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1. Human Chorionic Gonadotrophin (hCG)
Gonadotrophin hormones are produced by different tissues such as the pituitary and the placenta and their main function is gonadic regulation in ovaries and testicles. Hormones that belong to this family are: FSH ( follicle-stimulating hormone), LH (luteinizing hormone) and hCG (human chorionic Gonadotrophin). For the purpose of this project we are going to look deeper at the human chorionic Gonadotrophin (hCG).
Human Chorionic Gonadotrophin is a glycoprotein hormone which is produced after conception of the embryo. After hCG been produced several chemical reactions are taking place. One of them is the continued ovarian production of the hormone progesterone. This hormone helps to sustain the uterine lining and stops menstruation. The hCG can be detected in the pregnant women blood or urine and the amount of it can be used as a pregnancy test. It can be used as a tumor marker for testicular cancer because it can be locally produced and act within the testis. Excess amount of hCG in testis can indicate testicular cancer. Another use of hCG is to induce ovulation in the ovaries and testosterone production in testis when infertility problem is diagnosed. The hCG also plays a role in cellular differentiation and may activate apoptosis.
A study carried out by Almeida B.E. et al, (2010), supports that hCG binds to the same receptor, which is a transmembrane glycoprotein that belongs to the G-protein-coupled receptor superfamily and is present in the ovarian theca cells in females and in the testicular Leydig cells in males.
1.2 Recombinant human chorionic Gonadotrophin vs. urinary human Chorionic Gonadotrophin
Urinary HCG (uHCG) has been in use for decades; however, there are potential disadvantages associated with uHCG, including: interbatch inconsistencies; large quantities of starting material required for purification; possible contamination by other urinary proteins ( that is our subject) that may result in local, post-injection side-effects experienced by some patients; and a movement to avoid human source materials (The International Recombinant Human Chorionic Gonadotrophin Study Group, 2001). Recombinant HCG (rHCG) with high specific activity has become available. Recombinant HCG is produced in a Chinese hamster ovary cell line expressing the genes for the alpha and beta subunits of HCG, and the protein is purified using stepwise chromatography (Loumaye et al., 1996). The pharmacokinetic profile of rHCG is comparable to that of uHCG with linearity over a dose range of 500-20,000 IU and a terminal elimination half-life of approximately 30 h (Lathi and Milki, 2001; Trinchard-Lugan et al., 2002). A clinical trial carried out by Chang et al., 2001 compares two doses of rHCG and shows that 250 Î¼g is at least as effective as 5000 IU of uHCG for final follicular maturation without the higher incidence of adverse effects originally reported for the 500-Î¼g dose.
1.3 Structure of human Chorionic Gonadotrophin
The peptide structure of the hCG group of molecules was established by Bahl and colleagues in 1972  and confirmed and refined by Morgan and colleagues in 1975 (Figure â€‹(Figure1)) . It is a glycoprotein which consists of 244 amino acids and has a molecular weight of 36,000. Both the a- and the b-chains are composed of three loops held in place by a cystine knot of three disulfide bonds . The Î± subunit comprises of 92 amino acids and Î² subunit of 145.
Amino acid sequence of hCG Î±-subunit and Î²-subunit [16,17]. Digits indicated amino acid residue positions and N and O indicate the positions of N- and O-linked oligosaccharides
Oligosaccharides constitute approximately 25-30% of the molecular weight of regular hCG. The N- and O-linked oligosaccharide structures of regular hCG were first determined by Kessler and colleagues [65,66], and refined and confirmed by Mizouchi and Kobata in 1980 , further refined by Elliott and colleagues in 1997  and by Kobata and Takeuchi in 1999 [19There are 2 N-linked oligosaccharides on the Î±-subunit of hCG and 2 N-linked oligosaccharides on the Î²-subunit of hCG. There are also 4 O-linked oligosaccharides on the C-terminal peptide region of the Î²-subunit of hCG (Figure â€‹(Figure11).
