Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Base-sequence dependence of noncovalent complex formation and reactivity of benzo[a]pyrene diol epoxide with polynucleotides

The base-sequence selectivity of the noncovalent binding of (+/-)-trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyr ene (BPDE) to a series of synthetic polynucleotides in aqueous solutions (5 mM sodium cacodylate buffer, 20 mM NaCl, pH 7.0, 22 degrees C) was investigated. The magnitude of a red-shifted absorbance at 353 nm, attributed to intercalative complex formation, was utilized to determine values of the association constant Kic. Intercalation in the alternating pyridine-purine polymers poly(dA-dT).(dA-dT) (Kic = 20,000 M-1), poly(dG-dC).(dG-dC) (4200 M-1), and poly(dA-dC).(dG-dT) (9600 M-1) is distinctly favored over intercalation in their nonalternating counterparts poly(dA).(dT) (780 M-1), poly(dG).(dC) (1800 M-1), and poly(dA-dG).(dT-dC) (5400 M-1). Methylation at the 5-position of cytosine gives rise to a significant enhancement of intercalative binding, and Kic is 22,000 M-1 in poly(dG-m5dG).(dG-m5dC). In a number of these polynucleotides, values of Kic for pyrene qualitatively follow those exhibited by BPDE, suggesting that the pyrenyl residue in BPDE is a primary factor in determining the extent of intercalation. Both BPDE and pyrene exhibit a distinct preference for intercalating within dA-dT and dG-m5dC sequences. The catalysis of the chemical reactions of BPDE (hydrolysis to tetrols and covalent adduct formation) is enhanced significantly in the presence of each of the polynucleotides studied, particularly in the dG-containing polymers. A model in which catalysis is mediated by physical complex formation accounts well for the experimentally observed enhancement in reaction rates of BPDE in the alternating polynucleotides; however, in the nonalternating polymers a different or more complex catalysis mechanism may be operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potential of the neem tree (Azadirachta indica) for pest control and rural development

The neem tree (Azadirachta indica) contains promising pest-control substances found effective against many economically important pests. These materials are easy to process by village-level industries and easy to use by limited-resource farmers, thereby offering potential for crop protection and off-farm income generation. The tree’s numerous pharmacological and other complementary uses make it doubly attractive for incorporation in large-scale rural development efforts.     

Fluorescence and photoelectron studies of the intercalative binding of benz(a)anthracene metabolite models to DNA

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.     

Oxidative metabolism and protoplast culture

Summary Barley leaf blade protoplasts accumulate malonaldehyde, a product of lipid peroxidation, during culture. In addition, glutathione levels fall after protoplast isolation and the proportion of glutathione in the oxidized state rises. These data indicate oxidative stress after protoplast isolation and during culture. The cause of this phenomenon is revealed by data showing that the activities of enzymes associated with antioxidative processes including glutathione reductase and ascorbate peroxidase decrease after barley protoplast isolation. In contrast, protoplasts isolated from suspension cultured cells of bromegrass and soybean exhibit little evidence for oxidative stress and increased activities of glutathione reductase and ascorbate peroxidase. We suggest that an antioxidative response is associated with mitosis and colony formation from protoplasts, as exhibited by bromegrass and soybean. Conversely, failure of an antioxidative response is associated with low viability and absence of mitosis, as in barley. Increased viability of barley leaf protoplasts cultured on feeder layer cells is correlated with increased glutathione content and higher glutathione reductase activity.     

Ter-Seq: A high-throughput method to stabilize transient ternary complexes and measure associated kinetics

Regulation of biological processes by proteins often involves the formation of transient, multimeric complexes whose characterization is mechanistically important but challenging. The bacterial toxin CcdB binds and poisons DNA Gyrase. The corresponding antitoxin CcdA extracts CcdB from its complex with Gyrase through the formation of a transient ternary complex, thus rejuvenating Gyrase. We describe a high throughput methodology called Ter-Seq to stabilize probable ternary complexes and measure associated kinetics using the CcdA-CcdB-GyrA14 ternary complex as a model system. The method involves screening a yeast surface display (YSD) saturation mutagenesis library of one partner (CcdB) for mutants that show enhanced ternary complex formation. We also isolated CcdB mutants that were either resistant or sensitive to rejuvenation, and used surface plasmon resonance (SPR) with purified proteins to validate the kinetics measured using the surface display. Positions, where CcdB mutations lead to slower rejuvenation rates, are largely involved in CcdA-binding, though there were several notable exceptions suggesting allostery. Mutations at these positions reduce the affinity towards CcdA, thereby slowing down the rejuvenation process. Mutations at GyrA14-interacting positions significantly enhanced rejuvenation rates, either due to reduced affinity or complete loss of CcdB binding to GyrA14. We examined the effect of different parameters (CcdA affinity, GyrA14 affinity, surface accessibilities, evolutionary conservation) on the rate of rejuvenation. Finally, we further validated the Ter-Seq results by monitoring the kinetics of ternary complex formation for individual CcdB mutants in solution by fluorescence resonance energy transfer (FRET) studies.     

Sequence requirements for the termination of rolling-circle replication of plasmid pT181

Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.     

Detergent dissection of membrane proteins of Entamoeba histolytica and its effect on lymphokine release in in vitro.

Fifty-two amoebic liver abscess cases were assessed for the release of lymphokines (LMIF) using detergent dissected membrane proteins (DDMP) of axenic Entamoeba histolytica (NIH:200) obtained with sodium deoxycholate treatment. Lymphokines release by T lymphocytes in response to both DDMP and whole amoebic lysate (WAL) was tested by leukocyte migration inhibition test on blood samples from amoebic liver abscess cases. A significant increase was noted in the release of LMIF and 100% positivity was observed with DDMP compared to whole amoebic extract with a positivity of 73%. The difference between means of the above two with regards to release of LMIF was found to be highly significant (P less than 0.005). This shows the patients had high degree of leukocyte sensitization to surface antigens of E. histolytica compared to the whole amoebic lysate. These findings suggest that the antigens shed might have important role as a potent antigen in elicitation of CMI response in amoebic liver abscess cases.