Cold ethanol fractionation and heat inactivation of hepatitis a virus during manufacture of albumin from human plasma

The purpose of the present study was to examine the efficacy and mechanism of fraction IV cold ethanol fractionation and pasteurization (60°C heat treatment for 10h), involved in the manufacture of albumin from human plasma, in the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and the amount of virus in each fraction then quantified using a 50% tissue culture infectious dose (TCID₅₀). HAV was effectively partitioned from albumin during the fraction IV cold ethanol fractionation with a log reduction factor of 3.43. Pasteurization was also found to be a robust and effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.60 log TCID₅₀ to undetectable levels within 5 h of treatment. The log reduction factor achieved during pasteurization was≽4.76. Therefore, the current results indicate that the production process for albumin has sufficient HAV reducing capacity to achieve a high margin of virus safety.     

Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.     

Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana

Cold tolerance in plants is an ecologically important trait that has been under intensive study for basic and applied reasons. Determining the fitness benefits and costs of cold tolerance has previously been difficult because cold tolerance is normally an induced trait that is not expressed in warm environments. The recent creation of transgenic plants constitutively expressing cold tolerance genes enables the investigation of the fitness consequences of cold tolerance in multiple temperature environments. We studied three genes from the CBF (C-repeat/dehydration responsive element binding factor) cold tolerance pathway, CBF1, 2 and 3, in Arabidopsis thaliana to test for benefits and costs of constitutive cold tolerance. We used multiple insertion lines for each transgene and grew the lines in cold and control conditions. Costs of cold tolerance, as determined by fruit number, varied by individual transgene. CBF2 and 3 overexpressers showed costs of cold tolerance, and no fitness benefits, in both environments. CBF1 overexpressing plants showed no fitness cost of cold tolerance in the control environment and showed a marginal fitness benefit in the cold environment. These results suggest that constitutive expression of traits that are normally induced in response to environmental stress will not always lead to costs in the absence of that stress, and that the ecological risks of CBF transgene escape should be assessed prior to their use in commercial agriculture.     

Rapid responses to high temperature and desiccation but not to low temperature in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera,Tineidae)

A broad definition of rapid cold hardening (RCH) is that it is the process whereby insects increase their survival of a sub-zero temperature after a brief (h) pre-exposure to a less severe low temperature. The effects of various pre-treatments on survival of two h at -7.9 degrees C were investigated in the freeze tolerant sub-Antarctic caterpillar Pringleophaga marioni (Lepidoptera: Tineidae), the first time RCH has been investigated in a freeze tolerant arthropod. All caterpillars froze when exposed to -7.9 degrees C, and none of the low temperature pre-treatments (-5, 0, 5 and 15 degrees C, as well as -5 degrees C and 0 degrees C with a delay before freezing) nor slow cooling (0.1 degrees C/min) elicited any improvement in survival of -7.9 degrees C as compared to controls. However, high temperature treatments (25, 30 and 35 degrees C), desiccation and acclimation for 5 days at 0 degrees C did result in significant increases in survival of the test temperature, possibly as a result of heat shock protein production. Haemolymph osmolality was elevated only by the 35 degrees C pre-treatment. It is suggested that the unpredictable environment of Marion Island means that P. marioni must always be physiologically prepared to survive cold snaps, and that this year-round cold hardiness therefore supersedes a rapid cold hardening response.     

Temperature adaptation of proteins: engineering mesophilic-like activity and stability in a cold-adapted alpha-amylase

Two multiple mutants of a psychrophilic alpha-amylase were produced, bearing five mutations (each introducing additional weak interactions found in pig pancreatic alpha-amylase) with or without an extra disulfide bond specific to warm-blooded animals. Both multiple mutants display large modifications of stability and activity arising from synergic effects in comparison with single mutations. Newly introduced weak interactions and the disulfide bond confer mesophilic-like stability parameters, as shown by increases in the melting point t(m), in the calorimetric enthalpy DeltaH(cal) and in protection against heat inactivation, as well as by decreases in cooperativity and reversibility of unfolding. In addition, both kinetic and thermodynamic activation parameters of the catalyzed reaction are shifted close to the values of the porcine enzyme. This study confirms the central role of weak interactions in regulating the balance between stability and activity of an enzyme in order to adapt to the environmental temperature.     

The Carboxylesterase Gene Family from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Carboxylesterases hydrolyze esters of short-chain fatty acids and have roles in animals ranging from signal transduction to xenobiotic detoxification. In plants, however, little is known of their roles. We have systematically mined the genome from the model plant Arabidopsis thaliana for carboxylesterase genes and studied their distribution in the genome and expression profile across a range of tissues. Twenty carboxylesterase genes (AtCXE) were identified. The AtCXE family shares conserved sequence motifs and secondary structure characteristics with carboxylesterases and other members of the larger / hydrolase fold superfamily of enzymes. Phylogenetic analysis of the AtCXE genes together with other plant carboxylesterases distinguishes seven distinct clades, with an Arabidopsis thaliana gene represented in six of the seven clades. The AtCXE genes are widely distributed across the genome (present in four of five chromosomes), with the exception of three clusters of tandemly duplicated genes. Of the interchromosomal duplication events, two have been mediated through newly identified partial chromosomal duplication events that also include other genes surrounding the AtCXE loci. Eighteen of the 20 AtCXE genes are expressed over a broad range of tissues, while the remaining 2 (unrelated) genes are expressed only in the flowers and siliques. Finally, hypotheses for the functional roles of the AtCXE family members are presented based on the phylogenetic relationships with other plant carboxylesterases of known function, their expression profile, and knowledge of likely esterase substrates found in plants.     

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.     

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.