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1.
Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age)
have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained
in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics
(penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml;
fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO2: 95% air.
The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning
and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and
interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria,
rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive
staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.
This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute. 相似文献
2.
Summary Serial passage cultures of colonic epithelial cells from young rats have been maintained for more than 6 months in Eagle's
minimum essential medium buffered with HEPES (25 mM) and supplemented with 2.5% fetal bovine serum, 0.5 μg/ml insulin, 5.0 μg/ml transferrin, and antibiotics. The cells proliferated
in this medium with a population doubling time of approximately 53 h. The cells retained differentiated morphology as evidenced
by secretory activity and the presence of secretory granules, microvilli, tonofilaments, and desmosomal junctions. Further
cells at the fourth passage had normal karyotypes with 42 chromosomes and exhibited anchorage dependent growth. High concentrations
of fetal bovine serum (10 to 15%) exerted toxic effects on the colonic epithelial cell cultures.
Supported by National Cancer Institute Contract N01-CP-75914. 相似文献
3.
An electrophysiological freeze fracture assessment of cadmium nephrotoxicity in vitro 总被引:1,自引:0,他引:1
Debra J. Hazen-Martin Donald A. Sens John G. Blackburn Mary C. Flath Mary Ann Sens 《In vitro cellular & developmental biology. Plant》1989,25(9):791-799
Summary Human proximal tubule cell cultures exposed to doses of cadmium chloride (CdCl2) between 0.05 μg/ml and 0.5 μg/ml exhibited alterations in cell membrane structure and transport function. At these Cd concentrations,
cell numbers were not significantly altered from control values in either nonreplicating confluent, or actively replicating
subconfluent cultures. Transmission electron microscopy revealed few alterations in cultures treated with 0.05 μg/ml Cd. Tight
junctions were intact; organelles and myeloid body formation appeared normal. Freeze fracture analysis confirmed the integrity
of the tight junctions as well as increased numbers of vesicles or pits along the lateral cell membrane, indicating increased
endocytotic activity. Cells exposed to 0.1 μg/ml Cd were characterized by decreased numbers of microvilli and inhibited myeloid
body formation. Cd doses of 0.5 μg/ml elicited nuclear chromatin condensation, fragmented sealing strands in 5 to 10% of the
tight junction profiles, sparse microvilli, and inhibited myeloid body formation. Electrophysiologic assessments of transport
function by Ussing chamber analysis revealed decreases in transepithelial potentials for all three concentrations, with significant
differences at Cd concentrations of 0.5 to 0.1 μg/ml. Cells treated with 0.5 μg/ml Cd also exhibited slight decreases in electrical
resistance, consistent with the minimal fragmentation of sealing strands observed in freeze fracture replicas. Resistance
in cultures treated with 0.1 or 0.05 μg/ml Cd remained within control values and indicated that drops in potential difference
and short circuit current in these cells reflected true alterations in ion transport.
This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society
of Toxicology (SOT) and the Tissue Culture Association field at the 27th annual meeting of the SOT in Dallas, Texas in 1988.
This work was supported by the Johns Hopkins Center for Alternatives to Animal Testing. The Balzers Freeze Fracture Unit utilized
in these studies was provided by equipment grant S10 RR02329 from the National Institutes of Health, Bethesda, MD. 相似文献
4.
Pan H Feng J Cerniglia CE Chen H 《Journal of industrial microbiology & biotechnology》2011,38(10):1729-1738
Azo dyes are widely used in the plastic, paper, cosmetics, food, and pharmaceutical industries. Some metabolites of these
dyes are potentially genotoxic. The toxic effects of azo dyes and their potential reduction metabolites on Staphylococcus aureus ATCC BAA 1556 were studied. When the cultures were incubated with 6, 18, and 36 μg/ml of Orange II and Sudan III for 48 h,
76.3, 68.5, and 61.7% of Orange II and 97.8, 93.9, and 75.8% of Sudan III were reduced by the bacterium, respectively. In
the presence of 36 μg/ml Sudan III, the cell viability of the bacterium decreased to 61.9% after 48 h of incubation, whereas
the cell viability of the control culture without the dye was 71.5%. Moreover, the optical density of the bacterial cultures
at 10 h decreased from 0.74 to 0.55, indicating that Sudan III is able to inhibit growth of the bacterium. However, Orange
II had no significant effects on either cell growth or cell viability of the bacterium at the tested concentrations. 1-Amino-2-naphthol,
a metabolite common to Orange II and Sudan III, was capable of inhibiting cell growth of the bacterium at 1 μg/ml and completely
stopped bacterial cell growth at 24–48 μg/ml. On the other hand, the other metabolites of Orange II and Sudan III, namely
sulfanilic acid, p-phenylenediamine, and aniline, showed no significant effects on cell growth. p-Phenylenediamine exhibited a synergistic effect with 1-amino-2-naphthol on cell growth inhibition. All of the dye metabolites
had no significant effects on cell viability of the bacterium. 相似文献
5.
