Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Common wheat is one of the most important cereal crops in the world. The improvement of its yield and quality by the introduction of heterologous gene(s) is very significant. Avena sativa L. (2n = 42), belonging to the Avena tribe, possesses resistance to drought, coldness and many dis-eases. Its contents of proteins and fat in seed, especially lysine and unsaturated fatty acid are highest in crops, therefore it is regarded as healthy food. Sexual hybridization between wheat and Avena sativa…     

Asymmetric somatic hybridization between wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and <Emphasis Type="Italic">Avena sativa</Emphasis> L.

Protoplasts from cell suspensions of young-embryo-derived calli, whichwere non- regenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

A method of collecting water samples from immediately below an ice cover

A plexiglas box with cellular neoprene gaskets in two opposite faces which can be frozen in situ in a developing ice cover on a lake is described along with a water sampling system using evacuated glass tubes (Vacutainers) and blood collecting needles. This system allows collecting of water samples from directly beneath the ice without disturbing the ice cover. The box may be removed in an ice block at the end of the sampling period.     

Calorimetric studies of freeze-induced dehydration of phospholipids.

Differential scanning calorimetry (DSC) was used to determine the amount of water that freezes in an aqueous suspension of multilamellar dipalmitoylphosphatidylcholine (DPPC) liposomes. The studies were performed with dehydrated suspensions (12-20 wt% water) and suspensions containing an excess of water (30-70 wt% water). For suspensions that contained > or = 18 wt% water, two ice-formation events were observed during cooling. The first was attributed to heterogeneous nucleation of extraliposomal ice; the second was attributed to homogeneous nucleation of ice within the liposomes. In suspensions with an initial water concentration between 13 and 16 wt%, ice formation occurred only after homogeneous nucleation at temperatures below -40 degrees C. In suspensions containing < 13 wt% water, ice formation during cooling was undetectable by DSC, however, an endotherm resulting from ice melting during warming was observed in suspensions containing > or = 12 wt% water. In suspensions containing < 12 wt% water, an endotherm corresponding to the melting of ice was not observed during warming. The amount of ice that formed in the suspensions was determined by using an improved procedure to calculate the partial area of the endotherm resulting from the melting of ice during warming. The results show that a substantial proportion of water associated with the polar headgroup of phosphatidylcholine can be removed by freeze-induced dehydration, but the amount of ice depends on the thermal history of the samples. For example, after cooling to -100 degrees C at rates > or = 10 degrees C/min, a portion of water in the suspension remains supercooled because of a decrease in the diffusion rate of water with decreasing temperature. A portion of this supercooled water can be frozen during subsequent freeze-induced dehydration of the liposomes under isothermal conditions at subfreezing storage temperature Ts. During isothermal storage at Ts > or = -40 degrees C, the amount of unfrozen water decreased with decreasing Ts and increasing time of storage. After 30 min of storage at Ts = -40 degrees C and subsequent cooling to -100 degrees C, the amount of water associated with the polar headgroups was < 0.1 g/g of DPPC. At temperatures > -50 degrees C, the amount of unfrozen water associated with the polar headgroups of DPPC decreased with decreasing temperature in a manner predicted from the desorption isotherm of DPPC. However, at lower temperatures, the amount of unfrozen water remained constant, in large part, because the unfrozen water underwent a liquid-to-glass transformation at a temperature between -50 degrees and -140 degrees C.     

Resistance of selected strains of Pseudomonas aeruginosa to low-intensity ultraviolet radiation.

The resistances of 10 strains of Pseudomonas aeruginosa and other microorganisms to an ultraviolet (UV) intensity of 100 muW/cm2 were determined. Organisms were exposed in 2- or 15-ml saline suspensions contained in uncapped polyethylene bottles for increasing periods of time, and the surviving fractions were enumerated. Decimal reduction times were calculated by regression analysis, using the least-squares method. The 10 strains of P. aeruginosa, compared with Micrococcus radiodurans and Candida albicans, were very susceptible to low-intensity UV radiation. Results from this study showed that a UV intensity of 100 muW/cm2 penetrated saline suspensions up to 40 mm deep sufficiently to kill high levels of microbial cells, especially P. aeruginosa cells. These results allowed us to design a system for determining and monitoring the sterilization capability of low-intensity UV radiation. In our particular case, UV proved to be an efficient mode for sterilizing saline suspensions of P. aeruginosa in polyethylene bottles. The significance and application of these findings with regard to supporting UV as a sterilant are discussed.     

