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51.
Simian virus 40 T-antigen-related cell surface antigen: serological demonstration on simian virus 40-transformed monolayer cells in situ. 总被引:20,自引:9,他引:11 下载免费PDF全文
Simian virus 40 (SV40)-transformed monolayer cells were analyzed in situ by indirect immunofluorescence microscopy for the postulated cell surface location of SV40 T-antigen-related molecules. With antisera prepared against purified, sodium dodecyl sulfate-denatured SV40 T-antigen, positive surface staining was obtained when the cells had been treated with formaldehyde before immunofluorescence analysis. In contrast, living SV40-transformed cells analyzed in monolayer were surface fluorescence negative. The fixation procedure developed in this study combined with a double staining immunofluorescence technique allowed the simultaneous analysis of the same cells for the expression of both SV40 T-antigen-related surface antigen and nuclear T-antigen. The localization of SV40 T-antigen-related surface antigen on the outer surface of the plasma membrane of formaldehyde-fixed SV40-transformed cells was demonstrated directly by the protein A-mediated binding of Staphylococcus aureus bacteria on formaldehyde-fixed SV40-transformed cells precoated with antiserum against sodium dodecyl sulfate-denatured T-antigen. Both cell surface staining and S. aureus binding were found to be highly specific for SV40 T-antigen-related binding sites. These results indicate that T-antigen-related molecules in a cryptic form are located on the surface of SV40-transformed monolayer cells and can be detected in situ after modification of the cell surface architecture. 相似文献
52.
Ramachandra Reddy Dale Henning Harris Busch 《Biochemical and biophysical research communications》1981,98(4):1076-1083
The primary nucleotide sequence was reported earlier for U1 RNA (Reddy et al, (1974) J. Biol. Chem. , 6486–6494), an snRNA implicated in splicing of HnRNAs. In view of the presence of homologous pseudouridine (ψ) residues in 5′-ends of several highly conserved U-snRNAs and the recent report of modified bases in the U1 RNA structure (Branlant et al, (1980) Nucleic Acids Res. , 4143–4154) a study was made for the presence of ψ and other modified nucleotides in the 5′-end of the U1 RNA. Identification of ψ residues at positions 6 and 7, shows the 5′-sequence of U1 RNA is: m32, 2,7 GpppAm-Um-A-C-ψ-ψ-A-C-C-U-G-G-C-A-G-G-G-G-A-G-A-U-A-C. The ψ residues in place of U at positions 6 and 7 may affect the binding of U1 RNA at intron-exon splice junctions. 相似文献
53.
E L Lloyd M A Gemmell C B Henning D S Gemmell B J Zabransky 《International journal of radiation biology and related studies in physics, chemistry, and medicine》1979,36(5):467-478
Mammalian cells in culture have been shown here for the first time to be transformed by alpha irradiation. Mouse embryo (C3H 10T1/2) cells were transformed with 5.6 MeV alpha particles from a Tandem Van de Graaff machine. Malignant tumours were induced following inoculation of the transformed cells into syngeneic hosts. Unirradiated control cells failed to produce tumours. The morphology of the transformed foci was similar to that obtained by X-rays and chemicals but different from virally transformed cells. The transformation frequency increased approximately as the cube of the dose to a maximum of about 4 per cent ofthe surviving cells which occurred between 1.5 and 2.5 x 10(7) alpha particles per cm2 (205-342 rad). It appears that alpha particle irradiation may exert a direct effect on the genome of the cell to produce malignancy without any external immunological or hormonal influences. 相似文献
54.
