Indian Journal of Biochemistry & Biophysics

Minireview
Apolipoproteins and their role in different clinical conditions: an overview	73
M Irshad* and R Dubey

Papers
Variant of mitochondrial ribosomal protein s28 (mrps28) gene is differentially expressed in response to radiation in a cervical carcinoma derived cell line	81
G Gopal & T Rajkumar*

Temporal expression patterns of timeless in vg and cry^b mutants of Drosophila melanogaster	87
G Suthakar, P Subramanian* and T Manivasagam

Phospholipase C from two bacterial strains acts differently on pure phospholipids and membrane bound glycosylphosphatidylinositol (GPI) anchors	92
Arshi Rastogi, Tarun E Hutchinson and Ben M J Pereira*

Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom	100
Xianghu Liu, Xiaolong Xu, Jiexia Chen, Wenqi Liu and Qingliang Liu*

Exploring selectivity requirements for COX-2 versus COX-1 binding of 2-(5-phenyl-pyrazol-1-yl)-5-methanesulfonylpyridines using topological and physico-chemical parameters	106
Santanu Chakraborty, Chandana Sengupta and Kunal Roy*

Notes
Role of a protein inhibitor isolated from human renal stone matrix in urolithiasis	113
S Aggarwal, C Tandon¹, M Forouzandeh, S K Singla, R Kiran and R K Jethi*

Modulation of spontaneous electrical activity of freshly isolated 3-day embryonic chick ventricle by cAMP and cGMP	118
P Prakash* and O Tripathi

Proton NMR transverse relaxation time and membrane stability in wheat leaves exposed to high temperature shock	122
Shantha Nagarajan*, D K Joshi, Anjali Anand, A P S Verma and P C Pathak

Age-related changes in the elemental constituents and molecular behaviour of bone	127
D V Rai*, R Darbari and L M Aggarwal

Instructions to Authors	131

Minireview

Apolipoproteins and their role in different clinical conditions: An overview

Apolipoproteins or apoproteins are a group of proteins associated with lipoproteins in different proportions and play significant roles in several diseases. Different types of apolipoproteins, including apolipoproteins A, B, C, D, E, H and J and their subclasses have been reported, in addition to a few more apolipoproteins reported recently. These proteins have varied, but definite roles in normal physiology in our body. Moreover, their blood levels have strong association with clinical conditions during different diseases and are used as diagnostic and prognostic markers and to compute index of risk for some serious disease entities. Present article gives an overview of the structural features, physiological significance and diagnostic and clinical implications of apolipoproteins.

Keywords: Apoproteins, lecithin-cholesterol acyl transferase, lipoprotein(a), lipoprotein lipase, clinical implications.

Papers

Variant of mitochondrial ribosomal protein s28 (mrps28) gene is differentially expressed in response to radiation in a cervical carcinoma derived cell line

Gene expression before and after radiation treatment (10 Gy) in a cervical carcinoma derived cell line SiHa was studied. Differential display performed on non-irradiated and irradiated cells (10 Gy) using a combination of 3 anchor primers and 16 arbitrary primers yielded 20 differentially expressed fragments. Differential expression of one of the fragments differentially expressed in the irradiated sample was also confirmed in Northern blots. Cloning and sequencing of the fragment revealed it to be similar to a splice variant of the MRPS28 gene. Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using primers designed to amplify the splice variant and confirmed the expression. In conclusion, the study shows that the splice variant kDec03 of the MRPS28 gene is differentially expressed in response to radiation in SiHa cells.

Keywords: Cervix cancer, SiHa cells, radiation response, differential display, mitochondrial ribosomal proteins, MRPS28, kDec03 MRPS28, splice variants MRPS, reverse transcriptase polymerase chain reaction, differential expression.

Temporal expression patterns of timeless in vg and cry^b mutants of Drosophila melanogaster

The temporal expression patterns of timeless (tim) in Drosophila melanogaster at various time points were studied in intestine and salivary gland of wild type (WT), vestigial (vg) and cryptochrome-absent (cry^b) mutants under 12 hr:12 hr white light:darkness (LD) and 12 hr:12 hr blue light (450 nm):darkness (BD) conditions. At ZT 06 and ZT 10, tim expression was almost nil and at ZT 18 and ZT 22, the expression was most pronounced in WT and mutants, when compared to other time points. As vg flies have greatly reduced wings, their gross locomotor activity was poorer and levels of tim expression were also least than WT flies. The weaker expression of tim in cry^b flies suggested the significant role of blue light photoreceptor cryptochrome for a stronger synchronization of circadian clock. The expression patterns of tim in the salivary gland of larvae further suggested the presence of peripheral oscillators during the developmental stages.

