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Purification and biochemical characterization of pancreatic phospholipase A2 from the common stingray Dasyatis pastinaca
No full textMammalian sPLA2-IB are well characterized. In contrast, much less is known about aquatic ones. The aquatic world contains a wide variety of living species and, hence represents a great potential for discovering new lipolytic enzymes.
A marine stingray phospholipase A2 (SPLA2) was purified from delipidated pancreas. Purified SPLA2, which is not glycosylated protein, was found to be monomeric protein with a molecular mass of 14 kDa. A specific activity of 750 U/mg for purified SPLA2 was measured at optimal conditions (pH 8.5 and 40 °C) in the presence of 4 mM NaTDC and 8 mM CaCl2 using PC as substrate. The sequence of the first twenty first amino-acid residues at the N-terminal extremity of SPLA2 was determined and shows a close similarity with known mammal and bird pancreatic secreted phospholipases A2. SPLA2 stability in the presence of organic solvents, as well as in acidic and alkaline pH and at high temperature makes it a good candidate for its application in food industry. SPLA2 has several advantageous features for industrial applications. Stability of SPLA2 in the presence of organic solvents, and its tolerance to high temperatures, basic and acidic pH, makes it a good candidate for application in food industry to treat phospholipid-rich industrial effluents, or to synthesize useful chemical compounds.Personna
Study of the possible physiological effects of vitamin C on some markers of neutrophil activation in chronic hemodialysis patients.
No full textAbstract; Elevated oxidative status and reduced antioxidant defense systems are characteristic of end stage renal disease patients which may accelerate the prevalence of atherosclerosis and other chronic complications. The intradialytic activation and degranulation of neutrophils result in the release of neutrophil elastase which is one of the most powerful cytotoxic enzymes because of its biologic effects, together with reactive oxygen species and myeloperoxidase. CD 11b expression on granulocytes was reported to be significantly increased in end stage renal disease patients. Since the antioxidant system is severely impaired in uremic patients, the present study was designed to test the hypothesis that vitamin C supplementation might alleviate oxidative stress and modulate neutrophil activation markers in hemodialysis patients. In our study, we reported a significant decrease of plasma neutrophil elastase, reactive oxygen species, myeloperoxidase, CD11b expression on neutrophils among vitamin C supplemented group compared to unsupplemented group. Plasma total cholesterol and low density lipoprotein was significantly decreased while high density lipoprotein was significantly increased in vitamin C supplemented group compared to unsupplemented group. Collectively, the major finding of the present study was the strong association observed between vitamin C supplementation and reduction of all markers of neutrophil activation in hemodialysis patients.Persona
Solvent Effect on the Amine-Catalyzed Ring-Opening of Azlactone in Acetonitrile-Water Mixtures
No full textThe kinetics of teriethylamine-catalyzed solvolysis of azlactone (oxazolinone) in acetonitrile-water mixtures (10-50 %) in the temperature range (30-60 °C) have been studied. Non-linear plots of log kobs with the reciprocal of relative permittivity were obtained. The thermodynamic parameters H*, S* and G* have been determined; G* increases gradually as the mole fraction of the cosolvent increase, due to a complex quasi-mirror image compensation of H* and S*. The negative values of the entropy of activation and the non-linear relation of log kobs with the reciprocal of relative permittivity suggested selective solvation by the higher polar water molecules. The isokinetic temperature obtained indicated, that the reaction was enthalpic controlled. The presence of methoxy group in the para position retarded
the reaction by about three times of hydriogen. The rate was also decreased with increasing the organic cosolvent. The reactivity was analyzed using Kamlet-Taft solvatochromic parameters and were applied successfully in mixed aqueous -acetonitrile mixtures.King Saud Universit
Simple, Three-Component, Highly Efficient Green Synthesis of Thiazolo[3,2-a]pyridine Derivatives Under Neat Conditions
No full textGreen, highly efficient, three-component syntheses of thiazolo-[3,2-a]pyridine derivatives via reaction of malononitrile, aromatic aldehydes, and 2-mercaptoacetic acid with a catalytic amount of piperidine without solvent with molar ratios of 2:2:1.2 and 2:2.2:1, respectively, has been reporte
