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Heat shock proteins stage

Heat shock proteins stage

Detail Description

Heat shock proteins (HSP) are a family of proteins that are produced by cells in response to exposure to stressful conditions. HSPs are found in virtually all living organisms, from bacteria to humans. HSPs function primarily as molecular chaperones, facilitating the folding of other cellular proteins, preventing protein aggregation, or targeting improperly folded proteins to specific degradative pathways. Some HSPs are expressed at low levels under normal physiological conditions but show dramatically increased expression in response to cellular stress, others are constitutively expressed. Specific HSPs play a role in regulating apoptosis by interacting directly with key components of the apoptotic pathway.

 

Studies on stress proteins (HSP) include gene expression and the complex signal transduction pathways that control the regulation of stress genes; other studies focus on the role of stress proteins as molecular chaperones that regulate various aspects of protein folding and transport and on the role of stress proteins in human disease.

 

HSP heating and cooling stage (HSP H&C stage) in the 5-45°C range (+/-0.3°C accuracy) adapts the original designed for ultra-fast qPCR military applications to create the world’s fastest heater-cooler for live-cell imaging microscopy now controlling in real-time your favorite molecular processes to observe dynamic events linked to temperature on live and discover unthought mechanisms. You can change the temperature in less than 10 seconds, regardless of the delta between the two temperatures. It can be mounted to any upright and inverted microscope models very easy to install and control by user friendly software.

Examples of HSP applications:

1. The developmental cycle of drosophila depends on temperature, being of 8.5 to 9 days at 25°C and being slowed down at higher and lower temperatures because of suboptimal growth and is used as an animal model for research on human disease, including cancer, neurological disorders, and rare diseases. Fruit flies are heat shocked at 35–37 °C, whereas human or mouse cells are induced to make Hsps when the temperature is raised to several degrees above their normal body temperature of 37 °C, for instance 41–42 °C.

2. Functional studies on thermosensory neurons in C. elegans

3. The study of temperature sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. Ts mutations are typically missense mutations, which retain the function of a specific essential gene at standard (permissive) low temperature, lack that function at a defined high (nonpermissive) temperature, and exhibit partial (hypomorphic) function at an intermediate (semipermissive) temperature. Such mutants make possible the analysis of physiologic changes that follow controlle inactivation of a gene or gene product by shifting cells to a non-permissive temperature, offering a powerful approach to the analysis of gene function. While an extensive RNAi-based screen has identified many essential C. elegans genes required for proper development of the adult gonad, very little is known about the regulation of membrane dynamics during adult oogenesis. Given the known requirements for membrane trafficking in both germline development and eggshell formation, experiment to screen a large collection of temperature-sensitive, embryonic lethal C. elegans mutants is done for defects in both gonad development and eggshell production. TS mutant strains of C. elegans were maintained at the permissive temperature of 15°C. Embryonic phenotype characterization was performed by selecting L4 hermaphrodites grown at the permissive temperature and moving them to 26°C for a minimum of 6 hr before dissecting embryos into water. The dye permeability assay used to determine permeability barrier defects is described in Carvalho et al. Postembryonic phenotype characterization was performed by moving synchronized populations of L1 larvae to 26°C for several (3–5) days and scoring adult germline morphology.

4. Yeast cells:

  • Fission yeast temperature-sensitive mutants has contributed to the discovery of new genes involved in yeast biology. Temperature sensitive mutations are often missense mutations. Mutated proteins retain their functionality at low, permissive, temperature but at higher, restrictive temperature, they become inactive. Fission yeast biology is intrinsically sensitive to temperature.

  • Fission yeast cells were grown at 25°C for live imaging. Cells were shifted from 25°C to 5°C in less than 10 sec, inducing microtubule depolymerization. Microtubulerepolymerization occurred upon shifting back the temperature to 25°C.

  • Fission yeast cdc25-22 cells were were blocked at the G2/M transition for 4 hours at restrictive temperature (36.5 °C). Synchronous release into the cell cycle was achieved by a quick shift (10 seconds) into permissive temperature (25°C). Septation index is an indicator of the progression of fission yeast cells into the cell cycle. High synchrony was obtained as well when blocking nda3-KM311 mutants at 16°C and measuring septation index upon release to 32°C.

  • Recorded timelapses of exocytosis of yeast cells between 10°C and 30°C were analyzed via kymographs to determine the lifetimes of exocytic events at different temperatures.

5. Heat shock proteins have been recently recognized for their potential role in regulating immune responses.

- They are known to bind, in a noncovalent fashion, to immunogenic peptides.

- When tumor cells are exposed to high temperature, heat shock protein-peptide complexes are presented on the cell surface. These complexes can be recognized by antigen-presenting cells (dendritic cells) via major histocompatibility complex (MHC) class I molecules. Once dendritic cells have received this type of stimulus, they migrate to lymph nodes, where they prime T-cell lymphocytes to be cytotoxic toward cells that express the peptide-heat shock protein complex. High temperature has been shown to enhance the rate of dendritic cell migration.

- Heat shock proteins also induce dendritic cell maturation and proinflammatory cytokine release.

- Cell membrane localization of heat shock proteins also activates the innate immune system by activating natural killer (NK) cells.

6. Other sources of cellular stress such as viral infection, fever, hypoxia, and radiation exposure have been shown to up-regulate heat shock proteins as well. Because this process appears to occur naturally, there have been efforts to exploit the use of high temperature to produce tumor-derived vaccines and to augment the in vivo response to such vaccines. high temperature has also been reported to up-regulate a number of proinflammatory cytokines and adhesion molecules that facilitate immune cell trafficking across endothelial cells to gain access to the tumor interstitium. Additionally, shed heat shock proteins, with or without associated peptides, may act as chemokines to attract immune cells (particularly, macrophages) toward a region of tissue that has undergone heat stress. Also, HSP expression is modulated by different stimuli involved in all steps of atherogenesis including oxidative stress, proteolytic aggression, or inflammation.

 

Specific package and methods for different applications:

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