Chemistry

Nanotechnology is a rapidly growing field of science, which is particularly interesting for researchers since the early 90s of the last century has become a vital part of the modern technology. Nanomaterials are increasingly becoming a part of our regular lives (Hill and Julang, 2017). They are characterized by new characteristics that differ from those existing at the macro materials.

Therefore, nanomaterials are used in innovative products and processes (Fariq et al., 2017). Recently, application of nanomaterial extensively increased, because of high demands for the production of such materials. Classically, the nanoparticles are produced by chemical and physical methods (Stark et al., 2015), as these methods are costly, toxic and non-eco-friendly, scientists are looking forward to synthesizing low cost, non-toxic, eco-friendly nanoparticles (Singh et al., 2016; Sangeetha et al., 2017).

Biogenic synthesis of nanoparticles using organisms such as bacteria, fungus and plants emerged as a suitable alternative to the more complex physical and chemical synthetic procedures (Singh et al., 2016). Fungi have some advantages over other microorganisms because they are easy to handle, their nutritional requiems are simple, have a high wall-binding capacity, as well as their capabilities for the intracellular metal uptake (Bhattacharjee et al., 2017).

Silver nanoparticles are among the most widely-used metals, and are used as antimicrobial agents, water treatment, textile industries, sunscreen lotions …etc. (Raja et al., 2012). We assume that each kind of fungi could have its own machinery to reduce the metals through a production of a group of enzymes.

So, the synthesized nanoparticle by each kind of fungi could show a specific characteristic including definite shape and size that makes them effective in many applications, especially as antimicrobial agents. Therefore, the main aim of the present study depends on the wide survey of many fungal species that were isolated from Saudi habitats to investigate their potentiality to synthesize the silver-nanoparticles.

The physical characteristics of the newly produced nanoparticles will be studied using accurate and fine techniques including the X-Ray Diffraction (XRD), Fourier Transform InfraRed (FT-IR) and the transition electron microscopy (TEM). The antibacterial activity of the characterized silver nanoparticles will be studied against many medically-important bacteria, especially that are involving in human diseases such as Escherichia coli, Proteus vulgaris, Staphylococcus aureus and Pseudomonas aeruginosa.

The expected results of this research are the obtaining of new fungal species that have the ability to produce new AgNPs with the specific characteristic that could be used and a new antibiotic or antibacterial agents to control the bacterial infections especially those have a resistance to the classical chemical antibiotics

Excretion is the removal of waste products of metabolism from our body system.

Contents

Excretory functions

2 Component organs

2. 1 Lungs

2. 2 Kidneys

2. 3 Ureter

2. 4 Urinary bladder

2. 5 Urethra

3 Urine formation

4 Reasons For Excretion

The excretory system removes metabolic and liquid toxic wastes as well as excess water from the organism, in the form of urine, sweat, urea or bile. This is important so as to help maintain balance within the organism and prevent damage to m the body.

As your body performs the many functions that it needs in order to keep itself alive, it produces wastes. These wastes are chemicals that are toxic and that if left alone would seriously hurt or even kill you. For example, as your cells break down amino acids, they produce a dangerous toxin known as urea. The cells of your body excrete this urea into your blood Excretory organs Skin Skin is an excretory organ. The regulation of body temperature causes it to produce sweat which contain urea surplus water, salts and other waste .

Lungs

One of the main functions of the lungs is to diffuse gaseous wastes, such as carbon dioxide, from the bloodstream as a normal part of respiration

Kidneys

The kidney’s primary function is the elimination of waste from the bloodstream by production of urine. They perform several homeostatic (metabolic balance) functions such as:

1. Maintain volume of extracellular fluid
2. Maintain ionic balance in the blood
3. Maintain pH concentration of the blood.
4. Excrete toxic metabolic by-products such as urea, ammonia, and uric acid.