1.4 Function and regulation of hCG
Human chorionic gonadotropin interacts with the LHCG receptor and promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing fetus. Due to its highly-negative charge, hCG may repel the immune cells of the mother, protecting the fetus during the first trimester. Because of its similarity to LH, hCG can also be used clinically to induce ovulation in the ovaries as well as testosterone production in the testes. As the most abundant biological source is women who are presently pregnant, some organizations collect urine from pregnant women to extract hCG for use in fertility treatment. Human chorionic gonadotropin also plays a role in cellular differentiation/proliferation and may activate apoptosis.
One of the most important function is to prevent the normal involution of the corpus luteum at the end of cycle during pregnancy. (http://www.hcglab.com,)
Placental hCG replaces pituitary LH in controlling progesterone production at the initiation of pregnancy, from implantation of pregnancy (~3 weeks gestation) to 6 weeks of gestation. The syncytiotrophoblast cells make progesterone independent of hCG stimulation from 6 weeks gestation until term.
Research by multiple groups in the past 10 years shows a much more logical primary function on hCG, maintaining maternal blood supply to support hemochorial placentation and nutritional support of fetus (Figure â€‹(Figure6,6, Panel A - C) [96-101]. hCG maintains angiogenesis in the myometrial spiral arteries through the length of pregnancy acting on LH/hCG receptors on the spiral arteries [96-101]. It has also been shown that hCG promotes the fusion of villous cytotrophoblast cells to syncytiotrophoblast . Both of these biological functions are critical to efficient placentation in humans. This is the more logical prime function of regular hCG through the length of gestation.
Dissociation, cleavage and clearance
As an initial step, the dimers regular hCG and hyperglycosylated hCG are either slowly cleaved (nicked) or slowly dissociated into Î± and Î² subunits (Figure â€‹(Figure5).5). The dissociation half-time of dimers into subunits is 700 ± 78 hours at 37°C . Hyperglycosylated hCG dissociates much more rapidly that regular hCG (140 ± hours at 37°C ).
A macrophage or circulating leukocyte elastase protease cleaves hCG Î²-subunit at Val residue 44 (residue 44-45 cleavage) or Gly residue 47 (residue 47-48 cleavage) generating nicked hCG (Figure â€‹(Figure1)1) [39,78]. Nicked hCG is 30-fold less stable than regular hCG (dissociation half time 22 ± 5.2 hr. at 37°C ) rapidly dissociating releasing a free Î±-subunit and a nicked hCG free Î². Similarly, dissociated hCG Î²-subunit is rapidly cleaved by leukocyte elastase to make nicked hCG free Î². Both nicking and dissociation eliminate hCG hormone activity (shown by ability to bind corpus luteum hCG/LH receptor and to promote progesterone production ). As such the combination of nicking and dissociation, in either order, leads to the rapid deactivation and clearance of regular or hyperglycosylated hCG .
Nicked hCG or nicked hCG free Î², while cleaved at 44-45 or 47-48, remain structurally intact due to 5 disulfide linkages between the component peptides. Further degradation of hCG by leukocyte elastase leads to cleavage of Î²-subunit at Leu residue 92 . (we have to link this with the bacterial proteases)
Nicking and cleavage to make Î²-subunit core fragment continues throughout pregnancy deactivating hCG . In pregnancy urine samples, Î²-subunit core fragment concentration or the degradation end product concentration is relatively small in the first weeks of gestation, it equals hCG dimer concentration at 6-7 weeks of pregnancy. Î²-core fragment concentrations then exceeds dimer concentrations in urine thereafter, 7 weeks to term [14,89,90].
Nicked free ß-subunit is rapidly removed from the circulation. It may be degraded in the kidney to ß-core fragment, and excreted into urine. It is common to find residual nicked in serum or Î²-subunit core fragment in urine, the products of degradation, in the weeks following parturition of pregnancy or surgical evacuation of hydatidiform mole or tumor.
