What are intestinal enzymes called topoisomerases,how do digestive enzymes help,probiotic candida die off symptoms nausea,best digestive enzyme for breastfeeding jaundice - 2016 Feature

Digestion is needed to breakdown large insoluble molecules found in the bolus into smaller soluble molecules.  These molecules can then absorbed through the walls of the small intestine and onto the blood stream by diffusion.
The muscle before the bolus of food contract and the muscles after the bolus relax resulting in a wave of muscular contractions which pushes the bolus through the oesophagus. In the duodenum, the bile produced by the liver as well as the enzymes produced by the pancreas are released in the alimentary canal and they will mix with the food. The degree to which pathogenic bacteria are able to cause disease determines their virulence. In addition to its role in invasion, several lines of evidence indicate that the Shigella virulence factor IpaB is both necessary and sufficient to induce apoptosis in macrophages.
Another virulence factor encoded on the virulence plasmid is IcsA (intracellular spread, also called VirG). Neutrophils are essential for innate host defense against invading microorganisms, such as bacteria and fungi. Neutrophils are an essential component of the acute inflammatory response and the resolution of microbial infection. The concept of bacterial recognition is based on so-called pathogen associated molecular patterns (PAMPs), which are recognized by pattern recognition receptors (PRRs) (Gordon, 2002). Upon encountering bacteria, neutrophils engulf these microbes into a phagosome, which fuses with intracellular granules to form a phagolysosome (Lee, et al., 2003). The oxygen-independent mechanisms encompass the contents of the three neutrophil granule subsets: the azurophil, specific and gelatinase granules, which contain characteristic proteases, antimicrobial proteins and peptides enzymes (Borregaard and Cowland, 1997). The oxygen-independent mechanisms encompass the contents of the three neutrophil granule subsets: the azurophil, specific and gelatinase granules, which contain characteristic proteases, antimicrobial proteins and peptides, and enzymes. The importance of the oxygen-independent mechanism in is in two very rare inherited diseases, the Chediak-Higashi syndrome (Introne, et al., 1999) and Specific Granule Deficiency (Gombart and Koeffler, 2002). In the past, studies often focused on the effects of either the oxygen-dependent or oxygen-independent mechanisms. Macrophages phagocytose Shigella, but Shigella escapes from the phagolysosome of macrophages within minutes.
Shigella is alive within the phagocytic vacuole of neutrophils for up to one hour (Mandic-Mulec, et al., 1997).
How NE exactly contributes to the phagolysosomic retention of Shigella was shown in one study that assessed the ability of a human neutrophil granule extract (hNGE) to degrade different Shigella virulence factors (Weinrauch, et al., 2002). By specifically cleaving the Shigella virulence factors, NE possibly inhibits the interaction of Shigella with host proteins, thus preventing the escape of Shigella from the phagolysosome of neutrophils.
Despite their opposing specificity towards Shigella virulence factors NE and CG are neutral serine proteases of the same subfamily.
Chymotrypsin-like serine proteases combine a large group of diverse proteases, including chymotrypsin, trypsin, NE, and CG. Catalytic triads cleave a peptide bond between the carbonyl group of the N-terminal amino acid and the amide group of the C-terminal amino acid. The nucleophilic attack leads to conformational change in the vicinity of the carbonyl carbon atom of the substrate. An important step for the proper function of the catalytic triad is the correct binding and positioning of the substrate. The S1 pocket is thought to have an important role in the substrate specificity of the individual chymotrypsin-like serine proteases (Steitz and Shulman, 1982).
Besides common characteristics, specific residues differ in the S1 pocket of the individual proteases. Apart from the residue at the bottom of S1, other amino acids define the characteristics of the pocket through their side chains. The S1 pocket of chymotrypsin allows large, hydrophobic side chains to enter the pocket completely, whereas trypsin prefers long, negatively side chains. Yet, the model that substrate specificities are only determined by the S1 pockets is incomplete. As mentioned above, CG and NE belong to the same subfamily of chymotrypsin-like serine proteases.