The way the kidneys do this is with nephrons inside the glomeruli. There are over 1 million nephrons in each kidney, these nephrons act as filters inside the kidneys. The kidneys filter needed materials and waste, the needed materials go back into the bloodstream, and unneeded materials becomes urine and is gotten rid of. In some cases, excess wastes crystallize as kidney stones. They grow and can become a painful irritant that may require surgery or ultrasound treatments. Some stones are small enough to be forced into the urethra Urine formation

Within the kidney, blood first passes through the renal artery to the capillary formations called a glomerulus and is collected in the Bowman’s capsule which filters the blood from its contents—primarily food and wastes. After the filtration process, the blood then returns to collect the food nutrients it needs, while the wastes pass into the collecting duct, to the renal pelvis, and to the ureter, and are then secreted out of the body via the urinary bladder. What is a kidney stone? A kidney stone is a hard, crystalline mineral material formed within the kidney or urinary tract.

Kidney stones are a common cause of blood in the urine (hematuria) and often severe pain in the abdomen, flank, or groin. Kidney stones are sometimes called renal calculi. The condition of having kidney stones is termed nephrolithiasis. Having stones at any location in the urinary tract is referred to as urolithiasis, and the term ureterolithiasis is used to refer to stones located in the ureters.

The Human Skin

19. 3 Skin and Lungs as Accessory Excretory Organs

In addition to the urinary system, the skin, lungs and liver of vertebrates are accessory excretory organs.

1. Skin:

Human skin possesses glands for secreting two fluids on its surface, namely sweat from the sweat glands and sebum from sebaceous glands.

Sweat is a watery fluid containing in solution primarily contains sodium-chloride, urea,and excess water.

Vertical Section of the Skin Sebum is a wax-like secretion which helps to excrete some lipids such as waxes, sterols, other hydrocarbons and fatty acids on the skin.

2. Lungs:

Lungs which are the main respiratory organs of vertebrates, help to eliminate the entire volume of carbon dioxide produced in the body, as well as some moisture, during expiration.

The lungs maintain the blood-gas homeostasis through elimination of carbon dioxide. When lungs fail to eliminate enough carbon dioxide, the kidneys attempt to compensate. They change some of the carbon dioxide into sodium bicarbonate, which becomes part of the blood buffer system.

Summary

1. Excretion is the removal of nitrogenous waste products from the body.
2. Kidneys are the most important excretory organs of mammals. Through filtration, reabsorption and active transport, waste is remove, but kidneys conserve substances useful to the organisms.
3. In general, kidneys regulate the intake and the outflow of water and salts in the blood and help to maintain homeostasis.
4. Regulation of kidney function is achieved by certain hormones such as antidiuretic hormone, aldosterone and angiotensin.
5. Skin and lungs also act as accessory excretory organs.

Nitrogenous Waste

The liver also works by breaking down nitrogenous waste. Your different metabolic processes in your body are important to keep you alive, but they also create waste in the form of nitrogen.

The body must then transport the nitrogen waste out of your body. Your liver converts the nitrogenous waste into urea, which is then transported out of the body when you urinate.

Bile

Once toxins have been converted, the liver deposits the deactivated toxins into the bile. The liver excretes bile as a digestive aid for harder to digest fats, as well as a way to transport toxins out of the body. The bile is then stored in the gallbladder till needed. Once it is needed, it moves the duodenum and helps with the digestion and excretory process.

Human embryonic stem (hES) cells have the unique capability of differentiating into all cell types, leading to the development of an entire organism. As the integrity of ES cells is critical for the developing embryo, these cells have likely evolved mechanisms that detect and respond rapidly to adverse stimuli.

Indeed, hES cells have been shown to be highly sensitive to DNA damage, but the molecular mechanisms underlying this rapid death remain unclear. Caspases are critical mediators of apoptosis in mammalian cells, and a key protein that controls their activation is Bax, a proapoptotic member of the Bcl-2 family. While the main components of the apoptotic pathway have been identified, exactly how this pathway is regulated in various primary cells remains unclear.

Here, we examined the apoptotic pathway in hES cells and report a unique mechanism engaged by hES cells that can prime them to undergo rapid apoptosis inresponse to genotoxic damage.To visualize GFP-tagged Bax, 3-day colonies of hES cells were transfected with 2 mg of hBaxC3-EGFP (Addgene) with FuGENE HD transfection reagent.

