The conserved methionine residue of the metzincins: a site-directed mutagenesis study.

The metalloprotease clan of the metzincins derive their name from the presence of a conserved methionine residue that is located on the C-terminal side of the zinc-binding consensus sequence HEXXHXXGXXH. This methionine residue is located in a rather divergent part of the primary sequence but is structurally very well conserved. It is located under the pyramidal base of the three histidine residues that coordinate the catalytic zinc ion and is not involved in any direct contact with the metal nor the substrate. In order to clarify its role, this methionine residue (M226) of the protease C from Erwinia chrysanthemi has been mutated to various other amino acids. The mutants M226L, M226A, M226I were sufficiently stable to be isolated, while the mutants M226H, M226S and M226N could not be purified. The kinetic properties of these mutants were analysed. All mutants showed decreased activity, whereby increases in K(M) as well as decreases in k(cat) were observed. The M226L mutant and M226C-E189 K double mutant, which has the catalytic glutamic acid substituted as well, could be crystallised. The structure of the M226L mutant was determined to a resolution of 2.0 A and refined to R(free) of 0.20. The structure is isomorphous to the wild-type and does not show large differences, with the exception of a very small movement of the zinc-liganding histidine residues. The M226C-E189 K double mutant crystal structure has been refined to an R(free) of 0.20 at 2.1 A resolution. A small rearrangement of the zinc-liganding histidine residues can be detected, which leads to a slightly different zinc coordination and could explain the decrease in activity.     

Effects of diet,habitat, and phylogeny on the fecal microbiome of wild African savanna (Loxodonta africana) and forest elephants (L. cyclotis)

The gut microbiome, or the community of microorganisms inhabiting the digestive tract, is often unique to its symbiont and, in many animal taxa, is highly influenced by host phylogeny and diet. In this study, we characterized the gut microbiome of the African savanna elephant (Loxodonta africana) and the African forest elephant (Loxodonta cyclotis), sister taxa separated by 2.6–5.6 million years of independent evolution. We examined the effect of host phylogeny on microbiome composition. Additionally, we examined the influence of habitat types (forest versus savanna) and diet types (crop‐raiding versus noncrop‐raiding) on the microbiome within L. africana. We found 58 bacterial orders, representing 16 phyla, across all African elephant samples. The most common phyla were Firmicutes, Proteobacteria, and Bacteroidetes. The microbiome of L. africana was dominated by Firmicutes, similar to other hindgut fermenters, while the microbiome of L. cyclotis was dominated by Proteobacteria, similar to more frugivorous species. Alpha diversity did not differ across species, habitat type, or diet, but beta diversity indicated that microbial communities differed significantly among species, diet types, and habitat types. Based on predicted KEGG metabolic pathways, we also found significant differences between species, but not habitat or diet, in amino acid metabolism, energy metabolism, and metabolism of terpenoids and polyketides. Understanding the digestive capabilities of these elephant species could aid in their captive management and ultimately their conservation.     

Pre-Existing Hypoxia Is Associated with Greater EEG Suppression and Early Onset of Evolving Seizure Activity during Brief Repeated Asphyxia in Near-Term Fetal Sheep

Spontaneous antenatal hypoxia is associated with high risk of adverse outcomes, however, there is little information on neural adaptation to labor-like insults. Chronically instrumented near-term sheep fetuses (125 ± 3 days, mean ± SEM) with baseline PaO₂ < 17 mmHg (hypoxic group: n = 8) or > 17 mmHg (normoxic group: n = 8) received 1-minute umbilical cord occlusions repeated every 5 minutes for a total of 4 hours, or until mean arterial blood pressure (MAP) fell below 20 mmHg for two successive occlusions. 5/8 fetuses with pre-existing hypoxia were unable to complete the full series of occlusions (vs. 0/8 normoxic fetuses). Pre-existing hypoxia was associated with progressive metabolic acidosis (nadir: pH 7.08 ± 0.04 vs. 7.33 ± 0.02, p<0.01), hypotension during occlusions (nadir: 24.7 ± 1.8 vs. 51.4 ± 3.2 mmHg, p<0.01), lower carotid blood flow during occlusions (23.6 ± 6.1 vs. 63.0 ± 4.8 mL/min, p<0.01), greater suppression of EEG activity during, between, and after occlusions (p<0.01) and slower resolution of cortical impedance, an index of cytotoxic edema. No normoxic fetuses, but 4/8 hypoxic fetuses developed seizures 148 ± 45 minutes after the start of occlusions, with a seizure burden of 26 ± 6 sec during the inter-occlusion period, and 15.1 ± 3.4 min/h in the first 6 hours of recovery. In conclusion, in fetuses with pre-existing hypoxia, repeated brief asphyxia at a rate consistent with early labor is associated with hypotension, cephalic hypoperfusion, greater EEG suppression, inter-occlusion seizures, and more sustained cytotoxic edema, consistent with early onset of neural injury.     

Antenatal Dexamethasone after Asphyxia Increases Neural Injury in Preterm Fetal Sheep

Background and Purpose

Maternal glucocorticoid treatment for threatened premature delivery dramatically improves neonatal survival and short-term morbidity; however, its effects on neurodevelopmental outcome are variable. We investigated the effect of maternal glucocorticoid exposure after acute asphyxia on injury in the preterm brain.

Methods

Chronically instrumented singleton fetal sheep at 0.7 of gestation received asphyxia induced by complete umbilical cord occlusion for 25 minutes. 15 minutes after release of occlusion, ewes received a 3 ml i.m. injection of either dexamethasone (12 mg, n = 10) or saline (n = 10). Sheep were killed after 7 days recovery; survival of neurons in the hippocampus and basal ganglia, and oligodendrocytes in periventricular white matter were assessed using an unbiased stereological approach.

Results

Maternal dexamethasone after asphyxia was associated with more severe loss of neurons in the hippocampus (CA3 regions, 290±76 vs 484±98 neurons/mm², mean±SEM, P<0.05) and basal ganglia (putamen, 538±112 vs 814±34 neurons/mm², P<0.05) compared to asphyxia-saline, and with greater loss of both total (913±77 vs 1201±75/mm², P<0.05) and immature/mature myelinating oligodendrocytes in periventricular white matter (66±8 vs 114±12/mm², P<0.05, vs sham controls 165±10/mm², P<0.001). This was associated with transient hyperglycemia (peak 3.5±0.2 vs. 1.4±0.2 mmol/L at 6 h, P<0.05) and reduced suppression of EEG power in the first 24 h after occlusion (maximum −1.5±1.2 dB vs. −5.0±1.4 dB in saline controls, P<0.01), but later onset and fewer overt seizures.

Conclusions

In preterm fetal sheep, exposure to maternal dexamethasone during recovery from asphyxia exacerbated brain damage.