Introduction

Asoke K. Basu MVSc, PhD , Roxanne A. Charles DVM, MSc , in Ticks of Trinidad and Tobago - an Overview, 2017

Cellular organisms are divided in two groups known as prokaryotes and eukaryotes. The genetic material, dna (Dna) and ribonucleic acid (RNA) are bundled into structures called chromosomes. In the eukaryotic cell, the chromosomes are surrounded by a nuclear membrane forming a true nucleus. Eukaryotic cells incorporate a diverseness of organelles and a cytoskeleton, which is equanimous of microtubules, microfilaments, and intermediate filaments. The general structures of prokaryotic cells are a plasma membrane, cytoplasm, ribosomes, and genetic material in the form of Deoxyribonucleic acid and RNA. Unlike eukaryotes, the genetic textile in prokaryotes is not surrounded by a nuclear membrane and lies in a region called the nucleoid. A prokaryotic chromosome is circular but in eukaryotes, the chromosome is linear. Some cells have other structures such as cell wall, pili, and flagella. The cell components play a vital role in survival, growth, and reproduction of the cell.

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Origin of Life, RNA World and

P.One thousand. Higgs , in Encyclopedia of Evolutionary Biology, 2016

Factors Pointing to RNA

Cellular organisms on World today use three types of biopolymers ( Figure 1). DNA genes are transcribed by poly peptide enzymes to brand mRNAs. The mRNAs are translated past ribosomes (whose main component is RNA) to make proteins. Thus, DNA, RNA, and proteins are mutually dependent on one another for replication. The RNA World hypothesis is that modern organisms evolved from a simpler organisation that used simply RNA. In the RNA globe, RNA genes would be copied past RNA catalysts (Joyce, 2002).

Figure 1. Comparison of the DNA+RNA+Protein system used by modern organisms with the RNA-only organisation that could have existed in the RNA Globe.

In addition to logical simplicity, there are many other factors that advise this could be true. Although no cellular life forms accept genes fabricated of RNA, at that place are many viruses that employ RNA as genetic cloth. RNA and DNA encode genetic information in the same way. A strand of either nucleic acid can be a template on which a second strand is assembled. Pairing between complementary nucleotides ways that sequence information is passed on. When the 2nd strand acts as a template, a new copy of the original sequence is made. RNA viruses replicate by this two-step processes. RNA virus replication is catalyzed by protein enzymes (RNA polymerases) that are encoded by the viral genome. In the RNA Globe, information technology is envisaged that in that location were RNA polymerases that were 'ribozymes' (i.e., catalysts fabricated of RNA). There are no naturally occurring RNA polymerase ribozymes of this blazon, only at that place has been considerable progress in synthesizing polymerases in the laboratory, as we will discuss below. Nevertheless, there are many other types of naturally occurring ribozymes, and the power of RNA to be a catalyst is well established.

The starting time ribozymes discovered were self-splicing introns (Kruger et al., 1982), which splice themselves out of pre-mRNA sequences without the assistance of protein enzymes. For the RNA Globe hypothesis, the virtually fundamental naturally occurring ribozyme is ribosomal RNA. From the 3 dimensional structure of the ribosome, it is clear that the active site for the peptide bond reaction is made of RNA (Nissan et al., 2000). This suggests that the original ribosomes were made only of RNA, and that the ribosomal proteins that are present in modernistic ribosomes were more than recent additions. Protein synthesis also depends on transfer RNAs to decode the gene sequence and messenger RNAs that comprise the genetic sequence. The whole process of translation merely makes sense if RNAs preceded proteins.

The vast repertoire of catalytic functions that is carried out by mod proteins is impressive. Protein sequences are relatively small-scale and flexible compared to nucleic acids, and the amino acid side bondage in proteins contain a much larger range of chemical groups from which catalytic structures can be built than practise the bases in RNA. Although proteins may be more than efficient and more versatile catalysts than RNA, there appears to be no machinery in proteins equivalent to gratuitous base pairing in RNAs. Thus the information in an amino acid sequence cannot be passed directly from 1 protein to another. Also, the traditional view of nucleic acids as having a limited range of functions, is condign somewhat outdated (Breaker and Joyce, 2014), as new discoveries are made regarding nucleic acid functions in cells.

In some versions of the RNA Globe, information technology is envisaged that relatively circuitous organisms with many dissimilar kinds of RNA catalysts evolved earlier the origin of encoded protein sequences (Chen et al., 2007). In this view, the evolution of the ribosome and the genetic code would marking the cease of the RNA World. Proteins would and then brainstorm to take over well-nigh of the roles that were previously catalyzed by ribozymes. A slightly different moving-picture show is that amino acids and minor peptides were essential players alongside RNA all along (Li et al., 2013), and that there was never a very large repertoire of purely RNA catalysts. Using peptides as cofactors of ribozymes, or fifty-fifty having amino acids covalently linked to ribozymes, is compatible with an RNA World picture of early life, only it is important to remember that long protein sequences could non be encoded earlier the translation process arose.

The other side of the cofactor statement is that many modernistic proteins use either unmarried nucleotides or dinucleotides every bit cofactors that associate with the folded proteins and are essential for their part. This is often seen as further evidence for the RNA Earth (White, 1976), with the nucleotide cofactors being relics of an earlier stage of evolution.