Yasuhiro Tomooka Stephen E. Harris John A. McLachlan 《In vitro cellular & developmental biology. Plant》1985,21(4):237-244
Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained
in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically,
immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number
was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10
ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required
eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin,
and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free
media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides
a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells. 相似文献
6.
Hélio V. Nobre-Júnior Ricardo A. Oliveira Flavio D. Maia Marcelle A. S. Nogueira Manoel Odorico de Moraes Mary Anne M. Bandeira Geanne M. Andrade Glauce S. B. Viana 《Neurochemical research》2009,34(6):1066-1075
In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal
plant, Myracrodruon
urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells.
In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml)
reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an
increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly
decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic
increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented
by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented
an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from
apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for
tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of
TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated
neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our
findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as
Parkinson’s disease. 相似文献
7.
Wakae Fujimaki Janet R. Griffin Eugenie S. Kleinerman 《Cancer immunology, immunotherapy : CII》1993,36(1):45-51
The purpose of this study was to determine the effects of ibuprofen on the ability of liposome-encapsulated muramyl tripeptide phosphatidylethanolamine (L-MTP-PE) to activate human blood monocytes in vitro. We undertook these experiments because the major toxic side-effects following L-MTP-PE infusion, fever and chills, could be prevented when ibuprofen was given orally immediately before L-MTP-PE infusion. It was therefore important to determine whether ibuprofen interfered with the macrophage-activation properties of L-MTP-PE. Peripheral blood monocytes were isolated from normal donors, then incubated with L-MTP-PE in the presence or absence of ibuprofen. The cytotoxic properties of the monocytes were assessed by a radioisotope-release assay against A375 cells. Ibuprofen at dose levels of 40 µg/ml suppressed the generation of the cytotoxic phenotype but did not interfere with the killing process once the cells were activated. Interleukin-1 (IL-1) and tumor necrosis factor (TNF) production, as well as the mRNA expression of these cytokines, was suppressed by 40 µg/ml ibuprofen. Since IL-1 and TNF play a crucial role in the cytotoxic function of monocytes, these findings may explain the mechanism by which ibuprofen inhibited the generation of the cytotoxic phenotype by L-MTP-PE. By contrast, ibuprofen dose levels up to 10 µg/ml had no effect on the generation of monocyte-mediated cytotoxicity by L-MTP-PE and no effect on the production, secretion, or mRNA expression of TNF and IL-1. Therefore, we concluded that if ibuprofen is to be used to control the side-effects of L-MTP-PE, blood levels of up to 10 µg/ml are desirable. In two of three patients, we determined that an oral dose of 200 mg given immediately before L-MTP-PE infusion could achieve these desired blood levels. 相似文献
8.
Ten species of Aspergillus isolated from soil samples collected from different locations in the Indian Himalayan region have been studied for their
growth requirements and tricalcium phosphate solubilization at different temperatures. The Aspergillus species could grow at low temperature and tolerated a wide range of pH. Phosphate solubilization by various Aspergillus species ranged between 374 μg/ml (A. candidus) to 1394 μg/ml (A. niger) at 28°C, 33 μg/ml (A. fumigatus) to 2354 μg/ml (A. niger) at 21°C, 93 μg/ml (A. fumigatus) to 1452 μg/ml (A. niger) at 14°C, and 21 μg/ml (A. wentii) to 83 μg/ml (A. niger) at 9°C. At 21 and 28°C, phosphate solubilization showed a decrease within 4 weeks of incubation, whereas at 9°C and 14°C,
it continued further up to 6 weeks of incubation. In general, phosphate solubilization by different Aspergillus species was recorded at a maximum of 28°C or 21°C; biomass production was favored at 21°C or 14°C. Conversely, A. nidulans and A. sydowii exhibited maximum phosphate solubilization at 14°C and produced maximum biomass at 21°C. Data suggest that suboptimal conditions
(higher or lower temperature) for fungal growth and biomass production were optimal for the production of metabolites involved
in phosphate solubilization. Significant negative correlations were obtained between pH and phosphate solubilization for eight
species at 28°C, for seven at 21°C, and for nine at 14°C. Extracellular phosphatase activity was exhibited only in case of
A. niger, whreas intracellular phosphatase activity was detected in all species, the maximum being in A. niger. Statistically significant positive or negative correlations were obtained between phosphate solubilization and other parameters
in most cases at different temperatures. 相似文献
9.
