Is any difference between cells individual organisms or group of organism of any species?

Factors Pointing to RNA

Cellular organisms on Earth today use three types of biopolymers (Figure 1). DNA genes are transcribed by protein enzymes to make mRNAs. The mRNAs are translated by ribosomes (whose chief 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 system that used only RNA. In the RNA world, RNA genes would be copied by RNA catalysts (Joyce, 2002).

In addition to logical simplicity, there are many other factors that suggest this could be true. Although no cellular life forms have genes made of RNA, there are many viruses that use RNA as genetic material. 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 means that sequence information is passed on. When the second 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 World, it is envisaged that there were RNA polymerases that were ‘ribozymes’ (i.e., catalysts made of RNA). There are no naturally occurring RNA polymerase ribozymes of this type, but there 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 ability of RNA to be a catalyst is well established.

The first ribozymes discovered were self-splicing introns (Kruger et al., 1982), which splice themselves out of pre-mRNA sequences without the aid of protein enzymes. For the RNA World hypothesis, the most fundamental naturally occurring ribozyme is ribosomal RNA. From the three 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 modern ribosomes were more recent additions. Protein synthesis also depends on transfer RNAs to decode the gene sequence and messenger RNAs that contain the genetic sequence. The whole process of translation only makes sense if RNAs preceded proteins.

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 Also, the traditional view of nucleic acids as having a limited range of functions, is becoming somewhat outdated (Breaker and Joyce, 2014), as new discoveries are made regarding nucleic acid functions in cells.

In some versions of the RNA World, it is envisaged that relatively complex organisms with many different kinds of RNA catalysts evolved before the origin of encoded protein sequences (Chen et al., 2007). In this view, the evolution of the ribosome and the genetic code would mark the end of the RNA World. Proteins would then begin to take over most of the roles that were previously catalyzed by ribozymes. A slightly different picture is that amino acids and small 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 even having amino acids covalently linked to ribozymes, is compatible with an RNA World picture of early life, but it is important to remember that long protein sequences could not be encoded before the translation process arose.

The other side of the cofactor argument is that many modern proteins use either single nucleotides or dinucleotides as cofactors that associate with the folded proteins and are essential for their function. This is often seen as further evidence for the RNA World (White, 1976), with the nucleotide cofactors being relics of an earlier phase of evolution.

Viral genome and its functioning.

In cellular organisms, the major functions of the nucleic acids – replication, transcription, and translation – are distributed between two 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′ end). In translation, a protein 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 same 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 can fulfill both (+) and (−) functions (Table. 1.4).

Table 1.4. Structure of plant viral genomes (Harrison BD, 1982)

As can be seen, only a small number of viruses contain double-stranded information molecules, reading from which is done similar to cellular organisms. Most phytopathogenic viruses have one multifunctional RNA (+) strand. Its functions are storage and realization of information. In addition, the RNA (+) strand is an infectious molecule. After arrival of the virus particle containing an RNA (+) strand to the cell, the first stage is decapsulation, i.e. release of the RNA molecule from its protein coat. This process is carried out on the cell receptors by the proteases of the host plant. The next process is replication, catalyzed by the enzyme RNA-dependent RNA polymerase (replicase), which in most viruses is encoded by the own genome. The RNA of the tobacco mosaic virus (TMV), typical of this group, encodes four proteins (Figure 1.1).

Translation begins from the 5′ end of the molecule – synthesis of 126 kDa and 183 kDa proteins, replicase components. These proteins occur in the cell at early stages of the infection process; hence they are called early proteins. Another virus – turnip yellow mosaic virus – develops hybrid replicase in the infected cell: one of its components (115 kDa) is encoded by the viral genome, the other – a 45 kDa protein – by the host cell genome. Apparently, the host enzyme system is routinely used for replication of viral RNA, as for many plants infected with viruses a substantial increase 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 structure. The (−) strand serves as a matrix for synthesis of new molecules of the viral (+) RNA, which functions as mRNA in translation of the late viral protein cells in ribosomes. In TMV they consist of a 30 kDa transport protein and a 17.5 kDa structural protein of the coat (Figure 1.1). 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 after arrival to the cell, and disjunctive (separated) replication of a new generation of viruses occurs, similar rather to the factory conveyor than reproduction of cellular organisms (synthesis of individual components, sometimes occurring in different 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 particular, RNA transcriptase, and all these are covered with an additional coat containing lipids. Such a particle enters the cell like a “Noah's Ark” with its own enzymes. Further synthesis of the new generation of viruses is carried out not separately, but in one compartment.

