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Bacterial ? Amylase Production

Introduction
Starch is the primary repository of carbohydrates in evolved plants [1]. It is not only the prime form of a dietary sugar storage molecule but has extensive industrial applications in the manufacturing of packaging substances to the synthesis of alcohol [1]. The enzyme alpha amylase has the ability to degrade starch to simpler sugars with reducing ends [2]. They are termed as endohydrolases as they bring about the schism of -1, 4 D- glycoside linkage within the chain randomly [3].
This catalysing activity makes it a salient feature particularly in the food based and textile industries which have starch as an indispensable raw material that demands hydrolysis as a part of the production [4, 5]. Traditionally, amylases were introduced into the baking mixture or other places requiring the dextrinizing enzyme in the form of barley flour or malted wheat [6]. Alpha amylase contributes towards gas generation and gas retention in bread and give proper colour to the crust [6].
Fuelzyme is an industrially developed, genetically engineered alpha amylase obtained from a combination of three enzymes due to DNA shuffling of the parental enzymes [3]. It is beneficial due to its lower viscosity, activity maintained over a wide pH range and better thermostability [3]. It is of use in biodiesel or ethanol based industry and not suitable for use in food industries due to restrictions in downstream processing [3].
The potential enzyme manufacturers
The bacteria known to produce alpha amylase are Geobacillus stearothermophilus, Bacillus licheniformis and Bacillus amyloliquefacians [3]. The alpha amylase produced by Bacillus stearothermophillushas more ascendancy than that produced by Bacillus licheniformis due to greater specific activity and lesser pH required for optimum growth [7]. Bacillus subtilis strains are widely exploited owing to their biological features such as easy, evident growth in simple culture medium, lack of secretion of toxic substances and no appreciable pathogenicity [4]. They also possess the highly demanded ability of secreting the formed amylase in the growth medium [3].
Fungi of genus Aspergillus and Rhizopus have been known to synthesize this industrially important sugar producing enzyme [8].Amylases which are fungal in origin can replace malt amylases imparting undesirable colour to the food products but get fairly inactivated by heat making them less applicable in the bakery based industry [9].
Alpha amylases are also derived from anaerobes like Clostridium thermoamylolyticum which are known to be heat stable [10]. Clostidium thermohydrosulfuricum synthesized alpha amylase has exceeding starch hydrolysing characteristics at a pH of around 4 [10]. Amylase is also derived from the heat loving bacterium Bacillus diastaticus. However, its recovery using ammonium sulphate cannot be ensured without contamination [2].
A novel amylase enzyme has been discovered featuring in the bacterium Alicyclobacillus pohilae which increases the shelf life of bread [6]. Yoneda et al discovered a strain of B. subtilis which on genetic modification increased the amylase yield 170 times [4]. The Bacillus subtilis strain AB101 is a recombinant strain known to be the most superior amongst the organisms of the same species [4].
Inside the fermenter
The producers of the biocatalyst, that is, the micro-organisms in action are subjected to aerobic or anaerobic conditions depending upon the need[11].Clostridium spp are cultured under anaerobic conditions whereas Bacilli are grown in submerged media with proper aeration or on the surface of shallow medium[2, 11]. Fungi are cultured on solid media composed of wheat bran, rice bran or straw [8].
The solid medium may contain starch in crude form, that is, in the form of beans, potatoes, grains, corn, arrowroot or other starch rich plants [2, 8]. Starch in the liquid medium can be in the form of uncooked rice flour [8]. Starch in soluble form or maltodextrin is also added to the medium [10,11].The source of starch is not a critical consideration as the enzyme is synthesized using the proteins in the medium and starch is just a stimulator which can be added in the range of 0.2% to 10%[2].
The protein content of the medium should be high [2]. 4% protein based substances in the medium have found to be suitable for the formation of amylase and its further precipitation [2].The media contains yeast extract as supplement of minerals and vitamins [10, 11]. Calcium hydroxide is particularly added to the medium to prevent the inactivation of amylase and stabilize it [11]. Maltose aggravates the production whereas glucose acts as an inhibitor [10].
