CODEN : IJBBBQ ISSN : 0301-1208
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VOLUME 45 |
NUMBER 2 |
APRIL 2008 |
Minireview
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Cathepsins:
Fundamental Effectors of Endolysosomal Proteolysis |
75 |
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Papers
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Optimized conditions for high-level
expression and purification of recombinant human interleukin-2 in E. coli |
91 |
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Paromita Sengupta, Kalpana Meena, Rama Mukherjee, S K Jain and |
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Optimized expression, solubilization and purification of nuclear
inclusion protein b of Cardamom mosaic virus |
98 |
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T Jebasingh, T Jacob, M
Shah, D Das, |
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Optimized culture
conditions for bacteriocin production by Pediococcus
acidilactici LAB 5 and its characterization |
106 |
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Vivekananda
Mandal*, Sukanta Kumar Sen and Narayan Chandra Mandal |
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Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate
oxidase and peroxidase on to aryl amine glass beads affixed on plastic strip
for determination of triglycerides in serum |
111 |
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Genetic
screening in couples experiencing recurrent assisted procreation failure |
116 |
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Notes |
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Identification and partial characterization of
juvenile hormone esterase from cotton pest Dysdercus cingulatus |
121 |
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Mitochondrial complex I impairment and
differential carbon monoxide sensitivity of cytochrome c oxidase in wild type
and CMS II mutants of Nicotiana
sylvestris |
126 |
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130 |
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Instructions to Authors |
133 |
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——————
*Author
for correspondence
AUTHOR INDEX
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116 |
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98 |
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121 |
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75 |
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116 |
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98 |
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91 |
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|
98 |
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98 |
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116 |
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116 |
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91 |
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|
106 |
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106 |
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|
91 |
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|
111 |
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|
91 |
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121 |
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|
126 |
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|
75 |
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116 |
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|
111 |
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116 |
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|
106 |
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|
91 |
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|
98 |
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116 |
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|
116 |
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116 |
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|
116 |
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|
116 |
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|
116 |
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98 |
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116 |
MINIREVIEW
Indian
Journal of Biochemistry & Biophysics
Cathepsins: Fundamental Effectors of Endolysosomal Proteolysis
Sonia Guha and Harish Padh*
Department of Cell and Molecular Biology, B.
V. Patel Pharmaceutical Education and Research Development (PERD) Centre,
S.G. Highway, Thaltej, Ahmedabad 380054, India
Received 21 August 2007; revised 7 March 2008
Intracellular protein degradation is a universal feature of eukaryotic cells and vital for nutrition, protein turnover, intracellular signaling, development and other major physiological processes like antigen presentation and immunity. One of the major compartments of intracellular proteolysis is the endosome-lysosome system. The latter offers a highly orchestrated, vesicular pathway for protein transport and ultimate degradation in lysosomes. Though lysosomes are the classical organelles of complex, multi-enzymatic degradation, it is increasingly evident that endosomes conduct much more than mere transport functions. Endosomes contain significant levels of proteases like cathepsins and are sites of potent intracellular proteolysis. Further, discrete classes of endosomes harbor specific cathepsins and perform selective and exclusive functions. Hence, extra-lysosomal proteolytic machinery within the endocytic pathway enjoys spatial and temporal control over proteolytic functions. The review outlines the structural association and function(s) of major endolysosomal cathepsins.