Unlike cellular organisms, the viral genome experiences a deficit of information, as it can encode only several proteins. In some viruses, the molecule of nucleic acid contains not four reading frames, like the TMV RNA, but a larger number; however, the increase in the amount 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, have 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 information problem in different ways.

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

Fragmentation of the genome. On centrifugation of some viruses isolated from infested plants, in density gradient of cesium or sucrose, it was found 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 easily separated in gradients into separate fractions). Each separate 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 similar 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 short particles encodes the structural proteins of the coat. A fraction consisting of the long particles can infect plants and form a new RNA generation, but it is unstable and cannot survive outside a plant cell; the short particles are stable, they cannot infect plants and reproduce in them. Two components were found in the viruses of tobacco rattle, ring spot of raspberry, black ring spot of tomato, cucumber mosaic, etc., three in the cow pea mosaic virus, and four in the alfalfa mosaic virus.

Use of a helper. It was written earlier that shortage of information made some viruses use the host cell enzymes during replication and translation. There are viruses (suggested to be called virusoids) that have an RNA molecule consisting of several hundred nucleotides and are incapable of encoding more than one protein (for instance, structural protein of the coat). The virus receives the other proteins, necessary for intracellular maturation, from another virus, the helper; therefore, it cannot live 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-free plants and though it is covered with its own coat, it uses the early proteins encoded by the TNV genome.

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

I INTRODUCTION

Multi-cellular organisms require a patent vascular system for efficient transport of nutrients and metabolic by-products. The hemostatic system is designed to maintain blood within the vascular system in a fluid state under physiological conditions. It is primed to react quickly to vascular injury in an explosive manner to minimize blood loss, mainly during injuries and child birth. As a consequence, evolutionary pressure favors efficient hemostatic mechanisms.

While a number of hemostatic mechanism can be separated, a synergy between 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 by 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 after the reproductive period and thus have a low evolutionary pressure. Thrombosis can be viewed as accident of nature with insufficient time to adapt through evolution to advances of modern medicine and longevity. Already in 1886, Rudolf Virchow identified the predisposing factors for thrombosis (also known as Virchow's triad), including alterations in the vessel wall, alterations in normal blood flow, and alterations in the composition of blood. Thrombotic diseases present clinically on the arterial site as coronary artery disease leading to acute coronary syndrome and sudden cardiac death, 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 bleeding or thrombosis. This conclusion is derived from mouse models or human monogenetic traits (for textbook coverage of Thrombosis and Haemostasis see Coleman et al., 2001; Loscalzo and Schafer, 1998; and Michelson, 2002).

Thrombosis is the main cause of morbidity and mortality in the western world, pointing to a large 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 balloon angioplasty and stenting (see Chapter 8). To extend the benefits observed in acute 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 one of these programs.

Abstract

As multi-cellular organisms evolved from small clusters of cells to complex metazoans, biological tubes became essential for life. Tubes are typically thought of as mainly playing a role in transport, with the hollow space (lumen) acting as a conduit to distribute nutrients and waste, or for gas exchange. However, biological tubes also provide a platform for physiological, mechanical, and structural functions. Indeed, tubulogenesis is often a critical aspect 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). Here we review our current understanding of the genetic regulation, molecular processes, and physical forces involved in tubulogenesis and morphogenesis of the epidermal, digestive and excretory systems in C. elegans.

