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1.
James H. Eley 《Applied microbiology and biotechnology》1988,28(3):297-300
Summary Of a number of possible buffers only HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) supported continued and maximum growth of photoautotrophically grown Anacystis nidulans when grown in medium C and aerated with air (0.03% CO2). Cultures aerated with 1% CO2 in air had adequate buffering without the use of additional buffers in medium C. With either air or 1% CO2 grown cells, phosphate and TRICINE (N-tris-hydroxymethyl-methylglycine) buffers gave growth rates of 67% compared to HEPES buffer while complete inhibition of growth occurred with TRIS (tris-hydroxymethylaminomethane) buffer. 相似文献
2.
Euglena were cultured under 3 W m-2 constant white light. In culture medium, cells show immediate and long lasting step-down photophobic responses and photoaccumulation behavior to blue light if dim red light-adapted for 30 min. However, if cells are suspended in buffered, saltcontaining solutions (adaptation buffers), strong step-down photobehavior and photoaccumulation responses are not observed for several hours. These behaviors gradually increase in strength to reach a maximum after 6–12 h; after which a stable response is maintained. The relative rates of appearance and the relative strengths of the responses are influenced by the concentrations of Ca2+ and K+, but not H+ or Na+ ions, in the adaptation buffers. Expression of the stepdown photobehavior thus requires that the cells adapt to the chemical environment in which they are suspended.Abbreviations Hepes
N-2-hydroxypiperazine-N-2-ethanesulfonic acid
- Mes
2(N-morpholino)-ethanesulfonic acid
- Pipes
piperazine-N,N-bis (2-ethanesulfonic acid)
- Taps
tris(hydroxymethyl) methylaminopropanesulfonic acid
This work was supported, in part, by grant No. PCM-79-05320 from the U.S. National Science Foundation to B.D. 相似文献
3.
C. Anthony Poole Helen C. Reilly Michael H. Flint 《In vitro cellular & developmental biology. Plant》1982,18(9):755-765
Summary Chick embryo epiphyseal chondrocytes cultured in media containing HEPES, TES, and BES zwitterion buffers, used in combination
or independently, consistently developed cytoplasmic vacuoles. This cytoplasmic vacuolation was resolved when the zwitterion
buffered media was replaced by media containing bicarbonate:CO2 enriched air buffer. Vacuoles were infrequent or absent in cultures grown in bicarbonate:CO2 enriched air. Chondrocytes with an established extracellular matrix showed less vacuolation than fibroblastlike and polygonal
shaped cells that lacked such a matrix. The granular endoplasmic reticulum and Golgi dictyosomes of zwitterion buffered chondrocytes
were distended and contained a flocculent amorphous material. Cytoplasmic vacuoles (0.5 to 3.0 μm diam) formed by the fusion
and intracellular accumulation of Golgi vesicles and vacuoles also contained a flocculent material enhanced by ruthenium red.
Membrane bound extracellular vacuoles containing ruthenium red stained proteoglycan aggregates were common in the extracellular
matrix of zwitterion buffered cultures but were generally absent from bicarbonate treated cultures. Electron dense calcium
deposits seemed much larger and more numerous in the presence of zwitterion buffers.
It is suggested that HEPES, TES, and BES buffers, used alone or in combination, may adversely affect cell membrane systems,
and thus the transport or secretory mechanisms operative in cultured chondrocytes, or both, resulting in vacuole formation
and the intracellular accumulation of synthesized export material. Although the mechanism by which HEPES, TES, and BES induce
these changes remains unclear, the use of zwitterion buffers in biological preparations should be treated with caution.
This work forms part of a project on Connective Tissue Remodelling supported and financed by the Medical Research Council
of New Zealand, of which M. H. F. is a Career Fellow. 相似文献
4.
H. R. Massie H. V. Samis M. B. Baird 《In vitro cellular & developmental biology. Plant》1972,7(4):191-194
Summary N-2-Hydroxyethylpiperazine-N′-2-ethane sulfonic acid (HEPES) and N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)
have been found to be nontoxic substitutes for bicarbonate buffer in cell culture media. WI-38 embryonic human lung cells
have been carried beyond the 60th passage with both HEPES and TES buffers. 相似文献
5.
