CODEN : IJBBBQ ISSN : 0301-1208
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VOLUME 44 |
NUMBER 4 |
AUGUST 2007 |
Papers
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| Site-directed mutagenesis of conserved Thr407, Asp433, and Met464 residues in small subunit of Escherichia coli g-glutamyltranspeptidase |
197 |
| Huei-Fen Lo, Long-Liu Lin*, Pei-Jing Chen and Wei-Mou Chou | |
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Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli |
204 |
| Daling Zhu, Aihua Li, Jianguo Wang*, Ming Li and Taozhen Cai | |
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Azadirachta
indica leaf extract modulates initiation phase of murine forestomach
tumorigenesis |
209 |
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Metabolism of arachidonic acid in sheep uterus: In vitro studies |
216 |
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A Sai Padma*, S Agarwal, D Bharat Reddy, T Sandeep
Prasad and
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Effect of water deficit on carbohydrate status and
enzymes of carbohydrate metabolism in seedlings of wheat cultivars |
223 |
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Effect of UV-B and high visual radiation on
photosynthesis in freshwater |
231 |
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Vibrational dynamics and heat capacity in
polyglycine II |
240 |
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Abhishek Kumar Mishra, Mahendra Singh, Poonam Tandon*
and Vishwambhar Dayal Gupta |
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Notes |
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Purification and properties of an extracellular
pectin lyase produced by the strain of Penicillium oxalicum in solid-state fermentation |
247 |
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Purification and some properties of carbonic anhydrase from Elephas
trogontherii (Steppe elephant)
bone |
252 |
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Modulation of DMBA-induced biochemical changes by organoselenium
compounds in blood of rats |
257 |
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Instructions to Authors |
260 |
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——————
*Author
for correspondence
PAPERS
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 197-203
Site-directed mutagenesis of conserved Thr407, Asp433 and Met464
residues in small subunit of Escherichia coli g-glutamyltranspeptidase
1Department of Food and Nutrition, Hungkuang University, Shalu, Taichung
City, Taiwan
2Department of Applied Chemistry, National Chiayi University, Chiayi
City, Taiwan
3Graduate
Institute of Biotechnology, National Chiayi University, Chiayi City, Taiwan
Received 10 April 2007; revised 20 July 2007
Sequence comparison showed that residues Thr407,
Asp433, and Met464 in the small subunit of Escherichia coli g-glutamyltranspeptidase (EcGGT) were conserved
in the aligned enzymes. In this study, we further investigated the functional
significance of these conserved residues by site-directed mutagenesis. The
wild-type and mutant enzymes were overexpressed in the recombinant E.
coli M15 cells and purified to near
homogeneity by Ni2+-NTA resin. Except M464L, other mutants had shown
no GGT activity under enzyme assay conditions and activity staining.
Furthermore, mutations on these residues impaired the capability of
autocatalytic processing of the enzyme. Based on these observations, it is
concluded that these residues play an important role in the enzyme maturation.
Keywords:
Escherichia coli, g-Glutamyltranspeptidase, Site-directed mutagenesis, Autocatalytic processing
*E-mail: llin@mail.ncyu.edu.tw
Indian Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 204--208
Cloning, expression and characterization of
aerolysin fromAeromonas hydrophila in Escherichia coli
Daling Zhua,b, Aihua Lia, Jianguo Wanga, * , Ming Lia and Taozhen Caia
aThe Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
bGraduate School of the Chinese Academy of Sciences, Beijing 100039, China
Received 02 December 2006; revised June 2007
Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc.A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W)of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli bl21. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His·Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.
Keywords: Aeromonas hydrophila, Cloning, aerA gene,
Prokaryotic expression, Recombinant protein AerA-W, Hemolytic activity
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 209-215
Azadirachta indica
leaf extract modulates initiation phase of murine forestomach tumorigenesis
Subhash
Chander Gangar and Ashwani Koul*
Department
of Biophysics, Basic Medical Sciences Block, Panjab University, Chandigarh 160
014, India
Received 02 March 2007; revised 09 July 2007
The effects of aqueous Azadirachta indica leaf
extract (AAILE) on benzo(a)pyrene [B(a)P]-induced forestomach tumorigenesis,
B(a)P-DNA adduct formation and certain parameters of carcinogen
biotransformation system in mice have been reported earlier from our
laboratory. In this study, the effects of AAILE on the enzymes of B(a)P
biotransformation, which play crucial role in initiation of chemical
carcinogenesis ¾
aryl hydrocarbon hydroxylase (AHH) and
uridinediphosphoglucuronosyltransferase (UDP-glucuronosyltransferase) have been
evaluated in murine forestomach and liver. In addition, lipid peroxidation
(LPO) levels in forestomach as well as liver and the activities of tissue injury
marker enzymes ¾
lactate dehydrogenase, aspartate aminotransferase and
alkaline phosphatase in the serum have also been evaluated. Oral administration
of AAILE (100 mg/kg body wt for 2 weeks) reduces the AHH activity and enhances
the UDP-glucuronosyltransferase activity in both the tissues, suggesting its
potential in decreasing the activation and increasing the detoxification of
carcinogens. The LPO levels decrease upon AAILE treatment in the hepatic
tissue, suggesting its anti-oxidative and hence anti-carcinogenic effects.