Effect of misonidazole on formation of thymine damage by gamma rays

The effect of the radiosensitizer misonidazole (Ro-07-0582) on the formation of thymine base damage of the 5,6-dihydroxydihydrothymine-type by gamma rays was measured under aerobic and hypoxic conditions. HeLa cells, prelabeled with [methyl-3H]thymidine, were suspended in phosphate-buffered saline in the presence and absence of misonidazole. Concentrations of misonidazole up to 15 mM were used. The cell suspensions were irradiated at ice temperature with 60Co gamma rays. Dose-response curves under aerobic and hypoxic conditions showed a much depressed base damage formation under hypoxia, which was created by blowing a stream of nitrogen across the cell suspensions for 30 min on ice. The presence of misonidazole had little or no detectable effect under hypoxia. It is concluded that an effect on the level of formation of thymine base damage is not primarily responsible for the radiosensitization by misonidazole under hypoxic conditions.     

On the mechanism of injury to slowly frozen erythrocytes.

When cells are frozen slowly in aqueous suspensions, the solutes in the suspending solution concentrate as the amount of ice increases; the cells undergo osmotic dehydration and are sequestered in ever-narrowing liquid-filled channels. Cryoprotective solutes, such as glycerol, reduce the amount of ice that forms at any specified subzero temperature, thereby controlling the buildup in concentration of those other solutes present, as well as increasing the volume of the channels that remain to accommodate the cells. It has generally been thought that freezing injury is mediated by the increase in electrolyte concentration in the milieu surrounding the cells, rather than reduction of temperature or any direct action of ice. In this study we have frozen human erythrocytes in isotonic solutions of sodium chloride and glycerol and have demonstrated a correlation between the extent of damage at specific subzero temperatures, and that caused by the action at 0 degrees C of solutions having the same composition as those produced by freezing. The cell lysis observed increased directly with glycerol concentration, both in the freezing experiments and when the cells were exposed to corresponding solutions at 0 degrees C, showing that the concentration of sodium chloride alone is not sufficient to account quantitatively for the damage observed. We then studied the effect of freezing in anisotonic solutions to break the fixed relationship between solute concentration and the volume of the unfrozen fraction, as described by Mazur, P., W. F. Rall, and N. Rigopoulos (1981. Biophys. J. 653-675). We confirmed their experimental findings, but we explain them differently. We ascribe the apparently dominant effect of the unfrozen fraction to the fact that the cells were frozen in, and returned to, anisotonic solutions in which their volume was either less than, or greater than, their physiological volume. When similar cell suspensions were subjected to a similar cycle of increase and then decrease in solution strength, but in the absence of ice (at 20 degrees C), a similar pattern of hemolysis was observed. We conclude that freezing injury to human erythrocytes is due solely to changes that occur in the composition of their surrounding milieu, and is most probably mediated by a temporary leak in the plasma membrane that occurs during the thawing (reexpansion) phase.     

Cryobiology of Neurospora crassa. II. Alteration in freeze response of Neurospora crassa conidia by additives

Neurospora crassa conidia in aqueous suspensions were frozen and thawed in the presence of various agents. Colony counts with these treatments were compared with those of the following (a) unfrozen, agent-treated, (b) unfrozen water suspended, and (c) frozen, water suspended. It was found that dimethyl sulfoxide (0.5–20%) resulted in total protection against freeze damage. Glycerol and calcium chloride decreased survival as much as 90% with fast freeze. The latter agents have properties which should decrease the rate of outflow of cellular water during temperature lowering. The results are consistent with the proposal that intracellular ice crystal growth to membrane rupturing dimensions is the damaging freeze mechanism under these conditions.     

New water disinfection system using UVA light-emitting diodes

AIM: To evaluate the ability of high-energy ultraviolet A (UVA) light-emitting diode (LED) to inactivate bacteria in water and investigate the inactivating mechanism of UVA irradiation. METHODS AND RESULTS: We developed a new disinfection device equipped with high-energy UVA-LED. Inactivation of bacteria was determined by colony-forming assay. Vibrio parahaemolyticus, enteropathogenic Escherichia coli, Staphylococcus aureus and Escherichia coli DH5alpha were reduced by greater than 5-log(10) stages within 75 min at 315 J cm(-2) of UVA. Salmonella enteritidis was reduced greater than 4-log(10) stages within 160 min at 672 J cm(-2) of UVA. The formation of 8-hydroxy-2'-deoxyguanosine in UVA-LED irradiated bacteria was 2.6-fold higher than that of UVC-irradiated bacteria at the same inactivation level. Addition of mannitol, a scavenger of hydroxyl radicals (OH(*)), or catalase, an enzyme scavenging hydrogen peroxide (H(2)O(2)) to bacterial suspensions significantly suppressed disinfection effect of UVA-LED. CONCLUSION: This disinfection system has enough ability to inactivate bacteria and OH(*) and H(2)O(2) participates in the disinfection mechanism of UVA irradiation. SIGNIFICANCE AND IMPACT OF THE STUDY: We newly developed UVA irradiation system and found that UVA alone was able to disinfect the water efficiently. This will become a useful disinfection system.     

