Stress-induced accumulation and tissue-specific localization of dehydrins in Arabidopsis thaliana

Stress-induced accumulation of five (COR47, LTI29, ERD14, LTI30 and RAB18) and tissue localization of four (LTI29, ERD14, LTI30 and RAB18) dehydrins in Arabidopsis were characterized immunologically with protein-specific antibodies. The five dehydrins exhibited clear differences in their accumulation patterns in response to low temperature, ABA and salinity. ERD14 accumulated in unstressed plants, although the protein level was up-regulated by ABA, salinity and low temperature. LTI29 mainly accumulated in response to low temperature, but was also found in ABA- and salt-treated plants. LTI30 and COR47 accumulated primarily in response to low temperature, whereas RAB18 was only found in ABA-treated plants and was the only dehydrin in this study that accumulated in dry seeds.Immunohistochemical localization of LTI29, ERD14 and RAB18 demonstrated tissue and cell type specificity in unstressed plants. ERD14 was present in the vascular tissue and bordering parenchymal cells, LTI29 and ERD14 accumulated in the root tip, and RAB18 was localized to stomatal guard cells. LTI30 was not detected in unstressed plants. The localization of LTI29, ERD14 and RAB18 in stress-treated plants was not restricted to certain tissues or cell types. Instead these proteins accumulated in most cells, although cells within and surrounding the vascular tissue showed more intense staining. LTI30 accumulated primarily in vascular tissue and anthers of cold-treated plants.This study supports a physiological function for dehydrins in certain plant cells during optimal growth conditions and in most cell types during ABA or cold treatment. The differences in stress specificity and spatial distribution of dehydrins in Arabidopsis suggest a functional specialization for the members of this protein family.     

Hexagonal shaped ice spicules in frozen antifreeze protein solutions

In the presence of antifreeze proteins from both Antarctic and Arctic fishes, water freezes in the form of long c-axis spikes or spicular-like crystals. Transmission electron microscopy of the Pt/C replicas of the freeze fractured spicular ice in a small capillary revealed the presence of many hexagonally shaped structures whose cross-sectional dimensions were between 0.5 and 10 microm. Well-defined parallel faces were associated with most fractured and etched spicules. When fracture planes occurred near the tip of a spicule, well-defined pyramidal faces were apparent. Steps were sometimes associated with these pyramidal spicular crystal faces. On some of the replicas obvious roughening of certain crystal faces of the spicule was observed, suggesting that the antifreeze proteins may have adsorbed to those faces.     

Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves

Acclimation of winter oilseed plants in the cold (i.e. at temperatures >0 degrees C) followed by short exposure to sub-lethal freezing temperatures resulted in pronounced ultrastructural changes of leaf epidermal and mesophyll cells. The following major changes were observed upon acclimation at 2 degrees C: increased thickness of cell walls; numerous invaginations of plasma membranes; the appearance of many large vesicles localized in the cytoplasm in close proximity to the central vacuole; the occurrence of abundant populations of microvesicles associated with the endoplasmic reticulum (ER) cisternae or located in the vicinity of dictyosomes; and the occurrence of paramural bodies and myelin-like structures. In addition, large phenolic deposits were observed in the vicinity of the plasma membrane and membrane-bound organelles such as chloroplasts, large vesicles or cytoplasm/tonoplast interfaces. Transient freezing (-5 degrees C for 18 h) of the cold-acclimated leaves led to reversible disorganization of the cytoplasm and to pronounced structural changes of the cellular organelles. Chloroplasts were swollen, with the stroma occupying one half of their volume and the thylakoid system being displaced to the other half. Large phenolic aggregates disappeared but distinct layers of phenolic deposits were associated with mitochondrial membranes and with chloroplast envelopes. In frost-thawed cells recovered at 2 degrees C for 24 h, dictyosomes and dictyosome- or ER-derived small vesicles reappeared in the ribosome-rich cytoplasm. Aberrations in the structure of chloroplasts and mitochondria were less pronounced. Few phenolic deposits were seen as small grains associated with chloroplast envelopes and vesicle membranes. These observations demonstrate that plants undergo different changes in cell ultrastructure depending on whether they are subjected to chilling or freezing temperatures. Results are discussed in relation to membrane recycling and the possible role of phenolics during the first and second stages of plant acclimation at low temperature.     

