Evaluation of rapid molecular diagnostics for differentiating medicinal Kaempferia species from its adulterants

Accurate detection of unique herbs is crucial for herbal medicine preparation. Zingiberaceae species, which are important in Ayurvedic medicine of India, are often misidentified in Northeast (NE) Indian herbal markets. Kaempferia galanga (Zingiberaceae) is one of the major components of popular Ayurvedic drugs used for rheumatic diseases (i.e.,“Gandha Thailam” and “Rasnairandadi Kashayam”), contusions, fractures, and sprains. In NE India, herbal healers often misidentify plants from the Marantaceae family (e.g., Calathea bachemiana and Maranta leuconeura) as Kaempferia, which leads to adulteration of the medicinal herb. This misidentification of herbs occurs in NE India because Zingiberaceae plant barcoding information is inadequate. As a consequence, herbal medicine is not only therapeutically less effective but may also cause adverse reactions that range from mild to life-threatening. In this study, we used eight barcoding loci to develop “fingerprints” for four Kaempferia species and two species frequently mistaken for Kaempferia. The PCR and sequencing success of the loci matK, rbcL and trnH-psbA were found to be 100%;the combination of matK, rbcL, and trnH-psbA proved to be the ideal locus for discriminating the Kaempferia species from their adulterants because the combined loci showed greater variability than individual loci. This reliable tool was therefore developed in the current study for accurate identification of Kaempferia plants which can effectively resolve identification issues for herbal healers.     

Control of brown dog tick,Rhipicephalus sanguineus using assembly pheromone encapsulated in natural polymer,chitosan

In the current study, an attempt was made to encapsulate assembly pheromone using natural polymer, chitosan. Chitosan beads were prepared by incorporating assembly pheromone in conjunction with an acaricide, namely, deltamethrin. In the in vitro bioassay, the test beads attracted and killed 79 % of unfed larvae, 88 % of unfed nymphs and 61 % of unfed adults of the brown dog tick, Rhipicephalus sanguineus, in 24 h of exposure. Field trials were carried out to attract and kill the pre-parasitic environmental stages. The beads were dispersed onto specially designed devices and they were placed in infested kennels. The devices were observed after 10 days.     

Effect of <Emphasis Type="Italic">Gymnema montanum</Emphasis> leaves on red blood cell resistance to oxidative stress in experimental diabetes

The aim of the present study was to evaluate the protective effect of Gymnema montanum on red blood cell (RBC) membrane in diabetic rats during lipid peroxidation. Ethanol extract of G. montanum leaves (GLEt) was administered orally to alloxan-induced diabetic rats for 3 weeks, and the effects on blood glucose, insulin, lipid peroxidation markers, thiobarbituric acid reactive substances, hydroperoxides in plasma and antioxidant enzymes including superoxide dismutase, catalase and glutathione peroxidase activities in erythrocytes were studied. Administration of GLEt to diabetic animals at doses of 50, 100, and 200 mg/kg body weight lowered elevated blood glucose levels by 24, 35, and 66%, respectively, relative to untreated diabetic rats. In comparison, treatment with the known antidiabetic drug, glibenclamide (600 μg/kg body weight) decreased blood glucose concentrations by 51%. Plasma insulin concentrations were increased in the diabetic rat by 73% with GLEt (200 mg/kg body weight) and 45% with glibenclamide (600 μg/kg body weight). Although a significant decrease in the lipid peroxidation markers was observed in plasma on treatment with GLEt and glibenclamide, the RBC antioxidant levels were increased significantly in diabetic rats. Furthermore, erythrocytes from the GLEt-treated animals were found to be more resistant to H₂O₂-induced peroxidation than that of untreated diabetic animals. The chemical characterization of the polyphenolics of the extract showed the presence of gallic acid (5.29% w/w), resveratrol (2.2% w/w), and quercetin (16.6% w/w). The results of this study suggest that G. montanum may be useful for the control, management, and prevention of oxidative stress associated with diabetes.     

C-Terminal truncation affects subunit exchange of human αA-crystallin with αB-crystallin

In human lenses, C-terminal cleavage of αA-crystallin at residues 172,168, and 162 have been reported. The effect of C-terminal truncation of αA-crystallin on subunit exchange and heterooligomer formation with αB-crystallin and homooligomer formation with native αA-crystallin is not known. We have conducted fluorescence resonance energy transfer studies which have shown that the rates of subunit exchange of αA_1–172 and αA_1–168 with αB-wt were two-fold lower than for αA-wt interacting with αB-wt. The subunit exchange rate between αA_1–162 and αB-wt was six-fold lower. These data suggest that cleavage of the C-terminal residues could significantly affect heterooligomerization. On the other hand, the subunit exchange rates between αA-wt and the truncated αA-crystallins were either unchanged or only slightly decreased, which suggest that homooligomerization may not be significantly influenced by C-terminal truncation. The main conclusion from this study is that cleavage of C-terminal residues of αA-crystallin including the nine residues of the flexible tail is expected to significantly affect the formation of heteroaggregates. Reconstitution experiments showed that the presence of an intact C-terminus is essential for the formation of fully integrated heteroaggregates with equal proportion of αA and αB subunits.     

