In situ Reversible Aggregation of Extracellular Cellobiase in the Filamentous Fungus Termitomyces clypeatus

Samudra Prosad Banik,Swagata Pal,Shakuntala Ghorai,Sudeshna Chowdhury,Rajib Majumder,Soumya Mukherjee,Suman Khowala 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.5

Cellobiase (E.C. 3.2.1.21), is a widely exploited industrial glycosidase with a major role in biofuel industry. Its stability and shelf life are major bottlenecks in achieving a superior formulation for industry. In the filamentous fungus Termitomyces clypeatus, the enzyme is secreted in a co-aggregated form with sucrase; the separation of this co-aggregation results in substantial loss of the enzyme’s activity. The aim of the present study was to examine the mode of aggregation of the secreted cellobiase-sucrase coaggregate and its role in the stabilization of cellobiase. Transmission electron microscopy and dynamic light scattering of purified co-aggregates revealed reversible, concentration driven self-aggregation of the extracellular enzymes to form larger entities. However, the intracellular enzyme aggregates were rigid,non-interacting, and possessed a higher percentage of disulphide bonds. Circular dichroic spectra of the two coaggregates indicated no significant difference in secondary structures. Self-association increased the stability of extracellular aggregates towards heat by 1.5 fold, SDS by 4 ~ 7 fold, and chaotropic agents, by 1.5 ~ 2 fold, than the intracellular counterpart. The Km of extracellular aggregate varied between 0.29 and 0.45 mM as a result of spontaneous aggregation and disaggregation, whereas that of intracellular aggregate was 0.22 mM irrespective of its concentration status. In situ detection of cellobiase in native PAGE revealed two activity bands of the extracellular enzyme, which indicated a minimum of two active dissociated aggregate species, as compared to a single band for the intracellular enzyme. These studies are believed to improve the understanding of aggregation of the fungal glycosidases, which remains to be a blackbox, to increase the efficacy of these enzymes. Cellobiase (E.C. 3.2.1.21), is a widely exploited industrial glycosidase with a major role in biofuel industry. Its stability and shelf life are major bottlenecks in achieving a superior formulation for industry. In the filamentous fungus Termitomyces clypeatus, the enzyme is secreted in a co-aggregated form with sucrase; the separation of this co-aggregation results in substantial loss of the enzyme’s activity. The aim of the present study was to examine the mode of aggregation of the secreted cellobiase-sucrase coaggregate and its role in the stabilization of cellobiase. Transmission electron microscopy and dynamic light scattering of purified co-aggregates revealed reversible, concentration driven self-aggregation of the extracellular enzymes to form larger entities. However, the intracellular enzyme aggregates were rigid,non-interacting, and possessed a higher percentage of disulphide bonds. Circular dichroic spectra of the two coaggregates indicated no significant difference in secondary structures. Self-association increased the stability of extracellular aggregates towards heat by 1.5 fold, SDS by 4 ~ 7 fold, and chaotropic agents, by 1.5 ~ 2 fold, than the intracellular counterpart. The Km of extracellular aggregate varied between 0.29 and 0.45 mM as a result of spontaneous aggregation and disaggregation, whereas that of intracellular aggregate was 0.22 mM irrespective of its concentration status. In situ detection of cellobiase in native PAGE revealed two activity bands of the extracellular enzyme, which indicated a minimum of two active dissociated aggregate species, as compared to a single band for the intracellular enzyme. These studies are believed to improve the understanding of aggregation of the fungal glycosidases, which remains to be a blackbox, to increase the efficacy of these enzymes.

Evidence of an Alternative Route of Cellobiase Secretion in the Presence of Brefeldin A in the Filamentous Fungus Termitomyces clypeatus

( Banik Samudra Prosad ),( Swagata Pal ),( Sudeshna Chowdhury ),( Shakuntala Ghorai ),( Suman Khowala ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.4

Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 μg/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at 50oC, 93.3% activity at pH 9, 63.64% activity in the presence of 1M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe.

안구적출에 따른 위둔덕의 칼슘결합단백질의 재구축 및 상호 연관성

안병수,고길석,안명수,김경주,권안성,정명섭,박춘매,조병옥,김진우,Samudra Acharya,Parmeshwar Narayan Amatya,장인엽 朝鮮大學校 附設 醫學硏究所 2007 The Medical Journal of Chosun University Vol.32 No.1

Background: Superior colliculus is a part of midbrain, and participates in the visual reflexes, It receives afferent fibers from optic nerve, visual cortex, and spinotectal tract. After optic deprivation, the microscopic structure of the superior colliculus changed. Calcium-binding proteins (CBPs) Play an important role in the neuronal protection, differentiation and reorganization of the central nervous system, Objectives and Methods: The effects of neonatal retinal deafferentation on a CBPs, calbindm D-28k (CB), Parvalbumin (PB) and calretimn (CR), and the existence of colocalization between the CBPs were examined immunohistochemically in the rat superior colliculus. Results: On the experimental (contralateral to enucleation) side of superior colliculus, the number of CB-immunoreactive (IR) cells was reduced (77.4% compared to control), but not fibers. The number of PB-IR neurons and fibers was also reduced on the experimental side (88.5% compared to control), In the other hand, the CR-IR cells were dramatically increased (642% compared to control), but CR-IR fibers were markedly decreased on the experimental side. The colocalization between CB-CR and PV-CR was rarely observed in the superior colliculus Conclusion: These results suggest that the changes of retinotectal projection may alter the expressional pattern of CBPs in different manners; relatively stable in CB- and PV-IR neurons and plastic in CR-IR neurons.

