Raghbir Chand Gupta

Ribosome-inactivating proteins: current status and biomedical applications

Ribosome-inactivating proteins (RIPs) are mainly present in plants and function to inhibit protein synthesis through the removal of adenine residues from eukaryotic ribosomal RNA (rRNA). They are broadly classified into two groups: type I and type II. Type I RIPs are a diverse family of proteins comprising a single polypeptide chain, whereas type II RIPs are heterodimeric glycoproteins comprising an A-chain (functionally equivalent to a type I RIP) linked via a disulphide bond to a B chain, mediating cell entry. In this review, we describe common type I and type II RIPs, their diverse biological functions, mechanism of cell entry, stability in plasma and antigenicity. We end with a discussion of promising applications for RIPs in biomedicine.

Ribosome inactivating proteins from plants inhibiting viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

Cytology of five species of subfamily Papaveroideae from the Western Himalayas

During the present course, population-based meiotic studies were carried out on five species of subfamily Papaveroideae from selected localities of Kashmir and Himachal Pradesh in the Western Himalayas (India). Varied intraspecific chromosome counts were reported for the first time in Argemone mexicana and Meconopsis latifolia, both existing on 2n = 2x = 14. The x = 7, confirmed for the first time from the newly found diploid cytotype, is suggested to be the primary chromosomal basic number for the Meconopsis. Furthermore, meiotic course was noted to be normal in Argemone ochroleuca, it varied from normal to abnormal in the populations of A. mexicana and Papaver dubium whereas it was invariably found to be abnormal in all the populations of Meconopsis aculeata and M. latifolia. These anomalous taxa were marked with meiotic abnormalities in the form of cytomixis, chromosomal stickiness, unoriented bivalents, formation of laggards and bridges resulting in abnormal microsporogenesis, and production of heterogeneous-sized fertile pollen grains along with reduced pollen fertility.

Meiotic studies in some selected angiosperms from the Kashmir Himalayas

Abstract  As a part of our program to explore and evaluate genetic diversity of flowering plants of the Kashmir Himalayas, meiotic studies have been carried out on 150 wild species. Of these, Caltha alba (2n= 32), Delphinium roylei (2n= 16), D. uncinatum (2n= 16), Ranunculus palmatifidus (2n= 28), and Sedum heterodontum (2n= 14) have been cytologically worked out for the first time. New intraspecific diploid or polyploid cytotypes have been recorded for Alchemilla vulgaris (2n= 34, 96), Arabis amplexicaulis (2n= 16), Impatiens amphorata (2n= 14), I. racemosa (2n= 12), I. sulcata (2n= 16, 12), Meconopsis latifolia (2n= 14), Potentilla supina (2n= 14), Saxifraga cernua (2n= 16), Sium latijugam (2n= 24), and Vicatia coniifolia (2n= 44). Four species, Arabidopsis thaliana (2n= 10), Berberis vulgaris (2n= 28), Potentilla nubicola (2n= 14), and P. sericea (2n= 28), have been cytologically reported for the first time from India. A large number of meiotic abnormalities have been observed in most of these species, leading to a reduction in pollen fertility and production of heterogeneous‐sized pollen grains.

Structural heterozygosity and cytomixis driven pollen sterility in Anemone rivularis Buch.-Ham. ex DC. from Western Himalaya (India)

Meiotic studies have been carried out on 12 accessions of Anemone rivularis from different regions of Western Himalaya. The accessions from Chamba, Kullu and Kinnaur districts of Himachal Pradesh and Uttarkashi district of Uttarakhand showed a normal meiotic course leading to high pollen fertility (87–100%). However, the accession scored from Saat Tal, Uttarakhand showed structural heterozygosity for reciprocal translocations. This is the first report of occurrence of structural heterozygosity in the species. Out of the 604 analysed pollen mother cells (PMCs), 38.41% (232) showed quadrivalent formation at diakinesis and metaphase-I. Comparison of chiasma frequency was also performed among selected populations using one-way analysis of variance (ANOVA), which showed significant differences. The occurrence of cytomixis involving inter PMC transfer of chromatin material among neighbouring meiocytes (45.68%) was also observed. PMCs at anaphases/telophases showed laggards (31.25%) and chromatin bridges at A-I/T-I and A-II/T-II (21.02%) leading to abnormal microsporogenesis. Consequently, abnormal sporads such as triads (66.67%), polyads (3.33%) and triads with micronuclei (0.83%) were formed. Structural heterozygosity and inter PMC chromatin material transfer in the individuals of the species seem to be responsible for the reduction in pollen fertility (67.8%) and formation of pollen grains of heterogeneous sizes.

