S. Anuntalabhochai

Ion beam induced Deoxyribose Nucleic Acid transfer

We report our observations of the interaction of energetic ions with bacterial cells, inducing direct deoxyribose nucleic acid (DNA) transfer into Escherichia coli (E. coli). Argon- and nitrogen-ion beams were used to bombard the bacteria E. coli in a vacuum with energy of 26 keV and fluence in the range 0.5–4×1015 ions/cm2. Three DNA plasmids, pGEM2, pGEM-T easy, and pGFP, carrying different marker genes, were subsequently transferred (separately) into the appropriately ion-bombarded bacteria and successfully expressed. The results of this study indicate that ion beams with an energy such that the ion range is approximately equal to the cell envelope thickness, at a certain range of fluence, are able to generate pathways for macromolecule transfer through the envelope without irreversible damage.

A study of low-energy ion beam effects on outer plant cell structure for exogenous macromolecule transferring

Abstract The aim of this study was to investigate low-energy ion beam effects on inducing exogenous macromolecule transfer through cell walls into cells and the related dependence. The experiment focused on 20–30 keV Ar ion implantation in various plant tissues to doses ranging from 1014 to 1016 ions/cm2. Auxiliary 15–30 keV N ion implantation in the plant tissues as well as 25 keV Ar ion implantation in bacteria of E. coli was also carried out. The effects of ion beam bombardment on the outer cell structure and the ability of transferring large exogenous molecules of Trypan blue (TB) and plasmid DNA were investigated. Typical results show that the 20 keV Ar ion implantation only leads to retaining of the TB dye in the cell wall whereas the 30 keV Ar ion implantation can allow the dye to enter the cell. A discussion based on simulations of the ion implantation processes indicates that the cell wall composed of cellulose microfibrils is in a porous structure so that ions at certain low energies with appropriate doses can increase permeability of the cell wall and induce exogenous macromolecule transferring.

Ion penetration depth in the plant cell wall

Abstract This study investigates the depth of ion penetration in plant cell wall material. Based on the biological structure of the plant cell wall, a physical model is proposed which assumes that the wall is composed of randomly orientated layers of cylindrical microfibrils made from cellulose molecules of C6H12O6. With this model, we have determined numerical factors for ion implantation in the plant cell wall to correct values calculated from conventional ion implantation programs. Using these correction factors, it is possible to apply common ion implantation programs to estimate the ion penetration depth in the cell for bioengineering purposes. These estimates are compared with measured data from experiments and good agreement is achieved.

Characteristics of heavy ion beam-bombarded bacteria E. coli and induced direct DNA transfer

Abstract The goal of the work described here was to study ion beam interactions with bacteria and thus develop an understanding of the mechanisms involved in ion bombardment-induced direct gene transfer into bacterial cells. Ar ion beams at an energy of 26 keV and fluences ranging from 5×10 14 to 4×10 15 ions/cm 2 were used to bombard bacterial cells of Escherichia coli strain DH5α. The bacteria were able to survive the low-temperature and low-pressure treatment conditions for at least a few hours. The ion bombardment created novel crater-like structures on the surface of the bacterial cell envelope, as observed by scanning electron microscopy. Four variously sized DNA plasmids carrying the ampicillin resistance gene were transferred and expressed in E. coli cells bombarded with ion fluences of 1×10 15 and 2×10 15 ions/cm 2 . The dependence of the DNA transfer on the plasmid DNA size, ion fluence and incubation time all suggests that the ion beam-induced surface crater-like structures provide the pathway for the mechanism that is responsible for the ion beam-induced DNA transfer.

Some investigations of ion bombardment effects on plant cell wall surfaces

Abstract Recent developments in the field of ion beam bioengineering, for example our own work demonstrating ion beam-induced transfer of exogenous macromolecules into the interior cell region, have underscored the need for a better understanding of the effects of ion bombardment on the cell wall material. We describe here, our investigations of ion beam sputtering of plant cell wall material and ion beam-induced damage to the cell wall. The presently available ion implantation simulation programs are not adequate, and experimental results are not available, either. We have indirectly estimated the surface sputtering yield of plant cell wall composed of C6H12O6-compound by remodeling the cell wall material so as to use partial mass densities and surface binding energies in the available ion implantation programs. For bombardment with a 30-keV Ar-ion beam, the sputtering yield from the cell wall is estimated to be approximately 10 atoms/ion, which is somewhat greater than the value predicted by direct program simulation, but in good agreement with experimental results. We have also performed electron microscopy on the ion-bombarded cell walls. The micrographs show novel microcrater-like structures on the cell wall subsequent to ion bombardment, which could be the ion beam-generated pathways for exogenous macromolecule transfer.

Expression of OsSPY and 14-3-3 genes involved in plant height variations of ion-beam-induced KDML 105 rice mutants.