First Report of Alternaria alternata on Verbesina encelioides in Saudi Arabia
No full textIn winter 2009, severe foliar infections were observed on Verbesina encelioides in the greenhouse of the King Saud University at Riyadh (Saudi Arabia). Symptoms appeared first as greyish brown patches around the tips and margins of young leaves then extended towards the midrib, causing curling and shedding, of the leaves. Leaf pieces with lesions plated on potato dextrose agar (PDA) yielded fungal colonies with brown septate hyphae bearing straight 1-3 septate conidiophores, 50x3-6 µm in size. Conidia were obclavate to ellipsoidal with a short cylindrical pale-brown to light-brown beak with muriform septation and were usually solitary, occasionally in short chains. On potato carrot agar (PCA), mature conidia were 10-3x5-12 µm in size, showed 3-7 transverse septa, 1-5 longitudinal septa and were borne in chains with more than 5 elements. Based on these morphological characters, the fungus was identified as Alternaria alternata (Simmons, 1992), confirmed by the Indian Type Collection Centre of the Indian Agricultural Research Institute, New Delhi, and given I.D.No.7912.10. Healthy V. encelioides seedlings obtained from the Botanical Garden of the King Saud University were grown in steam-sterilized soil and used for pathogenicity tests. Detached leaves and whole plants were inoculated with a 5x105 ml-1 aqueous conidial suspension of the fungus (Pryor and Michailides, 2002). Detached leaves were maintained in a chamber at near 100% relative humidity for 3 days and inoculated plants were covered with a polythene bag and incubated at 28±2°C, in the glasshouse. Plants/leaves sprayed with sterile water served as controls. Disease symptoms developed within 3 days on inoculated leaves. Control plants remained unaffected. A. alternata was re-isolated from the lesions, thereby fulfilling Koch's postulates. To our knowledge this is the first report of a pathogenic A. alternata affecting Verbesina encelioides
Influence of backpack weight on school girls' balance and musculoskeletal pain
No full textBACKGROUND: School age children are in critical stage of their development. They might be carrying heavy backpacks and they are unable to voice their concern and problems. That leaves them more exposed to postural deviations, musculoskeletal problems and balance instability. OBJECTIVES: The aims of this study were to measure the backpack weight carried by Saudi school girls, to determine the influences of backpack loads on ear and shoulder posture level. determine the different influences of backpack weight on musculoskeletal pain and investigate the effectiveness of daily load of backpack on dynamic balance and postural stability. METHODS: A sample of 175 Saudi school girls age between 12-15 years recruited from random five public schools in Riyadh city. A questionnaire was used to evaluate musculoskeletal problems. Postural mirror was used to measure the level of the ears and the level of the shoulders. The balance master machine was used to measure the dynamic balance in three tests (limits of stability, Step /quick turn, Step up/over). RESULTS: The questionnaire showed significant number of girls complain of shoulder (43.43%), neck (32.6%) and back pain (40%), and majority of girls (87.4%) carried their backpack using a single strap backpack, who reported that their
backpacks were heavy. The results revealed that most of the students spend more than 4 hours watching television and using computer. There is a significant difference in shoulder level (P<0.000). A significant difference in the balance master variables with carrying and without backpack was reported. CONCLUSION: The results of this study concluded that carrying backpack that is 10.08% of body weight caused girl’s complain of shoulder, neck and back pain, induced postural changes and affected their dynamic balance. The backpack weight percentile is recommended to be reduced to 5% body weight.
KEY WORDS: Backpack, Musculoskeletal problems, Posture, Dynamic balance
Identification and Characterization of in-vitro Metabolites of Some Investigational Anti-Cancer Compounds (KM-1081 and KM-1082) Using Liquid Chromatography Ion Trap Mass Spectrometry
No full textMastersThe in-vitro metabolic profile of two investigational anti-cancer agents, 2-amino-5-(4-bromophenyl)-9-chloro-3-phenylpyrimido [4,5-c]quinolin-1(2H)-one (KM-1081), 2-amino-9-chloro-3,5-bis(4-chlorophenyl)pyrimido[4,5-c]quinolin-1 (2H)-one (KM-1082), using rat liver microsomes and hepatocytes is described. KM-1081 and KM-1082 are members of the Pyrimido [4,5-c] quinolin-1(2H)-ones, which is a new class of antimitotic agents. The two compounds exhibited a promising antitubular activity as part of a series of members of the same class newly synthesized and tested [1].
