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Produced by the phytopathogenic fungus Alternaria tenuis, it induces chlorosis in many plants, including lettuce, potato, cucumber, and spinach, but has little or no effect on others such as radish, tobacco, and corn (4, 5). The chlorosis results from a selective disruption of chloroplast function in that the amounts of lipids (6) and proteins (7) specific to chloroplasts are reduced and the ultrastructural alterations found are confined to the chloroplast (8). The sharp demarcation between sensitive and insensitive species suggested that chloroplasts from sensitive species might possess a specific receptor site for tentoxin. Because preliminary studies have shown that tentoxin inhibits phosphorylating electron transport in lettuce chloroplasts (9) and chloroplast coupling factor 1 (CF1) is directly associated with photophosphorylation (10), we investigated it as a potential receptor molecule for tentoxin. This paper presents evidence that CF1 from lettuce, a tentoxin-sensitive species, has a single binding site for tentoxin and that when tentoxin occupies this site, both CF1 ATPase and phosphorylating electron transport are inactivated. In contrast, CF1 from radish, an insensitive species, has a lower affinity for tentoxin than does lettuce CF1; tentoxin does not inhibit radish ATPase and only slightly inhibits photophosphorylation.MATERIALS AND METHODS Chloroplasts were prepared from leaves of lettuce (cv. Romaine) and spinach purchased locally and from seedlings of radish (cv. Comet) and other species grown in controlled environment chambers. One hundred grams of selected leaves were blended for 10 sec at 40 in 250 ml of a 0.4 M sucrose 0.05 M N-[tris(hydroxymethyl)methyl]glycine (Tricine) buffer, pH 8.0, (ST). The homogenate was filtered through eight layers of cheesecloth, and the chloroplasts were collected by centrifugation (1000 X g for 10 min). After washing in 40 ml of ST and an additional centrifugation, the chloroplast pellet was suspended in 10 ml of ST and adjusted to 1 mg of chlorophyll per ml (11).Electron transport was measured with an oxygen electrode using methods modified from those of Arntzen (9). Chloroplasts (35 ,g of chlorophyll) in ST were added to 3 ml of stirred reaction mixture at 280 followed by the addition of tentoxin. Next, the electrode was inserted during a brief cessation of stirring. Two minutes after the addition of tentoxin, the reaction chamber was illuminated with a tungsten lamp (15 klm/m2). The course of oxygen uptake was followed during the subsequent 1 min. Basal rates were measured in the presence of 3 mM ADP; complete rates were determined in the added presence of 5 mM NaH2PO4. Only preparations having a complete rate more than twice the basal rate were used.Coupling factor 1 was prepared from chloroplasts by the methods of Lien and Racker (12). Lettuce CF1 was judged to be pure by gel electrophoresis (13, 14) after chromatography on DEAE-Sephadex A-50 using a linear (NH4)2SO4 gradient (Step 3 of ref. 12). Radish CF1 of comparable purity was obtained after initial co...

BackgroundDengue, a mosquito-borne viral disease, poses a significant global public health risk. In tropical countries such as India where periodic dengue outbreaks can be correlated to the high prevalence of the mosquito vector, circulation of all four dengue viruses (DENVs) and the high population density, a drug for dengue is being increasingly recognized as an unmet public health need.Methodology/Principal findingsUsing the knowledge of traditional Indian medicine, Ayurveda, we developed a systematic bioassay-guided screening approach to explore the indigenous herbal bio-resource to identify plants with pan-DENV inhibitory activity. Our results show that the alcoholic extract of Cissampelos pariera Linn (Cipa extract) was a potent inhibitor of all four DENVs in cell-based assays, assessed in terms of viral NS1 antigen secretion using ELISA, as well as viral replication, based on plaque assays. Virus yield reduction assays showed that Cipa extract could decrease viral titers by an order of magnitude. The extract conferred statistically significant protection against DENV infection using the AG129 mouse model. A preliminary evaluation of the clinical relevance of Cipa extract showed that it had no adverse effects on platelet counts and RBC viability. In addition to inherent antipyretic activity in Wistar rats, it possessed the ability to down-regulate the production of TNF-α, a cytokine implicated in severe dengue disease. Importantly, it showed no evidence of toxicity in Wistar rats, when administered at doses as high as 2g/Kg body weight for up to 1 week.Conclusions/SignificanceOur findings above, taken in the context of the human safety of Cipa, based on its use in Indian traditional medicine, warrant further work to explore Cipa as a source for the development of an inexpensive herbal formulation for dengue therapy. This may be of practical relevance to a dengue-endemic resource-poor country such as India.

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