Microtome sectioning causes artifacts in rhizobox experiments

Experimental data on rhizosphere characteristics at high spatial resolution are required to improve our knowledge on phytoavailability of nutrients and pollutants. In numerous studies, sectioning using refrigerated microtomes has been employed to obtain thin soil layers at defined distances from the root surface. In this study, we assessed the effect of thin slicing and freezing on soil chemical characteristics. Two experimental soils were frozen at –20°C and sliced using a refrigerated microtome. In general, chemical changes relative to the non-sliced control were more pronounced as the trim thickness (thickness of a single slice) decreased. Maximum increases in pH and electrical conductivity (EC) for the smallest trim thickness used (20 m) were 0.9 units and 50%, respectively. Extractable fractions of P (0.5 M NaHCO₃) K, Mg, Mn, Na and Si (1 M NH₄NO₃) increased up to 40, 91, 19, 621, 50 and 100%, respectively. Based on these results, we suggest to use a trim thickness of 200 m. Apart from slicing, freezing (a prerequisite for the microtome technique) was found to bias soil chemical parameters. To circumvent microtome-related artifacts we present a home-made slicing device as a cost-effective alternative, which allows sectioning of non-frozen rhizosphere soil employing one single slice.     

A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30–40 kDa to overcome problems arising from rapid ¹H and ¹³C transverse relaxation. However, ¹H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D₂O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H₂O. As a side-effect partial C protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary ¹H^N- and ¹H-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D₂O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of C protonation of individual amino acids is determined quantitatively and transverse relaxation properties of ¹H^N and ¹⁵N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.     

A Simple Mathematical Model of Second-Messenger Mediated Slow Excitatory Postsynaptic Potentials

We have developed a novel and simple mathematical model of a slow excitatory postsynaptic potential (EPSP) based on an abstraction of the processes of activation, inactivation, and summation of a cAMP, protein kinase A (PKA)-dependent second-messenger cascade. The model describes the activation of receptors, G-proteins, and production of cAMP as the first stage and uses first-order, non-rate-limited kinetics. The second stage corresponds to the release of active, PKA catalytic subunit and can use first- or higher-order kinetics. The third stage represents simple phosphorylation of ion channels and is limited by the number of channels available. The decay of each stage is based on first-order, mass-action kinetics. These equations and some variations were solved numerically and values of the parameters were determined by fitting to a variety of experimental data from myenteric neurons of the guinea-pig ileum. The model produced a slow EPSP with a nonlinear stimulus-response relationship that resulted from the underlying kinetics of the signaling cascade. This system of equations is suitable for incorporation into a large-scale computer simulation, and the methodology should be generalizable to other pathways.     

The Israel National Skin Bank: Quality Assurance and Graft Performance of Stored Tissues

The Israel National Skin Bank (INSB) was founded jointly by the Israel Defense Forces (IDF) Medical Corps and the Ministry of Health in 1986. The prime purpose of the Skin Bank is to treat burn victims incurred at war or during mass casualty incidences. The INSB Protocol is comprised of international skin bank protocols and our previous and present research results. They provide the framework for selecting optimal guidelines for procurement, processing, preservation, storage and evaluation of transplantation performance of viable skin grafts. For evaluation and direct comparison of graft performance of glycerolized or cryopreserved skin stored for long periods, we have applied a mouse recipient model developed by us. This model assesses graft performance before the rejection process takes place. The in vivo design has inherent clinical relevance, which is especially appealing. Cryopreserved skin performed better than glycerolized skin (p > 0.027), but fresh skin performed significantly better than cryopreserved skin (p > 0.003), as analyzed by the Mann–Whitney non-parametric test. Then graft performance of skin specimens were cryopreserved by programmed or stepwise freezing and stored at -80°C or in liquid nitrogen for 1 and 6–10 months was evaluated. The average score of skin preserved by programmed freezing and stored in liquid nitrogen is the highest for both storage periods. This method has a highly significant advantage (p < 0.007) over the others for 6–10 months storage, evaluated by graft adherence. Several interaction factors determine the quality of cryopreserved skin. Highly significant is the interaction factor/'combined effect' of sample variability with the method of cryopreservation or with the storage period. Finally, the results of paired comparison of selected histology criteria of cryopreserved to fresh skin indicated that storage of skin for up to 5 years did not impair significantly its performance compared to fresh skin, whereas, after six years of storage, there was a highly significant (p < 0.001) impairment in skin quality. We offer a simplified in vivo model and analysis for cryopreserved skin graft performance, suggesting that the evaluation procedures, which are issues of great interest in skin banking, may help future skin banks to make informed choices and decisions regarding quality issues.     