Non-significant alterations have been observed in tissue injury marker enzymes
upon AAILE treatment, suggesting its safety at the given dose
. In conclusion,
AAILE appears to modulate initiation phase of carcinogenesis and may be
suggested as safe and an effective agent for chemoprevention.
Keywords: Aryl hydrocarbon
hydroxylase; Azadirachta indica;
Benzo(a)pyrene; Lipid
peroxidation; Tissue injury marker enzymes; UDP-glucuronosyltransferase
*Email :ashwanik@pu.ac.in, drashwanikoul@yahoo.co.in
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 216-222
Metabolism of arachidonic acid in sheep uterus: In vitro studies
A Sai Padmaa*, S Agarwalb, D Bharat Reddyb, T Sandeep Prasada and P Reddannab
a Department of Biochemistry, Bharatiya Vidya Bhavan’s Vivekananda College, Sainikpuri, Secunderabad 500 094, India
bSchool of Life Sciences, University of Hyderabad, Hyderabad 500 046, India
Received 24 January 2007; revised 22 May 2007
Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins ¾ PGF2α and prostacyclin derivative 6-keto PGF1α. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.
Keywords: Arachidonic acid metabosism, Lipoxygenase, Cyclooxygenase, Prostaglandins, HPETEs and HETEs
*E-mail: saipadmabhavans@yahoo.co.in
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 223-230
Effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism in seedlings of wheat cultivars
Kamaljit Kaur, Anil K Gupta and Narinder Kaur*
Department of Biochemistry and Chemistry, Punjab Agricultural University, Ludhiana 141 004, India
Received 09 February 2007; revised 14 May 2007
The effect of water deficit on carbohydrate status and enzymes of carbohydrate metabolism (a and b amylases, sucrose phosphate synthase, sucrose synthase, acid and alkaline invertases) in wheat (Triticum aestivum L.) was investigated in the seedlings of drought-sensitive (PBW 343) and drought-tolerant (C 306) cultivars. The water deficit was induced by adding 6% mannitol (water potential -0.815 Mpa) in the growth medium. The water deficit reduced starch content in the shoots of tolerant seedlings as compared to the sensitive ones, but increased sucrose content in the shoots and roots of tolerant seedlings, indicating their protective role during stress conditions. It also decreased the a -amylase activity in the endosperm of seedlings of both the cultivars, but increased a and b amylase activities in the shoots of tolerant ones. Sucrose phosphate synthase (SPS) activity showed a significant increase at 6 days of seedling growth (DSG) in the shoots of stressed seedlings of tolerant cultivar. However, SPS activity in the roots of stressed seedlings of sensitive cultivar was very low at 4 DSG and appeared significantly only at day 6. Sucrose synthase (SS) activity was lower in the shoots and roots of stressed seedlings of tolerant cultivar than sensitive ones at early stage of seedling growth. Higher acid invertase activity in the shoots of seedlings of tolerant cultivar appeared to be a unique characteristic of this cultivar for stress tolerance. Alkaline invertase activity, although affected under water deficit conditions, but was too low as compared to acid invertase activity to cause any significant affect on sucrose hydrolysis. In conclusion, higher sucrose content with high SPS and low acid invertase and SS activities in the roots under water deficit conditions could be responsible for drought tolerance of C 306.
Keywords:
Triticum aestivum L., Water deficit,
Acid invertase, Alkaline invertase, a
and b
-Amylase,
Sucrose phosphate synthase, Sucrose synthase, Sucrose, Starch
*E-mail: nkaur @rediffmail.com
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 231-239
Effect of UV-B and high visual radiation on
photosynthesis in freshwater
(Nostoc spongiaeforme) and marine (Phormidium
corium) cyanobacteria
Rupali Bhandari and Prabhat Kumar Sharma*
Department of Botany, Goa University, Goa 403 206, India
Received 11 September 2006; revised 17 May 2007
Human activity is causing depletion of ozone in
stratosphere, resulting in increased UV-B radiation and global warming.