In the developing granule, NE and CG are post-translationally processed to mature, active proteins. At sites of inflammation, neutrophils degranulate and discharge NE and CG mainly into the phagocytic vacuole and to a lesser extent into the extracellular space (Faurschou and Borregaard, 2003). Apart from their degradative properties, NE and CG are thought to be important regulatory tools in inflammatory processes (Wiedow and Meyer-Hoffert, 2005). As described above, NE is a key component in the resolution of a Shigella infection by neutrophils. The acid lowers the pH of the gastric juices to a value close to the pH optimum for pepsin. Some are harmless transients, others become part of the commensal flora that we a lifetime. In the environment Shigella can be found in brackwater but their only natural hosts are primates. Among those cases 99% of them are found in areas of the world with only limited medical support and poor sanitation causing low hygienic standards. Shigella traverses the epithelial barrier through specialized membranous epithelial cells, called M-cells (Wassef, et al., 1989). After reaching the epithelium it invades epithelial cells and is phagocytosed by resident macrophages.
Virulence depends on the resistance of the host and as well as on the invasiveness and toxicity of the bacteria.
First, mutant strains of Shigella that are invasive but do not express IpaB are not cytotoxic (Zychlinsky, et al., 1994). IcsAis essential for intra- and inter-cellular movement of Shigella (Bernardini, et al., 1989). During this time, Shigella primarily challenges two host innate defense cells: macrophages and neutrophils. PAMPs are microbial structures, which, upon interaction with elements of the host innate immune system, trigger the initiation of host protective responses accumulating in the clearance of the pathogen by phagocytic cells.
In the phagolysosome the bacteria are killed after exposure to enzymes, antimicrobial peptides and reactive oxygen species (ROS). Lysozyme, for instance, disrupts anionic bacterial surfaces, rendering the bacteria more permeable, whereas NE degrades virulence factors. Proteases, such as neutrophil elastase (NE), degrade bacterial proteins, including virulence factors (Weinrauch, et al., 2002).
However, a ROS function might also be to recruit K+ to the phagolysosome, allowing granule proteins to go from a highly organized intra-granule structure into solution (Reeves, et al., 2002). In contrast to that, Shigella is trapped within the phagolysosome of neutrophils and is eventually killed. Thus it is important to retain the bacteria within the vacuole to allow the ROS-dependent and -independent killing mechanisms to exert their functions on the bacterium.
The super-family of serine proteases contains peptidases with very diverse functions such as digestion, degradation, blood clotting, cellular and humoral immunity, fibrinolysis, fertilization, embryonic development, protein processing and tissue remodelling (Rawlings and Barrett, 1993). The common features among chymotrypsin-like serine proteases are the strictly conserved geometry in their catalytic triad and their overall structure.
Its structure is a classical example of the common fold of chymotrypsin-like serine proteases.
The catalytic residues (His57, Asp 102 and Ser195, yellow) and the enzyme residues that contact substrate residues are shown (blue). It is assumed that these varying amino acid compositions account for the different substrate specificities (Krem, et al., 1999). In enzymes with trypsin or chymotrypsin specificities, amino acid 216 is a glycine (figure 1.7). Val190 and 216 confine the S1 pocket of elastase to small alkyl side chains [adapted from (Berg JM, 2003)]. At the sites of infection they are mainly released into the phagocytic vacuole and to a lesser extend to the extracellular space to exert their specific an unspecific functions. NE and CG are very basic with a PI of > 9 and ~12, respectively, and through their arginine residues they are probably anchored to the negatively charged heparin and chondroitin sulfate proteoglycan matrix of the granule. The catalytic triad consisting of histidine (position 57), aspartate (102) and serine (195) is shown in purple. These proteolytic characteristics may be helpful for the neutrophil egress from the bloodstream and their subsequent transmigration through tissue. When exposed to the extracellular environment, the enzymes are either membrane-bound, part of neutrophil extracellular traps (NETs) (Brinkmann, et al., 2004), or freely released. NE specifically targets Shigella virulence factors but not proteins that are not associated with virulence or that are important for bacterial homeostasis (Weinrauch, et al., 2002). Humans take up Shigella from stools or soiled fingers of infected persons or from contaminated food and water. Although the macrophages phagocytose Shigella, the bacteria can escape from the phagolysosome to the cytoplasm within minutes (Finlay and Falkow, 1988;Maurelli and Sansonetti, 1988).