Rat liver microsomes (RLMs) and isolated rat hepatocytes were prepared in-house. KM-1081 and KM-1082 were incubated with rat liver microsomes and rat hepatocytes according to published methodologies. Purified incubation mixtures were analyzed by liquid chromatography-Ion trap mass spectrometry (LC-ITMS) to identify possible metabolic products. Mass spectrometric analysis of the incubated compounds revealed the presence of several peaks representing potential metabolites. These peaks
were proposed to be the results of N-deamination of KM-1081and KM-1082, as well as several other products. Screening of total ion current (TIC) chromatograms of RLMs incubations for KM-1081 show a peak eluting at ~ 54 minutes with a molecular ion peak at m/z 475 corresponding to KM-1081 and peak eluting at ~57 minutes with a molecular ion peak of m/z 460 is proposed to be of the N-deaminated KM-1081.
Additionally peaks with m/z of 460, 494.2 and 510.3 were found in the TIC chromatogram and were proposed to be enol form of the N-deaminated KM-1081, hydroxy reduced KM-1081 and dihydoxy reduced KM-1081, representing. The peaks appeared in a smaller concentration than the N-deaminated product. Screening of TIC chromatogram of rat liver microsomal incubations for KM-1082 showed a peak eluting at ~ 63 minutes with a molecular ion peak at m/z 465 corresponding to the KM-1082. A peak eluting at ~ 65 minutes with a molecular ion peak of 450 was, similar to KM-1081, proposed to be of the N-deaminated KM-1082. As with the case of KM-1081, peaks corresponding to enol form of N-deaminated KM-1082, Dihydroxylated KM-1082 and Trihydoxylated KM-1082 were found but with smaller concentration. Based on these results, the N-deaminated derivative of both compounds appears to be the major metabolic product of RLMs metabolism.
Screening of TIC chromatograms of rat hepatocytes incubations for both compounds showed the proposed N-deamination, albeit representing a higher proportion compared to that resulting from RLMs metabolism. These results suggest the extensive, though number-limited nature of the metabolism of KM-1081 and KM-1082
Enhancing the stability of recombinant human erythropoietin in albumin free formulations
No full textMastersErythropoietin (EPO) is α-glycoprotein hormone of 165 amino acids and molecular weight 35 KD that is 40 % glycosylated. EPO is mainly produced by renal peritubular interstitial cells in the kidney together with hepatocytes. Generally, serum EPO concentrations of 10 to 25 mI.U/ mL are necessary for maintaining hemoglobin levels within the normal range of 12 to 17 g/dL. The terminal half-life (t1/2) of EPO is 5 hours, which requires an average EPO production rate of 2 U/kg/day.
Circulating EPO level can increase up to 1000 fold because of the logarithmic increase in the number of producing cells. EPO acts on RBCs precursor cells in bone marrow (CFUe=colony-forming unit–erythroid and Erythroblast) and stimulate their differentiation, and maturation to red blood cells and prevent the apoptosis of those cells. The oxygen sensor within the renal cells detects the oxygen content of the blood and consequently regulates the amount of EPO released in the blood. So in response to hypoxia, additional EPO-producing peritubular
cells are recruited for production of EPO. When the hypoxic stimulus is removed, the recruited cells return to their non-secretary state.
EPO has various indications, including treatments of anemia associated with kidney diseases, anemia associated with bone marrow transplantation, iron deficiency anemia, cancer- or antitumor agent-related anemia, and AIDS-related anemia.
