The more adaptive to change, the more likely you are to survive: Protein adaptation in extremophiles.

Discovering how organisms and their proteins adapt to extreme conditions is a complicated process. Every condition has its own set of adaptations that make it uniquely stable in its environment. The purpose of our review is to discuss what is known in the extremophilic community about protein adaptations. To simplify our mission, we broke the extremophiles into three broad categories: thermophiles, halophiles and psychrophiles. While there are crossover organisms- organisms that exist in two or more extremes, like heat plus acid or cold plus pressure, most of them have a primary adaptation that is within one of these categories which tends to be the most easily identifiable one. While the generally known adaptations are still accepted, like thermophilic proteins have increased ionic interactions and a hardier hydrophobic core, halophilic proteins have a large increase in acidic amino acids and amino acid/peptide insertions and psychrophiles have a much more open structure and reduced ionic interactions, some new information has come to light. Thermophilic stability can be improved by increased subunit-subunit or subunit-cofactor interactions. Halophilic proteins have reversible folding when in the presence of salt. Psychrophilic proteins have an increase in cavities that not only decrease the formation of ice, but also increase flexibility under low temperature conditions. In a proof of concept experiment, we applied what is currently known about adaptations to a well characterized protein, malate dehydrogenase (MDH). While this protein has been profiled in the literature, we are applying our adaptation predictions to its sequence and structure to see if the described adaptations apply. Our analysis demonstrates that thermophilic and halophilic adaptations fit the corresponding MDHs very well. However, because the number of psychrophiles MDH sequences and structures is low, our analysis on psychrophiles is inconclusive and needs more information. By discussing known extremophilic adaptations and applying them to a random, conserved protein, we have found that general adaptations are conserved and can be predicted in proposed extremophilic proteins. The present field of extremophile adaptations is discovering more and more ways organisms and their proteins have adapted. The more that is learned about protein adaptation, the closer we get to custom proteins, designed to fit any extreme and solve some of the world's most pressing environmental problems.

In contrast to kindled seizures, the frequency of spontaneous epilepsy in the limbic status model correlates with greater aberrant fascia dentata excitatory and inhibitory axon sprouting, and increased staining for N-methyl-D-aspartate, AMPA and GABA(A) receptors.

This study determined whether there were differences in hippocampal neuron loss and synaptic plasticity by comparing rats with spontaneous epilepsy after limbic status epilepticus and animals with a similar frequency of kindled seizures. At the University of Virginia, Sprague-Dawley rats were implanted with bilateral ventral hippocampal electrodes and treated as follows; no stimulation (electrode controls; n=5): hippocampal stimulation without status (stimulation controls; n=5); and limbic status from continuous hippocampal stimulation (n=12). The limbic status group were electrographically monitored for a minimum of four weeks. Four rats had no recorded chronic seizures (status controls), and all three control groups showed no differences in hippocampal pathology and were therefore incorporated into a single group (controls). Eight limbic status animals eventually developed chronic epilepsy (spontaneous seizures) and an additional eight rats were kindled to a similar number and frequency of stage 5 seizures (kindled) as the spontaneous seizures group. At the University of California (UCLA) the hippocampi were processed for: (i) Niss1 stain for densitometric neuron counts; (ii) neo-Timm's histochemistry for mossy fiber sprouting; and (iii) immunocytochemical staining for glutamate decarboxylase, N-methyl-D-aspartate receptor subunit 2, AMPA receptor subunit 1 and the GABA(A) receptor. In the fascia dentata inner and outer molecular layers the neo-Timm's stain and immunoreactivity was quantified as gray values using computer image analysis techniques. Statistically significant results (P<0.05) showed the following. Compared to controls and kindled animals, rats with spontaneous seizures had: (i) lower neuron counts for the fascia dentata hilus, CA3 and CA1 stratum pyramidale; (ii) greater supragranular inner molecular layer mossy fiber staining; and (iii) greater glutamate decarboxylase immunoreactivity in both molecular layers. Greater supragranular excitatory mossy fiber and GABAergic axon sprouting correlated with: (i) increases in N-methyl-D-aspartate receptor subunit 2 inner molecular layer staining; (ii) more AMPA receptor subunit 1 immunoreactivity in both molecular layers; and (iii) greater outer than inner molecular layer GABA(A) immunoreactivity. Furthermore, in contrast to kindled animals, rats with spontaneous seizures showed that increasing seizure frequency per week and the total number of natural seizures positively correlated with greater Timm's and GABAergic axon sprouting, and with increases in N-methyl-D-aspartate receptor subunit 2 and AMPA receptor subunit 1 receptor staining. In this rat limbic status model these findings indicate that chronic seizures are associated with hippocampal neuron loss, reactive axon sprouting and increases in excitatory receptor plasticity that differ from rats with an equal frequency of kindled seizures and controls. The hippocampal pathological findings in the limbic status model are similar to those in humans with hippocampal sclerosis and mesial temporal lobe epilepsy, and support the hypothesis that synaptic reorganization of both excitatory and inhibitory systems in the fascia dentata is an important pathophysiological mechanism that probably contributes to or generates chronic limbic seizures.

Design and construction of a long-term continuous video-EEG monitoring unit for simultaneous recording of multiple small animals.

In recent years several new rat models of human limbic/mesial temporal lobe epilepsy have been described [1,2,4-7,11,15-17]. Unlike earlier models such as kindling in which the seizures are induced by an exogenous stimulus, these new models are characterized by seizures that occur spontaneously at random intervals. Although the spontaneity of the seizures makes these models more like human epilepsy, documentation of these seizures by direct observation is highly inefficient, and sub-behavioral electrographic seizures could be missed. Continuous paper EEG and video recording have been used [5-7,15], but these techniques are resource intensive. The slow paper speed required by long-term paper recordings limits the ability to differentiate between true seizure activity and electrical artifact. Subtle behavioral seizures are likely to be missed during rapid review of video recordings alone [16]. Ambulatory cassette EEG recordings have been used [3], but the systems require expensive proprietary hardware, and the systems have limited channels for recording (8-16). To improve the utility of the models, we developed a long-term EEG/video monitoring system to detect the electrographic seizures and document their behavioral accompaniment. The system is based on commercially available components, including a computerized EEG seizure detection system that was initially developed for human seizure monitoring [8,9,13]. Seizures are reliably detected and the data are reduced so that 24 h of recording can be reviewed in 30-90 min. Although the computer program is accurate, special care must be taken in system design and construction to reduce sources of electrical artifact that can cause false detections when multiple animals are recorded simultaneously on a single EEG machine. During data review it is necessary to differentiate between electrical artifact induced by animal activity from true seizure activity by key EEG patterns. Certain seizure patterns (less than 3 hz. low amplitude) will not be detected by the seizure detection program, but the system is highly effective for typical limbic seizures and may be useful for the animal models of absence epilepsy [12,14]. It can also be used as a continuous or intermittent EEG/physiological recording device for experiments that examine animals' spontaneous behavior and the EEG correlate (e.g. sleep/wake cycles, learning and memory tasks).