Patterns of sequence conservation in presynaptic neural genes

Background  

The neuronal synapse is a fundamental functional unit in the central nervous system of animals. Because synaptic function is evolutionarily conserved, we reasoned that functional sequences of genes and related genomic elements known to play important roles in neurotransmitter release would also be conserved.     

Nonsequence-specific DNA recognition: a structural perspective

Chromosomal proteins that form essential architectural components of chromatin bind and bend DNA with an intrinsic low degree of sequence preference. Comparisons made between two recently determined structures of high mobility group (HMG) protein-DNA complexes and other nonsequence-specific protein-DNA complexes reveal the structural basis of this important mode of DNA binding.     

Hormonal control of enzyme activity during the plasma membrane transformation of uterine epithelial cells

Ultrastructural and light microscopic catalytic histochemical methods were used to study the distribution and changes in distribution of four phosphatase enzymes; alkaline phosphatase, 5'-nucleotidase, thiamine pyrophosphatase and adenosine triphosphatase in uterine epithelial cells in response to the ovarian hormones, oestrogen, progesterone or a combination of both used in different regimes on ovariectomised rats. Reaction product for all four enzymes was clearly localised in the epithelial cells, especially with oestrogen priming. However, the four enzymes showed markedly different patterns of organisation of reaction product in response to other hormonal treatments.Our findings clearly show that the expression of these enzymes is under ovarian hormonal control. However, while all of the enzymes are upregulated by oestrogen, the response to progesterone is variable, which can upregulate or downregulate different enzymes. The findings are particularly obvious at the electron microscopic level on the apical plasma membrane of the uterine epithelial cells, which was the main focus of our study.     

Exploration of the DTrp-NMeLys motif in the search for potent somatostatin antagonists

Previous studies from this laboratory demonstrated that N-methylation at Lys(5) residue in somatostatin octapeptide antagonist analogues increased the GH release inhibition potency by as much as 300%. We have now further investigated N-methylation of this Lys(5) residue in conjunction with a number of N- and C-terminal modifications previously found to give highly potent somatostatin receptor antagonists. Synthetic analogues were tested in a functional assay for their ability to inhibit somatostatin-inhibited GH release from rat pituitary cells in culture and to displace 125I-labeled somatostatin from CHO cells transfected with the five known human somatostatin receptors. Several interesting observations resulted from the study. Replacement of liphophilic Nal(8) at the C-terminus with a hydrophilic His(8) resulted in the increased affinity and selectivity for type 2 receptor to give the most potent antagonist analogue yet discovered (K(i), 1.5 nM), although in the rat pituitary cells inhibitory activity on somatostatin inhibited GH release decreased somewhat. A His(3) substitution within the cyclic portion of the analogues retained pituitary cell potency and affinity for type 2 receptor as did substitution with Bip(8) and Fpa(1). Replacement of Cpa(1) with Iph(1) did not effect the affinity for type 2 receptor significantly, but did decrease the effects on rat cell GH release. Iph(3) within-ring substitution increased the selectivity for sst(2) appreciably although the affinity for that receptor was considerably decreased. Substitution of Npa(3) resulted in good selectivity for sst(2) receptor. Replacement of Nal(8) with D-Trp(8) also increased the selectivity for type 2 receptor. Use of a 'bivalent ligand' approach in which two peptides were joined by 4,4'-biphenyldicarbonyl as a spacer destroyed the affinity for all the subtypes, however, the bivalent ligand formed with the Ahp spacer displayed significant affinity and high selectivity for the type 2 receptor.     

Changes in plasma membrane Ca-ATPase and stromal interacting molecule 1 expression levels for Ca2+ signaling in dystrophic mdx mouse muscle

The majority of the skeletal muscle plasma membrane is internalized as part of the tubular (t-) system, forming a standing junction with the sarcoplasmic reticulum (SR) membrane throughout the muscle fiber. This arrangement facilitates not only a rapid and large release of Ca(2+) from the SR for contraction upon excitation of the fiber, but has also direct implications for other interdependent cellular regulators of Ca(2+). The t-system plasma membrane Ca-ATPase (PMCA) and store-operated Ca(2+) entry (SOCE) can also be activated upon release of SR Ca(2+). In muscle, the SR Ca(2+) sensor responsible for rapidly activated SOCE appears to be the stromal interacting molecule 1L (STIM1L) isoform of STIM1 protein, which directly interacts with the Orai1 Ca(2+) channel in the t-system. The common isoform of STIM1 is STIM1S, and it has been shown that STIM1 together with Orai1 in a complex with the partner protein of STIM (POST) reduces the activity of the PMCA. We have previously shown that Orai1 and STIM1 are upregulated in dystrophic mdx mouse muscle, and here we show that STIM1L and PMCA are also upregulated in mdx muscle. Moreover, we show that the ratios of STIM1L to STIM1S in wild-type (WT) and mdx muscle are not different. We also show a greater store-dependent Ca(2+) influx in mdx compared with WT muscle for similar levels of SR Ca(2+) release while normal activation and deactivation properties were maintained. Interestingly, the fiber-averaged ability of WT and mdx muscle to extrude Ca(2+) via PMCA was found to be the same despite differences in PMCA densities. This suggests that there is a close relationship among PMCA, STIM1L, STIM1S, Orai1, and also POST expression in mdx muscle to maintain the same Ca(2+) extrusion properties as in the WT muscle.     

