Management of collagen vascular diseases in childhood.

Collagen vascular diseases seen in children include systemic, discoid and neonatal lupus, dermatomyositis, scleroderma, juvenile rheumatoid arthritis, and, in rare cases, Sjogren's syndrome. Although these diseases are uncommon in children, when seen, they are associated with significant morbidity. This review describes the clinical features of each condition and provides an overview of treatment options now available. These include numerous systemic treatments which can be used as steroid-sparing agents.

Physical properties of a single-motif erythrocyte spectrin peptide: a highly stable independently folding unit.

Spectrin is a long flexible rod-like actin cross-linking protein mostly comprised of many tandem homologous 106-residue motifs. In this study, the conformational stability and physical properties of a single homologous motif peptide, alpha1, were evaluated and compared to intact spectrin monomers and alphabeta heterodimers. It is interesting that while spectrin dimers elongate by about 3-fold in low ionic strength buffers relative to their size in physiological buffers, the single-motif peptide does not show significant changes in secondary structure in 10 mM phosphate buffer compared with isotonic buffer. This single-motif peptide is monomeric in physiological buffer as demonstrated by equilibrium sedimentation studies, and its hydrodynamic radius determined by gel filtration and dynamic light scattering of about 2.2 nm is consistent with an elongated rod-like shape. Unfolding of the single-motif peptide in urea solutions was similar to unfolding of intact heterodimers. Differential scanning calorimetry analyses showed that this single motif undergoes a reversible two-state transition with a Tm of 53 degrees C and an enthalpy of 65 kcal/mol in physiological buffer. Thermal stability was unaffected by ionic strength changes, but was decreased below physiological pH. These data show that this 13 kDa spectrin motif is a monomeric, highly stable, triple-helical, independently folding protein building block with physical characteristics that define many of the structural properties of the 526 kDa spectrin heterodimer. In contrast, interactions between adjacent motifs are probably responsible for spectrin's molecular flexibility and elasticity.

Mapping the human erythrocyte beta-spectrin dimer initiation site using recombinant peptides and correlation of its phasing with the alpha-actinin dimer site.

Human erythroid spectrin dimer assembly is initiated by the association of a specific region near the N-terminal of beta-spectrin with a complementary region near the C-terminal of alpha-spectrin (Speicher, D. W., Weglarz, L., and DeSilva, T. M. (1992) J. Biol. Chem. 267, 14775-14782). Both spectrin subunits consist primarily of tandem, 106-residue long, homologous, triple-helical motifs. In this study, the minimal region of beta-spectrin required for association with alpha-spectrin was determined using recombinant peptides. The start site (phasing) for construction of dimerization competent beta-spectrin peptides was particularly critical. The beginning of the first homologous motif for both beta-spectrin and the related dimerization site of alpha-actinin is approximately 8 residues earlier than most spectrin motifs. A four-motif beta-spectrin peptide (beta1-4+) with this earlier starting point bound to full-length alpha-spectrin with a Kd of about 10 nM, while deletion of these first 8 residues reduced binding nearly 10-fold. N- and C-terminal truncations of one or more motifs from beta1-4+ showed that the first motif was essential for dimerization since its deletion abolished binding, but beta1+ alone could not associate with alpha-monomers. The first two motifs (beta1 2+) represented the minimum lateral dimer assembly site with a Kd of about 230 nM for interaction with full-length alpha-spectrin or an alpha-spectrin nucleation site recombinant peptide, alpha18-21. Each additional motif increased the dimerization affinity by approximately 5-fold. In addition to this strong inter-subunit dimer association, interactions between the helices of a single triple-helical motif are frequently strong enough to maintain a noncovalent complex after internal protease cleavage similar to the interactions thought to be involved in tetramer formation. Analysis of hydrodynamic radii of recombinant peptides containing differing numbers of motifs showed that a single motif had a Stokes radius of 2.35 nM, while each additional motif added only 0.85 nM to the Stokes radius. This is the first direct demonstration that spectrin's flexibility arises from regions between each triple helical motif rather than from within the segment itself and suggests that current models of inter-motif connections may need to be revised.

Monoclonal antibodies to a TCR idiotope modulate the superantigen-induced responses of encephalitogenic rat T cells.

