Use of indirect immunofluorescence and western blotting to assess the role of circulating antimyocardial antibodies in dogs with dilated cardiomyopathy.

The presence of circulating anti-heart IgG and IgM autoantibodies was assessed by indirect immunofluorescence and by probing Western blot transfers of normal canine myocardial proteins separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis with samples of sera from dogs with dilated cardiomyopathy (n = 27) and from healthy control dogs (n = 20). No difference was demonstrated between the two groups using either method. Sera taken from the affected dogs at three monthly intervals throughout the course of the disease showed no change at all in the pattern of reactivity.

Morphological, histochemical, and myosin isoform analysis of the diaphragm of adult horses, Equus caballus.

The horse provides an interesting model for study of the structure and function of the mammalian diaphragm. Multiple regions of diaphragm from seven adult horses were prepared for histochemistry, immunocytochemistry, myosin heavy chain electrophoresis, and native myosin electrophoresis. Two additional adults were dissected to demonstrate myofiber and central tendon morphology and stained for acetylcholinesterase to demonstrate motor endplates.

Neonatal development of the diaphragm of the horse, Equus caballus.

The diaphragm of neonatal horses is significantly different from the diaphragm of adult horses in terms of histochemical fiber type composition, myosin heavy chain isoform, and native myosin isoform composition. There is a significant increase in the percentage of type I fibers present in the diaphragm with increasing age from birth through about seven months postnatal age. A possible lack of postural tone in the hiatal region of the neonatal diaphragm is suggested to account for increased incidence of vomiting or aspiration pneumonia in younger horses.

Atomic structure of the MAP kinase ERK2 at 2.3 A resolution.

The structure of the MAP kinase ERK2, a ubiquitous protein kinase target for regulation by Ras and Raf, has been solved in its unphosphorylated low-activity conformation to a resolution of 2.3 A. The two domains of unphosphorylated ERK2 are farther apart than in the active conformation of cAMP-dependent protein kinase and the peptide-binding site is blocked by tyrosine 185, one of the two residues that are phosphorylated in the active enzyme.

Determinants of plasma cholesterol responsiveness to diet.

Plasma cholesterol change, or 'responsiveness', to dietary saturated fat modification has long been acknowledged. The present study sought to determine the specific, predicted response of each cholesterol subfraction to known dietary manipulations. Two metabolically controlled diets, one with a low polyunsaturated:saturated fat (low P:S) ratio, and one with a high P:S ratio were fed in a crossover design to sixty-seven normolipidaemic subjects pooled from six foregoing metabolic studies.

Regulation of the MAP kinase cascade.

The MAP kinase cascade is regulated by many hormones and growth factors and its activation leads to changes in properties of cytoplasmic, membrane-associated, and nuclear proteins. The MAP kinases themselves are activated by MEKS. MEKs lie at a point of convergence for multiple upstream signals, mediated by distinct protein kinases, Raf, MEK kinase, and Mos, all of which have MEK kinase activity.

Replacement laminoplasty in selective dorsal rhizotomy: possible protection against the development of musculoskeletal pain.

The authors present a retrospective review of 35 patients who underwent selective dorsal rhizotomy between 1990 and 1992. The first 15 patients underwent laminectomy from L1 to the sacrum without replacement of the posterior elements. The subsequent 20 patients had the laminae replaced during wound closure.

The interaction of SV40 small tumor antigen with protein phosphatase 2A stimulates the map kinase pathway and induces cell proliferation.

Interaction with SV40 small tumor antigen (small t) compromised the ability of multimeric protein phosphatase 2A to inactivate the mitogen-activated protein kinase ERK1 and the mitogen-activated protein kinase kinase MEK1. Transient expression of small t in CV-1 cells activated MEK and ERK but did not affect Raf activity. Small t stimulated the growth of quiescent CV-1 cells almost as effectively as did serum.

Regulation and properties of extracellular signal-regulated protein kinases 1, 2, and 3.

The extracellular signal-regulated kinases ERK1 and ERK2 are 43- and 41-kd enzymes activated by many extracellular cues. They lie within a protein kinase cascade that is used to achieve many cellular responses. In addition to the wide variety of regulatory contexts in which they are activated, they phosphorylate important regulatory proteins, including receptors, transcription factors, cytoskeletal proteins, and other protein kinases.

Crystallization and preliminary X-ray studies of extracellular signal-regulated kinase-2/MAP kinase with an incorporated His-tag.

The extracellular signal-regulated kinase ERK2, a member of the protein kinase superfamily, phosphorylates a variety of cellular proteins in response to extracellular signals. ERK2 expressed in Escherichia coli as a fusion protein with the sequence Ala-His6 at the N terminus has low basal activity and very low levels of phosphate incorporation, but can be fully activated. The Ala-His6 ERK2 as expressed in the unphosphorylated form has been crystallized in space group P2(1).

Histochemical and myosin composition of vampire bat (Desmodus rotundus) pectoralis muscle targets a unique locomotory niche.

The vampire bat pectoralis muscle contains at least four fiber types distributed in a nonhomogeneous pattern. One of these fiber types, here termed IIe, can be elucidated only by adenosine triphosphatase (ATPase) histochemistry combined with reactions against antifast and antislow myosin antibodies. The histochemical and immunohistochemical observations indicate a well-developed specialization of function within specific regions of the muscle.

