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 WELCOME TO CELLULAR AND MOLECULAR BIOLOG Y! Welcome to the BHSc. Program! As a first-­‐›‡ƒ”…Ǥ•–—†‡–ǡ›‘—™‹ŽŽ„‡–ƒ‹‰ƒ…‘—”•‡‡–‹–Ž‡†Dz‡ŽŽ—Žƒ”ƒ†‘Ž‡…—Žƒ”
‹‘Ž‘‰›dzȋͳͲ͸ȌǤHere, we are providing a summary of the content covered during first term. Overall, the course will focus on intracellular and intercellular modes of communication. The two perspectives may seem unrelated at first; however, upon further progression of the course, you will discover that they are deeply interconnected. OVERVIEW: CELLULAR A ND MOLECULAR B IOLOGY Cellular communication is essential for the survival of multi-­‐cellular organisms as it facilitates the specialization of function. Dr. Rangachari covers the general mechanisms of extracellular communication. Additionally, Dr. Seidlitz focuses on the molecular aspectȄgene expression (intracellular communication) through DNA replication, transcription, and translation. Fundamentally, cellular communication refers to the processes by which cells recognize and respond to a variety of signals. Just like any other communication process, this comprises of the following steps: Steps of Cellular Communication Synthesis Ȉ A s ignal (any messenger molecule carrying biological meaning) is produced Storage (optional) Release Ȉ The messenger molecule is released Ȉ The messenger molecule could be stored or made on-­‐
demand, depending on their function Effect Ȉ The released molecule binds to receptors and elicits a response Termination Ȉ The response must be terminated in order to ensure that it does not last indefinitely In molecular communication, the same principles are applied to genetic expression processes: Molecular Communication: Genetic Expression DNA Replication Ȉ The signal (genetic information) is synthesized via DNA replication DNA Transcription & Translation Ȉ DNA stores genetic information Ȉ The signal (genetic information) is released into the cytoplasm by transcribing DNA into RNA, and then translated into proteins Proteins Ȉ Proteins produce a response by acting as enzymes or receptors that alter cellular mechanisms Degradation Ȉ Eventually, the RNA and protein molecules are degraded, terminating the response This summary outlines each of the above mechanisms, starting with molecular biology in relation to cellular communication. MOLECULAR BIOLOGY: G ENOMIC E XPRESSION The central dogma of molecular biology states that the sequence of molecular information is always linear: DNA is transcribed into RNA, and then translated to a protein. However, some exceptions exist, such as the reverse transcription of RNA to DNA and RNA replication. The following explains the main concepts of genomic expression: DNA: Fundamentals Deoxyribonucleic acid (DNA) is the universal genetic code; it stores the genetic information for eukaryotic cells. It mediates cellular behavior via the following mechanisms: 1) DNA Replication DNA replication is semi-­‐conservative: after each replication process, 2 DNA copies are produced, each with 1 original strand and 1 new strand. Once replication begins, the 2 strands are pulled apart and appropriate nucleotide bases are added by DNA polymerase to form complementary strands. 2) Transcription Transcription is the process of forming a RNA transcript from DNA. The DNA is unwound, and transcription factors guide polymerase to the promoter region. RNA polymerase adds complementary bases to the template strand. 3) Translation mRNA leaves the nucleus and acts as a template for protein production. Two ribosomal subunits attach to the mRNA; tRNA brings complementary amino acids to the mRNA. A peptide bond connects each amino acid, forming the protein. 4) Post-­‐ translational Modifications Before proteins function, they are further modified in the endoplasmic reticulum and Golgi body. This ensures that the proteins are properly folded and have the correct conformation required for their function. After modification, proteins act as enzymes, messenger molecules, or receptors that not only alter intracellular functions, but also participate in extracellular communication! EXTRACELLULAR COMMUNICATION: L IGANDS A ND RECEPTORS To reiterate, proteins can form either a receptor (a site where molecules can bind) or a ligand (a signaling molecule that binds to receptors). Together, the receptor and ligand provide the basis for extracellular communication principles: x The receptor can be in 1 of 2 forms: activated (elicits a response) or inactivated (creates no response). Depending on their type, messenger molecules can bind to either the inactive or active form. x The molecule-­‐receptor binding process is stochastic (random). One can only predict the probability of finding an active or inactive receptor at a given time. x An equilibrium exists between the active and inactive receptor conformations. x The various types of ligands influence equilibrium. The net effect of the ligands determines the equilibrium position: Ligand type Agonist Partial agonist Antagonist Inverse agonist Description A ligand that binds to the active receptor form and elicits a response Binds to active receptors slightly more than inactive receptors No preference for inactive or active receptor form; binds to both inactive and active sites to inhibit agonist action Changes receptor