group of lysine. Such isopeptide bonds, either within or between protein
polypeptide chains are formed in many biological processes. However, before we started our work no enzymes were known to be
capable of specifically splitting isopeptide bonds in proteins. The isopeptidase activity we detected was specific for isopeptide
bonds. The enzyme was termed destabilase. Here we report the first purification of destabilase, part of its amino acid sequence,
isolation and sequencing of two related cDNAs derived from the gene family that encodes destabilase proteins, and the detection
of isopeptidase activity encoded by one of these cDNAs cloned in a baculovirus expression vector. The deduced mature protein
products of these cDNAs contain 115 and 116 amino acid residues, including 14 highly conserved Cys residues, and are formed
from precursors containing specific leader peptides. No homologous sequences were found in public databases.
Received: 9 April 1996 / Accepted: 17 May 1996 相似文献
2.
Zhong Qiao Shao Shihe Mu Runhong Wang Hua Huang Shiteng Han Jun Huang He Tian Shuwei 《Molecular biology reports》2011,38(1):503-509
The Cag Type IV secretion apparatus proteins in Helicobacter pylori can mediate the injection of effector CagA protein into eukaryotic target cells. Although this apparatus forms an important
pathway for bacterium–host interaction, its assembly process in vivo is poorly understood, and the proteins which contribute
to break the bacterial cell walls in Cag-PAI have not yet been identified. The cagγ gene in Cag-PAI is a unique member that contains a conserved SLT catalysis domain, which makes it an attracting question
whether cagy gene has the capacity to digest the bacterial cell wall. In the current study, therefore, the cagγ gene was cloned from the H. pylori NCTC 11637 and expressed in Escherichia coli, and its lytic effect on cell walls in vitro was observed. Results indicated that Cagγ protein has a lytic activity against
bacterial cell walls. An allelic-exchange mutant (Δcagγ) was further constructed to investigate the relationship between Cagγ
and effector CagA translocation. These results suggested that Cagγ contributed to the assembly of Cag Type IV secretion apparatus
by digesting the peptidoglycan meshwork of bacterial cell walls. 相似文献
3.
4.
Brüsehaber E Böttcher D Musidlowska-Persson A Albrecht D Hecker M Doderer K Bornscheuer UT 《Applied microbiology and biotechnology》2007,76(4):853-859
Pig liver esterase (PLE) is probably the most important carboxyl esterase in organic synthesis and is commercially obtained
by extraction of the animal tissue. However, problems occur in its application due to the presence of several isoenzymes (α-,
β- and γ-PLE). The functional expression of the γ-isoenzyme was already shown and differences in the enantioselectivity compared
to the commercial preparations were confirmed. The amino acid and nucleotide sequences of the α- and β-PLE are still unknown.
In this work, putative sequences of the α-isoenzyme were identified from a commercial PLE preparation by 2D gel electrophoresis,
digestion with proteases and analysis using Matrix-assisted laser desorption/ionization–time of flight (TOF) and electrospray
ionisation quadrupole–TOF mass spectrometry. Based on these results, three amino acid exchanges were introduced into the gene
encoding γ-rPLE by site-directed mutagenesis, and the proteins were expressed in E. coli Origami (DE3). The produced PLE mutants were characterised with respect to their substrate specificity and enantioselectivity.
No significant differences in the activity towards methyl butyrate were found, but several variants showed substantially enhanced
enantioselectivity in the resolution of (R,S)-1-phenyl-2-butyl acetate with E = 100 for the best mutant V236P/A237G. 相似文献
5.
Michikatsu Sato Machiko Watanabe Hiroto Nagano Yoshiaki Yagi 《Biotechnology letters》1994,16(7):703-708
A simple and specific recovery method for α-cyclodextrin (α-CD) was developed by employing co-digestion of CD reaction mixtures
with CGTase fromBacillus ohbensis and α-glucosidase. The combination of CGTase fromB. ohbensis and α-glucosidase, such as α-amylase, β-amylase, or glucoamylase was examined for the selective degradation of β-and γ-CD
in the CD reaction mixture formed by CGTase fromB. macerans. The co-digestion of the CD mixture with Taka-amylase and the CGTase resulted in α-CD and maltodextrins, the combination
with β-amylase resulted in α-CD and maltose, and that with glucoamylase resulted in α-CD and glucose. The conditions of selective
degradation of β- and γ-CD by co-digestion with the CGTase and glucoamylase were optimized as follows: the incubation pH,
5.5; incubation temperature, 50°C; CGTase concentration, 15 u/g of substrate; glucoamylase, 10 u/g of substrate; substrate
concentration, 10% (w/v); the incubation time was fixed for 18 hr from the stand point of operation convenience.