Keywords: Endosome, Cathepsins,
Intracellular proteolysis
*E-mail: perd@perdcentre.com
PAPERS
Indian
Journal of Biochemistry & Biophysics
Optimized conditions for high-level
expression and purification of recombinant human interleukin-2 in E. coli
Paromita Sengupta1,2, Kalpana Meena1,
Rama Mukherjee1, S K Jain2 and Kapil Maithal1,*
1Dabur
Research Foundation, 22, Site IV, Sahibabad,
2Center
for Biotechnology,
Received 11 October 2007; revised 20 February 2008
Interleukin-2 (IL-2), a potent cytokine has been used in anti-cancer therapy for over a decade now. IL-2, originally identified as a growth factor for T lymphocytes is a 15 kDa hydrophobic glycoprotein that induces the activation, clonal proliferation and differentiation of T and B-lymphocytes and enhances the cytotoxicity of monocytes and natural killer (NK) cells. Here, we report a simple method for the cloning, high-level expression and purification of IL-2 protein, which can be easily extended to other bioactive therapeutic proteins. The IL-2 gene was amplified from human spleen cDNA and cloned in a prokaryotic (E. coli) expression system. An optimal expression of the IL-2 protein was determined by varying the expression conditions like temperature, inducer concentration and duration of induction. The protein was expressed as inclusion bodies and a panel of reagents including detergents, urea and guanidine hydrochloride were used to solubilize it. After solubilization, the protein was renatured and subjected to a single step gel-filtration chromatography to yield immuno-bioactive IL-2 protein with >99% purity.
Keywords: Interleukin-2,
Inclusion bodies, Cytokines, Refolding
*E-mail: maithalk@rediffmail.com
Indian
Journal of Biochemistry & Biophysics
Vol. 45, April 2008, pp.98-105
Optimized expression, solubilization and purification of nuclear inclusion protein b of Cardamom mosaic virus
T Jebasingh, T Jacob, M Shah, D Das,
Received 5 September 2007; revised 28 February 2008
All RNA viruses encode an RNA-dependent RNA polymerase (RdRP) that is
required for replication of the viral genome. Nuclear inclusion b (NIb) gene codes for the RdRp in Potyviridae
viruses. In this study, expression, solubilization and purification of NIb
protein of Cardamom mosaic virus (CdMV) is reported. The objective of the
present study was to express and purify the NIb protein of CdMV on a large
scale for structural characterization, as the structure of the RdRp from a
plant virus is yet to be determined. However, the expression of NIb protein
with hexa-histidine tag in Escherichia
coli led to insoluble aggregates. Out of all the approaches [making
truncated versions to reduce the size of protein; replacing an amino acid
residue likely to be involved in hydrophobic intermolecular interactions with a
hydrophilic one; expressing the protein along with chaperones; expression in
Origami cells for proper disulphide bond formation, in E. coli as a fusion with maltose-binding protein (MBP) and in Nicotiana tabacum] to obtain the RdRp in a soluble form, only expression in E. coli as a fusion with MBP and its
expression in N. tabacum were
successful. The NIb expressed in plant or as a fusion with MBP in E. coli can be scaled up for further
work.
Keywords: RNA-dependent
RNA polymerase, Nuclear inclusion b protein, Cardamom mosaic virus, Inclusion
bodies, Expression, Solubilization
*Email: abayamba@gmail.com
Indian
Journal of Biochemistry & Biophysics
Vol. 45, April 2008, pp.106-110
Optimized culture conditions for bacteriocin production by Pediococcus acidilactici LAB 5 and its characterization
Vivekananda Mandal1*, Sukanta Kumar Sen2 and Narayan Chandra Mandal#2
1P.G.
Department of Botany,
2Microbiology
Laboratory, Department of Botany, Visva-Bharati, Santiniketan 731 235,
Received 30 Octobe 2007; revised 28 February 2008
A strain of Pediococcus acidilactici LAB 5 was isolated from vacuum-packed fermented meat product, in order to obtain a novel bacteriocin from food-grade organisms. Optimized culture conditions for bacteriocin production in different media (viz., MRS, TGE, TGE + buffer, TGE + Tween 80, and TGE + Tween 80 + buffer) and at different temperatures and pH conditions were reported. TGE + Tween 80 + buffer medium was found to be most effective for bacteriocin production (about 2,400 AU/ ml) by this strain, when incubated at 37°C for 24 h. Bacteriocin, partially purified by adsorption-desorption method showed molecular mass of 10.3 kDa and produced prominent inhibition zone in activity gel. It showed significant storage stability both at high as well as in low temperatures for up to 6 months and retained its activity in a number of organic solvents, except in 2-mercaptoethanol. The treatment with amylase and lysozyme did not change its activity, but it lost its activity on proteinase K treatment. Antibacterial efficacy of bacteriocin was proved against some food spoilage and human pathogenic bacteria like Enterococcus, Leuconostoc, Listeria, Staphylococcus and Streptococcus.