Superantigens, such as staphylococcal enterotoxins, activate T lymphocytes by linking MHC class II molecules on antigen presenting cells to the V beta element of the TCR. Through this effect on T cells, superantigens may influence the immune response and autoimmune disease. In fact, superantigens may activate or anergize cells involved in the production of experimental allergic encephalomyelitis. Lewis rats recognize an encephalitogenic epitope in myelin basic protein (MBP) residues 68-88 through an MHC class II I-A restricted process using TCR V beta 8.2. The F30 murine mAb reacts with an encephalitogenic idiotope (Id) on the TCR of V beta 8.2+ encephalitogenic Lewis rat T cells. In the present study it was demonstrated that the same mAb anti-Id inhibited the proliferation and IL-2 secretion induced by staphylococcal enterotoxins A, B and E in a V beta 8.2+ encephalitogenic Lewis rat T cell line specific for guinea pig MBP peptide 68-88. The mAb anti-Id did not inhibit the response of control T cells similarly derived but inhibited V beta 8.2- and recognizing MBP peptide 87-99. Control anti-Id failed to inhibit the response of either cell line. These findings imply that the specific antigen and superantigen react with TCR in a manner similar enough to be inhibited by the same anti-Id. The mechanism may involve the induction of anergy by the anti-Id, interference/steric hindrance by the reaction of anti-Id with TCR and possibly a little direct reaction of anti-Id with superantigens. Anti-Id directed immunotherapy may have a role in modulating the damage of inflammatory demyelination induced by both specific antigens and superantigens.

Functional characterization of recombinant human red cell alpha-spectrin polypeptides containing the tetramer binding site.

Spectrin, a heterodimer composed of alpha and beta subunits, interacts with itself head-to-head to form tetramers in the erythrocyte membrane cytoskeleton. The NH2-terminal region of alpha-spectrin, encompassing the alpha I 80-kDa domain, was expressed in Escherichia coli. In addition to the correctly initiated polypeptide, four smaller polypeptides were produced by initiation at internal codons. Only the full-length polypeptide was able to bind to spectrin dimers, beta monomers, and to a recombinant polypeptide containing the COOH terminus of beta-spectrin. The head-to-head interaction with beta-spectrin was also retained by a recombinant polypeptide containing the NH2-terminal 158 amino acids of the alpha subunit. Deletion of the first 27 or 49 NH2-terminal amino acids abolished binding of this polypeptide to the beta monomer. The phasing used to design these recombinant polypeptides was based on a conformational model recently refined by Speicher et al. (Speicher, D. W., DeSilva, T. M., Speicher, K. D., Ursitti, J. A., Hembach, P., and Weglarz, L. (1993) J. Biol. Chem. 268, 4227-4235), where the structural unit begins and terminates around residue 30 of the repeat unit. The binding properties, mobility on gel filtration, and circular dichroism data of the recombinant polypeptides indicated that most polypeptides were able to assume their native conformation.

A blood pressure independent index of aortic distensibility.

A non-invasive Doppler ultrasound technique for the assessment of aortic compliance based on the in vivo measurement of pulse wave velocity along the thoraco-abdominal aortic pathway is described. An approach for correcting for the effect of blood pressure on aortic compliance is considered. The derivation of an index of distensibility, Cp, which is independent of blood pressure is provided and applied to data collected from 58 normal, healthy volunteers. Medical disorders such as atherosclerosis, diabetes mellitus and familial hypercholesterolaemia have all been shown to affect arterial distensibility. We suggest that the clinical measurement of Cp may be a useful, non-invasive tool for assessing such patients' susceptibility to atheromatous arterial disease as well as for monitoring their response to therapy.

Location of the human red cell spectrin tetramer binding site and detection of a related "closed" hairpin loop dimer using proteolytic footprinting.

Head-to-head association of two spectrin alpha beta heterodimers to form tetramers involves the formation of two equivalent alpha-beta complexes. The sites on the alpha subunit N-terminal region and beta subunit C-terminal region that form these alpha beta complexes have been identified using protease footprinting and direct binding assays. The existence of a similar previously hypothesized internal head-to-head alpha beta interaction in dimers was also demonstrated. The discrete regions of both subunits that are protected from proteolysis in tetramers and dimers are not due to the laterally associated subunit since head-to-head complexes of a univalent alpha peptide with a univalent beta peptide show similar protection of the same sites. These sites are unshielded immediately after monomers assemble side-to-side to form heterodimers, demonstrating that reconstituted dimers are initially in an "open" conformation. Conversion of open dimers to a closed form through formation of the internal head-to-head alpha beta association, as demonstrated by restoration of protease protection, occurred on a time scale of hours at 0 degrees C. Analysis of peptide binding affinities as well as isolation and sequence analysis of head-to-head alpha beta noncovalent complexes further defined the regions required for association on both subunits. These regions are homologous to the 106-residue repetitive motif that comprises most of both chains. An algorithm designed to improve prediction accuracy of multiple homologous motifs was used to model the conformation of spectrin repetitive motifs as well as the contact regions. In this model, the separate alpha and beta binding sites are incomplete complementary parts of a triple stranded folding unit. Formation of the alpha beta head-to-head complex produces a triple stranded conformational unit that is slightly different from other homologous motifs in the protein. Most hemolytic anemia mutations that are known to disrupt tetramer association are located in the mapped regions, including several mutations that induce a conformational change in the paired subunit.