Alternative Splicing

Also observed in many cellular organisms, alternative splicing allows production of transcripts having the potential to encode different proteins with different functions from the same gene (Fig. 3.7). The sequence of the mRNA is not changed as with RNA editing; rather the coding capacity is changed as a result of alternative splice sites. Alternative splicing is regulated by cellular and viral proteins that modulate the activity of the splicing factors U1 and U2, both of which are components of the spliceosome. The spliceosome is made up of the snRNAs (small 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 point sites. Serine/arginine-rich proteins, as well as heterogeneous nuclear ribonucleoproteins, play a key role in splice site recognition. Alternative splicing (1) increases the virus’ ability to encode several proteins in a given transcript (e.g., adenoviruses and retroviruses can encode ~12 different peptides from one pre-mRNA), (2) is a mechanism to regulate early and late expression for viruses (e.g., papillomaviruses), and (3) splicing is coupled to export of mRNA out of the nucleus. While only mature, spliced mRNA transcripts are exported out of the nucleus, hepadnaviruses and retroviruses are able to export 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. It is capable of associating with numerous cellular spliceosome subunits, such as U1 and U6 snRNAs, and can inhibit cellular gene expression by blocking the spliceosome component recruitment and its transition to the active state.

Both conservation and evolution of viral splice site sequences allow for improved adaptation to the host, and ensure recognition by the host’s splicing machinery. Therefore, viruses can induce preferential induction 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.

Transcription

The genes of all cellular organisms are composed of double-stranded DNA (some viruses have single-stranded DNA genomes and others even RNA genomes) and the first step in their expression is transcription. Transcription involves using one of the two strands of DNA as a template to make an RNA copy by an enzyme called RNA polymerase. All RNA polymerases synthesize an RNA chain from the 5′ end to the 3′ end while reading the template strand of the DNA in the 3′–5′ direction. The RNA molecules are synthesized from specific starting sites on the DNA and also terminate at specific sites. The sites where RNA polymerase (using accessory factors) recognizes the beginning of a transcriptional unit are termed promoters. In higher organisms, the unit of transcription is almost always a single gene. However, in prokaryotes, the transcriptional unit may contain 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 common mechanism in bacteria is the binding of a repressor protein to a specific region of the DNA near the promoter which then blocks transcription. The sequence to which the repressor protein binds is termed an ‘operator’, a term which has given its name to the transcriptional unit called an ‘operon’. In bacteria, an operon may contain one or more genes, all under the control of the single operator. Another mechanism for regulating gene expression is the binding of a regulatory protein to the DNA which activates transcription. Such positive control is widespread in eukaryotic genes. It is not uncommon for genes to be under more than one form of regulation, nor is it uncommon, in bacteria, for some regulatory proteins to be both repressors and activators for different 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 place can vary quite dramatically between different organisms. Also not 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 than one million bases). The great length of these transcripts results from the fact that the protein-encoding genes of eukaryotes often have several introns (noncoding sequences) interspersed within the coding sequences (exons), and these are transcribed as a unit. Such genes are sometimes referred to as ‘split genes’. In genes containing introns, then, one part of gene expression is the processing of the transcript to remove these introns. Indeed, in eukaryotes, most transcripts from protein-encoding genes need three distinct processing steps to be converted into mRNA: capping, splicing, and tailing. Capping involves adding a modified guanosine to the 5′ end of the pre-mRNA. It is this cap that allows the RNA to be recognized by 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 also coupled to transcription. The splicing process in eukaryotic pre-mRNA is complex and involves ribonucleoprotein particles called ‘spliceosomes’ that contain various protein 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 will result in a frameshifted message). Splicing is also regulated, and some genes have transcripts that can be spliced in more than one way (alternative splicing) to yield more than one protein from a single gene. Alternative splicing pathways are particularly prevalent in the transcripts from genomes of small animal viruses but occur in other genomes also.

