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91.
B. Delhotal F. Lemonnier M. Couturier C. Wolfrom M. Gautier A. Lemonnier 《In vitro cellular & developmental biology. Plant》1984,20(9):699-706
Summary The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver
cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5, mmol·1−1 and 27.5 mmol·I−1 glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than
fibroblasts. At Day 6, increases were observed in [3H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing
5.5, mmol·1−1 glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day
6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values
were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics
of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5
mmol·1−1. Several possible explanation for the stimulation of cell growth in fructose medium were discussed.
This work was supported by grants for the Institut National de la Santé et de la Recherche Médicale (ATP 82-79-114) and the
Unité d'Enseignement et de Recherche, Le Kremlin-Bicêtre, Université Paris-Sud (C. R. 848). 相似文献
92.
Summary Growth characteristics of human esophageal epithelial cells have been determined in primary explant and serial culture. Normal
human esophagus was obtained from donor patients in a heart/lung transplantation program; tissue obtained at autopsy (6 to
22 h after death) was not viable. When mucosal specimens (1.5 mm2) were explanted on a plastic surface and attached with a plasma clot, 35% of explants detached from the surface within 48
h. The addition of epsilon amino caproic acid (EACA) to the culture medium increased explant attachment of 93% (P<0.001). Outgrowth kinetics were similar in both the presence and absence of EACA. No advantage of human serum over nonhuman
sera was observed in primary culture. Esophageal epithelium could be frozen in 10% dimethyl sulfoxide without affecting growth
kinetics. Addition of dexamethasone (DEX) significantly altered esophageal cell morphology in primary culture and increased
viability on serial culture. Studies of pH revealed an optimum at pH 7.4 with significantly decreased growth occuring at 6.8
and no growth at 6.2.
Esophageal cells in primary explant cultures could be released by trypsin and passaged two additional times with an eightfould
increase in total number. An increased rate of attachment and multiplication was observed for cells plated on a collagen substrate
compared to platic.
The addition of EACA and DEX to the culture media and the subculture on a collagen substrate provide a method for the isolation
and serial cultivation of human esophageal cells from biopsy-sized specimens of normal esophageal epithelium.
Supported in part by Grant AM—14121 of the United States Public Health Service. A preliminary report of this work appeared
in Clin. Res. 30: 93A; 1982. 相似文献
93.
George E. Milo G. Adolph Ackerman Ronald L. Sanders 《In vitro cellular & developmental biology. Plant》1984,20(12):899-911
Summary Human lung epithelial cells have been isolated and maintained in pure culture and characterized during their time in culture.
Any residual fibroblasts were removed by selective trypsinization within the first 48 h in culture and the residual epithelial
cells from the primary culture grew to confluent density. The epithelial cells at Passage 2 or greater were serially subpassaged
when cultures reached ca. 80% confluency. This procedure permitted us to conduct biochemical and structural studies of starting
materials and subsequent population doublings.
Electron microscope evaluation of both initial monolayers and cell suspensions showed cultures to be composed of a single
cell type. These cells had microvilli on their free or apical surface. Subsequent population doubling level 1 up to 5 exhibited
the same structures. They contained lamellar inclusions, which are typical of Type II alveolar epithelial cells. Fetal lung
(age 18 to 20 wk) cell suspensions processed for electron microscopy before culturing showed cells to be undifferentiated,
epithelial-like with small microvilli along cell borders, and with desmosomes at cell junctions. Lamellar inclusions were
not observed in these cells. Ultrastructural studies of the cultured epithelial cells demonstrated that the lamellar inclusions
had a slightly positive reaction when tested for acid phosphatase. Phospholipid analysis of these lung epithelial cells showed
a phospholipid composition consistent with that found in surfactant-containing Type II cells. Cultured epithelial cells stained
with phosphine 3-R demonstrated a green fluorescent cytoplasm and nucleus with brightly fluorescent yellow-orange perinuclear
particles. The preceding characterization of these cells leads us to conclude that they exhibit structural and biochemical
features commensurate with Type II epithelial cells from human lung. Moreover, these selection techniques applied to the isolation
of human lung Type II cells from the tissue permit us to study the differentiative function of these cells routinely under
conditions of growth in vitro.
This work was supported in part by grants from EPA, R 806638-01 and 131-640-1599A1 相似文献
94.
Hybrid recombinant human leukocyte interferon inhibits differentiation in murine B-16 melanoma cells 总被引:1,自引:0,他引:1
P B Fisher H Hermo D R Prignoli I B Weinstein S Pestka 《Biochemical and biophysical research communications》1984,119(1):108-115
We have investigated the effects of recombinant human leukocyte interferons (IFN-alpha A and IFN-alpha D) and various hybrid recombinant human leukocyte interferons on differentiation in B-16 mouse melanoma cells. Inhibition of both spontaneous and melanocyte hormone stimulated differentiation was observed with one hybrid construct, IFN-alpha A/D (Bgl) consisting of amino acids 1 to 62 from IFN-alpha A and amino acids 64 to 166 from IFN-alpha D. In contrast, the parental human interferons, IFN-alpha A and IFN-alpha D, when used alone or in combination, as well as other hybrid human leukocyte interferons, did not cause significant inhibition of melanogenesis in B-16 mouse cells. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) also inhibited B-16 differentiation and the combination of TPA with IFN-alpha A/D (Bgl) or mouse L-cell interferon was synergistic in delaying melanogenesis. These studies indicate that the IFN-alpha A/D (Bgl) hybrid that exhibits antiviral activity on mouse cells can also inhibit differentiation of murine cells. 相似文献
95.