The most favourable pH of the medium of fermentation for alpha amylase production is 6.5 to 7.0 [10].Temperature is not allowed to exceed 90-C in case of strains of B. subtilis and B. amyloliquefaciens while in case of B. licheniformis, it is allowed to reach 110-C [12]. The most fitting temperature range for most organisms is between 50-C to 70-C [2]. Hence, a fermentation temperature of 65-C is maintained [2]. The time for which a cycle of fermentation is run is from 12hrs to 24hrs though amylase activity is observed within 4hrs from the start of fermentation [2]. Maximum yield is obtained after 12hrs [2].
Downstream processing, purification, confirmation and assays
The enzyme is released into the medium [10]. The efforts and cost involved in disrupting the cells to get the protein product is eliminated [10].
The cells are removed by filtration and the extraneous media components are precipitated out by calcium chloride [10].Centrifugation can also be carried out for removal of solids [2].
Dialysis is a preferred step to be carried out prior to concentration and precipitation as this process ensures increased purity of the final product [2].
The solution containing the enzyme thus formed is concentrated and put forth for refinement on starch that is granular in nature [10]. Potency is increased by concentrating the solution [2].
Precipitation is carried out using organic solvents, miscible in water such as dioxane, acetone, ethyl methyl ketone, ethanol, methanol, n-propanol, isopropyl alcohol and tertiary butyl alcohol [2]. One or two volumes of the solvent serve the purpose [2].
Filtration and centrifugation is carried out after precipitation to remove the precipitated amylase [2]. The final precipitated product is dried [2]. Drying can be brought about at room temperature or at 65-C under vacuum on calcium chloride without affecting the stability of amylase obtained [2].
Chromatography techniques are also employed to bring about purification post refinement and removal of starch [10].
SDS polyacrylamide gel electrophoresis carried out of the purified sample helps determine the molecular weight with a protein marker run along [10].
Assays to determine amylase activity
Two assays are conducted:
The first assay is a modification of Fuwa method wherein starch is stained with iodine [13]. The decrease in intensity of blue colour per ml of enzyme is determined [13]. The temperature and pH of the system are altered to determine the optimum activity [13].
The second assay is based on the principle of colorimetry [13]. The protocol followed is that of Nelson reducing-sugar assay wherein 100?l of relevantly diluted amylase is incubated with 1.9 ml of starch for 10 minutes at a fixed temperature followed by addition of Nelson Reagent D to give the colour [13]. Spectrophotometer is used to determine the colour intensity [13].
Modifying the enzyme and the way of producing it for better yields and economics
A Bacillus strain has been subjected to manipulation following the principles of genetic engineering and plasmid expression vectors such as coliphage 105 for enhanced alpha amylase production [4]. The gene coding for the protein of interest is interpolated under the transcriptional influence of a promoter of phage origin [4]. Addition of antibiotic is essential to selectively promote the proliferation of the plasmid bearers [4]. It is also important for retention of the genetically recombinant cells over extended periods of time in a continuous culture overcoming vulnerability of plasmid to curing [4]. The cloned genes get over expressed in association with the pro-phage based vectors with high stability, apt location on the chromosome and conducive regulation by the phage system [4].
To make the baking process more efficient, alpha amylase obtained from Bacillus subtilis and Bacillus stearothermophillusis modified to reduce its thermostability as its application in the food industry requires[9].The modification brought about is acylation employing acylating agents such as the monocarboxylic acid anhydrides, ethanoic, propanoic, butanoic, valeric and even benzoic anhydrides [9]. Dicarboxylic and cyclic anhydrides do not serve the purpose [9]. The quantity of acylating agent incorporated to bring about the reaction is not crucial but the amount has to be enough to acylate at least fifty percent of the free amino acids of the amylase molecule [9]. The original enzyme is introduced to acylation in association with starch to prevent its degeneration during the process [9].