The transcripts of protein-encoding genes from prokaryotes do not require processing to be functional; therefore, the transcripts of these genes are mRNAs. Also, as mentioned above, some transcriptional units in prokaryotes contain information from several contiguous genes. The mRNAs produced from such units are said to be ‘polycistronic’, in contrast to ‘monocistronic’ mRNA, which carries information for only one gene product. In Escherichia coli, over 70% 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 product is an RNA molecule, but even here processing is involved, and in this case processing occurs in both prokaryotes and eukaryotes. (Therefore, the only major class of RNA that can be used directly as transcribed is mRNA from prokaryotes.) The only genes we shall discuss here whose final 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 process 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 not related to that which splices eukaryotic mRNA. Some of the introns in rRNA and tRNA are self-splicing (and self-splicing introns are also known in a few bacteriophage mRNAs). Self-splicing introns (a particular kind of self-splicing intron) are widely found in nature and they are the only type found in bacteria and bacteriophage. In both eukaryotes and prokaryotes, tRNAs and rRNAs are made initially as longer precursors and all must be cut to their final size. In addition, tRNAs contain many modified bases (and in some cases the final conserved CCA sequence at the 3′ end must be added enzymatically). Modification of rRNAs is less extensive.

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

DNA damage exists in all cellular organisms. While DNA damage is distinguished from mutation, mutation can result from unrepaired DNA. While most DNA damage can be repaired, such repair systems are not 100% efficient. Un-repaired DNA damage accumulates in non-replicating cells, such as neurons or myocytes of adult mammals, and can cause aging. DNA damage can be subdivided into two types: (1) endogenous damage caused by reactive oxygen species (ROS) that are derived from metabolic byproducts and (2) exogenous damage caused by radiation (UV, X-ray, gamma), hydrolysis, plant toxins, and viruses. 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.

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 then acquisition of new cells. A virus life cycle consequently is successful only to the extent that those three steps are productively completed. Many things can go wrong in the course of the viral life cycle such that productive infection is never achieved (Fig. 7.1), 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.

Phage-resistance mechanisms encoded by bacteria (bacterial resistance) serve to limit phage host range. Though often viewed mainly as blocks on phage adsorption, there are a number of additional bacteria, 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 have been extensively reviewed especially among lactic acid bacteria (LAB; Allison and Klaenhammer, 1998; Daly et al., 1996; Dinsmore and Klenhammer, 1995; Forde and Fitzgerald, 1999; Garvey et al., 1995; Hill, 1993; Klaenhammer and Fitzgerald, 1994). Phages, in turn, employ numerous resistance‐countering and therefore host-range expanding adaptations, as are also 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.

Bacterial resistance mechanisms are usually differentiated into adsorption blocks (Section IV), phage-genome uptake blocks (Section V.A), restriction modification (Section V.B), and abortive infections (Section VI). More recently, CRISPR mechanisms have been added to this list (Section V.C). Here we employ a similar but more broadly applicable scheme which emphasizes phage versus bacterium survival (Fig. 7.3). We find this approach to be more 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 can be viewed largely in terms of survival following phage encounter. This functioning occurs within natural environments (Abedon, 2010; Thingstad et al., 2008; Weinbauer, 2004), industrial ferments (Bogosian, 2006; plus above 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, above all, to assure bacterial survival, but at the same time plays a predominant role in defining phage host range.

3.5 Apoptosis

Cell death in a multi-cellular organism can occur by two distinct mechanisms: apoptosis and necrosis. The former can be 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 chains [1,12,51]. So, the internucleosomal cleavage has been shown to accompany apoptosis occurring in a wide 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. Concerning 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 [1-3,52-54]. 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 artificial photosynthesis and solar-energy conversion system under laboratory conditions. For example, synthetic dyes have been used to dope BLMs and corresponding photoresponses investigated [2,5-6]. Experimental findings indicate that electron mediator-doped BLMs can accelerate the photoinduced electron transfer across membranes, and enhance the photoelectric conversion efficiency [2,4]. Fullerenes, in particular C60 as a modifier of BLM, have attracted much interest in the study of photoelectric conversion because of the affinity of these molecules for electrons and also, because of their highly hydrophobic properties for doping BLMs [6-9]. However, past experiments on the photoelectric property of C60 modified BLMs were mostly conducted on the conventional planar BLMs [6,8], the defect of which is the fragility, thus precludes protracted investigations and practical 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 [2,8,10-12]. In the present work, a simpler method for forming s-BLMs for photoelectric conversion studies is reported. S-BLMs are easily self-assembled on ITO (indium-tin oxide) conducting glass, and the photoelectric properties of the lipid bilayer, as well as C60 modified BLM are systematically studied. The mechanism of the facilitation effect by C60 on the photoinduced electron transfer across the BLM, as well as the potential application of s-BLMs in photodynamic therapy is discussed [12,15,18,19].

