Bioactive agents like secondary metabolites and peptides are gaining much interest while addressing the issues of agricultural and health threats including pests and pathogens and in drug development. Among the plant bioactive peptides, cyclotides being disulphide rich, stable, resistant and having ability to graft epitopes on it or allow sequence variations in its different loops that are very important regarding biological application and research interests for drug development and delivery. The present study was therefore focused on evaluating the bioactive potential of cyclotide bearing indigenous plants including Viola odorata, Viola tricolor, Viola hybrid, Petunia, Clitoria ternatea, pansy F1, Panicum vigatum, Panicum laxam, Panicum maximum and Hamelia patens. The extracts were prepared in phosphate buffer saline (PBS) and protein extraction buffer (PEB). Ptunia and MCOTI-I both separately showed highest DPPH activity (antioxidant) due toas the activity was reduced significantly after treating with Proteinase K. Hamelia possessed highest reducing power, hemolytic, thromobolytic and antimicrobial activites. Mutagenic responses of the (Ames test) medicinal plants were not significant. DNA demage protection assays were also done on all protein extracts including MCOTI-I. Cyclotide genes were also isolated from the selected plant’s DNA using specific primers. It was found that the chimeric arrangement of cliotide gene (from Clitoria plant) as most attractive with conserved sequences in cyclotide and Albumin-1 domains and little intronic variations. Moreover, for cyclotide-protein interaction studies, Texas Red-DBCO amine dye was synthesized to label MCOTI-I-AziF with p-azidophenylalanine Uaa in origami DE3. Optimizations were first done by using the MCOTI-I and its label form MCOTI-I-Lys-TxRd. The click reaction to bind TxRd-DBCO with MCOTI-I-AziF was finally done in buffered guianidinum HCl at a molar ratio of 1:100 and analysed through LC/MS and MCOTI-I-AziF-TxRd-DBCO was confirmed by mass spectrometry. For binding assay FRET analysis was done with Trypsin-EGFP, saturation was achieved with KD value 29.7 ± 1.08 nM. Optimization of same amount of MCOTI-I inteins (using westernblot) was taken in 3-4 h. The IPTG induction avoided the overnight incubations and unwanted backgrounds. The MCOTI-I a cyclotide was expressed for the first time in yeast cells. Current research opens new understandings towards the bioactive xii potential of cyclotide bearing plants with/without peptide content, genetics of cyclotides genes from indegenous plants, optimizations of expression and in vitro labeling studies of MCOTI-I for optical studies regarding drug development and targeting.
جواں جذبوں اور امکانات کا شاعر ڈاکٹر رحمت علی شادؔ جینے کے ڈھنگ تیری جدائی سکھا گئی اور یاد تیری مجھ کو ہے شاعر بنا گئی شہرِ فرید میں جنم لینے والے ڈاکٹر فہد ملک ایک خوش اخلاق،خوش لباس، خوش اطوار اور خوبرو نوجوان ہیں۔ جن کی پہچان کے دو بڑے حوالے ہیں۔ ایک یہ کہ وہ ایک فرض شناس میڈیکل ڈاکٹر ہیںاور دوسرا معتبر حوالہ یہ بھی ہے کہ وہ پاک پتن کے ابھرتے ہوئے ایک عمدہ لب و لہجے کے نووارد شاعر ہیں۔ شہرِ فرید کی ادبی روایت میں ایک خوش گوار اضافہ ثابت ہونے والے فہد ملک کے کلام میں فنی و فکری ہر دو طرح کی جھلکیاں بخوبی دیکھی جاسکتی ہیں۔ ڈاکٹر فہد ملک کی شاعری میں سادگی اور سلاست کا نصر نمایاں ہے۔ وہ کبھی لفاظی ،ثقیل اور بھاری بھرکم تراکیب کے چکر میں نہیں پڑتے ان کو جو بات کہنا ہوتی ہے بڑے سادہ اور موثر انداز میں کہہ دیتے ہیں۔ انھوں نے غزل کے ساتھ نظم بھی لکھی ہے جہاں انھوں نے نہ صرف خیال اور موضوع کو بہتر انداز میں بیان کیا ہے بل کہ عام فہم استعارات و تشبیہات ،اضافتیں، اشارے کنایے اور روایتی علامات بھی استعمال کی ہیں۔ ان کے خیالات منفرد اور لہجہ زود فہم ہے۔ وہ کوئی بھی خیال پیش کرتے ہیں تو اس کو الجھاتے نہیں۔بل کہ کھول کر بیان کر دیتے ہیں۔کسی بھی شاعر کے کلام میں سہلِ ممتنع کا استعمال ایک عمدہ خوبی سمجھا جاتا ہے۔ کیوں کہ بڑے بڑے موضوعات اور بڑی بڑی باتوں کو چند موزوں الفاظ میں بیان کر دینا کوئی آسان بات نہیں۔ چھوٹے چھوٹے مصرعوں میں ایک مکمل اور بھرپور مضمون کو بیان کرنا غیر معمولی بات ہے۔ فہد ملک کی شاعری میں بھی سہلِ ممتنع کی متعدد مثالیں موجود ہیں۔ سہلِ ممتنع کا انداز لیے...