Influences of temperature, oxidative stress, and phosphorylation on binding of heat shock proteins in skeletal muscle fibers

Heat shock proteins (HSPs) help maintain cellular function in stressful situations, but the processes controlling their interactions with target proteins are not well defined. This study examined the binding of HSP72, HSP25, and αB-crystallin in skeletal muscle fibers following various stresses. Rat soleus (SOL) and extensor digitorum longus (EDL) muscles were subjected in vitro to heat stress or strongly fatiguing stimulation. Superficial fibers were "skinned" by microdissection and HSP diffusibility assessed from the extent of washout following 10- to 30 min exposure to a physiological intracellular solution. In fibers from nonstressed (control) SOL muscle, >80% of each HSP is readily diffusible. However, after heating a muscle to 40°C for 30 min ～95% of HSP25 and αB-crystallin becomes tightly bound at nonmembranous myofibrillar sites, whereas HSP72 bound at membranous sites only after heat treatment to ≥44°C. The ratio of reduced to oxidized cytoplasmic glutathione (GSH:GSSG) decreased approximately two- and fourfold after heating muscles to 40° and 45°C, respectively. The reducing agent dithiothreitol reversed HSP72 binding in heated muscles but had no effect on the other HSPs. Intense in vitro stimulation of SOL muscles, sufficient to elicit substantial oxidation-related loss of maximum force and approximately fourfold decrease in the GSH:GSSG ratio, had no effect on diffusibility of any of the HSPs. When skinned fibers from heat-treated muscles were bathed with additional exogenous HSP72, total binding increased approximately two- and 10-fold, respectively, in SOL and EDL fibers, possibly reflective of the relative sarco(endo)plasmic reticulum Ca(2+)-ATPase pump densities in the two fiber types. Phosphorylation at Ser59 on αB-crystallin and Ser85 on HSP25 increased with heat treatment but did not appear to determine HSP binding. The findings highlight major differences in the processes controlling binding of HSP72 and the two small HSPs. Binding was not directly related to cytoplasmic oxidative status, but oxidation of cysteine residues influenced HSP72 binding.     

Interval time-place learning in young children

While previous research has investigated the ability of animals to learn the spatial and temporal contingencies of biologically significant events (known as time-place learning), this ability has not been studied in humans. Children ranging from 5 to 10 years old were tested on a modified interval time-place learning task using a touchscreen computer. Results demonstrate the children were able to quickly learn both the timing and the sequence of this task. Despite a lack of anticipation on baseline trials, the children continued to follow the spatio-temporal contingencies in probe sessions where these contingencies were removed. Performance on the probe sessions provide strong evidence that the children had learned the spatio-temporal contingencies. Future research is needed to determine what age-related changes in iTPL occur. Furthermore, it is argued that this procedure can be used to extend interval timing in research in children, including, but not limited to, investigation of scalar timing with longer durations than have previously been investigated.     

Distinct cortical circuit mechanisms for complex forelimb movement and motor map topography

Cortical motor maps are the basis of voluntary movement, but they have proven difficult to understand in the context of their underlying neuronal circuits. We applied light-based motor mapping of Channelrhodopsin-2 mice to reveal a functional subdivision of the forelimb motor cortex based on the direction of movement evoked by brief (10?ms) pulses. Prolonged trains of electrical or optogenetic stimulation (100-500?ms) targeted to anterior or posterior subregions of motor cortex evoked reproducible complex movements of the forelimb to distinct positions in space. Blocking excitatory cortical synaptic transmission did not abolish basic motor map topography, but the site-specific expression of complex movements was lost. Our data suggest that the topography of?movement maps arises from their segregated output projections, whereas complex movements evoked by prolonged stimulation require intracortical synaptic transmission.     

Inactivation of Pyruvate Dehydrogenase Kinase 2 by Mitochondrial Reactive Oxygen Species

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H₂O₂) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H₂O₂ derives from superoxide (O₂^˙̄), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O₂^˙̄ production, such as may occur under nutrient excess and low ATP demand, the increase in O₂^˙̄ and H₂O₂ may provide feedback signals to modulate mitochondrial metabolism.