Keywords: Antibacterial, Bacteriocin,
Cystibiotics, Pediococcus acidilactici
*Email: mandal_vivek@yahoo.co.in
Indian
Journal of Biochemistry & Biophysics
Vol. 45, April 2008, pp.111-115
Co-immobilization of lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase on to aryl amine glass beads affixed on plastic strip for determination of triglycerides in serum
Minakshi and C
Biochemistry
Research Laboratory, Department of Biochemistry & Genetics, MD University,
Rohtak 124 001,
Received 9 April 2007; revised 10 January 2008
Commercial
lipase, glycerol kinase (GK), glycerol-3-phosphate oxidase (GPO) and peroxidase
(POD) have been
co-immobilized covalently on to arylamine glass beads affixed on a plastic
strip through diazotization with a conjugation yield of 89.1 mg/g support and
64.1% retention of specific activity. The co-immobilized enzymes showed maximum
activity at pH 7.5, when incubated at
40°C for 20 min. The strip was employed for determination of serum
triglycerides (Tgs). The minimum detection limit of the method was 0.20 mM/L. The recovery of added Tgs was
88.0%. Within day and between day coefficient of variations were <7.0 % and
<11.0%, respectively. A good correlation (r = 0.982) was observed between
total serum Tgs values obtained by present method and the most commonly used
enzymic colorimetric method, employing free enzymes. Among the various serum
substances tested at their physiological concentrations, only cholesterol,
ascorbic acid and bilirubin caused 30%, 15%, and 20% inhibition of strip-bound
enzymes, respectively. The strip lost 50% of its activity after 150 regular
uses over a period of 33 days, when stored in reaction buffer at 4°C. The
method reported here has the advantage over other existing methods, as it
provides higher sensitivity, better stability and reusability of co-immobilized
enzymes and is also economical.
Keywords: Triglyceride, Lipase, Glycerol kinase, Glycerol-3-phosphate
oxidase, Peroxidase, Co-immobilization, Arylamine glass beads, Serum, Plastic
strip
*E-mail:
pundircs@rediffmail.com
Indian
Journal of Biochemistry & Biophysics
Vol. 45, April 2008, pp. 116-120
Genetic screening in couples experiencing recurrent assisted procreation failure
Rima Dadaa*, R Kumara, M B Shamsia
, M Tanwara, D Pathaka, S Venkatesha,
M Kumara, H Singha, K Singha, M Aronc,
R Kumarb, G Singhe, R K Sharmad and
N P Guptab
aLaboratory of Molecular Reproduction and Genetics, Department of Anatomy; bDepartment of Urology;
cDepartment of Pathology, All India Institute of Medical Sciences,
dAssisted Reproductive Technique Centre, Army Research and
eDepartment
of Pathology, Airforce Central Medical Establishment,
Received 7 May 2007; revised 29 January 2008
Infertility is a major health problem affecting about 10-20% of couples in the reproductive age group. Male factor is assumed to be responsible in about 50% cases of infertility. The origin of reduced testicular sperm function is unknown in about 50-70% of cases and for such couples assisted reproduction techniques (ART) are a boon. Male infertility is often due to poor semen quality and may be associated with genetic defects. ART has revolutionized management of infertility and intracytoplasmic sperm injection (ICSI) is the ART procedure of choice in 60-80% cases. Despite major technological advancements and professional expertise in ART, the success rate and carry-home live birth rate of ICSI is low (18-25%). This study was aimed to understand the genetic etiopathology of recurrent ART failure. For this, 110 couples with 3 or more failed ART cycles were recruited. A detailed history was taken and only idiopathic ART failure cases were enrolled for this study. They were subjected to cytogenetic and Yq microdeletion analysis. Genetic abnormalities were detected in 19 couples. Since a large number (18.2%) cases harboured genetic abnormalities, it is important for all couples opting for ART to undergo a thorough genetic analysis to prevent recurrent emotional, physical and financial stress.