Paul M. Mbugua A. A. Welder D. Acosta 《In vitro cellular & developmental biology. Plant》1988,24(8):743-752
Summary Primary cultures of spontaneously beating myocardial cells isolated from neonatal rat hearts were used to screen the cardiotoxic
effects of Jamesoni's mamba (Dendroaspis jamesoni) venom and components isolated from the venom by gel filtration and ion exchange chromatography. Cardiotoxicity was evaluated
on the basis of leakage of lactate dehydrogenase (LDH), changes in morphology, cell membrane lysis, cellular viability, and
alterations in spontaneous beating activity. The whole venom caused dose- and time-dependent leakage of LDH, disruption of
the cell monolayer, decreases in viability, and inhibition of beating activity. Gel filtration of the venom yielded eight
fractions (DjI to DjVIII). DjI (30 μg/ml), DjII (20 μg/ml), and DjV (20 μg/ml) caused significant (P<0.001) leakage of LDH, extensive morphologic damage, and decreases in viability. At lower concentrations DjI to DjVIII caused
progressive inhibition of spontaneous beating activity. The main fraction (DjV), which was the most toxic, was further separated
into 14 polypeptides (Dj1 to Dj14) by ion-exchange chromatography using Bio-Rex 70. Based on the ability to induce LDH leakage,
produce morphologic damage, lyse cell membranes, and arrest beating activity, four categories of polypeptides were identified:
cardiotoxins, Dj1 and Dj2; cardiotoxinlike polypeptides, Dj3 to Dj8; less active membrane lytic polypeptides, Dj9 to Dj13;
and membrane lytic polypeptide, Dj14.
This study was supported in part by the Fulbright Scholar Program (1986–1987) and the Burroughs Wellcome Fund. D. A. is a
Burroughs Wellcome Scholar in Toxicology. 相似文献
10.
The purpose of the present study was to investigate whether didanosine (ddI) directly causes morphological and ultrastructural
abnormalities of dorsal root ganglion (DRG) neurons in vitro. Dissociated DRG cells and organotypic DRG explants from embryonic 15-day-old Wistar rats were cultured for 3 days and then
exposed to ddI (1 μg/ml, 5 μg/ml, 10 μg/ml, and 20 μg/ml) for another 3 days and 6 days, respectively. Neurons cultured continuously
in medium served as normal controls. The diameter of the neuronal cell body and neurite length were measured in dissociated
DRG cell cultures. Neuronal ultrastructural changes were observed in both culture models. ddI induced dose-dependent decreases
in neurite number, length of the longest neurite in each neuron, and total neurite length per neuron in dissociated DRG cell
cultures with 3 days treatment. There were no morphological changes seen in organotypic DRG cultures even with longer exposure
time (6 days). But ddI induced ultrastructural changes in both culture models. Ultrastructural abnormalities included loss
of cristae in mitochondria, clustering of microtubules and neurofilaments, accumulation of glycogen-like granules, and emergence
of large dense particles between neurites or microtubules. Lysosome-like large particles emerged inconstantly in neurites.
ddI induced a neurite retraction or neurite loss in a dose-dependent manner in dissociated DRG neurons, suggesting that ddI
may partially contribute to developing peripheral neuropathy. Cytoskeletal rearrangement and ultrastructural abnormalities
caused by ddI in both culture models may have a key role in neurite degeneration. 相似文献
11.