Shigella escapes the phagosome of both cells but while Shigella replicates within epithelial cells it induces apoptosis in macrophages probably by activation of caspase-1 (Casp-1).
Bacterial components and proteins that mediate adhesion, invasion, toxicity, and evasion of host immune cells are termed virulence factors (Mayer-Scholl, et al., 2004). Second, microinjection of IpaB into the cytosol of macrophages efficiently triggers apoptosis (Chen, et al., 1996). As described above, macrophages phagocytose Shigella but are not capable to retain the bacterium within their phagolysosome and kill it. They are the most abundant white blood cells (60%) but only have a short half-life if not activated.
Molecules signaling for neutrophil infiltration are predominantly the aminoterminal formylated methionin bacterial peptide (fMLP), IL-8 and TNF-α from macrophages and epithelial cells, and C5a, C3a and C4a from the complement cascade (Burg and Pillinger, 2001). The arsenal of cytotoxic agents has been traditionally divided into either oxygen- independent or -dependent mechanisms (figure 1.2).
In the Chediak-Higashi syndrome, neutrophils contain giant granules resulting from specific and azurophil granule fusion. In turn, a constituent of the azurophil granules, myeloperoxidase, generates hypochlorous acid (HOCl) from hydrogen peroxide. The relative contribution of ROS to these two different mechanisms is very intriguing, yet it seems premature to draw conclusions as to whether ROS contribute directly to microbial killing or only serve as activators of granule proteins (Roos and Winterbourn, 2002).
One neutrophil protease, neutrophil elastase (NE), seems essential to keep Shigella in the vacuole.
The common feature of serine proteases is the occurrence of a highly reactive serine residue in their catalytic center.
These sites are composed of different amino acids and form structural pockets, which interact with the amino acid side chains of the substrate (figure 1.5). The sat position 214 is highly conserved among the chymotrypsin-like serine proteases and contributes to the S1 binding (Perona and Craik, 1995). The positively charged arginine side chain at position P1 of the substrate is attracted by the negatively charged aspartate 189 located at the bottom of the S1 specificity pocket. Trypsin, for example, prefers to cleave after an aor lysine at the P1 position of the substrate. Since the glycine side chain consists only of hydrogen, it allows large substrate side chains to the base of the pocket. Transferof specificity requires the additional exchange of amino acidsin at least two distal segments of the enzyme, none of whichdirectly contacts substrate (Hedstrom, et al., 1992). One of the specific functions of NE is the degradation of virulence factor of Shigella and other Gram-negative enteropathogenic bacteria. The residues of the catalytic triad are the same positions (His57, Asp102 and Ser195) and their overall fold consists of two ß-barrels.
Protein synthesis seems restricted to the differentiating neutrophil, since no transcription, at least of the NE gene (Fouret, et al., 1989), is observed in neutrophils after they have left the bone marrow. Upon activation, neutrophils discharge their granule contents into the bacteria containing vacuole or to the extracellular space. The same study indicates that NE and CG act as effectors in the endotoxic shock cascade downstream of TNFα. Therefore, NE is likely to modify the interaction of Shigella with host cells, as seen in macrophages or epithelial cells upon Shigella uptake.
61% of all fatalities attributable to shigellosis involve children less than 5 years of age as systemic complications of shigellosis occur frequently in children (CDC, 2005;WHO, 2005).
Shigella is closely related to Escherichia and is occasionally considered as one strain of the E.
They are located in the epithelium covering the gut-associated lymphoid tissue (GALT) (Kraehenbuhl and Neutra, 1992).
Unlike in the epithelial cells, Shigella rapidly induces macrophage apoptosis (Zychlinsky, et al., 1992).
They damage the colonic mucosa by breaking tight junctions to reach bacteria in the intestinal lumen.