The human EPO gene was cloned in 1983, allowing for clinical development of recombinant human EPO (rhEPO). Endogenous EPO and rhEPO share the same amino acid sequence, with slight differences in the sugar profile. The cloning of the EPO gene allowed for the commercial production of the rhEPO. EPO alfa (Epogen®; Amgen Inc., USA) was the first rhEPO commercialized in the United States in 1989, followed by EPO beta in 1990 (NeoRecormon®); F. Hoffmann-La Roche Ltd., Switzerland) in Europe. EPO alfa and beta, both produced using Chinese hamster ovary cells system, This EPO has minor structural differences but the same physiological effects.
An EPO omega produced in baby hamster kidney cells differs from previous EPO in the glycosylation profile. EPO delta (Dynepo®, Shire plc, Basingstoke, UK), was produced from an engineered human fibrosarcoma cell line HT1080 in 2007. EPO is considered as the world best selling drug with annual sales in the range of 10 to 12 billion $ per year.
EPO, however, is not stable in an aqueous solution. Even at very low temperatures
(-80°C), a substantial decrease in activity can be observed. As a consequence, EPO lose its native physiochemical properties and physiological activity. The denaturation of EPO is generally irreversible, thus EPO cannot recover its native properties, once denatured. Furthermore, the high tendency of EPO to adsorption leads to aggregation via a denaturation process, and administration of the denatured protein causes hypersensitivity.
There are many reasons for the decrease in the activity of the EPO. These include the following:
o EPO has low stability and degraded in the aqueous solution
o Adsorption of the drug on the inner surface of the wall of the syringe especially if the dose of the drug is only a few micrograms.
o Destruction of the EPO by catalytic effects of the surface of the ampoules due to the presences of traces of heavy metals, and/or atmospheric oxygen.
Under these circumstances, various attempts have been made to develop a method to increase the stability of EPO in the aqueous solution and prevent its adsorption on the inner surface of the wall of container. Some protein formulations solved the denaturation with a lyophilization method. However, lyophilization increases manufacturing costs, involves an increased risk due to mechanical troubles, lyophilized products are inconvenient since they have to be reconstituted prior injection, and a large capacity freeze-dryer is required for processing. Another approach is to use stabilizers to enhance the stability of the EPO. Protein stabilizers include surfactants, polysaccharides, serum albumin, amino acids, and salts.
The use of human serum albumin as stabilizer suffers high risk of viral contamination, in addition to, more susceptibility to allergic reactions. In Europe, use of materials from human and animal sources as pharmaceutical additives is increasingly restricted. Based on that, it is clear that more studies for the optimization of stable EPO formulae free from human serum albumin are highly demanded. The primary objective of this work is to formulate EPO in formula(e) free from blood-derived proteins and show improved stability. This formula will rule out the risk of viral contamination and ensure biological activity. Several stabilizing agents will be used to replace blood-derived component, albumin or purified gelatin.
The specific work undertaken in this thesis is briefly described below:
Chapter two: Assessment of different factors affecting the stability of erythropoietin in parenteral solutions.
In this chapter, different EPO formulae were prepared, The composition of each formula was set to vary in a way to allow studying the effect of presence and type of the amino acids and the presence and type of surfactant on the stability profile of EPO using accelerated stability testing at two different elevated temperature (25oC, 40oC) stored for 60 days. Bradford method, ELISA, and SDS gel electrophoresis were used as analytical methods. The outcomes of the results highlighted that the presence of surfactant is necessary to avoid the initial adsorption of EPO on the walls of vials and reduces the ability of the protein to aggregate. The type of surfactant is a critical factor as Tween 20 was superior to Poloxamer 118. Amino acids have a role in the stabilization of EPO. The presence of glycine was found to be critical factor and the combination of glycine with acidic amino acid such as glutamic acid was better than the combination with neutral or basic amino acids. F4 (containing glycine 6mg/ml, glutamic acid 1.5 mg/ml, Tween 20 0.1 mg/ml, and mannitol 5 mg/ml) is considered the optimum formula among all the prepared formulae as it kept high content of EPO after two months storage in both elevated temperatures and it showed minimum aggregation and degradation levels.
Chapter three: Encapsulation of erythropoietin in nanoparticles.
