Screening and Media Optimization for Enhancing L-asparaginase Production, an Anticancer Agent, from Different Filamentous Fungi in Solid State Fermentation.

Aim: The aim of present study was to screen new potent fungal isolates and microorganisms possessing extracellular L-asparaginase production capacity. In addition, optimization of cultural and environmental conditions required for enzyme production will be carried out for the highest Lasparaginase producer in solid state fermentation (SSF) technique using agro-industrial residues. Study Design: Screening and physiological studies on the formation of L-asparaginase by Trichoderma viride F2 in order to obtain the optimum cultural and environmental conditions required for enzyme production. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between July 2013 and June 2015. Methodology: Optimization of physical and nutritional parameters for enzyme production was investigated. Various locally available agro-industrial residues have been screened individually or as mixtures for L-asparaginase production. The combination of Rice husk (RH) with wheat bran (WB) (3:2) proved to be an efficient mixture for enzyme production as it gave the highest enzyme activity (71.87±3.19 U/g-ds) when compared to individual RH (66.71±2.76 U/g-ds) or WB (62.28±2.13 U/g-ds) substrates. Results: Maximal L-asparaginase production (113.43±5.11 U/g-ds) by T. viride F2 was obtained with moisture content of 75%, an inoculums size of 1 x 108 spores/ml and an initial medium pH of 5.0 when incubated at 28ºC for four days. Presence of Tween 20 enhanced enzyme production by 1.19 folds. Glucose (1.0%), Casein (1.5%) and MgCl2 (0.05%) were found to be the best carbon, organic nitrogen and ion sources, respectively. Supplementation of the medium with NaNO3 (0.15%) as an inorganic nitrogen source further increased L-asparaginase production. Under these optimized conditions, L-asparaginase production by T. viride F2 was maximum with a yield of 276.5±13.4 U/g-ds in SSF, which was more than 19-fold enhancement in enzyme activity as compared to that obtained in the basal medium (SmF) (14.23±0.87 U/ml). Conclusion: The results suggest that choosing a suitable substrate coupled with optimization of different parameters can improves enzyme production markedly. Moreover, the production of Lasparaginase from a process based on RH and WB as substrates in SSF is economically attractive due to abundant substrates availability in agriculture-based countries with cheaper cost.

Effect of some antioxidants on the prostate of adult and aged albino rats: a histological and immunohistochemical study.

Effect of some antioxidants on the prostate of adult and aged albino rats. Twenty-five adult and twenty-five aged male albino rats were divided into four groups: group I (control group) group II (zinc sulphate treated group), group III (vitamin E) & group IV (vitamin C) zinc administered in doses of 0.2673 mg for adult rat and 0.693 mg for aged rat, vitamin E administered in doses of 0.973 mg for adult rat and 2.52 mg for aged rat, vitamin C administered in doses of 1.215mg for adult rat and 3.15 mg for aged rat. The prostate glands were processed and stained by H&E, Masson trichrome & immunoreaction of androgen receptor for light microscopic examinations. Morphometric analysis for collagen fibers and immunoreaction area percent was performed and statically analyzed. Zinc showed improvements, in which decrease in number of mucosal fold and increase in immunoreactions of nuclear androgen receptor in ventral lobe also, Decrease fibrosis and increase in immunoreactions of nuclear androgen receptor in dorsolateral lobe. vitamin E showed improvements, in which decrease in number of mucosal fold, decrease size of acini, decrease of epithelial heights and increase in immunoreactions of nuclear androgen receptor in ventral lobe also, decrease of epithelial heights, decrease fibrosis and increase in immunoreactions of nuclear androgen receptor in dorsolateral lobe. vitamin C there were improvements, in which decrease in number of mucosal fold, dilatation of acini and slightly increase in immunoreactions of nuclear androgen receptor in ventral lobe also, rarified collagenous fibers and increase in immunoreactions of nuclear androgen receptor in dorsolateral lobe. It also ameliorated blood vessels congestion. Zinc, vitamin E and vitamin C exerted no harmful effects on adult prostate but ameliorated effects against aged prostate.

Purification, Kinetic Properties and Antitumor Activity of L-Glutaminase from Penicillium brevicompactum NRC 829.

Aim: The aims of the present study were to purify and characterize L-glutaminase from Penicillium brevicompactum NRC 829; and to evaluate the antitumor activity of the purified enzyme against different tumor human cell lines. Study Design: Testing of antitumor activity of L-glutaminase, purified from a filamentous fungal strain, against four different tumor human cell lines. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between January 2011 and February 2012. Methodology: P. brevicompactum NRC 829 was grown and maintained on modified Czapek Dox agar (MCD) medium. Cell-free extract was directly used as the source of crude enzyme. L-glutaminase was purified by heat treatment for 20 min at 50ºC, followed by gel filtration on Sephadex G-100 and G-200 columns. Results: An intracellular L-glutaminase from Penicillium brevicompactum NRC 829 was purified to homogeneity (162.75 fold) with an apparent molecular mass (Mr) of 71 kDa. The purified enzyme showed its maximal activity against L-glutamine when incubated at pH 8.5 at 50ºC for 30 min. The purified enzyme retained about 92 % of its initial activity after incubation at 70ºC for 30 min indicating the thermo-stability nature of this enzyme. The highest activity was reported towards its natural substrate, L-glutamine, with an apparent Km value of 1.66 mM. The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 63.3μg/ml. Conclusion: L-glutaminase purified from Penicillium brevicompactum NRC 829 is a potential candidate in food and pharmaceutical industries.

