Animal mortality and illegal poison bait use in Greece.

The present study describes the use of poison baits against so-called pest species in Greece and explores various aspects of this illegal practice. Data were collected from 2000 to 2016, and a total of 1015 poisoning incidents in rural areas causing the death of 3248 animals were examined. In 58.7% of investigated cases, the motives remained unknown; in the remaining cases, human-wildlife conflicts and retaliatory actions among stakeholders (e.g., hunters vs. livestock breeders) were found to be the main reasons for poison bait use. The target animals for these actions were mainly mammalian carnivores, and stray canids, all of which were blamed for livestock and game losses. Avian scavengers were the wildlife species most affected by secondary poisoning (30% of the wildlife fatalities), whereas shepherd dogs accounted for 66.4% of domestic animal losses. Toxicological analyses showed that a wide range of chemical substances were used, mostly legal or banned pesticides (e.g., carbamates, organophosphates, and organochlorines) and potassium cyanide. Furthermore, the widespread trafficking of black marketed insecticides was also recorded, with methomyl (in powder form) and carbofuran being most common. The majority of poisoning events (72%) took place outside protected areas, while in approximately 73.4% of them, no official reporting to the competent authorities was made. Overall, the study highlights the significant impact of illegal poison bait use on wildlife in Greece and addresses its extreme socioeconomic complexity. The need for an integrated national anti-poison strategy is discussed.

Early ischaemic preconditioning of spinal cord enhanced the binding profile of heat shock protein 70 with neurofilaments and promoted its nuclear translocation after thoraco-abdominal aortic occlusion in pigs.

OBJECTIVE(S): Heat shock protein 70 (Hsp70) is detected in substantial amounts in normal neurons and this basal content may protect a cell against harmful conditions without the need for additional synthesis. Herein, we investigate the potential protective role of these basal levels of Hsp70, in an early ischaemic preconditioning (IPC) experimental model, suggesting a possible role of this protein as a first window of protection. DESIGN, MATERIAL AND METHODS: Forty-two pigs were used in an experimental thoraco-abdominal aortic occlusion model. Twelve animals (two groups) were used for neurological evaluation. The remaining 30 animals (five groups) were used for immunoprecipitation and immunohistochemical studies. These were performed to study the binding relationship of Hsp70/cytoskeleton elements and the cellular distribution of Hsp70, respectively. RESULTS: The IPC + ischaemia-group showed significant better neurologic scores compared with those of the ischaemia group, indicating a protective role for IPC (P = 0.003). The immunoprecipitations demonstrated that early IPC increased significantly the binding profile of Hsp70/neurofilaments (P = 0.025). In addition, translocation of Hsp70 into the nucleus was observed, which was conserved until the sustained ischaemia. CONCLUSIONS: These results indicate that Hsp70 may have an important role in early IPC of the spinal cord, by protecting neurofilaments and by ensuring the functionality and the integrity of the nucleus, at the time the intensive insult begins.

HSC70 interactions with SV40 viral proteins differ between permissive and nonpermissive mammalian cells.

SV40 belongs to a group of DNA tumor viruses which induce the expression of the 70 Kd heat shock proteins, but the meaning of this induction remains unclear. Investigating the role of hsc70 in the SV40 life cycle, we found that the protein translocates to the nucleus late in infection of permissive CV1 cells, in contrast to infected nonpermissive BALB/3T3 and NIH/3T3 cells in which hsc70 remains cytoplasmic. Moreover, the pattern of hsc70 nuclear staining was diffused and clearly distinguishable from that observed after heat shock. In addition hsc70 late in infection coimmunoprecipitated with the viral capsid protein VP1, suggesting a role in the process of viral packaging. Interactions of hsc70 with the early viral oncoprotein T antigen were observed only in nonpermissive cells, indicating that the binding of the above proteins is specific to cells that do not support viral propagation. Finally, treatment of permissive CV1 cells with interferon gamma, a known antiviral cytokine, resulted in hsc70 binding to T antigen. Our results suggest that the role of hsc70 in the process of SV40 infection is directly related to the ability of the host cells to support viral propagation and is clearly different between permissive and nonpermissive cell lines.

Aggregation of hsp70 and hsc70 in vivo is distinct and temperature-dependent and their chaperone function is directly related to non-aggregated forms.

