Microwave-assisted technology for the clearing and staining of arbuscular mycorrhizal fungi in roots

The use of microwave irradiation as a source of energy to clear and stain intra-radical arbuscular mycorrhizal fungi propagules has been tested on a variety of indigenous and cultivated herbaceous plants. The aim of the study was to evaluate the efficiency of microwave irradiation on root softening, fungi tissue staining, and preservation of DNA integrity for subsequent molecular analyses. The proposed methodology has been adapted from the standard procedures used to detect and quantify mycorrhizal root colonization levels. Using a domestic microwave oven, tissue clearing and staining required together between 30 s and 1.5 min of microwave treatment to be completed, depending the diameter size of the roots. The well-performing chemical stains tested were acid fuchsin, trypan blue, and aniline blue. The acid fuchsin clearing and staining processes, as performed, were also demonstrated to preserve DNA integrity for further molecular analyses. Irradiation by microwaves has been used with success in our laboratory within the frame of several studies. It offers considerable time saving over traditional method, reducing processing times from several hours to a few minutes while decreasing considerably the amount of chemicals and energy required to perform analyses.     

Salt-assisted acid hydrolysis of chitosan to oligomers under microwave irradiation

The effect of inorganic salts such as sodium chloride on the hydrolysis of chitosan in a microwave field was investigated. While it is known that microwave heating is a convenient way to obtain a wide range of products of different molecular weights only by changing the reaction time and/or the radiation power, the addition of some inorganic salts was shown to effectively accelerate the degradation of chitosan under microwave irradiation. The molecular weight of the degraded chitosan obtained by microwave irradiation was considerably lower than that obtained by traditional heating. Moreover, the molecular weight of degraded chitosan obtained by microwave irradiation assisted under the conditions of added salt was considerably lower than that obtained by microwave irradiation without added salt. Furthermore, the effect of ionic strength of the added salts was not linked with the change of molecular weight. FTIR spectral analyses demonstrated that a significantly shorter time was required to obtain a satisfactory molecular weight by the microwave irradiation-assisted inorganic salt method than by microwave irradiation without inorganic salts and conventional technology.     

Guest editorial

Conclusion The introduction of microwave technology in the laboratory is a slow process, but we are sure that the point of no return will be passed in 1990. Forced by environmental defence groups, we start to realize that minimalization of reagent usage in the laboratory is urgently needed. This is especially the case with reagents which are unpleasant or dangerous for laboratory personnel (such as formalin), or which are harmful to the environment (heavy metals). Perhaps administration and laboratory management will see their responsibilities and take steps to make it possible for the laboratory personnel to achieve this. It is clear to us that microwave technology can play a key role here.From whom information regarding the Boerhaave Course on Microwaves, 15–19 October, can be obtained.     

Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin.

Tandemly repeated DNA families appear to undergo concerted evolution, such that repeat units within a species have a higher degree of sequence similarity than repeat units from even closely related species. While intraspecies homogenization of repeat units can be explained satisfactorily by repeated rounds of genetic exchange processes such as unequal crossing over and/or gene conversion, the parameters controlling these processes remain largely unknown. Alpha satellite DNA is a noncoding tandemly repeated DNA family found at the centromeres of all human and primate chromosomes. We have used sequence analysis to investigate the molecular basis of 13 variant alpha satellite repeat units, allowing comparison of multiple independent recombination events in closely related DNA sequences. The distribution of these events within the 171-bp monomer is nonrandom and clusters in a distinct 20- to 25-bp region, suggesting possible effects of primary sequence and/or chromatin structure. The position of these recombination events may be associated with the location within the higher-order repeat unit of the binding site for the centromere-specific protein CENP-B. These studies have implications for the molecular nature of genetic recombination, mechanisms of concerted evolution, and higher-order structure of centromeric heterochromatin.     

