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Amylase Activity In Germinating Barley

Amylase is a calcium dependent enzyme which hydrolyzes complex carbohydrates at alpha 1,4-linkages to form maltose and glucose. Amylase is an enzyme found in the germinating seeds. Imbibition process causes the release of growth plant hormone gibberelin which stimulates the synthesis of amylase. The activity of the amylase enzyme is affected by many factors such as temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators. In germinating barley, the food reserves are stored in the endosperm. The cotyledons store food for the use of embryo in the form of starch. Amylase enzyme breaks down starch into maltose, a chain of two glucose molecules Maltose then breaks down into glucose by the enzyme glucosidase. Glucose then enters the glycolytic pathway where it is used for the production of ATP and carbon molecules for biosynthesis. Glucose is used for the growth of plumule and radicle. This process is also known as the germination process. The emergence of plumule and radicle indicate that the seeds have germinated. In germinated seeds, the blue colour of the Benedict’s solution change to brick-red precipitate indicating the presence of glucose while maintaining the yellowish-brown colour of the iodine solution indicating the absence of starch. However, in non-germinated seeds, the yellowish-brown colour of the iodine solution change to blue black indicating the presence of starch while maintaining the blue colour of the Benedict’s solution indicating the absence of glucose. Most of the time, when all the starch have been used up, the seedling capable of undergoing photosynthesis to produce energy and carbon.
Hypothesis The higher the amylase activity, the higher the rate of seed germination. This is observed by a higher change in length of plumule and radicle. Hence, when performing the Benedict’s test, the concentration of brick-red precipitate is higher seedlings and the solution remains blue for the dormant seed.
Aim The aim of the experiment was to extract amylase from barley and to use it for the catalysis of a biochemical reaction hence investigating the amylase activity during seed germination.
Materials and methods Ten germinating seeds were taken and using a paper towel, the germinants were patted dry and the weight of the germinating seeds were recorded. Next, using a mortar and pestle, the 10 germinating seeds were crushed into a puree. Slowly adding 10 ml of buffer, the germinating seeds were further crushed for two minutes. This will allow the amylase to go into the solution. The crushed seeds was filtered into a 100 ml beaker and the amylase extract was poured into a measuring cylinder. The volume of amylase extract was recorded. A five-fold dilution of the latter was done by pipetting 5 ml of the amylase extract and adding 20 ml of buffer to make up a total volume of 25 ml. This mixture is called the diluted amylase extract. A control was then done by adding 5 ml of the diluted amylase extract in a test tube and placing it in a water bath at 80o C for 10 minutes. When the 10 minutes have elapsed the control was removed and allow to cool to room temperature.
Next the activity of amylase per mass of germinating barley tissue is to be determined. For this, onto ceramic plates, one drop of iodine was placed into 21 wells. A reaction mixture was then prepared by adding 5 ml buffer and 1 ml of 0.5% starch solution in a test tube. Then using a pasteur pipette, one drop of the reaction mixture was removed and added to one drop of the iodine. The iodine turned blue black. This was done to ensure the presence of starch in the reaction mixture. The previously made diluted amylase extract is thoroughly remix and 1 ml of the latter was added to the reaction mixture. The mixture is called amylase reaction mixture. (As soon as the amylase reaction mixture was prepared, reaction started. Amylase started to break down starch into simple sugars). Immediately, starting with well 0 on the ceramic plate, one drop of amylase reaction mixture was added to the iodine using a pasteur pipette. At one minute interval, another drop of the amylase reaction mixture was added to another well. This was repeated until the achromic point was reached. When the achromic point had been reached, the time elapsed was recorded.
Once the achromic point was reached, the amylase reaction mixture was kept for the determination of maltose. (Note: Benedict’s reagent gives a red-yellow precipitate of cuprous oxide when boiled with maltose. This reaction does not occur with starch.) In a test tube, 2 ml of the amylase reaction mixture and 2 ml of Benedict’s reagent was added. A control reaction mixture was also prepared by adding 5 ml buffer and 1 ml of 0.5% starch solution but without the amylase extract. 2 ml of the control reaction mixture was then added in a test tube along with 2 ml of Benedict’s reagent. Both the Benedict’s reagent tubes were placed in a water bath at 80oC for 10 minutes and then examined for presence of cuprous oxide precipitate.
All of the above steps were then repeated but with dormant seeds and seedlings. All data were then recorded for further investigation.