Elsa J. B. Murray Samuel S. Murray Khai K. -T. Tram David B. N. Lee 《Experimental cell research》1994,215(2)
Parathyroid hormone (PTH) and PTH-related peptide (PTH-rP) bind to a common receptor and initiate second-messenger cascades that stimulate bone turnover and hypercalcemia. However, PTH is more potent than PTH-rP in inducing bone resorption and coupled bone metabolism in intact tissue, suggesting that these proteins elicit dissimilar postreceptor responses. We compared the effects of PTH and PTH-rP on osteoblastic retraction, an early event that must occur before the osteoclast can achieve access to the underlying bone mineral and begin resorption. MC3T3-E1 mouse osteoblasts were incubated in vehicle or 4.8 nM PTH or PTH-rP with or without 1 mM dibutyryl cAMP (Bt2cAMP). Morphologic changes were observed from 0 to 120 min. PTH caused marked retraction within minutes, which was not enhanced by Bt2cAMP. PTH-rP or Bt2cAMP induced slower, more modest retraction than PTH. The combined effect of PTH-rP plus Bt2cAMP was greater than that of PTH-rP, but less than that of PTH. PTH-rP and PTH had similar effects on cAMP generation. Thus, compared to PTH, PTH-rP induces less osteoblastic retractile response, exposing less bone surface to osteoclastic resorption. This may account for its lower hypercalcemic potency in vivo and contribute to its relative inability to stimulate coupled bone resorption and formation. 相似文献
6.
Brian Colman 《Journal of phycology》1978,14(4):434-437
The claim that Chlorella sp. (CCAP 211/8p), sometimes referred to as C. fusca, Shihira and Krauss, does not excrete glycolate has been reexamined. Chlorella sp. grown on 5% CO2in air, excreted glycolate when incubated in light in 10 mM bicarbonate. Excretion ceased 30–60 min after transfer of the cells to air and no excretion could be detected with air-grown cells or with cells grown on 5% CO2in media buffered at pH 8.0. Incubation with 10 mM isonicotinyl hydrazide, a glycolate pathway inhibitor, caused excretion in air-grown cells and stimulated excretion in CO2-grown cells indicating that both the rate of glycolate synthesis and metabolism is higher in CO2grown cells than in air-grown cells. Enhanced glycolate synthesis and excretion in CO2-grown cells is correlated with law photosynthetic rate in 10 mM bicarbonate, and the photosynthetic rate of these cells doubles over a period of 2–2.5 h after initial transfer from high CO2to bicarbonate. This correlation of photosynthetic induction with cessation of glycolate excretion is similar to that reported in a bluegreen alga and thought to occur in other green algae. These results indicate that glycolate excretion and its regulation in this species of Chlorella is not different from that in other algae. 相似文献
7.
R J Bull 《Life sciences》1979,24(4):323-335
The importance of HCO3?/CO2 to the maintenance of stable metabolic conditions in rat cerebral cortex slices has been investigated. Replacement of bicarbonate buffered media with glycylglycine or phosphate buffered salines resulted in an increased lability of the cytochrome redox potential of brain slices as measured by dual wavelength spectroscopy. Depletion of reduced cytochrome in the electron transport chain was associated with changes in the metabolic responses of tissues to electrical stimulation or elevated concentrations of potassium. This appears primarily as a loss of the late reductive responses of the tissue nicotinamide adenine dinucleotides NAD(P)H to these stimuli and decreased lactic acid output of the tissues. The effects could be largely reversed in a combined glycylglycine and HCO3?/CO2 buffered media. It is suggested that although the reduction of NAD(P) in response to stimulation may be substantially located in the cytosol, it is also modulated by the mitochondrial redox potential. It is suggested that the lability of the redox potential in the absence of HCO3?/CO2 may be related to depletion of TCA cycle intermediates normally replaced by CO2 fixation. 相似文献
8.
Most animal cell culture media can be buffered using bicarbonate and high pressure CO2 in a closed system. However, in an open system, the pH of the culture media increases continuously due to the marked difference
in CO2 pressure between the culture media and the atmosphere. Therefore, it is important to measure the exact pH of the culture
media in an intact closed system. In this study, a pH measurement method was developed using visible light. The pH was calculated
from light absorbance by the cells and by the culture media. This method was successfully applied to both suspension and anchorage-dependent
cell cultures. 相似文献
9.
Background
Induction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates.Methodology/Principal Findings
We tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro.Conclusions/Significance
We conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions. 相似文献10.