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Plant parasite microorganisms

Yu. T. Dyakov , S.Five. Zinovyeva , in Comprehensive and Molecular Phytopathology, 2007

Viral genome and its operation.

In cellular organisms, the major functions of the nucleic acids – replication, transcription, and translation – are distributed betwixt 2 types of molecules: double-stranded DNA and single-stranded RNA. In replication, the DNA molecule is unzipped and on each strand a complementary second strand is constructed using the enzyme DNA-polymerase. In transcription, the enzyme RNA-polymerase builds an RNA strand (minus strand) complementary to the Dna strand (plus strand) in a definite direction (from 5′ end of the molecule towards 3′ terminate). In translation, a poly peptide molecule is synthesized from individual amino acids, using ribosomes, on the RNA molecule as a matrix, in the opposite direction (from 3′end towards 5′ end).

In viruses, the aforementioned type of molecule (RNA in most phytopathogenic viruses) performs all the three functions: replication, transcription, and translation. Besides, in most viruses the nucleic acids are single-stranded, and tin can fulfill both (+) and (−) functions (Table. 1.4).

Table one.iv. Construction of plant viral genomes (Harrison BD, 1982)

Genome Number of viruses Percent
Single-stranded Dna 26 4
Double-stranded DNA 13 2
Unmarried-stranded Deoxyribonucleic acid (plus strand) 470 76
Unmarried-stranded DNA (minus strand) 85 fourteen
Double-stranded RNA 26 + all fungal viruses 4

As tin can be seen, only a small number of viruses contain double-stranded information molecules, reading from which is done like to cellular organisms. About phytopathogenic viruses have one multifunctional RNA (+) strand. Its functions are storage and realization of information. In improver, the RNA (+) strand is an infectious molecule. Afterwards inflow of the virus particle containing an RNA (+) strand to the cell, the start stage is decapsulation, i.e. release of the RNA molecule from its protein glaze. This process is carried out on the cell receptors by the proteases of the host institute. The next process is replication, catalyzed past the enzyme RNA-dependent RNA polymerase (replicase), which in virtually viruses is encoded by the own genome. The RNA of the tobacco mosaic virus (TMV), typical of this group, encodes 4 proteins (Figure i.1).

Figure 1.1. TMV genome and TMV genome-encoded proteins.

Translation begins from the v′ cease of the molecule – synthesis of 126 kDa and 183 kDa proteins, replicase components. These proteins occur in the cell at early on stages of the infection process; hence they are called early proteins. Another virus – turnip yellow mosaic virus – develops hybrid replicase in the infected cell: 1 of its components (115 kDa) is encoded past the viral genome, the other – a 45 kDa poly peptide – past the host cell genome. Manifestly, the host enzyme system is routinely used for replication of viral RNA, as for many plants infected with viruses a substantial increment in the synthesis of RNA-dependent RNA polymerase is typical.

RNA polymerase builds an RNA (−) strand complementary to the (+) strand of the viral RNA. This process results in formation of a replicative form of the viral RNA in the cell, represented partially and completely by a double-stranded construction. The (−) strand serves as a matrix for synthesis of new molecules of the viral (+) RNA, which functions every bit mRNA in translation of the tardily viral protein cells in ribosomes. In TMV they consist of a 30 kDa ship protein and a 17.5 kDa structural poly peptide of the coat (Effigy i.1). The final process, encapsulation, consists of maturation of the whole particle – self-assembly of the structural protein molecules on the surface of the RNA molecule.

Thus, in viruses with the RNA (+) strand, the viral particle disappears later arrival to the jail cell, and disjunctive (separated) replication of a new generation of viruses occurs, similar rather to the manufactory conveyor than reproduction of cellular organisms (synthesis of individual components, sometimes occurring in dissimilar compartments of the cell, and assembly of the whole particles).

In viruses with the RNA (−) strand, this strand is non-infectious, because it cannot function as a matrix RNA. Therefore, in addition to protein-coated RNA molecules, the particle also contains enzymes, in item, RNA transcriptase, and all these are covered with an boosted coat containing lipids. Such a particle enters the cell like a "Noah's Ark" with its ain enzymes. Further synthesis of the new generation of viruses is carried out not separately, but in one compartment.

Different cellular organisms, the viral genome experiences a deficit of data, as information technology tin can encode only several proteins. In some viruses, the molecule of nucleic acrid contains not four reading frames, similar the TMV RNA, only a larger number; nonetheless, the increase in the corporeality of information needs to be accompanied by an increase in the length of the information molecule, while the single-stranded molecules, typical of most viruses, accept no structural rigidity of the double-stranded molecules, and with an increased length they lose the structure necessary for recognition by enzymes. Different viruses approach the shortage of data trouble in unlike ways.

1.

Multifunctionality of the viral proteins. In POTY viruses (Y virus of potato and related viruses) the ca. x kb genome contains a single long open reading frame (ORF) translated into a large polyprotein (340–370 kDa), which is divided into 10 viral proteins in co-translation and post-translation, using ain proteases. Almost all the proteins are multifunctional, i.due east. contain several domains providing diverse functions. For instance, the capsid protein (CP) is responsible for aphid manual, cell-to-cell and systemic send, and virus assembly; protein HC-Pro for aphid transmission, systemic movement, papain-like cysteine proteinase, and synergism in combined infections.

2.