Modification is also carried out with respect to the process of deriving the enzyme [14]. An organosilicon polymer is introduced in the fermentation medium at the beginning itself which is known to be water insoluble at the temperature to which fermentation is subjected and soluble in water at a lower temperature [14]. The organosilicon is a copolymer composed of dimethyl silicon and propylene oxide [14]. The approved concentration of the copolymer is one part of it for every 4000 parts of the fermentation medium [14]. The copolymer addition is greatly applauded due to the enhanced yield and reduction in cost due to lack of requirement of special antifoam agents [14]. Better downstream processing and recovery is ensured as no oily residues otherwise created by the antifoam agents are produced [14].
Conclusion
Amylases yielded from the different organisms differ significantly with respect to features such as molecular weight, optimum temperature and pH requirement, enzyme activity and affinity towards calcium ions [13]. This broadens the scope of their appositeness. For e.g. amylase produced by a novel strain of Bacillus stearothermophilus has high affinity for calcium ions which makes its inclusion in detergents and use in presence of chelating agents and soft water apropos [13].
Mixed culture system with varying proportion of various species and strains of genus Aspergillus and Rhizobium have shown to give better yields than pure cultures of the same [7]. The enzyme once purified can be immobilized using befitting carriers like ceramics, organic polymers and glass [13]. Immobilization can be brought about by crosslinking, entrapment, adsorption and bonding [13].
Research is carried out to modify the amino acid sequence in the enzyme active site of amylase for enhanced activity [12]. For e.g. Bacillus licheniformis mutant G475R has the amino acids methionine, tryptophan and aspargine replaced by valine, histidine, alanine and serine [12].
Use of antibiotics as a selective pressure for harnessing recombinant techniques increases the expense of downstream processing since presence of antibiotics ordinarily in food products is not appreciable [4].Hence alternative way of selective isolation need to be worked out.
Properties such as specificity towards the substrate, its binding to it, the cleavage of the substrate, activity and stability profile with respect to pH, stability at higher temperature, towards oxidation and low calcium ion concentration and its activity alone and in combination can be altered [3].
References
[1] Myers Alan M. and James Martha Graham. Isolation of Su1, a starch debranching enzyme, the product of the maize gene Sugary 1. Februaury 7, 2006. US 6,995,300 B2.
[2] Tetrault Philip A., Lafayette West and Stark Egon. Process for preparing alpha amylase. November 30, 1954. US 2,695,863.
[3] Nedwin Glenn F., Sharma Vivek and Shetty Jayarama K. Alpha amylase blend for starch processing and method of use thereof. October 1, 2013. US 8,545,907 B2.
[4] Leung Yun Chung, Lo Wai Hung and Errington Jeffery. Method for production of alpha-amylase in recombinant Bacillus. June 23, 2009. US 7,550,281 B2.
[5] Leung Yun Chung, Lo Wai Hung and Errington Jeffery. Method for production of alpha-amylase in recombinant Bacillus. December 12, 2002. US 0187541 A1.
[6] Lucie Parenicova. Alpha Amylase. July 31, 2013. EP 2620,496 A1.
[7] Carrol et al. Alpha-Amylase mixtures for starch liquefaction. July 8, 1987. EPO 252730 A2.
[8] Kitakyushu Foundation. 2012. JP20100293378. EPO.
[9] Brumm Phillip J. Reduced-stability alpha-amylase and process for its production. December 10, 1987. EPO 273268 A2.
[10] Zeman Nancy W. Novel thermostable, aciduric alpha-amylase and method for its production. September 23, 1986. US 4613570.
[11] Katkocin et al. Novel thermostable, aciduric alpha-amylase and method for its production. November 25, 1987. EPO131253 B1.
[12] Robert M. Caldwell, Colin Mitchinson and Traci H Ropp. Mutant ? amylase. 2001. US 6211134 B1.