Beta Lactam Antibiotics: Examples and Uses

The beta-lactam antibiotics for their extensive scale of actions are preferred most among antimicrobial factors. The penicillins and cephalosporins are the two categories of this β – lactam antibodies that are extraordinarily less toxic to organisms.(1) At present ,the β-lactam groups of antibiotics are the highest frequently used universal antibiotics .(2)
Cellular membrane of most bacteria enclosed by a cell wall but an extra outermost layer seen on some of them. The periplasmic space in gram negative bacteria is the cavity in the middle of the cell membrane and the cell wall. Periplasm instead of a clearly defined periplasmic space is retained by most gram positive bacteria .(3)
But peptidoglycan is the greatest significant element of the cell wall that linked as a new cell by way of the metabolic absorption in periplasm is a polymer made of N-acetyl muramic acid alternating with N-acetyl glucosamine.Arises of the bacterial cell that is actually a process of peptidoglycan synthesis where accumulation of 5 amino acids to N-acetyl muramic acid is one of the leading phases. A precursor of peptidoglycan that conducted by a cell wall acceptor crossway the cell membrane in the periplasm and developed by linking N-acetyl glucosamine to the N-acetyl muramic acid . Generous crosslinking occurs for two key enzymes (trans peptidase and D-alanyl carboxypeptidase) and for the capacity to bind penicillins and cephalosporins, they are recognized as the penicillin binding proteins. B4
Development of cell wall by cross linking of a number of films of peptidoglycan grounds numerous layers and a much denser cell wall in gram positive bacteria than gram negative bacteria. Beta-lactam ring attach enzymes to cross-link peptidoglycans, that is a chemical structure which is available in the beta-lactam antibiotics consist of all penicillins and cephalosporins. Synthesis of bacterial cell wall is prevented by the affect of beta-lactam when transpeptidase and D-alanyl carboxypeptidase enzymes are attaching there by means of cross-linking and cause deterioration of bacterial cell wall.b5
As a bactericidal agents the antibiotic-penicillin binding protein complex of beta-lactam antibiotics excites autolysin discharge that have the capability of digest cell wall that left after bursting a cell. Generally, excessive inner osmotic pressure possessed by gram positive bacteria and in a low osmotic pressure enclosed atmosphere , cells those are lack of a usual and rigid cell wall are burst out.b6
There are many different types of methods of that bacteria became reistance to beta-lactam antibiotics. Transformation is one of the most important mechanisms among them and in the mean time of this process transfer of chromosomal genes between bacterium happens. Due to the death of a a resistance gene in a bacterium releasing of naked DNA in surrounding environment happens. a process known as homologous transformation and by this method the resistance gene in the host bacteria transferred from the naked DNA to the chromosome. the segment of the host DNA have been remodelled by resistance genes results altered penicillin binding proteins production by coding for cross-linking enzymes. But still cross linking of the peptidoglycan layers of the cell wall happens due to these altered penicillin binding proteins and reduces affinity for beta-lactam antibiotics and the bacterium became resistance. In penicillin-resistant S. pneumonia, this process caused the acquirement of genes from other naturally arising penicillin-resistant Streptococcus species.
Bacteria grow into resistant to beta-lactam antibiotics by one more significant system is by the construction of enzymes capable of deactivating or altering the drug formerly it has a chance to apply its outcome on the bacteria. peni
The first human gammaretrovirus that is Xenotropic murine leukemia virus-related virus (XMRV) and responsible for chronic fatigue syndrome and prostate cancer (PC) have been revealed in recent times. Gammaretroviruses family is famous for their capability to activate cancer in the infested hosts. Analyzing study showed that vaccine-induced XMRV Env -specific binding and neutralizing antibodies (NAb) titers had limited span but highly changeable. in antibody levels, the different incidence stated for XMRV in a number of prostate cancer and chronic fatigue syndrome cohorts can be explained by the reasonably fast diminution . (2)
Monoclonal antibodies (mAbs) have exceptional therapeutic applications in ophthalmology and can be used therapeutically by binding to molecular objects with high specificity. Tumour necrosis factor (TNF), epithelial growth factor receptor, vascular endothelial growth factor (VEGF) receptor, basic fibroblast growth factor receptor, platelet-derived growth factor, and cluster of differentiation antigens repressed by a number of single-agent therapies. Existing and future mAbs in contrast to different cytokines were evaluated for ocular disease treatment and two anti-VEGF mAbs( bevacizumab and ranibizumab), and three anti-TNF agents (infliximab, etanercept, and adalimumab), instruct ocular neovascularization and intraocular inflammation. Other mAbs showed positive results for ocular lymphoma or ocular inflammation but Ranibizumab is the only FDA-approved therapy. Intravenous application of mAbs has established satisfactory toxicity profiles, while intraocular injection may decrease the chances of systemic complications . To develop the excellence and extent of responses is the challenge for the future by merging biologic therapies while lessening side effects. 2i
Leading causes of death in the world for coronary syndromes, stroke and other ischaemic arterial diseases . Therapy involves with medical actions correlating thrombolysis, antiplatelet drugs, and the re-opening of the coronary artery by angioplasty. In ischaemic cardiovascular diseases, platelet initiation is a acute phase . Chimeric Fab, c7E3 or abciximab is the only one recombinant antithrombotic antibody currently used in therapy and obstructs the ultimate phase of platelet aggregation. Subendothelium matrix activation by other platelet receptors have been recognized as prospective targets for the improvement of antithrombotic antibodies .2ii
In drug development, insulin-like growth factor receptor I (IGF-IR) is becoming an attractive target. IGF-IR owed confined homology to insulin receptor and its specificity permits to distinguish between the two receptors. Recently there are some ongoing on IGF-IR and ongoing clinical trials on anti-IGF-IR monoclonal antibodies and combined treatments. 2iii

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