Shigella virulence factors are encoded on a 220 kb virulence plasmid which is essential for Shigella pathogenicity (Sansonetti, et al., 1982). It is localized to the outer membrane (120 kDa form) and is secreted through a type V system (90 kDa form). They are terminally differentiated cells, incapable of cell division, and synthesize very low levels of RNA and protein. They are found on all microorganisms, which allows a limited number of receptors to recognize PAMPs, and importantly they are essential for microbial survival, therefore no escape mutants can be generated (Mukhopadhyay, et al., 2004). Both of these systems probably collaborate in killing microbes (Roos and Winterbourn, 2002). Specific Granule Deficiency is characterized by the absence of specific granules and defensins. Besides killing bacteria inside the phagolysosomes, neutrophils can also degranulate and release antimicrobial factors into the extracellular space (Faurschou and Borregaard, 2003). Neutrophils with pharmacologically inhibited or genetically inactive NE allow the escape of wildtype Shigella into the cytoplasm. The same extract did not affect proteins important for Shigella homeostasis such as outer-membrane protein A (OmpA), maltose-binding protein (MBP) or recombinase A (recA), which are outer-membrane, periplasmic and cytosolic proteins, respectively. Apart from that, serine proteases are very diverse and divided into evolutionary unrelated clans (Barrett and Rawlings, 1995). The serine side chain forms a hydrogen bond with the imidazole ring of histidine and this histidine shares a hydrogen with aspartate. This interaction as well as five enzyme-substrate hydrogen bonds at positions P1 and P3 and glycine 193 help to position the scissile peptide bond (red) for the nucleophilic attack by the polarized hydroxyl group of Ser 195 (red arrow).
This is because a negatively charged aspartate (Asp 189) is located at the bottom of the trypsin S1 pocket.
On the contrary, in elastase-like enzymes the side chains of the amino acids at position 190 and 216 exclude large and bulky side chains to enter the S1 pocket (figure 1.7). It is notable that studies on the different S1 sites of the chymotrypsin-like serine proteases have been carried out with peptide but not with full-length protein substrates. It is intriguing that CG does not cleave these effectors since the two proteins share many characteristics. In addition to that, NE and CG show a striking structural similarity beyond their overall fold (figure 1.8). This N-terminal processing seems essential for activity of the protein, since recombinant expression of full-length NE or mature NE starting with a methionine did not yield active proteins (Li and Horwitz, 2001).
A minimal proportion of the active proteins is also localized on the surface of unstimulated neutrophils (Owen, et al., 1995). These plasma-derived inhibitors belong to the family of serpins (serine protease inhibitors) and are highly abundant extracellular proteins (Shapiro, 2002). In accordance with the observed specificity of NE for virulence factors of Gram-negative bacteria, mice deficient in NE have impaired survival following infections with gram-negative pathogens. Indeed, in neutrophils with pharmacologically inhibited or genetically inactive NE, Shigella can escape into the cytoplasm and its survival rate is increased (Weinrauch, et al., 2002). All Shigellae species are able to cause bacterial dysentery, in which the diarrhea is not only mucopurulent but also bloody (Sansonetti, 1992). These complications include acute renal failure, hemolytic-uremic syndrome, toxic megacolon and neurological ea(Goldfarb, et al., 1982). M-cells are the only port of entry across the epithelium for Shigella, because Shigella cannot invade colonocytes through their apical membrane (Mounier, et al., 1992). In an apoptotic process, a cell synthesizes the molecules responsible for its own death (Arends and Wyllie, 1991). Together with IL-8 secreted from the invaded epithelial cells, they signal for PMN (polymorphonuclear leukocyte or neutrophils). To resolve the Shigella infection, neutrophils need to be recruited to the site of infection. Neutrophils are generated from the pluripotent haematopoietic stem cells in the bone marrow and are characterized by multi-lobed nuclei and abundant granules in the cytoplasm, which contain host-defense molecules (Mayer-Scholl, et al., 2004). Physiologically circulating neutrophils in the blood contact the endothelium and transiently interact with it, a phenomenon termed rolling. The severity of the symptoms in these diseases underlines the fundamental role of granule proteins in host defense.
Hydrogen peroxide is bactericidal only at high concentrations, therefore a variety of secondary oxidants have been proposed to account for the destructive capacity of the neutrophils (Hampton, et al., 1998). The cells can also generate neutrophil extracellular traps (NETs), which are composed of granule and nuclear constituents that kill bacteria extracellularly (Brinkmann, et al., 2004).