[13] Kindle et al. Thermostable alpha amylase having a low requirement for m calcium ions, derived frombacillus microorganism. July 15, 1986. US 4600693 A.
[14] Wynes Robert A., Llyod Norman E., Linton and Iowa. Process for the preparation of bacterial alpha-amylase. December 3, 1968. US 3414479.
Reference table
Sr. No.
Inventor
Company name
Year
Title
Number
1
Myers Alan M. and James Martha Graham
Iowa State Research Foundation, Inc ., Ames US
2006
Isolation of Su1, a starch debranching enzyme, the product of the maize gene Sugary 1
US 6,995,300 B2
2
Tetrault Philip A., Lafayette West and Stark Egon
Purdue Research Foundation, Lafayette, Indiana
1954
Process for preparing alpha amylase.
US 2,695,863
3
Nedwin Glenn F., Sharma Vivek and Shetty Jayarama K
Danisco US Inc., Palo Alto, CA (US)
2013
Alpha amylase blend for starch processing and method of use thereof.
US 8,545,907 B2
4
Leung Yun Chung, Lo Wai Hung and Errington Jeffery
The Hongkong Polytechnique University, Kowloon (HK)
2009
Method for production of alpha-amylase in recombinant Bacillus.
US 7,550,281 B2
5
Leung Yun Chung, Lo Wai Hung and Errington Jeffery
The Hongkong Polytechnique University, Kowloon (HK)
2002
Method for production of alpha-amylase in recombinant Bacillus.
US 0187541 A1
6
Lucie Parenicova
DSM IP Assets B.V.
2013
Alpha Amylase.
EPO 2,620,496 A1
7
Carrol et al.
NOVO Industries
1987
Alpha-Amylase mixtures for starch liquefaction.
EPO 252730 A2
8
Kitakyushu Foundation
2012
. Production method of amylase by using mixed bacterial culture of Aspergillus bacterium and Rhizopus bacterium.
JP20100293378
9
Brumm Phillip J
Enzyme Bio-systems Ltd.
1987
Reduced-stability alpha-amylase and process for its production.
EPO 273268 A2
10
Zeman Nancy W
CPC International Inc., Englewood Cliffs, NJ.
1986
Novel thermostable, aciduric alpha-amylase and method for its production.
US 4613570
11
Katkocin et al.
CPC International Inc., Englewood Cliffs, NJ.
1987
Novel thermostable, aciduric alpha-amylase and method for its production.
EPO131253 B1
12
Robert M. Caldwell, Colin Mitchinson and Traci H Ropp
Genecor International, Inc.
2001
Mutant ? amylase.
US 6211134 B1
13
Kindle et al
Corning Glass Works, Corning, N.Y.
1986
Thermostable alpha amylase having a low requirement for m calcium ions, derived from bacillus microorganism.
US 4600693 A
14
Wynes Robert A., Llyod Norman E., Linton and Iowa
Standard Brands Incorporated, New York
1968
Process for the preparation of bacterial alpha-amylase.
US 3414479

Smoking and Alveolar Damage

Lakshmi Mohanadas
Cigarette smoking is one of the major cause for many of the lung diseases. Smoking is a practice of burning a dried plant and inhaling the smoke. It is one of most common form of recreational drug usage.
People who smoke, get addicted to smoking which leads to gradual damage to the body. The substances that are inhaled causes reactions at the nerve endings in the central nervous system. These reactions are similar to that caused by the naturally occurring hormones, dopamine and endorphins, which are the main contributors of the sensation of pleasure known as ‘being high’. The level of being high can vary from mild stimulus (generally caused by nicotine) to intense euphoria (caused by heroin, cocaine).
On combustion, active substances are released into the body through the inhalation of the smoke. These active substances are a mixture of aerosol particles and gases which mainly contain nicotine. This then gets absorbed into the blood stream.