Fragmentation of the genome. On centrifugation of some viruses isolated from infested plants, in density slope of cesium or sucrose, it was establish that they constitute a mixture of larger and smaller particles (solutions of cesium chloride or sucrose are layered in a centrifuge test tube from more concentrated to less concentrated solutions, the specimen being studied is put on the top and is centrifuged; the mixture of particles in the specimen, differing by molecular weight, is hands separated in gradients into dissever fractions). Each split fraction is not infectious or is slightly infectious, while the mixture is highly infectious. The ratio of larger and smaller particles in a plant is usually constant. It was found that though the particles are coated with the like protein molecules, their RNA differ in the structure and encoded proteins. For instance, the RNA contained in the long particles encodes early proteins, enzymes, while the RNA in the brusk particles encodes the structural proteins of the glaze. A fraction consisting of the long particles tin infect plants and form a new RNA generation, just it is unstable and cannot survive exterior a found cell; the brusque particles are stable, they cannot infect plants and reproduce in them. Ii components were found in the viruses of tobacco rattle, band spot of raspberry, blackness ring spot of love apple, cucumber mosaic, etc., three in the moo-cow pea mosaic virus, and four in the alfalfa mosaic virus.

3.

Use of a helper. It was written before that shortage of information made some viruses use the host cell enzymes during replication and translation. There are viruses (suggested to exist called virusoids) that accept an RNA molecule consisting of several hundred nucleotides and are incapable of encoding more than one protein (for case, structural protein of the coat). The virus receives the other proteins, necessary for intracellular maturation, from another virus, the helper; therefore, it cannot alive in the cells not infected with the helper such as the satellite virus (SV), satellite of tobacco necrosis virus (TNV). It never occurs in TNV-gratis plants and though information technology is covered with its own coat, it uses the early proteins encoded by the TNV genome.

4.

Helper-contained replication of a short ring RNA molecule that contains no data on the structural protein. Such molecules (viroids) can self-replicate in a constitute and crusade serious diseases (spindle tuber disease, etc.).

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ORALLY BIOAVAILABLE GLYCOPROTEIN IIB/IIIA ANTAGONISTS: A NEGATIVE CASE STUDY

DIETMAR A. SEIFFERT M.D. , JEFFREY T. BILLHEIMER Ph.D. , in Target Validation in Drug Discovery, 2007

I INTRODUCTION

Multi-cellular organisms require a patent vascular system for efficient ship of nutrients and metabolic by-products. The hemostatic system is designed to maintain blood within the vascular system in a fluid state under physiological weather condition. It is primed to react apace to vascular injury in an explosive manner to minimize blood loss, mainly during injuries and kid nativity. As a issue, evolutionary pressure favors efficient hemostatic mechanisms.

While a number of hemostatic machinery tin be separated, a synergy betwixt all systems is required for efficient hemostasis. Hemostatic mechanisms include the vessel wall (vascular contraction to reduce blood flow/loss), exposure of tissue factor to initiate blood coagulation, platelets (primary hemostasis past adhesion to areas of injury and providing pro-coagulant surfaces), the coagulation system (leading to fibrin formation and platelet stimulation), the anti-coagulant system (shutting down coagulation enzymes), and the fibrinolytic system (removal of blood clots and initiation of wound healing).

Thrombosis is a pathological extension of hemostasis leading to blood clots in the vasculature. Thrombotic events mainly occur afterwards the reproductive period and thus have a depression evolutionary pressure. Thrombosis can exist viewed as blow of nature with insufficient time to adapt through development to advances of modernistic medicine and longevity. Already in 1886, Rudolf Virchow identified the predisposing factors for thrombosis (as well known as Virchow'south triad), including alterations in the vessel wall, alterations in normal blood flow, and alterations in the limerick of claret. Thrombotic diseases nowadays clinically on the arterial site as coronary artery illness leading to acute coronary syndrome and sudden cardiac expiry, cerebrovascular diseases including transient ischemic attacks and strokes, peripheral arterial occlusive diseases, and on the venous site as deep vein thrombosis and pulmonary embolism.

A functional hemostatic system is essential for survival and alterations in either procoagulant-, anticoagulant-, and fibrinolytic systems or platelet number or functional responsiveness are associated with either haemorrhage or thrombosis. This conclusion is derived from mouse models or homo monogenetic traits (for textbook coverage of Thrombosis and Haemostasis encounter Coleman et al., 2001; Loscalzo and Schafer, 1998; and Michelson, 2002).

Thrombosis is the primary crusade of morbidity and mortality in the western world, pointing to a big unmet medical need that is incompletely served by existing therapies. Parenteral GP IIb/IIIa antagonists show promise in the treatment of patients undergoing coronary revascularization procedures, including airship angioplasty and stenting (see Chapter viii). To extend the benefits observed in astute treatment to chronic atherothrombotic diseases, a number of pharmaceutical companies pursued oral glycoprotein IIb/IIIa programs. This review focuses on antagonists that underwent extensive clinical evaluations in Phase Iii trials and is written by two scientists involved in the preclinical and clinical development of i of these programs.