Immunolocalization of laminin during postnatal development of the testis, epididymis and vas deferens of albino rat

Disorders of testicular function may have their origins in fetal or early life as a result of abnormal development. Laminin-1 is emerging as the key molecule in early embryonic basement membrane assembly. Accumulating evidence supported the idea that extracellular matrix [ECM] molecules and mesenchymal cells might influence Sertoli and spermatogenic cell functions. Detecting the changes in the distribution and prevalence of laminin-1 assembly during postnatal development of the testis, epididymis and vas deferens in albino rats. Thirty male albino rats were used and divided into six groups [n= 5 each] according to the age [postnatal day]. These were one day, 1 week, 2 weeks, 3 weeks, 4 weeks, and 8 weeks postnatal. Specimens were fixed and processed, sectioned and stained by hematoxylin and eosin and immunohistochemical stain for laminin-1. The area percent of positive laminin immunostaining was measured and results were statistically analyzed. at day one postnatal, the testis was formed of solid un-canalized cords of seminiferous tubules with abundant laminin expression in the cells of the cords. With advancement of development the cords were luminized and the laminin expression declined to involve the basement membrane and the apical portions of the Sertoli cells at the 8[th] week postnatal. The epididymis at postnatal day one had a small diameter and narrow lumen and laminin expression involved the cytoplasm of the epithelial lining. As development proceeded the expression became confined to the apical portion, the site of stereocilia together with its presence in the basement membranes. The same pattern of changes in laminin expression together with morphological appearance was detected in the vas deferens. The present study was able to demonstrate a change in the distribution as well as the prevalence of laminin-1 immunoreactivity within the testis, epididymis and vas. During the period of postnatal development starting at postnatal day one up to 8 weeks postnatal. This would reflect an essential role for laminin in early postnatal period of development

Purification, Characterization and Antitumor Activity of L-asparaginase from Penicillium brevicompactum NRC 829.

Aim: The aims of this study were to attempt to extract, purify and characterize of Lasparaginase, an antitumor agent, from Penicillium brevicompactum NRC 829. Study Design: Testing of antitumor activity of L-asparaginase against four different tumor human cell lines. Place and Duration of Study: Department of Microbial Chemistry, Genetic Engineering and Biotechnology Division, National Research Centre (NRC), Cairo, Egypt, between June 2010 and November 2011. Methodology: Penicillium brevicompactum NRC 829, a local isolated strain from Culture Collection of the National Research Centre of Egypt, was grown and maintained on modified Czapek Dox medium. The fresh fungal biomass was thoroughly ground with washed cold sand. The cell contents were extracted with cold 0.1M Tris-HCl pH 8.0, thereafter, the slurry obtained was centrifuged at 5500 rpm for 15 min and the supernatant was directly used as the source of enzyme. The purification of L-asparaginase from crudeenzyme extracts of P. brevicompactum was achieved by a sequential multi-steps process starting by heat treatment for 20 min at 50ºC, followed by gel filtration on Sephadex G-100 column, and the most active fractions of L-asparaginase were dialyzed out, lyophilized and then loaded on a Sephadex G-200 column. Results: An intracellular glutaminase-free-L-asparaginase from Penicillium brevicompactum NRC 829 was purified to homogeneity with an apparent molecular mass (Mr) of 94 kDa. The purified enzyme was 151.12 fold with a final specific activity of 574.24 IU/mg protein and about 40% yield recovery. The purified L-asparaginase showed its maximal activity against L-asparagine when incubated at pH 8.0 at 37ºC for 30 min. The enzyme was more stable at alkaline pH than the acidic one and thermally stable up to 60 min at 50-60ºC. L-asparaginase was highly specific for its natural substrate, L-asparagine with a Km value of 1.05 mM. The activity of L-asparaginase is activated by mono cations and various effectors including K+, Na+, 2-mercaptoethanol (2-ME), and reduced glutathione (r-GSH), whereas it is moderately inhibited by various divalent ions including Hg2+, Cu2+, and Ag+. Results indicated the involvement of sulfhydryl group(s) in the enzyme active site(s). The purified enzyme inhibited the growth of human cell line hepatocellular carcinoma (Hep-G2), with IC50 value of 43.3μg/ml. Conclusion: L-asparaginase purified from Penicillium brevicompactum NRC 829 is a potential candidate for medical applications.

