Indian Journal of Experimental Biology

Review Articles
Opioid peptides, opioid receptors and mechanism of down regulation Kirti Chaturvedi	5

Role of lipid and lipoprotein metabolizing enzymes in the development of atherosclerosis Ramaswamy Subramanian, Manisha Ramaswamy & Kishor M Wasan	14

Papers
Image reconstruction of optical attenuation coefficient variation in biological tissues Susamma Chacko, D Kumar & Megha Singh	26

Role of apoptosis in photodynamic sensitivity of human tumor cell lines Seema Gupta, B S Dwarakanath, K. Muralidhar & Viney Jain	33

Effect of oophorectomy on expression of calcium sensing receptor mRNA in rat duodenal mucosa M Mehrotra, S K Gupta, S Tiwari, A Agarwal, K Kumar, P K Awasthi & M M Godbole	41

Involvement of GABA-A receptor chloride channel complex in isolation stress-induced free choice ethanol consumption in rats Pramod K Mediratta, Prabha Mahajan, Krishna K Sharma, Rishabh Bhandari & Shitla P Dubey	47

Differential organization of a LINE–1 family in Indian pygmy field mice B Chatterjee, M Bahadur, S Srivastava & T Sharma	53

Protective effect of Pongamia pinnata flowers against cisplatin and gentamicin induced nephrotoxicity in rats Annie Shirwaikar, S Malini, S Chandrika Kumari	58

Production of thiophenes from callus cultures of Tagetes patula L. and its mosquito larvicidal activity T Rajasekaran, G A Ravishanakar & B Obul Reddy	63

Establishment of embryonic cultures and somatic embryogenesis in callus culture of guggul-Commiphora wightii (Arnott.) Bhandari Sandeep Kumar, S S Suri, K C Sonie & K G Ramawat	69
Notes
Effect of phenol on ultra structure and plasmid DNA of Xanthomonas oryzae pv. oryzae N Mohan & A Mahadevan	78

Predation and searching efficiency of a ladybird beetle, Coccinella septempunctata Linnaeus in laboratory environment Omkar & Shefali Srivastava	82

Protective effect of Lawsonia alba Lam., against CCl₄ induced hepatic damage in albino rats Manoj Bhandarkar & Aqueel Khan	85

Effect of onion (Allium cepa Linn.) and garlic (Allium sativum Linn.) on plasma triglyceride content in Japanese quail (Coturnix coturnix japonicum) V Girish Kumar, K P Surendranathan, K G Umesh, D R Gayathri Devi & M R Sandhya Belwadi	88

Enhanced esterase activity in salivary gland and midgut of Aedes aegypti mosquito infected with dengue-2 virus D T Mourya, M S Rohankhedkar, P Yadav, V Dighe & D N Deobagkar	91

Alterations in polypeptides pattern in malaria vector Anopheles stephensi, fed upon immunized blood causing fecundity reduction Monika Gulia, S K Gakhar & T Adak	94

Announcements
XVIth Annual Conference of National Environmental Science Academy on Emerging Pollutants: Impact on Health, Agriculture and Environment; Indo-US Workshop on Molecular Biology and Biotechnological Applications of Mycorrhizal Fungi and National Congress on Molecular Symbiosis	2

Author Index

Announcements

XVIth Annual Conference of National Environmental Science Academy on Emerging Pollutants:

Impact on Health, Agriculture and Environment

Organised by the National Environmental Science Academy, in collaboration with the Indian Society of Remote Sensing, Kolkata Chapter, and Central Pollution Control Board, the conference will be held at the Lady Brabourne College, Kolkata and will cover the following topics: 1) Ecological imbalance and restoration, 2) Applications of remote sensing towards management of natural resources, 3) Land and people: some indications, 4) Management of chemical/biological/agricultural warefare, 5) Control measures for abatement, and 6) Ethical issues. For further information kindly contact either the Organising Secretary, XVIth NESA, Dr Amit Krishna De [35 Garpar Road, Kolkata 700 009. Telefax: 033-2402551 (O), 3503887 (R). E-mail: amitkde@satyam.net.in or amitkde_2000@yahoo.com] or the Convener, XVIth NESA, Dr Aditi Nag Chaudhuri [Department of Chemistry, Lady Brabourne College, Kolkata 700 017. Phone: 033-2448120 (O), 4625927 ®. E-mail: odditybio@yahoo.com]

Indo-US Workshop on Molecular Biology and Biotechnological Applications of Mycorrhizal Fungi