Asymmetric somatic cell hybridization in plants

As part of an investigation into whether it would be possible to use UV radiation as a suitable pretreatment of the donor cells in asymmetric hybridization experiments, the effects of this treatment on sugarbeet (Beta vulgaris L.) protoplast DNA have been determined and compared with those of gamma radiation. Both nuclear and mitochondrial DNAs have been examined. The dose ranges chosen had previously been determined to be potentially applicable for fusion experiments. Pulsed field gel electrophoresis and standard agarose gel electrophoresis have been used in combination with laser scanning densitometry to gain an insight into the precise nature and degree of DNA damage resulting from irradiation. It was observed that UV radiation introduced substantial modifications to sugarbeet DNA. Double-strand breaks were detected, the number of which was found to be directly proportional to the dose applied. Such breaks indicate that UV radiation results in substantial chromosome/chromatid fragmentation in these cells. Chemical modifications to the DNA structure could be revealed by a significant reduction in DNA hybridization to specific mitochondrial and nuclear DNA probes. Following gamma irradiation at equivalent biological doses (i.e. those just sufficient to prevent colony formation) much less damage was detected. Fewer DNA fragments were produced indicating the presence of fewer double-strand breaks in the DNA structure. In comparison to UV treatments, DNA hybridization to specific probes following gamma radiation was inhibited less. For both treatments, mitochondrial DNA appeared more sensitive to damage than nuclear DNA. The possibility that DNA repair processes might account for these differences has also been investigated. Results indicate either that repair processes are not involved in the effects observed or that DNA repair occurs so fast that it was not possible to demonstrate such involvement with the experimental system used. The general relevance of such processes to asymmetric cell hybridization is discussed.     

The microwave oven: a novel means of decontaminating parasitological specimens and glassware

This study was conducted to determine the effects of microwave radiation on developmental and infective stages of Eimeria nieschulzi, Strongyloides ratti, and Taenia taeniaeformis. Fecal samples and laboratory preparations containing these three parasites were subjected to microwave radiation for brief periods in a microwave oven, and then in vitro and/or in vivo assessments of viability were made for each organism and preparation. Our results showed that microwave irradiation is extremely effective in killing or preventing development of helminth and protozoan parasites without unduly distorting eggs or developmental stages. Therefore, microwaves may prove useful for decontaminating diagnostic samples or sterilizing contaminated materials in the laboratory and thus for reducing risk to laboratory personnel from parasites of public health importance.     

Significance of dimers to the size of newly synthesized DNA in UV-irradiated Chinese hamster ovary cells.

DNA synthesized after UV irradiation is smaller than that in unirradiated cells even when pulse-labeling times are increased to compensate for the overall reduction in the rate of DNA replication. By isolating newly replicated DNA, incubating it with dimer-specific endonuclease from Micrococcus luteus, and analyzing it on alkaline sucrose gradients, we have been able to demonstrate that this DNA is synthesized in segments corresponding in size to the interdimer distance on the parental strand. In addition, the same DNA analyzed on neutral gradients shows no reduction in molecular weight as a result of UV irradiation and/or endonuclease digestion. Our data are thus inconsistent with the presence of "gaps" in newly synthesized DNA opposite the dimers on the parental strand. We suggest that if such gaps are produced as a result of delayed synthesis around dimers, they are filled before the growing point reaches the next dimer.     

Effect of Caffeine on Postreplication Repair in Human Cells

DNA synthesized shortly after ultraviolet (UV) irradiation of human cells is made in segments that are smaller than normal, but at long times after irradiation the segments made are normal in size. Upon incubation, both the shorter and the normal segments are elongated and joined by the insertion of exogenous nucleotides to form high molecular weight DNA as in nonirradiated cells. These processes occur in normal human cells, where UV-induced pyrimidine dimers are excised, as well as in xeroderma pigmentosum (XP) cells, where dimers are not excised. The effect of caffeine on these processes was determined for both normal human and XP cells. Caffeine, which binds to denatured regions of DNA, inhibited DNA chain elongation and joining in irradiated XP cells but not in irradiated normal human or nonirradiated cells. Caffeine also caused an alteration in the ability to recover synthesis of DNA of normal size at long times after irradiation in XP cells but not in normal cells.     

