A de novo 2.3 kb structural variant in MITF explains a novel splashed white phenotype in a Thoroughbred family.

Splashed white in horses is characterized by extensive white patterning on the legs, face and abdomen and may be accompanied by deafness. To date, seven variants in microphthalmia-associated transcription factor (MITF) and two variants in Paired Box 3 (PAX3) have been identified to explain this phenotype. A splashed white Thoroughbred stallion, whose sire and dam were not patterned, was hypothesized to have a de novo variant leading to his white coat pattern. A whole-genome sequencing candidate gene approach identified two single nucleotide variants (SNVs) in SOX10, four SNVs in MITF and a 2.3 kb deletion in MITF with the alternative allele present in this stallion but absent in the other 18 horses analyzed. All six SNVs were annotated as modifiers and were not further considered. The deletion in MITF (NC_009159.3:g.21555811_21558139delinsAAAT) encompasses exon 9 encoding a part of the helix-loop-helix domain required for DNA binding. Sanger sequencing and parentage testing confirmed that this deletion was a de novo mutation of maternal origin. Consistent with the published nomenclature, we denote this likely causal variant as SW8. Genotyping three of this stallion's offspring identified SW8 only in the nearly all-white foal that was confirmed deaf by brainstem auditory evoked response testing. This foal was also a compound heterozygote for dominant white variants (W20/W22), but to date, W variants alone have not been connected to deafness. SW8 marks the fourth de novo MITF variant in horses reported to cause white patterning. The link between deafness and all MITF variants with and without other variants impacting melanocyte development and function needs to be further explored.

Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction.

Synaptotagmins are synaptic vesicle-associated, phospholipid-binding proteins most commonly associated with Ca(+2)-dependent exocytotic and Ca(+2)- independent endocytotic events. Synaptotagmin III is a 63.2-kD member of the synaptotagmin homology group; one of its characteristic properties is the ability to bind divalent cations and accessory proteins promiscuously. In the cytosolic portion of this protein, a flexible seven-amino acid linker joins two homologous C2 domains. The C2A domain binds to phospholipid membranes and other accessory proteins in a divalent cation-dependent fashion. The C2B domain promotes binding to other C2B domains, as well as accessory proteins independent of divalent cations. The 3.2 A crystal structure of synaptotagmin III, residues 295-566, which includes the C2A and C2B domains, exhibits differences in the shape of the Ca(+2)-binding pocket, the electrostatic surface potential, and the stoichiometry of bound divalent cations for the two domains. These observations may explain the disparate binding properties of the two domains. The C2A and the C2B domains do not interact; synaptotagmin, therefore, covalently links two independent C2 domains, each with potentially different binding partners. A model of synaptotagmin's involvement in Ca(+2)-dependent regulation of membrane fusion through its interaction with the SNARE complex is presented.

Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs.

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Structure of the protein kinase Cbeta phospholipid-binding C2 domain complexed with Ca2+.

BACKGROUND: Conventional isoforms (alpha, beta and gamma) of protein kinase C (PKC) are synergistically activated by phosphatidylserine and Ca2+; both bind to C2 domains located within the PKC amino-terminal regulatory regions. C2 domains contain a bipartite or tripartite Ca2+-binding site formed by opposing loops at one end of the protein. Neither the structural basis for cooperativity between phosphatidylserine and Ca2+, nor the binding site for phosphatidylserine are known. RESULTS: The structure of the C2 domain from PKCbeta complexed with Ca2+ and o-phospho-L-serine has been determined to 2.7 A resolution using X-ray crystallography. The eight-stranded, Greek key beta-sandwich fold of PKCbeta-C2 is similar to that of the synaptotagmin I type I C2 domain. Three Ca2+ ions, one at a novel site, were located, each sharing common aspartate ligands. One of these ligands is donated by a dyad-related C2 molecule. A phosphoserine molecule binds to a lysine-rich cluster in C2. CONCLUSIONS: Shared ligation among the three Ca2+ ions suggests that they bind cooperatively to PKCbeta-C2. Cooperativity may be compromised by the accumulation of positive charge in the binding site as successive ions are bound. Model building shows that the C1 domain could provide carboxylate and carbonyl ligands for two of the three Ca2+ sites. Ca2+-mediated interactions between the two domains could contribute to enzyme activation as well as to the creation of a positively charged phosphatidylserine-binding site.

Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution.

The evolutionarily conserved SNARE proteins and their complexes are involved in the fusion of vesicles with their target membranes; however, the overall organization and structural details of these complexes are unknown. Here we report the X-ray crystal structure at 2.4 A resolution of a core synaptic fusion complex containing syntaxin-1 A, synaptobrevin-II and SNAP-25B. The structure reveals a highly twisted and parallel four-helix bundle that differs from the bundles described for the haemagglutinin and HIV/SIV gp41 membrane-fusion proteins. Conserved leucine-zipper-like layers are found at the centre of the synaptic fusion complex. Embedded within these leucine-zipper layers is an ionic layer consisting of an arginine and three glutamine residues contributed from each of the four alpha-helices. These residues are highly conserved across the entire SNARE family. The regions flanking the leucine-zipper-like layers contain a hydrophobic core similar to that of more general four-helix-bundle proteins. The surface of the synaptic fusion complex is highly grooved and possesses distinct hydrophilic, hydrophobic and charged regions. These characteristics may be important for membrane fusion and for the binding of regulatory factors affecting neurotransmission.

Supporting the bereaved relative: reflections on the actor's experience.

For several months 1 and a team of fellow performers were involved, as simulated bereaved relatives, with a research project based in hospitals in the north-west of England. The project, the European Donor Hospital Education Programme, EDHEP (UK), was set up in order to provide a research input into the already established EDHEP in Europe. The programme is designed to develop communication and interpersonal skills among health care workers in Intensive Care/Therapy Units, especially in the breaking of bad news of death to near relatives and in asking consent for the removal of tissues and/or organs for transplantation. The associated research was initiated by an international grouping of clinical psychologists with the purpose of assessing the efficacy of the training schedule Results are expected soon from the main body of research, some aspects of which provide analytical data drawn from encounters between health care professionals and actors as simulated relatives. Here, however, the experiences and feelings of the 'relatives' themselves are described, discussed and reflected upon.

Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C.

C2 domains are found in many proteins involved in membrane traffic or signal transduction. Although C2 domains are thought to bind calcium ions, the structural basis for calcium binding is unclear. Analysis of calcium binding to C2 domains of synaptotagmin I and protein kinase C-beta by nuclear magnetic resonance spectroscopy revealed a bipartite calcium-binding motif that involves the coordination of two calcium ions by five aspartate residues located on two separate loops. Sequence comparisons indicated that this may be a widely used calcium-binding motif, designated here as the C2 motif.

Effect of acute allograft rejection on exercise hemodynamics in patients who have undergone cardiac transplantation.

The effect of acute allograft rejection on exercise hemodynamics was evaluated in 8 consecutive cardiac allograft recipients (group 1) when the right ventricular endomyocardial biopsy showed evidence of allograft rejection (R), and when no evidence of rejection (NR) was present. A separate group of 10 cardiac transplant recipients (group 2) with no evidence of rejection on biopsy done at the end of the first and second year post-transplantation served as controls. The exercise hemodynamics were abnormal in both groups in both studies with a moderate increase of the pulmonary artery wedge pressure to a mean of 17.2 (NR) and 19.4 mm Hg (R) in group 1 (p = not significant [NS]) and 20.1 and 21.2 mm Hg in group 2 (p = NS), a mild increase of the mean right atrial pressure to a mean of 10 mm Hg (NR) and 10 mm Hg (R) in group 1 (p = NS), 11.9 mm Hg and 12.5 mm Hg in group 2 (p = NS), and a moderate increase of the arteriovenous oxygen content difference to a mean of 8.5 (NR) and 8.4 vol percent (R) in group 1 and 8.3 and 8.0 vol percent in group 2. No significant difference was observed between the two studies of the same group in any of the hemodynamic parameters except for the heart rate in group 1 (from 91 +/- 16 to 97 +/- 16 beats/min [p < 0.05] with and without evidence of allograft rejection, respectively). In conclusion, heart transplant recipients do not usually manifest further exercise hemodynamic deterioration during mild to moderate rejection.

Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.

C2 domains are regulatory sequence motifs that occur widely in nature. Synaptotagmin I, a synaptic vesicle protein involved in the Ca2+ regulation of exocytosis, contains two C2 domains, the first of which acts as a Ca2+ sensor. We now describe the three-dimensional structure of this C2 domain at 1.9 A resolution in both the Ca(2+)-bound and Ca(2+)-free forms. The C2 polypeptide forms an eight-stranded beta sandwich constructed around a conserved four-stranded motif designated as a C2 key. Ca2+ binds in a cup-shaped depression between two polypeptide loops located at the N- and C-termini of the C2-key motif.

Isolation of a cDNA clone for murine catalase and analysis of an acatalasemic mutant.

We have investigated the genetic control of murine catalase expression by analyzing catalase transcription and translation products from the tissues of control (Csa) and acatalasemic (Csb) mouse strains. Csb animals possess nearly normal catalase enzyme activity levels in liver, while displaying approximately 20 and 1% of normal activity levels in kidney and red blood cells, respectively. Immunoblot analyses of catalase in these tissues have revealed reduced levels of immunologically reactive catalase protein in Csb kidney and red blood cells, paralleling the reduction of catalase enzyme activity in these tissues. In order to determine the molecular basis for Csb acatalasemia, we have isolated a cDNA clone for murine catalase and have used this probe to analyze Csa and Csb genomic DNA and catalase mRNA. These studies have revealed: 1) no restriction fragment length polymorphisms between Csa and Csb genomic DNAs; 2) no differences in the levels of Csa and Csb catalase mRNA within a single tissue; and 3) no differences in the sizes of Csa and Csb catalase mRNAs. These observations suggest that the genetic defect that produces the tissue-specific reduction of catalase expression in Csb mice is not due to a marked rearrangement of DNA within the Csb catalase structural gene. Furthermore, the Csb mutation does not act at the level of gene transcription or mRNA stability, but rather at the level of mRNA translation and/or catalase protein turnover.

Comparison of Fick and dye cardiac outputs during rest and exercise in 1,022 patients.

The Fick and indicator-dilution techniques for measurement of cardiac output (CO) were compared at rest in 1,022 patients and in 786 during exercise. Duplicate measurements of dye CO at rest revealed that 92.7% fell within 10% of the line of identity and 99% within 20%. For the resting Fick and dye comparisons, 44.6% were within 10% of the identity line and 74.7% within 20%. When mean CO was less than 4.4 L/min, dye CO was higher than Fick. This relationship persisted for CO between 4.4 and 7.4 whereas for above 7.4 L/min, Fick was higher than dye. During exercise, 50.2% of the Fick and dye comparisons fell within 10% and 77.1% within 20% of the line of identity. There was a systematic difference between the two methods during exercise with dye CO higher than Fick CO. This study agrees with Fick and dye comparison studies with 74.7% and 77.1% of values within 20% of the identity line during rest and exercise, respectively. However, these results differ from others in that dye CO was higher than Fick CO for low and normal values whereas Fick was greater for the higher CO values. The overall agreement between the two methods in a large group of patients with diverse cardiac diseases over a broad spectrum of CO values supports use of either method for clinical studies.

Digital subtraction coronary angiography using high-pass temporal filtration: a comparison with cineangiography.