Summary Histone kinase activity was purified from human polymorphonuclear leukocytes by ammonium sulphate precipitation of a 180 000 × g supernatant, followed by DEAF-cellulose chromatography and gelfiltration. On DEAE-cellulose cAMP dependent kinase activity eluted in two peaks, I and III, at 1.2 mmho and 6.5 mmho, respectively. Catalytic subunit (C) from both peaks had Mr 33 000, 3.0S. Regulatory subunit (R) from peak I and III both had Mr 33 000 upon gelfiltration, but sedimented at 2.8–3.0S and 3.0–3.2S, respectively. R2 and R4 subunits were identified. The R-C dimer from peak I and III sedimented at 4.8S and (4.8)–5.1S, respectively. The holoenzyme from peak I had Mr 165 000, 6.7S, which suggest a R2C2 structure, while that of peak III sedimented at 6.7S, but eluted at Mr 330 000 (2R2C2) by gelfiltration.The K
m
app
for peak I and III enzymes were, respectively: histone IIA 0.5 mg/ml (both forms), ATP 18 m and 23 m, and cAMP 5 × 10–8
m and 6.3 × 10–8
m. Both enzymes had pH optimum 6.7–6.9 and were equally sensitive to Ca2+ temperature and protein kinase inhibitor. The substrate specificity was histone VS histone IIA = histone VIS casein > phosvitin. Peak I enzyme, but not peak III enzyme, was dissociated by histone and high ionic strength and reassociation of R and C subunits were facilitated by ATP-Mg. It is concluded that peak I and III enzymes represent type I and II cAMP dependent protein kinases, respectively. Type I comprises 20–30% of cAMP dependent protein kinase activity and is absent from the 180 000 × g supernatant of gently disrupted cells.Purified catalytic subunit had K
m
app
(ATP) 20 m with rabbit muscle glycogen synthase I as substrates. Synthase I from rabbit muscle and human leukocytes were phosphorylated by catalytic subunit to synthase D (ratio of independence less than 0.07).cAMP independent histone kinase activity eluted in one peak (Peak II) at 3 mmho. The enzymatic activity sedimented at 3.4S and eluted from gelfiltration with Mr 78 000. K
m
app
for ATP was 78 m and for histone IIA 0.5 mg/ml. The enzyme was sensitive to temperature, but less sensitive than cAMP dependent protein kinase to Ca2+, and insensitive to protein kinase inhibitor. The substrate specificity was histone IIA > histone VS = histone VIS, while casein and phosvitin were poor substrates. Glycogen synthase I was not phosphorylated. The cAMP independent histone kinase activity comprised 15% of the total histone kinase activity in a crude homogenate of leukocytes. Its physiological substrate is unknown.Abbreviations AR
activity ratio for cAMP dependent protein kinase
- cAMP
adenosine cyclic 3:5-monophosphate
- cIMP
inosine cyclic 3:5-monophosphate
- cGMP
guanosine cyclic 3:5-monophosphate
- Glucose-6-P
glucose-6-phosphate
- DDT
dithiothreitol
- EGTA
ethylene glycol-bis-(-aminoethylether)-N, N-tetraacetic acid
- PMSF
phenylmethylsulfonylfluoride
- PKI
protein kinase inhibitor
- RI
ratio of independence for glycogen synthase
- SDS
sodium dodecyl sulphate 相似文献
55.
Dr. Henning Schmalbruch 《Cell and tissue research》1979,204(2):187-200
Summary The volume and surface area of mitochondria and sarcoplasmic reticulum in fast and slow twitch fibres of the cat triceps surae muscle were determined from thin sections. The width of the Z-line and the array of glycogen granules identified fast and slow twitch fibres.The relative volume occupied by mitochondria was largest in slow twitch gastrocnemius fibres. Fast twitch fibres showed the greatest scatter of mitochondrial content. This corresponds with the fact that motor units of the fast twitch type differ most with respect to resistance to fatigue.The relative volume of the sarcoplasmic reticulum was twice as large in fast as in slow twitch fibres. The volume fraction occupied by longitudinal tubules of the reticulum was the same in fast and slow twitch gastrocnemius fibres but was only half as large in the slow twitch soleus fibres. This difference may be related to post-tetanic potentiation: this property is present in all gastrocnemius fibres but is absent in soleus fibres.The specific tetanic force is 3 to 5 times smaller in slow twitch gastrocnemius than in slow twitch soleus fibres or fast gastrocnemius fibres. There was, however, no detectable morphological difference that might be related to this difference in force.Freeze fractures demonstrated directly that, in soleus fibres, terminal cisternae and longitudinal tubules of the reticulum were scarce as compared to gastrocnemius fibres. The plasma membranes of some gastrocnemius fibres displayed square arrays of 60-nm particles; these arrays were absent in other gastrocnemius fibres and in all soleus fibres. They probably characterize plasma membranes of fast twitch fibres.This study was supported by grants from the Danish Medical Research Council. I wish to thank Mrs. M. Bjærg for valuable technical help 相似文献
56.