Leaves of the C3 plants Brassica oleracea L., Datura suaveolens Humb. & Bonpl. ex Willd., Helianthus annuus L. and Nicotiana tabacum L. with open stomata were exposed in a leaf chamber in the dark to CO2 concentrations varying from 1 to 20% in air. When they were transferred back into CO2-free air, CO2 was rapidly released. It originated from dissolved CO2 and from the bicarbonate in the chloroplast stroma, since vacuoles are acidic and chloroplasts contain carbonic anhydrase which rapidly liberates CO2 from bicarbonate. The data were fitted to a model which accounts for the CO2/bicarbonate equilibrium in buffers with different CO2 concentrations and initial pH values. From this, pH values and CO2-dependent pH changes in the chloroplast stroma were calculated. The full range of external CO2 concentration caused acidic shifts up to 1 pH unit. The best fits of the data points were obtained with stromal buffer concentrations ranging from 45 to 65 mM and stromal pH values at low CO2 between 7.5 and 7.9. Calculated buffer capacities ranged from 23 to 31 mM H+ per pH unit. The work shows that measurements of solubilized CO2 are useful to investigate proton buffering and pH regulation in the chloroplast stroma of intact leaves.Abbreviation Chl
chlorophyll
This work was supported by Sonderforschungsbereich 251 of the University of Würzburg and the Volkswagenstiftung grant I-67762. We are very grateful to Dr. V. Oja for helpful advice. 相似文献
11.
Five hydrogen ion buffers were compared for their usefulness in regulating pH in a model oligotrophic, moderately acidic (pH 6.0) algal growth medium. These were 3,3-dimethylglutaric acid (DMGA), tricarbaliylic acid (TCA), trans-aconitic acid (tAA), N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) and 2-(N-morpholino) ethanesulfonic acid (MES). All buffers (2.5 mM) except HEPES limited the reduction of pH in a NH4+-based medium during growth of Chrysochromulina breviturrita Nich. to less than 0.12 units, compared with more than 2 units in an unbuffered medium. Long term growth of C. breviturrita in these media was significantly inhibited (P < 0.05) by TCA and tAA. MES was able to control pH with the minimum amount of NaOH (1.0 mM) added to the medium to adjust to pH 6.0. Four of five bacterial isolates were capable of utilizing tAA as a sole organic-C source, and no isolate could metabolize HEPES or MES. No significant differences (P > 0.05) were found in the maximum growth rates of six algal species (from five classes) in a medium with or without MES buffer, although significantly greater cell yields of Ochromonas danica Prings. were obtained in the buffered medium. MES (pK4=6.15) was considered to be the most useful buffer in the pH range 5.0–6.5, due to its biological inertness, buffering capacity, the minimal requirement for excess base to adjust pH and its minimal metal complexing ability. 相似文献
12.
Recent work showed that chloroplast thylakoid membranes stored in 100 mM KCl-containing media have delocalized energy coupling consistent with a rapid equilibration of the proton gradient between the proton-producing redox steps and the lumen bulk phase (Beard and Dilley 1986). Thylakoids stored in low salt media showed localized energy coupling. A related thylakoid membrane property is the occurrence of sequestered, metastable, acidic domains, associated with pK
a
7.5 amine groups. For low salt-stored membranes the domain protons appear to be in the direct (localized) diffusion pathway of protons involved in energizing ATP formation, whereas in thylakoids stored in high KCl, domain protons equilibrated with the lumen during the development of the ATP energization threshold (Theg et al. 1988). This work tested whether the 100 mM KCl storage treatment did or did not cause the dissipation of the metastable acidic domain protons in the dark, storage period. By three criteria, it was found that the 100 mM KCl storage treatment had only a slight tendency to dissipate the acidic domain protons into alkaline media under dark conditions. Storage in KCl does not cause the dissipation of the acidic domains in the dark, but allows domain protons to equilibrate with the lumen after the redox system begins turning over, but before the ATP energization threshold pH is reached. These results must be considered in models of how the thylakoid structure can accommodate metastable acidic domains and how such domain protons diffuse to the CF0-CF1 complexes in energy coupling.Abbreviations PSII
photosystem 2
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- MES
2(N-morpholine) ethanesulfonic acid
- Taps
N-tris (hydroxymethyl)-methyl-3-amino-propanesulfonic acid
- EPPS
N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid
- gram D
gramicidin D
This research supported in part by grants from the USDA and NSF. 相似文献
13.
Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin 总被引:6,自引:0,他引:6
Ihara H Denhardt DT Furuya K Yamashita T Muguruma Y Tsuji K Hruska KA Higashio K Enomoto S Nifuji A Rittling SR Noda M 《The Journal of biological chemistry》2001,276(16):13065-13071
Osteopontin is an RGDS-containing protein that acts as a ligand for the alpha(v)beta(3) integrin, which is abundantly expressed in osteoclasts, cells responsible for bone resorption in osteopenic diseases such as osteoporosis and hyperparathyroidism. However, the role of osteopontin in the process of bone resorption has not yet been fully understood. Therefore, we investigated the direct function of osteopontin in bone resorption using an organ culture system. The amount of (45)Ca released from the osteopontin-deficient bones was not significantly different from the basal release from wild type bones. However, in contrast to the parathyroid hormone (PTH) enhancement of the (45)Ca release from wild type bones, PTH had no effect on (45)Ca release from organ cultures of osteopontin-deficient bones. Because PTH is located upstream of receptor activator of NF-kappaB ligand (RANKL), that directly promotes bone resorption, we also examined the effect of RANKL. Soluble RANKL with macrophage-colony stimulating factor enhanced (45)Ca release from the bones of wild type fetal mice but not from the bones of osteopontin-deficient mice. To obtain insight into the cellular mechanism underlying the phenomena observed in osteopontin-deficient bone, we investigated the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the bones subjected to PTH treatment in cultures. The number of TRAP-positive cells was increased significantly by PTH in wild type bone; however, no such PTH-induced increase in TRAP-positive cells was observed in osteopontin-deficient bones. These results indicate that the absence of osteopontin suppressed PTH-induced increase in bone resorption via preventing the increase in the number of osteoclasts in the local milieu of bone. 相似文献
14.
Günter A. Peschek 《Archives of microbiology》1979,123(1):81-92
The effect of growth conditions on aerobic and anaerobic hydrogenase activities of Anacystis nidulans was studied. It was found that the two hydrogenase activities both of which were confined to the particulate fraction of cell-free extracts correlated in an opposite way with growth temperature: The algae were always grown photoautotrophically in presence of H2 but after growth at 25° C a significant oxyhydrogen reaction contrasted with negligible photoreduction rates while the opposite was true after growth at 40°C. A similar correlation between incubation temperature and induction of the respective hydrogenase activity was also observed with resting cells.Kinetic analysis of the two different types of hydrogenase — catalysed reactions with Anacystis membranes yielded the following Michaelis-Mentenparameters: K
M=55 M H2 and v
max=0.12 mol H2 per min and mg protein for the oxyhydrogen reaction, and K
M=170 M H2 and v
max=0.3 mol H2 per min and mg protein for the photoreductions. Also the dependences of oxyhydrogen and of photoreduction activities on pH and on temperature were measured; both pH and temperature profiles were found to be markedly different for each type of H2-supported reaction.The results are discussed as pointing to the possible occurrence of two functionally distinct hydrogenase enzymes which can be synthesized by Anacystis in response to the conditions of induction.Abbreviations BO
p-benzoquinone
- CAP
chloramphenicol
- chl
chlorophyll
- cytc
horse heart cytochrome c
- DCMU
3-(34-dichlorophenyl)-1,1-dimethylurea
- DCPIP
2,6-dichlorophenolindophenol
- fd
ferredoxin
- FeCy
ferricyanide
- MB
methylene blue
- MV
methyl viologen
- HEPES
N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid
- MES
2-(N-morpholino)-ethanesulfonic acid
- PIPES
piperazine-N,N-bis-(2-ethanesulfonic acid)
- tricine
N-tris-(hydroxymethyl)-methylglycine
- Tris
tris-(hydroxymethyl)-aminomethan 相似文献
15.
Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO2 fluxes in cell suspensions using mass spectrometry. Addition of H13CO3
− to cell suspensions in the dark caused a transient increase in the CO2 concentration in the medium far in excess of the equilibrium CO2 concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once
equilibrium between the Ci species had been achieved, a CO2 efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected
nor does this species demonstrate a capacity to take up CO2 by active transport. Photosynthetic O2 evolution and the release CO2 in the dark depend on HCO3
− uptake since both were inhibited by the anion exchange inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS).
The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA
inhibitor, markedly inhibited CO2 efflux in the dark, indicating that CO2 efflux was dependent upon the intracellular dehydration of HCO3
−. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which
maintains the equilibrium between CO2 and HCO3
− and thus causes a subsequent release of CO2 to the external medium.
Received: 20 September 1999 / Accepted: 25 October 1999 相似文献
16.
