Targeting glycolytic isoenzymes
Glycolytic pathway serves as the principal energetic source for the cell. The higher dependency of cancer cells upon glycolytic metabolism for the production of ATP  provides a loftier motive to target glycolytic enzymes. Many isoforms of these enzymes have been found to be specifically expressed in tumor cells and are being exploited as a potential candidates to be used as drug targets. The transport of glucose across the plasma membrane is regulated by various isoforms of glucose transporters (GLUT1-14 or SLC2A1-14). GLUT1, GLUT3 and GLUT4 are found to be expressed at higher levels in cancer cells compared to normal cells . GLUT3 and other transporters could be targeted by the use of specific antibodies or drugs like Phloretin or Ritonavir causing the cells to starve by blocking their nutrient uptake through these transporters.
Another important metabolic enzyme of the glycolytic pathway is hexokinase (HK), which regulates the first rate limiting step of glucose metabolism. Cancer cells have been found to be heavily dependent on hexokinases isoforms, like the hexokinase 2 (HK2) . The specific expression of HK2 in adipose tissue and skeletal muscles provides an opportunity to target HK2 without having the risk of affecting other tissues. Compounds like Methyl Jasmonate isolated from plants have shown to disrupt the association between mitochondria and hexokinases (HKI
Types of Tissue and Muscle
Muscle Muscle is one of the four fundamental types of tissue present in animals. It is a soft tissue primarily responsible to produce movement of a body part. Its cells convert the chemical energy of ATP into the mechanical energy of motion and exert a useful pull on another tissue. More specifically, muscle contraction serves the following overlapping functions: movement and contractile move of body contents in the course of respiration, circulation, digestion, defecation, urination, and childbirth. Stability by resisting the pull of gravity. Communication. As well as the control of body openings and passages. And finally, producing around 85% of our body heat, which is vital for the metabolism (Carey Carpenter, 2007).
There are three histological types of muscle—cardiac, smooth and skeletal—which differ in appearance, physiology, and function.
The cardiac muscle is essentially limited to the heart, though it extends slightly into the nearby blood vessels. It is involuntary and striated because of the regular arrangement of their actin and myosin filaments. Its cells are much shorter, so they are commonly called myocytes, and are mononuclated (Saladin, 2003). The myocytes assemble branches of adjoining cells and form a network by attaching to each other in attachment points called intercalated discs that provide strong mechanical adhesions between adjacent cells. Smooth muscle lacks striations and is involuntary. Smooth muscle cells are usually long and spindle-shaped, and each fusiform cell has a single and centered nucleus (Purves et al., 2004). Small amounts of smooth muscle are found in the iris of the eye and in the skin, but most of it, called visceral muscle, forms layers in the walls of the digestive, respiratory, and urinary tracts, blood vessels, the uterus, and other viscersa (Alberts et al., 2008). The skeletal muscle type is of special interest of this thesis and will be further studied in the following sections.
Skeletal muscles are, as the name implies, are bound to the skeleton by means of tendons, which means that they are volitional. It is composed of both muscular tissue and connective. A skeletal muscle cell (muscle fiber) is about 10 to 100m in diameter and 30 cm long. It is surrounded by a sparse layer of areolar connective tissue called the endomysium, which allows room for blood capillaries and nerve fibers to reach each muscle fiber. Muscle fibers are grouped in bundles called fascicles, which are visible to the naked eye as parallel strands. Each fascicle is separated from neighboring ones by a connective tissue sheath called the perimysium, usually somewhat thicker than the endomysium. The muscle as a whole is surrounded by still another connective tissue layer, the epimysium. The epimysium grades imperceptibly into connective tissue sheets called fasciae, deep fasciae between adjacent muscles and a superficial fascia (hypodermis) between the muscles and skin. It is described as striated and voluntary (Saladin, 2003).
The skeletal muscle tissue consists of long and cylindrical cells called muscle fibers, which are actually huge single cells that form during development by the fusion of many separate cells, called myoblasts. Each cell contains multiple nuclei adjacent to the plasma membrane, and they are about 10 to 100m in diameter and 30 cm long. The bulk of the cytoplasm inside is made up of myofibrils, which is the name given to the basic contractile elements of the muscle cell. Within each myofibril are thin actin filaments and thick specific muscle isoforms of myosin II filaments (Alberts et al., 2008). Myosin filaments are bundles of molecules with globular heads and polypeptide tails. Actin filaments consist of two chains of actin monomers twisted together. They are wrapped by chains of the polypeptide tropomyosin and studded at intervals with another protein, troponin.