Most part of the content was presented in poster session at the 7th International Cyclodextrin Symposium, Tokyo, April 1994. 相似文献
6.
Peroxisome proliferator-activated receptor gamma (PPARγ) activation by its ligands reportedly inhibits monocyte function.
However, because the concentrations of PPARγ ligands used in previous studies were higher than typically expected to activate
PPARγ, we clarified whether PPARγ ligands influence monocyte function and cell viability of the human monocyte cell line THP-1.
We determined tumor necrosis factor-alpha (TNF-α) release as a monocyte function and cell viability using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide. Both troglitazone and 15-deoxy-Δ12,14-prostaglandin J2 (15-d-PGJ2) seemed to inhibit phorbol ester-induced TNF-α release from THP-1 cells. On the other hand, neither pioglitazone
nor rosiglitazone inhibited phorbol ester-induced TNF-α release. Because the cytotoxicity of troglitazone and 15-d-PGJ2 was
significantly (p<0.05, Tukey–Kramer) stronger than that of pioglitazone and rosiglitazone, the inhibition of TNF-α release seemed to parallel
the lack of cell viability. We concluded that PPARγ ligands did not directly inhibit TNF-α release in THP-1 cells.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
7.
N-acetylglucosaminyltransferase (GnT)-IV catalyzes the formation of the GlcNAcβ1-4 branch on the GlcNAcβ1-2Manα1-3 arm of the
core structure of N-glycans. Two human GnT-IV isozymes (GnT-IVa and GnT-IVb) had been identified, which exhibit different expression profiles
among human tissues and cancer cell lines. To clarify the enzymatic properties of the respective enzymes, their kinetic parameters
were determined using recombinant full-length enzymes expressed in COS7 cells. The K
m of human GnT-IVb for UDP-GlcNAc was estimated to be 0.24 mM, which is 2-fold higher than that of human GnT-IVa. The K
m values of GnT-IVb for pyridylaminated (PA) acceptor sugar chains with different branch numbers were 3- to 6-fold higher than
those of GnT-IVa. To compare substrate specificities more precisely, we generated recombinant soluble enzymes of human GnT-IVa
and GnT-IVb with N-terminal flag tags. Both enzymes showed similar substrate specificities as determined using fourteen PA-sugar
chains. They preferred complex-type N-glycans over hybrid-types. Among the complex-type N-glycans tested, the relative activities of both enzymes were increased in proportion to the number of GlcNAc branches on
the Man α1-6 arm. The Man α1-6 arm of the acceptors was not essential for their activities because a linear pentasaccharide
lacking this arm, GlcNAcβ1-2Manα1-3Manβ1-4GlcNAcβ1-4 GlcNAc-PA, was a substrate for both enzymes. These results indicate that
human GnT-IVb exhibits the same acceptor substrate specificities as human GnT-IVa, although GnT-IVb has lower affinities for
donors or acceptors than GnT-IVa. This suggests that GnT-IVa is more active than GnT-IVb under physiological conditions and
that it primarily contributes to the biosynthesis of N-glycans. 相似文献
8.
Alesutan I Sopjani M Munoz C Fraser S Kemp BE Föller M Lang F 《The Journal of membrane biology》2011,240(3):151-158
Connexins provide intercellular connections that allow passage of ions and small organic molecules. They clamp the cell membrane
potential and cellular ion composition to that of neighboring cells. The cell membrane potential and ion composition of an
energy-depleted cell could thus be maintained despite its compromised Na+/K+ activity. By the same token, however, the breakdown of ion gradients in that cell imposes an additional challenge to the
neighboring cells, which may jeopardize their survival. Thus, timely closure of connexins may be critically important for
the survival of those cells. Energy depletion stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase
that senses energy depletion and stimulates several cellular mechanisms to enhance energy production and to limit energy utilization.
The present study explored whether AMPK regulates connexin 26. To this end, cRNA encoding connexin 26 was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (α1β1γ1), of the constitutively active γR70QAMPK (α1β1γ1[R70Q]) or of the inactive mutant αK45RAMPK (α1[K45R]β1γ1). Connexin 26 activity was determined in dual-electrode voltage-clamp experiments. Moreover, connexin 26
abundance was determined in the oocyte cell membrane by chemiluminescence and confocal microscopy. As a result, connexin 26-mediated
current and connexin 26 protein abundance were significantly decreased by coexpression of γR70QAMPK and, to a lower extent, by wild-type AMPK but not by αK45RAMPK. In conclusion, AMPK is a potent regulator of connexin 26. 相似文献
9.