Tubulin and Neurofilament Proteins Are Transported Differently in Axons of Chicken Motoneurons

SUMMARY 1. We previously showed that actin is transported in an unassembled form with its associated proteins actin depolymerizing factor, cofilin, and profilin. Here we examine the specific activities of radioactively labeled tubulin and neurofilament proteins in subcellular fractions of the chicken sciatic nerve following injection of L-[³⁵S]methionine into the lumbar spinal cord.2. At intervals of 12 and 20 days after injection, nerves were cut into 1-cm segments and separated into Triton X-100-soluble and particulate fractions. Analysis of the fractions by high-resolution two-dimensional gel electrophoresis, immunoblotting, fluorography, and computer densitometry showed that tubulin was transported as a unimodal wave at a slower average rate (2–2.5 mm/day) than actin (4–5 mm/day). Moreover, the specific activity of soluble tubulin was five times that of its particulate form, indicating that tubulin is transported in a dimeric or small oligomeric form and is assembled into stationary microtubules.3. Neurofilament triplet proteins were detected only in the particulate fractions and transported at a slower average rate (1 mm/day) than either actin or tubulin.4. Our results indicate that the tubulin was transported in an unpolymerized form and that the neurofilament proteins were transported in an insoluble, presumably polymerized form.     

豚鼠主动脉前庭自发性慢反应电位去极离子流的初步分析

为研究主动脉前庭自发慢反应电位的去极离充性质,利用豚鼠的离体以及心脏,常规玻璃微电极细胞内记录方法和离子通道组断剂,观测最大舒张电位（ＭＤＰ）、０相除极幅度（ＡＰＡ）、０相最大除极速度（Ｖｍａｘ）、４个自动除极速度（ＶＤＤ）、复极５０％（ＡＰＤ５０）和９０％（ＡＰＤ９０）的时间以及自发放电频率（ＲＰＦ）。结果发现：⑴０．５μｍｏｌ／Ｌ尼索地平（Ｎｉｓ）可使该慢电位的ＡＰＡ、Ｖｍａｘ、ＶＤＤ明显减小     

A chamber for the simulation of radiation freezing of plants

Frost injury to plants can occur following episodic radiation frosts. In the UK this is particularly important to spring sown crops such as potatoes. Most laboratory based frost studies simulate freezing using either conductive or convective freezing chambers. Such frost tests do not simulate overnight freezing events adequately. A freezing chamber based on radiative cooling is described which mimics overnight radiative freezing. The chamber is rectangular in design (1 m × lm × 2 m high) with a radiative cooling plate at the top of the chamber cooled to -40°C to -45°C using HFC coolants, which acts as a cold black body. The sides of the chamber are also cooled to variable temperatures down to -5°C in order to prevent the chamber walls radiating to the plant material during testing. Using thermocouples to measure air temperature and plant temperature the chamber has been characterised to simulate the radiative cooling conditions found in the UK during autumn and spring. Exotherm detection upon plant freezing is simplified by virtue of the reduction in temperature fluctuation normally experienced at the plant surface during natural freezing. Radiation frosts and subsequent frost damage to potatoes have been recorded in the temperature range -4°C to –5°C. The equipment is recommended for studies of frost damage to plants normally caused by episodic radiation frost events.