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81.
The slime-forming bacterium Methylobacterium sp. was isolated from a Finnish paper machine and its exopolysaccharide (EPS) was produced on laboratory scale. Sugar compositional analysis revealed a 100% galactan (EPS). However, FT-IR showed a very strong peak at 1611 cm(-1) showing the presence of pyruvate. Analysis of the pyruvate content revealed that, based on the sugar composition, the EPS consists of a trisaccharide repeating unit consisting of D-galactopyranose and [4,6-O-(1-carboxyethylidene)]-D-galactopyranose with a molar ratio of 1:2, respectively. Both linkage analysis and 2D homo- and heteronuclear 1H and 13C NMR spectroscopy revealed the following repeating unit: -->3)-[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)[4,6-O-(1-carboxyethylidene)]-alpha-D-Galp-(1-->3)-alpha-D-Galp-(1-->. By enrichment cultures from various ground and compost heap samples a polysaccharide-degrading culture was obtained that produced an endo acting enzyme able to degrade the EPS described. The enzyme hydrolysed the EPS to a large extent, releasing oligomers that mainly consisted out of two repeating units. 相似文献
82.
Gudipaty L Munetz J Verhoef PA Dubyak GR 《American journal of physiology. Cell physiology》2003,285(2):C286-C299
Interleukin (IL)-1beta is a proinflammatory cytokine that elicits the majority of its biological activity extracellularly, but the lack of a secretory signal sequence prevents its export via classic secretory pathways. Efficient externalization of IL-1beta in macrophages and monocytes can occur via stimulation of P2X7 nucleotide receptors with extracellular ATP. However, the exact mechanisms by which the activation of these nonselective cation channels facilitates secretion of IL-1beta remain unclear. Here we demonstrate a pivotal role for a sustained increase in cytosolic Ca2+ to potentiate secretion of IL-1beta via the P2X7 receptors. Using HEK-293 cells engineered to coexpress P2X7 receptors with mature IL-1beta (mIL-1beta), we show that activation of P2X7 receptors results in a rapid secretion of mIL-1beta by a process(es) that is dependent on influx of extracellular Ca2+ and a sustained rise in cytosolic Ca2+. Moreover, reduction in extracellular Ca2+ attenuates approximately 90% of P2X7 receptor-mediated IL-1beta secretion but has no effect on enzymatic processing of precursor IL-1beta (proIL-1beta) to mIL-1beta by caspase-1. Similar experiments with THP-1 human monocytes and Bac1.2F5 murine macrophages confirm the unique role of Ca2+ in P2X7 receptor-mediated secretion of IL-1beta. In addition, we report that cell surface expression of P2X7 receptors in the absence of external stimulation also results in enhanced release of IL-1beta and that this can be repressed by inhibitors of P2X7 receptors. We clarify an essential role for Ca2+ in ATP-induced IL-1beta secretion and indicate an additional role of P2X7 receptors as enhancers of the secretory apparatus by which IL-1beta is released. 相似文献
83.
Heparin and its derivatives bind to HIV-1 recombinant envelope glycoproteins, rather than to recombinant HIV-1 receptor, CD4 总被引:1,自引:0,他引:1
We have employed a direct radiolabel binding assay to investigate the
interaction between3H-heparin and recombinant envelope glycoproteins,
rgp120s, derived from several different isolates of HIV-1. Comparable
dose-dependent binding is exhibited by rgp120s from isolates IIIB, GB8, MN
and SF-2. Under identical experimental conditions the binding of3H- heparin
to a recombinant soluble form of the cellular receptor for gp120, CD4, is
negligible. The binding of3H-heparin to rgp120 is competed for by excess
unlabeled heparin and certain other, but not all, glycosaminoglycan and
chemically modified heparins. Of a range of such polysaccharides tested,
ability to compete with3H-heparin for binding was strictly correlated with
inhibition of HIV-1 replication in vitro. Those possessing potent
anti-HIV-1 activity were effective competitors, whereas those having no or
little anti-HIV-1 activity were poor competitors. Scatchard analysis
indicates that the K d of the interaction between heparin and rgp120 is 10
nM. Binding studies conducted in increasing salt concentrations confirm
that the interaction is ionic in nature. Synthetic 33-35 amino acid
peptides based on the sequence of the V3 loop of gp120 also bind to heparin
with high affinity. V3 loop peptides that are cyclized due to terminal
cysteine residues show more selective binding than their uncyclized
counterparts. Overall, these data demonstrate further that heparin exerts
its anti-HIV-1 activity by binding to the envelope glycoprotein of HIV-1,
rather than its cellular receptor, CD4. This study confirms that the V3
loop of gp120 is the site at which heparin exerts its anti- HIV-1 activity.
