X-ray, spectroscopic and normal-mode dynamics of calexcitin: structure-function studies of a neuronal calcium-signalling protein.

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

Haematologic determinants of left atrial spontaneous echo contrast in mitral stenosis.

OBJECTIVES: To determine if a relationship exists in mitral stenosis, in patients with either sinus rhythm or atrial fibrillation, between left atrial spontaneous echo contrast and the haematologic indices haematocrit, red cell concentration, mean corpuscular volume, platelet count and volume. METHODS: Left atrial spontaneous echo contrast severity was graded on a scale of 0-4 in 163 patients with symptomatic mitral stenosis (84 patients in sinus rhythm, 79 patients in atrial fibrillation) undergoing transesophageal echocardiography, cardiac catheterization and full blood examination as part of assessment prior to balloon mitral valvuloplasty. RESULTS: In sinus rhythm, spontaneous echo contrast grade was negatively correlated with cardiac index (r=-0.33), mitral valve area (r=-0.25) and mitral regurgitation grade (r=-0.22) and positively correlated with haematocrit (r=0.24) and red cell concentration (r=0.25). Spontaneous echo contrast grade was not correlated with left atrial diameter or mean corpuscular volume. In atrial fibrillation, spontaneous echo contrast grade was also negatively correlated with mitral valve area (r=-0.25) and mitral regurgitation (r=-0.36) but was positively correlated with left atrial diameter (r=0.34) and was not correlated with cardiac index, haematocrit or red cell concentration. There was no correlation between spontaneous echo contrast grade and platelet variables in either group. CONCLUSIONS: Natural variation in red cell concentration in patients with symptomatic mitral stenosis was an independent predictor of the severity of left atrial spontaneous echo contrast in sinus rhythm, but no relationship between red cell concentration and spontaneous echo contrast grade was evident in atrial fibrillation.

Balloon mitral valvuloplasty at Monash Medical Centre: follow up of 201 procedures over an 11-year period.

Over a period of 11 years from 1988 to 1999, 201 patients underwent balloon mitral valvuloplasty (BMV) at Monash Medical Centre, Australia. Before BMV,133 patients (66%) were symptomatic with minimal activity or at rest. BMV increased mitral valve area and cardiac output, and reduced transmitral, left atrial and pulmonary pressures, with infrequent procedural complications (<8%). At the initial 3-month follow up after BMV, symptoms were absent or minimal in 178 patients (89%), with 85% remaining event free at 12 months. At long-term follow up (median: 30 months; range: 0-129 months), cumulative event-free survival was 73% after 5 years. After BMV, 37 patients (18%) underwent mitral valve surgery, while a repeat BMV was performed in three patients (1.5%). The results of this series provide additional data for the growing body of evidence which suggests that BMV is a relatively safe and effective procedure for producing long-term benefit in patients with symptomatic mitral stenosis.

Phosphate binders do not compromise the copper status of adult renal disease patients on hemodialysis.

The effects of phosphate binder therapies (calcium carbonate, calcium acetate, or aluminum hydroxide) on copper status were assessed in 88 adult patients with end-stage renal disease on hemodialysis. Subjects were divided into two groups based on binder therapy compliance as specified by medical criteria established by the Health Care Financing Administration. Binder compliant subjects (n = 62), aged 59.7+/-12.2 yr, maintained serum phosphorus concentrations of 3.5-6.0 mg/dL. Noncompliant subjects (n = 26), aged 54.2+/-13.8 yr, had serum phosphorus concentrations > 6.0 mg/dL. Mean plasma and red blood cell (RBC) copper, serum ceruloplasmin, and erythrocyte superoxide dismutase activity did not differ between binder compliant and noncompliant subjects. Mean RBC copper, plasma copper, and serum ceruloplasmin concentrations of hemodialysis subjects were within normal limits, despite copper intakes of less than two-thirds of the lower limit of the Estimated Safe and Adequate Daily Dietary Intakes for copper. The phosphate binder therapies (calcium carbonate, calcium acetate, and/or aluminum hydroxide) had no effect on the copper status of patients with end-stage renal disease on hemodialysis.