50 years thymine dimer

Fifty years ago thymine dimer was discovered in the Biochemical and Biophysical Laboratory of Delft Technological University, The Netherlands, by one of the authors of this review (Beukers) as the first environmentally induced DNA lesion. It is one of the photoproducts formed between adjacent pyrimidine bases in DNA by UV irradiation, currently known as cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts. Major lesions found in DNA after in vitro or in vivo UV irradiation are the cis-syn cyclobutane thymine dimer and the thymine-cytosine (6-4) photoproduct. Even after 50 years the study of photo-induced DNA lesions is still going on as is illustrated by the hundreds of papers published every year and the millions hits when browsing the internet for dimer-related information. Living organisms possess efficient and different mechanisms to repair detrimental lesions in their DNA. A unique mechanism to repair CPDs is reversion by either direct interaction with light of short wavelength or by enzymatic photoreactivation. Photophysical mechanisms that induce and reverse molecular bonds in biological macromolecules have been a main focus of research of the group in Delft in the middle of the last century. This review describes the break-through results of these studies which were the result of intense interactions between scientists in the fields of physics, organic chemistry and biochemistry. Philosophically, the "view" of the group in Delft was very appealing: since in nature photolesions are induced in DNA by the sun, how is it possible that repair of these lesions could be accomplished by the same energy source. Evolutionary, it is hardly possible to think of a more efficient repair mechanism.     

Depression, antidepressants, and peripheral blood components

The biological attributes of affective disorders and factors which are able to predict a response to treatment with antidepressants have not been identified sufficiently. A number of biochemical variables in peripheral blood constituents have been tested for this purpose, as a consequence of the lack of availability of human brain tissue. At first, the biological attributes of mental disorders were sought at the level of concentrations of neurotransmitters and their metabolites or precursors. Later on, attention shifted to receptor systems. Since the 1990s, intracellular processes influenced by an illness or its treatment with psychopharmaceuticals have been at the forefront of interest. Interest in biological predictors of treatment with antidepressants has reappeared in recent years, thanks to new laboratory techniques which make it possible to monitor cellular processes associated with the transmission of nerve signals in the brain. These processes can also be studied in plasma and blood elements, especially lymphocytes and platelets. The selection of the qualities to which attention is paid can be derived from today's most widely discussed biochemical hypotheses of affective disorders, especially the monoamine hypothesis and the molecular and cellular theory of depression. Mitochondrial enzymes can also play an important role in the pathophysiology of depression and the effects of antidepressants. In this paper, we sum up the cellular, neurochemical, neuroendocrine, genetic, and neuroimmunological qualities which can be measured in peripheral blood and which appear to be indicators of affective disorders, or parameters which make it possible to predict therapeutic responses to antidepressant administration.     

Formation of nascent DNA molecules during inhibition of replicon initiation in mammalian cells.

X-irradiation of mammalian cells with moderate doses (100-1000 rads) inhibits the initiation of DNA replicons. This inhibition is observed as depressed amounts of radioactivity at low molecular weights when the DNA from the cells is analysed by velocity sedimentation in alkaline sucrose gradients at 30 min after irradiation. There is no detectable effect on chain elongation and joining of those molecules that do initiate replication; this is indicated by the presence of the same amounts of radioactivity in nascent DNA molecules of high molecular weights from control and irradiated cells. The labeling of DNA molecules that initiated replication before irradiation continues unhindered for more than 60 min after irradiation, which is observed as peaks of radioactivity at high S values in alkaline sucrose gradients from irradiated cells. These data indicate that DNA replication in mammalian cells proceeds by continuous joining of nascent molecules that initiate almost simultaneously at origins at various distances from one another. Some of the interorigin distances are much greater than others, implying that large replicons make up a significant component of mammalian DNA.     

Action of ultrasonic irradiation on the DNA of cultivated bovine epithelial lens cell

Cultivated epithelial lens cells have been submitted to a 20 kHz continuous ultrasonic irradiation. At relativity high intensity, destruction of the cells was observed. At lower intensities, where no cell destruction appeared, the molecular weight of the single strand DNA of these cells was monitored to determine whether breakage of DNA molecules was induced by the ultrasound. No breakage of the single strand DNA was observed for intensities below 0.1 and 0.4 W cm-2 with irradiation times of less than 20 minutes and 15 seconds, respectively. These intensities and their corresponding irradiation time are higher than those used in current diagnostic practice.     