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Nematode Models of Evolution and Disease

Daniel D. Shaye , Martha C. Soto , in Current Topics in Developmental Biological science, 2021

Abstruse

Equally multi-cellular organisms evolved from pocket-sized clusters of cells to circuitous metazoans, biological tubes became essential for life. Tubes are typically idea of equally mainly playing a role in ship, with the hollow space (lumen) acting as a conduit to distribute nutrients and waste, or for gas exchange. However, biological tubes as well provide a platform for physiological, mechanical, and structural functions. Indeed, tubulogenesis is frequently a critical attribute of morphogenesis and organogenesis. C. elegans is made up of tubes that provide structural support and protection (the epidermis), perform the mechanical and enzymatic processes of digestion (the buccal cavity, pharynx, intestine, and rectum), transport fluids for osmoregulation (the excretory system), and execute the functions necessary for reproduction (the germline, spermatheca, uterus and vulva). Hither we review our current understanding of the genetic regulation, molecular processes, and concrete forces involved in tubulogenesis and morphogenesis of the epidermal, digestive and excretory systems in C. elegans.

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Replication and Expression Strategies of Viruses

Sephra Rampersad , Paula Tennant , in Viruses, 2018

Alternative Splicing

Also observed in many cellular organisms, alternative splicing allows product of transcripts having the potential to encode different proteins with different functions from the same cistron ( Fig. 3.seven). The sequence of the mRNA is non changed as with RNA editing; rather the coding capacity is changed as a consequence of alternative splice sites. Alternative splicing is regulated by cellular and viral proteins that modulate the activeness of the splicing factors U1 and U2, both of which are components of the spliceosome. The spliceosome is fabricated upwardly of the snRNAs (southwardmall nuclear RNAs) U1, U2, U4, U5, and U6, together with various regulatory factors. Activation of the spliceosome is facilitated by cis-acting signals in the mRNA sequence. Some of these signals include donor splice sites (5′ terminus), acceptor splice sites (3′ terminus), polypyrimidine tracts, and branch betoken sites. Serine/arginine-rich proteins, likewise equally heterogeneous nuclear ribonucleoproteins, play a key part in splice site recognition. Culling splicing (1) increases the virus' ability to encode several proteins in a given transcript (eastward.g., adenoviruses and retroviruses tin can encode ~12 different peptides from one pre-mRNA), (2) is a mechanism to regulate early and late expression for viruses (due east.yard., papillomaviruses), and (3) splicing is coupled to consign of mRNA out of the nucleus. While only mature, spliced mRNA transcripts are exported out of the nucleus, hepadnaviruses and retroviruses are able to consign nonspliced mRNA transcripts out of the nucleus for translation. On the other hand, the NS1 protein (nonstructural protein 1) of influenza viruses can interact with multiple host cellular factors via its effector- and RNA-binding domains. Information technology is capable of associating with numerous cellular spliceosome subunits, such as U1 and U6 snRNAs, and tin inhibit cellular factor expression by blocking the spliceosome component recruitment and its transition to the active land.

Figure 3.7. Alternative splicing. Alternative splicing is mutual in parvovirus pre-mRNA transcript processing and allows for the generation of dissimilar proteins from a specific nucleotide sequence on the viral mRNA strand. Dotted lines indicate alternative splice sites.

Both conservation and development of viral splice site sequences allow for improved adaptation to the host, and ensure recognition by the host's splicing mechanism. Therefore, viruses can induce preferential consecration of viral mRNA splicing by the cellular splicing machinery. Knowledge concerning the coordination between cellular and viral genome splicing comes from adenoviruses and retroviruses, but only limited data are available for other viruses, for example, influenza viruses.

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Gene Expression

J. Parker , in Brenner'due south Encyclopedia of Genetics (Second Edition), 2001

Transcription

The genes of all cellular organisms are equanimous of double-stranded DNA (some viruses accept single-stranded Dna genomes and others even RNA genomes) and the starting time step in their expression is transcription. Transcription involves using one of the two strands of Dna as a template to make an RNA re-create by an enzyme called RNA polymerase. All RNA polymerases synthesize an RNA concatenation from the v′ end to the three′ cease while reading the template strand of the Deoxyribonucleic acid in the iii′–5′ direction. The RNA molecules are synthesized from specific starting sites on the Deoxyribonucleic acid and also terminate at specific sites. The sites where RNA polymerase (using accessory factors) recognizes the start of a transcriptional unit are termed promoters. In higher organisms, the unit of measurement of transcription is almost ever a unmarried gene. However, in prokaryotes, the transcriptional unit may comprise several contiguous genes. These genes are often related in function and/or belong to one pathway.

Transcription is a target of several regulatory mechanisms. These can serve to repress or activate transcription, or lead to premature termination. One mutual mechanism in leaner is the bounden of a repressor protein to a specific region of the DNA near the promoter which and then blocks transcription. The sequence to which the repressor protein binds is termed an 'operator', a term which has given its proper name to the transcriptional unit called an 'operon'. In leaner, an operon may contain 1 or more genes, all under the command of the single operator. Another mechanism for regulating gene expression is the binding of a regulatory protein to the Deoxyribonucleic acid which activates transcription. Such positive command is widespread in eukaryotic genes. Information technology is non uncommon for genes to be under more than than one form of regulation, nor is it uncommon, in bacteria, for some regulatory proteins to be both repressors and activators for dissimilar genes. Attenuation is another form of transcriptional regulation, but in this case the transcript is terminated early in elongation. The mechanism by which attenuation takes identify can vary quite dramatically between different organisms. Besides non all regulatory molecules are proteins; regulatory RNA can also play a role.