UV radiation and potential biological effects beneath the perennial ice cover of an antarctic lake

High-resolution spectral scans of solar ultraviolet radiation (UVR) were obtained directly beneath the 4.0–5.0 m thick, perennial ice cover of Lake Hoare, South Victoria Land, Antarctica. Both UVA (320–400 nm) and UVB (280–320 nm) radiation were detectable beneath the ice using a diver-deployed, underwater scanning spectroradiometer which permitted accurate measurement in the 280–340 nm range, while avoiding effects of surface shading and/or hole effects. UVR at wavelengths <310 nm was not detectable below the ice. This lower wavelength UVB appears to penetrate the Lake Hoare ice to depths of no more than 1.5 m during relatively cloud-free austral summer days. Based upon estimated biologically effective UVR dosages and DNA dosimeter data, exposure of benthic and planktonic microbes to the UVR encountered immediately beneath the ice is unlikely to inhibit microbial metabolism. Although waters of oligotrophic antarctic lakes are highly transparent to UVR, the thick, high scattering and optically dense ice covers on many of these lakes offers organisms a degree of protection largely unavailable in temperate and tropical systems. Thinning or complete loss of these overlying ice covers is likely to have major consequences for the structure of antarctic lake microbial communities.     

Abiotic formation of bioorganic compounds in space--preliminary experiments on ground and future exobiology experiments in space.

Simulation experiments on ground have shown that "amino acid precursors", which give amino acids after acid-hydrolysis, can be formed when an ice mixture simulating ice mantles of interstellar dust particles (lSDs) is irradiated with high energy particles or UV light. It is strongly suggested that such bioorganic compounds were delivered by comets for the first biosphere on the Earth. It is of great interest to confirm this hypothesis in actual space conditions, such as in an exposed facility of JEM. Fundamental designs for such exobiology experiments in earth orbit (EEEO) will be discussed.     

Staining Osteoid Seams in Thin Slabs of Undecalcified Trabecular Bone

For qualitative and quantitative study of osteoid seams in trabecular bone, 5-7 mm thick slabs were cut from the bodies of fresh, frozen, undecalcified human vertebrae. After washing out the bone marrow and soft tissue in a jet stream of water, the slabs were stained in 0.5% aqueous basic fuchsin for 30-40 hr at 18-20 C. The specimens were then trimmed by sawing off both overstained surfaces, to make a 2 mm slab which was submerged in 50% ethanol (2 or 3 changes of 10-30 min each), until the nonosteoid trabeculae became pale pink. The slab was allowed to dry in air. Osteoid seams are stained dark red and are well differentiated under a dissecting microscope with reflected illumination, either dry or immersed in water. This method permits the various types of trabculae to be separately studied in the same specimen     

Resistance and recovery studies on ultraviolet-irradiated spores of Bacillus pumilus.

A spore suspension model and a procedure for recovering ultraviolet (UV)-irradiated spores of Bacillus pumilus were investigated. A most-probable-number tube dilution method using double-strength Trypticase soy broth was found to be superior to the agar plate method for recovering optimal numbers of spores irradiated with sublethal doses of UV energy. Aqueous suspensions of B. pumilus survived UV doses up to 108,000 ergs/mm2 as determined by a most-probable-number recovery and estimation procedure. Resistance and stability data were consistent and reproducible, indicating the dependability of this method for recovering UV-damaged spores. The procedures used to collect information concerning resistance characteristics for two strains of B. pumilus are discussed.     

The great Cenozoic ice sheet

Ocean-floor sediments contain a record of Quaternary continental glaciations in which global cooling prevails over a 100,000 year cycle and the intensity of the last glacial maximum is as strong as any of the earlier glaciations. Yet, the areal extent of Quaternary glacial drift in North America and Eurasia during the last glaciation is much less extensive than during several earlier glaciations. In an attempt to explore this problem, two versions of a Great Cenozoic Ice Sheet were reconstructed. The minimum version transformed a frozen bed beneath high interior ice domes and along southern margins during the last glaciation into a thawed bed during more extensive earlier glaciations. This allowed thinner ice to cover a larger area with little change in ice volume, thereby satisfying both the continental and oceanic records of Quaternary glaciation. The maximum version extended frozen basal conditions beneath high interior ice domes and along southern margins, so that a greater areal extent could only be accommodated by thicker ice. A greater ice volume is required in this case, and would seem to put the Great Cenozoic Ice Sheet in the late Tertiary, where some evidence for global cooling on a 400,000 year cycle exists and might provide the longer timespan needed to produce a larger ice sheet.     