In this section we show how elastase and other proteases and glycosidase convert 3 forms of hCG Î²-subunit in serum and urine into 13 forms (Figure â€‹(Figure3).3). Different secreted and degraded forms of hCG may best mark different conditions (Table 1). We emphasize again, it is important when considering a total hCG assay and the structure and degradation findings presented here, to select an assay detecting all form of hCG Î²-subunit. Regular hCG may mark a normal pregnancy, hyperglycosylated hCG may best mark gestational trophoblastic diseases, hyperglycosylated hCG free Î² may best mark a Down syndrome pregnancy, nicked hCG missing Î²CTP may best mark clearing hCG at parturition or following termination, and urine Î²-subunit core fragment ay best mark non-trophoblastic malignancies.
The dissociation, degradation and clearance pathways of regular hCG [39,78-87]. Metabolic clearance half-times (MCR) are those published by Wehman and colleagues [85-88]. Similar degradation and clearance pathways are predicted for hyperglycosylated hCG and hyperglycosylated hCG free Î² with an end product of urine Î²-subunit core fragment.
3. airborne bacteria and Bacterial enzymes
Bacteria are microscopic organisms of various shapes, constituting the smallest and most elemental form of plant life. They are almost everywhere in nature and multiply rapidly. Bacteria consist of single cells with a nucleus and typically a tough cell wall.
Airborne bacteria are usually a reflection of man's activity. Staphylococci are found in all individuals, but with variations in carriage between age groups and populations. Aerosol particles shed from the human body are, first, droplets expelled from the respiratory tract through the nose and mouth. They consist of liquid which may surround infective material and second, skin squames which are constantly being lost from the skin surface. When urine is collected a small sample of urine is placed on CLED agar plates and incubated at 37oC which mimics the temperature of the body. Any microorganisms that are present in the urine sample grow over the next 24 to 48 hours as small circular colonies. The size, shape, and color of these colonies help to identify which bacteria are present, and the number of colonies indicates the quantity of bacteria originally present in the urine sample (at this point need to mention about CFU) . Observation of the colonies on the agar plate will give the total number and determining how many types have grown in a mixed culture. A good clean sample should be collected for the test but often there are cases of contamination due to an infection or due to contamination from the skin picked up during the urine collection.
colonies from each type of bacteria present that appears significant in number or type, will be smeared on a slide, dry , and stained with dyes, performing a test called a gram stain. the microorganisms are examined under the microscope. Different types of bacteria will exhibit characteristic colors and shapes. For instance, the bacterium Escherichia coli, which causes the majority of urinary tract infections, will appear as pink gram-negative rods under the microscope. Lactobacillus, which is a common vaginal contaminant in women's urine samples, will appear as thin purplse gram-positive rods. Some of the bacteria, such as Lactobacillus, are easy for an experienced laboratorian to identify, are nonpathogenic, and do not require any further investigation. Others, such as gram-negative rods, represent groups of similar bacteria and will require additional testing to determine exactly which type of bacteria is present. If there is no or little growth on the agar after 24 to 48 hours of incubation, the urine culture is considered negative for pathogens and the culture is complete. Biochemical tests are used to identify which bacteria are present and susceptibility testing is done to identify antimicrobial agents that inhibit the growth of the bacteria.
3.2 Bacterial enzyme function
Inside the wall is the cell matter, which contains enzymes that help break down food for digestion and build cell parts. When the bacteria collide with a food source, the enzyme punctures the outer shell allowing the nucleus to enter the food source and devour it. An enzyme is the catalyst that allows the bacteria to function. Without one, the other would not exist.