During cigarette burning, incomplete combustion occurs due to which carbon monoxide (CO) is produced which reduces the ability of blood to carry oxygen molecules. Cigarette smoke contains reactive oxygen species (ROS) and reactive nitrogen species (RNS) such as hydrogen peroxide, superoxide and hydroxyl radical, peroxynitrite(2). These species causes severe damage to alveolar epithelial cells. Smoking is the major cause to pulmonary emphysema, pulmonary fibrosis, COPD (Chronic obstructive pulmonary disease).
Pulmonary emphysema is the gradual damage of alveolar cells which progressively leads to short breath. It a type of COPD in which lung tissue is broken down with increasing an inflammatory response. Pulmonary fibrosis results due to the buildup of fibroblast in the small airways which eventually blocks the airway making it difficult to breath and causes eventual degradation of lung tissue due to less air supply.
Smoking also increases the risk of lung cancer in an individual. The compounds inhaled during the smoking process have the tendency to alter the DNA of a person which can cause DNA alterations which can be passed onto the next generation of the smoker.
The major part of the lungs that get affected by smoking is the alveoli. Alveoli are small balloon-like air sacs which act as the sites for gas exchange in the lungs. These sacs gets inflated and deflated during inhalation and exhalation.

Fig1 : Diagram of an alveoli

Fig 2: Lung filled with alevoli
Alveoli are surrounded by a mesh of capillaries which bring carbon dioxide from rest of the body to exhale and absorb the oxygen that is inhaled. They are formed of epithelial cells and extracellular matrix. Some of them consist of Pores of Kohn which help in alveoli spacing. They are also composed of collagen and elastic fibers which help the alveoli cells to stretch in order to get filled up with air during inhalation and relax back to its original state during exhalation.
Generally there are three type of alveolar cells : type I, type II and macrophages. Type I cells are generally composed of squamous cells and form the wall of the alveoli. Type II cells , also known as great alveolar cells, produce pulmonary surfactant that reduces the surface tension of the membrane and increases the capacity of gaseous exchange. Macrophages helps in attacking the foreign harmful materials.
Type II cells plays a major role in the repair of lung tissue. They are known as the progenitors of Type I cells as they repair the endothelium of the alveoli when it gets damaged by moving to the denuded area (2,5,6) and proliferating into new cell Type II cells which can differentiate into Type I cells, thereby restoring the damage.
Cigarette smoking has severe effects on these alveolar cells in the lungs which can lead to diseases like pulmonary emphysema, pulmonary fibrosis and lung cancer. Due to cigarette smoking alterations are caused to the alveolar epithelial cells (Type I and Type II), which can increase the epithelial permeability, decrease the surfactant production of Type II cells, produce the cytokines and growth factors which causes inflammatory responses in the lungs. Also there is an increase in the cell death by either apoptosis or necrosis depending on the quantity of smoke components inhaled, which is because just active smoking is not harmful but passive smoking can also damage a person’s lung.
Cigarette smoking has a number of adverse effects on the lungs. One of these effects in the generation of excessive oxidative stress. As mentioned earlier, cigarette smoke contains nearly 4000 different chemicals which are mainly carcinogenic (3). Among these major constituents are ROS (reactive oxygen species) and RNS( reactive nitrogen species) (2,3). A single puff of cigarette smoke contains 1017 oxidant molecules out of which 1014 are ROS such as nitrogen oxide and superoxide radical which when in the lungs, reacts immediately to form reactive peroxynitrite. Also the smoke contains hydroquinones which after redox cycle forms superoxide radical and hydrogen peroxide. This hydrogen peroxide and hydroquinones can enter the cell’s nucleus and can cause oxidative DNA damage. Another factor contributing to the oxidative stress is the production of iron from ferritin which can trigger the phagocytes to release ROS (2). It also reduces the levels of anti-oxidants in the blood.
