Ontogenetic features of autonomic homeostasis in children according to the data of variation pulsometry

A method of variation pulsometry was used to determine the vegetative homeostasis state in 20 children aged from 4 to 14. Normotonic type of the cardiac rhythm regulation was observed in a group of children aged 4-5. In children aged 12-14 the effect of sympathetic area of the vegetative nervous system decreased while the effect of the parasympathetic one increased. The tension index, effect of the humoral regulation channel and the degree of cardiac rhythm control centralization decreased.     

Structuro-functional characteristics of aldolase from rabbit muscles in diabetes

The structural peculiarities of rabbit muscle aldolase accompanying enhancement of the aldolase activity in diabetes are described from the data of tryptophan phosphorescence at the room temperature and fluorescence polarization. It is shown that the pathology-concomitant conformational changes occur in both the hydrophobic part and NAD-binding site of the enzyme. The character of the structural changes in the hydrophobic part of the protein in diabetes and an increase in the enzymic activity are similar to that observed in normal aldolase after its interaction with NADH and are believed to be associated with the enhancement of the rigidity in the Trp-147 environment.     

Stabilizing effect of oligomerization on aldolase protomers in rabbit muscles

It is shown by the fluorimetric analysis that with the 1,2 M MgCl2-induced dissociation of rabbit muscle aldolase the tertiary structure of the resulted protomers (subunits) remains practically unchanged. Significant changes in the protomeric enzyme are provoked by subsequent addition of urea up to the concentration of 2,3 M, and are, evidently, manifested in a significant decrease in regularity of the hydrophobic part of aldolase and in possible transition of its Trp-147 into more polar environment. This transition is reflected in the longwave shift of the protein fluorescence maximum (lambda max) by 13 nm (from 320 to 333 nm). But the joint action of MgCl2 and urea does not lead to complete unfolding of the resulted protomeric enzyme. More deep structural alterations in the subunits occur on acidic dissociation, and lambda max shift in this case reaches 342 nm. Structural changes caused by MgCl2 and urea are concomitant with the increase of fluorescence quenchibility with NADH. Here a short-wave lambda max shift, being usually observed in native aldolase fluorescence quenching, is not registered. This mean that the photoselection of protein fluorophores does not occur. The results thus obtained produce an evidence that oligomerization endows aldolase protomers with enhanced stability.     

Effect of NADH on the catalytic function and reactivity of thiols from rabbit muscle aldolase

NADH has a corresponding binding site in aldolase, and can activate the reaction of the aldole cleavage of the substrate (fructose-1,6-bisphosphate). Unlike the considerable protection by the substrate, the similar effect of NADH on the sulphydryl enzyme groups is less pronounced, and may be attributed to single cysteine residue. The functionally related and spatially separated binding sites for NADH and substrate are suggested.     

Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS

Background

The blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), and blood-cerebrospinal fluid barrier (BCSFB) control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease.

Methodology/Principal Findings

Evans Blue (EB) dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage.

Conclusions/Significance

Results suggest that the BSCB is compromised in areas of motor neuron degeneration in ALS mice at both early and late stages of the disease.     

Comparative study of the structural characteristics of aldolase in rabbit muscles in normal states and in alloxan diabetes

It is shown that the activity of aldolase synthesized in rabbit muscles under diabetes is higher than that at normal state. This fact is probably a result of some structural alterations in NAD-binding site with Trp-291 and -311 in it which overlaps a considerable part of C-terminal region of the protein. The hydrophobic part of the enzyme containing Trp-147 under diabetes seems to remain unaltered. This consideration is based on the longwave shift in aldolase fluorescence lambda max (from 320 to 324 nm) under this pathology, suggesting a transition of Trp-291 and -311 into more polar environment and is confirmed by the disappearance of the difference in lambda max in the NADH presence. The NADH-originated shift in lambda max position for the both proteins ended at the same wave-length at 314 nm. The position of lambda max at 324 nm resulting from possible structural modification of NAD-binding site under diabetes correlates with an increase in the Stern-Volmer quenching constant value (from 4359 to 7500 M-1 for aldolase under normal and diabetic states, respectively). These quenching data evidence in favour of the suggestion on the existence of two classes of tryptophanyls in the aldolase molecule.     

Characterization of the isozymes of pyruvate dehydrogenase phosphatase: implications for the regulation of pyruvate dehydrogenase activity

The activity of mammalian pyruvate dehydrogenase complex (PDC) is regulated by a phosphorylation/dephosphorylation cycle. Dephosphorylation accompanied by activation is carried out by two genetically different isozymes of pyruvate dehydrogenase phosphatase, PDP1c and PDP2c. Here, we report data showing that PDP1c and PDP2c display marked biochemical differences. The activity of PDP1c strongly depends upon the simultaneous presence of calcium ions and the E2 component of PDC. In contrast, the activity of PDP2c displays little, if any, dependence upon either calcium ions or E2. Furthermore, PDP2c does not appreciably bind to PDC under the conditions when PDP1c exists predominantly in the PDC-bound state. The stimulatory effect of E2 on PDP1c can be partially mimicked by a monomeric construct consisting of the inner lipoyl-bearing domain and the E1-binding domain of E2 component. This strongly suggests that the E2-mediated activation of PDP1c largely reflects the effects of co-localization and mutual orientation of PDP1c and E1 component facilitated by their binding to E2. Both PDP1c and PDP2c can efficiently dephosphorylate all three phosphorylation sites located on the alpha chain of the E1 component. For PDC phosphorylated at a single site, the relative rates of dephosphorylation of individual sites are: 2>site 3>site 1. Phosphorylation of sites 2 or 3 in addition to site 1 does not have a significant effect on the rates of dephosphorylation of individual sites by PDP1c, suggesting a random mechanism of dephosphorylation. In contrast, there is a significant decrease in the overall rate of dephosphorylation of pyruvate dehydrogenase by PDP2c under these conditions. This indicates that the mechanism of dephosphorylation of PDC phosphorylated at multiple sites by PDP2c is not purely random. These marked differences in the site-specificity displayed by PDP1c and PDP2c should be particularly important under conditions such as starvation and diabetes, which are associated with a great increase in phosphorylation of sites 2 and 3 of pyruvate dehydrogenase.     

Structural transitions in polycytidylic acid: proton buffer capacity data

The pH-dependences of proton buffer capacity of poly(C) were computed on the basis of the literature data. In these curves there were observed four peaks: two narrow and two wide ones. The first narrow peak reflects the process of cooperative formation of double helices, which is induced by protonation of the N3 atom of nucleotide bases. The first wide peak is assigned to noncooperative process of poly(C) double helices protonation at the N3 nitrogen atom. It is proposed that the second wide peak corresponds to noncooperative protonation of the neutral cytosine bases at the oxygen atom. This reaction causes cooperative dissociation of the poly(C) double helices. The second narrow peak reflects the dissociation process.