In this chapter, EPO loaded solid lipid nanoparticles and polymeric nanoparticles were prepared using double emulsion method (w/o/w) with least process related stress on the
encapsulated drug. The produced nano-particles were evaluated for their particle size, size distribution and particle charge. The particle shape was examined by scanning electron microscope. The encapsulation efficiency and the in vitro release profile of EPO from the particles were assessed using Bradford methods. The EPO stability during encapsulation was checked by SDS-Gel Electrophoresis. The results showed that polymeric nanoparticles composed of PLGA and PEG 6000 loaded with EPO (F18) was superior over all other prepared formulae. The particle size diameter was 225.9±3.8 nm. SEM images of F18 revealed spherical in shape having smooth regular surfaces with no aggregates. The sizes observed from SEM micrographs were consistent with those obtained from particle size analyzer. Entrapment efficiency for F18 was 33.3 %, and SDS gel image showed neither aggregation nor degradation occurred to EPO during encapsulation process by the applied method. Finally, all the formulae showed a burst drug release with values around 50% cumulative drug released. F18 release was slowly increased over 24 hours to a maximum of 82%.
Chapter four: Assessment of the in vivo biological activity of erythropoietin in different parenteral formulae.
In this chapter, parallel comparative in-vivo bioassay was designed to evaluate the biological activity of different EPO formulae achieved best in-vitro attributes. Balb C mice were used and flow-cytometer method was applied to measure the reticulocyte to red blood cell ratio for all the selected treatment. Pure EPO, and marketed product Eprex® were used as positive controls. The result showed that F4 showed good stability in-vitro proved to be active in-vivo and the ingredient of the formula did not affect the physiological activity of the EPO. EPO loaded nano particles (F18) suspended into PBS were able to maintain the physiological activity
of EPO for 2 weeks after single S C injection compared with pure and marketed EPO formulae. It was concluded that ELISA is a useful method for the detection of the EPO activity only if no aggregation or degradation was detected in the samples
The Attitude of Male and Female Teachers and Students towards Teachers' Feedback on the Writings of Secondary School Students in Rafha City, Saudi Arabia
No full textThis study aims to investigate male and female teachers' and students'
attitudes towards teachers' feedback on the writings of secondary school
students in Rafha city. The population of the study is 480 students (240
males and 240 females) and 50 teachers ( 25 males and 25 females). In
this study, five instruments are used to accomplish the objectives of the
study:1) Students' questionnaire; 2)Teachers' questionnaire; 3)Students'
interviews; 4)Teachers' interviews; and 5)Class observation.
The analysis of the results indicates that students and teachers have
positive attitudes towards teachers' feedback on students' writings. A ttest
analysis show no differences between male and female teachers and
students attitudes toward teachers' feedback. The results reveal that
students understand their teachers' feedback as they meant by their
teacher.
A major conclusion of the study reveals that teacher's feedback is a
very important technique to improve students' writing. Therefore,
teachers should be involved in training courses to show them the
importance of feedback and equip them with effective feedback strategies
and recommended principles
Towards Developing Automatic Name-Entity-Recognition System for Arabic Text
No full textName Entity Recognition (NER) has emerged as a Natural Language Processing (NLP) technology that is effective and can provide high value to several different kinds of application such as Information Extraction (IE), Information Retrieval (IR), Question Answering (QA), text clustering, etc. NER is responsible for the identification of proper names in text and their classification as different types of named entity such as people, locations, and organizations. There are two main approaches to NER, one is based on linguistic knowledge in particular grammar rules and hence called rule-based, while the other is based on machine learning techniques.
We aim in this research to build Name Entity Recognition automatic system for the Arabic language using Machine Learning (ML) approaches; these approaches have many models such as Maximum Entropy (ME), Decision Tree (DR), Support vector Machines (SVM), and Hidden Markov Model (HMM). Among these models we used HMM to build our system for the reason that it relies on the context structure. ML approaches provide us the ability to work with unrestricted domain, and to adapt the suitable machine learning with the nature and the difficulties of some characteristic of the Arabic language. The Arabic language does not exhibit differences in orthographic case; whereas the English language mixes case texts, therefore, there is some obvious clue such as initial capitalized letters to indicate the presence of a name constituent