Plant regeneration from alginate-encapsulated shoot tips of Spilanthes acmella (L.) Murr., a medicinally important and herbal pesticidal plant species

This article demonstrates the plantlet regeneration from alginate-encapsulated shoot tips of Spilanthes acmella. Shoot tip explants excised from in vitro proliferated shoots were encapsulated in calcium alginate beads. The best gel complexation for encapsulation of shoot tips was achieved using 3% sodium alginate and 100 mM calcium chloride. Maximum percent response for the conversion of encapsulated shoot tips into plantlets was obtained on growth regulator-free full-strength liquid MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium. The addition of MS nutrients in alginate matrix was found to have pronounced effect on shoot and root emergence from alginate beads. Encapsulated shoot tips could be stored at low temperature (4°C) up to 60 days. Plantlets regenerated from encapsulated shoot tips were acclimatized successfully. The present synthetic seed technology could be useful in large-scale propagation as well as short-term conservation and germplasm distribution and exchange of Spilanthes acmella. S. K. Singh and M. K. Rai contributed equally to this work.     

Utilization of an amphipathic leucine zipper sequence to design antibacterial peptides with simultaneous modulation of toxic activity against human red blood cells

The toxicity of naturally occurring or designed antimicrobial peptides is a major barrier for converting them into drugs. To synthesize antimicrobial peptides with reduced toxicity, several amphipathic peptides were designed based on the leucine zipper sequence. The first one was a leucine zipper peptide (LZP); in others, leucine residues at the a- and/or d-position were substituted with single or double alanine residues. The results showed that LZP and its analogs exhibited appreciable and similar antibacterial activity against the tested gram-positive and gram-negative bacteria. However, the substitution of alanine progressively lowered the toxicity of LZP against human red blood cells (hRBCs). The substitution of leucine with alanine impaired the binding and localization of LZP to hRBCs, but had little effect on the peptide-induced damage of Escherichia coli cells. Although LZP and its analogs exhibited similar permeability, secondary structures, and localization in negatively charged membranes, significant differences were observed among these peptides in zwitterionic membranes. The results suggest a novel approach for designing antibacterial peptides with modulation of toxicity against hRBCs by employing the leucine zipper sequence. Also, to the best of our knowledge, the results demonstrate that this sequence could be utilized to design novel cell-selective molecules for the first time.     

Desiccation induced changes in osmolytes production and the antioxidative defence in the cyanobacterium Anabaena sp. PCC 7120

Cells of Anabaena sp. PCC 7120, a low desiccation tolerant cyanobacterium, was subjected to prolonged desiccation and effect of loss of water was examined on production of osmolytes, and antioxidant response as well as on overall viability in terms of photosynthetic activity. During dehydration (22 h), the organism maintained about 98.5 % loss of cellular water, yet cells remained viable as about 30 % of photosynthetic O₂-evolution activity resumed upon hydrating (1 h) such cells. In desiccated state, cyanobacterial cells accumulated osmolytes within 1 h though their contents decreased thereafter. The highest levels of trehalose (179 nmol mg⁻¹ protein), sucrose (805 nmol mg⁻¹ protein) and proline (23.2 nmol mg⁻¹ protein) were attained within 1 h. Chlorophyll a and carotenoid contents also increased within 1 h but phycocyanin level showed opposite trend. The oxygen-evolving activity declined in desiccated cyanobacterial biomass while rehydration led to instant recovery, indicating that cells protect the photosynthetic machinery against desiccation. Notwithstanding, activities of antioxidant enzymes (catalase, peroxidase and superoxide dismutase) attained their peaks after 3 h of desiccation, though within 10 min of rehydration, their levels returned back close to basal activities of the cultured cells. We propose that onset of osmolyte production in conjunction with upshift of antioxidant enzymes apparently protects the cyanobacterial cells from desiccation stress.     

Inhibition of HDAC6 Deacetylase Activity Increases Its Binding with Microtubules and Suppresses Microtubule Dynamic Instability in MCF-7 Cells

The post-translational modification of tubulin appears to be a highly controlled mechanism that regulates microtubule functioning. Acetylation of the ϵ-amino group of Lys-40 of α-tubulin marks stable microtubules, although the causal relationship between tubulin acetylation and microtubule stability has remained poorly understood. HDAC6, the tubulin deacetylase, plays a key role in maintaining typical distribution of acetylated microtubules in cells. Here, by using tubastatin A, an HDAC6-specific inhibitor, and siRNA-mediated depletion of HDAC6, we have explored whether tubulin acetylation has a role in regulating microtubule stability. We found that whereas both pharmacological inhibition of HDAC6 as well as its depletion enhance microtubule acetylation, only pharmacological inhibition of HDAC6 activity leads to an increase in microtubule stability against cold and nocodazole-induced depolymerizing conditions. Tubastatin A treatment suppressed the dynamics of individual microtubules in MCF-7 cells and delayed the reassembly of depolymerized microtubules. Interestingly, both the localization of HDAC6 on microtubules and the amount of HDAC6 associated with polymeric fraction of tubulin were found to increase in the tubastatin A-treated cells compared with the control cells, suggesting that the pharmacological inhibition of HDAC6 enhances the binding of HDAC6 to microtubules. The evidence presented in this study indicated that the increased binding of HDAC6, rather than the acetylation per se, causes microtubule stability. The results are in support of a hypothesis that in addition to its deacetylase function, HDAC6 might function as a MAP that regulates microtubule dynamics under certain conditions.     

Nickel Uptake by Pseudomonas aeruginosa: Role of Modifying Factors

Pseudomonas aeruginosa cells growing in minimal medium were 40-fold more sensitive to Ni²⁺ than cells growing in enriched medium, suggesting a possible protective role of medium ingredients. Likewise, cells pre-grown in enriched medium showed a high K _m (6.15 mM) and increased Ni²⁺ uptake (950 nmol mg⁻¹ protein, 1h) over cells pre-sown in minimal medium (K _m , 0.48 mM; 146 nmol mg⁻¹ protein, 1 h). The overall pattern indicates that cells pre-grown in enriched medium were characterized by having lowered affinity towards Ni²⁺ than those with minimal medium background. The enhanced Ni²⁺ uptake by enriched medium-grown cells can be correlated with the improved metabolic state of the cells. Ni²⁺ uptake was optimum at neutrality (pH 7.0). A major Ni²⁺ transport system was competitively inhibited by Mg²⁺, Zn²⁺, Cd²⁺, or Co²⁺ (400 μM each). Noticeably, a minor Ni²⁺ transport pathway was still operative even in the higher concentration range of Mg²⁺ (4 mM and 40 mM). The stimulation of Ni²⁺ uptake monitored in the presence of different carbon sources (0.5% wt/vol, each) showed the sequence: glucose (1.6-fold) > phenol = gallic acid (1.5-fold). Succinate, in comparison, reduced Ni²⁺ uptake (0.5-fold) possibly because of its acting as a metal chelator as well. Sensitivity of Ni²⁺ transport towards methyl viologen, azide, 2-4 DNP, and DCCD suggested that transport was energy-linked. Received: 13 January 1998 / Accepted: 21 May 1998