Key words: Circadian, Drosophila melanogaster, cryptochrome, timeless, vestigial, temporal expression

Phospholipase C from two bacterial strains acts differently on pure phospholipids and membrane bound glycosylphosphatidylinositol (GPI) anchors

Phospholipase C (PLC) was purified to homogeneity from the culture filtrate of Bacillus cereus (65-fold, 540 U/mg protein) and B. thuringiensis (76-fold, 306 U/mg protein) by conventional techniques of enzyme purification. The purified enzymes have the molecular mass of 34 kDa and 38 kDa respectively, as determined by SDS-PAGE. Both the PLCs exhibited identical sensitivity to pH, temperature, cations, anions and inhibitors like glutathione and
p-chloromercuribenzoate. PLC-Bc showed a preference for phosphatidylinositol, while PLC-Bt favoured phosphatidylcholine as the substrate. Although both the enzymes were able to hydrolyze pure phosphatidylinositol, distinct differences were observed in their activity on phosphatidylinositol-anchored membrane proteins. PLC-Bc cleaved and released alkaline phosphatase, a GPI-anchored marker enzyme from microsomal membranes to a greater extent, than PLC-Bt. Experiments with sperm membranes, followed by SDS-PAGE revealed that the pattern of proteins released from their GPI-anchors by PLC-Bc and PLC-Bt were dissimilar. Although some proteins were cleaved in common by both PLCs, some others including a prominent 57 kDa protein were resistant to PLC-Bt, but sensitive to cleavage by PLC-Bc. The type of modification in the GPI anchor, special environment on membranes, and relative charge of host plasma membrane to the charge of PLC may be the factors that are responsible for the differential action of two enzymes.

Keywords: Phospholipase C, Bacillus cereus, Bacillus thuringiensis, phospholipids, microsomal membrane, sperm plasma membrane, glycosylphosphatidylinositol (GPI) anchor, alkaline phosphatase, ATPase, inhibitors.

Effects of metal ions and an inhibitor on the fluorescence and activity of acutolysin A from Agkistrodon acutus venom

Acutolysin A, a protein isolated from the venom of Chinese Five-pace snake (Agkistrodon acutus) has shown marked hemorrhagic and proteolytic activities. In the present study, the effects of metal ions and an inhibitor EDTA on the fluorescence and function of autolysin A have been studied, by following fluorescence and activity measurements. Acutolysin A contains a Ca²⁺-binding site, which provides it with important structural stability, and a Zn²⁺-binding site, which is essential for its enzymatic activities. The removal of metal ions in acutolysin A by incubation with EDTA results in irreversible inhibition and complete denaturation, and a marked decrease in its fluorescence intensity. The fluorescence intensity of acutolysin A is also decreased in the presence of Cu²⁺, Co²⁺, Mn²⁺or Mg²⁺, but does not change in the presence of Ca²⁺, Cd²⁺,or Tb³⁺. Caseinolytic activity of acutolysin A is enhanced by Co²⁺, Ca²⁺ and Mg²⁺, but is partly inhibited by Cu²⁺, Mn²⁺ and Tb³⁺, and completely inhibited by Cd²⁺. Both Zn²⁺and Co²⁺ recover the loss of activity of the protein caused by Cd²⁺.