National Congress on Molecular Symbiosis

Organised by the School of Life Sciences, Jawaharlal Nehru University, New Delhi, the workshop and congress will include the topics on: (1) State-of- the Art of the subject; (2) Molecular Taxonomy of Symbiotic Fungi; (3) Signaling Mechanisms in Mycorrhizal Symbiosis; (4) Molecular Genetics of Mycorrhizal Fungi; (5) Biotechnological Applications of the Fungi including Medicinal Plants and Orchids; (6) Relevance and Impact of Genetic Engineering; and (7) Interface of Science, Industry, Management and Corporate Sectors, etc. For further details, please contact: Dr Ajit Varma, Professor of Microbial Technology, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India. Phone: 011-6704511 (Direct line); 011-6107676/6167557 ext.2511; E-mail: ajitvarma73@hotmail.com

Review Articles

Opioid peptides, opioid receptors and mechanism of down regulation

Biogenesis of various endogenous opioid peptides, anatomical distribution and the characteristics of multiple receptors with which they interact provides an opportunity for understanding the role of opioid systems and mechanism of opioid tolerance. Cellular and anatomical distribution of opioid receptor and their function is important for identification of neuronal systems and local network involved in initiation of drug action and subsequent development of adaptations resulting from repeated drug use. The details concerning discovery and progress in endogenous opioid peptide research and their distribution in brain have been described in this review. This review also describes opioid receptors, their distribution and mechanism of down regulation, which may be one of the causes for tolerance to opioids. Agonist induced down regulation and recent evidence for involvement of ubiquitin/proteasome system in this process has been discussed.

Role of lipid and lipoprotein metabolizing enzymes in the

development of atherosclerosis

Cardiovascular disease is the primary cause of mortality in developed and developing nations. With an increase in the aging population, there is a surge in the incidence of atheroscleortic cardiovascular diseases. One of the most common and lethal manifestations of atherosclerosis is coronary heart disease, accounting for 50% of the atherosclerotic cardiovascular diseases in men and women younger than 75 years. Peripheral arterial diseases, manifested mainly as intermittent claudication constitute approximately 10% of the atherosclerotic cardiovascular events. According to the American Heart Association 2001 Heart and Stroke Statistical Update, atherosclerosis accounts for 75% of all deaths due to cardiovascular diseases. Therefore, atherosclerosis continues to remain the primary cause of health concern for the population at large. The aim of this review is to discuss the role of enzymes that are involved in the metabolism of lipid and lipoproteins in the development of atherosclerosis.

Papers

Image reconstruction of optical attenuation coefficient variation in

biological tissues

A procedure for non-invasive imaging of the optical attenuation coefficient variation of in vivo thick organs/tissues is developed. The laser back-scattered surface profiles at various locations of human forearm, by multi-probe reflectometer, are measured. These profiles are matched by iterative procedure, with that as obtained by Monte Carlo simulation and the corresponding values of attenuation coefficient (equal to the sum of absorption and reduced scattering coefficients) are determined. By interpolation of this data a 100 x 100 grid is constructed and after median filtering of this data a color-coded image of the variability of the optical attenuation coefficient of the forearm is obtained. These images in different subjects show variation due to change in overall tissue composition and blood pooling. This non-invasive imaging procedure may help in identifying the diseased affected regions in healthy tissues and in application of photodynamic therapy.

Role of apoptosis in photodynamic sensitivity of human tumour cell lines

Photodynamic therapy (PDT) using a photosensitizer, such as haematoporphyrin derivative (HpD), in conjunction with visible light is a promising new modality to treat localized cancer. Cell death caused by PDT (through the generation of reactive oxygen species) can occur either by apoptosis (interphase death or as a secondary event following mitosis) and/or necrosis depending on the cell type, concentration and intracellular localization of the sensitizer, and the light dose. Since, apoptosis induced by PDT treatment plays an important role in determining the photodynamic efficacy, in the present work we have investigated the role of apoptotic cell death in relation to the observed differences in sensitivity to HpD-PDT between a human glioma cell line (BMG-1) carrying wild-type tumour suppressor gene p53 and a human squamous carcinoma cell line (4451) with mutated p53. HpD (photosan-3; PS-3) -PDT induced apoptosis was studied by: [A] flow-cytometric analysis of DNA content (sub G0/G1 population); [B] phosphatidylserine externalization (Annexin-V +ve cells); [C] cell size and cytoskeleton reorganization (light-scatter analysis); and [D] fluorescence microscopy (morphological features). PS-3-PDT induced a significantly higher level of apoptosis in BMG-1 cells as compared to 4451 cells. This was dependent on the concentration of PS-3 as well as post-irradiation time in both the cell lines. At 2.5 g/ml of PS-3 the fraction of BMG-1 cells undergoing apoptosis (60%) was nearly 6 folds higher than 4451 cells (10%). In BMG-1 cells the induction of apoptosis increased with PS-3 concentration up to 5 g/ml (>80%). However, a decrease was observed at a concentration of 10 g/ml, possibly due to a shift in the mode of cell death from apoptosis to necrosis. In 4451 cells, on the other hand, the increase in apoptosis could be observed even up to 10 g/ml of PS-3 (60%). Present results show that the higher sensitivity to PS-3-PDT in glioma cells arise on account of a higher level of apoptosis and suggest that induction of apoptosis is an important determinant of photodynamic sensitivity in certain cell types.