However, impact of these climatic changes on the aquatic organism (especially
marine) is not fully understood. Here, we have studied the effect of excess
UV-B and visible radiation on photosynthetic pigments, fatty acids content,
lipid peroxidation, nitrogen content, nitrogen reductase activity and membrane
proteins, induction of mycosporine-like amino acids (MAAs) and antioxidant
enzymes superoxide dismutase (SOD) and ascorbate peroxidase (APX) in freshwater
(Nostoc spongiaeform) and marine (Phormidium
corium) cyanobacteria. UV-B
treatment resulted in an increase in photosynthetic pigments in Nostoc and decrease in Phormidium, but high light treatment
caused photobleaching of most of the pigments in both the species. Unsaturation
level of fatty acids of both total and glycolipids remained unchanged in both
the cyanobacteria, as a result of
UV-B and high light treatments. Saturated fatty acids of total and glycolipids
declined slightly in Nostoc by both
the treatments, but remained unchanged in Phormidium.
No changes in the unsaturated lipid content in our study probably suggested
adaptation of the organism to the treatments. However, both treatments resulted
in peroxidation of membrane lipids, indicating oxidative damage to lipids
without any change in the level of unsaturation of fatty acid in the cell
membrane. Qualitative and quantitative changes were observed in membrane
protein profile due to the treatments. Cyanobacteria were able to synthesize
MAAs in response to the UV-B treatment. Both treatments also increased the
activities of SOD and APX. In conclusion, the study demonstrated induction of
antioxidants such as SOD and APX under visible light treatment and screening
pigment (MAAs) under UV-B treatment, which might protect the cyanobacteria from
oxidative damage caused by high light and UV-B radiation.
Keywords: Antioxidant enzymes, Cyanobacteria, Fatty acids,
High light, Mycosporine-like amino acids, Photosynthetic active radiation,
Photosynthetic pigments, Ultraviolet-B radiation
*Email: prabhat_k_sharma@rediffmail.com
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 240-246
Vibrational dynamics and heat capacity in polyglycine II
Abhishek Kumar Mishraa, Mahendra Singhb, Poonam Tandona* and Vishwambhar Dayal Guptaa
aDepartment of Physics, University of Lucknow, Lucknow 226 007, India
bBrahmanand P.G. College , Kanpur 208 004, India
Received29 September 2006;revised 21 June 2007
The earlier works on the vibrational dynamics of polyglycine II (PG II) suffer from several infirmities, which not only suppress the dynamical nature of normal modes, but also lead to several incorrect assignments and interactive constants of the potential field. In this study, we have re-examined the phonon dispersion profiles of PG II using Higgs method for evaluation of phase-related normal modes and have attempted to remove the infirmities, as far as possible. The Wilson’s GF matrix method combined with the Urey-Bradley force field has been used for normal mode analysis. This potential field leads to correct assignments of Raman, infrared and inelastic neutron scattering frequencies. Characteristic features of the dispersion curve (v versusd /p plot), such as repulsion and regions of high density-of-states have been interpreted. In addition, the heat capacity as a function of temperature has been obtained via density-of-states. It agrees well with the experimental data and is being reported for the first time.
Keywords: Polyglycine II, Density-of-states, Phonon
dispersion, Heat capacity, Urey-Bradley force field
*E-mail: poonam_tandon@yahoo.co.uk
NOTES
Indian
Journal of Biochemistry & Biophysics
Vol. 44, August 2007, pp. 247-251
Purification and properties of an extracellular pectin lyase produced
by the strain of Penicillium oxalicum
in solid-state fermentation
Sangeeta Yadav*1 and N V Shastri
Department of Biochemistry, RTM Nagpur University, Amravati Road, Nagpur 440 010, India
Received 09 May 2006; revised 07 May 2007
A pectin lyase (PNL, EC 4.2.2.10) produced extracellularly by the strain of Penicillium oxalicum in solid-state fermentation medium containing deoiled mandarin orange peel meal was purified to apparent homogeneity by a protocol that included ammonium sulfate precipitation, DEAE-Sephadex A-50 and Sephadex G-100 chromatography. The enzyme had molecular mass of 50 kD, as determined by SDS- PAGE and showed optimum pH and temperature at 8.0 and 50° C respectively. It had an isoelectric point (pI) of 5.0 and showed a Km of 1.1 mg/ml of citrus pectin. The enzyme was strongly inhibited by Mo4+, Ag+ and Pb2+ and also by polyphenolic compounds, in particular tannic acid.