Selective coronary angiograms were obtained using a real-time high-pass temporal filtration digital subtraction technique with videotape storage and display and compared to simultaneously recorded 35-mm cineangiograms for 32 stenotic lesions in 15 patients. Both methods were evaluated by three independent observers using caliper measurement of percent diameter reduction for each lesion. There was a good correlation between the two imaging methods for individual observers, though considerable variability was seen, r = .73, standard error of estimate (SEE) = 9.1%. The average severity of stenosis and the interobserver variability were similar between methods. This digital subtraction technique for selective coronary angiography compares favorably with a conventional film-based technique for evaluation of coronary stenoses and offers advantages of real-time image processing, limited tolerance to patient motion, and relatively small digital memory requirements. In addition to further improvements in image quality, more objective computer-aided scoring methods are needed to reduce the variability in lesion analysis.

Left main coronary artery occlusion.

Patients with complete occlusion of the left main coronary artery are candidates for massive myocardial infarction and sudden death and are thought to have a uniformly poor prognosis. Complete occlusion of the left main coronary artery was identified in 2 male patients among 2,546 patients undergoing cardiac catheterization over a period of 14.5 years in our institution. Both patients had angina pectoris. Left ventricular end-diastolic pressure was markedly elevated in one, and the ejection fraction was moderately to markedly reduced in both. Significant collateral flow to the left coronary system from the right coronary artery was present in both patients. Our study supports previous reports that left main coronary artery occlusion is rarely encountered during cardiac catheterization.

Cross-sectional study of the effects of immunosuppressive drugs on chronic periodontal disease in man.

Oral examinations were performed on 102 patients receiving immunosuppressive drug therapy following renal transplantation. A further 111 control patients matched for age and sex, were also examined. The severity of dental caries of the 2 groups was compared by considering their decayed, missing and filled teeth (DMF-T), and the severity of periodontal disease was compared in terms of pocket depths, Plaque, Calculus and Sulcular Bleeding Indices, and Russell's Periodontal Index. When comparing the 2 groups, note was made of restorations involving the gingival margins, the presence of partial dentures and recent and current antibiotic therapy. No significant differences were found between the groups with regard to age, sex distribution, plaque levels, DMF and Russell's Periodontal Index. The immunosuppressed group had significantly less calculus, fewer restorations involving the gingival margins and significantly lower mean pocket depth. However, they did have more gingival recession than the controls and also a higher Sulcus Bleeding Index. All differences seen were maintained even when patients from both groups on antibiotics were eliminated from the analysis. Within the immunosuppressed group no relationship was found between the length of time the drugs were taken and the periodontal variables. The study indicates that patients on immunosuppressive therapy show no change in susceptibility to destructive periodontal disease.

Hemodynamic and clinical comparison of the Hancock modified orifice and standard orifice bioprostheses in the aortic position.

Bioprosthetic aortic valve replacement in patients with a small aortic root has been associated with postoperative transvalvular gradients. A modified orifice Hancock xenograft bioprosthesis has been developed and is purported to increase significantly the effective orifice area (as evaluated by in vitro testing) compared to the standard orifice Hancock bioprosthesis. To assess the in vivo differences, we compared 481 patients with standard orifice prostheses with 156 patients with modified orifice prostheses. Postoperative catheterization was performed in 24 patients with modified orifice (valve diameters 19 to 25 mm) with 14 with standard orifice valves (valve diameters 21 to 25 mm). Actuarial rates of survival, valve failure, endocarditis, and thromboembolism did not differ significantly between the two subgroups. Peak aortic valve gradients on the whole were less in the modified orifice subgroup than in the standard origice subgroup (12 +/- 1 torr versus 20 +/- 6 torr [mean +/- SEM]), but the difference was not statistically significant (p greather than 0.05). The calculated in vivo aortic valve areas were slightly, but insignificantly, greater in the modified orifice subgroup than in the standard orifice subgroup (p greater than 0.05). These in vivo data partially corroborate the in vitro findings of increased effective orifice area and internal-to-external diameter ratio for the modified orifice bioprosthesis. The hemodynamic differences between the two valve types are small, however, and the putative clinical advantages inherent in the use of the modified orifice bioprosthesis remain to be completely defined.