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1.
Mature osteoclasts, multinucleated giant cells responsible for bone resorption, are terminally differentiated cells with a
short life span. Recently, we have demonstrated that osteoclast apoptosis is regulated by ERK activity and Bcl-2 family member
Bim. In this paper, we summarize the methods we used to study osteoclast apoptosis in vitro and in vivo. Using adenovirus and retrovirus vectors, we were able to introduce foreign genes into osteoclasts and examine their effects
on osteoclast survival in vitro. In addition, we established the modified methods for in situ hybridization and BrdU labeling of bone sections from mice
to study osteoclast survival in vivo. The detailed methods described here could be useful for studying the biological process in bone. 相似文献
2.
Assembly of DNA into chromatin allows for the formation of a barrier that protects naked DNA from protein and chemical agents
geared to degrade or metabolize DNA. Chromatin assembly occurs whenever a length of DNA becomes exposed to the cellular elements,
whether during DNA synthesis or repair. This report describes tools to study chromatin assembly in the model systemSaccharomyces cerevisiae. Modifications to anin vitro chromatin assembly assay are described that allowed a brute force screen of temperature sensitive (ts) yeast strains in order to identify chromatin assembly defective extracts. This screen yielded mutations in genes encoding
two ubiquitin protein ligases (E3s):RSP5, and a subunit of the Anaphase Promoting Complex (APC),APC5. Additional modifications are described that allow for a rapid analysis and anin vivo characterization of yeast chromatin assembly mutants, as well as any other mutant of interest. Our analysis suggests that
thein vitro andin vivo chromatin assembly assays are responsive to different cellular signals, including cell cycle cues that involve different
molecular networks.
Published: July 3, 2003 相似文献
3.
To assess the role of genes required for skin organogenesis, tissue regeneration and homeostasis, we have established in vitro
skin equivalents composed of primary cells or cell lines, respectively. In these organotypic cocultures keratinocytes generate
a normal epidermis irrespective of the species and tissue origin of fibroblasts. The combination of cells derived from mouse
and human tissues facilitates the identification of the origin of compounds involved in epidermal tissue reconstitution and
thus the precise analysis of growth regulatory mechanisms.
Published: April 12, 2004 相似文献
4.
5.
In this study, Dendrobium Sonia 17 plantlets were used to induce in vitro flowering. Inflorescences were induced and rooting was inhibited in the half-strength Murashige and Skoog medium containing
20 μM N
6-benzyladenine (BA). The medium with high P and low N contents was effective to induce inflorescences while the medium with
low P and high N contents was only effective to promote forming of shoots. In addition, the induced in vitro inflorescences were able to multiply and maintain without exhibiting a distinctive vegetative phase. Different morphologies
of in vitro flowers such as incomplete flower structures, abnormal and unresupinated in vitro flowers were observed. 相似文献
6.
In adults, hematopoiesis occurs in bone marrow (BM) through a complex process with differentiation of hematopoietic stem cells
(HSCs) to immune and blood cells. Human HSCs and their progenitors express CD34. Methods on hematopoietic regulation are presented
to show the effects of the chemokine, stromal-derived growth factor (SDF)-1α and the neuropeptide, substance P (SP). SDF-1α
production in BM stroma causes interactions with HSCs, thereby retaining the HSCs in regions close to the endosteum, at low
oxygen. Small changes in SDF-1α levels stimulate HSC functions through direct and indirect mechanisms. The indirect method
occurs by SP production, which stimulates CD34+ cells, supported by ligand-binding studies, long-term culture-initiating cell assays for HSC functions, and clonogenic assays
for myeloid progenitors. These methods can be applied to study other hematopoietic regulators. 相似文献
7.
Caenorhabditis elegans is an attractive model system for determining the targets of neuroactive compounds. Genetic screens in C. elegans provide a relatively unbiased approach to the identification of genes that are essential for behavioral effects of drugs
and neuroactive compounds such as alcohol. Much work in vertebrate systems has identified multiple potential targets of ethanol
but which, if any, of those candidates are responsible for the behavioral effects of alcohol is uncertain. Here we provide
detailed methodology for a genetic screen for mutants of C. elegans that are resistant to the depressive effects of ethanol on locomotion and for the subsequent behavioral analysis of those
mutants. The methods we describe should also be applicable for use in screening for mutants that are resistant or hypersensitive
to many neuroactive compounds and for identifying the molecular targets or biochemical pathways mediating drug responses.
Published: June 8, 2004. 相似文献
8.