The process of introducing nucleic acids into eukaryotic cells by nonviral methods is defined as transfection. Using various chemical, lipid or physical methods, this gene transfer technology is a powerful tool to study gene function and protein expression in the context of a cell. Development of reporter gene systems and selection methods for stable maintenance and expression of transferred DNA have greatly expanded the applications for transfection.

Assay-based reporter technology, together with the availability of transfection reagents, provides the foundation to study mammalian promoter and enhancer sequences, trans-acting proteins such as transcription factors, mRNA processing, protein:protein interactions, translation and recombination events (Groskreutz and Schenborn, 1997).

Transfection is a method that neutralizes or obviates the issue of introducing negatively charged molecules (e.g., phosphate backbones of DNA and RNA) into cells with a negatively charged membrane. Chemicals like calcium phosphate and DEAE-dextran or cationic lipid-based reagents coat the DNA, neutralizing or even creating an overall positive charge to the molecule.

This makes it easier for the DNA:transfection reagent complex to cross the membrane, especially for lipids that have a “fusogenic” component, which enhances fusion with the lipid bilayer. Physical methods like microinjection or electroporation simply punch through the membrane and introduce DNA directly into the cytoplasm. Here we describe the striking observation that healthy undifferentiated hES cells maintain Bax in its preactivated state at the Golgi.

This is in contrast to other cell types in which Bax is typically present in an inactive form in the cytosol. Our results also highlight the fact that the apoptotic machinery undergoes dynamic changes even at early stages of differentiation.While undifferentiated hES cells have constitutively active Bax and undergo rapid apoptosis in response to DNA damage, just 2 days of differentiation induced significant changes suchthat Bax was no longer active, and the cells were no longer highly sensitive to DNA damage.

This could be manifested with even greater complexity in vivo as cells during early embryogenesis undergo rapid proliferation and differentiation.

The protein fraction from 70 percent saturation of recrystallised ammonium sulphate was found to have the maximum protein content (19.6 mg/g flower) and hence it was selected for further studies and is abbreviated as PAF in the present study.

Characterization of the selected PAF by Native PAGE and SDS PAGE

The selected PAF was characterized by column chromatography. This showed a single peak and was further characterized in native PAGE and SDS page. The results are shown in Plate the SDS-PAGE analysis of the ammonium sulphate precipitated protein extract showed 15 different protein bands with good visibility in CBB R250 staining method.

• A – Standard Protein Markers;
• B – PAGE;
• C– SDS

PAGE Each fraction showed a number of major and minor bands indicating several proteins. Of the several bands obtained in 70 per cent saturation of ammonium sulphate, the major band alone was eluted. In order to find out the presence of subunits in this band, it was further subjected to SDS PAGE.

The results showed one major protein indicating the absence of subunits. The molecular weight of this protein was found to be 99 KD when compared to the standard molecular markers.

Fifty percent effective dose of selected protein fraction of Plumeria alba The free radical scavenging capacity of PFC was tested by its ability to bleach the stable DPPH.

The DPPH (2,2 diphenyl -1- picryl hydrazyl) radical scavenging activity was carried out using different concentrations of PFC (Figure). The radical scavenging activity was found to be dose dependent. Figure Percentage Free Radical Scavenging Activity of Selected protein fraction of Plumeria alba The protein fraction of Plumeria flower extract showed the dose dependent DPPH radical scavenging activity.

From the graph, the 50 percent effective concentrations of these were found to be 28 µg and 35 µg and used in the further studies. Free radicals and their scavenging systems play important role in the healing of normal and delayed types of wounds. The dose response curve of DPPH radical scavenging activity

Ascorbic acid Plant sampleDPPH RadicalScavenging 9(%) of the extract and standards showed that at the highest concentration (0.5mg ml-1) the scavenging effect of the methanolic extract reached 9.3% (Afolayan et al., 2008). Shyuret al. (2005) also reported that the scavenging activity for free radicals of 1,1diphyryl-2-picrylhydrazyl (DPPH) has been widely used to evaluate the antioxidant activity of natural products from plants.

The antioxidant activities of the leafy vegetables of India were measured in different systems of assays such as DPPH assay, super oxide radical scavenging assay, hydroxyl radical scavenging assay and lipid peroxidation assay and IC50 values were calculated (Dasgupta and De 2007).

