A protocol for micropropagation of Alpinia galanga

Emerging buds of rhizome of Alpinia galanga Willd produced shoots and roots simultaneously when cultured in MS medium supplemented with kinetin 3.0 mg l^-1. Each explanted shoot bud produced 8 shoots in average and roots simultaneously within 8 weeks. Shoot proliferation could be continued even after a year by transferring each divided shoot explant to the same medium. Regenerated plantlets could be sucessfully transferred to soil where they grew well within 10–12 weeks with 80% survivality. This revised version was published online in June 2006 with corrections to the Cover Date.     

Discrete differences between strains of different Rhizobium spp. for competitive nodule occupancy on beans

Rhizobium etli strain TAL182 and R. leguminosarum bv phaseoli strain 8002, both of which produce melanin pigment, were tested for their nodulation competitiveness on beans by paired inoculation with two strains which do not produce melanin: R. tropici strain CIAT899 and Rhizobium sp. strain TAL1145. An assay was developed to distinguish nodules formed by the melanin-producing and non-producing strains. Strain TAL182 had discrete competitive superiority over CIAT899 and TAL1145 for nodulation of beans. Nodulation competitiveness was not correlated with the ability to produce melanin pigment or the host range of the Rhizobium strains tested.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gillmore 402, Honolulu, HI 96822, USA     

Germacrane and eudesmane derivatives from Calea reticulata

Calea reticulata afforded in addition to known compounds two new sesquiterpenes. These were germacrane and eudesmane derivatives, identified as germacra-4(15),5,10(14)-trien-1-one and 6-epi-β-verbesinol coumarate, respectively.     

Four guaianolides,a eudesmanolide and a germacranolide from Ursinia saxatilis

An investigation of Ursinia saxatilis afforded in addition to known compounds a new eudesmanolide derived from ursialpinolide, a germacranolide rel     

The cytoplasmic domain of neuropilin-1 regulates focal adhesion turnover

Though the vascular endothelial growth factor coreceptor neuropilin-1 (Nrp1) plays a critical role in vascular development, its precise function is not fully understood. We identified a group of novel binding partners of the cytoplasmic domain of Nrp1 that includes the focal adhesion regulator, Filamin A (FlnA). Endothelial cells (ECs) expressing a Nrp1 mutant devoid of the cytoplasmic domain (nrp1^cytoΔ/Δ) migrated significantly slower in response to VEGF relative to the cells expressing wild-type Nrp1 (nrp1^+/+ cells). The rate of FA turnover in VEGF-treated nrp1^cytoΔ/Δ ECs was an order of magnitude lower in comparison to nrp1^+/+ ECs, thus accounting for the slower migration rate of the nrp1^cytoΔ/Δ ECs.     

Habitat distribution modelling and reinforcement of Elaeocarpus serratus L. - A threatened tree species of Assam,India for improvement of its conservation status

Elaeocarpus serratus L., commonly known as ‘rudraksh’ referred in the Ayurveda as a wonderful plant for strengthening body constitutions, has been recognized as a threatened plant of Assam, India. Traditionally, rudraksh beads, its bark and leaves are used to cure various ailments like stress, anxiety, depression, nerve pain, epilepsy, migraine, lack of concentration, asthma, hypertension, arthritis and liver diseases. The population stock of the species has been depleting very fast in its natural habitat due to rapid habitat fragmentation and changing climate altering the structural and functional integrity of the plant. Hence, conservation of E. serratus L. with proper scientific investigation to prevent from extinction in its wild habitat is urgently needed. The present study was emphasized with the specific objectives to study the distribution and population status, predication of suitable sites through ENM, standardization of macropropagation methods and reinforcement/reintroduction into the suitable wild habitat to improve conservation status. In the present investigation E. serratus L. was reported in few locations of Assam and Arunachal Pradesh with population sizes of mean density, frequency of occurrence and abundance in relation to other associated species as 0.333, 13.922 and 2.215 respectively. For improving the conservation status, potential area and habitat for reinforcement was predicted using Maximum Entropy (MaxEnt) distribution modelling algorithm. Subsequently, macropropagation protocol was standardized through seed germination and air-layering; saplings were raised and 1050 saplings were reintroduced to the wild habitats selected on the basis of ecological niche modelling. Survival rate was found significantly high as 68%, suggesting that our approach is effective for changing population status and to conserve the plant.     

Detection of an intermediate during unfolding of bacterial cell division protein FtsZ: loss of functional properties precedes the global unfolding of FtsZ

Using environment-sensitive fluorescence of 1-anilinonaphthalene-8-sulfonic acid, polarization of fluorescein 5'-isothiocyanate-labeled FtsZ, and far-UV circular dichroism spectroscopy, the chemical unfolding of FtsZ was found to proceed through two steps. The first step of the urea-induced unfolding produced an intermediate, which then unfolded at higher concentrations of urea. The intermediate state contains native-like secondary structure and much less tertiary structure compared with the native state. It is distinct from the native state as well as from the unfolded state. Similar to urea-induced unfolding of FtsZ, thermal unfolding of FtsZ also occurs in two steps. The midpoints for the first and second thermal unfolding transitions were found to be 38 +/- 4 and 77 +/- 5 degrees C, respectively. Further, the functional properties of FtsZ are extremely sensitive to urea, guanidium chloride, and sodium dodecyl sulfate. For example, 50% inhibition of the FtsZ assembly and GTP hydrolysis occurred at 0.1 and 0.2 m of urea, respectively. FtsZ lost its functional properties before any significant perturbation in the secondary or tertiary structure was detected by using several fluorescence techniques and far UV-CD indicating preferential local unfolding of the functional region(s). In addition, the unfolded FtsZ regains its ability to polymerize fully upon removal of urea. The data taken together suggest that FtsZ unfolds reversibly through a multistep process, and local responses that inhibit functional properties precede the global transition of FtsZ to the unfolded state.     

Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2-terminal kinase-mediated apoptosis in response to noscapine

We have previously discovered the opium alkaloid noscapine as a microtubule interacting agent that binds to tubulin, alters the dynamics of microtubule assembly, and arrests mammalian cells at mitosis (Ye, K., Ke, Y., Keshava, N., Shanks, J., Kapp, J. A., Tekmal, R. R., Petros, J., and Joshi, H. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1601-1606; Ye, K., Zhou, J., Landen, J. W., Bradbury, E. M., and Joshi, H. C. (2001) J. Biol. Chem. 276, 46697-46700; Zhou, J., Panda, D., Landen, J. W., Wilson, L., and Joshi, H. C. (2002) J. Biol. Chem. 277, 17200-17208). Here we show that noscapine does not compete with paclitaxel for tubulin binding and can efficiently inhibit the proliferation of both paclitaxel-sensitive and paclitaxel-resistant human ovarian carcinoma cells (i.e. the parental cell line 1A9 and two derivative cell lines, 1A9PTX10 and 1A9PTX22, which harbor beta-tubulin mutations that impair paclitaxel-tubulin interaction (Giannakakou, P., Sackett, D. L., Kang, Y. K., Zhan, Z., Buters, J. T., Fojo, T., and Poruchynsky, M. S. (1997) J. Biol. Chem. 272, 17118-17125). Strikingly, these cells undergo apoptotic death upon noscapine treatment, accompanied by activation of the c-Jun NH(2)-terminal kinases (JNK). Furthermore, inhibition of JNK activity by treatment with antisense oligonucleotide or transfection with dominant-negative JNK blocks noscapine-induced apoptosis. These findings thus indicate a great potential for noscapine in the treatment of paclitaxel-resistant human cancers. In addition, our results suggest that the JNK pathway plays an essential role in microtubule inhibitor-induced apoptosis.     

Spatial relationship between the prodan site,Trp-214, and Cys-34 residues in human serum albumin and loss of structure through incremental unfolding

Prodan (6-propionyl-2-(dimethylamino)-naphthalene), a competitive inhibitor of warfarin binding to human serum albumin (HSA) at drug site I, was used to determine the inter- and intradomain distances of HSA. The fluorescence resonance energy transfer (FRET) distances between prodan and Trp-214, prodan and 7-(diethyl amino)-4-methylcoumarin 3-maleimide (CM)-modified Cys-34, and Trp-214 and CM-Cys-34 were determined to be 25.5 +/- 0.5 A, 33.1 +/- 0.8 A, and 32.4 +/- 1 A, respectively. FRET analysis showed that low concentration of palmitic acid (5 microM) increased the interdomain distance between the Trp-214 in domain II and CM-Cys-34 in domain I by approximately 5 A without perturbing the secondary structure of HSA and the immediate environment of Trp-214. Palmitic acid (5 microM) increased the prodan fluorescence by increasing the quantum yield of bound prodan without altering the tryptophan environment. However, palmitic acid (>10 microM) decreased the prodan fluorescence and increased the tryptophan fluorescence. Our results indicate that the high affinity palmitic acid binding site is located at the interface of domains I and II. On the basis of our measurements, a schematic model representing the drug site-1, Trp-214, and Cys-34 along with the palmitic acid sites has been constructed. In addition, prodan fluorescence, FRET, and ligand binding were used to monitor guanidine hydrochloride-induced denaturation of HSA. An analysis of the equilibrium unfolding data suggests that HSA undergoes a two-state unfolding transition with no detectable intermediate. However, kinetic analysis using multiple probes and thermal denaturation studies showed that the unfolding of the prodan site in HSA preceded the unfolding of tryptophan environment. In addition, the separation of domain I and II occurred before the global unfolding of the protein. The data support the idea that HSA loses its structure incrementally during its unfolding.     

Determination of the size and chemical nature of the stabilizing "cap" at microtubule ends using modulators of polymerization dynamics

The size and chemical nature of the stabilizing cap at microtubule (MT) ends has remained enigmatic, in large part because it has been difficult to detect and measure it directly. By pulsing steady-state suspensions of bovine brain microtubules (MTs) with trace quantities of [gamma(32)P]GTP and sedimenting the MTs through 50% sucrose cushions to reduce background contaminating (32)P to negligible levels, we were able to detect a small number of (32)P molecules that remain stably bound to the MTs (a mean of 25.5 molecules of (32)P per MT). Analysis of the chemical form of the stably bound (32)P by thin-layer chromatography revealed that it was all (32)P-orthophosphate ((32)P(i)). The (32)P(i) was determined to be located at the MT ends because colchicine and vinblastine, drugs that suppress tubulin incorporation into the MT by binding specifically at MT ends, reduced the quantity of the stably bound (32)P(i). Taxol, a drug that stabilizes MT dynamics by binding along the MT surface rather than at the ends, did not affect the stoichiometry of the bound (32)P(i). If the bound (32)P is equally distributed between the two ends, each end would contain 12-13 molecules of (32)P(i). Beryllium fluoride (BeF(3-)) and aluminum fluoride (AlF(4-)), inorganic phosphate analogues, suppressed the dynamic instability behavior of individual MTs and, thus, stabilized them. For example, BeF(3-) (70 microM) reduced the MT shortening rate by 2.5-fold and decreased the transition frequency from the growing or the attenuated state to rapid shortening by 2-fold. The data support the hypothesis that the stabilizing cap at MT ends consists of a single layer of tubulin GDP-P(i) subunits. The data also support the hypothesis that the mechanism giving rise to the destabilized GDP-tubulin core involves release of P(i) rather than hydrolysis of the GTP.