Squamous cell carcinoma antigen as a tumor marker in patients with hepatocellular carcinoma

Hepatocellular carcinoma [HCC] is considered the fifth most common cancer in the world. Owing to its increased incidence in the last decade and the expected further increase in the next 2 decades, HCC is arousing great interest. HCC commonly develops on cirrhotic livers and therefore, surveillance programs have been suggested to identify early HCC, at a stage suitable for surgical or interventional therapy and has a better clinical outcome. The only serologic marker used in clinical practice is alpha-fetoprotein [alpha-FP], but its sensitivity is poor. Hence, the investigation of new markers is required. To assess the clinical utility of squamous cell carcinoma antigen [SCCA] as a non invasive marker in the early diagnosis of HCC and whether the association of alpha-FP and SCCA could improves the diagnostic power. This study is conducted on 65 newly diagnosed hepatic focal lesion cases from those attending the Tropical Medicine Department, Cairo University Hospitals [Group I] as well as 20 age and sex matched healthy control subjects [Group II]. Group I was further subdivided into la [49 HCC proved untreated patients] and Ib [16 patients with Cirrhosis only] according to their histopathological findings. All patients were subjected to full history taking, clinical examination, laboratory investigations [including liver function test, hepatitis markers, alpha-FP and SCCA serum levels], triphasic abdominal CT and pathological examination. Group I included 42 males [64.7%] and 23 females [35.3%] with ages ranging between 42-70 years [60.7111.28], of them 16 patients had HBV [24.6%], 37 patients had HCV [56.9%] and 12 patients [18.4%] had mixed HBV and HCV infection. Group I was further subdivided into group la which included 49 HCC proved patients and group Ib which included 16 patients with regeneration nodules [cirrhosis only] according to their histopathologic findings. Group II [control] included 20 age and sex matched healthy subjects. Mean levels of serum alpha-FP and SCCA in group Ia was significantly higher when compared with group Ib [p<0.0005 for both of them]. At a cutoff of serum alpha-FP 200 ng/mL, the sensitivity was 35% and the specificity was 100% while at a cutoff >400ng/mL, the sensitivity decreased to 7.6%. On using the receiver operator curve [ROC], to improve the specificity and sensitivity of alpha-FP and SCCA, the cutoff value of 40ng/ml and 0.55ug/L yielded a sensitivity of 67.2% and 61.2% respectively and specificity of 100% [best cutoff]. When combined sensitivity of them was calculated at the best-chosen cutoff values, sensitivity improved to 87.7% with specificity of 100%.Combined use of alpha-FP and SCCA in the screening of patients with hepatic focal lesions may increase the chance of diagnosis of HCC patients

Retinal toxicity to triamcinolone acetonide's vehicle: an electrophysiology and electron microscopy study

To assess rabbit retinal toxicity to triamcinolone acetonide vehicle, benzyl alcohol, when injected intravitreally. This prospective comparative experimental study included 24 pigmented rabbits assigned into 2 groups; group 1 [experiment, n=12] received intravitreal 0.1ml of benzyl alcohol [BA], and group 2 [control, n=12] received intravitreal 0.1ml of balanced salt solution [BSS]; all injections were done in the right eyes. Clinical examinations were done on both eyes of all available rabbits before injection, at 1 and 3 hours post injection, and together with electroretinograms [ERGs] at 3, 7, 14, 28 and 42 days following injections. Three rabbits from each group were killed at 7, 14, 28 and 42 days and both eyes were sent for either light or electron microscopic examination for quantitative morphometric measurements. Mean amplitudes of ERG a and b waves of the BA-injected eyes were 6.42 +/- 9.02 micro v and 11.18 +/- 15.18 micro v at 3 days respectively, which was significantly reduced compared to the BSS-injected eyes [30.87 +/- 8.22 micro v and 57.90 +/- 13.38 micro v, respectively; p<0.01 t-test] and the non-injected contralateral eyes [36.20 +/- 7.85 micro v and 64.10 +/- 9.36 micro v, respectively; p<0.01 t-test]. These ERG responses continued to be significantly reduced in the BA-injected eyes [p<0.01 t-test] of the unkilled rabbits up to 6 weeks. The mean ganglion cell count [at a magnification of X200 in a standard frame of 4740.32 micro m[2]] were significantly reduced [p<0.005 t-test] in the BA-injected eyes [8.42 +/- 2.4] compared to the BSS- and non-injected eyes [16.42 +/- 3.9 and 16.5 +/- 4.2, respectively]. The mean inner nuclear layer [INL] and outer nuclear layer [ONL] thickness were significantly reduced [p<0.005 t-test] in the BA-injected eyes [3.78 +/- 0.96 micro m and 11.77 +/- 1.29 micro m, respectively] compared to the BSS- [6.1 +/- 0.92 micro m and 21.82 +/- 0.95 micro m, respectively] and non-injected eyes [7.05 +/- 1.9 micro m and 22.49 +/- 1.0/micro m, respectively]. Electron microscopy showed intracellular irreversible changes in the GCL, INL, ONL, and PRL at 6 weeks in group 1, with no significant changes in group 2. There was no significant rise in the intraocular pressure or clinical evidence of increased lens density during the study period. Triamcinolone Acetonide's Vehicle, benzyl alcohol, produced severe irreversible ERG and structural damage to rabbit neurosensory retinal following intravitreal injection