Application of sodium-dikegulac reduced plant height with associated increase in branch and leaf number and root biomass inC. roseus (L.) G. DON. Chlorophyll content reduced significantly after first month of 100 and 250 μg/ml DK application. However, such
reduction was replaced by significant rise after forth month in 250 μg/ml DK application and fifth month in 100 μg/ml DK application
followed by appreciable decline only in 250 μg/ml DK treatment but 100 μg/ml DK maintained higher level till harvest. Total
sugar content was significantly high during forth and fifth month stage of growth after DK application. Amino acid content
was higher during third to fifth month in 100 μg/ml DK treatment and during third to forth month in 250 μg/ml DK treatment.
Tryptophan, on the other hand showed higher content at the fifth month stage of growth after application of DK in both the
concentrations. Leaf and root dry weight as well as total alkaloid content were highest in 100 μg/ml DK application. DK, therefore,
appears to be a potential chemical for increasing biomass and alkaloid content inC. roseus. 相似文献
12.
The inhibitory effect of silibinin on ochratoxin A (OTA)-mediated apoptosis on primary rat hepatocytes was investigated. Rat
hepatocytes were prepared by two different methods: the classical enzymatic digestion method by collagenase perfusion and
a new EDTA-perfusion method. The EDTA-perfusion method yielded hepatocytes, which were stably cultivated without DNA fragmentation
for up to 96 h, whereas the collagenase-prepared hepatocytes showed apoptosis events as early as from the start of preparation
even in the absence of OTA. Treatment with 12.5 μmol/l OTA of cultured hepatocytes prepared under ETDA perfusion developed
DNA-laddering after 24–36 h. Lipopolysaccharide (LPS) of 0.1 up to 12.5 μg/ml showed no apoptotic DNA-effects under these
conditions. A low concentration of 26 μmol/l silibinin given prior to OTA slightly prevented OTA-mediated DNA-laddering, whereas
a five times higher concentration of silibinin (130 μmol/l) completely inhibited OTA-mediated apoptosis. Under the same conditions,
caspase-3 activity in hepatocytes increased in a time-dependent manner under OTA exposure within 12–24 h but was blocked by
130 μmol/l silibinin. In contrast, LPS incubation for 12 and 24 h did not alter caspase-3 activity. To measure viability of
OTA-/LPS-treated hepatocytes, the MTT-test and Live/Dead kit were applied. The results demonstrated that the used OTA concentration
of 12.5 μmol/l only moderately decreased viability for up to 24 h but showed cytotoxic effects depending on longer incubation
times (≥36 h). In contrast, LPS up to 12.5 μg/ml exhibited no cytotoxic effects up to 48 h. In summary, our results showed
contrasting effects on apoptosis in primary rat hepatocytes by OTA (produces apoptosis) versus LPS (produces no apoptosis),
also depending on the method of hepatocyte preparation. Silibinin at 130 μmol/l showed significant hepatoprotective and antiapoptotic
effects against OTA-mediated cell damage on cultured rat hepatocytes. 相似文献
13.
David Kirk Susumu Kagawa Gudrun Vener K. Shankar Narayan Y. Ohnuki Lawrence W. Jones 《In vitro cellular & developmental biology. Plant》1985,21(3):165-171
Summary A serum-free medium (HMRI-2) has been developed for the outgrowth and subculture of epithelial cells from normal adult human
ureter and bladder. Medium HMRI-2 consists of Ham’s MCDB 152 with double the amounts of the essential amino acids in Stock
1, low Ca2+ (0.06 mM) and is supplemented with epithelial growth factor, 5 ng/ml; transferrin, 5 μg/ml; insulin, 5 μg/ml; ethanolamine and phosphoethanolamine,
0.1 mM each; hydrocortisone, 2.8×10−6
M; and bovine pituitary extract, 126 μg protein/ml. The cultured cells showed ultrastructural markers of epithelial cells (prekeratin
fibers, tonofilaments, surface microvilli with glycocalyx), exhibited ABO antigens, and had a normal human diploid karyotype.
Primary cultures could be subcultured and also cryopreserved in HMRI-2 in liquid nitrogen. Cells in mass cultures showed a
population doubling time of 40.5±4.5 h and had a maximum in vitro life span of 20 to 25 population doublings. It was observed
that primary outgrowths, secondary cultures, and even cryopreserved cells all retained the capacity to respond to high Ca2+ and serum by differentiation and desquamation. This study has resulted in the availability of easily obtainable serum-free
epithelial cultures from normal adult human ureter and bladder. The useful in vitro life span of these cultures may be important
in future studies of carcinogenesis.