conformation to inactive form, preventing agonist & partial agonist from binding to active receptors Effect on equilibrium Shifts equilibrium towards the active form, increasing the overall response Shifts equilibrium slightly towards the active form, resulting in little increase in the overall response No net effect in equilibrium; only inhibits agonist action and decreases overall response Shifts equilibrium towards the inactive form, decreasing the overall response In general, drug molecules act as ligands and produce their effects by occupying receptors. The following properties determine its mode of action: x Potency: The drug concentration needed to produce an effect. A potent drug requires a smaller concentration to produce the same response than a less potent drug. x Efficacy: The capacity of a drug to produce an effect. When 2 drugs are at an equal concentration, the more efficacious drug will produce a greater response than the other. x Affinity: The drug's ability to bind to the desired receptors. EXAMPLES OF MESSENGER MOLECULES There are hundreds of different messenger molecules within the body. Here are a few examples discussed in the course to demonstrate how the mechanisms mentioned above bring about various biological and physiological changes: Messenger Molecule Synthesis Storage Release Response Termination Examples Histamine Enzymatic synthesis Stored in granules (secretory vesicles), which are found inside mast cells Stored in granules within neurons Physical (ex: UV) and chemical stimuli (ex: allergens) trigger degranulation: granules fuse into the membrane of mast cells & release contents Transported into vesicles & fused into cell membrane for release (exocytosis) Various physiological responses. Ex: asthma, pain, itching, allergies Enzymatic degradation Allergy medications prevent adverse physiological effects by inhibiting histamine activity Acetylcholine (ACh) Noradrenaline (NE) 2 different receptors: Muscarinic, and Nicotinic to elicit different responses Acts on specific GPCRs (more on this later) Diffusion, cellular uptake, enzymatic degradation Nerve gases or insecticides produce toxic effects by inhibiting ACh uptake & causing excessive ACh levels Cocaine, steroids, and some antidepressants produce their effects by inhibiting NE uptake THE AUTONOMIC NERVOUS SYSTEM Out of the messenger molecules mentioned above, ACh and NE are neurotransmitters that play an integral role in the autonomic nervous system (ANS). This will be explained in greater detail in Part II of the summary. EXTRACELLULAR COMMUNICATION: L IGANDS A ND RECEPTORS All of the messenger molecules shown above are examples of constitutive expression (continuously expressed). However, synthesis of some messenger molecules can be inducible (dependent of external stimuli). These molecules are not stored, but released instantly. Here are some examples of on-­‐demand messenger molecules: Lipophilic Molecules Steroid Hormones Eicosanoids Production ƒ Made on-­‐demand in endocrine glands ƒ Constitutive or inducible types of production Gases Cannabinoids ƒ Made on-­‐demand Nitric Oxide (NO) ƒ Constitutive or inducible types of production Release ƒ Released to the bloodstream ƒ Released extracellularly upon stimuli ƒ Released intracellularly or extracellularly upon stimuli ƒ Released extracellularly upon stimuli Effects ƒ Turns on intracellular gene transcription ƒ Essential in intracellular metabolism ƒ Many varied physiological effects, ex: fever, gastric mucus production, etc. ƒ Acts on GPCRs (more on this later) ƒ NO causes synthesis of cyclic GMP in muscle cells, causing smooth muscle relaxation & erection Termination Applications ƒ Given to organ transplant patients as immuno-­‐suppressant ƒ Intracellular transport or enzymatic degradation ƒ Drugs inhibit eicosanoid ƒ Endocannabinoids found in production to prevent marijuana and chocolate inflammation & fever, etc ƒ Produce euphoric sensations ƒ Ex: Aspirin ƒ Viagra prolongs erection by inhibiting cGMP uptake SIGNAL TRANSDUCTION: G-­‐PROTEIN COUPLED RECEPTORS As mentioned above, many messenger molecules that are released extracellularly can have a variety of intracellular effects. This signal transduction is done using second messengers, molecules that relay signals from cellular surface receptors to target intracellular molecules. For instance, one of the most abundant receptors found in the body is the G-­‐protein coupled receptor (GPCR). GPCR activation by an extracellular signaling molecule results in the synthesis of cyclic AMP, a second messenger. cAMP in turn activates another protein, causing a whole cascade of enzyme activation p rocesses and ultimately resulting in cellular responses. In other words, signal transduction results in amplification; just 1 extracellular signal is enough to activate over 109 intracellular molecules! GENOMIC CONTROL: E XTRACELLULAR TO I NTRACELLULAR COMMUNICATION Signal transduction can also influence genetic expression at various control points. For example, enzymes called gene regulatory proteins (GRPs) can either activate or inhibit transcription by binding to gene regulatory sequences in DNA. Steroids, for instance, inhibit the activity of repressory GRPs, thereby turning on gene transcription and altering cellular activity. METHODOLOGIES Numerous