The majority of genes encode proteins, and the RNA transcript must then be used as (or processed to become) a messenger RNA (mRNA). As mentioned above, eukaryotic transcriptional units are almost always single genes, but some transcripts from protein-encoding genes (particularly from animals) can be very long (more i million bases). The peachy length of these transcripts results from the fact that the poly peptide-encoding genes of eukaryotes often have several introns (noncoding sequences) interspersed inside the coding sequences (exons), and these are transcribed every bit a unit of measurement. Such genes are sometimes referred to as 'dissever genes'. In genes containing introns, so, one part of cistron expression is the processing of the transcript to remove these introns. Indeed, in eukaryotes, about transcripts from protein-encoding genes demand three singled-out processing steps to be converted into mRNA: capping, splicing, and tailing. Capping involves calculation a modified guanosine to the 5′ cease of the pre-mRNA. It is this cap that allows the RNA to be recognized past the translational machinery of the cell as an mRNA. The RNA splicing process removes introns and joins the exons together. Tailing involves cutting the transcript at a specific site downstream of the region encoding the protein and polyadenylating the newly created 3′ end.

These processing events are coupled to transcription. Capping takes place very soon after transcription has started. At least in the higher eukaryotes, where genes may have, in the extreme, many large introns, splicing is too coupled to transcription. The splicing process in eukaryotic pre-mRNA is complex and involves ribonucleoprotein particles called 'spliceosomes' that contain various poly peptide factors and small nuclear RNA molecules (snRNPs or 'snurps'). Splicing involves recognition of specific sites on the RNA and very precise cleavage and ligation of the RNA (since an error of a single nucleotide volition consequence in a frameshifted message). Splicing is also regulated, and some genes have transcripts that can be spliced in more one mode (alternative splicing) to yield more than ane protein from a unmarried cistron. Alternative splicing pathways are peculiarly prevalent in the transcripts from genomes of small beast viruses simply occur in other genomes also.

The transcripts of protein-encoding genes from prokaryotes do not crave processing to be functional; therefore, the transcripts of these genes are mRNAs. Also, equally mentioned above, some transcriptional units in prokaryotes incorporate information from several contiguous genes. The mRNAs produced from such units are said to exist 'polycistronic', in contrast to 'monocistronic' mRNA, which carries information for only ane gene product. In Escherichia coli, over lxx% of the mRNA is monocistronic and about 30% is polycistronic (with about 6% containing the information from four or more genes).

For some genes, the final production is an RNA molecule, simply even here processing is involved, and in this case processing occurs in both prokaryotes and eukaryotes. (Therefore, the only major course of RNA that tin can be used straight as transcribed is mRNA from prokaryotes.) The only genes we shall discuss here whose last product is RNA are genes encoding transfer RNA (tRNA) and genes encoding ribosomal RNA (rRNA). In both prokaryotes and eukaryotes, some of both types of genes may contain introns. Although the procedure by which these introns are removed involves excising the intron and ligating the exons, and is called 'splicing', the machinery which performs these reactions is non related to that which splices eukaryotic mRNA. Some of the introns in rRNA and tRNA are self-splicing (and self-splicing introns are as well known in a few bacteriophage mRNAs). Cocky-splicing introns (a particular kind of self-splicing intron) are widely found in nature and they are the only type plant in bacteria and bacteriophage. In both eukaryotes and prokaryotes, tRNAs and rRNAs are made initially equally longer precursors and all must be cut to their last size. In improver, tRNAs contain many modified bases (and in some cases the final conserved CCA sequence at the three′ cease must be added enzymatically). Modification of rRNAs is less extensive.

All these RNAs, whether they are informational intermediates like mRNA or final products of factor expression like tRNA and rRNA, are used in the next step of gene expression: translation.

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DNA Damage Responses in Atherosclerosis

Kenichi Shimada , ... Moshe Arditi , in Biological DNA Sensor, 2014

Dna impairment exists in all cellular organisms. While DNA damage is distinguished from mutation, mutation can issue from unrepaired Dna. While about DNA damage can be repaired, such repair systems are not 100% efficient. Un-repaired Deoxyribonucleic acid damage accumulates in non-replicating cells, such as neurons or myocytes of adult mammals, and tin can cause aging. Dna damage tin be subdivided into two types: (1) endogenous harm acquired by reactive oxygen species (ROS) that are derived from metabolic byproducts and (two) exogenous damage acquired by radiations (UV, X-ray, gamma), hydrolysis, found toxins, and viruses. Electric current data suggest that increased oxidative stress is a major characteristic of hypercholesterolemia-induced atherosclerosis and that oxidative stress is most likely associated with Dna damage in the development of cholesterol-induced plaques. This chapter critically addresses the extent to which the DNA damage, the sensing of it, and Dna damage repair are involved in the pathogenesis of atherosclerosis.

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Advances in Applied Microbiology

Paul Hyman , Stephen T. Abedon , in Advances in Practical Microbiology, 2010

I Introduction

Viruses are obligate intracellular parasites of cellular organisms. As such, their basic life cycle involves cooption of cellular metabolism toward production of new virus particles, release of those particles from their cellular confines, and and then acquisition of new cells. A virus life bike consequently is successful only to the extent that those three steps are productively completed. Many things tin go wrong in the course of the viral life cycle such that productive infection is never accomplished ( Fig.   seven.1), and we can describe these phenomena in terms of a virus' host range, which for bacteriophages (phages) is an assortment of susceptible bacteria types. Additionally relevant is the range of bacterial types to which phages can transduce DNA.