Inclusion of xylan in a medium for the enumeration of total culturable rumen bacteria. 总被引:6,自引:6,他引:0 下载免费PDF全文
The influence of the inclusion of xylan in a medium for enumeration of total culturable rumen bacteria was investigated. Maximum colony numbers were obtained on a medium, GCSX-2, which contained 0.033% each glucose and cellobiose and 0.067% each soluble starch and xylan. This medium gave higher colony counts than either medium 98-5 of Bryant and Robinson (J. Dairy Sci. 44:1446-1456, 1961), medium 98-5 of Chung and Hungate (Appl. Environ. Microbiol. 32:649-652, 1976), containing an added lucerne (hemicellulose + cellulose) fiber substrate, or medium GCSX-2 with the added lucerne (hemicellulose + cellulose) fraction. The time of collection of rumen fluid influenced the colony counts on the media containing the lucerne fiber substrate but was without effect on medium GCSX-2. 相似文献
57.
Isolated thylakoid membranes are damaged during freezing in dilute salt solutions, as shown by the inactivation of photochemical thylakoid reactions. After freezing, a number of membrane proteins were found in the particle-free supernatant. Up to 5% of the total membrane protein was solubilized by freezing, and the pattern of released proteins as seen in sodium dodecyl sulfate gel electrophoretograms was influenced by the nature of the solutes present. Membranes protected by sucrose did not release much protein during freezing. Concentrated salt solutions caused protein release also in the absence of freezing. Among the proteins released were ferredoxin—NADP+ reductase, plastocyanin and coupling factor CF1. Subunits of CF1 were found in different proportions in the supernatants of thylakoid suspensions after freezing in the presence of different salts. Cyclic photophosphorylation was largely inactivated before significant protein release could be detected.It is suggested that protein release is the final consequence of the non-specific suppression of intramembrane ionic interactions by the high ionic strength created in the vicinity of the membranes by the accumulation of salts during slow freezing. Salt effects on water structure and alterations of nonpolar membrane interactions by the incorporation of (protonated) lipophilic anions from organic salts into the membrane phase during freezing may also be involved. 相似文献
58.
59.
R Chen I Hindennach U Henning 《Hoppe-Seyler's Zeitschrift für physiologische Chemie》1978,359(12):1807-1810
The sequence of the cyanogen bromide fragments of one of the major outer membrane proteins of E. coli B/r has been established with the aim of elucidating the primary structure of this protein. Separation of all fragments on one molecular sieve column was achieved upon citraconylation of these fragments. Overlapping peptides were obtained by digestion of the protein, or a cyanogen bromide fragment arising from incomplete cleavage, with trypsin or Staphylococcus aureus protease. 相似文献
60.
Triton WR 1339 was found to contain a high molecular weight fraction with globular polymers of ˜ 105 D and a diameter of ˜80 Å (‘macrotriton’) and a low molecular weight fraction (‘microtriton’). The intracellular distribution of subfractions of [3H]Triton WR 1339 was followed by cell fractionation and by gel chromatography techniques in parallel with electron microscopy and autoradiography. Macrotriton is selectively stored in lysosomes and all evidence supports a slowly working endocytotic uptake mechanism. Microtriton permeates quickly into the cells and is rapidly and efficiently released into the bile. The data presented suggest some intracellular leaking of lysosomal contents due to the action of Triton WR 1339. 相似文献