Microwave facilitation of domperidone antagonism of apomorphine-induced stereotypic climbing in mice

The dopaminergic agonist apomorphine produced dose-dependent stereotypic climbing behavior in mice housed in cages with vertical bars. This drug effect was competitively inhibited by systemic pretreatment with the centrally acting dopaminergic antagonist haloperidol but not by microwave irradiation (2.45 GHz, 20 mW/cm2, CW, 10 min) nor by systemic pretreatment with domperidone, a dopaminergic antagonist that only poorly penetrates the blood-brain barrier (BBB). Yet when mice were systemically pretreated with domperidone and then subjected to microwave irradiation (as above), the apomorphine effect was significantly reduced. Microwave irradiation also facilitated antagonism of the apomorphine effect by low and otherwise ineffective systemic pretreatment doses of haloperidol. Apomorphine-induced stereotypic climbing behavior was also reduced by domperidone administered intracerebrally, which bypassed the BBB. Exposure of intracerebral domperidone-pretreated animals to microwave irradiation failed to increase the degree of antagonism. These findings indicate that microwave irradiation can facilitate central effects of domperidone, a drug which acts mainly in the periphery. One possible explanation for these findings is that microwave irradiation alters the permeability of the BBB and increases the entry of domperidone to central sites of action.     

Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts

It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.     

Ionizing radiation damage to the folded chromosome of Escherichia coli K-12: sedimentation properties of irradiated nucleoids and chromosomal deoxyribonucleic acid.

The structures of the membrane-free nucleoid of Escherichia coli K-12 and of unfolded chromosomal deoxyribonucleic acid (DNA) were investigated by low-speed sedimentation on neutral sucrose gradients after irradiation with 60Co gamma rays. Irradiation both in vivo and in vitro was used as a molecular probe of the constraints on DNA packaging in the bacterial chromosome. The number of domains of supercoiling was estimated to be approximately 180 per genome equivalent of DNA, based on measurements of relaxation caused by single-strand break formation in folded chromosomes gamma irradiated in vivo and in vitro. Similar estimates based on the target size of ribonucleic acid molecules responsible for maintaining the compact packaging of the nucleoid predicted negligible unfolding due to the formation of ribonucleic acid single-strand breaks at doses of up to 10 krad; this was born out by experimental measurements. Unfolding of the nucleoid in vitro by limit digestion with ribonuclease or by heating at 70 degrees C resulted in DNA complexes with sedimentation coefficients of 1,030 +/- 59S and 625 +/- 15S, respectively. The difference in these rates was apparently due to more complete deproteinization and thus less mass in the heated material. These structures are believed to represent intact, replicating genomes in the form of complex-theta structures containing two to three genome equivalents of DNA. The rate of formation of double-strand breaks was determined from molecular weight measurements of thermally unfolded chromosomal DNA gamma irradiated in vitro. Break formation was linear with doses up to 10 krad and occurred at a rate of 0.27 double-strand break per krad per genome equivalent of DNA (1,080 eV/double-strand break). The influence of possible nonlinear DNA conformations on these values is discussed.     

Stable loss of global DNA methylation in the radiation-target tissue--a possible mechanism contributing to radiation carcinogenesis?

Radiation-induced lymphomagenesis and leukemogenesis are complex processes involving both genetic and epigenetic changes. Although genetic alterations during radiation-induced lymphoma- and leukemogenesis are fairly well studied, the role of epigenetic changes has been largely overlooked. Rodent models are valuable tools for identifying molecular mechanisms of lymphoma and leukemogenesis. A widely used mouse model of radiation-induced thymic lymphoma is characterized by a lengthy "pre-lymphoma" period. Delineating molecular changes occurring during the pre-lymphoma period is crucial for understanding the mechanisms of radiation-induced leukemia/lymphoma development. In the present study, we investigated the role of radiation-induced DNA methylation changes in the radiation carcinogenesis target organ--thymus, and non-target organ--muscle. This study is the first report on the radiation-induced epigenetic changes in radiation-target murine thymus during the pre-lymphoma period. We have demonstrated that acute and fractionated whole-body irradiation significantly altered DNA methylation pattern in murine thymus leading to a massive loss of global DNA methylation. We have also observed that irradiation led to increased levels of DNA strand breaks 6 h following the initial exposure. The majority of radiation-induced DNA strand breaks were repaired 1 month after exposure. DNA methylation changes, though, were persistent and significant radiation-induced DNA hypomethylation was observed in thymus 1 month after exposure. In sharp contrast to thymus, no significant persistent changes were noted in the non-target muscle tissue. The presence of stable DNA hypomethylation in the radiation-target tissue, even though DNA damage resulting from initial genotoxic radiation insult was repaired, suggests of the importance of epigenetic mechanisms in the development of radiation-related pathologies. The possible role of radiation-induced DNA hypomethylation in radiation-induced genome instability and aberrant gene expression in molecular etiology of thymic lymphomas is discussed.     