In most regions of the myofibril, each thick myosin filament is surrounded by six thin actin filaments, and conversely, each thin actin filament sits within a triangle of three thick myosin filaments. The myofibril consists of repeating units, called sarcomeres, which are the units of contraction, built up of approximately 2.2 microns in length. Each sarcomere is made of overlapping filaments of actin and myosin, which create a distinct band pattern. As the muscle contracts, the sarcomeres shorten, and the appearance of the band pattern changes. Each sarcomere is bounded by Z-lines, which are structures that anchor the thin actin filaments. Centered in the sarcomere is the A-band, which contains all the myosin filaments. The H-zone and the I-band, are regions where actin and myosin filaments do not overlap in the relaxed muscle. The dark stripe within the H-zone is called the M-band; it contains proteins that help hold the myosin filaments in their regular arrangement. The bundles of myosin filaments are held in a centered position within the sarcomere by a protein called titin. Cardiac muscle and smooth muscle also contain sarcomeres, although the organization is not as regular as that in skeletal muscle (Alberts et al., 2008; Purves et al., 2004.).
The plasma membrane, called the sarcolemma has tunnel-like infoldings called transverse (T) tubules that penetrate through the fiber and emerge on the other side. The function of a T tubule is to carry an electrical current from the surface of the cell to the interior when the cell is stimulated. Most other organelles of the cell, such as mitochondria and smooth endoplasmic reticulum (ER), are located between adjacent myofibrils. The sarcoplasm also contains an abundance of glycogen, which provides stored energy for the muscle to use during exercise, and a red pigment called myoglobin, which binds oxygen until it is needed for muscular activity. The smooth ER of a muscle fiber is called sarcoplasmic reticulum (SR). It forms a network around each myofibril, and alongside the T tubules it exhibits dilated sacs called terminal cisternae. The SR is a reservoir for calcium ions; it has gated channels in its membrane that can release a flood of calcium into the cytosol, where the calcium activates the muscle contraction process (Saladin, 2003).
For contraction to occur, an action potentials spreads from the end plate and when it reaches the T tubules, it continues down them into the sarcoplasm. Action potentials open voltage-regulated ion gates in the T tubules. These are physically linked to calcium channels in the terminal cisternae of the sarcoplasmic reticulum (SR), so gates in the SR open as well and calcium ions diffuse out of the SR, down their concentration gradient and into the cytosol. The calcium ions bind to the troponin of the thin filaments. The troponin-tropomyosin complex changes shape and shifts to a new position. This exposes the active sites on the actin filaments and makes them available for binding to myosin heads; the myosin heads must have an ATP molecule bound to it to initiate the contraction process. Myosin ATPase, an enzyme in the head, hydrolyzes this ATP. The energy released by this process activates the head, which “cocks” into an extended, high-energy position. The head temporarily keeps the ADP and phosphate group bound to it. The cocked myosin binds to an active site on the thin filament. Myosin releases the ADP and phosphate and flexes into a bent, low-energy position, tugging the thin filament along with it. This is called the power stroke. The head remains bound to actin until it binds a new ATP. Upon binding more ATP, myosin releases the actin. It is now prepared to repeat the whole process—it will hydrolyze the ATP, recock (the recovery stroke), attach to a new active site farther down the thin filament, and produce another power stroke (Saladin, 2003).
Classes of muscle fibers
Not all muscle fibers are metabolically alike or adapted to perform the same task. Some respond slowly but are relatively resistant to fatigue, while others respond more quickly but also fatigue quickly. Indeed, skeletal muscles can be divided into fast and slow twitch fibers and its myosin heavy chain (MHC) isoform expression.
Type I (MHC-I): Also called slow oxidative (SO) or slow-twitch. These fibers have relatively abundant mitochondria, myoglobin, and blood capillaries, and therefore a relatively deep red color. They are well adapted to aerobic respiration, which does not generate lactic acid. Thus, these fibers do not fatigue easily. However, in response to a single stimulus, they exhibit a relatively long twitch, lasting about 100 milliseconds (msec).