Matrix-Assisted Laser Desorption/Ionization – Time of Flight Mass Spectra (MALDI-TOF MS)

MALDI-TOF mass spectra were used for the analysis of peptide mass fingerprinting and MS/MS ion search identification of the in-gel trypsin digested protein fragments (Figure 5), selected peptide masses were submitted to Mascot (http://www.matrixscience.com) for SwissProt databases search. There was no conclusive match in peptide mass fingerprinting, since MS/MS ion search program was selected for further identification.

The MS/MS ion search in the NCBIProt database revealed that, WRKY transcription factor WRKY24-like isoform X1 with the protein score of 81 (Protein score is 10*Log(P), where P is the probability that the observed match is a random event. Protein scores greater than 80 are significant

Health Cl Extremely flammable Cl Very toxic by inhalation: syrnptoms usually occur within a few hours of exposure D Phosphine is irritating to the mucous membranes of the nose, mouth, throat and espiratory tract 0 Inhalation may result in weakness, chest tightness and pain, dry mouth, cough, sickness, vomiting, diarrhoea, chills, muscle pain, headache, dizziness, ataxia, confusion and lung damage. These symptoms may develop 2-3 days after exposure 0 Severe poisoning may result in increased heart rate, low blood pressure, convulsions, coma, heart damage and death.

These symptoms usually within 4 days but may be delayed up to 1-2 weeks C] Exposure to the eyes or skin may cause Irritation 0 Long-term exposure may cause anaemla, bronchltls, gastrointestinal disorders, peech and motor problems, toothache, weakness, weight loss, swelling and damage of the jaw bone and spontaneous fractures 0 Phosphine has not been associated with cancer 0 Phosphine is not likely to cause reproductive or developmental effects Environment 0 Dangerous for the Environment 0 Inform Environment Agency of substantial release incidents Prepared by L Assem & M Takamiya Institute of Environment and Health Cranfield University 2007 Version 1 Background Phosphine is a colourless gas, which is slightly heavier than air.

It usually smells of garlic or rotting fish due to the presence of ontaminants but pure phosphine is odourless. is extremely flammable and highly reactive with air, copper and copper-containing alloys. exposed to higher levels of phosphine, although occupational incidents involving exposure to phosphine are rare, and safety levels are in place to protect employees. Phosphine is rarely found in nature. Small amounts can be formed during the breakdown of organic matter, although it is rapidly degraded. Phosphine is released into the air via emissions from various manufacturing processes and from the use of metal (magnesium, aluminium and zinc) phosphide umigants and pesticides, which release phosphine on contact with water or acid.

The major uses of phosphine are as a fumigant during the storage of agricultural products such as nuts, seeds, grains, coffee and tobacco, and in the manufacture of semi-conductors. Phosphine is also used in the production of some chemicals and metal alloys and is an unintentional by-product in the illegal manufacture of the drug methamphetamine. Inhalation is the most likely route of exposure to phosphine, although ingestion of metal phosphides may also occur. Symptoms are non-specific and include irritation of the espiratory tract, headaches, dizziness, abdominal pain, sickness, and vomiting. convulsions, damage to the lungs, heart, liver and kidney, and death. Long-lasting effects of single dose exposure are unlikely, most symptoms clearing within a month.

Long-term exposure to phosphine, while unlikely to occur, can cause bronchitis, gastrointestinal, visual, speech and motor problems, toothache, swelling of the Jaw, anaemia and spontaneous fractures. Children exposed to phosphine will have the same symptoms of poisoning as adults. Phosphine is not likely to cause harm to the nborn child as acute effects are not known to cause developmental effects. Phosphine is rapidly broken down in the environment and it is very unlikely that the general population will be exposed to sufficient levels of phosphine to cause health effects. However, people may be exposed to very small amounts of phosphine present in air, food and water. Phosphine has not been associated with cancer and has not reviewed by the International Agency for Research on Cancer.