Cytomorphological studies and HPTLC fingerprinting in different plant parts of three wild morphotypes of Datura metel L. "Thorn Apple" from North India.

Datura metel L. (Solanaceae) with its trade name “Thorn Apple” is a herb that exists in tropical, warm temperate regions of the old world, throughout the hills of India, up to an altitude of 8000 ft. The objective of the present study is to investigate the morphological and phytochemical variabilities in the wild taxa of D. metel. On the basis of 17 phenotypic traits three morphotypes I, II and III were identified, and highly significant variations were seen among them. The three morphotypes of the species, with quite distinct flower colours, that is, purple, yellow and white, had the same diploid chromosome number n=12. The pharmacological activities of D. metel are mainly attributed to the presence of two tropane alkaloids, namely hyoscyamine and scopolamine. For the systematic quantification of hyoscyamine and scopolamine in different plant parts such as, leaves, roots and seeds of these three wild morphotypes (I, II and III) of D. metel, the high performance thin layer chromatography (HPTLC) technique was employed. Better resolution was achieved by using chloroform:acetone:diethyl amine (50: 40: 10 v/v/v) as a mobile phase. Quantitative, densitometric evaluation of the plate was performed in the absorbance/reflectance mode at 530 nm. The average recovery of hyoscyamine and scopolamine was 97.4 and 96.8%, respectively, showing the excellent reproducibility of the method. The calibration curves were linear in the range of 1000–4000 ng for hyoscyamine and 500-2000 ng for scopolamine, respectively. The method was simple, precise, specific, sensitive, accurate and could be used for routine analysis as well as quality control of raw materials and herbal formulations. The present study has established a link between cytomorphological variations and chemical characterization for the first time and was also helpful in discovering the best genotype with richer active constituents for future herbal formulations. Key words: Cytotypes, datura metel L, morphotypes, high performance thin layer chromatography (HPTLC), hyoscyamine, scopolamine

Cytomorphological studies of genus Saxifraga L. from Western Himalaya

Cytomorphological studies have been carried out for analysing the cyto-genetic diversity in 14 populations covering 5 species belonging to the genus Saxifraga L. (fam. Saxifragaceae) from Western Himalaya. New intraspecific cytotypes in Saxifraga diversifolia Wall. ex Ser. (n = 8 and n = 16), S. filicaulis Wall. ex Ser. (n = 8) and S. siberica Linn. (n = 16) are added on world-wide basis. Cytotypes of S. flagellaris Willd. (n = 8), S. ligulata Wall. (n = 17) and S. siberica Linn. (n = 8) although worked out earlier from other countries are being reported for the first time from India. Further, comparison of the different cytotypes studied at present for Saxifraga diversifolia Wall. ex Ser and S. siberica Linn. show significant variation in their morphology, and also increased size for morphphological features in the polyploids. The course of meiosis has been observed to be abnormal in all the populations of S. filicaulis Wall. ex Ser., S. flagellaris Willd. and S. siberica Linn., but in S. diversifolia Wall. ex Ser. it exhibited patterns of normal to abnormal behaviour in different populations.

Cytomictic anomalous male meiosis and 2n pollen grain formation in Mertensia echioides Benth. (Boraginaceae) from Kashmir Himalaya.

Presently Mertensia echioides Benth. (Boraginaceae) collected from Kashmir Himalaya, India, is cytologically analyzed for the first time revealing 2n = 2x = 24 (diploid). Interestingly we found 4.3–6.2% syncytic meiocytes/PMCs with 2n = 4x = 48 (tetraploid) in addition to normal meiocytes (2n = 24) during male meiosis. These comparatively larger PMCs (pollen mother cells) lead to the formation of fertile giant 2n pollen grains. A frequency of 6.4–13.3% PMCs shows transfer of chromatin material at prophase-I and, therefore, results in aneuploid meiocytes. Whole chromatin transfer by the process of cytomixis could also have led to the formation of tetraploid cells. Translocation heterozygosity is also evident in the form of multivalents in 12–17% diploid (2x) meiocytes at diakinesis and metaphase-I and is reported for the first time in this species. The syncytes formed depict open chain hexavalent and quadrivalent formation in the three populations with different frequencies. Moreover chromatin stickiness at metaphase-I is observed in 45% of PMCs in population-1 (P-1). Syncyte or unreduced PMC formation leading to unreduced fertile gametes is here speculated to act as a possible way out for infraspecific polyploidization in the species.

