The effect of acute heat exposure on rat pituitary corticotroph activation: the role of vasopressin

The increased ambient temperature affects the function of hypothalamic-pituitary-adrenal (HPA) axis. Since the correlation among vasopressin (VP), adrenocorticotropic hormone (ACTH) and corticosterone (CORT) responses to various stressors have been long recognized, the aim of this study was to reveal the aforementioned hormones production and morphology of the pituitary gland after exposure to acute heat. Rats were exposed to high ambient temperature (38 °C) for 20 or 60 minutes. The circulating hormones were determined by an ELISA test or chemiluminescence's method. The results obtained show the elevation in ACTH and CORT secretion depending on the duration of heat exposure. The VP concentration increased only after prolonged exposure to heat (60 min). The pituitary morphology was examined by routine and fluorescent immunohistochemistry as well as electron microscopy. Observed changes in the anterior and posterior pituitary well corresponded to circulating hormones, regarding the volume density of ACTH-immunopositive cells, percentage of ACTH immunopositive area v. total area and number of VP-immunopositive containing varicose fibers per total area. Acute heat exposure also induced changes in shapes of ACTH-immunopositive cells. Cells appeared stellate with numerous slender cytoplasmic processes and degranulated, which is the most obvious after 20 min. In addition, immunopositivity of endothelial and anterior pituitary cells for VP suggests its influence on ACTH secretion.     

Testing the species pool hypothesis for mire vegetation: exploring the influence of pH specialists and habitat history

The Evolutionary species pool hypothesis (ESPH) predicts that historically more common habitats will be richer in species because they have had greater opportunity for the evolution of suitably adapted species. We explored the relationship between mire species richness and pH, an important environmental variable in mires, in two regions that differ in habitat pH distribution: the West Carpathians and Bulgaria. Mire habitats in both the West Carpathians and Bulgaria demonstrate support for the ESPH prediction that habitats with more common pH values host more species. We also explored the influence of habitat history by examining the distribution of generalists and specialists along gradients of habitat pH, using extensive community-level vegetation data from European mires in these two regions. We found a striking pattern with the distribution of pH-specialists having three distinct peaks in both regions, whereas the total species pool peaked in near neutral pH habitats in both regions. Because the peaks in specialist richness do not correspond to regional pH distribution patterns, we hypothesize that historical explanations may be important, and that habitats currently rich in pH-specialists may have historically acted as pleniglacial refugia for many mire species. Our findings support the general predictions of the ESPH, but further suggest that historical processes such as patterns of glacial refugia, may significantly influence contemporary species distributions and the diversity of plant species in mire habitats.     

The role of the S-S bridge in retroviral protease function and virion maturation

Retroviral proteases are translated as a part of Gag-related polyproteins, and are released and activated during particle release. Mason-Pfizer monkey virus (M-PMV) Gag polyproteins assemble into immature capsids within the cytoplasm of the host cells; however, their processing occurs only after transport to the plasma membrane and subsequent release. Thus, the activity of M-PMV protease is expected to be highly regulated during the replication cycle. It has been proposed that reversible oxidation of protease cysteine residues might be responsible for such regulation. We show that cysteine residues in M-PMV protease can form an intramolecular S-S bridge. The disulfide bridge shifts the monomer/dimer equilibrium in favor of the dimer, and increases the proteolytic activity significantly. To investigate the role of this disulfide bridge in virus maturation and replication, we engineered an M-PMV clone in which both protease cysteine residues were replaced by alanine (M-PMV(PRC7A/C106A)). Surprisingly, the cysteine residues were dispensable for Gag polyprotein processing within the virus, indicating that even low levels of protease activity are sufficient for polyprotein processing during maturation. However, the long-term infectivity of M-PMV(PRC7A/C106A) was noticeably compromised. These results show clearly that the proposed redox mechanism does not rely solely on the formation of the stabilizing S-S bridge in the protease. Thus, in addition to the protease disulfide bridge, reversible oxidation of cysteine and/or methionine residues in other domains of the Gag polyprotein or in related cellular proteins must be involved in the regulation of maturation.     