Workers employed as fumigators, pestcontrol operators, transport workers and those involved in the production or use of hosphine and metal phosphides (welding, metallurgy, semi-conductors), may be General information: Page 2 of 5 PHOSPHINE – GENERAL INFORMATION Production and Uses Phosphine is present in emissions from some industrial processes such as the manufacture of some chemicals and metal alloys of metal phosphides) and as a catalyst and in the production of polymers The main uses of phosphine are as a chemical dopant in the manufacture of semiconductors for the electronics industry, and in the fumigation (in the form of metal phosphides) of stored agricultural products such as cereal grains and tobacco. Phosphine is also used as a condensation catalyst and in the manufacture of some polymers. Zinc phosphide is used as a rodenticide in the form of a pellet or as a paste mixed with food. Small amounts of phosphine are produced in the production of chemicals such as phosphonium halide and acetylene gas. General information: Page 3 of 5 Frequently Asked Questions What is phosphine? Phosphine is a colourless gas which is highly flammable and explosive in air.

Pure phosphine is odourless, although most commercially available grades have the odour of garlic or decaying fish. Small amounts of phosphine can occur naturally, formed uring the anaerobic degradation of organic matter. Phosphine is corrosive towards metals, in particular copper and copper-containing alloys. What is phosphine used for? A major use of phosphine is as a semi-conductor doping agent by the electronics industry. Metal (aluminium, magnesium and zinc) phosphides, which release phosphine on contact with moisture and acid, are used as rodenticides and fumigates during storage of agricultural commodities such as grain e. g. cereals, and tobacco. Phosphine is also used as a catalyst and in the production of polymers.

How does phosphine get into the environment? Small amounts of phosphine occur naturally during the decomposition of phosphorouscontaining organic matter e. g. in marsh gas. Emissions and effluents from the manufacture of some chemicals and metal alloys, as well the production or use of phosphine and metal phosphides (welding, metallurgy, semi-conductors, rodenticides and fumigants), release phosphine into the air. How will I be exposed to phosphine? It is unlikely that the general population will be exposed to significant amounts of phosphine, since it is degraded quickly in the environment; the half-life of phosphine in the air is about one day or less.

However, people may be exposed to very small amounts by inhaling air, drinking water and eating food containing phosphine. Workers involved with industries and processes where phosphine is used, e. g. fumigation and pest control, may be exposed to higher levels of phosphine. People living nearby sites where phosphine is being used may also be exposed to small amounts of phosphine in the air. Phosphine gas does not present a risk of secondary contamination, although solid phosphides may pose some risk. Absorption though the skin is not considered a significant route of exposure. If there is phosphine in the nvironment does not always lead to exposure. Clearly, in order for phosphine to cause any adverse health effects you must come into contact with it.

You may be exposed by breathing, eating, or drinking the substance or by skin contact. Following exposure to any chemical, the adverse health effects you may encounter depend on several factors, including the amount to which you are exposed (dose), the way you are exposed, the duration of exposure, the form of the chemical and if you are exposed to any other chemicals. Exposure to phosphine or metal phosphides can be irritating to the respiratory tract nd can cause weakness, chest pain and tightness, dry mouth, cough, sickness, vomiting, diarrhoea, chills, muscle pain, headache, dizziness, ataxia and confusion. Severe cases may lead to lung damage, convulsions, damage to the heart, liver and kidney, and death.

General information: Page 4 of 5 Long-term exposure to low levels of phosphine can cause anaemia, bronchitis, gastrointestinal problems, visual, speech and motor problems, toothache, swelling of the Jaw and spontaneous fractures. Can phosphine cause cancer? The Governmental Committee on Mutagenicity recently reviewed the available data n carcinogenicity of phosphine and concluded that it did not cause cancer in animal studies. Phosphine has not been reviewed by the International Agency for Research on Cancer (‘ARC), and the US Environmental Protection Agency (US EPA) considers phosphine as not classifiable as to human carcinogenicity, due to inadequate animal studies and a lack of human tumour data. Does phosphine affect children or damage the unborn child?

