Activity of Ca2+/Calmodulin-Dependent Protein Kinase II Following Ischemia: A Comparison Between CA1 and Dentate Gyrus in a Hippocampal Slice Model

Abstract: Both CA1 and dentate gyrus regions of the hippocampal slice exhibit an irreversible loss of synaptic transmission after exposure to in vitro ischemic conditions (buffer without oxygen and glucose). However, after shorter durations of ischemia (8–10 min) the CA1 region shows an irreversible loss of synaptic responses, whereas the dentate gyrus region completely recovers synaptic responses upon reoxygenation. To determine biochemical mechanisms underlying this differential susceptibility, we have examined changes in Ca²⁺/calmodulin-dependent protein kinase II (CaM-KII) and cyclic AMP-dependent protein kinase activities in homogenates from CA1 and dentate gyrus regions of the hippocampal slice after increasing durations of in vitro ischemia. Time-dependent changes in CaM-KII activities were correlated with changes in electrophysiological responses. CA1 homogenates from slices exposed to 1 min of ischemia showed significant increases in CaM-KII activity, whereas there was no significant change in kinase activity in dentate homogenates after 1 min of ischemia. However, after longer durations of ischemia (5, 10, and 20 min) we found a time-dependent reduction in CaM-KII activity in both CA1 and dentate gyrus regions, whereas no change was detected in cyclic AMP-dependent protein kinase activity. Irreversible depression of CaM-KII activity was seen at shorter durations of ischemia (10 min) in the CA1 region than in dentate region (20 min), which correlated with irreversible effects on synaptic responses. Immunoblot analysis showed that the decrease in CaM-KII activity was not due to degradation of CaM-KII protein. However, the microtubule-associated protein MAP2, known to be a substrate for the Ca²⁺-dependent proteases (calpains), showed extensive proteolysis evident after 90 min of reoxygenation after ischemia.     

Litter‐dwelling beetles (Insecta: Coleoptera) can survive in clear‐cutting during subsequent soil ploughing

1. A 2‐year study of litter dwelling beetles was conducted in different mature pine stands and clear‐cuts in Lithuania using the litter sifting method. We hypothesized that clear‐cutting and subsequent ploughing would increase species diversity and the abundance of beetles, and also would encourage the immediate appearance of early‐successional beetle species replacing late successional species in the clear‐cuts.
2. We did not confirm a hypothesis regarding increase in the number of species and abundance of beetles in clear‐cuts.
3. Our hypothesis regarding the immediate appearance of early successional species and disappearance of late‐successional species in clear‐cuts was confirmed.
4. We also revealed that subsequent soil ploughing in clear‐cuts did not accelerate this process, which was linked to the possibility of late successional species surviving in the undisturbed spaces between the strips of ploughed soil.
5. The present study shows that late‐successional forest litter‐dwelling coleopteran species of old pine forests have a better chance of survival in a ploughed clear‐cut, at least in northern Europe, than has been reported in other studies.

     

Neutron Reflectometry Study of the Conformation of HIV Nef Bound to Lipid Membranes

Nef is an HIV-1 accessory protein that directly contributes to AIDS progression. Nef is myristoylated on the N-terminus, associates with membranes, and may undergo a transition from a solution conformation to a membrane-associated conformation. It has been hypothesized that conformational rearrangement enables membrane-associated Nef to interact with cellular proteins. Despite its medical relevance, to our knowledge there is no direct information about the conformation of membrane-bound Nef. In this work, we used neutron reflection to reveal what we believe are the first details of the conformation of membrane-bound Nef. The conformation of Nef was probed upon binding to Langmuir monolayers through the interaction of an N-terminal His tag with a synthetic metal-chelating lipid, which models one of the possible limiting cases for myr-Nef. The data indicate that residues are inserted into the lipid headgroups during interaction, and that the core domain lies directly against the lipid headgroups, with a thickness of ∼40 Å. Binding of Nef through the N-terminal His tag apparently facilitates insertion of residues, as no insertion occurred upon binding of Nef through weak electrostatic interactions in the absence of the specific interaction through the His tag.     

A phylogeny reconstruction of the Dendrophylliidae (Cnidaria,Scleractinia) based on molecular and micromorphological criteria,and its ecological implications

Recent molecular phylogenetic studies have shown that most traditional families of zooxanthellate shallow‐water scleractinians are polyphyletic, whereas most families mainly composed of deep‐sea and azooxanthellate species are monophyletic. In this context, the family Dendrophylliidae (Cnidaria, Scleractinia) has unique features. It shows a remarkable variation of morphological and ecological traits by including species that are either colonial or solitary, zooxanthellate or azooxanthellate, and inhabiting shallow or deep water. Despite this morphological heterogeneity, recent molecular works have confirmed that this family is monophyletic. Nevertheless, what so far is known about the evolutionary relationships within this family, is predominantly based on skeleton macromorphology, while most of its species have remained unstudied from a molecular point of view. Therefore, we analysed 11 dendrophylliid genera, four of which were investigated for the first time, and 30 species at molecular, micromorphological and microstructural levels. We present a robust molecular phylogeny reconstruction based on two mitochondrial markers (COI and the intergenic spacer between COI and 16S) and one nuclear (rDNA), which is used as basis to compare micromorphogical and microstructural character states within the family. The monophyly of the Dendrophylliidae is well supported by molecular data and also by the presence of rapid accretion deposits, which are ca. 5 μm in diameter and arranged in irregular clusters, and fibres that thicken the skeleton organized in small patches of a few micrometres in diameter. However, all genera represented by at least two species are not monophyletic, Tubastraea excluded. They were defined by traditional macromorphological characters that appear affected by convergence, homoplasy and intraspecific variation. Micromorphogical and microstructural analyses do not support the distinction of clades, with the exception of the organization of thickening deposits for the Tubastraea clade.