Using a series of chemical and physiological inhibitors, it was shown that NE was the protease responsible for the observed cleavage of virulence factors. The clans differ in their overall structure and the succession of the catalytic residues in their primary sequences (Krem and Di Cera, 2001). The respresentation was generated using PYMOL (DeLano, 2002) and is based on the crystallization of chymotrypsin by (Pjura, et al., 2000).


The resulting geometric arrangement allows the three amino acids to act together in a nucleophilic attack and to cleave the peptide bond of the substrate (see also figure 1.6). The peptide bond is cleaved and the carbonyl group of P1 is transiently attached to the serine side chain, whereas the amide group of P1’ is bound to the histidine imidazole ring. In most of these proteases, including NE and CG, the β-sheets are connected by two surface loops and the disulfide bond Cys191-Cys220(Perona and Craik, 1997). The aspartate can interact with the long and positively charged side chains of arginine and lysine (figure 1.6). This structural observation is in accordance with the preference of NE for valine as P1residue in peptide substrates (Harper, et al., 1984), since valine only has a small alkyl side chain.
The recognition of these protein substrates likely involves other substrate-enzyme interactions outside of the binding pockets (Perona and Craik, 1995).
This surface expression is increased when the neutrophils are exposed to chemoattractants such as fMLP (Owen, et al., 1995). The specificity of NE for virulence factors holds also for other Gram-negative pathogens such as Salmonella and Yersinia (Weinrauch, et al., 2002). If our innate immunity is compromised, the harmless microorganisms can quickly become serious or even fatal threats to our health. Following passage across M-cells, the microorganism interacts with two different host cells: epithelial cells and macrophages. Accordingly, macrophages infected with Shigella show the two cardinal signs of apoptosis: specific morphological changes and fragmentation of nuclear DNA into multimers of approximately 200 bp. This is a prerequisite for the polar movement of Shigella in mammalian cells, including bacterial spreading between epithelial cells.
Molecules mediating this are leucocyte (L), platelet (P) and endothelial (E) selectins, which permit interaction between neutrophils, and neutrophils and endothelial cells (Janeway, et al., 2001). The importance of ROS for antimicrobial activity is validated by the susceptibility to infections of patients suffering from chronic granulomatous disease, a condition where the NADPH oxidase complex is inactive (Dinauer, et al., 2000). Clan members are subdivided into families based on sequence homology (Barrett and Rawlings, 1995;Rawlings and Barrett, 1993). The histidine transfers the proton of the serine to the amide group of P1’ and the former C-terminal part of the substrate can dissociate from the enzyme. In contrast to that, chymotrypsin contains an uncharged serine at the bottom of its S1 pocket. The RMSD describes the difference in localization of the α-atoms at similar positions in the structure. However, the carboxyl prodomains do not seem to be important for proper targeting and enzymatic activity (Gullberg, et al., 1995).
The correct distribution of NE in the granules and on the surface seems important to prevent neutrophil deficiencies (neutropenia). Interestingly, CG shows an antimicrobial activity that is independent of its enzymatic function (Bangalore, et al., 1990). Furthermore, CG, another abundant granule protease, does not degrade Shigella virulence proteins, although it is homologous to NE and their crystal structures are almost identical.
In addition, among the microorganisms that humans encounter each day are those whose survival depends on their ability to cause cellular damage to their host. This region encodes the invasion plasmid antigens (ipa) operon, the membrane expression of ipas (mxi) and surface presentation of invasion plasmid antigens (spa) operons, as well as other, independently expressed genes. Together these data suggest a model where IpaB is secreted into the macrophage cytosol during infection. After exposure of circulating neutrophils to chemoattractants (IL-8, fMLP, C5a, LTB4), members of the β2-integrin family mediate the conversion of the rolling state to a state of tight stationary adhesion (Burg and Pillinger, 2001). NE and CG are members of one subfamily of the chymotrypsin-like clan (Lesk and Fordham, 1996). Upon binding of the substrate, the histidine ring positions the serine side chain and polarizes the hydroxyl group of this side chain by transiently binding its hydrogen.