MAP kinase becomes stably activated at metaphase and is associated with microtubule-organizing centers during meiotic maturation of mouse oocytes.

Using antisera generated against sequences conserved between the ERK1- and the ERK2-encoded species of mitogen-activated protein (MAP) kinases of the rat, species of approximate M(r) 42 and 44 kDa were identified in mouse oocytes. When oocytes underwent meiotic maturation, both species displayed a retarded electrophoretic mobility, consistent with modification by phosphorylation. The slow-migrating forms first appeared after the oocytes had entered metaphase, and their appearance was sensitive to inhibitors of protein synthesis or phosphorylation.

Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro.

Extracellular signal-regulated protein kinases (ERK) 1 and 2 and mutants of each were expressed in bacteria with a hexahistidine tag and purified using nickel-chelate chromatography. Basal activity of wild type ERK2 was approximately 2 nmol/min/mg. Self-catalyzed phosphorylation occurred in vitro on the major physiological site of tyrosine phosphorylation in an intramolecular reaction.

Phosphorylation of the TAL1 oncoprotein by the extracellular-signal-regulated protein kinase ERK1.

Alteration of the TAL1 gene is the most common genetic lesion found in T-cell acute lymphoblastic leukemia. TAL1 encodes phosphoproteins, pp42TAL1 and pp22TAL1, that represent phosphorylated versions of the full-length (residues 1 to 331) and truncated (residues 176 to 331) TAL1 gene products, respectively. Both proteins contain the basic helix-loop-helix motif, a DNA-binding and protein dimerization motif common to several known transcriptional regulatory factors.

Low-density lipoprotein cholesterol responsiveness to diet in normolipidemic subjects.

Both apolipoprotein E genotype (apo E) and diet predict very-low-density (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) levels. In a retrospective pooled analysis of six studies, we sought to identify the predictors of VLDL-C and LDL-C change, or "responsiveness," to a diet crossover. "Response" to diet was studied in 67 normolipidemic subjects of common apo E genotype.

Gender differences in lipoprotein responses to diet.

Gender and dietary saturated fat remain two strong predictors of coronary heart disease susceptibility. In a retrospective meta-analysis of five studies, we analyzed the impact of gender and diet composition on lipoprotein change, or 'response' (delta) in 63 normolipidemic subjects fed two contrasting, metabolically controlled diets. One diet had a low polyunsaturated to saturated fatty acid ratio (low P:S) and the second, a high P:S ratio in a crossover design.

NGF and other growth factors induce an association between ERK1 and the NGF receptor, gp140prototrk.

As detected by coimmunoprecipitation from PC12 cells, NGF induces rapid association between ERK1 (a growth factor-activated serine/threonine protein kinase) and gp140prototrk NGF receptors. In contrast, no such association is found with the closely related ERK2. Anti-trk immunocomplexes generated from NGF-treated cells also contain protein kinase activity that shares many properties with soluble ERK1.

Xanthomatized atypical T cells in a patient with mycosis fungoides and hyperlipidemia.

Background: Lipoprotein-T-cell interactions are being reported with increasing frequency, and there is evidence that lipoproteins play a role in immunoregulation. We describe a patient with mycosis fungoides and hyperlipidemia who developed xanthomatization in one preexisting plaque. The case is unique in that some of the lipidized cells were atypical T cells.

Met and hepatocyte growth factor/scatter factor signal transduction in normal melanocytes and melanoma cells.

The proto-oncogene c-MET encodes a transmembrane tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF stimulates the proliferation and motility of various cell types. Because HGF/SF is also a melanocyte mitogen, we investigated the biological role of HGF/SF, including c-Met expression, activation and signal transduction, in normal and malignant human melanocytes.

Activation of p42 MAP kinase and the release of oocytes from cell cycle arrest.

Clam oocytes are arrested naturally at the G2/M border in meiosis and contain an inactive 42 kDa ERK/MAP kinase, p42MAPK. Following fertilization, p42MAPK is rapidly phosphorylated on tyrosine residues and concomitantly activated. Both tyrosine phosphorylation and activation of p42MAPK begin within 2-3 min of fertilization, peak at approximately 15 min, then rapidly decline and disappear around the end of meiosis I.

Modulation of the dynamic instability of tubulin assembly by the microtubule-associated protein tau.

Microtubule-associated proteins (MAP), such as tau, modulate the extent and rate of microtubule assembly and play an essential role in morphogenetic processes, such as axonal growth. We have examined the mechanism by which tau affects microtubule polymerization by examining the kinetics of microtubule assembly and disassembly through direct observation of microtubules using dark-field microscopy. Tau increases the rate of polymerization, decreases the rate of transit into the shrinking phase (catastrophe), and inhibits the rate of depolymerization.

Influence of dietary fat, apolipoprotein E phenotype, and sex on plasma lipoprotein levels.

Background: The "Western" diet, sex, and apolipoprotein (Apo) E polymorphism have been implicated as codeterminants of lipid levels.

Methods And Results: In a retrospective analysis, we evaluated the combined impact of dietary fat, sex, and Apo E phenotype on lipoprotein levels in 67 subjects fed two contrasting, metabolically controlled diets: one a "Western" diet, with a low polyunsaturated to saturated (P:S) fatty acid ratio and the other a "therapeutic" diet, with a high P:S ratio. The high P:S diet compared with P:S diet exerted a far stronger predictive influence on lipoprotein concentrations than Apo E phenotype, sex, or the latter two factors combined.