Keywords: Acutolysin A; fluorescence; metal ion; Agkistrodon acutus, zinc-metalloproteinases, EDTA, snake venom

Exploring selectivity requirements for COX-2 versus COX-1 binding of 2-(5-phenyl-pyrazol-1-yl)-5-methanesulfonylpyridines using topological and physico-chemical parameters

Considering the current need for development of selective cyclooxygenase-2 (COX-2) inhibitors, an attempt has been made to explore physico-chemical requirements of 2-(5-phenyl-pyrazol-1-yl)-5-methanesulfonylpyridines for binding with COX-1 and COX-2 enzyme subtypes and also to explore the selectivity requirements. In this study, E-states of different common atoms of the molecules (calculated according to Kier & Hall), first order valence connectivity and physico-chemical parameters (hydrophobicity p, Hammett s and molar refractivity MR of different ring substituents) were used as independent variables along with suitable dummy parameters in the stepwise regression method. The best equation describing COX-1 binding affinity [n = 25, Q² = 0.606, R_a²= 0.702, R² = 0.752, R = 0.867, s = 0.447, F = 15.2 (df 4, 20)] suggests that the COX-1 binding affinity increases in the presence of a halogen substituent at R₁ position and a p-alkoxy or p-methylthio substituent at R₂ position. Furthermore, a difluoromethyl group is preferred over a trifluoromethyl group at R position for the COX-1 binding. The best equation describing COX-2 binding affinity [n = 32, Q² = 0.622, R_a²= 0.692, R² = 0.732, R = 0.856, s = 0.265, F = 18.4 (df 4, 27)] shows that the COX-2 binding affinity increases with the presence of a halogen substituent at R₁ position and increase of size of R₂ substituents. However, it decreases in case of simultaneous presence of 3-chloro and 4-methoxy groups on the phenyl nucleus and in the presence of highly lipophilic R₂ substituents. The best selectivity relation [n = 25, Q² = 0.455, R_a²= 0.605, R² = 0.670, R = 0.819, s = 0.423, F = 10.2 (df 4, 20)] suggests that the COX-2 selectivity decreases in the presence of p-alkoxy group and electron-withdrawing para substituents at R₂ position. Again, a trifluoro group is conducive for the selectivity instead of a difluoromethyl group at R position. Furthermore, branching may also play significant role in determining the selectivity as evidenced from the connectivity parameter.

Keywords: QSAR, COX-2 inhibitors, COX-1 binding, 2-(5-phenyl-pyrazol-1-yl)-5-methanesulfonylpyridines, topological parameters, physico-chemical parameters, non-selective non-steroidal anti-inflammatory drugs

Role of a protein inhibitor isolated fromhuman renal stone matrix in urolithiasis

The role of biomolecule(s) from renal stone matrix in urolithiasis was investigated. The ability of a particular fraction (>10 kDa fraction) isolated from the EDTA extract of powdered human renal stones to influence calcium oxalate monohydrate (COM) crystal growth was studied. The most potent inhibitor of COM crystal growth obtained from >10 kDa fraction was purified by various chromatographic techniques and SDS-PAGE, etc. and was found to have a molecular mass of 36 kDa. The urine and serum samples obtained from normal persons were found to be more potent in inhibiting the growth of COM crystals as compared to the kidney-stone patients. Polyclonal antibodies were raised against this inhibitor and were employed to determine the concentration of 36 kDa inhibitor in urine and serum samples of normal persons and kidney-stone patients.

Modulation of spontaneous electrical activity of freshly isolated 3-day embryonic chick ventricle by cAMP and cGMP

Effects of cyclic nucleotides 8-Bromo-cAMP and 8-Bromo-cGMP (membrane permeable analogs of cAMP and cGMP) were examined on action potential (AP) configuration and rate of spontaneous firing of the freshly isolated 3-day embryonic chick ventricle (ECV) to assess the role of l-type slow Ca²⁺ channels in upstroke of AP and spontaneous electrical activity (pacemaker potential). The 3-day ECV exhibited prominent automaticity and spontaneous APs characterized by maximum upstroke velocity (+V_max), maximum diastolic potential (MDP), overshoot (E_ov), AP duration at -20 mV (APD₂₀) and cycle length (CL) of 33.09±3.18 V/sec, -63.77±1.17 mV, 17.40±0.91 mV, 51.20±3.05 m sec and 795±150 m sec, respectively (n= 10 preparations). 8-Br-cAMP (1 mM) caused significant increase in E_ov and APD₂₀ (37% and 56%, respectively, p<0.01), but failed to produce any stimulatory effect on +V_max and MDP. Surprisingly, 8-Br-cAMP produced negative chronotropic effect on spontaneous firing (automaticity) and enhanced the CL significantly by 43% (p<0.05). 8-Br-cGMP, however, had no effect on AP configuration and the rate of spontaneous firing. The present findings with 8-Br-cAMP suggest that l-type slow Ca²⁺ channels do not contribute to upstroke of AP and pacemaker potential of spontaneously firing freshly isolated 3-day ECV. The negative chronotropic effect of 8-Br-cAMP suggests that the ionic mechanism underlying pacemaker potential is [Ca]_i-dependent. However, the lack of any effect of 8-Br-cGMP on spontaneous electrical activity of freshly isolated 3-day ECV indicates that cGMP does not modulate the basal Ca²⁺ channel activity in young embryonic myocardium.