Moreover, it reveals that high affinity binding to heparin is shared by all
four rgp120s examined, despite amino acid substitutions within the V3 loop.
相似文献
84.
N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells 总被引:5,自引:3,他引:2
The potential of insect cell cultures and larvae infected with recombinant
baculoviruses to produce authentic recombinant glycoproteins cloned from
mammalian sources was investigated. A comparison was made of the N-linked
glycans attached to secreted alkaline phosphatase (SEAP) produced in four
species of insect larvae and their derived cell lines plus one additional
insect cell line and larvae of one additional species. These data survey
N-linked oligosaccharides produced in four families and six genera of the
order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of
Autographa californica and Bombyx mori nucleopolyhedroviruses was purified
from cell culture medium, larval hemolymph or larval homogenates by
phosphate affinity chromatography. The N-linked oligosaccharides were
released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic
acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by
fluorescence imaging. The oligosaccharide structures were confirmed with
exoglycosidase digestions. Recombinant SEAP produced in cell lines of
Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx
mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni ,
H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that
were structurally identical to the 10 oligosaccharides attached to SEAP
produced in T.ni cell lines. The oligosaccharide structures were all
mannose-terminated. Structures containing two or three mannose residues,
with and without core fucosylation, constituted more than 75% of the
oligosaccharides from the cell culture and larval samples.
相似文献
85.
Rapid detection of salmonellae in poultry with the magnetic immuno-polymerase chain reaction assay. 总被引:7,自引:3,他引:4 下载免费PDF全文
A C Fluit M N Widjojoatmodjo A T Box R Torensma J Verhoef 《Applied microbiology》1993,59(5):1342-1346
Rapid detection of salmonellae in chicken meat was accomplished by using the magnetic immuno-polymerase chain reaction assay (MIPA). A direct polymerase chain reaction assay performed with chicken meat spiked with Salmonella typhimurium resulted in poor sensitivity (approximately 10(7) CFU/g of meat). The use of immunoseparation with a Salmonella serogroup B-specific monoclonal antibody improved the sensitivity, but enrichment was required for the detection of low levels of contamination. Enrichment for 6 h in either buffered peptone water, lactose broth containing tergitol-7, or selenite-cystine broth resulted in the detection of an initial inoculum of 100 CFU per g of meat. Enrichment of the salmonellae present on 25 g of spiked chicken meat for 24 h in either buffered peptone water or selenite-cystine broth before detection by the MIPA yielded a detection limit of approximately 0.1 CFU/g of meat. A detection limit of approximately 1 CFU/g of meat was obtained when the spiked meat was stored at -20 degrees C before enrichment for 24 h and analysis with the MIPA. Although the MIPA was developed for S. typhimurium, a MIPA in which a panel of six monoclonal antibodies specific for Salmonella serogroups A through E was used detected the presence of 0.1 CFU of Salmonella enteritidis per g of chicken meat. These data indicate that the method is applicable to other commonly isolated serotypes. 相似文献
86.
Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay. 总被引:13,自引:6,他引:7 下载免费PDF全文
A C Fluit R Torensma M J Visser C J Aarsman M J Poppelier B H Keller P Klapwijk J Verhoef 《Applied microbiology》1993,59(5):1289-1293
A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h. 相似文献
87.