We used non-denaturing gradient analysis of cell extracts before and after heat treatment of the cells and showed that hsp70 and hsc70 aggregate in vivo in a temperature-dependent fashion. Their aggregation profiles were found to be clearly distinguishable and sensitive to ATP depletion. Pore exclusion limit electrophoresis showed that these two proteins are mainly found in autoaggregated forms including dimers, trimers and oligomers. The addition of denatured luciferase to the cell extracts reversed the aggregation of both proteins towards their non-aggregated forms. Immunoprecipitation and Western-blot analysis showed that the non-aggregated form is the only one bound to denatured luciferase. Our results suggest that aggregated hsp70 and hsc70 represent predominantly self-associated molecules unable to exert chaperone activity. The cochaperone hsp40 was also found to be aggregated and, on addition of denatured luciferase, its aggregation was reversed to a non-aggregated state. Immunoprecipitation analysis indicated that hsp40 forms a complex with the non-aggregated form of hsc70 and denatured luciferase. These results confirm previous in vitro studies and support the suggestion that in vivo cytosolic hsp70 and hsc70 exist mainly in an oligomer-monomer equilibrium which is dependent on the environmental temperature, the levels of ATP and the presence of denatured proteins.

Pharmacological modulation of heat shock factor 1 by antiinflammatory drugs results in protection against stress-induced cellular damage.

The activation of heat shock genes by diverse forms of environmental and physiological stress has been implicated in a number of human diseases, including ischemic damage, reperfusion injury, infection, neurodegeneration, and inflammation. The enhanced levels of heat shock proteins and molecular chaperones have broad cytoprotective effects against acute lethal exposures to stress. Here, we show that the potent antiinflammatory drug indomethacin activates the DNA-binding activity of human heat shock transcription factor 1 (HSF1). Perhaps relevant to its pharmacological use, indomethacin pretreatment lowers the temperature threshold of HSF1 activation, such that a complete heat shock response can be attained at temperatures that are by themselves insufficient. The synergistic effect of indomethacin and elevated temperature is biologically relevant and results in the protection of cells against exposure to cytotoxic conditions.

Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic myocardial recovery.

Heat shock treatment induces expression of several heat shock proteins and subsequent post-ischemic myocardial protection. Correlations exist between the degree of stress used to induce the heat shock proteins, the amount of the inducible heat shock protein 70 (HSP70) and the level of myocardial protection. The inducible HSP70 has also been shown to be protective in transfected myogenic cells. Here we examined the role of human inducible HSP70 in transgenic mouse hearts. Overexpression of the human HSP70 does not appear to affect normal protein synthesis or the stress response in transgenic mice compared with nontransgenic mice. After 30 min of ischemia, upon reperfusion, transgenic hearts versus nontransgenic hearts showed significantly improved recovery of contractile force (0.35 +/- 0.08 versus 0.16 +/- 0.05 g, respectively, P < 0.05), rate of contraction, and rate of relaxation. Creatine kinase, an indicator of cellular injury, was released at a high level (67.7 +/- 23.0 U/ml) upon reperfusion from nontransgenic hearts, but not transgenic hearts (1.6 +/- 0.8 U/ml). We conclude that high level constitutive expression of the human inducible HSP70 plays a direct role in the protection of the myocardium from ischemia and reperfusion injury.

The hsc70 gene which is slightly induced by heat is the main virus inducible member of the hsp70 gene family.

We have found that SV40 infection of CV1 cells induces the synthesis of a 72 kDa protein that upon molecular cloning was shown to be the product of the hsc70 gene. The above gene product was found to be mainly virus inducible, in contrast to the hsp70 gene product which was mainly heat inducible. The two genes were found to be cell cycle regulated in a distinctively different manner.

Constitutive expression of heat-shock protein 70 in mammalian cells confers thermoresistance.

A 70-kDa heat-shock-protein (hsp 70) expression vector which contains the human hsp 70 gene linked to the human beta-actin promoter, was constructed and used to transfect CV1 monkey cells. Stably transfected CV1 clones were isolated which constitutively synthesized increased amounts of hsp70 at normal temperature. It is shown that these clones are resistant to elevated temperature. This finding indicates that hsp70 is involved in the protection of the cells against a lethal heat treatment and maybe responsible for the phenomenon of thermotolerance.

Specific inhibition of simian virus 40 protein synthesis by heat and arsenite treatment.

The effects of heat treatment of CV1 cells infected with simian virus 40 (SV40) on viral and cellular protein synthesis were investigated by one-dimensional and two-dimensional polyacrylamide gel electrophoresis. A 12-h heat treatment during the late phase of the viral life-cycle inhibits VP1 synthesis. No inhibition of normal cellular proteins is apparent, but heat-shock proteins are strongly induced and accumulate in the cells. Inhibition of VP1 synthesis in infected cells is demonstrated to occur also after arsenite treatment, another agent known to induce heat-shock proteins. Northern blot analysis of cytoplasmic RNA demonstrated a decrease in the abundance of late SV40 mRNAs thus showing that the inhibition occurs at the transcriptional or immediately post-transcriptional level. Cumulative labeling with [3H]thymidine of viral DNA showed that the decrease in the abundance of late mRNAs is not due to a blocking of viral DNA synthesis. Immunofluorescence microscopy and immunoprecipitation analysis show that heat and arsenite treatments also affect the synthesis of T antigen. These results suggest that heat-shock proteins may play a role in the inhibition of SV40 virus gene functions.