experimental procedures are used to perform structural and functional analyses on biological systems. Here is a summary of the main methodologies covered throughout the course: Methodology Polymerase Chain Reaction (PCR) Gel electrophoresis Organ baths CONCLUSION Description Amplification of DNA using heat cycles Applications Structural analysis: Replicate a single DNA molecule into millions of copies, allowing us to identify the primary DNA sequence. A sample is separated according to size and electric charge. A tissue sample is placed inside a solution. Electronic probes are used to measure responses of the tissue to various chemical stimuli. Structural analysis: Separation, identification, and sequence analysis of DNA, RNA, and protein molecules. Also used to test levels of enzymes present within a sample. Functional analysis: Test how the sample responds to various physical and chemical stimuli. This mind map summarizes the processes of cellular communication. Essentially, extracellular signals are transduced into intracellular signalling processes to alter cellular behaviour. This occurs by directly influencing intracellular functions or altering genomic expression. Changes in gene expression affect protein synthesis, thereby influencing the enzymes involved in this cyclic process. Drugs often target various points in this communication process to alter specific cellular functions; we provided you with many examples, ranging from Asprin and allergy medications to Viagra. As you can see, this extremely complex and intricate system opens up enormous implications in terms of scientific discoveries and inventions. This is what makes the study of cellular and molecular biology so fascinating! BOTTLENECK CONCEPT: THE AUTONOMIC NERVOU S SYSTEM Out of the topics previously discussed, one concept you may find difficult is the autonomic nervous system. Since our group struggled to understand this idea at first, we thought the explanation given below may be helpful. The neurotransmitters acetylcholine (ACh) and noradrenaline (NE) play important roles in the autonomic nervous system (ANS), a component of the peripheral nervous system. The autonomic nervous system has 2 subdivisions: parasympathetic nervous system (PNS) and sympathetic nervous system (SNS). The two pathways often have opposite effects on the body: 1) The PNS has calming effects, ex: the slowing of heartbeat and bronchi constriction. 2) The SNS has arousing effects, ex: pupil dilation and bronchi dilation. Various drug molecules influence the autonomic nervous system by altering the activities of ACh and NE. However, our group found this idea a little difficult to understand, for the following reasons: 1) How can 1 drug molecule alone have both parasympathetic and sympathetic effects on the body? For example, sarin is a neurotoxin that inhibits breakdown of ACh molecules. It has sympathetic effects (vasoconstriction) on the arterioles and veins, but parasympathetic effects in the bladder and bronchi (constricted bronchi & bladder contractions). 2) How does the body maintain a balance between the opposing forces of the PNS and SNS? But after brainstorming this puzzling concept as a group, we were able to come up with a way to understand it better. Less dominant autonomic tone Blocks = parasympathetic or sympathetic innervations at the body site Acetylcholine Predominant autonomic tone We decided to think of the balance between the PNS and SNS as a see-­‐saw. Each body site has both parasympathetic and sympathetic innervations, however it is the net overall balance between the two that determines the predominant automatic tone (the heavier side of the see-­‐saw). Each of the above blocks represents parasympathetic and sympathetic influences onto organs. Think of each body site as being comprised of different "blocks", or different sets of parasympathetic and sympathetic innervations. A see-­‐saw is very difficult to balance perfectly. Therefore, in each body site, one side of the see-­‐saw is always naturally heavier than the other. For example, the natural predominant tone of the eyes is parasympathetic. However, arterioles, veins, and sweat glands both have a sympathetic predominant tone. ACh is the neurotransmitter used at both the PNS and SNS. Think of ACh as a block that always sits only on the predominant side of the see-­‐saw. Therefore: 1) Excess ACh shifts the see-­‐saw more to the predominant side, thus exaggerating the predominant autonomic tone. This is why sarin produces such a wide variety of effects; it increases ACh levels and hence exaggerates the predominant tone at each body site. 2) ACh inhibition shifts the see-­‐saw towards the less predominant side, thereby decreasing the predominant tone. NE is found only at the sympathetic nervous system. So NE sits only on the sympathetic side of the see-­‐saw. Hence: 1) Excess NE shifts the see-­‐saw towards the sympathetic side, exaggerating the sympathetic tone. 2) NE inhibition shifts the see-­‐saw towards the parasympathetic side, decreasing the sympathetic tone. That's how acetylcholine and noradrenaline mediate the activities of the autonomic nervous system! We hope this helped you understand the connections between the nervous system and the concepts of molecular biology! Good luck in your first year! :)