The impact of privacy protections on recruitment in a multicenter stroke genetics study.

The authors reviewed the recruitment of stroke-affected sibling pairs using a letter-based, proband-initiated contact strategy. The authors randomly sampled 99 proband enrollment forms (Phase 1) and randomly sampled 50 sibling reply cards (Phase 2). The sibling response rate was 30.6%, for a pedigree response rate of 58%. Of the siblings who replied, 96% authorized further contact. Median time from proband enrollment to pedigree DNA banking, which required 3+ probands, was 134 days.

Characterization, receptor mapping and blood-brain barrier transcytosis of antibodies to the human transferrin receptor.

Monoclonal antibodies to the human transferrin receptor were screened for binding to capillary vessels in human, monkey, rabbit and rat brain tissue. Two antibodies were selected that bind both human and monkey but not rabbit or rat microvessels. With recombinant fragments of the human receptor, both antibodies were shown to bind to a region of the extracellular portion of the receptor that is relatively variable among species. Binding, which was characterized by using purified receptor and K562 cells, was not reduced by excess transferrin, indicating that the antibodies bind the receptor at a site different from that of transferrin. When the antibodies were radiolabeled and injected i.v. into cynomolgous monkeys, they distributed selectively to brain but not to other organs or tissues. The antibodies were found almost exclusively in the brain parenchyma, rather than the capillaries, indicating that they had transcytosed the blood-brain barrier. These results show that antibodies to the human transferrin receptor cross the blood-brain barrier and may be useful for noninvasive delivery of therapeutic proteins to the central nervous system.

Protein and peptide binding and stimulation of in vitro lysosomal proteolysis by the 73-kDa heat shock cognate protein.

Lysosomal degradation of intracellular proteins during serum withdrawal is stimulated by a member of the 70-kDa heat shock protein (hsp70) family (Chiang, H.-L., Terlecky, S. R., Plant, C. P., and Dice, J. F. (1989) Science 246, 382-385). This hsp70, isolated by affinity chromatography with RNase S-peptide-Sepharose, is referred to as the 73-kDa peptide recognition protein (prp73). We now report that prp73 binds to several proteins and peptides whose degradative rates are increased during serum withdrawal. prp73 also binds to the pentapeptide, KFERQ, and more weakly to most modified RNase S-peptide derivatives with a single amino acid substitution within the KFERQ sequence. Taken together, these results suggest that prp73 binds to a variety of proteins at peptide regions biochemically related to KFERQ. Three lines of evidence indicate that prp73 is the heat shock cognate protein of 73 kDa (hsc73): (a) among five hsp70s tested, hsc73 binds to RNase S-peptide most avidly, (b) both prp73 and hsc73 also bind to RNase A and aspartate aminotransferase but not to ovalbumin, lysozyme, or ubiquitin, and (c) both prp73 and hsc73 promote uptake and degradation of [3H] RNase S-peptide by lysosomes in vitro, while three other hsp70s are without activity in this assay.

Upper airway function in Ondine's curse.

Congenital central alveolar hypoventilation (Ondine's curse) is a rare disorder with absence of automatic control of ventilation but preservation of voluntary breathing. Phrenic pacing, used to treat this condition, is usually not successful without a tracheotomy. We performed fiberoptic videoendoscopy on an affected infant airway. During wakefulness and ventilation by a negative pressure ventilator, the airway was normal, but with sleep onset, passive inspiration produced phasic epiglottic collapse. This case demonstrates the importance of central control in maintaining upper airway patency.

Targeting specific proteins for lysosomal proteolysis.

A class of cytosolic proteins has been identified that are degraded faster (have shorter half-lives) in human diploid fibroblasts deprived of serum. In RNase A, a model protein used for these studies, a pentapeptide comprising amino acids 7-11, Lys-Phe-Glu-Arg-Gln or KFERQ, is responsible for its enhanced degradation. The cytosolic proteins that are degraded faster during serum deprivation are recognized by an antiKFERQ antibody and, therefore, probably contain variations of the KFERQ motif. These cytosolic proteins are degraded in lysosomes. Transport into lysosomes in vitro is stimulated by ATP and the heat shock cognate protein of 73 kDa (hsc73).

A pathway of lysosomal proteolysis mediated by the 73-kilodalton heat shock cognate protein.

Cultured IMR-90 diploid human lung fibroblasts respond to withdrawal of serum or growth factors by increasing protein degradation. This increase, due to enhanced transfer of proteins into lysosomes, is specific for a class of intracellular proteins containing peptide sequences biochemically related to Lysine-Phenylalanine-Glutamate-Arginine-Glutamine (KFERQ). This peptide motif is recognized by an intracellular protein which facilitates its transfer into lysosomes in vitro and presumably, in vivo. We called this protein the peptide recognition protein of 73-kilodaltons (prp73). We have shown prp73 to be the constitutive member of the heat shock 70kD family (hsc73) by a variety of criteria. Furthermore, our reconstitution of this pathway of lysosomal degradation in vitro has provided insight in to the molecular mechanisms and requisite biochemical components.

