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191.
William Clay Bracken John Carey Marilyn Emerson Holtzer Alfred Holtzer 《Biopolymers》1988,27(8):1223-1237
A method is described for preparation of a species of β tropomyosin that is sulfhydryl-blocked at C36 and disulfide-cross-linked at C190. Five steps are involved: (1) Rabbit skeletal muscle tropomyosin, comprising αα and αβ species, is oxidized with ferricyanide, disulfide-cross-linking both species at C190. (2) The product is treated with iodoacetamide, blocking the only remaining free sulfhydryl, i.e., C36 of the β-chains. (3) The C36-blocked, C190-cross-linked product is reduced with dithiothreitol (DTT), unfolded in urea, and α and β chains separated by ion-exchange chromatography. (4) The C36-blocked β chains are refolded by dialysis. (5) The refolded, C36-blocked ββ species are cross-linked at C190 by ferricyanide oxidation. The resulting C36-blocked, C190-cross-linked ββ product is separated from contaminating species—mostly completely blocked β-chains and multichain cross-linked molecules—by size-exclusion chromatography in denaturing (guanidinium chloride) solvent. The five-step process and the final product were monitored by titration of free sulfhydryls and by NaDodSO4/polyacrylamide gel electrophoresis (PAGE). Thermal unfolding curves from CD are reported for the resulting pure, C36-blocked, C190-cross-linked ββ species and for its DTT-reduction product, the noncross-linked C36-blocked species. The latter shows almost the same thermal unfolding transition as intact, noncross-linked ββ species. The former shows a pretransition similar to, but larger in extent than, the one well known to occur in the analogous case of C190-cross-linked αα tropomyosin. These unfolding transitions are compared with one another and with that previously reported for doubly cross-linked (at C36 and C190) ββ species. These comparisons are made in the light of current physical models for coiled-coil unfolding equilibria. It is concluded that although no extent model is demonstrably satisfactory, any successful model must include strain at the cross-link, loop entropy, and regional nonuniformities as essential parts of the physics. 相似文献
192.
Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes 总被引:4,自引:0,他引:4
Elayne A. Bornslaeger William T. Poueymirou Peter Mattei Richard M. Schultz 《Experimental cell research》1986,165(2):507-517
Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase. 相似文献
193.
Females homozygous for the maternal-effect mutation abo (2-44.0) produce a large fraction of eggs which arrest during embryogenesis. Increasing doses of defined heterochromatic regions inherited by offspring of abo mothers from their fathers function zygotically to bring about a partial rescue of the abo-induced embryonic lethality. Another property of the abo mutation is that the severity of the maternal effect decreases when an abo stock is maintained in homozygous condition for a number of generations. Here, we show that the factors which change in homozygous abo stocks to result in the decrease in maternally induced embryonic lethality, act zygotically, dominantly and additively. More importantly, we show that the X and second chromosomes, but not the Y and third chromosomes, derived from homozygous abo stocks are, when inherited from males, more effective in promoting zygotic rescue of the abo-induced lethality than are the equivalent chromosomes derived from an abo stock maintained in heterozygous condition. The chromosomal locations of the factors maintained in the homozygous condition. The chromosomal locations of the factors altered in homozygous stock, as well as their behavior, strongly suggest that the same heterochromatic elements that are responsible for rescuing embryos from the abo-induced maternal effect are altered in homozygous abo flies in such a way that the maternal effect itself is less severe. 相似文献
194.
195.
Symbiotic Relationship of Bacteroides cellulosolvens and Clostridium saccharolyticum in Cellulose Fermentation 总被引:1,自引:0,他引:1 下载免费PDF全文
William D. Murray 《Applied microbiology》1986,51(4):710-714
In coculture, Bacteroides cellulosolvens and Clostridium saccharolyticum fermented 33% more cellulose than did B. cellulosolvens alone. Also, cellulose digestion continued at a maximum rate 48 h longer in coculture. B. cellulosolvens hydrolyzes cellulose and supplies C. saccharolyticum with sugars and a growth factor replaceable by yeast extract. Alone, B. cellulosolvens exhibited an early cessation of growth which was not due to nutrient depletion, low pH, or toxic accumulation of acetic acid, ethanol, lactic acid, H2, CO2, cellobiose, glucose, or xylose. However, a 1-h incubation of B. cellulosolvens spent-culture medium with C. saacharolyticum cells starved for growth factor allowed a resumption of B. cellulosolvens growth. The symbiotic relationship of this naturally occurring coculture is one of mutualism, in which the cellulolytic microbe supplies the saccharolytic microbe with nutrients, and in turn the saccharolytic microbe removes a secondary metabolite toxic to the primary microbe. 相似文献
196.