This work was supported by a grant from the National Cancer Institute (R01CA25089), Bethesda, MD. 相似文献
14.
Robert A. Harper B. Allen Flaxman 《In vitro cellular & developmental biology. Plant》1981,17(5):393-396
Summary Primary cell cultures of normal rabbit epidermal cells (keratinocytes) were established without the use of enzymatic techniques.
Six experiments were carried out on cells from six different rabbits. When these cells were exposed to methotrexate (MTX)
for 24 h at 1 μg/ml, proliferation, as measured by cells entering mitosis, was significantly inhibited (P<0.05) in only one experiment. When the dose of MTX was elevated to 100 μg/ml, only two experiments showed significant inhibition
of mitosis. This minimal inhibition of mitosis by MTX was contrasted by the dramatic inhibitory effect of this antimetabolite
on DNA synthesis. At 1 μg/ml MTX for 24 h, DNA synthesis, as measured by [3H]deoxyuridine uptake, was inhibited >95%. We can conclude that under certain conditions, the rabbit keratinocyte may represent
a normal cell type that is inherently resistant to the antiproliferative effects of methotrexate.
The research was supported by National Cancer Institute Grant CA 11536. 相似文献
15.
16.
Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine
bone marrow. The in vitro growth of colony-forming units–granulocyte/macrophage (CFU-GM), burst forming units–erythroid (BFU-E) and colony-forming
units–mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 μg/ml) suppressed all of the different
progenitor cells by 100%. A comparison of the dose–response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar
patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing
doses of VRB. The appearence of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity
was seen in cultures exposed to 0.05 and 0.075 μg/ml; and a marked loss at the dose of 0.1 μg/ml. Our results show that VRB
has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected
at a similar dose (0.025 μg/ml), suggesting that the stroma is more resistant to this drug.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
17.
In vitro activity of a new triazole antifungal agent,Sch 56592, against clinical isolates of filamentous fungi 总被引:6,自引:0,他引:6
Sch 56592 is a new triazole derivative that possesses potent, broad-spectrum antifungal activity. We evaluated the in vitroactivity
of Sch 56592 compared with that of itraconazole, amphotericin B and 5-fluorocytosine against 51 clinical isolates of filamentous
fungi, including Aspergillus flavus(10), A. fumigatus(12), Fusariumspp. (13), Rhizopus spp. (6), Pseudallescheria boydii(5),
and one isolate each of Acremoniumspp., A. niger, A. terreus, Paecilomycesspp., and Trichodermaspp. In vitrosusceptibility
testing was performed using the microdilution broth method outlined in the NCCLS 27-A document. Sch 56592 was highly active
against A. flavus(MIC90, 0.25 μg/ml), A. fumigatus(MIC90, 0.12 μg/ml), P. boydii(MIC50, 1 μ/ml) and Rhizopusspp (M1C50, 1 μg/ml). By comparison with itraconazole, Sch 56592 was four- to eight-fold more active against isolates of Aspergillusand
both compounds showed equipotent in vitroactivity against P. boydiiand Rhizopusspp. Sch 56592 was four- to 16-fold more active
than amphotericin B against Aspergillusspp. and P. boydiiand both antifungal drugs displayed similar activity against Rhizopusspp.
Overall, Sch 56592 showed good in vitroactivity against all isolates tested (MIC, ≤ 2 μg/ml) except isolates of Fusarium(MIC
range, 1–>4 μg/ml). On the basis of these data Sch 56592 has promising activity against Aspergillus spp. and other species
of filamentous fungi that are likely to be encountered clinically. Additional in vitroand in vivostudies are warranted.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
18.