An efficient micropropagation protocol was established for Capsicum chinense Jacq. cv. Umorok, a pungent chilli cultivar. Shoot-tip explants were cultured on Murashige and Skoog (MS) medium containing
cytokinins (22.2–88.8 μM 6-benzylaminopurine, BAP, 23.2–93.0 μM kinetin, Kin, or 22.8–91.2 μM zeatin, Z) alone or in combination
with 5.7 μM indole-3-acetic acid (IAA). Maximum number of shoots were induced on medium containing 91.2 μM Z or 31.1 μM BAP
with 4.7 μM Kin. The separated shoots rooted and elongated on medium containing 2.5 or 4.9 μM indole-3-butyric acid (IBA).
Axillary shoots were induced from in vitro raised plantlets by decapitating them. The axillary shoot-tip explants were used for further multiple shoot buds induction.
A maximum of about 150 plantlets were obtained from a single seedling. Hardened and acclimatized plantlets were successfully
established in the soil. 相似文献
9.
This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots
was observed on MS augmented with 3.0 mg dm−3 N6-benzylaminopurine (BAP) and 0.05 mg dm−3 α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the
highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots
with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original
explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm−3 indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets
were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in
leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants. 相似文献
10.
Experimental modulation of capsule size is an important technique for the study of the virulence of the encapsulated pathogen
Cryptococcus neoformans. In this paper, we summarize the techniques available for experimental modulation of capsule size in this yeast and describe
improved methods to induce capsule size changes. The response of the yeast to the various stimuli is highly dependent on the
cryptococcal strain. A high CO2 atmosphere and a low iron concentration have been used classically to increase capsule size. Unfortunately, these stimuli
are not reliable for inducing capsular enlargement in all strains. Recently we have identified new and simpler conditions
for inducing capsule enlargement that consistently elicited this effect. Specifically, we noted that mammalian serum or diluted
Sabouraud broth in MOPS buffer pH 7.3 efficiently induced capsule growth. Media that slowed the growth rate of the yeast correlated
with an increase in capsule size. Finally, we summarize the most commonly used media that induce capsule growth in C. neoformans.
Published: March 3, 2004 相似文献
11.
The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression
process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the
molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation,
and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream
acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular
interleukin-1β. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead
seabream testicular acidophilic granulocytes and permits their quantification.
Published: June 29, 2004. 相似文献
12.
M. Chandrika Thoyajaksha V. Ravishankar Rai K. Ramachandra Kini 《Biologia Plantarum》2008,52(4):735-739
In the present study, a polymerase chain reaction (PCR)-based method namely inter simple sequence repeat (ISSR) was employed
to assess genetic stability in tissue culture-derived Dictyospermum ovalifolium plantlets. To study genomic stability of micropropagated plants, 14 individuals were randomly tagged among a population of
2500 regenerants and were compared with single donor mother plant. A total of 51 clear and reproducible bands ranging from
200 bp to 2.1 kb were scored corresponding to an average of 3.64 bands per primer. Two of the 51 bands were polymorphic (3.92
%) among 14 individuals, thus indicating the occurrence of low level genomic variation in the micropropagated plants. Cluster
analysis indicates that genetic similarity values were 0.978 which allows classification of the plants to distinct groups.
Further an attempt was made to reintroduce the micropropagated plants into their natural habitat. Over one thousand six hundred
fifty plants were successfully established. 相似文献
13.
Salmonella enterica serovar Typhimurium is capable of swarming over semi-solid surfaces. Although its swarming behavior shares many readily observable
similarities with other swarming bacteria, the phenomenon remains somewhat of an enigma in this bacterium since some attributes
skew away from the better characterized systems. Swarming is quite distinct from the classic swimming motility, as there is
a prerequisite for cells to first undergo a morphological transformation into swarmer cells. In some organisms, swarming is
controlled by quorum sensing, and in others, swarming has been shown to be coupled to increased expression of important virulence
factors. Swarming in serovar Typhimurium is coupled to elevated resistance to a wide variety of structurally and functionally
distinct classes of antimicrobial compounds. As serovar Typhimurium differentiates into swarm cells, thepmrHFIJKLM operon is up-regulated, resulting in a more positively charged LPS core. Furthermore, as swarm cells begin to de-differentiate,
thepmr operon expression is down-regulated, rapidly reaching the levels observed in swim cells. This is one potential mechanism
which confers swarm cells increased resistance to antibiotics such as the cationic antimicrobial peptides. However, additional
mechanisms are likely associated with the cells in the swarm state that confer elevated resistance to such a broad spectrum
of antimicrobial agents.
Published: September 26, 2003 相似文献
14.