H2AX: 유전자 손상 보호자

아마티아 파머셔 나라얀,아카랴 사무드라,유호진 朝鮮大學校 附設 醫學硏究所 2007 The Medical Journal of Chosun University Vol.32 No.1

The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 10-30 minutes of DNA damage and forms foci at break sites. Histone H2AX has a role in suppressing genomic instability and cancer. Phosphorylated H2AX (gamma-HZAX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.

Antimicrobial and Anti-Inflammatory Effects of Cecropin A(1-8)–Magainin2(1-12) Hybrid Peptide Analog P5 against Malassezia furfur Infection in Human Keratinocytes

Ryu, Sunhyo,Choi, Soon-Yong,Acharya, Samudra,Chun, Young-Jin,Gurley, Catherine,Park, Yoonkyung,Armstrong, Cheryl A,Song, Peter I,Kim, Beom-Joon The Society for Investigative Dermatology, Inc 2011 The Journal of investigative dermatology Vol.131 No.8

The lipophilic fungus Malassezia furfur (M. furfur) is a commensal microbe associated with several chronic diseases such as pityriasis versicolor, folliculitis, and seborrheic dermatitis. Because M. furfur-related diseases are difficult to treat and require prolonged use of medications, the treatment for M. furfur-related skin diseases is supposed to gain control over M. furfur growth and the inflammation associated with it, as well as to prevent secondary infections. In this study, we investigated the antifungal and anti-inflammatory effects of cecropin A(1-8)–magainin 2(1-12) hybrid peptide analog P5 on M. furfur. The minimal inhibitory concentration of P5 against M. furfur was 0.39 μM, making it 3–4 times more potent than commonly used antifungal agents such as ketoconazole (1.5 μM) or itraconazole (1.14 μM). P5 efficiently inhibited the expression of IL-8 and Toll-like receptor 2 in M. furfur-infected human keratinocytes without eukaryotic cytotoxicity at its fungicidal concentration. Moreover, P5 significantly downregulated NF-κB activation and intracellular calcium fluctuation, which are closely related with enhanced responses of keratinocyte inflammation induced by M. furfur infection. Taken together, these observations suggest P5 may be a potential therapeutic agent for M. furfur-associated human skin diseases because of its distinct antifungal and anti-inflammatory action.

Realistic Simulations on Reverse Junction Characteristics of SiC and GaN Power Semiconductor Devices

Guannan Wei,Yung C. Liang,Ganesh S. Samudra 전력전자학회 2012 JOURNAL OF POWER ELECTRONICS Vol.12 No.1

This paper presents a practical methodology for realistic simulation on reverse characteristics of Wide Bandgap (WBG) SiC and GaN p-n junctions. The adjustment on certain physic-based model parameters, such as the trap density and photo-generation for SiC junction, and impact ionization coefficients and critical field for GaN junction are described. The adjusted parameters were used in Synopsys Medici simulation to obtain a realistic p-n junction avalanche breakdown voltage. The simulation results were verified through benchmarking against independent data reported by others.

Realistic Simulations on Reverse Junction Characteristics of SiC and GaN Power Semiconductor Devices

Wei, Guannan,Liang, Yung C.,Samudra, Ganesh S. The Korean Institute of Power Electronics 2012 JOURNAL OF POWER ELECTRONICS Vol.12 No.1

This paper presents a practical methodology for realistic simulation on reverse characteristics of Wide Bandgap (WBG) SiC and GaN p-n junctions. The adjustment on certain physic-based model parameters, such as the trap density and photo-generation for SiC junction, and impact ionization coefficients and critical field for GaN junction are described. The adjusted parameters were used in Synopsys Medici simulation to obtain a realistic p-n junction avalanche breakdown voltage. The simulation results were verified through benchmarking against independent data reported by others.

Order of N Complexity Transform Domain Adaptive Filters : Software and Hardware Implementation

A. Najafi,B. Farhang Boroujeny,G. S. Samudra 대한전자공학회 1993 JTC-CSCC : Joint Technical Conference on Circuits Vol.- No.-

Realistic Simulation on Reverse Characteristics of SiC/GaN p-n Junctions for High Power Semiconductor Devices

Guannan Wei,Yung C. Liang,Ganesh S. Samudra 전력전자학회 2011 ICPE(ISPE)논문집 Vol.2011 No.5

This paper presents a practical methodology for realistic simulation on reverse characteristics of Wide Bandgap (WBG) SiC/GaN p-n junctions. The adjustment on certain physic-based model parameters, such as the trap density and photo-generation for SiC junction, and impact ionization coefficients and critical field for GaN junction are described. The adjusted parameters were used in Synopsys Medici simulation to obtain a realistic p-n junction avalanche breakdown voltage. The simulation results were verified through benchmarking against independent data reported by others.

CT-Guided Percutaneous Biopsy of Intrathoracic Lesions

Hira Lal,Zafar Neyaz,Alok Nath,Samudra Borah 대한영상의학회 2012 Korean Journal of Radiology Vol.13 No.2

Percutaneous CT-guided needle biopsy of mediastinal and pulmonary lesions is a minimally invasive approach for obtaining tissue for histopathological examination. Although it is a widely accepted procedure with relatively few complications, precise planning and detailed knowledge of various aspects of the biopsy procedure is mandatory to avert complications. In this pictorial review, we reviewed important anatomical approaches, technical aspects of the procedure, and its associated complications.