A selective pathway for degradation of cytosolic proteins by lysosomes.

A lysosomal pathway of proteolysis is selective for cellular proteins containing peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). This pathway is activated in confluent cultured cells that are deprived of serum growth factors and in certain tissues of fasted animals. We have reconstituted this lysosomal degradation pathway in vitro. Transport into lysosomes requires a KFERQ-like sequence in the substrate protein and uptake and/or degradation is stimulated by ATP. A member of the heat shock 70 kDa protein family, the 73 kDa constitutive heat shock protein, binds to KFERQ-like peptide regions within proteins and, in some as yet unidentified manner, facilitates transfer of the proteins into lysosomes. Several possible mechanisms of selective protein transport into lysosomes are discussed.

Metallic clips as a time/cost saving adjunct to head and neck surgery.

Metal surgical clips are commonly used in head and neck surgery today. In this study of 10 patients, 69 vessels in the neck were ligated with tantalum clips and 68 vessels were ligated with silk ties. The times for cutting and ligating each vessel were recorded and the results show a 31-second timesavings by clipping the vessel as opposed to tying. The use of metal clips on vessels is found to be both safe and effective, while reducing operative time.

Early home management of patients with Pierre Robin sequence.

Robin Sequence is a well-described disorder with a reported incidence of 1 in 2000 births. It is characterized by glossoptosis and micrognathia often associated with a cleft palate. Respiratory obstruction at the level of the tongue occurs in many affected children. Prior to routine intensive care unit management, it was the primary factor in the mortality reported in patients with moderately severe to severe deformity. Treatment of this obstruction is controversial and may include a long stay in an intensive care setting. We report two cases that demonstrate properly placed and constructed nasopharyngeal tubes allow a non-surgical treatment with early discharge of the patient and without the risks and morbidity of surgery or the complications of tracheostomy.

A common mechanism for posttranslational activation of plasma membrane receptors?

The process for posttranslational acquisition of ligand binding function is remarkably similar for three receptors with dissimilar structures, namely, the insulin, epidermal growth factor, and acetylcholine receptors. These receptors lack the ability to bind ligand immediately after translation, but slowly (t1/2 = 30-45 min) acquire this capacity while in the endoplasmic reticulum. This activation step occurs with similar kinetics for all three receptors and, in each case, required N-linked glycosylation. Several lines of evidence suggest a common mechanism for the acquisition of ligand binding function that involves the rearrangement of metastable disulfide bonds formed during or immediately after translation. This process precedes subunit assembly of both insulin and acetylcholine receptors, which also occurs in the endoplasmic reticulum. The posttranslational processing steps leading to the acquisition of ligand binding function may be an example of a more general process affecting cell surface proteins.

Actinomycosis of the middle ear.

Actinomycosis is an uncommon infection of the middle ear. Only 21 cases of actinomycosis of the middle ear have been reported in the English literature prior to this paper. The offending organism is Actinomyces israelii, which is an anaerobic, filamentous organism that is difficult to grow in culture. The infection is chronic and is seldom diagnosed prior to tympanomastoidectomy. The identification of small, yellow, glue-like masses, which are called sulfur granules, is often the key to making the diagnosis of actinomycosis of the middle ear. Following tympanomastoidectomy, penicillin is given orally for 3-6 months.

Post-translational changes in tertiary and quaternary structure of the insulin proreceptor. Correlation with acquisition of function.

Tertiary and quaternary structural changes that occur during post-translational processing of the insulin proreceptor were examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S]methionine, labeled insulin receptor species, isolated by immuno- and insulin-affinity adsorption, were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis under conditions where intra- and intermolecular disulfide bonds remained intact or were cleaved by reduction. Reducing SDS-polyacrylamide gel electrophoresis confirmed that the insulin receptor is synthesized as a long-lived (t1/2 = 3 h) proreceptor precursor of 210 kDa which undergoes proteolytic cleavage and carbohydrate maturation to form the alpha- and beta-subunits of the mature receptor. The proreceptor acquires insulin binding activity through a subtle structural change (t1/2 = 45 min) detected only by an autoimmune antibody specific for an epitope of the active insulin binding site. Analysis of insulin receptor species by nonreducing SDS-polyacrylamide gel electrophoresis revealed that the proreceptor undergoes two additional structural changes not detected by reducing SDS-polyacrylamide gel electrophoresis. The proreceptor is synthesized as a monomer (M1) with an apparent molecular mass of 170 kDa that is converted by disulfide rearrangement to another monomeric form of 190-kDa apparent molecular mass (M2). N-Linked glycosylation is required for this transition, since aglycoproreceptor, synthesized in the presence of tunicamycin, does not undergo any detectable tertiary or quaternary structural changes. M2 self-associates to form a disulfide-linked proreceptor dimer (D) which is subsequently proteolytically processed, forming the mature, disulfide-linked alpha 2 beta 2 receptor tetramer. The mature receptor was distinguished from the three proreceptor species (M1, M2, and D) by its cell surface location and its ability to bind tightly to wheat germ agglutinin-agarose, indicating the presence of complex oligosaccharide chains. Subcellular fractionation indicated that both the M1 to M2 and M2 to D conversions occur in the endoplasmic reticulum. Separation of the nonreduced proreceptor species into "active" and "inactive" forms by affinity chromatography on insulin-agarose revealed that neither the transition of M1 to M2, nor of M2 to D, is correlated with the acquisition of insulin binding function. Rather, during its life-time, the M2 species acquires insulin binding activity and an epitope recognized by a binding site specific autoimmune antibody through a subtle structural change not detected by reducing or nonreducing SDS-polyacrylamide gel electrophoresis.