Type II (MHC-II): Also called fast glycolytic (FG) or fast-twitch. They are well adapted for quick responses but not for fatigue resistance. They are rich in enzymes of the phosphagen and glycogen–lactic acid systems. Their sarcoplasmic reticulum releases and reabsorbs Ca2 quickly, which partially accounts for their quick, forceful contractions. They are relatively pale (white fibers). These fibers produce twitches as short as 7.5 msec, but because of the lactic acid they generate, they fatigue more easily than SO fibers.
Some authorities recognize two subtypes of FG fibers called types MHC-IIA and MHC-IIB. Type IIB is the common type just described, while IIA, or intermediate fibers, combine fast-twitch responses with aerobic fatigue-resistant metabolism. Type IIA fibers, however, are relatively rare except in some endurance-trained athletes (Saladin, 2003). Notably, human skeletal muscle does not contain MHCIIb (Spangenburg and Booth, 2003; Schiaffino and Reggiani, 1994; Smerdu et al., 1994). In addition, ‘‘hybrid’’ fibers containing two MHC isoforms (i.e., type I/IIA, IIAX, IIXB) can also be present in muscle (Schiaffino and Reggiani, 1994; Staron and Pette, 1993).
The fiber types can be differentiated histologically by using stains for certain mitochondrial enzymes and other cellular components, like using immunohistochemical procedures with antibodies against the specific MHC isoforms (Schiaffino et al., 1989; Lucas et al., 2000). All muscle fibers of one motor unit belong to the same physiological type. Nearly all muscles are composed of both SO and FG fibers, but the proportions of these fiber types differ from one muscle to another.
Vertebrate skeletal myogenesis proceeds through three stages: determination of the muscle progenitor cells, called myoblasts; proliferation and in some cases migration of myoblasts; and their terminal differentiation into mature muscle by fusing to form multinucleated myotubes (Buckingham et al., 2003; Shi and Garry, 2006).
Muscle tissuesare derived from themesodermallayer of embryonicgerm cellsin a process known asmyogenesis. All muscles are derived fromparaxial mesoderm .The paraxial mesoderm is divided along the embryo’s length intosomites, corresponding to thesegmentationof the body.Muscle cells come from two cell lineages in the myotome somite, the epimere and hypomere, which formepaxialandhypaxialmuscles, respectively. Most muscles are hypaxial. During development,myoblasts either remain in the somite to form muscles associated with the vertebral column or migrate out into the body to form all other muscles. Myoblast migration is preceded by the formation ofconnective tissueframeworks, usually formed from the somaticlateral plate mesoderm.Myoblasts follow chemical signals to the appropriate locations, where they fuse into elongate skeletal muscle cells (Sweeney, 1997).
Muscle fibersform from the fusion ofmyoblastsinto multi-nucleated fibers calledmyotubes. In the early development of an embryothese myoblasts will proliferate if enoughfibroblast growth factor(FGF) is present, without differentiating. When these factors are depleted, the myoblasts cease division and secretefibronectinonto theirextracellular matrix and bind to it through _5β1 integrin, their major fibronectin receptor (Menko and Boettiger 1987; Boettiger et al. 1995).
The second stage involves the alignment of the myoblasts together into chains and subsequently into myotubes. This step is mediated by cell membrane glycoproteins, including several cadherins and CAMs (Knudsen 1985: Knudsen et al. 1990). Recognition and alignment between cells takes place only if the two cells are myoblasts. However, identity of the species is not critical (Yaffe and Feldman, 1965).
The third stage is the actual cell fusion itself. In this stage,calciumions are critical for development (Shainberg et al. 1969; David et al. 1981). Fusion is mediated by a set of metalloproteinasescalledmeltrins (e.g., c-Met).Myocyte enhancer factors(MEFs) promote myogenesis.Serum response factor(SRF) plays a central role during myogenesis, being required for the expression of striated alpha-actin genes (Wei et al., 1998).Expression of skeletalalpha-actinis also regulated by theandrogen receptor; steroids can thereby regulate myogenesis (Vlahopoulos et al., 2005).
The specific extracellular signals that induce determination of each group of myoblasts are expressed only transiently. These signals trigger production of intracellular factors that maintain the myogenic program after the inducing signals are gone. We discuss the identification and functions of these myogenic proteins, and their interactions, in the next several sections.