Effigy vii.1. Possible phage infection and bacterial survival outcomes as shown from the perspective of both phage biological science and bacterial mechanisms of phage resistance. The curved, thicker bars to the above-left and bottom-left are representations of filamentous phage virions, whose productive infections issue in chronic release. Tailed phages, shown in a higher place-correct and bottom-right, all display lytic productive infections. Functions primarily associated with phage virions (i.eastward., adsorption and release) are shown as curt dashed lines. Longer dashed lines are those leading directly to bacterium expiry. Notation that technically, as defined here, uptake blocks exercise not give rise to bacterial infections, since phage genomes do not accomplish the bacterial cytoplasm and hence we bypass "Devastation Infection" in the diagram. Nonetheless, uptake blocks from the bacterium's perspective may exist viewed as a grade of brake since phages are inactivated without loss of bacterium viability. Pseudolysogeny* we define equally a nonproductive, nondestructive, nonphage-genome reproductive, nonlysogenic, and nonchronic phage infections (Abedon, 2009b; Miller and Day, 2008). Depending on the phage, productive infections may result in either chronic virion release (via budding or, more than typically, extrusion) or, more likely, occur via lysis. Productive infections are illustrated every bit increases in phage numbers. Not shown are reductions in either infection vigor or phage productivity, as presented in Figs.   7.2 and 7.3, respectively.

Phage-resistance mechanisms encoded by bacteria (bacterial resistance) serve to limit phage host range. Though often viewed mainly equally blocks on phage adsorption, at that place are a number of additional leaner, prophage, and, perhaps most typically, plasmid-encoded mechanisms which interfere with phage infections (Fig.   7.2). Collectively, these mechanisms have been described as making up the "Bacteriophage 'Resistome'" (Hoskisson and Smith, 2007), and they accept been extensively reviewed peculiarly amongst lactic acrid bacteria (LAB; Allison and Klaenhammer, 1998; Daly et al., 1996; Dinsmore and Klenhammer, 1995; Forde and Fitzgerald, 1999; Garvey et al., 1995; Colina, 1993; Klaenhammer and Fitzgerald, 1994). Phages, in plough, use numerous resistance‐countering and therefore host-range expanding adaptations, equally are as well discussed in these reviews. See also Ackermann and DuBow (1987), Nieradko and Los (2006), and Weinbauer (2004) for further explorations of phage host range and bacterial resistance.

Figure seven.2. Bacterial resistance equally a function of phage infection stages. Darker shading to right is indicative of greater levels of reduction in phage fettle. Not indicated is the degree to which host fitness is impacted (for that, meet Fig.   7.3). Abbreviations employed include "vir" for "virulent" (significant phage virulence toward host bacteria; see Section   II.D), ↑ and ↓ significant increased and decreased, respectively, and CRISPR, which is as defined in the text (Section   V.C). Unsaid is that more virulent phages, such as phage T4, irreversibly destroy bacteria earlier in infections than do less virulent phages, such as phage λ. Encounter Dinsmore and Klenhammer (1995) for a like representation.

Bacterial resistance mechanisms are usually differentiated into adsorption blocks (Section   4), phage-genome uptake blocks (Section   5.A), restriction modification (Section   5.B), and abortive infections (Department   Six). More than recently, CRISPR mechanisms accept been added to this listing (Section   V.C). Here nosotros utilise a similar but more broadly applicable scheme which emphasizes phage versus bacterium survival (Fig.   7.iii). We find this approach to be more than applicable to our interest in phage–host ecological interaction (Abedon, 2006, 2008a,b, 2009a, 2010; Abedon and LeJeune, 2005; Breitbart et al., 2005; Hyman and Abedon, 2008) since phage functioning is primarily a product of infection success while bacterial functioning tin can exist viewed largely in terms of survival following phage encounter. This functioning occurs inside natural environments (Abedon, 2010; Thingstad et al., 2008; Weinbauer, 2004), industrial ferments (Bogosian, 2006; plus in a higher place for LAB ferments), in the course of phage employment to combat nuisance and pathogenic bacteria (phage therapy; e.g., Balogh et al., 2010; Goodridge, 2010; O'Flaherty et al., 2009), etc., and often is antagonistic in terms of phage versus bacterium success. Bacterial resistance thus serves, in a higher place all, to assure bacterial survival, just at the aforementioned fourth dimension plays a predominant role in defining phage host range.