Keywords: Chromosomal abnormalities,
Yq microdeletion, Assisted reproduction, AZF, Infertility, Assisted
Reproduction (ART), Intracytoplasmic sperm injection (ICSI)
*E-mail: rima_dada@rediffmail.com
NOTES
Indian
Journal of Biochemistry & Biophysics
Vol. 45, April 2008, pp.121-125
Identification and partial characterization of juvenile hormone esterase from cotton pestDysdercus cingulatus
U Gayathri Elayidam* and D Muraleedharen
Department of
Zoology,
Received 22 May 2007; revised 29 January 2008
Juvenile hormone esterase (JHE), a selective enzyme that hydrolyzes the methyl ester of insect juvenile hormone plays an important role in regulating metamorphosis in nymphs as well as reproduction in adults. Studies on JH degradation provide insight into the possibilities of physiological disruption in the insects. In the present study, the JH degrading enzyme, JHE from the cotton pest Dysdercus cingulatus (Heteroptera) is characterized. Electrophoretic analysis of haemolymph during various developmental stages showed the JHE bands prominent only on the final day of 5th instar nymph, and the esterase substrate specificity confirmed the presence of JHE isoforms. In an attempt to clone cDNA of JHE gene from the final instar nymphs, mRNA isolated from fat bodies was coupled with JHE gene-specific primers and the cDNA was synthesized using RT-PCR. The PCR amplified cDNA showed the presence of JHE isoforms in D. cingulatus.
Keywords: Dysdercus cingulatus, Juvenile Hormone Esterase, isoforms, PCR, cDNA
*Email: gayathri_elayidam@yahoo.com
Indian
Journal of Biochemistry & Biophysics
Vol. 45, April 2008, pp.126-129
Mitochondrial
complex I impairment and differential carbon monoxide sensitivity of cytochrome
c oxidase in wild type and CMS II
mutants of Nicotiana sylvestris
R M Naik
Received 25 July 2007; revised 12
February 2008
Plant mitochondria unlike
their animal counterpart have some unique features with highly branched
respiratory chain. The present work was undertaken in order to investigate the
effect of loss/dysfunction of plant mitochondrial complex I on the relative
flux of electrons through alternative oxidase (AOX) and cytochrome oxidase.
Loss of a major subunit of mitochondrial complex I in cytoplasmic male sterile
II (CMS II) mutant of Nicotiana
sylvestris caused respiratory redox perturbations, as evident from the
differential CO sensitivity of cytochrome oxidase. The leaf segments of CMS II
mutant when exposed to CO under dark aerobic condition were insensitive to the
inhibition of cytochrome oxidase, as against the wild type (WT). The
differential CO response of WT and CMS II mutants appeared to be due to
differences in the redox state of cytochrome a3 (cyt a3),
the terminal electron acceptor during in
situ respiration. Cyt a3 appeared to be more in its oxidized
form in CMS II and hence unable to form cyt a3-CO complex.
Pre-treatment of CMS II leaves with 2,4-dinitrophenol, an uncoupler of
oxidative phosphorylation increased the CO response. The slight increase in
rotenone-insensitive respiration of CMS II could be attributed partly to
enhanced flux of electrons through cytochrome pathway to compensate for the
loss of phosphorylation site and partly through AOX, which was induced by
nitrate.