The culm length of two semidwarf rice mutants (PKOS1, HyKOS1) obtained from low-energy N-ion beam bombardments of dehusked Thai jasmine rice (Oryza sativa L. cv. KDML 105) seeds showed 25.7% and 21.5% height reductions and one spindly rice mutant (TKOS4) showed 21.4% increase in comparison with that of the KDML 105 control. A cDNA-RAPD analysis identified differential gene expression in internode tissues of the rice mutants. Two genes identified from the cDNA-RAPD were OsSPY and 14-3-3, possibly associated with stem height variations of the semidwarf and spindly mutants, respectively. The OsSPY gene encoded the SPY protein which is considered to be a negative regulator of gibberellin (GA). On the other hand, the 14-3-3 encoded a signaling protein which can bind and prevent the RSG (repression of shoot growth) protein function as a transcriptional repressor of the kaurene oxidase (KO) gene in the GA biosynthetic pathway. Expression analysis of OsSPY, 14-3-3, RSG, KO, and SLR1 was confirmed in rice internode tissues during the reproductive stage of the plants by semi-quantitative RT-PCR technique. The expression analysis showed a clear increase of the levels of OsSPY transcripts in PKOS1 and HyKOS1 tissue samples compared to that of the KDML 105 and TKOS4 samples at the age of 50-60 days which were at the ages of internode elongation. The 14-3-3 expression had the highest increase in the TKOS4 samples compared to those in KDML 105, PKOS1 and HyKOS1 samples. The expression analysis of RSG and KO showed an increase in TKOS4 samples compared to that of the KDML 105 and that of the two semidwarf mutants. These results indicate that changes of OsSPY and 14-3-3 expression could affect internode elongation and cause the phenotypic changes of semidwarf and spindly rice mutants, respectively.

Combined quantum-mechanics/molecular-mechanics dynamics simulation of A-DNA double strands irradiated by ultra-low-energy carbon ions

Ion beams have been widely applied in a few biological research fields such as radioactive breeding, health protection, and tumor therapy. Up to now many interesting and impressive achievements in biology and agriculture have been made. Over the past several decades, scientists in biology, physics, and chemistry have pursued investigations focused on understanding the mechanisms of these radiobiological effects of ion beams. From the chemical point of view, these effects are due to the ion irradiation induced biomolecular damage, direct or indirect. In this review, we will present a chemical overview of the direct effects of ion irradiation upon DNA and its components, based on a review of literature combined with recent experimental results. It is suggested that, under ion bombardment, a DNA molecule undergoes a variety of processes, including radical formation, atomic displacement, intramolecular bond-scissions, emission of fragments, fragment recombination and molecular crosslink, which may lead to genetic mutations or cell death. This may help to understand the mechanisms of the radiobiological effects caused by ion irradiation, such as radiation breeding and tumor therapy.

Molecular simulations of ultra-low-energy nitrogen ion bombardment of A-DNA in vacuum

For investigating mechanisms involved in low-energy ion beam induced mutation, besides experiments using low-energy and low-fluence ions to bombard naked DNA, molecular simulations were carried out as an effort towards the insight in molecular interactions between ions and DNA. In the current study, Monte Carlo (MC) and molecular dynamics (MD) simulations were applied. The results of MC simulations provide some clues about the interaction energies and sites of preference of N-ion bombardment on an A-DNA short duplex strand. MD simulations of a single N-ion moving towards the same DNA strand with different linear velocities corresponding to bombardment energies of 0.1, 1, 10 and 100 eV revealed information about changes in bond lengths and visibly distorted structures of bombarded nucleotides. The simulations demonstrated that ion-bombardment-induced DNA change in structure was not a random but preferential effect.

Developmental and Phylogenetic Characteristics of Stellantchasmus falcatus (Trematoda: Heterophyidae) from Thailand

This study aimed to investigate the infection status, worm development, and phylogenetic characteristics of the intestinal trematode, Stellantchasmus falcatus. The metacercariae of S. falcatus were detected only in the half-beak (Dermogenus pusillus) out of the 4 fish species examined. Their prevalence was 90.0%, and the intensity of infection was 919 metacercariae on average. Worms were recovered from 33 (97.1%) of 34 chicks that were experimentally infected with 200 S. falcatus metacercariae each, and the average recovery rate was 43.0%. The body size and inner organs of S. falcatus quickly increased in the experimental chicks over days 1-2 post-infection (PI). In addition, ITS2 sequence data of this parasite were analyzed to examine the phylogenetic relationships with other trematodes using the UPGMA method. The results indicated that the ITS2 sequence data recorded from trematodes in the family Heterophyidae appeared to be monophyletic. This study concluded that D. pusillus serves as a compatible second intermediate host of S. falcatus in Thailand and that S. falcatus can develop rapidly in the experimental chicks. Data collected from this study can help to close the gap in knowledge regarding the epidemiology, biology, and phylogenetic characteristics of S. falcatus in Thailand.

Overexpression of OsRab7B3, a small GTP-binding protein gene, enhances leaf senescence in transgenic rice

Rab family proteins are small GTP-binding proteins involved in intracellular trafficking. They play critical roles in several plant development processes. Different expression patterns of 46 Rabs in the rice genome were examined in various rice tissues and in leaves treated with plant growth regulators and under senescence conditions. One of the OsRab genes, OsRab7B3, closely associated with senescence in expression pattern, was chosen for functional analysis. Expression of sGFP under the control of the OsRab7B3 promoter increased in leaves when ABA and NaCl were applied or when kept in dark. In transgenic rice overexpressing OsRab7B3, the senescence-related genes were upregulated and leaf senescence was significantly enhanced under dark conditions. Moreover, leaf yellowing occurred earlier in the transgenic plants than in the wild type at the ripening stage. Hence it is suggested that OsRab7B3 act as a stress–inducible gene that plays an important role in the leaf senescence process.