Location of Pakistan has great geo-political and geo-strategic significance in the South Asian region. Pakistan played an important role for the strategic goals of the United States during the ruling periods of President Zia-ul-Haq and President Musharraf. As a frontline state Pakistan received economic and military assistance, during the period of President Zia-ul-Haq, for supporting US interests against Soviet invasion in Afghanistan. After 9/11, due to its strategic location, Pakistan became the ally of the United States against terrorism and the biggest beneficiary of US economic assistance in the region. However, as a result of these policies Islamic extremism, sectarian violence and ethnic cleavages cropped up in the society, shattering social and cultural values. The purpose of this research paper is to highlight the key role played by Pakistan as a frontline state and its impact on the Pakistani society. This research paper follows descriptive and analytical methods.
Cellulase constitute the third largest group of enzymes employed in the industry for the processing of food, textile, detergents, paper and pulp and conversion of agricultural biomass into bio fuel and other useful products. The present study therefore was focused on production of cellulases from Trichoderma harzianum for employing in various applications. Trichoderma harzianum after qualitative screening on carboxymethyl cellulose agar and cellulose Azure was utilized for the production of endoglucanase, exoglucanase and β- glucosidase. Mandel`s mineral salt medium was found to be suitable for the production of cellulases in submerged culture at 120rpm. Significant improvements in specific activities of cellulases were recorded in the Mandel’s salt medium supplemented with 3% wheat bran and 0. 06% lactose as an inducer and addition of Tween 80 further facilitated cellulase production. Maximum production of all the enzymes was achieved between 72 -96 hours of fermentation in most of the production parameter notable activities were achieved at 96 hours. The enzyme production was favorable at temperature of 25°C and 30°C with optimum production reported at 30°C with significant specific activities of endoglucanase (137.3U/mg), exoglucanase (80.63 U/mg) and β-glucosidase (106.88 U/mg). pH 5.0 brought significant production of endoglucanase (139.26 U/mg), exoglucanase(90.12 U/mg) and β-glucosidase (109.88U/mg). Purification was carried out on Sephadex G-100, which revealed two distinct peaks of enzyme activity. Lyophilized fractions with 425.7% yield for endoglucanase, 464. 6% exoglucanase and 468% β-glucosidase were subjected to SDS-PAGE for the determination of molecular weights that resulted in separation of protein into five bands of 95KDa, 75 KDa, 65KDa, 60KDa and 20KDa. Characterization of crude and purified cellulases indicated maximum residual activities of crude cellulases between 35-55°C with maximum activities attained at 45°C. The temperature optima of purified cellulases shifted from 45°C to 50°C for endoglucanse and exoglucanase and to 55°C for β-glucosidase. Study of residual activity of crude and purified cellulases in varying pH revealed that the enzymes have a broad pH range both crude and purified fractions were most active at pH5- _______________________________________________________________________________ Production, Purification & Characterization of Cellulases from Trichoderma harzianum in Sub merged Condition & their Applications. 6. 5 with maximum residual activities retained at pH 5.5. Metal ions like Mn2+, Zn2+, Ca2+, Mg2+, K, and Na had stimulatory effect on the activity of crude as well as purified cellulases Fe2+ ion neither inhibited nor had a stimulatory effect. Lead, Hg2+ and Cu2+ had maximum inhibitory effect. Co2+ inhibited endoglucanase and exoglucanase activity while a slight inhibition was reported in case of β-glucosidase. Enzyme activities of purified enzymes were stimulated by mercaptoethanol. SDS and EDTA had a slight inhibitory effect. Crude enzyme were supplemented in a high fiber formulated poultry feed. Seven dietary treatments comprising of four dietary doses of cellulases only, two dietary mixed doses with cellulases and proteases and a dietary dose without enzyme supplementation that was kept as a control were fed to birds over a period o f 43 days. Enzyme supplementation resulted in increase in average weight gain of all the treatments when compared with control. 156g more weight than control group (A) was recorded in group E (16ml cellulase /kg feed) on 43rd day. A decrease in feed conversion ratio was recorded in all the treatment groups that were linear with increasing enzyme doses. With increasing cellulase dose a decrease in viscosity was noted. In vitro maximum viscosity (1.29 cP) was recorded in control group (A) and minimum was in group E (0.712 cP). Similar results were reported for In vivo viscosity studies of digesta from live birds with maximum figure (1. 089 cP) in control group (A) and maximum reduction was attained in group E (0.521). Dry matter digestibility in control group (80.58%) was less than in all treatment groups with maximum reported in group G (91.08%). Deinking of different type of waste paper resulted in increase in color units of all treated pulps over their respective controls. An average of 2% increase in brightness index of all treated pulps was achieved. Image analysis revealed reduction of residual ink (%) in all the enzyme treated hand sheets. The crude enzyme was found to be stable in all five commercial detergents used, with 90% of residual activity retained after 1 hour of incubation at 40 °C in detergent A. More than 40% residual activity was retained in rest of the detergents after one hour. The crude enzyme was active in removing/fading different type of stains and along with detergent the cleaning power was enhanced further. The crude preparation of cellulases was also successful in biostoning of denim bringing out an even faded look of the garment with an additional advantage of negligible back staining.