Muscle-specific transcription factors
Satellite cells and proliferating myoblasts is characterized by the expression of Pax-genes, more specifically Pax7 and Pax3, which are transcription factors that regulate proliferation. Back to the developmental stage, in the lateral portion of the somite, which forms the hypaxial muscles, factors from the surrounding environment induce the Pax3 transcription factor. In the absence of other inhibitory transcription, Pax3 then activates the genes encoding two muscle-specific transcription factors, Myf5 and MyoD. In the medial region of the somite, which forms the epaxial muscles, MyoD is induced through a slightly different pathway.
Pax7 residing satellite cells proliferating stage and Pax7 knockout mice completely lack satellite cells (Seale et al., 2000). Cells expressing Pax7 is believed to be important for the regeneration of myoblasts. Also the expression of Pax3 has negative effect on the differentiation and it has been shown that this transcription factor is down-regulated in muscle by ubiquitination and proteasomal degradation before differentiation can proceed (Boutet et al., 2007). Pax genes have been shown to regulate the proliferation and survival even of certain cancers such as melanoma (Muratovska et al., 2003). Pax transcription factors are downregulated upon further differentiation and other proteins predominate.
Meanwhile Pax3 _____, Pax7 appears only in muscle stem cells (___).
Muscle cells come from two cell lineages in the somite. In both instances, paracrine factors instruct the myotome cells to become muscles by inducing them to synthesize the MyoD proteins (Maroto et al. 1997; Tajbakhsh et al. 1997). Or also called the myogenic bHLH (basic helix-loop-helix) proteins. The proteins of this family all bind to similar sites on the DNA and activate muscle-specific genes (e.g. the muscle-specific creatine phosphokinase gene by binding to the DNA immediately upstream from it, or the chicken muscle acetylcholine receptor) (Lassar et al. 1989; Piette et al. 1990). MyoD and Myf5 belong to this family and are particularly important for muscle differentiation .
Their important role during differentiation is supported by the MyoD-/-/Myf-5-/- mice lacking fully developed skeletal muscle (Rudnicki et al., 1993). Myf-5 promotes myoblasts’ proliferation and is required for the cells to initiate differentiation (Ustanina et al., 2007). Absence of MyoD inhibits differentiation in cell culture and the protein is therefore considered to be a positive regulator of the process (Sabourin et al., 1999). While Pax3 is found in several other cell types, the myogenic bHLH proteins are specific for muscle cells. Any cell making a myogenic bHLH transcription factor such as MyoD or Myf5 is committed to becoming a muscle cell.
Myogenic regulatory factors (proper name?)
Later than the MyoD proteins expression during differentiation, the myogenin and the myogenic regulatory factor 4 (MRF4) are present. Mice with deleted myogenin in developing myoblasts can start the differentiation process but they cannot move and die soon after birth (Hasty et al., 1993). Among other things, this suggests that myogenin is required at a later stage of the process. Similarly, MRF4 is important for the growth of muscle tissue (Rhodes and Konieczny, 1989). It is noted that the MEF2 family of transcription factors also regulate differentiation (Olson et al., 1995), but their functions are not described in this context.
In summary, Pax transcription factors help to sustain it from the stem cell stage of satellite cells, MyoD and Myf-5 act as myogenic determinants in the myoblast’s diet, and myogenin and MRF4 are known as regulators of the later differentiation and muscle fiber formation (Pallari, 2011).
Although some factors that induce differentiation remain unknown, some growth factors and signaling molecules have been shown to regulate the process. Notch signaling is important in animal embryonic development, in that it participates in the cell fate determination (Alberts et al., 2008). Notch has a dual role of myoblasts in that the protein has an inhibitory effect on myoblasts differentiation (Shawber et al., 1996) and simultaneously stimulates their proliferation (Conboy and Rando, 2002). The chemoattractant SDF-1 also has an inhibitory effect on myoblasts differentiation and stimulates their proliferation by activation of the PKCζ (ÖdemiÅŸ et al., 2007). Moreover, differentiation requires the expression and activity of cyclindependent kinase (Cdk) inhibitors, such as p21 and p27, critical for the withdrawal of myoblasts from the cell cycle (Kitzmann and Fernandez, 2001). To fully understand the factors that regulate, activate and inhibit satellite cells and their differentiation requires even much research (Kuang and Rudnicki, 2007; Shi and Garry, 2006.).
 developmental biology Scott gilbert
 developmental biology Scott gilbert