The addition of physiological concentrations of zinc (25-200 (Μg/dL) to Dulbecco’s Modified Eagle’s Medium containing tibiae
from 19-d chick embryos resulted in a concentration-dependent increase in tibial content of tartrate-resistant acid phosphatase
(TRAP) and an increase in bone resorption, as measured by tibial calcium release. This increase in bone resorption was additive
to the resorptive effect resulting from the addition of 10-9-10-7
M parathyroid hormone (PTH), but was not additive to similar effects produced by the addition of 10-9-10-7
M prostaglandin E2 (PGE2). An inhibitor of prostaglandin synthesis, flurbiprofen (10-6
M), did not influence the effect of zinc on bone resorption. However, the addition of 2,6-pyridinedicarboxylic acid (10-3
M, 2,6-PDCA), a chelator of zinc, did attenuate the effects of zinc, as did the addition of an inhibitor of DNA replication
(hydroxyurea, 10-3
M). Hydroxyurea also attenuated the bone resorptive response to PGE2, but had no influence on the effects of PTH.
These results indicate that physiological concentrations of zinc alter bone resorptive rates in vitro by a mechanism that
is dependent on DNA replication. 相似文献
17.
18.
The enzyme acetyl-CoA: 17-O-deacetylvindoline 17-O-acetyltransferase which terminates vindoline biosynthesis has been isolated from Catharanthus roseus leaves, further characterized and purified to homogeneity by three step column chromatography and subsequent preparative isoelectric focusing. Kinetic properties concerning the enzyme reaction are discussed. Five multiple forms of the acetyl-transferase could be observed, each consisting of two subunits. This enzyme is now the best characterized of the enzymes involved in vindoline biosynthesis.Abbreviations DTE
dithiothreitol
- EDTA
ethylenediamine-tetraacetic acid
- HEPES
N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid
- IEF
isoelectric focusing
- KPi
potassium phosphate
- Mr
rel.molecular mass
- PEG
polyethylene glycol
- SDS-PAGE
sodium dodecylsulfate polyacrylamide gel electrophoresis
- Tris
2-amino-2-(hydroxymethyl)-1,3-propandiol 相似文献
19.
Lawrence G. Raisz M.D. Jack Y. Vanderhoek Ph.D. Hollis A. Simmons Barbara E. Kream Ph.D. K.C. Nicolaou Ph.D. 《Prostaglandins & other lipid mediators》1979,17(6):905-914
Prostaglandin synthesis by fetal rat bones was examined by thin-layer chromatography of culture media after preincubation with labeled arachidonic acid. Cultures in rabbit complement (non-heat inactivated serum) were compared with cultures in heat-inactivated serum or cultures treated with indomethacin. The major complement-dependent products were PGE2, PGF2α and 6-keto-PGF1α, the metabolite of prostacyclin (PGI2). Since PGI2 had not been previously identified in bone its ability to stimulate bone resorption was tested. Repeated addition of PGI2 stimulated release of previously incorporated 45Ca from fetal rat long bones in both short-term and long-term cultures at concentrations of 10−5 to 10−9M. Because of the short half life of PGI2 in solution at neutral pH, we tested a sulfur analog, thiaprostacyclin (S-PGI2) which was found to be a stimulator of bone resorption at concentrations of 10−5 to 10−6M. These studies suggest that endogenous PGI2 production may play a role in bone metabolism. Since vessels produce PGI2 it is possible that PGI2 release may be responsible for the frequent association between vascular invasion and resorption of bone or calcified cartilage in physiologic remodeling and pathologic osteolysis. 相似文献
20.
Graham A. Couche Mary Ann Pfannenstiel Kenneth W. Nickerson 《Applied microbiology and biotechnology》1986,24(2):128-133
Summary
Bacillus thuringiensis var.israelensis protein toxin radiolabeled with14C-iodoacetamide was bound to latex beads. The efficiency of attachment was not affected by the temperature or length of incubation or by the presence of several different buffers, salts, or organic solvents. However, attachment decreased dramatically at pH values >8.5 or in the presence of detergents (anionic, neutral, or cationic). Protein concentrations 2–3 mg/m2 of bead surface led to a progressively decreasing efficiency of protein adsorption. With minor variations, these findings should also be applicable to the attachment of numerous other proteins of diagnostic significance.Abbreviations BSA
bovine serum albumin
- Bti
Bacillus thuringiensis var.israelensis
- EDTA
ethylenediaminetetraacetic acid
- HEPES
N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid
- LAT
latex agglutination test
- PAGE
polyacrylamide gel electrophoresis
- SDS
sodium dodecylsulfate
- TCA
trichloroacetic acid
- Tris
Tris(hydroxymethyl)aminomethane
- TTAB
tetradecyltrimethylammonium bromide 相似文献