Keywords: cAMP, cGMP, action potential, embryonic chick ventricle, spontaneous electrical activity, Ca²⁺ channel, chronotropic effect

Proton NMR transverse relaxation time and membrane stability in wheat leaves exposed to high temperature shock

Electrolyte leakage from leaves and NMR transverse relaxation time (T₂) of leaf water were used to differentiate between heat-tolerant (NIAW 845) and susceptible (HD 2428) wheat (Triticum aestivum L.) cultivars. The leaves were exposed to high temperature shock in the range 30 to 55°C and the damage caused, when evaluated by the two approaches was in close agreement. The critical temperature of injury leading to loss of membrane integrity was lower (39.1°C) for susceptible cultivar, compared to tolerant cultivar (44.2°C). Component analyses of NMR data revealed the existence of two fractions of cellular water in leaf tissues, namely, bound and free bulk water with distinct relaxation times. A dramatic reduction in the proportion of free water and a corresponding increase in bound water was observed in response to increase in temperature. This change in proportion occurred around 38°C and 43°C in HD 2428 and NIAW 845 respectively. The high temperature induced irreversible damage to cellular membrane integrity led to loss of compartmentation of cellular water fractions. The tolerant cultivar maintained its membrane integrity and cell water compartmentation until a temperature of 43°C and susceptible could maintain it only until 38°C.

Key words: NMR transverse relaxation time, wheat leaves, membrane stability, high temperature shock, electrolyte leakage, heat tolerant, heat susceptible

Age-related changes in the elemental constituents and molecular behaviour of bone

Age-related changes in bone composition within the age groups of 30, 60, 120 and 180 days in rats have been studied using thermogravimetery, elemental analysis and energy dispersive X-ray (ED X-ray). The structural changes in rats the bone samples were monitored by IR spectroscopy. The main constituents of hydroxyapatite, namely Ca, P and their oxides were analyzed. Organic changes, such as C and N contents in the matrix were found to have a predominant role in the initial development of the bone. An incremental increase in the mineral content of bone with advancing age was also observed. Elemental composition (C and N contents) was observed to be independent of age at the initial stages. The amount of Ca and its oxide content was found to increase, and the P and its oxides showed a decreasing trend, with the advancing age. IR spectra revealed that the mineral phase comprised both amorphous and crystalline hydroxyapatite, even at maturity; the amorphous content being higher at the earlier stages (14.09%, at 30 days), but was gradually replaced by crystalline component with advancing age (63.09% at 180 days). The present data may be useful in explaining the ageing phenomenon and helpful in understanding the bone growth and remodeling.

Keywords: bone, rat, energy dispersive X-ray, elemental analysis, hydroxyapatite, IR spectra, age-related changes

Aggarwal L M 127

Aggarwal S 113

Anand A 122

Chakraborty S 106

Chen J 100

Darbari R 127

Dubey R 73

Forouzandeh M 113

Gopal G 81

Hutchinson T E 92

Irshad M 73

Jethi R K 113

Joshi D K 122

Kiran R 113

Liu Q 100

Liu W 100

Liu X 100

Manivasagam T 87

Nagarajan S 122

Pathak P C 122

Pereira Ben M J 92

Prakash P 118

Rai D V 127

Rajkumar T 81

Rastogi A 92

Roy K 106

Sengupta C 106

Singla S K 113

Subramanian P 87

Suthakar G 87

Tandon C 113

Tripathi O 118

Verma A P S 122

Xu X 100

ISSN : 0301-1208		CODEN : IJBBBQ
VOLUME 42	NUMBER 2	APRIL 2005