William F. Fett Stanley F. Osman Marshall L. Fishman T. S. Siebles III 《Applied microbiology》1986,52(3):466-473
Eighteen plant-pathogenic and three non-plant-pathogenic pseudomonads were tested for the ability to produce alginic acid as an exopolysaccharide in vitro. Alginate production was demonstrated for 10 of 13 fluorescent plant-pathogenic pseudomonads tested with glucose or gluconate as the carbon source, but not for all 5 nonfluorescent plant pathogens and all 3 non-plant pathogens tested. With sucrose as the carbon source, some strains produced alginate while others produced both polyfructan (levan) and alginate. Alginates ranged from <1 to 28% guluronic acid, were acetylated, and had number-average molecular weights of 11.3 × 103 to 47.1 × 103. Polyfructans and alginates were not elicitors of the soybean phytoalexin glyceollin when applied to wounded cotyledon surfaces and did not induce prolonged water soaking of soybean leaf tissues. All or most pseudomonads in rRNA-DNA homology group I may be capable of synthesizing alginate as an exopolysaccharide. 相似文献
197.
Regional Kinetic Constants for Blood–Brain Barrier Pyruvic Acid Transport in Conscious Rats by the Monocarboxylic Acid Carrier 总被引:1,自引:1,他引:0
The present investigation using labeled pyruvate describes the regional distribution and kinetics of the monocarboxylic acid carrier at the blood-brain barrier of conscious rats. The experimental procedure involved the arterial injection of a single bolus of 200 microliter containing [1-14C]pyruvate, [3H]water, and varying concentrations of unlabeled pyruvate into the common carotid via an indwelling externalized catheter. The hemisphere ipsi-lateral to the injection and rostral to the midbrain was removed and dissected into five regions. A kinetic analysis revealed no significant regional differences in Km values with an overall average of 1.37 mM. However, there was regional variation in the density of the monocarboxylic acid carrier as indicated by varied levels of the kinetic constant Vmax. The cortex showed the highest Vmax value of 0.42 +/- 0.08 mumol/min/g whereas values for the caudate/putamen, thalamus/hypothalamus, and remaining portion of hemisphere ranged significantly lower at 0.22-0.27 mumol/min/g. The Vmax for the hippocampus was intermediate at 0.37 +/- 0.12 mumol/min/g. The nonsaturable carrier described kinetically by KD had an overall average of 0.034 ml/min/g. The present study confirms quantitatively previous results suggesting a variable regional distribution of the monocarboxylic acid carrier. 相似文献
198.
199.
Summary The tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) was examined for its ability to induce endogenous retrovirus from a high-passage clone
of Kirsten sarcoma virus-transformed Balb/c (K-Balb) mouse cells. TPA activated virus in a concentration-dependent manner
(0.0016 to 4.0 μM). Exposure to 1mM actinomycin D inhibited virus induction, suggesting that cellular RNA synthesis is required de novo by this inducer. A broad-spectrum
neutralizing antibody to murine type C virus, gp70, was shown to neutralize the infectivity of the induced virus. The activated
virus had the host range of the xenotropic Balb virus:2, and after removal of the inducer, the activated state decayed rapidly.
TPA stimulated DNA, RNA, and protein synthesis in K-Balb cells, indicating that the mechanism of inducation may be different
from that of previously identified virus inducers. The effects observed using the well-defined K-Balb system offer an opportunity
to study the modulation of retrovirus gene expression by TPA.
This work was conducted while the authors were with the Biological Carcinogenesis Program, Frederick Cancer Research Facility,
Frederick, MD 21701, and was supported under Contract NO1-CO-75380 with the National Cancer Institute, National Institutes
of Health, Bethesda, MD 20205. 相似文献
200.
William D. Meek Walter L. Davis 《In vitro cellular & developmental biology. Plant》1986,22(12):725-737
Summary The potent fungal metabolite cytochalasin D (CD) and cationized ferritin (CF) are used in combination to test for negative
charge distribution on blebs (knobs). Two established human epithelial cell lines, WISH and HeLa, that display blebs in various
phases of the cell cycle or under certain culture conditions (37,46) are investigated. CD alone, applied at a low concentration
(1.0 μg/ml) and for a short time period (3 min), causes blebs to appear as the prevalent surface feature. These are filled
mainly with free ribosomes. Additionally, feltlike mats, presumed to be disorganized, compacted microfilaments, are formed
directly beneath the cell membrane. These are especially evident in the cortical cytoplasm below the blebs or bleb clusters.
CF (0.345 mg/ml), applied for a 5-min period after CD administration (1.0 μg/ml) for 3 min, appears along the surface of microvilli,
at the base of blebs, and in vesicles beneath the bleb clusters. In some cases, microfilaments (6 nm in diameter) are closely
related to the vesicles. CF does not preferentially bind to the apical cell membrane of blebs. Above areas of the subplasmalemmal
microfilaments, CF membrane binding is apparent, even under circumstances where the filaments are disorganized by cytochalasin
treatment. These results seem to show the following: (a) bleb membranes are different from the remainder of the cell and do
exhibit a loss of negative charge and (b) surface charge may be dependent on the presence or structural integrity of membrane-related
6-nm microfilaments.
The support of this research by a grant from the Baylor College of Dentistry and The Oklahoma College of Osteopathic Medicine
and Surgery is gratefully acknowledged. The assistance of Dr. J. H. Martin, Department of Pathology, Baylor University Medical
Center, is also greatly appreciated. 相似文献