Here, we introduce the photoelectric method used for analyzing the apoptosis of the nucleoli of human breast cancer cells (MCF-7 line) induced by 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-tin oxide). The photoelectric behavior of the “cell” and the nucleolus-related biological behavior, apoptosis, were investigated. Compared with the traditional techniques used to estimate apoptosis, such as the morphological observation and the agarose gel electrophoresis, the photoelectric analytical method of apoptotic system may provide a rapid and sensitive way to evaluate the nucleus apoptosis in earlier 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 have been three electrodes, as shown in Fig. 2. The working electrode was made of ITO-coated glass with the area of 2.0 cm2. 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 light 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 zero, which was a consideration from the previous study [2,13]. The dark current (light off) was first measured, and then the light current was measured with the light on. The light-induced current was determined as the difference between the two measured values.

Taxol is the agent that can cause apoptosis and during the apoptosis, nuclear DNA will degrade into oligonucleosome chains. In this experiment, the degradation of the nuclear DNA was verified by the examination of the gel electrophoresis and fluorescence microscopy study of the total DNA. After the nucleoli were incubated with Taxol for 20 min, the chromatin began to condense around the nuclear periphery. The peripheral chromatin ring began to condense into discrete masses after 40 min, and then faint DNA ladder emerged. After incubating for 1 hour, the chromatin masses blabbed of from the nuclear surface and became apoptotic bodies. During this time, the DNA ladder became distinct. These results indicated that the decreasing tendency of the photoelectric current had close relationship with the cleavage of the chromatin into oligonucleosome chains. The specificity of Taxol on nuclear DNA also displayed that the photoelectric current of the ITO/s-BLM/MCF-7 nucleus assemblage is mainly dictated by the nucleoli. Nucleoli skeleton is known to be essential not only in maintaining nucleoli structure, but also 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 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 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. One widely observed property of apoptotic cells is the cleavage of the DNA into fragments at sites separated by the internucleosomal spacing. The cleavage of nuclear DNA resulted in the damage of DNA molecules as photo-induced electron-transferring bridge. So, the photoelectric current decreasing was in accordance with the cleavage of the nucleoli. In this connection, Gao, Luo and their associates [40,51] have carried out experiments of s-BLMs without or with fullerene C60 that have 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, have been found to be limiting factors of the transmembrane photocurrent. Additionally, the facilitation effect of C60 doped BLMs on the photoinduced electron transfer across the BLM has been considered.

The s-BLM/cytosol nucleoli assemblage responded to white light (200-800 nm). Electron transfer along the DNA double helix and along nuclear skeleton is invoked in our interpretation. This novel photoelectric analytical method may be 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 property of apoptotic cells is the cleavage of the DNA into fragments at sites separated by the internucleosomal spacing. The cleavage of nuclear DNA resulted in the damage of DNA molecules as photo-induced electron-transferring bridge. If so, a decreasing of the photoelectric current would be detected in accordance with the cleavage of the nucleoli, as evidence of apoptosis. In the present paper, our findings using supported planar lipid bilayers (s-BLMs), based on combined methods of cyclic voltammetry and photochemistry [8,18] are described below.

The photoconductance of C60-containing BLM is higher than that of undoped BLM, as calculated from the slope of the I/V characteristics [9]. The above data indicate that C60 doped in the BLM accelerates the photoinduced electron transfer process across membrane self-assembled on the ITO support. 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 be interpreted as the electron transporting effect 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 across 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. It seems evident that this novel self-assembled ITO/BLM probes will be a promising tool for the study of light-induced properties of biomembranes (e.g., photodynamic therapy) and the development of biomimetic photoelectric devices (see Fig. 2 Middle).