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An assay system that measures the enzymatic activities (17 alpha-hydroxylase, 17,20-desmolase, and 17 beta-hydroxysteroid dehydrogenase) in the delta 4 pathway of testosterone biosynthesis using rat and human testicular homogenate was examined. This system involves the simultaneous separation of the steroid intermediates by a three-step TLC procedure. The observed Rf values were 0.78 for progesterone (P), 0.59 for 17 alpha-hydroxyprogesterone (17 alpha-HP), 0.70 for androstenedione (A), 0.5 for testosterone, 0.64 for dihydrotestosterone, and 0.45 for 3 alpha, 17 beta-androstanediol. The identification of these steroid intermediates was further accomplished by acetylation and rechromatography of the representative samples along with the authentic standards and by recrystallization to constant specific activity until three consecutive crystallizations were within +/- 5% of the mean value. Incubation time up to 30 min and increasing protein concentrations showed a linear relationship with respect to these three enzymatic activities. The optimum temperature for these enzymatic activities varied from 32 to 34 degrees C, with a sharp decline between 37 and 40 degrees C. The Michaelis constants (Km) for the rat testis homogenate samples were 0.17 microM for P, 0.22 microM for 17 alpha-HP, and 2.5 microM for A, while for the human testis the Km values were 1.2, 2.2, and 2.3 microM, respectively, for these substrates. The concentrations of the endogenous steroid substrates present in these homogenate samples did not alter the Km or Vmax values. The effect of human chorionic gonadotropin (hCG) in vitro on these steroidogenic enzyme activities was also studied. In the rat testis, 10 IU of hCG produced a significant rise in all the three enzyme activities whereas in the human testis 10 and 30 IU of hCG showed no significant change in any of these enzymatic activities. However, 100 IU of hCG resulted in a significant increase in 17 alpha-hydroxylase and 17,20-desmolase activities in the human testis. These studies suggest that this assay system for the measurement of these enzymatic activities using a testicular homogenate sample provides consistent and reproducible results. Based on the sensitivities of the measurements and our experience with testicular biopsy technique, we conclude that a routine testicular biopsy in the human should provide sufficient tissue to run these enzymatic assays. 相似文献
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Pankaj K. Sikka Kenneth E. McMartin 《In vitro cellular & developmental biology. Animal》1996,32(5):285-291
Summary Anin vitro model to establish primary and subcultures of rat kidney proximal tubule (RPT) cells is described. After excising the kidneys
and separating the cortex, the cortical tissue is digested with the enzyme DNAse-collagenase (Type I) resulting in a high
yield of viable RPT Cells. The isolated RPT cells are then seeded onto rat tail collagen-coated surfaces and grown to confluency
in a serum-free, hormonally defined medium. The cell yield can be increased by transfering the conditioned medium on Day 1
to more rat tail collagen-coated surfaces. RPT cell attachment and morphology was better on rat tail collagen-coated surfaces
than on bovine collagen Type I coated surfaces. The culture medium was a 1∶1 mixture of Ham’s F-12 and Dulbecco’s modified
Eagle’s medium supplemented with bovine serum albumin, insulin, transferrin, selenium, hydrocortisone, triiodothyronine, epidermal
growth factor, and glutamine. The RPT cells became confluent in 7–10 d, at which point they could be subcultured by trypsinizing
and growth in the same medium. In some studies, 10 ng/ml cholera toxin was added to the culture medium. We could passage the
RPT cells up to 14 times in the presence of cholera toxin. The cells were investigated for activity of several markers. The
cells were histochemically positive for alkaline phosphatase and γ-glutamyl transpeptidase activity and synthesized the intermediate
filament pankeratin. The RPT cells displayed apically directed sodium-dependent active glucose transport in culture. Hence,
the RPT cells retain structural and functional characteristics of transporting renal epithelia in culture. This rat cell culture
model will be a valuable tool for substrate uptake and nephrotoxicity studies. 相似文献
4.
Stefanie Bunse Sakshi Garg Stephan Junek Dirk Vogel Nariman Ansari Ernst H. K. Stelzer Erin Schuman 《PloS one》2013,8(12)
Cadherins, Ca2+-dependent adhesion molecules, are crucial for cell-cell junctions and remodeling. Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca2+-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca2+-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca2+-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins. 相似文献
5.
Arora Sakshi Singh Amarjeet Kumar Chaudhary Bhupendra 《Plant Cell, Tissue and Organ Culture》2020,141(3):511-531
Plant Cell, Tissue and Organ Culture (PCTOC) - In vitro regeneration of cotton seems to be highly genotype dependent and typically ensues through the extraordinarily complex mechanism of somatic... 相似文献
6.
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) is the clinical syndrome of persistent lung inflammation caused by various direct and indirect stimuli. Despite advances in the understanding of disease pathogenesis, few therapeutic have emerged for ALI/ARDS. Thus, in the present study we evaluated the therapeutic potential of ethyl gallate (EG), a plant flavanoid in the context of ALI using in vivo (BALB/c) and in vitro models (human monocytes). Our in vivo data supports the view that EG alleviates inflammatory condition in ALI as significant reduction in BALF neutrophils, ROS, proinflammatory cytokines and albumin levels were observed with the single i.p of EG post LPS exposure. Also, histochemical analysis of mice lung tissue demonstrated that EG restored LPS stimulated cellular influx inside the lung airspaces. Unraveling the mechanism of action, our RT-PCR and western blot analysis suggest that enhanced expression of HO-1 underlies the protective effect of EG on ROS level in mice lung tissue. Induction of HO-1 in turn appears to be mediated by Nrf2 nuclear translocation and consequent activation and ablation of Nrf2 activity through siRNA notably abrogated the EG induced protective effect in LPS induced human monocytes. Furthermore, our results indicate that EG generated moderate amounts of H2O2 could induce Nrf2 translocation in the in vitro systems. However, given the insignificant amount of H2O2 recorded in the injected material in the in vivo system, additional mechanism for EG action could not be excluded. Nevertheless our results highlight the protective role of EG in ALI and provide the novel insight into its usefulness as a therapeutic tool for the treatment of ALI. 相似文献
7.
Trametes ljubarskyi produces multiple laccase isozymes under various physicochemical conditions. During co-cultivation condition Rhodotorula mucilaginosa showed inter-specific interactions with T. ljubarskyi and hypersecretion of laccases; however, the underlying molecular mechanism is less-known. The analysis of proteomics data of co-cultivated cultures revealed the mechanism of metabolic coupling during fungal-yeast interactions. The results suggested high score GO terms related to stimulus-response, protein binding, membrane components, transport channels, oxidoreductases, and antioxidants. The SEM studies confirmed the cellular communication and their inter-specific interactions. This study allows us to deepen and refine our understanding of fungal-yeast symbiotic interaction; further, it also establishes a mutual relation by metabolic coupling for 10-fold higher laccase isozyme secretion (6532 U/ml). The purified laccase isozymes showed acidic pH optima (pH 3–4), higher thermo-stability (60 °C), and broad enzyme kinetics (Km) values. Our study also provides an in-depth understanding of laccase isozymes and their potential to degrade synthetic dyes, which may help the fungi to survive in an adverse environment. 相似文献
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