Cigarette smoke contains both gaseous and particle (tar) components which sensitively affect the Type II alveolar cells. Severe destruction to these cells lead to pulmonary emphysema. The hydroxyl radical released during smoking activates poly(ADP-ribose) polymers (PARP), a DNA repair enzyme, which causes in decreased levels of nicotinamide adenine dinucleotide (NAD) altering the ATP synthesis leading to cell death (4). The bronchial epithelial cells reacts to cigarette smoke by producing xanthine and hypoxanthine, which damages the genetic information of the cells(2).
The damages caused eventually leads to cell death by either apoptosis or necrosis, which depends on the magnitude of smoke inhaled. A recent study showed that at low levels of aqueous cigarette extracts, cells undergo apoptosis and at high levels they die by necrosis. These cell deaths are caused due to the effect of ROS on them. The in vitro test of cell apoptosis was supported by in vivo study on rats which were exposed to cigarette smoke for approximately 100 days and showed bronchial epithelial cell death.

Fig 3: Cell death of type II alveolar epithelial cells
Chronic inhibition of the vascular endothelial growth factor receptor induced apoptosis in alveolar endothelial cells, followed by pulmonary emphysema (fig 3). Exposure to smoke can also induce stress-induced senescence which prevents epithelial cell proliferation. Thus, cigarette smoke not only by induces epithelial cell death, but also inhibits epithelial repair processes. Studies have shown that cigarette smoking can cause the enlargement of alveolar spaces and increase the epithelial permeability which can also lead to diseases like hypoxia, emphysema, COPD.
For small damages in the lung, the lung tissue undergoes various repair mechanisms such as: chemotaxis, proliferation, production of extracellular matrix, remodeling of extracellular matrix and DNA repair mechanisms. Studies indicate that smoking cigarettes can inhibit these repair mechanisms in the lung tissue. Most of the repair mechanisms are carried out by the Type II alveoli cells, Pores of Kohn and mesenchymal cells. Type II cells generally repair the damaged Type I cells by migrating to the affected area, proliferating to new Type II cells and then act as a progenitor cell for the production of Type I cells.
The alveolar wall have holes that could provide channels between adjacent alveoli which are known as Pores of Kohn. These are traditionally known as pathways for collateral ventilation, but due to the small size, they are occluded with surfactant which probably precludes them from functioning in this capacity. These Pores of Kohn represent injuries that might have been repaired. They equalize pressure in adjacent alveoli cells and thereby helps to prevent the collapse of lung (7,9).

Fig 4: Scanning electron micrograph of normal mouse lung. (A) Normal young adult (2 mo) C57/Bl6 mouse. (B) Normal old (24 mo) C57/Bl6 mouse. Alveolar pores can be readily observed in both preparations. They are increased in size and number in the old mouse lung. (1)
Also it has been observed that due to emphysema induced by cigarette smoking these pores enlarge in response to the disease thereby reducing the effect of the disease. The number and size of Pores of Kohn are dependent on the age of the person (shown in figure 4). With an increase in number and size of pores, alveolar size also increases with age which is termed “senile emphysema.”
Mesenchymal cells also plays crucial role in the repair of lung tissue. These cells send signals which initiate development and maintenance of lung branching. Also alveolar mesenchymal cells store and release retinoic acid which are responsible for the formation of secondary septation. These cells are the major contributors of the extracellular matrix macromolecules. In-vitro bioassay models have been made to understand the repair mechanism of mesenchymal cells. Fibroblasts, type of mesenchymal cell, produces the extracellular macromolecule fibronectin which mediates the interaction between cells and extracellular matrix.

Fig 5: Cigarette smoke inhibition of fibroblast chemotaxis.(1)
In-vitro models of mesenchymal cells are developed by culturing fibroblast/myofibroblast in three-dimensional collagen gels which shows their ability to contract (8,11). This ability of fibroblast is inhibited by the components of cigarette smoke causing them to lose their ability to heal wounds by contracting the affected area.
