Essay: Tight junctions (intercellular junction)

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  • Subject area(s): Science essays
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  • Published: June 18, 2021*
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  • Tight junctions (intercellular junction)
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Introduction:

Tight junctions serve to maintain and protect the layers of intestinal epithelium from the external environment. One unique thing of this intercellular junction is that it not only regulates extracellular transport but also, it contains basal and apical compartments that fabricate selectively permeable obstruction in the paracellular pathway. The structure of these protein complexes prevent harmful pathogens and macromolecules from harming the intestinal cavity (Berkes et al., 2007).

Bacteria arise from food borne illnesses that can greatly affect the intestinal tract – increasing the chances of various toxins that can lead to symptoms like nausea and diarrhea. A great example would be consuming food that is not fully cooked properly, which is a consequence of food poisoning. There are various times where the intestinal tract has problems with its permeability, in which it is unable to distinguish essential molecules with bacteria it comes into contact with. This disturbance can inhibit functionality in the aspect of the TJ selectivity and can lead to bigger problems such as human diseases.

Any disruption in the cytoskeletal area or interaction with tight junction proteins such as claudins and occludins. Both TJ proteins are responsible for communicating with cell to cell interactions by their extracellular loops. Essentially, occludins regulate the signaling. While, claudins play a crucial role in the regulation in the permeability paracellular barrier, but some contain receptors specifically for bacterial toxins like Clostridium Perfringens Enteroxtin (CPE) that produce strains. The structural component of CPE’s has a binding domain at the C terminus and a localized active N terminus that corresponds with its essential role as a enterotoxin (Berkes et al., 2003). Claudin-1 and claudin-4 can interact with CPE fragments at the COOH terminus, thus causing structural changes in the tight junctions (C-CPE). A morphological change that exists is when the protein complex is degraded and bound onto claudins and are localized at the membrane area assembling an active pore. Degradation can severely affect the functionality of the tight junctions making them inactive. Furthermore, electrolytes are loss and a large flow of intestinal fluid can occur when an active pore forms and disrupts the selective barrier. With the use of a transepithelial electrical resistance assay (TEER), it can reveal any specific disruptions and bacterial pathogens that increase paracellular flux (Spitz et al.,1995).

As stated earlier, CPE damage causes food poisoning also known as a gratrointestinal disease; therefore, is the root cause of diarrhea and inflammation. It is important to know in this study how bacteria affects intestinal epithelium and how it is responsible for such diseases. Additionally, what steps are needed in order to prevent future occurring problems of food-borne pathogens. We hypothesize that bacteria is the source of inhibiting tight junction function by displacing occludins snf degrading claudins to form active proes, which in turn causes food poisoning symptoms.

Experimental Design:

The TER will be used to determine the function of TJ and paracellular pathway flux under three conditions: MFCK-I cells that are treated with C-CPE, MDCK-I cells treated with bacteria x (Bx), and lastly a baseline MDCK-I cell that gets no treatment. The significance of this method is to assay the plasma membrane epithelial resistance and functional barrier. A disrupted barrier in the tight junctions would be inefficient and will have very low resistance. Meanwhile the TER will measure how much resistance is occurring, the measurements of the paracellular pathway will be able to specify the size of molecules, especially larger ones, that are passing through the barriers (Stevenson et al., 1988).

Madin-Darby canine kidney strain I (MDCK-I) epithelial cell will be essential in carrying out this experiment because of the cultured components that contain Tjs like claudin-4 and claudin-1 (Stevenson et al., 1988). As stated earlier, importance of C-CPE’s is that they can degrade and bind onto target proteins. Meaning that when MDCK-I cells are incubated and bonded onto claudin -4, degradation will be apparent, thus disrupting the function of tight junctions. MDCK-I will be attained from the American Type Culture Collection and grown on a plate at a certain density (Sonoda et al., 1999). Cells will be measured by their conditions consistently and closely through a EVOM Epithelial Voltmeter. The TER will be measured after a Bx or CPE has been incorporated with the target cell for eight hours. This process will be given with various Bx doses for five times (Min et al., 2015).

The same controls will be used to measure the paracellular pathway flux in addition to incorporating an impermeable membrane tracer in each control. Fluorescein isothiocyanate-dextrals (FITC-dextrans) from Sigma Chemical Company are the specific tracers that will be used as fluorescent probes to observe the strength of permeability. Before conducting this portion of the experiment, MDCK-I cells will be prepared the same way as mentioned earlier, but for 24 hours. This will allow 4k, 10k, and 40k masses to be dissolved fully in a P buffer. The 4k FITC-dextran that contains minimal amount of P buffer will be placed in the apical region, while the larger amount of P buffer will be placed in the basal region. This step will be repeated for the 10k and 40k masses to avoid bias and efficient results. A fluorometer will be used to check the intensity of the fluorescence that takes place after three hours (Sonoda et al., 1999). An unpaired t-test will be performed and if the results conclude in an increase of paracellular pathway flux and a decrease in TER, then this proves that the function of the tight junctions have been interfered.

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