Eva Mattsson Liesbeth Verhage J. Rollof A. Fleer J. Verhoef H. van Dijk 《FEMS immunology and medical microbiology》1993,7(3):281-288
Abstract Cytokines play a major role in the pathophysiology of septic shock. In this study, human periferal blood monocytes were stimulated with peptidoglycan and teichoic acid, purified from a strain of Staphylococcus epidermidis . Polymyxin B (PM-B) was added to avoid the effects of possible contamination with endotoxin. Tumour necrosis factor-α (TNF), interleukin-1β (IL-1), and interleukin-6 (IL-6) in the supernates were measured by enzyme-linked immunosorbent assays. Peptidoglycan and teichoic acid induced TNF, IL-1, and IL-6 in a concentration-dependent manner. Teichoic acid was a weaker inducer than peptidoglycan, especially for IL-1. Lipopolysaccharide from an E. coli strain was used as a control, being 100–1000 times more potent than peptidoglycan and teichoic acid. 相似文献
88.
OBJECTIVE: To ascertain the opinions of Alberta physicians about the acceptance of active euthanasia as a medical act (the "medicalization" of active euthanasia) and the reporting of colleagues practising active euthanasia, as well as the sociodemographic correlates. DESIGN: Cross-sectional survey of a random sample of Alberta physicians, grouped by site and type of practice. SETTING: Alberta. PARTICIPANTS: A total of 2002 (46%) of the licensed physicians in Alberta were mailed a 38-item questionnaire in May through July 1991; usable responses were returned by 1391 (69%). RESULTS: Although only 44% of the respondents considered active euthanasia morally "right" at least 70% opted to medicalize the practice if it were legal by restricting it to be performed by physicians and to be taught at medical sites. Even though active euthanasia is criminal homicide in Canada, 33% of the physicians stated that they would not report a colleague participating in the act of anyone, and 40% and 60% stated that they would not report a colleague to medical or legal authorities respectively. Acceptance or rejection of active euthanasia as a medical act was strongly related to religious affiliation and activity (p < 0.01). CONCLUSIONS: This survey about active euthanasia revealed profound incongruities in the opinions of the sample of Alberta physicians concerning their ethical and social duties in the practice of medicine. These data highlight the need for relevant modifications of health education policies concerning biomedical ethics and physicians'' obligations to society. 相似文献
89.
Unusual pattern of bacterial ice nucleation gene evolution 总被引:5,自引:0,他引:5
Edwards AR; Van den Bussche RA; Wichman HA; Orser CS 《Molecular biology and evolution》1994,11(6):911-920
Bacterial ice nucleation activity (INA+ phenotype) can be traced to the
product of a single gene, ina. A remarkably sparse distribution of this
phenotype within three bacterial genera indicates that the ina gene may
have followed an unusual evolutionary path. Southern blot analyses, coupled
with assays for ice-nucleating ability, revealed that within four bacterial
species an ina gene is present in some strains but absent from others.
Results of hybridization experiments using DNA fragments that flank the ina
gene suggested that the genotypic dimorphism of ina may be anomalous. A
phylogenetic analysis of 16S ribosomal RNA gene sequences from a total of
14 ina+ and ina- bacterial strains indicated that the ina+ bacteria are not
monophyletic but instead phylogenetically interspersed among ina- bacteria.
The relationships of ina+ bacteria inferred from ina sequence did not
coincide with those inferred from the 16S data. These results suggest the
possibility of horizontal transfer in the evolution of bacterial ina genes.
相似文献
90.
J G Loeber J Verhoef J P Burbach A Witter 《Biochemical and biophysical research communications》1979,86(4):1288-1295
A method for the separation and subsequent quantification of endorphins and related peptides was developed. Separation of the peptides was achieved by high pressure liquid chromatography on a reversed phase column. By virtue of the high resolving capacity of this system peptides differing in only one amino acid residue could be separated easily. For quantification of the isolated peptides specific radioimmunoassay systems were used. The combination of the two techniques was applied to determine specifically a number of endorphin-like peptides in rat pituitary gland. For the first time the presence of des-tyrosine-endorphins in addition to known endorphin fragments is demonstrated. 相似文献