The effect of Type I interferon on bone resorption was studied by measuring its effect on parathormone-stimulated calcium release from neonatal murine calvaria in vitro. A pure human recombinant leukocyte interferon hybrid of the A and D subtypes was used, which has high antiviral activity on mouse cells. Calcium release was inhibited in a dose dependent fashion with 50% inhibition at about 10(-10) M or 600 U/ml, and the inhibition was reversible. The presence of interferon was required before or during the activation phase of the resorptive response, when the formation of osteoclasts from precursor cells would occur. When added to actively resorbing bone it had no effect. The data suggest that Type I interferon can inhibit the parathormone-regulated development of active osteoclasts, possibly by inhibiting osteoclast precursor differentiation. 相似文献
96.
Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus 总被引:2,自引:0,他引:2
Neurons producing growth hormone-releasing factor have been characterized and analyzed by immunohistochemistry in the hypothalami of human fetuses, neonates, infants and adults, using two antibodies against human pancreatic GRF (hpGRF). One of the antibodies recognized both the hpGRF(1-40)OH and hpGRF(1-44)NH2 in the mid portion (between the 28th and 39th amino acid), the other one specifically recognized the C-terminal end of hpGRF(1-44)NH2. These two antibodies stain a single neuronal system with cell bodies mainly located in the infundibular (arcuate) nucleus, and in the ventromedial and lateralis tuber nuclei. These neurons project to the median eminence where they give numerous endings in contact with portal vessels. These neurons are distinct from those containing LH-RH, somatostatin, CRF or pro-opiocortin. In fetuses, neurons immunoreactive with hpGRF antibodies are first detected at the 29th week. They display a neuroblastic aspect which persists after birth. Immunoreactive fibers are detectable in the median eminence after the 31st week. These results demonstrate that the infundibular nucleus plays a major role in control of GH secretion in man and that secretion of GRF appears late during fetal life; this suggests that the first stages of differentiation and development of GH producing cells in the human fetus do not depend on hypothalamic GRF secretion. 相似文献
97.
The kinetics of decay in absorbance at 610 nm in the reaction of cysteine with ceruloplasmin was biphasic under anaerobic conditions. Admission of oxygen to the bleached ceruloplasmin restored the blue color to about 75 % of the original value. However, under aerobic or anaerobic conditions an initial bleaching corresponded to a 25 % decrease in blue color. This change was irreversible and remained after removal of excess cysteine from the reaction mixture by dialysis. There was no correlation between transient and steady-state kinetic parameters. Circular dichroism measurements showed a characteristic reduction in the negative band at 450 nm, which is specific for type 1b copper. Isolation and further studies on cysteine-modified ceruloplasmin with a lower A610/A280 ratio showed < 10% reduction in enzyme activity toward p-phenylenediamine and o-dianisidine. Evidence is also presented that ceruloplasmin catalyzes the oxidation of cysteine with a one-electron reduction of oxygen and the formation of superoxide ion, which is then converted to H2O2 by ceruloplasmin. The effect of superoxide dismutase and catalase also confirms the presence of superoxide and H2O2. In sum, these data show that a permanent reduction of type 1b copper occurred when cysteine was used as a substrate. We conclude that there is a single electron transfer from cysteine directly to oxygen using one specific copper of ceruloplasmin, type 1b. 相似文献
98.
The dietary antagonism between copper and molybdate salts prompted a study of the inhibition of copper enzymes by thiomolybdate (TM). TM strongly inhibited the oxidase activity of five copper oxidase with I50% values in the 1-5 microM range. The mechanism of the TM effect on the copper oxidase, ceruloplasmin (Cp) (E.C. 1.16.3.1), was studied in detail. In Vmax vs. E plots, TM gave parallel data suggesting irreversibility but a large number of TM molecules per Cp were required. The inhibition of Cp by TM could not be reversed by dialysis. Isolation of TM-inhibited Cp on Sephadex G-10 did not yield any active Cp molecules. Cu(II) did not restore any inhibited oxidase activity. Gel electrophoresis supported the covalent binding of Cp by TM without any extensive change in protein structure. EPR results confirmed that Cu(II) is reduced to Cu(I) after reaction with TM. However, the Mo(VI) in MoS4(2-) did not change in oxidation number. Analysis of the TM-Cp compound accounted for all six Cu atoms as found in native Cp. The data suggest the covalent binding of sulfide to Cp copper. TM also inhibited the activity of ascorbate oxidase, cytochrome oxidase, superoxide dismutase, and tyrosinase. However, no inhibition of carbonic anhydrase, a zinc enzyme, was observed at 1 mM TM. 相似文献
99.
The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix. 相似文献
100.
Isolation and complete nucleotide sequence of the gene for bovine parathyroid hormone 总被引:7,自引:0,他引:7
The structure of the bovine parathyroid hormone (PTH) gene has been analyzed by Southern blot hybridization of genomic DNA and by nucleotide sequence analysis of a cloned PTH gene. In the Southern analysis, several restriction enzymes produced single fragments that hybridized to PTH cDNA suggesting that there is a single bovine PTH gene. The restriction map of the cloned gene is the same as that determined by Southern blot analysis of bovine DNA. The sequence of 3154 bp of the cloned gene has been determined including 510 bp and 139 bp in the 5' and 3' flanking regions, respectively. The gene contains two introns which separate three exons that code primarily for: (i) the 5' untranslated region, (ii) the pre-sequence of preProPTH, and (iii) PTH and the 3' untranslated region. The gene contains 68% A + T and unusually long stretches of 100- to 150-bp sequences containing alternating A and T nucleotides in the 5' flanking region and intron A. The 5' flanking region contains two TATA sequences, both of which appear to be functional as determined by S1 nuclease mapping. Compared to the rat and human genes, the locations of the introns are identical but the sizes differ. Comparable human and bovine sequences in the flanking regions and introns are about 80% homologous. 相似文献