Enzymes as a whole are the 'take charge' catalysts. They each perform a very specific action but do not become part of the action
Enzymes work by breaking apart large complex substrates into smaller, more readily absorbed nutrients that the bacteria can utilize. Enzymes from different sources have a specific temperature and pH range at which they are optimally effective, which is an important consideration when choosing an enzyme product. Enzymes are classified by the substrate they work on. For example, proteases work on proteins, breaking them down into amino acids and peptides. Cellulases break down cellulose, the major undigestible component of plant cell walls, into simpler sugars. Only very small quantities of enzymes are needed to change very large quantities of substrate.
3.3 different types of enzymes in bacteria
Several studies carried out by know in microbiology field shows that all bacteria produce their own specific enzymes. Enzymes produce by bacteria can be grouped into 5 basic types: coagulases, kinases, hyaluronidase, collagenase, and proteases.
3.3.1 Bacterial Coagulases
Coagulases produced by bacteria initiate the clotting of blood fibrinogen inside the blood vessels of human. The liver produces the plasma protein fibrinogen which is easily converted to fibrin through the chemical action of coagulase. The bacteria can use the fibrin clot to cover themselves in such a way that they are protected from the immune defenses of human. Species under the genus Staphylococcus are good examples of bacteria that produce coagulases. (Konopka and Gedney 2003; Wilson et al. 2002)
3.3.2 Bacterial Kinases
The human body has this physiological mechanism to isolate an infected portion of a body from the rest of the body by creating temporary blood clots which block the movement of bacteria toward the other parts of the body (Wilson et al. 2002; Lehman 2003). Unfortunately, some strains of bacteria produce kinases that dissolve the blood clots allowing them to be released from the site of infection. Widely known bacterial kinases include the staphylokinase produced by staphylococci (e.g. Staphylococcus aureus) and the streptokinase (a.k.a fibrinolysin) produced by streptococci (e.g. Streptococcus pyogenes).
3.3.3 Bacterial Hyaluronidase
Cells in the connective tissues of humans are joined together in place by hyaluronic acid, a special kind of polysaccharide found between the cells. Certain species of bacteria especially the streptococci produce hyaluronidases to dissolve the polysaccharides that bind the cells together. The dissolving action is believed to be associated in the blackening of infected wounds and to help the bacteria spread from the initial infection site towards other body parts. Clostridia species that cause gas gangrene utilize the enzyme when they infect their hosts. (Konopka and Gedney 2003; Wilson et al. 2002)
3.3.4 Bacterial Collagenase
Clostridia species not only produce hyaluronidases but also produce collagenase (Wilson et al. 2002) that breaks down protein collagen which is the main component of connective tissues into its constituent peptides and amino acids.
3.3.5 Bacterial Proteases
The body produces a class of antibodies called IgA antibodies that inhibit the adherence of pathogenic microbes to our mucosal surfaces (Wilson et al. 2002; Lehman 2003). There are certain bacterial strains that produce IgA proteases that destroy the IgA antibodies. If they successfully wiped away the antibodies, they can now penetrate the mucosa and infection then begins. Examples of bacteria that produce IgA proteases are Neisseria gonorrhoeae and N. meningitides, the causative agents for gonorrhea and meningococcal meningitis respectively.
Proteases, also known as proteinases or proteolytic enzymes, are a large group of enzymes. Proteases belong to the class of enzymes known as hydrolases, which catalyse the reaction of hydrolysis of various bonds with the participation of a water molecule. A secreted bacterial protease may also act as an exotoxin, and be an example of a virulence factor in bacterial pathogenesis. Bacterial exotoxic proteases destroy extracellular structures.
4 Matrix- assisted laser desorption ionization time of flight mass spectropmetry ( MALDI-TOF MS)
Protein Sequencing by Mass Spectrometry (MS) MS has become an important tool for determining the amino acid sequence of peptides and proteins. The MS technique involves creation and detection of charged peptide and protein ions in the gas phase. Soft ionization techniques like electrospray ionization (ESI, Fenn et al., 1989) and matrix-assisted laser desorption/ ionization (MALDI, Karas & Hillenkamp, 1988) are used to produce intact peptide and protein ions in the gas phase. The mass to charge ratio (m/z) of these ions can be rapidly and accurately measured allowing such applications like fast evaluation of the correctness of the sequence of peptides and proteins, checking the presence of post-translational modifications, and application of bioinformatics-assisted peptide-mass fingerprinting (PMF, Henzel et al., 1993; James et al., 1993; Mann, Hojrup, & Roepstorff, 1993; Pappin, Hojrup, & Bleasby, 1993; Yates et al., 1993) methods.