Keywords: Nicotiana sylvestris, Cytochrome
oxidase, Alternative oxidase, Carbon monoxide sensitivity, Nitrate reductase
induction,Mitochondrial complex I
*Email: rajeevnaik2@rediffmail.com
Indian Journal of Biochemistry & Biophysics
Vol. 45, April 2008, pp. 130-132
SFRR-India Satellite Meeting Report 2008 The Society for Free Radical Research (SFRR) ‑ India
satellite meeting 2008 on the theme “Free
radicals and antioxidants in human health, gene regulation and signal transduction”
was held at All India Institute of Medical Sciences (AIIMS), New Delhi during
11-12 February 2008, organized by the Department of Biochemistry, AIIMS, under
the secretarial steering guidance of Prof. D N Rao. The meeting was successful
with the participation of more than 330 delegates representing various academic
research institutes and universities, both within the country and abroad. The
Chief Guest was Dr. N K Ganguly, former Director General of Indian Council of
Medical Research (ICMR),
The inaugural lecture was delivered by Dr. Nilanjana Maulik
(University of Connecticut Medical School, USA) on how reactive oxygen species
regulate vascular angiogenesis through the expression of angiogenic genes, such
as VEGF, FGF, etc. and its redox signaling mechanisms. Prof. V P Menon (
Session-II started with Dr. K B Sainis’s [Bhabha Atomic
Research Centre (BARC), Mumbai] lecture on how chlorophyllin, a green coloring
plant product modifies lymphocyte homeostasis and lymphophemic condition.
Subsequently, Dr. Maitree Suttajit from
Session-III had lectures presented by eminent speakers viz. Dr. Hari S Sharma (Univ Medical Centre, Rotterdam, Netherlands), Dr. K L Khanduja (PG Institute of Medical Education and Research Institute, Chandigarh), Dr. Sandip K Bandyopadhyay (Dr. B C Roy PG Institute of Basic Medical Sciences, Kolkata), Dr. Irfan Rahman (Univ of Rochester Medical Centre, New York) and Dr. Malini Laloraya (Rajiv Gandhi Centre for Biotechnology, Thiruvanathapuram) on (i) how density DNA microarray chip analysis helps in identifying the upregulatory genes such as VEGF and ECM proteins to correct ventricular hypertrophy, (ii) efficacy of α-tocopherol succinate either alone or in combination with all trans-retinoic acid inducing apoptosis of cancer cells through upregulation of ROS and onco proteins, (iii) the healing activity of two traditional spice plant products in drug-induced gastric ulcer in animals, (iv) how dietary polyphenols act as safer nutraceuticals in chromatin remodeling by downregulating the ROS activity, and (v) how O2- anion helps in embryo implantation generated from peri-implanting embryos as well as from uteri, respectively.
In Session–IV, Dr. Chandan K Sen (Ohio State University
Medical Centre,
Dr. Vijay Kumar (International Centre for Genetic Engineering
and Biotechnology,
Day‑II started with Session–VII and VIII parallely. Dr.
Helmut Sies (
In Session‑VIII, Dr. D Shashikiran (Univ of Louisiana
at
Dr. Gopal C Kundu (National Centre for Cell Science, Pune)
started Session-IX with his lecture on the role of two important
transcriptional factors, their crosstalk, and how they regulate hypoxia/
reoxygenation in breast cancer progression. Dr. Molly Jacob (
Two Sessions (VI on day-1 and X
on day-2) were dedicated to presentations by students. There were twenty-six
such presentations with the duration of seven minutes each by the students from
various Institutes/Universities across the country. Of these, six were selected
for ‘best oral presentation’ awards. Among the 138 posters displayed, six were
awarded the ‘best poster’ awards. At the end, valedictory address was given by
the special guest Dr. R C Deka, (Dean, AIIMS,
·
More
emphasis on mid-career scientists presentation.
·
Senior
Faculty lectures should be minimized.
·
More
time for poster presentation.
·
Best
talks were invited as full manuscript to be published in Indian Journal of
Biochemistry & Biophysics, a CSIR peer-reviewed journal as Special
Issue.
The SFRR-India acknowledges the support provided by the funding agencies, both National and International, and the Industries through sponsorship.
Prof. D N Rao
Dept of Biochemistry
AIIMS,