Post-translational acquisition of insulin binding activity by the insulin proreceptor. Correlation to recognition by autoimmune antibody.

The post-translational acquisition of ligand binding activity by the insulin receptor was examined in 3T3-L1 adipocytes. In pulse-chase experiments with [35S] methionine, labeled receptor species were separated into "active" and "inactive" forms by affinity chromatography on insulin-agarose and then were characterized and quantitated. It was found that the newly translated high molecular weight proreceptor lacks the capacity to bind insulin. The acquisition of binding activity is relatively slow (t1/2 = 45 min) and occurs prior to conversion of the proreceptor to the mature alpha- and beta-subunits by proteolytic cleavage and maturation of its N-linked oligosaccharide chains (t1/2 = 3 h). Glycosylation appears to be required for this activation since the aglycoproreceptor, synthesized in the presence of tunicamycin, does not acquire insulin binding activity. However, once the proreceptor has acquired ligand binding activity, removal of its N-linked oligosaccharide chains with endoglycosidase H has no effect on the ability of the proreceptor to bind insulin. The modification of the proreceptor to bind insulin. The modification of the proreceptor that gives rise to insulin binding activity most likely involves a conformational change in the binding domain. A human autoimmune antibody that recognizes only the active insulin binding site does not interact with the inactive proreceptor, whereas a rabbit polyclonal antireceptor antibody recognizes all forms. Thus, the autoimmune antibody must recognize a new epitope created during conversion of the inactive proreceptor to the active form.

Post-translational acquisition of ligand binding- and tyrosine kinase-domain function by the epidermal growth factor and insulin receptors.

The epidermal growth factor receptor (EGFR) and insulin receptor undergo slow post-translational modification by which they acquire hormone binding and tyrosine kinase (EGFR) function. The half-time for acquisition of EGF or insulin binding activity is 30-40 min and of tyrosine kinase activity (EGFR), is 10-15 min. Tunicamycin, an inhibitor of N-linked oligosaccharide addition, blocks acquisition of both EGF and insulin binding activity. With EGFR, activation precedes acquisition of resistance to endoglucosaminidase H (t1/2 approximately equal to 75 min), a medial Golgi event. Treatment of active high mannose receptor with endo H generates fully active aglyco-receptor; thus, core oligosaccharide addition is a prerequisite for activation, but not for EGF binding per se. EGFR is activated in and translocated from the endoplasmic reticulum (ER) slowly (t1/2 approximately equal to 75 min). Since translocation rate equals the rate for acquisition of endo H resistance, transit from the ER is rate limiting for EGFR maturation. Tunicamycin inhibits exit from the ER parallel to its effect on acquisition of binding activity. Insulin proreceptor, a 210 kDa high-mannose glycopolypeptide, acquires insulin binding function (t1/2 approximately equal to 45 min) then is proteolytically cleaved (t1/2 approximately equal to 3 hr) into subunits of the mature alpha 2 beta 2 receptor. Modification giving rise to insulin binding activity is due to a conformational change in the binding domain, since human autoimmune antibody recognizes only the active species, while rabbit polyclonal antibody recognizes all forms. Newly-translated EGF proreceptor lacks a functional tyrosine domain capable of autophosphorylation; 30-40 min after translation, while still in the ER, tyrosine kinase activity is acquired. Since the kinase domain is cytoplasmic, the receptor may become phosphorylated on tyrosine before reaching the plasma membrane.

Duplex pulsed Doppler echocardiography in mitral regurgitation.

The accuracy of duplex pulsed Doppler echocardiography (d-PDE) for detecting mitral regurgitation was evaluated in 35 patients undergoing d-PDE, cardiac auscultation, and left ventriculography. With three transducer positions, the overall d-PDE sensitivity was 95%, specificity was 100%, positive predictive value was 100%, negative predictive value was 94%, and diagnostic accuracy was 97% compared with ventriculography. This technique was superior to auscultation (sensitivity 74%, specificity 94%, positive predictive value 93%, negative predictive value 75%, diagnostic accuracy 83%). No false-positive d-PDE results occurred, but discordant false-negative results occurred frequently among the three transducer positions. If discordant negative results are considered to be false negative, then d-PDE is both sensitive and specific when mitral regurgitation is defined as systolic spectral broadening in any one transducer position.