Effect of oophorectomy on expression of calcium sensing receptor

mRNA in rat duodenal mucosa

Calcium sensing receptor (CaR) in duodenal mucosa may be involved in active calcium absorption. Estrogen deficiency results in decreased intestinal calcium absorption. Effects of bilateral oophorectomy (OVX) have been studied on calcium homeostasis, bone mineral density (BMD) and CaR mRNA levels in duodenal mucosa at 4 weeks in adult female Sprague Dawley rats and compared with those in sham-operated and control group. There was no significant change in serum corrected calcium, inorganic phosphorous, calcidiol and intact parathyroid hormone in all the three groups. OVX rats had a significant decline in serum estrogen (E2) levels and alkaline phosphatase. They also had a significant decrease in BMD (DXA) at lumbar spine in vivo, and proximal and distal tibia in vitro while there was no significant change in serum E2 and BMD parameters in sham-operated and control rats. Northern blot analysis revealed no significant change in the CaR mRNA expression in duodenal mucosa in all three groups. The results suggests that CaR mRNA expression in duodenal mucosa is not affected by physiological circulating concentrations of estradiol in rats.

Involvement of GABA-A receptor chloride channel complex in isolation stress-induced free choice ethanol

consumption in rats

Pramod K Mediratta, Prabha Mahajan, Krishna K Sharma, Rishabh Bhandari & Shitla P Dubey

The present study revealed the effect of diazepam, a benzodiazepine, and progesterone, a pregnane precursor of neurosteroids, which act via modulating GABA-A chloride channel complex on the isolation stress-induced free choice ethanol consumption in adult rats. Isolation stress for 24 hr over a period of 6 days produced a significant increase in ethanol consumption, which persisted during the 6-day recovery period. Pretreating the animals with diazepam (5 mg/kg, ip), or progesterone (5 mg/kg, ip), blocked the isolation stress-induced increase in ethanol consumption. Bicuculline (2 mg/kg, ip), a GABA-A receptor antagonist significantly attenuated the effect of both diazepam and progesterone on stress-induced modulation of ethanol consumption. Isolation stress also caused an increase in total fluid consumption, which was antagonised by both diazepam and progesterone. Like ethanol consumption, this effect of diazepam and progesterone on isolation stress-induced increase in total fluid consumption was attenuated by bicuculline. Neither diazepam nor progesterone produced an increase in ethanol consumption in non-stressed rats. However, unlike diazepam, progesterone administration to non-stressed rats caused a significant increase in total fluid consumption. Results of the present study thus show that GABAergic mechanisms may be playing an important role in isolation stress-induced increase in ethanol consumption.

Differential organization of a LINE–1 family in

Indian pygmy field mice

Southern blot hybridization analysis of genomic DNAs digested with restriction endonuclease EcoR I and Ava II from Mus musculus domesticus, Mus booduga and Mus terricolor with a cloned repetitive DNA fragment of Mus booduga as a probe showed difference in restriction pattern of this DNA in these three species. Further Southern analysis of the BamH I digested genomic DNAs from these species hybridized with cloned DNA fragment as a probe and sequencing of the cloned DNA revealed that this 252 bp cloned DNA fragment is a part of BamHI repeat element of genus Mus and is 87 % homologous to the contiguous portion of the Mus musculus domesticus LINE–1 element. The species specific fragment pattern generated by different restriction endonucleases using this DNA as a probe revealed difference in the organization of LINE-1 repetitive element in the three species of genus Mus.