An overview of chromosome and basic numbers diversity in cytologically investigated polypetalous genera from the Western Himalayas (India)

Intensive exploration and evaluation of cytomorphological diversity has been carried out on 380 species of 127 genera belonging to 28 families of sub-class Polypetalae of flowering plants from Kashmir (Jammu and Kashmir) and Kangra and Sirmaur districts (Himachal Pradesh) of the Western Himalayas. The cytological investigations of these species over a period of three years revealed new and varied chromosome numbers for 100 species globally and 50 species in India, making a substantial addition to the knowledge of the genera to which these species belong. To obtain a comprehensive cytological picture of each of these genera, chromosomal data have been updated by compiling the literature on previous chromosomal numbers and supplementing it from the present studies. The final form is now ready to show the status both at global and Indian levels for various parameters like total number of taxonomically known species, number of cytologically determined species along with intraspecifically added number of cytological taxa, presently inferred basic numbers, level and frequency of polyploids, and information on number of species per genus carrying inter- and intraspecific euploid and aneuploid variability at the genus level. Of the total 127 genera, 39 genera have 75% or more cytologically worked out species. Addition of cytotypes in many cytologically known species has resulted in an enhanced number of chromosomal races/cytological taxa over such species, in the majority of genera, justifying the ever growing need to make population based intensive studies of any plant species. The data show that monobasic and dibasic genera are less common than tribasic and polybasic ones. Genera with x = 8 are most common, followed by x = 7 and x = 6. Of 127 genera, 47 genera exhibit polyploidy of up to 25%; 22 genera have 26–50%; 15 genera have 51–70%; and 26 genera have 76–100% polyploidy, while 17 genera lack polyploidy altogether. Interspecific and/or intraspecific euploid cytotypes such as diploids plus polyploids or with “polyploid series” are present in most of the 107 genera. Intraspecific aneuploid chromosome numbers are also shown by 100 genera. Since these genera belong to different families, so no generalization can be made at family level. However, at genus level chromosomal observations show the active role of various evolutionary processes responsible for chromosomal diversity in the majority of these genera distributed in the Western Himalayas of India.

Anticonvulsant activity and acute neurotoxic profile of Achyranthes aspera Linn.

ETHNOPHARMACOLOGICAL RELEVANCE Root powder of Achyranthes aspera Linn. (A. aspera) belongs to family Amaranthaceae is used in Indian traditional medicine for the management of epilepsy and its efficacy is widely acclaimed among the different rural communities. AIM OF THE STUDY The present study was aimed to establish the possible anticonvulsant effect of A. aspera methanolic root extract using acute anticonvulsant models and to evaluate the acute toxicity and neurotoxic potential A. aspera extract. MATERIAL AND METHODS A. aspera methanolic extract was standardized with respect to betaine using HPTLC. The maximal electroshock (MES), pentylenetetrazol (PTZ), picrotoxin and bicuculline induced seizure models were used to evaluate the anticonvulsant potential of standardized A. aspera root extract. The GABA content in cortex and hippocampus of extract treated mice was evaluated using HPLC. Moreover, the animals were also evaluated for acute toxicity study and neurotoxicity test. RESULTS A significant enhancement in the seizure threshold was observed by A. aspera extract (5 and 10mg/kg) treated mice in PTZ, picrotoxin and bicuculline models as compared to saline treated mice respectively, whereas the extract failed to show protection in MES induced seizures. Moreover, A. aspera treatment (5 and 10mg/kg) significantly enhances the GABA levels in hippocampus and cortex as compared to saline treated group. A. aspera root extract was devoid of any sign of acute toxicity as well as neurotoxicity. CONCLUSIONS A. aspera root extract exhibits significant anticonvulsant effect by facilitation of GABAergic neurotransmission in the brain.