Figure 7.3. Scheme for classifying phage infections and bacterial resistance. Shown are full general categories besides equally the more specific mechanisms of restriction modification (Section   V.B) and CRISPR (Section   V.C). Mechanisms of phage resistance can be a consequence of bacteria mutation, phage mutation, bacteria encoding of specific factors, or environmental causes, though here we emphasize bacteria mutation and factor encoding. "Restriction" is used equally both a more than general term (with a modified historical connotation; run into Department   V), that is, blocks on phage infection which act postphage attachment and prephage takeover of host metabolism, and as a more specific term, equally in phage DNA restriction by restriction endonucleases (Section   Five.B). Annotation that with lysogeny blocks nosotros are assuming that the phage has irreversibly committed to a lysogenic infection prior to exposure to such mechanisms. Reduced phage productivity—which too can be applied to lysogenic or chronic infections (though in which case bacteria survive)—refers to mechanisms which wearisome phage population growth, though if express to burst size reduction or latent period extension can also be described more than specifically as a reduced infection vigor (Fig.   7.ii; Section   6.B). We distinguish between lysis delays, as shown under that heading (lower-left quadrant), and failures to lyse as shown nether bootless infection (Section   VI; lower-right quadrant), but actually are not enlightened of any absolute blocks on phage lysis, specially since many phage-infected bacteria might be expected to eventually spontaneously deteriorate in such a manner that intracellular virion release is inevitable. Note that in this chapter we emphasize phage-encounter blocks (Department   IV.A), receptor loss (Department   Four.B), uptake blocks (Department   V.A), brake modification (Section   Five.B), CRISPR (Section   V.C), and abortive infections (Section   VI).

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Planar Lipid Bilayers (BLMs) and Their Applications

A. Ottova , ... H.T. Tien , in Membrane Science and Technology, 2003

three.v Apoptosis

Cell death in a multi-cellular organism can occur by 2 distinct mechanisms: apoptosis and necrosis. The former can exist distinguished from the latter by a number of characteristics, such as nuclear chromatin condensation, plasma membrane blabbing, cell shrinkage, nuclear fragmentation into apoptotic bodies, and the degradation of the nuclear Dna into oligonucleosome bondage [one,12,51]. So, the internucleosomal cleavage has been shown to accompany apoptosis occurring in a broad variety of cell types and the Dna electrophoresis is used extensively for identifying the process. Cell-free nucleus apoptosis is a new way for evaluating apoptotic effects. Recently, photoelectric behavior of mammalian cells was found having bioanalytical significance. Apropos this, photoelectric effects of bilayer lipid membranes (BLMs) and electron mediator modified BLMs have been extensively studied, on account of their potential applications in understanding the mechanism of natural photosynthesis, and in developing photoelectric devices [i-3,52-54]. Mimicking the functionalities in the natural photosynthesis, which are represented by photoactive groups, electron donors and acceptors [4], various, attempts have been made to realize the bogus photosynthesis and solar-energy conversion system nether laboratory conditions. For example, synthetic dyes have been used to dope BLMs and respective photoresponses investigated [two,5-6]. Experimental findings indicate that electron mediator-doped BLMs can accelerate the photoinduced electron transfer across membranes, and heighten the photoelectric conversion efficiency [2,4]. Fullerenes, in detail C60 as a modifier of BLM, have attracted much interest in the report of photoelectric conversion because of the analogousness of these molecules for electrons and also, considering of their highly hydrophobic properties for doping BLMs [half-dozen-9]. All the same, past experiments on the photoelectric property of C60 modified BLMs were generally conducted on the conventional planar BLMs [half-dozen,8], the defect of which is the fragility, thus precludes protracted investigations and applied applications. In contrast, BLMs self-assembled on the solid support (dubbed as s-BLM) showed much more stability, and exhibited electrochemical and photoelectric conversion properties. This kind of s-BLMs has many applications in the area of membrane biophysics and in the development of biosensors [ii,eight,10-12]. In the present work, a simpler method for forming s-BLMs for photoelectric conversion studies is reported. S-BLMs are easily cocky-assembled on ITO (indium-tin oxide) conducting glass, and the photoelectric properties of the lipid bilayer, as well equally Cthreescore modified BLM are systematically studied. The mechanism of the facilitation effect by C60 on the photoinduced electron transfer across the BLM, too every bit the potential application of s-BLMs in photodynamic therapy is discussed [12,xv,18,19].

Here, we introduce the photoelectric method used for analyzing the apoptosis of the nucleoli of human chest cancer cells (MCF-7 line) induced past Taxol (paclitaxel, an anticancer drug). The cell-free MCF-7 nucleoli are deposited on self-assembled bilayer lipid membranes (BLMs) on ITO conducting glass (ITO = indium-can oxide). The photoelectric beliefs of the "jail cell" and the nucleolus-related biological behavior, apoptosis, were investigated. Compared with the traditional techniques used to judge apoptosis, such as the morphological ascertainment and the agarose gel electrophoresis, the photoelectric belittling method of apoptotic system may provide a rapid and sensitive mode to evaluate the nucleus apoptosis in before time.

The photoelectric current measurements were performed using a model 600 voltage analyzer (CH instruments Inc., USA). The light source was a Xe light (USHID Inc., Japan) with the light intensity of 121.4   mW   cm−2. A super thin cell made of glass was used as the photoelectric cell in which there accept been 3 electrodes, as shown in Fig. ii. The working electrode was made of ITO-coated glass with the area of two.0   cmtwo. After the ITO glass was mounted, the width of the cell was 0.5   mm. The counter electrode was platinum metal, and the reference electrode was an Ag/AgCl electrode. In experiments, ITO conducting glass electrode was mounted in the calorie-free path, and the entire ITO window was shined in the light path. The voltage of between the reference electrode and the working electrode was set to zip, which was a consideration from the previous study [2,13]. The nighttime current (light off) was commencement measured, and and then the light current was measured with the low-cal on. The low-cal-induced current was determined equally the deviation between the two measured values.