Protective effect of Pongamia pinnata flowers against cisplatin and gentamicin induced nephrotoxicity in rats

Ethanolic extract of flowers of Pongamia pinnata was studied for its protective effect against cisplatin and gentamicin induced renal injury in rats. When the extract (300 & 600 mg kg1) was administered orally for 10 days following cisplatin (5 mg kg1 ip) on day 5, toxicity of cisplatin, as measured by loss of body weight, elevated blood urea and serum creatinine declined significantly. Similarly in gentamicin (40 mg kg-1 s.c) induced renal injury, the extract (600 mg kg-1) normalized the raised blood urea and serum creatinine levels. Reversal of cisplatin and gentamicin renal cell damage as induced by tubular necrosis ie, marked congestion of the glomeruli with glomerular atrophy, degeneration of tubular epithelial cells with casts in the tubular lumen and infiltration of inflammatory cells in the interstitium was confirmed on histopathological examination. In the preventive regimen, co-administration of the extract with gentamicin significantly prevented the renal injury both functionally and histologically. Ethanolic extract of flowers had a marked nitric oxide free radical scavenging effect, suggesting an antioxidative property. Two flavonoids, known for their antioxidant activity viz. kaempferol and 3, 5, 6, 7, 8-pentamethoxy flavone were isolated from the extract. The results suggested that the flowers of Pongamia pinnata had a protective effect against cisplatin and gentamicin induced renal injury through antioxidant property.

Production of thiophenes from callus cultures of Tagetes patula L.

and its mosquito larvicidal activity

Calli were initiated from leaf tissues of T. patula, on Murashige and Skoog’s medium supplemented with 2-4 dichlorophenoxacetic acid (2,4-D) and kinetin (Kn). The maximum accumulation of biomass was recorded on 6th week at 1.11g. dry wt/culture. Maximum thiophene content was recorded on 4th week at 0.008 % on dry wt basis in callus cultures of T.patula subjected to nutrient stress. Nitrogen stress induced 3-fold increase in thiophene production level in six weeks (0.024 % on dry weight basis). The best hormonal supplementation required for thiophenes production was found to be 2,4-D (2.0 mg L1) and kinetin (2.0 mg L1). The thiophenes produced in callus cultures of T.patula showed larvicidal effect against mosquito larvae.

Establishment of embryonic cultures and somatic embryogenesis in callus culture of guggul-Commiphora wightii (Arnott.) Bhandari

Somatic embryogenesis in callus cultures of Commiphora wightii (Arnott.) Bhandari was achieved. Though the frequency of explants producing embryonic culture was low, immature zygotic embryos were the only suitable explants to produce embryonic callus after reciprocal transfers on media containing 2,4,5-trichlorophenoxy acetic acid (0.1 mg1-1) and kinetin (0.1mg1-1) or devoid of growth regulators. All other media failed to produce embryonic callus. Embryonic cells were small, densely filled with cytoplasm and isodiametric as compared to non-embryonic cells, which were large, elongated and vacuolated. Maximum growth of embryonic callus was recorded on modified MS medium (MS-2 medium) supplemented with BA (0.25 mg1-1) and IBA (0.1 mg1-1). MS-2 salts supported higher growth of callus as compared to tissues grown on B5 medium containing same concentrations of plant growth regulators. Exogenous medium nutrients had no effect on somatic embryo development whereas plant growth regulators had little effect. Asynchronously growing embryos formed plantlets regularly which were successfully transferred to the field conditions.

Notes

Effect of phenol on ultra structure and plasmid DNA of

Xanthomonas oryzae pv. oryzae

Most phenolic substances of plant origin are toxic to microorganisms and they confer some degree of protection to plants against phytopathogens. Xanthomonas oryzae pv. oryzae, bacterial blight pathogen of rice (Oryza sativa) was treated with phenol (monohydroxy benzene) and its effects on the morphology and cytological changes of the bacterium were studied. Total lysis of cells occurred with 5 mM conc of phenol while at 2 mM conc, the cell walls became rough and cell contents started shrinking. Plasmids isolated from both treated (2 mM) and control cells did not show any marked difference under electron microscope except that they differed in their quantity and might influence pathogenicity.

Predation and searching efficiency of a ladybird beetle,

Coccinella septempunctata Linnaeus in

laboratory environment

The predation and searching efficiency of fourth instar of predatory C. septempunctata at various densities of mustard aphid, Lipaphis erysimi (Kaltenbach) and predator was investigated under laboratory conditions. The feeding rate of predatory stage decreased at increased prey- and predator densities. Highest percent (92.80%) prey consumption was observed at initial prey density and lowest percent (40.86%) prey consumption at highest prey density by the fourth instar, though the total prey consumption increased with increase in either prey- or predator densities. Similarly, the individual prey consumption was also highest at initial predator density and lowest at highest predator density owing to the mutual interference between the predators at higher densities. The area of discovery (searching efficiency) also decreased with increase in prey- and predator densities. Handling time of predator was highest at lower prey densities, which decreased with increased prey densities. The highest percentage of prey consumption at the prey density of 50 revealed that 1:50 predator-prey ratio was the best to reduce the pest population.