Seonghun Kim Doo-Byoung Oh Ohsuk Kwon Hyun Ah Kang 《Applied microbiology and biotechnology》2010,88(4):893-903
Sialidases can be used to transfer sialic acids from sialoglycans to asialoglycoconjugates via the trans-glycosylation reaction
mechanism. Some pathogenic bacteria decorate their surfaces with sialic acids which were often scavenged from host sialoglycoconjugates
using their surface-localized enzymes. In this study, we constructed an in vitro trans-sialylation system by reconstructing
the exogenous sialoglycoconjugate synthesis system of pathogens on the surfaces of yeast cells. The nanH gene encoding an extracellular sialidase of Corynebacterium diphtheriae was cloned into the yeast surface display vector pYD1 based on the Aga1p–Aga2p platform to immobilize the enzyme on the surface
of the yeast Saccharomyces cerevisiae. The surface-displayed recombinant NanH protein was expressed as a fully active sialidase and also transferred sialic acids
from pNP-α-sialoside, a sialic acid donor substrate, to human-type asialo-N-glycans. Moreover, this system was capable of attaching sialic acids to the glycans of asialofetuin via α(2,3)- or α(2,6)-linkage.
The cell surface-expressed C. diphtheriae sialidase showed its potential as a useful whole cell biocatalyst for the transfer of sialic acid as well as the hydrolysis
of N-glycans containing α(2,3)- and α(2,6)-linked sialic acids for glycoprotein remodeling. 相似文献
10.
A simple structured model for biomass and extracellular enzyme production with recombinant Saccharomyces cerevisiae YPB-G 总被引:1,自引:0,他引:1
A simple structured model is proposed for simulating batch cultivation data on growth, substrate utilization, and heterologous
enzyme production of recombinant Saccharomyces cerevisiae YPB-G. The enzyme is a fusion protein displaying α-amylase and glucoamylase activities. Cell growth is modulated mainly by
intracellular substrate and ethanol concentrations. Intracellular substrate concentration is evaluated by means of the extracellular
substrate and biomass concentrations. Extracellular α-amylase and glucoamylase activities are taken to depend on biomass concentration.
The nine parameters of the proposed model are determined using nonlinear estimation techniques, and the model is validated
against experiments not used in parameter determination. The model developed simulates glucose consumption, cell mass, α-amylase
and glucoamylase production in a batch system. Simulation and experimental results are found to be in good agreement. Journal of Industrial Microbiology & Biotechnology (2002) 29, 111–116 doi:10.1038/sj.jim.7000281
Received 07 January 2002/ Accepted in revised form 22 May 2002 相似文献
11.
Efficient production of recombinant barley α-amylase has been achieved in Aspergillus niger. The cDNA encoding α-amylase isozyme 1 (AMY1) and its signal peptide was placed under the control of the Aspergillus nidulans glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and the A. nidulans trpC gene terminator. Secretion yields up to 60 mg/l were obtained in media optimised for α-amylase activity and low protease
activity. The recombinant AMY1 (reAMY1) was purified to homogeneity and found to be identical to native barley AMY1 with respect
to size, pI, and immunoreactivity. N-terminal sequence analysis of the recombinant protein indicated that the endogenous plant signal
peptide is correctly processed in A. niger. Electrospray ionisation/mass spectrometry gave a molecular mass for the dominant form of 44 960 Da, in accordance with the
loss of the LQRS C-terminal residues; glycosylation apparently did not occur. The activities of recombinant and native barley
α-amylases are very similar towards insoluble and soluble starch as well as 2-chloro-4-nitrophenol β-d-maltoheptaoside and amylose (degree of polymerisation = 17). Barley α-amylase is the first plant protein efficiently secreted
and correctly processed by A. niger using its own signal sequence.
Received: 22 August 1997 / Received revision: 21 November 1997 / Accepted: 29 November 1997 相似文献
12.
PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes 总被引:2,自引:0,他引:2
The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility
complex (MHC) class I molecules. Recent evidence indicates that an interferon-γ (IFN-γ)-inducible PA28 activator complex enhances
the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined
the primary structures of the mouse PA28 α- and β-subunits. The deduced amino acid sequences of the α- and β-subunits were
49% identical. We also determined the primary structure of the mouse PA28 γ-subunit (Ki antigen), a protein of unknown function
structurally related to the α- and β-subunits. The amino acid sequence identity of the γ-subunit to the α- and β-subunits
was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the α- and β-subunits
(designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-γ-inducible subunits of PA28 are encoded outside
the MHC. The gene coding for the γ-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a γ-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-γ-inducible α- and β-subunits emerged by gene duplication
from a γ-subunit-like precursor.
Received: 11 March 1997 相似文献
13.
Miki Watanabe Sulaiman Sheriff Theresa A. Ramelot Nijiati Kadeer Junho Cho Kenneth B. Lewis Ambikaipakan Balasubramaniam Michael A. Kennedy 《International journal of peptide research and therapeutics》2011,17(4):281-299
After severe burn injury, proinflammatory cytokine levels are elevated in serum and skeletal muscle, which in turn increases
protein breakdown and decreases protein synthesis. In this study, C2C12 mouse skeletal muscle cell line myotubes were exposed
to proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) as an in vitro cell-line model of catabolic
response to burn injury and then treated with des-acyl ghrelin (DAG), a 28 amino acid polypeptide hormone thought to inhibit
protein breakdown and increase protein synthesis, to assess its therapeutic potential. Nuclear magnetic resonance-based metabonomics
was used to monitor metabolic activity of C2C12 myotubes under four treatment conditions: (1) control, (2) TNF-α/IFN-γ (TI),
(3) DAG (DA), and (4) TNF-α/IFN-γ followed by DAG (TIDA) to assess the effect of DAG treatment on cellular metabolic response
during basal or catabolic conditions. Twelve metabolites showed significant changes in concentrations following treatments
in the hydrophilic cell extracts. Lactate (P < 10−4) and citrulline (P < 10−9) increased with TNF-α/IFN-γ treatment, indicating increased protein degradation, and returned to control levels in the TIDA
group. Adenosine nucleotide levels had decreased trends in TI myotubes that returned to baseline levels after DAG treatment
(P < 10−4). Guanidinoacetate and pantothenate, metabolites involved in protein synthesis and cell proliferation, had increased concentration
trends following DAG treatment in both the DA and TIDA groups. Our metabonomics analysis provides further evidence that DAG
counteracts the catabolic response caused by elevated muscle TNF-α/IFN-γ cytokine levels following severe burns and can play
a potential therapeutic role in treatment of burn injury. 相似文献
14.
Masłyk M Kochanowicz E Zieliński R Kubiński K Hellman U Szyszka R 《Molecular and cellular biochemistry》2008,312(1-2):61-69
Since Svf1 is phosphoprotein, we investigated whether it was a substrate for protein kinase CK2. According to the amino acid
sequence Svf1 harbours 20 putative CK2 phosphorylation sites. Here, we have reported cloning, overexpression, purification
and characterization of yeast Svf1 as a substrate for three forms of yeast CK2. Svf1 serves as a substrate for both the recombinant
CK2α (K
m 0.35 μM) and CK2α′ (K
m 0.18 μM) as well as CK2 holoenzyme (K
m 1.1 μM). Different K
m values argue that CK2β(β′) subunit has an inhibitory effect on the activity of both CK2α and CK2α′ towards surviving factor
Svf1. Reconstitution of α′2ββ′ isoform of CK2 holoenzyme shows that β/β′ subunits have regulatory effect depending on the kind of CK2 catalytic subunit.
This effect was not observed in the case of α2ββ′ isoform, which may be due to interaction between Svf1 and regulatory CK2β subunit (shown by co-immunoprecipitation experiments).
Interactions between CK2 subunits and Svf1 protein may have influence on ATP as well as ATP-competitive inhibitors (TBBt and
TBBz) binding. CK2 phosphorylates up to six serine residues in highly acidic peptide K199EVIPESDEEESSADEDDNEDEDEESGDSEEESGSEEESDSEEVEITYED248 of the Svf1 protein in vitro. Presented data may help to elucidate the role of protein kinase CK2 and Svf1 in the regulation
of cell survival pathways. 相似文献
15.