To release the N-terminal part of the substrate from the serine side chain, a water molecule is needed and the same steps are repeated as described before. Thus, uncharged, aromatic side chains of phenylalanine, tyrosine and tryptophan fit into this hydrophobic pocket.
A RSMD of zero means that the structures are identical in conformation (Maiorov and Crippen, 1994).
Finally, the 267 amino acid NE precursor is processed to its 218 amino acid mature form and the CG 255 amino acid precursor results in a 226 amino acid mature CG protein. Therefore we raised the question why NE but not CG targets virulence factors and how NE distinguishes virulence factors from other bacterial proteins. The host response usually leads to the resolution of the infection within five to seven days. For example, the cell wall antigens, also known as O-antigens, of Shigella and E.coli are distinct. IpaB binds to and activates caspase-1, an event that simultaneously induces apoptosis and activates the pro-inflammatory cytokines IL-1β and IL-18.
Neutrophils adhere to the endothelium and secretory vesicles of the neutrophils are mobilized.
The barrels form a cleft in which the catalytic triad and the substrate-binding sites are located (Perona and Craik, 1997).
The serine hydroxyl group is now more nucleophilic and can attack the carbonyl group of the scissile bond.
The S1 pocket of CG, for example, shows similarity to the chymotrypsin pocket (Harper, et al., 1984).
In addition, NE is present in cystic fibrosis airways (Delacourt, et al., 2002) and induces excess mucus production in chronic obstructive pulmonary disease (COPD) (Shapiro, 2002).
A pathogen must enter a host and multiply sufficiently to establish itself or to be transmitted to a new susceptible host. However, without proper medical treatment the diarrhea can be life threatening to some persons, especially young children and the elderly. The O-antigen is the outer polysaccharide portion of the lipopolysaccharide (LPS) and consists of repeating sugar units. In order to move intra- and intercellularly, Shigella utilizes the cytoskeleton of the host cells (Makino, et al., 1986).
Purified NE also degrades virulence factors from other Gram-negative pathogens such as Salmonella and Yersinia (Weinrauch, et al., 2002). Aspartate supports the orientation of the histidine side chain and improves its proton acceptor qualities by electrostatic interactions. The carbonyl group binds the hydroxl group of the water and the P1-serine ester bond is cleaved. The S1 pocket of CG can harbor large and bulky side chains whereas only amino acids with small alkyl side chains fit into the S1 pocket of NE. All cases of CN and 75% of SCN cases are caused by mutations in the NE gene (Horwitz, et al., 1999). To this end, we first analyzed the substrate for a NE recognition motif in the primary or higher order structures.
This treatment involves rehydration as well as application of electrolytes and antibiotics.
Once infected, the cells secrete the cytokine interleukin-8 (IL-8) to recruit neutrophils (also called polymorphonuclear leukocyte, PMNs) to the site of infection. The type III secretion apparatus is composed of at least 30 proteins and is conserved among enteropathogenic bacteria (Cornelis and Van Gijsegem, 2000).
During migration through the tissue neutrophils cleave off or shed their selectins and proteases are liberated from the different granule subsets degrading vascular basement membranes and the intercellular matrix (Faurschou and Borregaard, 2003). The apparent specificity of NE for virulence factors is further supported by the fact that NE does not target the type III secretion apparatus itself nor does it cleave secreted Shigella proteins not associated with virulence. The N-terminal substrate dissociates, the histidine-serine hydrogen bond is reestablished and the enzyme is prepared for a new catalytic cycle (Berg JM, 2003). These mutations are thought to interfere with the correct targeting of NE from the trans-golgi network to the granules and the plasma membrane (Horwitz, et al., 2004). In most cases the pathogen-induced damage is not serious but a proportion of hosts will suffer from disease or even be killed.
Interestingly, cathepsin G (CG), another abundant granule protease with a high degree of homology to NE, does not degrade Shigella virulence proteins. Secondly, we addressed the question of the NE specificity by a functional analysis of NE mutants. These mutants were generated on the basis of a structural comparison of NE and CG and were tested for their ability to cleave the Shigella virulence factors IpaB and IcsA.



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