Children who ingest metal phosphides or inhale phosphine gas are expected to have similar symptoms as adults, e. g. sickness, vomiting, headache, dizziness, in severe ases leading to damage to the lungs, heart, liver and kidney and death. There is no evidence to suggest that maternal exposure to phosphine affects the health of the unborn child. What should I do if I am exposed to phosphine? It is very unlikely that the general population will be exposed to a level of phosphine high enough to cause adverse health effects. This document from the HPA Centre for Radiation, Chemical and Environmental Hazards reflects understanding and evaluation of the current scientific evidence as presented and referenced in this document.

Human embryonic stem (hES) cell has a unique ability of differentiating into all cell types, which leads to the development of the whole organism. As the integrity of ES cells is crucial for the developing embryo, these cells have likely evolved a mechanism that will detect and respond to adverse stimuli.

Indeed, hES cell has been shown to be highly sensitive to DNA damage, but the molecular mechanisms underlying this rapid death remain unclear. Caspases are critical mediators of apoptosis in the cells of mammals, and is considered a key protein that is responsible for controlling their activation is Bax, a Bcl-2 family proapoptotic member.

While the main components of the apoptotic pathway have been known and identified, exactly how this pathway is functioning and regulated in various primary cells is still unclear. The study examined the apoptotic pathway in the hES cells and also reported a unique mechanism of hES cells that can help them to undergo apoptosis in response to the genotoxic damage.

To visualize GFP-tagged Bax, the 3-day colonies of hES cells were transfected with 2 mg of hBaxC3-EGFP from Addgene with FuGENE HD transfection reagent from Roche. Transfection is the process of by which nucleic acids is introduce to the eukaryotic cells by methods that are nonviral. With the help of some various methods such as chemical, physical, lipid methods, this technology of gene transfer technology is a very powerful tool to investigate the gene function and the expression of protein of a cell.

Assay-based reporter technology, and with the availability of transfection reagents will actually give and provides the foundation necessary to study sequences of mammalian promoter and enhancer, the trans-acting proteins such as transcription factors, processing of mRNA, the interactions of different proteins, recombination, and translation events.

In general, transfection is a method or protocol used to neutralize or obviate the issue of introducing the negatively charged molecules such as phosphate backbone of DNA and RNA into the negatively charged membrane of the cells. In addition, chemicals such as calcium phosphate and cationic lipid-based reagents that coat the DNA, neutralize or even creates an overall positive charge to the molecule is also used.

This makes it easier for the DNA to transfection reagent complex to cross the said membrane, especially for lipids which has a fusogenic component that further enhances the fusion with the different lipid bilayer. Other methods such as physical methods like microinjection or electroporation have also been used that is simply punch through the membrane and will introduce DNA directly into the cytoplasm.

In this study, they describe the striking feature of the healthy undifferentiated hES cells, which maintain Bax in its preactivated state at the Golgi that is in contrast to other cell types. The results also highlight the fact that the apoptotic machinery undergoes a dynamic change even if its an early stages of differentiation.

Protein misfolding can be an aspect of several different human disorders, including cystic fibrosis, Alzheimer’s disease, and atherosclerosis. Many times, the misfolded protein is a membrane protein. In fact, a type of diabetes insipidus results from a mutation in the G-protein-coupled vasopressin 2 receptor that prevents the protein from making it to the cell membrane

• Describe how this type of receptor would normally get targeted to the membrane (from the beginning of translation)
• then propose one mechanism by which the mutation could cause a lack of proper targeting.

One of the most important protein’s target is G protein-coupled receptors, several signaling mechanisms depend on this type of receptor to change both internal and external stimuli to the intracellular responses. Basically, one of the G couple receptor subfamilies is G-coupled Vasopressin-2- Receptor (V2R), and this receptor is going through a strict quality control process at the endoplasmic reticulum, which presents the only correctly folded protein to gets through the secretory pathway.

The primary function of the V2 receptor is to activate the attached G protein that bound to the ? subunit then phosphorylated to GTP. The G protein couple receptor then activates the enzyme adenylate cyclase that catalyzes the reaction in the ER and forms cAMP from ATP. After that, cAMP acts as a second messenger and activates a protein kinase that phosphorylates the integral membrane proteins on the cell surface. Moreover, the secretory pathway organelles’ and the plasma membrane both are first introduced into the Endoplasmic reticulum, and the co-translationally proteins that can cross the ER are synthesized by the ribosome first then binds by chaperones to gets moves to the ER surface using GTP that allows them to move toward the receptor then release it.