Keywords: Pectins, Pectin lyases,
Pectin transeliminases, Penicillium oxalicum, Solid-state fermentation
*Email: sangeeta_rahul@rediffmail.com
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp. 252-256
Purification and some properties of carbonic
anhydrase from Elephas trogontherii
(Steppe elephant) bone
Yaşar Demir1, Hayrunnisa Nadaroğlu2 and Nazan Demir 3*
1Department of Chemistry, Faculty of Education
2Department of Food Technology, Oltu Profession High Scholl
3Department of Chemistry, Faculty of Science, Atatürk University,
25240, Erzurum, Turkey
Received 07 November 2006; revised
18 July 2007
Four isoenzymes of
carbonic anhydrase (CA) were purified from Elephas trogontherii (steppe elephant) bone (approx 0.3-0.5 million years old) from
different locations (outer peripheral, cytosolic, inner peripheral and
integral) using Sepharose 4B-L-tyrosine sulphanilamide affinity chromatography
and their kinetics properties were investigated and compared with known CA
isoenzymes. The purification degree of CAs was monitored by SDS-PAGE.
Purification fold for outer peripheral, inner peripheral, cytosolic and
integral CA was 395.6, 652.8, 1091 and 429.3 and the molecular mass (as
determined by gel filtration chromatography) was 37, 36, 35, and 39 kDa,
respectively.The optimal
temperature for isozymes was 10-20, 30, 30 and 60°
C and optimal pH was between 7.5-11, 7.5-10, 7.5-10 and 7.5 respectively. Km
values (at optimum pH and 20°
C) for p-nitrophenyl
acetate as substrate were 4.83, 6.80, 4.525 and 3.86 mM and the Vmax values for the same
substrate were 0.00097, 0.0149, 0.00249 and 0.00072 μmol/L*min,
respectively. I50 values
of isoenzymes for the inhibitors of CA ¾
sulphanilamide, KSCN, acetazolamide and NaN3
were also determined.
Keywords: Elephas
trogontherii,
Bone, Carbonic anhydrase, Kinetics properties, Steppe elephant
*E-mail: demirn@yahoo.com
Indian
Journal of Biochemistry & Biophysics
Vol.
44, August 2007, pp.257-259
Modulation of DMBA-induced biochemical changes by organoselenium compounds in blood of rats
I Ozdemir1, Z Selamoglu2, B Ates1,
Y Gok1 and I Yilmaz1*
1Department of Chemistry and 2Department
of Biology, Inonu University, 44280 Malatya, Turkey
Received 25 April 2006; revised 19 July 2007
The
protective role of two synthetic organoselenium compounds 1-isopropyl-3-methylbenzimidazole-2-selenone
(SeI) and 1, 3-di-p-methoxybenzylpyrimidine-2-selenone
(SeII) was examined against the 7,12-dimethylbenz[a]anthracene (DMBA)-induced
changes in biochemical parameters in blood of rats. Albino Winstar rats (150-200
g body wt) were treated with single dose of DMBA (50 mg/kg body wt) and
organoselenium compounds (25 m
mol/kg) for 4 weeks at two
days internal. Blood was taken from the anaesthetized rats ventricle from their
hearts for biochemical analysis. Administration of DMBA resulted in elevation
of urea, uric acid and creatinine levels as well as AST, ALT and LDH activities
and decrease in levels of total proteins, albumin and globulin. SeI and SeII
caused a significant (p<0.05) decrease in urea, uric acid and creatinine
levels and alanine aminotransferase (ALT); aspartate aminotransferase; (AST)
and lactate dehydrogenase (LDH) activities and significantly increased the
levels of total protein and albumin (p<0.05). These organoselenium compounds
are likely to be beneficial in human health.
Keywords: Biochemical parameters, 7,12-Dimethylbenz [a]anthracene or DMBA, Oxidative stress, Organoselenium compounds
*E mail: iyilmaz@inonu.edu.tr
AUTHOR INDEX
Agarwal S
216
Ates B
257
Bhandari R
231
Cai T
204
Chen P J
197
Chou W M
197
Demir N
252
Demir Y
252
Gangar S C
209
Gok Y
257
Gupta A K
223
Gupta V D
240
Kaur K
223
Kaur N
223
Koul A
209
Li A
204
Li M
204
Lin L L
197
Lo H F
197
Mishra A K
240
Nadaroğlu H
252
Ozdemir I
257
Prasad T S
216
Reddanna P
216
Reddy D B
216
Sai Padma A
216
Selamoglu Z
257
Sharma P K
231
Shastri N V
247
Singh M
240
Tandon P
240
Wang J
204
Yadav S
247
Yilmaz I 257
Zhu D 204