Measurements of alkali-labile DNA damage and protein-DNA crosslinks after 2450 MHz microwave and low-dose gamma irradiation in vitro

In vitro experiments were performed to determine whether 2450 MHz microwave radiation induces alkali-labile DNA damage and/or DNA-protein or DNA-DNA crosslinks in C3H 10T(1/2) cells. After a 2-h exposure to either 2450 MHz continuous-wave (CW) microwaves at an SAR of 1.9 W/kg or 1 mM cisplatinum (CDDP, a positive control for DNA crosslinks), C3H 10T(1/2) cells were irradiated with 4 Gy of gamma rays ((137)Cs). Immediately after gamma irradiation, the single-cell gel electrophoresis assay was performed to detect DNA damage. For each exposure condition, one set of samples was treated with proteinase K (1 mg/ml) to remove any possible DNA-protein crosslinks. To measure DNA-protein crosslinks independent of DNA-DNA crosslinks, we quantified the proteins that were recovered with DNA after microwave exposure, using CDDP and gamma irradiation, positive controls for DNA-protein crosslinks. Ionizing radiation (4 Gy) induced significant DNA damage. However, no DNA damage could be detected after exposure to 2450 MHz CW microwaves alone. The crosslinking agent CDDP significantly reduced both the comet length and the normalized comet moment in C3H 10T(1/2) cells irradiated with 4 Gy gamma rays. In contrast, 2450 MHz microwaves did not impede the DNA migration induced by gamma rays. When control cells were treated with proteinase K, both parameters increased in the absence of any DNA damage. However, no additional effect of proteinase K was seen in samples exposed to 2450 MHz microwaves or in samples treated with the combination of microwaves and radiation. On the other hand, proteinase K treatment was ineffective in restoring any migration of the DNA in cells pretreated with CDDP and irradiated with gamma rays. When DNA-protein crosslinks were specifically measured, we found no evidence for the induction of DNA-protein crosslinks or changes in amount of the protein associated with DNA by 2450 MHz CW microwave exposure. Thus 2-h exposures to 1.9 W/ kg of 2450 MHz CW microwaves did not induce measurable alkali-labile DNA damage or DNA-DNA or DNA-protein crosslinks.     

Isolation of subtelomeric DNA sequences labelling sheep and goat chromosome ends

Two techniques that make it possible to isolate telomere DNA are presented, using sheep as an example. The first technique is based upon the screening of a sheep BAC library with PCR amplified DNA segments preserved from high-power laser beam irradiation. Twenty-three BACs hybridising to 13 subtelomeric regions in sheep and goats were obtained (out of 27 in the sheep complement), of which 13 recognised more than one region, telomeric or not. Twenty-three microsatellites were isolated from these BACs and 22 were genetically mapped on the sheep international genetic map, always consistently with the cytogenetical localisation in 17 cases out of 22. These results are discussed. The second technique is based upon the selective cloning of subtelomeric enriched DNA. Preliminary results were obtained by this approach.     

Saccharomyces cerevisiae wine yeast populations in a cold region in Argentinean Patagonia. A study at different fermentation scales

AIMS: To study the diversity and dynamics of indigenous Saccharomyces wine populations during Malbec spontaneous fermentation, a representative Patagonian red wine, at both industrial and laboratory scale. METHODS AND RESULTS: Two molecular techniques, including restriction fragment length polymorphism of mitochondrial (mt) DNA and polymorphism of amplified delta interspersed element sequences, were used for characterization of indigenous yeasts at strain level. The mtDNA restriction patterns showed the major discriminative power; however, by combining the two molecular approaches it was possible to distinguish a larger number of strains and, therefore, draw more representative conclusions about yeast diversity. Although a great diversity of wild Saccharomyces cerevisiae strains was observed, only nine represented more than half of the total Saccharomyces yeast biota analysed; five of these were common and took over the Malbec must fermentation in both vinifications. CONCLUSIONS: Many different indigenous S. cerevisiae strains were identified; nevertheless, the dominant strains in both industrial and laboratory vinification processes were just a few and the same. SIGNIFICANCE AND IMPACT OF THE STUDY: Small-scale fermentation appears to be a valuable tool in winemaking, one especially helpful in evaluating microbiological aspects of as well as possible interactions between inoculated selected strains and native strains.