A rapid and efficient plant regeneration protocol for a wide range of alfalfa genotypes was developed via direct organogenesis. Through a successive excision of the newly developed apical and axillary shoots, a lot of adventitious
buds were directly induced from the cotyledonary nodes when hypocotyl of explants were vertically inserted into modified Murashige
and Skoog (MS) medium supplemented with 0.025 mg dm−3 thidiazuron (TDZ) and 3 mg dm−3 AgNO3. When the lower part of shoots excised from explants were immersed into the liquid medium with 1.0 mg dm−3 α-naphthaleneacetic acid (NAA) for 2 min, and then transferred to hormone free half-strength MS medium, over 83.3 % of the
shoots developed roots, and all plantlets could acclimatize and establish in soil. The protocol has been successfully applied
to eight genotypes, with regeneration frequencies ranging from 63.8 to 82.5 %. 相似文献
15.
We developed a rapid mutagenesis method based on a modification of the QuikChange® system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 – 22 bp to achieve melting temperatures greater than 80°C. 2: the final concentrations of both primers were increased to 5–10 ng/µl and the final concentration of template to 1–2 ng/µl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest. 相似文献
16.
Leukocyte endothelial cell interaction is a fundamentally important process in many disease states. Current methods to analyze
such interactions include the parallel-plate flow chamber and intravital microscopy. Here, we present an improvement of the
traditional intravital microscopy that allows leukocyte-endothelial cell interaction to be studied from the time the leukocyte
makes its initial contact with the endothelium until it adheres to or detaches from the endothelium. The leukocyte is tracked
throughout the venular tree with the aid of a motorized stage and the rolling and adhesive behavior is measured off-line.
Because this method can involve human error, methods to automate the tracking procedure have been developed. This novel tracking
method allows for a more detailed examination of leukocyte-endothelial cell interactions.
Published: August 27, 2004. 相似文献
17.
Teleost gut associated lymphoid tissue (GALT) consists of leucocyte populations located both intraepithelially and in the
lamina propria with no structural organization. The present study aims to assess different protocols for the isolation of
GALT cells from an important fish species in the Mediterranean aquaculture, the gilthead seabream. Mechanical, chemical and
enzymatic treatments were assayed. Nylon wool columns and continuous density gradients were used for further separation of
cell subpopulations. Light microscopy and flow cytometry showed that the highest density band (HD) consisted of a homogeneous
lymphocytic population, whereas the intermediate density band (ID) corresponded to epithelial and secretory cells and some
lymphocytes. Respiratory burst activity of total cell suspensions revealed very low numbers of potential phagocytic cells,
reflecting results from light microscopy and reports in other teleost species. The present data set up the basis for future
functional characterization of GALT in seabream. 相似文献
18.
D. Skálová M. Dziechciarková A. Lebeda E. Křístková B. Navrátilová 《Biologia Plantarum》2008,52(4):775-778
Embryo-rescue was used to facilitate interspecific hybridization of Cucumis anguria L. and C. zeyheri Sond. Embryos were excised from developing fruits at one week intervals for six weeks after hand pollination. Medium containing
coconut water was the most suitable for initial germination, and a medium with ascorbic acid was the best for embryo development
and plant recovery. Viable plants were obtained from embryos and these plants showed morphological characteristics different
from both parents. The analysis of the leucine aminopeptidase (LAP) locus revealed three hybrid types, H1.1, H1.2 and H2. 相似文献
19.
Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated
interference (RNAi) in cultured cells ofDrosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the
dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing
it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved
in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis
of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated
equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness
of the technique.
Published: June 15, 2003 相似文献
20.
In the present study, in vitro regeneration system for a recalcitrant woody tree legume, Leucaena leucocephala (cvs. K-8, K-29, K-68 and K-850) from mature tree derived nodal explants as well as seedling derived cotyledonary node explants
was developed. Best shoot initiation and elongation was found on full-strength Murashige and Skoog (MS) medium supplemented
with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 100 mg dm−3 glutamine, 20.9 μM N
6-benzylamino-purine (BAP) and 5.37 μM 1-naphthalene acetic acid (NAA). Rooting was induced in half-strength MS medium containing
2 % (m/v) sucrose, 100 mg dm−3 myoinositol, 14.76 μM indole-3-butyric acid (IBA) and 0.23 μM kinetin. The cultivar K-29 gave the best response under in vitro conditions. Rooted plantlets were subjected to hardening and successfully transferred to greenhouse. Further, somatic embryogenesis
from nodal explants of cv. K-29 via an intermittent callus phase was also established. Pronounced callusing was observed on full-strength MS medium containing
3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 40.28 μM NAA and 12.24 μM BAP. These calli were transferred to induction medium and maximum number of globular
shaped somatic embryos was achieved in full-strength MS medium fortified with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 15.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5.0 μM BAP and 1.0 mM proline. Moreover, an increase in endogenous
proline content up to 28th day of culture in induction medium was observed. These globular shaped somatic embryos matured in full-strength MS medium
with 3 % (m/v) sucrose, 100 mg dm−3 myoinositol, 10.0 μM BAP, 2.5 to 5.0 μM IBA and 0.5 mM spermidine. 相似文献