K. Vihko Marjo Seppänen Tiina Henttinen Juha Punnonen Seija Grénman Reijo Punnonen 《Cancer immunology, immunotherapy : CII》1997,43(6):368-374
The biology and pathogenesis of vulvar carcinoma are poorly understood at present. In order to understand this disease better,
we have used recently developed squamous cell carcinoma lines of the vulva as models. Two cell lines originating from two
individuals (UM-SCV-1A and UM-SCV-6) were cultured in vitro in 10% fetal calf serum. The effects of interleukins 10 and 13,
interferons α and γ, granulocyte/macrophage-growth-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), and transforming
growth factor β (TGFβ) on the proliferation of the cells was investigated by using radioactively labelled uridine as tracer.
In addition, an investigation on the molecular structure of extracted cellular DNA was carried out to investigate whether
programmed cell death (apoptosis) would be inducible by any of the factors. In UM-SCV-1A cells, interleukin-10 (IL-10) and
interleukin-13 (IL-13) caused an approximately 12-fold decrease in DNA synthesis in cells cultured for 72 h (P<0.001), while GM-CSF had no significant effect. TGFβ showed a significant inhibitory effect on deoxyuridine incorporation
(P<0.001), which was 2.0- and 4.2-fold at 48 h and 72 h, respectively. TFGα showed a 1.2-fold inhibitory effect on DNA synthesis
at 48 h (P<0.01) and a 1.5-fold inhibition at 72 h (P<0.05). Interferon γ (IFNγ) showed an inhibitory effect on DNA synthesis (1.3-fold; P<0.01). In UM-SCV-6 cells, both IL-10 and IL-13 showed inhibitory effects on deoxyuridine incorporation (1.3- and 1.4-fold
at 48 h, respectively; P<0.001) that were even more pronounced at 72 h (2.4- and 2.5-fold respectively; P<0.001). IFNγ caused a 3.6-fold inhibition of DNA synthesis by UM-SCV-6 cells at 72 h (P<0.001). Both TFGβ and TNFα inhibited uridine incorporation (3.0- and 1.6-fold at 48 h, respectively; 2.7-fold at 72 h for
both factors). GM-CSF inihibited DNA synthesis by UM-SCV-6 cells 1.3- 2.0-fold at 48 h and 72 h, respectively. In dose/response
analyses, the effect of INFα on DNA synthesis was inhibitory in both cell lines at 48 h, while stimulatory effects were observed
at 72 h. Electrophoretic analyses of DNA isolated from cells cultured in the presence or absence of different factors did
not reveal DNA fragmentation. All cytokines, with the exception of IFNα, showed inhibitory effects on DNA synthesis by vulvar
carcinoma cells. Of the factors studied, the recently described interleukins 10 and 13 showed potent inhibition of cell growth,
encouraging further investigation on the molecular mechanisms of the observed inhibition. Apoptosis does not seem to be induced
in the two vulvar carcinoma cell lines by any of the cytokines studied.
Received: 26 March 1996 / Accepted: 5 December 1996 相似文献
16.
17.
Kyoung Ah Kang In Kyung Lee Rui Zhang Mei Jing Piao Ki Cheon Kim Sang Young Kim Taekyun Shin Bum Joon Kim Nam Ho Lee Jin Won Hyun 《Cell biology and toxicology》2011,27(2):83-94
The radioprotective effect of geraniin, a tannin compound isolated from Nymphaea tetragona Georgi var. (Nymphaeaceae), against γ-radiation-induced damage was investigated in Chinese hamster lung fibroblast (V79-4)
cells. Geraniin recovered cell viability detected by MTT test and colony formation assay, which was compromised by γ-radiation,
and reduced the γ-radiation-induced apoptosis by the inhibition of loss of the mitochondrial membrane potential. Geraniin
protected cellular components (lipid membrane, cellular protein, and DNA) damaged by γ-radiation, which was detected by lipid
peroxidation, protein carbonyl formation, and comet assay. Geraniin significantly reduced the level of intracellular reactive
oxygen species generated by γ-radiation, which was detected using spectrofluorometer, flow cytometer, and confocal microscope
after 2′,7′-dichlorodihydrofluorescein diacetate staining. Geraniin normalized the superoxide dismutase and catalase activities,
which were decreased by γ-radiation. These results suggest that geraniin protects cells against radiation-induced oxidative
stress via enhancing of antioxidant enzyme activities and attenuating of cellular damage. 相似文献
18.