Smoke also affects the enzyme lysyl oxidase which is involved in the maturization and repair of elastic fibers which affects the polymerization and production of elastin.
Emphysema is also exacerbated by starvation, shortage of oxygen supply, which is followed by intratracheal elastase infusion (12). Studies were conducted on mouse that indicate that T-cell–derived factors drive lung cell apoptosis which play a major role in starvation-induced emphysema (10). It has been made evident that smoke promotes starvation and inhibits the refeeding ability (counter action of the cells to starvation) and thereby worsen emphysema.
Studies have shown that smoke inhibits airway epithelial cell chemotaxis and proliferation(14,13). When alveolar epithelial cells are cultured on collagen gel, gel contraction happens suggesting epithelial cells participate in closure of alveolar cells but smoke inhibits this mechanism(shown in figure 5).
COPD not only causes emphysema but also develop peribronchiolar fibrosis. It is developed in small bronchiolar airways and is characterized by the deposition of fibroblast and myofibroblast together with extracellular matrix to produce fibrillar collagen. This collagen contracts and narrows the small airways.
This fibroblast contraction was studied by Wang and colleagues (16) by evaluating the effect of smoke on three-dimensional collagen gels as a function of cell-density. Cigarette smoke inhibited contraction of three-dimensional collagen gels at low density of fibroblast. At a higher density, collagen gel contraction is stimulated by smoke. Wang and colleagues showed that, at increased density of fibroblast, latent transforming growth factor (TGF)-β is activated (fig 6). This activated TGF-β contracts the three-dimensional collagen gel by stimulation of fibroblast. This regulation of the tissue damage is carried out through the paracrine mechanisms(1).

Fig 6: Cigarette smoke modulation of repair by paracrine mechanisms: effect of cell density. (Upper panel) At low cell density, cigarette smoke inhibits fibronectin production and because fibronectin stimulates contraction, smoke inhibits contraction as well. Any effect of smoke in activating transforming growth factor (TGF)-β is inconsequential as the concentrations of TGF-β are too low to elicit a response. *Loss of stimulation causes inhibition in the presence of smoke. (Lower panel) At higher cell density, sufficient TGF-β is produced by the many cells in the local milieu to elicit a response. Smoke-induced activation of TGF-β results in augmented contraction, even in the face of direct inhibition of fibronectin production.(1)
As described earlier smoking can also affect DNA, as a result acquired genetic abnormalities are also developed. Cancer caused by cigarette smoking is also believed to be developed by somatic cell mutations. These mutations can also cause COPD. Cigarette smoke has the ability to damage DNA by several mechanisms (15) which can inhibit apoptosis (13, 17) which protects from unwanted, genome altered cell growth. As a result DNA repair gets initiated which leads to cell recovery and subsequent proliferation. This could lead to the accumulation of cells that have altered gene function and causes impaired repair responses within the lung of smokers .
Different strategies are developed to restore repair mechanism in the lung tissue. Studies on adult rats demonstrate that all-trans-retinoic acid can induce new alveolar wall formation (18,19). But limited studies on human volunteers has demonstrated safety but no alveolar repair. It can be concluded that different direct and indirect repair mechanisms are altered by cigarette smoking. The progression of diseases caused by lung tissue damage can be slowed by understanding and creating novel techniques which can target the repair responses in the lung.
References:
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Uhal BD. Cell cycle kinetics in the alveolar epithelium. Am J Physiol 1997; 272:L1031-1045.
Wright JL. The importance of ultramicroscopic emphysema in cigarette smoke-induced lung disease. Lung 2001;179:71–81.
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Kim HJ, Liu XD, Kobayashi T, Conner H, Wen FQ, Kohyama T, Fang QH, Abe S, Bitterman PB, Rennard SI. Cigarette smoke induces reversible DNA damage in human fetal lung fibroblast cultured in three-dimensional collagen gels. Am J Respir Crit Care Med 2003;167: A489.
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