Myofibrillar protein degradation in the chicken. 3-Methylhistidine release in vivo and in vitro in normal and genetically muscular-dystrophic chickens
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F. Bradley Hillgartner Anne S. Williams James A. Flanders Dexter Morin Robert J. Hansen 《The Biochemical journal》1981,196(2):591-601
Myofibrillar protein degradation was measured in 4-week-old normal (line 412) and genetically muscular-dystrophic (line 413) New Hampshire chickens by monitoring the rates of 3-methylhistidine excretion in vivo and in vitro. A method of perfusing breast and wing muscles was developed and the rate of 3-methylhistidine release in vitro was measured between 30 and 90min of perfusion. During this perfusion period, 3-methylhistidine release from the muscle preparation was linear, indicating that changes in 3-methylhistidine concentration of the perfusate were the result of myofibrillar protein degradation. Furthermore, the viability of the perfused muscle was maintained during this interval. After 60min of perfusion, ATP, ADP and creatine phosphate concentrations in pectoral muscle were similar to muscle freeze-clamped in vivo. Rates of glucose uptake and lactate production were constant during the perfusion. In dystrophic-muscle preparations, the rate of 3-methylhistidine release in vitro (nmol/h per g of dried muscle) was elevated 2-fold when compared with that in normal muscle. From these data the fractional degradation rates of myofibrillar protein in normal and dystrophic pectoral muscle were calculated to be 12 and 24% respectively. Daily 3-methylhistidine excretion (nmol/day per g body wt.) in vivo was elevated 1.35-fold in dystrophic chickens. Additional studies revealed that the anti-dystrophic drugs diphenylhydantoin and methylsergide, which improve righting ability of dystrophic chickens, did not alter 3-methylhistidine release in vitro. This result implies that changes in myofibrillar protein turnover are not the primary lesion in avian muscular dystrophy. From tissue amino acid analysis, the myofibrillar 3-methylhistidine content per g dry weight of muscle was similar in normal and dystrophic pectoral muscle. More than 96% of the 3-methylhistidine present in pectoral muscle was associated with the myofibrillar fraction. Dystrophic myofibrillar protein contained significantly less 3-methylhistidine (nmol/g of myofibrillar protein) than protein from normal muscle. This observation supports the hypothesis that there may be a block in the biochemical maturation and development of dystrophic muscle after hatching. Free 3-methylhistidine (nmol/g wet wt.) was elevated in dystrophic muscle, whereas blood 3-methylhistidine concentrations were similar in both lines. In summary, the increased myofibrillar protein catabolism demonstrated in dystrophic pectoral muscle correlates with the increased lysosomal cathepsin activity in this tissue as reported by others. 相似文献
19.
Denture stomatitis is often treated with antifungal agents but recurrences or new episodes are common, and certain episodes
can be resistant. New triazoles, such as posaconazole and voriconazole, may represent useful alternatives for management.
In vitro activities of amphotericin B, nystatin, miconazole, fluconazole, itraconazole, posaconazole and voriconazole against
150 oral Candida (101 C. albicans, 18 C. tropicalis, 12 C. glabrata, 11 C. guilliermondii, 4 C. parapsilosis, 2 Saccharomyces cerevisiae, 1 C. dubliniensis and 1 C. krusei) from 100 denture wearers were tested by the CLSI M27-A3 method. Resistant isolates were retested by Sensititre YeastOne
and Etest. Most antifungal agents were very active. However, 4 C. glabrata (33.3%), 2 C. tropicalis (11.1%), 6 C. albicans (5.6%) and 1 C. krusei were resistant to itraconazole. Posaconazole was active against 143 yeast isolates (95.3%): 6 C. albicans (5.9%) and 1 C. tropicalis (5.6%) were resistant. Geometric mean MICs were 0.036 μg/ml for C. parapsilosis, 0.062 μg/ml for C. albicans, 0.085 μg/ml for C. tropicalis, 0.387 μg/ml for C. guilliermondii and 0.498 μg/ml for C. glabrata. Voriconazole was active against 148 isolates (98.7%) with geometric mean MICs ranging from 0.030 μg/ml for C. parapsilosis, 0.042 μg/ml for C. albicans, 0.048 μg/ml for C. tropicalis, 0.082 μg/ml for C. guilliermondii, to 0.137 μg/ml for C. glabrata. Only 2 C.
albicans (2%) were resistant to voriconazole showing cross-resistance to other azoles. Posaconazole and voriconazole have excellent
in vitro activities against all Candida isolates and could represent useful alternatives for recalcitrant or recurrent candidiasis. 相似文献
20.
Masahiro Matsuda Shiro Shigeta Koichi Okutani 《Marine biotechnology (New York, N.Y.)》1999,1(1):68-73
A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral
activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration
is 1.4 μg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for
both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration
is 11.0 μg/ml; for another, 2.9 μg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated
derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity
of these products was noted against host cells at the 50% cytotoxic concentration of 100 μg/ml, except that naturally occurring
sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8–21 μg/ml.
Received May 1, 1998; accepted July 24, 1998. 相似文献