M. Loui Thomas Rajan A. Badwe Ramakant K. Deshpande Urmila C. Samant Shubhada V. Chiplunkar 《Cancer immunology, immunotherapy : CII》2001,50(4):218-225
The mechanism responsible for tissue specific localization of γδ T cell subsets is not well understood. In order to explain
the sequestration of specific γδ T cell subsets in the peripheral blood and tumor tissue of patients with esophageal cancer,
we examined the function and expression of adhesion molecules on these cells. A hierarchy in the expression of adhesion molecules
was observed. In vitro activated γδ T cells showed dominant expression of LFA-1 (CD11a), VLA-α4 (CD49d), intermediate expression
of VLA-α5 (CD49e) and L-selectin (CD62L), but low expression of CD44v6 and αEβ7 (CD103). It was observed that the γδ T cells use LFA-1, L-selectin and CD44v6 to bind to squamous cell carcinoma (SCC) cells,
whereas they adhere to fibroblast cells using LFA-1, VLA-α4 and VLA-α5. Vδ1 T cell subsets from the peripheral blood γδ T
cells utilize a larger array of adhesion molecules, namely LFA-1, VLA-α4, VLA-α5, L-selectin and αEβ7, to bind to SCC cells compared to the restricted usage of LFA-1, L-selectin and CD44v6 by the Vδ2 T cells. Flow cytometric
analysis of tumor infiltrating lymphocytes from the esophageal tumors confirmed the selective accumulation of Vδ1+γδ T cells in the tumor compartment. It thus appears that adhesion molecules expressed on these lymphocytes play an important
role in the recruitment and retention of Vδ1 T cells in the tumor milieu.
Received: 27 November 2000 / Accepted: 1 March 2001 相似文献
19.
Katarzyna Grzelkowska-Kowalczyk Wioletta Wieteska-Skrzeczyńska 《Cellular & molecular biology letters》2010,15(1):13-31
The aim of this study was to compare the effects of TNF-α, IL-1β and IFN-γ on the activation of protein kinase B (PKB), p70S6k, mitogen-activated protein kinase (MAPK) and p90
rsk
, and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose
uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-α abolished this effect. Glucose uptake in cells differentiated in the
presence of 10 ng/ml IFN-γ increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I.
IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-α nor IFN-γ influenced basal protein synthesis, but both
cytokines prevented the IGF-I effect. 10 ng/ml IL-1β did not modify either the basal or IGF-I-dependent glucose uptake and
protein synthesis. With the exception of TNF-α causing an 18% decrease in the level of PKB protein, the cellular levels of
PKB, p70S6k, p42MAPK, p44MAPK and p90
rsk
were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal
value after 40 min of IGF-I treatment), p42MAPK (a 2.81-fold increase after 50 min), and the activation of p70S6k and p90
rsk
, manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-α or IFN-γ, this IGF-I-mediated
PKB and p70S6k phosphorylation was significantly diminished, and the increase in p42MAPK and p90
rsk
phosphorylation was prevented. The basal p42MAPK phosphorylation in C2C12 cells treated with IFN-γ was high and comparable with the activation of this kinase by IGF-I. Pretreatment
of myogenic cells with IL-1β did not modify the IGF-I-stimulated phosphorylation of PKB, p70S6k, p42MAPK and p90
rsk
. In conclusion: i) TNF-α and IFN-γ, but not IL-1β, if present in the extracellular environment during C2C12 myoblast differentiation,
prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-α- and IFN-γ-induced IGF-I resistance of protein synthesis
could be associated with the decreased phosphorylation of PKB and p70S6k. iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-γ is PKB independent. iv) The similar effects
of TNF-α and IFN-γ on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement
of both of these cytokines in protein loss in skeletal muscle. 相似文献
20.
Marvin S. Peterson Myoung-Dong Kim Ki-Cheol Han Ji-Hyun Kim Jin-Ho Seo 《Biotechnology and Bioprocess Engineering》2002,7(1):52-55
Flow cytometric techniques were used to investigate cell size, protein content and cell cycle behavior of recombinantSaccharomyces cerevisiae strains producing human lysozyme (HLZ). Two different signal sequences, the native yeastMFα1 signal sequence and the rat α-amylase signal sequence, were used for secretion of HLZ. The strain containing the rat α-amylase
signal sequence showed a higher level of internal lysozyme and lower specific growth rates. Flow cytometric analysis of the
total protein content and cell size showed the strain harboring the native yeast signal sequence had a higher total protein
content than the strain containing the rat α-amylase signal sequence. Cell cycle analysis indicated that the two lysozyme
producing recombinant strains had an increased number of cells in the G2+M phase of the yeast cell cycle compared with the host strain SEY2102. 相似文献