Matrix Assisted Laser Desorption/Ionization (MALDI) is a method that allows for the vaporization and ionization of non-volatile biological samples from a solid phase directly into a gas phase. The sample is suspended, or dissolved, in a matrix. Matrices are small organic compounds that are co-crystallized with the samples. The presence of a matrix seems to spare the sample from degradation, resulting in the detection of intact molecules as large as 1 million Dalton. The first reports demonstrating successful MALDI Tof-MS biochemical analysis were published in the late 1980s from the labs of Tanaka et al.  and Karas and Hillenkamp .
4.2 Principle of MALDI
The general principle of MALDI-Tof-MS revolves around the rapid photo-volatilization of a sample embedded in a UV-absorbing matrix followed by time-of-flight mass spectrum analysis. The choice of the matrix is crucial for success in MALDI experiments. Typically, peptides or proteins are generally solubilized in a 0.1% aqueous trifluoroacetic acid (TFA). One microliter of this solution is mixed (usually directly on the probe tip) with a saturated aqueous solution of matrix and the solvent allowed to evaporate to form crystals. After MALDI, the desorbed ions are mainly analyzed using a time-of-flight mass spectrometer (Tof-MS) working in the linear or reflectron mode. (reference: Marvin et al)
4.3 time of flight mass analyzer
MALDI makes use of short pulses of laser light to induce the formation of intact gaseous ions. The molecules of the sample are not directly exposed to laser light, but are homogeneously embedded in matrix, which consists of small organic molecules. The matrix molecules strongly absorb the laser light to allow for very efficient energy transfer to the sample. Depending on their mass-to-charge ratio m/z, the ions have different velocities when they leave the acceleration zone and enter a field-free flight tube. After a time-of-flight the ions impact onto an ion detector which is often formed by two microchannel plates connected in series. The detector produces a signal (proportional to the number of ions arriving at the detector), which is processed by an ADC converter. The ADC is connected with a computer, in which the resulting MALDI spectrum can be stored and processed (e.g. for smoothing, etc.)
Aim of the project and hypothesis.
The purpose of this project is to investigate if bacterial enzymes that can be present in the urine can nick and degrade Human Chorionic Gonadotrophin (hCG) hormone. hCG is produced during pregnancy and is one of the most important hormones for regulating homeostasis. This hormone provides information about the state of pregnancy, but can also indicate the presence of tumor. Nicking or degradation can deactivate the hormone and hence presenting means of detecting hCG. Several studies have failed to recognize the pathways that dissociate, nick and degrade hCG and beta-subunit molecules in pregnancy.
Enzymes can nick and degrade hCG. A study carried out by Cole et al (1997), states that Gonadotrophin beta- subunit nicking enzyme (GBNE) which is an arginine-specific metalloprotease having a noticeable molecular weight of between 150-430 KDa, nicks the beta subunit of hCG . The presence of this enzymes though indicates a reproductive cancer or its associated tumors Nicking occurs by the action of proteolytic enzymes in the placental, cancer tissue or in the circulation. Proteolytic enzyme elastase was previously shown to digest hCG. In this project we test the hypothesis that if the proteolytic enzyme elastase can digest hCG, then bacterial proteolytic enzymes can also lead to the same result in nicking of hCG.
After proteolysis with a specific protease (e.g. elastase), proteins of different amino acid sequence produce a series of peptides masses, which can be detected by MALDI-Tof-MS. (ref: marvin et al, 2003)