Comparison of detection methods for Acarus siro (Acari: Acaridida: Acarididae) contamination in grain

Acarus siro L. 1758 (Acari: Acaridida: Acarididae) is an important pest of stored grain because it contaminates the grain by allergens and transfers pathogenous microorganisms. Rapid detection of contamination enables to intercept an early grain infestation by the pest. In this study, we compared the usability and efficiency of various detection approaches. Under laboratory conditions, grain samples of various sizes were infested by different levels of the following contaminants: eggs, adults, and feces of A. siro. The samples were analyzed by enzyme-linked immunosorbent assay (ELISA) by using anti-A. siro polyclonal antibody (immunochemical method), extracted in Berlese-Tullgren funnels, sieved, and processed by filth-flotation (conventional methods). The adults or juveniles of A. siro could be detected by all the three tested conventional methods and ELISA with detection limits in the range from 221 to 1,157 mites/kg grain. Eggs were detected by filth-flotation only; the detection limit was 1,950 eggs/kg grain. The feces of A. siro were detectable by ELISA test, only. ELISA enabled the detection of the feces with the minimal threshold level of 1.04 microg feces/g grain; it means the assay allowed to trace less than one metabolically active mite per gram of grain. The study thus demonstrated that reliable A. siro detection in grain can only be achieved by combining different detection methods. European Union and U.S. administratives dictate zero or near-zero tolerance level for mite infestation in stored products. This demand is difficult to fulfill, because every detection method is limited by its detection limit; thus, it is hard to reliably detect infestation levels lower than obtained detection limits. This methodical limitation is discussed in context with the determined detection limits of the tested methods.     

Thermodynamic benchmark study using Biacore technology

A total of 22 individuals participated in this benchmark study to characterize the thermodynamics of small-molecule inhibitor-enzyme interactions using Biacore instruments. Participants were provided with reagents (the enzyme carbonic anhydrase II, which was immobilized onto the sensor surface, and four sulfonamide-based inhibitors) and were instructed to collect response data from 6 to 36 degrees C. van't Hoff enthalpies and entropies were calculated from the temperature dependence of the binding constants. The equilibrium dissociation and thermodynamic constants determined from the Biacore analysis matched the values determined using isothermal titration calorimetry. These results demonstrate that immobilization of the enzyme onto the sensor surface did not alter the thermodynamics of these interactions. This benchmark study also provides insights into the opportunities and challenges in carrying out thermodynamic studies using optical biosensors.     

Interphase microtubules determine the initial alignment of the mitotic spindle

In the fission yeast Schizosaccharomyces pombe, interphase microtubules (MTs) position the nucleus [1, 2], which in turn positions the cell-division plane [1, 3]. It is unclear how the spindle orients, with respect to the predetermined division plane, to ensure that the chromosomes are segregated across this plane. It has been proposed that, during prometaphase, the astral MT interaction with the cell cortex aligns the spindle with the cell axis [4] and also participates in a spindle orientation checkpoint (SOC), which delays entry into anaphase as long as the spindle is misaligned [5-7]. Here, we trace the position of the spindle throughout mitosis in a single-cell assay. We find no evidence for the SOC. We show that the spindle is remarkably well aligned with the cell longitudinal axis at the onset of mitosis, by growing along the axis of the adjacent interphase MT. Misalignment of nascent spindles can give rise to anucleate cells when spindle elongation is impaired. We propose a new role for interphase microtubules: through interaction with the spindle pole body, interphase microtubules determine the initial alignment of the spindle in the subsequent cell division.     

The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling

Although the Arabidopsis thaliana genome contains genes encoding 20 mitogen-activated protein kinases (MAPKs) and 10 MAPK kinases (MAPKKs), most of them are still functionally uncharacterized. In this work, we analyzed the function of the group B MAPK kinase, MKK3. Transgenic ProMKK3:GUS lines showed basal expression in vascular tissues that was strongly induced by Pseudomonas syringae pv tomato strain DC3000 (Pst DC3000) infection but not by abiotic stresses. The growth of virulent Pst DC3000 was increased in mkk3 knockout plants and decreased in MKK3-overexpressing plants. Moreover, MKK3 overexpression lines showed increased expression of several PR genes. By yeast two-hybrid analysis, coimmunoprecipitation, and protein kinase assays, MKK3 was revealed to be an upstream activator of the group C MAPKs MPK1, MPK2, MPK7, and MPK14. Flagellin-derived flg22 peptide strongly activated MPK6 but resulted in poor activation of MPK7. By contrast, MPK6 and MPK7 were both activated by H(2)O(2), but only MPK7 activation was enhanced by MKK3. In agreement with the notion that MKK3 regulates the expression of PR genes, ProPR1:GUS expression was strongly enhanced by coexpression of MKK3-MPK7. Our results reveal that the MKK3 pathway plays a role in pathogen defense and further underscore the importance and complexity of MAPK signaling in plant stress responses.