Thin sectioning of slice preparations for immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue. Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle. Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm. We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections. The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed. We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice. This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice. A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT). This is then quickly frozen with crushed dry ice and the resulting wafer can be position easily for cryostat sectioning. Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.     

Cryobiology of neurospora crassa. I. Freeze response of Neurospora crassa conidia

Neurospora crassa conidia were frozen and thawed in water suspensions at various rates and with different minimum temperatures. Colony counts of the experimental conidia were compared with those of controls, which were taken as 100% survival. The data revealed that (1) survivals were near 100% after fast thaw (400 °/min) regardless of the freeze rate, (2) percentage of survival was inversely related to freeze rate when combined with slow thaw, (3) slow thaw (0.5 °/min) was damaging, and (3) the rates of freeze-thaw affected the system only in the −5 to −20 ° interval. The damaging freeze conditions were those which favor ice crystal growth. It is suggested that rupture of the membrane by ice crystals seems to be the plausible mechanism of damage in freezing and thawing N. crassa conidia.     

Protection of Normal, Lysogenic, and Pyocinogenic Strains Against Ultraviolet Radiation by Bound Acriflavine

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.     

A cryoprotection method that facilitates cutting frozen sections of whole monkey brains for histological and histochemical processing without freezing artifact

Cutting frozen sections of large (greater than 60 cc) blocks of monkey brain using the conventional procedures of infiltration with 30% sucrose as a cryoprotectant before freezing with pulverized dry ice often produces unacceptable levels of freezing artifact (FA) caused by displacement of tissue by ice crystals. Experiments investigating FA utilized perfusion-fixed brains from 46 monkeys and spanned combinations of cryoprotectants (glycerol, sucrose), freezing methods (dry ice or -75 degrees C isopentane), and fixatives (10% formalin, Karnovsky's or Timm's). The effects were evaluated by rating of FA severity in frozen sections of whole monkey brains. Minor FA appears as enlarged capillaries, more serious FA as large vacuoles, and both first appear midway between the periphery and center of the block. Stronger fixatives increased the severity of freezing artifact. The best method for eliminating FA was graded infiltration with up to 20% glycerol and 2% DMSO (in buffer or fixative), followed by rapid freezing in -75 degrees C isopentane. Although using a glycerol-DMSO infiltration before conventional freezing with pulverized dry ice or using conventional sucrose infiltration before freezing in isopentane gave better results than sucrose infiltration and dry-ice freezing, only the combination of glycerol-DMSO infiltration and freezing in isopentane produced consistently excellent results and virtually eliminated freezing artifact. To determine the effect of freezing with dry ice or isopentane on the rate of cooling in large blocks of CNS tissue, thermocouples were embedded in an 80-cc block of albumin-gelatin and frozen with the two methods. The rate of cooling (-3.5 degrees C/min) was twice as fast using isopentane.     

Principles and biotechnological applications of bacterial ice nucleation.

Certain aerobic, Gram-negative bacteria, including the epiphytic plant pathogen, Pseudomonas syringae, possess a membrane protein that enables them to nucleate crystallization in supercooled water. Currently, these ice-nucleating (IN) bacteria are being used in snow making and have potential applications in the production and texturing of frozen foods, and as a replacement of silver iodide in cloud seeding. A negative aspect of these IN bacteria is frost damage to plant surfaces. Thus, of the various types of biological ice nucleators, bacteria have been the subject of most research and also appear relevant to the anticipated practical uses. The intent of this review is to explain the identification and ecology of the ice-nucleating bacteria, as well as to discuss aspects of molecular biology related to ice nucleation and consider existing and potential applications of this unique phenomenon.     

VISUALIZATION OF FREEZING DAMAGE

Freeze-cleaving can be used as a direct probe to examine the ultrastructural alterations of biological material due to freezing. We examined the thesis that at least two factors, which are oppositely dependent upon cooling velocity, determine the survival of cells subjected to freezing. According to this thesis, when cells are cooled at rates exceeding a critical velocity, a decrease in viability is caused by the presence of intracellular ice; but cells cooled at rates less than this critical velocity do not contain appreciable amounts of intracellular ice and are killed by prolonged exposure to a solution that is altered by the presence of ice. As a test of this hypothesis, we examined freeze-fractured replicas of the yeast Saccharomyces cerevisiae after suspensions had been cooled at rates ranging from 1.8 to 75,000°C/min. Some of the frozen samples were cleaved and replicated immediately in order to minimize artifacts due to sample handling. Other samples were deeply etched or were rewarmed to -20°C and recooled before replication. Yeast cells cooled at or above the rate necessary to preserve maximal viability (~7°C/min) contained intracellular ice, whereas cells cooled below this rate showed no evidence of intracellular ice.