Analysis of human red cell spectrin tetramer (head-to-head) assembly using complementary univalent peptides.

The mass-driven assembly of spectrin dimers to form tetramers involves two equal head-to-head alpha-beta associations and requires at least 30 degrees C for interconversion to occur readily. In this paper, the properties of tetramer formation were investigated using two complementary univalent peptides (the alpha I domain and beta monomers). Since the alpha I domain lacks an essential nucleation site required for side-to-side (lateral) heterodimer assembly [Speicher et al. (1992) J. Biol. Chem. 267, 14775-14782], these two peptides can only assemble head-to-head at a single site. This head-to-head assembly readily occurs at lower temperatures, indicating the temperature barrier for dimer-tetramer interconversion is caused by a conformational constraint of the dimer. This constraint, a closed hairpin loop, is released when the laterally associated partner is removed. The univalent alpha I-beta binding affinity at 37 degrees C (Ka = 1.4 x 10(5) M-1) is similar to the dimer-tetramer association constant at the same temperature. As the temperature is decreased from 37 to 0 degrees C, the alpha I-beta binding affinity increases about 32-fold. In contrast with head-to-head associations involving dimers, the second-order rate constants of two complementary univalent peptides (i.e., alpha I and beta) are dramatically higher, and the estimated activation energy (about 50 kJ mol-1) is about 5-fold lower. An open dimer conformation is an obligatory high-energy intermediate required for dimer-tetramer interconversion, and opening the dimer hairpin loop contributes about 190 kJ mol-1 to the activation energy for tetramer association.(ABSTRACT TRUNCATED AT 250 WORDS)

New trichoverroids from Myrothecium verrucaria isolated by high speed countercurrent chromatography.

Three new double-bond isomers of the trichothecene trichoverrins (3, 4a and 4b) have been isolated, principally through the use of high speed countercurrent chromatography, which proved to be a powerful tool in the separation of these closely related structural isomers.

Properties of human red cell spectrin heterodimer (side-to-side) assembly and identification of an essential nucleation site.

The antiparallel side-to-side association of spectrin alpha and beta monomers is a two-step process which occurs in seconds even at 0 degrees C and at low concentrations. Assembly involves initial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homologous segments or repeats. Three repeats in the nucleation site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in alpha-actinin. The adjacent actin binding domain on the beta subunit and the adjacent EF hand motifs on the alpha subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both subunits. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Heterozygotic mutations involving either nucleation site are predicted to affect allele incorporation into the mature membrane skeleton.

The effectiveness of oral clonidine as a sedative/anxiolytic and as a drug to blunt the hemodynamic responses to laryngoscopy.

STUDY OBJECTIVE: To determine the effects of oral clonidine premedication on sedative, anxiolytic, and hemodynamic responses during the immediate preoperative period, laryngoscopy/intubation, and postanesthetic recovery. DESIGN: Randomized double-blind assignment to one of four treatment groups (clonidine 0.1 mg, clonidine 0.2 mg, triazolam 0.25 mg, or placebo); n = 10 per group. SETTING: Inpatient surgery in a university-staffed tertiary center. PATIENTS: Forty ASA physical status I and II adults of both sexes scheduled for a variety of procedures requiring general anesthesia. INTERVENTIONS: Anxiety and sedation scored on ordinal scale at time of treatment and 90 minutes later, just prior to anesthetic induction. Standardized induction protocol with automated hemodynamic monitoring at 1-minute intervals and a 45-second laryngoscopy to ensure a vigorous stress response. MEASUREMENTS AND MAIN RESULTS: Triazolam and both doses of clonidine increased sedation at 90 minutes both absolutely and compared with a placebo. Clonidine 0.2 mg decreased anxiety absolutely at 90 minutes but no more than a placebo. Clonidine 0.2 mg decreased systolic, mean, and diastolic blood pressures (BPs) but not heart rate (HR) at 90 minutes. Clonidine 0.2 mg also blunted the increase in systolic blood pressure (SP) [but not in diastolic blood pressure (DP) or HR] that accompanied laryngoscopy. There were no treatment differences in postanesthetic hemodynamics or duration of recovery. CONCLUSIONS: Oral clonidine 0.2 mg was effective in reducing the level of behavioral and hemodynamic responses preoperatively and in blunting systolic hypertension produced by prolonged laryngoscopy.