Taxol is the agent that can cause apoptosis and during the apoptosis, nuclear Dna volition degrade into oligonucleosome bondage. In this experiment, the degradation of the nuclear DNA was verified past the examination of the gel electrophoresis and fluorescence microscopy study of the total Deoxyribonucleic acid. After the nucleoli were incubated with Taxol for xx   min, the chromatin began to condense around the nuclear periphery. The peripheral chromatin ring began to condense into discrete masses afterwards xl   min, and then faint Deoxyribonucleic acid ladder emerged. After incubating for 1 hr, the chromatin masses blabbed of from the nuclear surface and became apoptotic bodies. During this fourth dimension, the Dna ladder became distinct. These results indicated that the decreasing trend of the photoelectric current had close relationship with the cleavage of the chromatin into oligonucleosome chains. The specificity of Taxol on nuclear Deoxyribonucleic acid too displayed that the photoelectric current of the ITO/s-BLM/MCF-7 nucleus aggregation is mainly dictated by the nucleoli. Nucleoli skeleton is known to be essential non simply in maintaining nucleoli structure, simply too in energy transfer. The apoptosis of nucleoli is accompanied by the disassembly of the nuclear lamina, which leads to the damage of nuclear skeleton, and thereby a decreasing of the photoelectric electric current as well. The decreasing tendency of the photoelectric current of the apoptotic nucleoli is related to the cleavage of the chromatin. In the interpretation of the photoelectric response of nucleoli, the possibility has been considered that the DNA double helix, which contains a stacked array of heterocyclic base of operations pairs, could be a suitable medium for electron transfer over long distance [12,51]. So the nuclear DNA can serve as an "electric wire" for photo-induced electron transfer by "hopping" from base to base. 1 widely observed property of apoptotic cells is the cleavage of the Deoxyribonucleic acid into fragments at sites separated by the internucleosomal spacing. The cleavage of nuclear Deoxyribonucleic acid resulted in the harm of Deoxyribonucleic acid molecules as photo-induced electron-transferring bridge. And then, the photoelectric current decreasing was in accordance with the cleavage of the nucleoli. In this connectedness, Gao, Luo and their associates [xl,51] have carried out experiments of s-BLMs without or with fullerene C60 that take been self-assembled on indium-tin oxide (ITO) glass. The photoelectric properties of the ITO supported planar lipid bilayers were studied. The light intensity of irradiation, bias voltage, and the concentration of donors, take been found to be limiting factors of the transmembrane photocurrent. Additionally, the facilitation effect of Csixty doped BLMs on the photoinduced electron transfer across the BLM has been considered.

The s-BLM/cytosol nucleoli assemblage responded to white lite (200-800   nm). Electron transfer along the DNA double helix and along nuclear skeleton is invoked in our estimation. This novel photoelectric belittling method may exist useful and could provide a rapid and sensitive technique in evaluating apoptosis by photodynamic therapy (PDT). The apoptotic response appears to be a function of both the photosensitizer and the cell line. One widely observed holding of apoptotic cells is the cleavage of the Dna into fragments at sites separated past the internucleosomal spacing. The cleavage of nuclear Deoxyribonucleic acid resulted in the impairment of DNA molecules every bit photo-induced electron-transferring bridge. If so, a decreasing of the photoelectric current would be detected in accordance with the cleavage of the nucleoli, equally testify of apoptosis. In the nowadays paper, our findings using supported planar lipid bilayers (s-BLMs), based on combined methods of circadian voltammetry and photochemistry [eight,18] are described below.

The photoconductance of C60-containing BLM is college than that of undoped BLM, as calculated from the slope of the I/V characteristics [9]. The above data indicate that C threescore doped in the BLM accelerates the photoinduced electron transfer procedure beyond membrane self-assembled on the ITO back up. As has been described above, the bilayer lipid membrane formed on the ITO substrate prevents the transmembrane electron transfer, thus reduces the intensity of the generated photocurrent. The comparatively higher photoconductivity of bilayer lipid membranes doped with fullerenes can exist interpreted as the electron transporting result of fullerenes. Once photoexcited, the fullerene in its long lived triplet state is reduced by the electron donors in the solution and forms the radical anion C60 −. Since fullerenes are free to move about within the lipid bilayer environment, due to their geodesic structure, molecular dimensions and highly hydrophobic properties, a subsequent electron transfer from a photoproduced anion to another fullerene in its photoexcited state occurs. This electron transporting effect of C60 propagates the electron flux from the donors in the solution through the membrane towards the ITO electrode, which acts as an electron acceptor. Thus, experimental findings have shown that a BLM self-assembled on metal electrode blocked the electron transfer beyond the electrode and solution. The present photoelectric conversion experiment indicates that photoinduced electron could transfer across BLMs self-assembled on ITO conducting glass and the mediators doped in BLMs could facilitate this transmembrane photoinduced electron transfer. Since the ITO/BLM probes possess biological compatibility, therefore biomaterials could be embedded and their photoresponse properties investigated. Information technology seems evident that this novel self-assembled ITO/BLM probes will be a promising tool for the study of calorie-free-induced properties of biomembranes (e.grand., photodynamic therapy) and the development of biomimetic photoelectric devices (see Fig. 2 Middle).

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