The soluble proteins and the integral membrane proteins as I mentioned above, can be targeted through the ER and then translocated by the same mechanism.Further, several mutations occurring in the transmembrane region which affect the structure of the protein. These are multiple mutation sites, such as mutations occurring in the amino acid residues which was acting as a causative agent for human disease. Also, there are other mutations occurring on the single site position that will affect the translation mechanism and cause many human diseases like: cystic fibrosis, Alzheimer’s disease, and atherosclerosis that proves the function of V2R protein which plays important role in the translation during the protein folding process.

One of the mechanism that may a reason for lacking the proper target protein is when the mutations of the CFTR gene occurring and affect the function of the chloride ion channels and cause defect in the protein sequences which lead to the production of diseases and misfolded of the proteins that are unable to recognize their functional destinations. Otherwise, Lack of the stop signals is another issue that prevents the protein from getting into the surface of the ER, also called the non-stop decay cellular pathway, because lack of this stop signals prevents mRNA from synthesis and translate the proteins, these consider as a point mutation that inhibits the essential stop codons. 30 pts. Describe the experiment shown in figure 3 from the paper we discussed in class (Miller et al, 2003).

In your description, consider the following questions: Why did they do it? How did they do it? What did they learn?  Diagram the results that you would expect to see in Lanes T, 1, 2, 3, and 4, if the amino acid signal DID in the protein Gap1p was mutated to random amino acids and tell why; and (C) Give two possible (different) results that might occur if the amino acid signal LxxLE in Bet1p was mutated to the amino acids DID (which are the signal in Gap1p).

Diagram the results expected in Lanes T, 1, 2, 3, and 4 and explain why you predicted this result for each case.left20840701.A001.A4467225201739500The experiment was performed to study the role of cargo binding domain of Sec24p in the process of protein sorting. To perform this study, both mutant and wild types subunit Sec23/24p and Sec23/24L616W were harvested from microsomal membranes. The immunoblotting assay performed to quantify cargo molecules using radio-labeled secondary antibodies. Comparison of the mutant subunit with wild type illustrated omission of some molecules in mutant one, these molecules were cargo protein molecules. While some of them are packed in a way that similar to the wild-type. It meant that there were some signals that remained unaffected although mutation was there.

Unexpectedly, it was found that in these unaffected molecules of mutation in Sec23/24p, packaging was better than the wild type. Further, they found the proteins that were highly affected had Bet1p and Gap1p/Sys1p chimera, because they completely depending on the Sys1p di-acidic of COPII vesicles. If the amino acid signal DID in the protein Gap1p was mutated to random amino acids, the resulting bands would be seen as in figure 1. A because the amino acids might be present in all the lanes. And Gap1p is required for di-acidic motifs to fuse together with the COPII vesicles at the mutated domain. Also, Sys1 peptide is not involved in Sec 23/24p hence its mutation is not contained in a di-acidic motif. If the signal LxxLE was mutated to the DID, the expected Bet1p packaging would be shown in figure 2.

A as not be seen in lanes containing Bet1p because the amino acid signal LxxLE does not exit after mutating to amino acids DID. The second probability for the Bet1p would be expected to bind with the vesicle for packaging when it interacts with di-acidic motifs which is needed. Explain the experiment shown in Figure 5C from the Shen et al. (2018) paper on the phosphorylation of CDC25 that we discussed in class.

In addition, be sure to also address the following: A) why did they do this experiment (the central issue); B) what did they learn; and C) what is another control experiment they could have done? D) Would these results change if you added a constitutively active form of LKB1 to the reaction? If so, how and why? A) Overall, the main purpose of this paper is to explain how the division of cells is highly regulated such that cells that fail to pass some specific stage-based tests cannot advance to the proceeding stages.