Survivin inhibition induces human neural tumor cell death through caspase-independent and -dependent pathways

Survivin inhibits apoptosis during development and carcinogenesis and is absent in differentiated cells. To determine whether survivin inhibition induces cell death in neural tumor cells, survivin antisense oligonucleotides (SAO) were administered to a human neuroblastoma (MSN) and an oligodendroglioma (TC620) resulting in a dose-dependent reduction in survivin protein. Although 74% of the SAO-treated MSN cells were trypan blue(+), PARP cleavage or activated caspase-3 was not observed. However nuclear translocation of AIF occurred and XIAP increased dramatically. Co-administration of z-Val-Ala-Asp(OMe)-fluoromethyl ketone (zVAD-fmk) with SAO did not inhibit cell death suggesting a caspase-independent mechanism of cell death. Propidium iodide (PI) staining revealed multiple large macronuclei with no apoptotic bodies supporting a role for survivin in cell division. By contrast, while 70% of the SAO-treated TC620 cells were trypan blue(+), PARP was cleaved, cells were TUNEL(+) and PI-staining revealed macronuclei and numerous apoptotic bodies. Co-treatment of the TC620 cells with SAO and zVAD-fmk blocked cell death. While no macronuclei or apoptotic bodies were observed there was a two-fold increase in metaphase cells. Our results suggest that survivin inhibition decreases the viability of human neural tumor cells and as a result of mitotic catastrophe, cell death can be initiated by either a classic apoptotic mechanism or a caspase-independent mechanism.     

Micropropagation ofUraria picta, a medicinal plant, through axillary bud culture and callus regeneration

Summary Micropropagation ofUraria picta, a leguminous herb, was achieved through axillary bud culture and nodal callus culture. Bud break was best when nodes were cultured on Murashige and Skoog (1962) (MS) medium supplemented with 2.6 μM α-naphthalene acetic acid and 4.4 μM N⁶-benzyladenine. Optimum shoot multiplication was observed in adenine sulphate at 2.47 μM concentration. Competent callus was initiated around the nodal ring of the explant on the basal medium supplemented with cytokinins and auxin (α-naphthalene acetic acid and N⁶-benzyladenine), which regenerated into new profusely growing shoots on transferring to 0.13 μM N⁶-benzyladenine. Shoots elongated to 5 node length with 1.11 μM N⁶-benzyladenine were rooted on half-strength MS basal medium. The rooted plants were successfully established with 80% survival. About 400 such plants were transferred to the field.     

Effect of nicotine on glycosaminoglycan metabolism in rats.

Cigarette smoking has been established as a major risk factor for atherosclerosis and also for lung cancer. Nicotine is one of the major components of cigarette smoke which is believed to be partly responsible for the deleterious effect of cigarette smoke. There was significant alteration in the concentration of glycosaminoglycans (GAG) in rats exposed to cigarette smoke. Administration of nicotine to rats has been found to decrease many of GAG fractions in the aorta, liver and heart and increase in the lungs. The increase in GAG now observed in lung tissue in rats administered nicotine and those exposed to cigarette smoke may be involved in the increased incidence of lung cancer in smokers. Increased activity of many of GAG hydrolysing enzymes indicates increased degradation of GAG. Sulphate metabolism in the liver is also significantly altered by nicotine. Thus administration of nicotine to rats caused alteration in the metabolism of GAG which are similar to those observed on exposure of rats to cigarette smoke, indicating that nicotine content of the tobacco smoke may partly be responsible for the effect on GAG observed on exposure to cigarette smoke.     

On the mechanism of a mutated and abnormally functioning gamma-aminobutyric acid (A) receptor linked to epilepsy

A recent report indicates that a lysine-to-methionine mutation (K289M) in the gamma2 subunit of a human gamma-aminobutyric acid neurotransmitter receptor, the GABA(A) receptor, is linked to generalized epilepsy with febrile seizures [Baulac et al. (2001) Nat. Genet. 28, 46-48]. This mutation caused a decreased current response to GABA [Baulac et al. (2001) Nat. Genet. 28, 46-48]. Here we determine changes that occur in the mechanism of opening and closing of transmembrane channels formed by the GABA(A) receptor as a result of this mutation. The K289M mutation was introduced into the gamma2L subunit of the rat GABA(A) receptor, and the mutated subunit was coexpressed with the alpha1 and beta2 subunits in HEK293 cells. Transient kinetic techniques suitable for investigating reactions on cell surfaces with a microsecond-to-millisecond time resolution [Hess, G. P., and Grewer, C. (1998) Methods Enzymol. 291, 443-473] were used. They allow one to determine not only the channel-opening probability and rates of receptor desensitization but also the opening and closing rates of the mutated GABA(A) receptor channel. The channel-opening equilibrium constant of the mutated receptor was found to be 5-fold lower than that of the wild type. We calculated that this decrease in the channel-opening equilibrium accounts for the dysfunction of the mutated receptor. We discuss how a knowledge of the mechanism of the mutated receptor indicates an approach for alleviating this dysfunction.