Protective effect of Lawsonia alba Lam., against CCl4

induced hepatic damage in albino rats

The oral administration in varying doses of aqueous suspension of extract of L. alba, bark extract to rats for 10 days afforded good hepatoprotection against CCl4 induced elevation in serum marker enzymes, serum bilirubin, liver lipid peroxidation and reduction in total serum protein, liver glutathione, glutathione peroxidase, glutathione-s-transferase, glycogen, superoxide dismutase and catalase activity. The results suggest hepatoprotective and antioxidant activity of extract of L. alba bark.

Effect of onion (Allium cepa Linn.) and garlic (Allium sativum Linn.) on plasma triglyceride content in Japanese quail

(Coturnix coturnix japonicum)

V Girish Kumar, K P Surendranathan, K G Umesh, D R Gayathri Devi & M R Sandhya Belwadi

Dietary onion and garlic caused an increase in the level of plasma triglyceride which could be due to insulin like activity of dietary alliums and other factors that promote lipogenesisi in growing stages. Changes in the plasma triglyceride level in the control group due to change in age and sex were also noted. The triglyceride level was more in female birds when compared to males of similar age group. The plasma trigelyceride level increased with age in both sex except for the level being similar in the 6 and 9-week old females and 3 and 6-week old male birds. The results suggest that the effects of alliums in growing and adult stages may be different which needs further study.

Enhanced esterase activity in salivary gland and midgut of Aedes aegypti mosquito infected with dengue-2 virus

Mosquitoes were infected by intrathoracic inoculation. About 95% head squashes were positive for dengue virus antigen on the 15th post infection day (PID). Esterase activity was determined in the homogenates prepared from the salivary glands and midguts on different PIDs of dengue virus inoculated and control mosquitoes showed that it was consistently higher in the virus-infected batches.

Alterations in polypeptides pattern in malaria vector Anopheles stephensi, fed upon immunized blood

causing fecundity reduction

Changes in polypeptides pattern of haemolymph, midgut, ovary and salivary glands of female mosquito A.stephensi were studied when fed upon anti-mosquito haemolymph antibodies. The expression of almost all polypeptides was reduced in haemolymph and ovary of the immune fed mosquitoes as compared to control. However, there was no significant difference in case of midgut and salivary glands. Seven polypeptides 100, 90, 84, 80, 62, 19 and 12.5 kDa were absent in haemolymph and five 92, 90, 80, 60 and 55 kDa were absent in ovaries. Changes in the polypeptide pattern have been correlated with the fecundity reduction due to immunized blood feeding.

Adak T	94	Malini S	58
Agarwal A	41	Mediratta Pramod K.	47
Awasthi P K	41
		Mehrotra M	41
Bahadur M	53	Mohan N	78
Bhandari Rishabh	47	Mourya D T	91
Bhandarkar Manoj	85	Muralidhar K	33

Chacko Susamma	26	Obul Reddy B	63
Chatterjee B	53	Omkar	82
Chaturvedi Kirti	5
		Rajasekaran T	63
Deobagkar D N	91	Ramaswamy Manisha	14
Dighe V	91	Ramawat K G	69
Dubey Shitla P.	47	Ravishanakar G A	63
Dwarakanath B S	33	Rohan Khedkar M S	91

Gakhar S K	94	Sandhya Belwadi M R	88
Gayathri Devi D R	88	Sharma Krishna K	47
Godbole M M	41	Sharma T	53
Gulia Monika	94	Shirwaikar Annie	58
Gupta S K	41	Singh Megha	26
Gupta Seema	33	Sonie K C	69
		Srivastava S	53
Jain Viney	33	Srivastava Shefali	82
		Subramanian Ramaswamy	14
Khan Aqueel	85	Surendranathan K P	88
Kumar D	26	Suri S S	69
Kumar Girish V	88
Kumar K	41	Tiwari S	41
Kumar Sandeep	69
Kumari Chandrika S	58	Umesh K G	88

Mahadevan A	78	Wasan Kishor M	14
Mahajan Prabha	47
		Yadav P	91

ISSN : 0019-5189		CODEN : IJEB (A6) 41(1) 1-98 (2003)
VOLUME 41	NUMBER 1	JANUARY 2003