In this particular experiment, HeLa cells were subjected to chemicals that are known to activate the enzyme AMPK. Also, the main catabolic processes that are involved to generate energy for cells to transition from G1/G2 were determined by the application of radiochemical approaches, the experiment required to approve how highly conserved cellular energy sensor can significantly delay mitosis entry and activation of AMP-activated protein kinase (AMPK). Further, Wee 1 family inactivates the cell cycle G2/M Phase which is controlled by Cdc3/cyclic B (mitotic cyclin-dependent kinase complex). They found that AMPK-dependents phosphorylation of CDC25C arranges a metabolic control point for the M-phase transition and the cell cycle phase G2.

Also, they learned that suppression of Wee 1 or acute induction partially reinstates mitosis ingress in the circumstance of activated protein kinase (AMPK). This experiment showed that when Phosphorylates Cdc25 is in a distributive and disordered state, it results into ultra-sensitivity in protein phosphorylation. For another control experiment, they might try exposing the U2OS (cell line) clones conditionally exhibiting Cdc25A etoposide. That will help to test whether Cdc25A degradation is significant for the G2 control point or not.D) If they add an active form of LKB1 to the reaction, the results will not change, because in cells LKBI activity inhibits AMPK activation in response to different stimulations. Also, LKBI is lost upon consistent isolation and therefore no effects of phosphates observed.20 pts.

Briefly describe the role of cyclin-CDK in the cell cycle and give an example of positive and negative control in this system. Also, describe how cell cycle regulation links to the stimulation of apoptosis at the molecular level. In the beginning, the cell cycle depends on many basic factors that control the regulation process starting from the signal transcription molecules, growth factors and the Cyclin Dependent Kinases enzymes include the checkpoints which control the transition process between the cell cycle phases by binding to the cyclin proteins CDKs then phosphorylate other proteins to transfer from one phase to another.

The role of transcription factors is to turn on the signals for gene expression, DNA replication, and cell divisions. As an example of CDKs, cyclin-dependent kinase 1 CDK1 is a cell division cycle protein homolog 2, that has a primary role in human cancer cells because CDK1 rather than any types of CDKs is fatal to the mutated version of MYC- dependent cancer that leads to a depletion of oncogenes like (Fos and Jun) in human cancer cells. Fos and Jun are combined forms of the transcription factor called AP1 that activates the delayed response genes such as cyclin D and CDK4. Recent studies reveal that the reason for the MYC breast cancer cells duplation is targeting CDF1 exhibit any other CDKs cell lines. Also, CDK1 inhibition can control and target the cancer cells in human and both phosphorylation and expression of MYC during the cell cycle process.

To initiate intracellular signaling pathways and stimulate the cell cycle entry, mitogens substance bind to the cell surface receptor with the activation form of GTPase Ras that activates MAP kinase cascade. That will lead to the expression of encoding gene of the transcription regulatory protein like MYC. Moreover, E2F transcription factor is a target for cyclin D and CDKs that stimulate proteins expression to initiate S phase, also E2F regulated by the tumor suppressor gene Rb (Retinoblastoma protein). At early G1 Phase, Rb protein combined with E2f to form the histone deacetylases protein that remains chromatic condensed, then cyclin kinase phosphorylates Rb protein, causing it to disassociate from E2F. That allows recruitment of histone acetylates, which decondense the chromatin and helps transcription complexes to form in G1 and S phase and to form a positive feedback.

For the negative control, if there are no growth factors present to stimulate the synthesis of Cyclin D in the new cell, Rb phosphorylated will turn off, and Rb will rebind to E2F, then the cell cycle will stop and that will lead to many negative results like prevent it to bind with DNA, or DNA damage and cells will return to the G0 phase. Apoptosis is a consequence of DNA damage; if the damage is not repaired, the cell cycle will divert towards apoptosis, otherwise, if the cell has DNA defects and fail to undergo apoptosis, that will change to the cancer cell. G1 phase regulators such as P53 and E2F are essential to promote the cell regulations and eliminate any damage or abnormal changes during the cell cycle.

P53 has a primary function to prevent any mutation in DNA during cell progression, so any defect in P53 will lead to cancer, and it’s usually mutated in cancer cells. Further, Rb protein considers as a tumor suppressor and also promotes apoptosis. Additionally, most human cancers have inactive Rb protein, either mutated Rb or the non-phosphorylated (inactivated form) of Rb protein.