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R J Darling

Publications and source records attributed to R J Darling.

8 recordsLinked to original sources
PubMed2001

Intracellular folding pathway of the cystine knot-containing glycoprotein hormone alpha-subunit.

Three of the five disulfide bonds in the glycoprotein hormone alpha-subunit (GPH-alpha) form a cystine knot motif that stabilizes a three-loop antiparallel structure. Previously, we described a mutant (alpha(k)) that contained only the three knot disulfide bonds and demonstrated that the cystine knot was necessary and sufficient for efficient GPH-alpha folding and secretion. In this study, we used alpha(k) as a model to study the intracellular GPH-alpha folding pathway. Cystine knot formation proceeded through a 1-disulfide intermediate that contained the 28-82 disulfide bond. Formation of disulfide bond 10-60, then disulfide bond 32-84, followed the formation of 28-82. Whether the two non-cystine knot bonds 7-31 and 59-87 could form independent of the knot was also tested. Disulfide bond 7-31 formed rapidly, whereas 59-87 did not form when all cysteine residues of the cystine knot were converted to alanine, suggesting that 7-31 forms early in the folding pathway and that 59-87 forms during or after cystine knot formation. Finally, loop 2 of GPH-alpha has been shown to be very flexible, suggesting that loop 2 does not actively drive GPH-alpha folding. To test this, we replaced residues 36-55 in the flexible loop 2 with an artificially flexible glycine chain. Consistent with our hypothesis, folding and secretion were unaffected when loop 2 was replaced with the glycine chain. Based on these findings, we describe a model for the intracellular folding pathway of GPH-alpha and discuss how these findings may provide insight into the folding mechanisms of other cystine knot-containing proteins.

Amino Acid Motifs↗
PubMed2000

Functional contributions of noncysteine residues within the cystine knots of human chorionic gonadotropin subunits.

Human chorionic gonadotropin (hCG) is a heterodimeric member of a family of cystine knot-containing proteins that contain the consensus sequences Cys-X(1)-Gly-X(2)-Cys and Cys-X(3)-Cys. Previously, we characterized the contributions that cystine residues of the hCG subunit cystine knots make in folding, assembly, and bioactivity. Here, we determined the contributions that noncysteine residues make in hCG folding, secretion, and assembly. When the X(1), X(2), and X(3) residues of hCG-alpha and -beta were substituted by swapping their respective cystine knot motifs, the resulting chimeras appeared to fold correctly and were efficiently secreted. However, assembly of the chimeras with their wild type partner was almost completely abrogated. No single amino acid substitution completely accounted for the assembly inhibition, although the X(2) residue made the greatest individual contribution. Analysis by tryptic mapping, high performance liquid chromatography, and SDS-polyacrylamide gel electrophoresis revealed that substitution of the central Gly in the Cys-X(1)-Gly-X(2)-Cys sequence of either the alpha- or beta-subunit cystine knot resulted in non-native disulfide bond formation and subunit misfolding. This occurred even when the most conservative change possible (Gly --> Ala) was made. From these studies we conclude that all three "X" residues within the hCG cystine knots are collectively, but not individually, required for the formation of assembly-competent hCG subunits and that the invariant Gly residue is required for efficient cystine knot formation and subunit folding.

Chorionic Gonadotropin↗
PubMed2000

Cystine knot mutations affect the folding of the glycoprotein hormone alpha-subunit. Differential secretion and assembly of partially folded intermediates.

The common glycoprotein hormone alpha-subunit (GPH-alpha) contains five intramolecular disulfide bonds, three of which form a cystine knot motif (10-60, 28-82, and 32-84). By converting each pair of cysteine residues of a given disulfide bond to alanine, we have studied the role of individual disulfide bonds in GPH-alpha folding and have related folding ability to secretion and assembly with the human chorionic gonadotropin beta-subunit (hCG-beta). Mutation of non-cystine knot disulfide bond 7-31, bond 59-87, or both (leaving only the cystine knot) resulted in an efficiently secreted folding form that was indistinguishable from wild type. Conversely, the cystine knot mutants were inefficiently secreted (<25%). Furthermore, mutation of the cystine knot disulfide bonds resulted in multiple folding intermediates containing 1, 2, or 4 disulfide bonds. High performance liquid chromatographic separation of intracellular and secreted forms of the folding intermediates demonstrated that the most folded forms were preferentially secreted and combined with hCG-beta. From these studies we conclude that: (i) the cystine knot of GPH-alpha is necessary and sufficient for folding and (ii) there is a direct correlation between the extent of GPH-alpha folding, its ability to be secreted, and its ability to heterodimerize with hCG-beta.

Alanine↗
PubMed2000

Autonomic nervous system evaluation of patients with vasomotor rhinitis.

OBJECTIVE: To demonstrate the utility of quantitative neurological laboratory testing of autonomic nervous system dysfunction and to apply this methodology to further study the relation of chronic vasomotor (nonallergic) rhinitis to the autonomic nervous system. METHODS: It has been suspected that vasomotor rhinitis is due either to a hyperactive parasympathetic nervous system or an imbalance between it and the sympathetic nervous system. The exact relation has not been determined. Recently neurological laboratories have been developed in which a battery of tests can be performed to determine reactivity of the autonomic nervous system. RESULTS: Autonomic nervous system testing was performed on 19 patients with symptoms fulfilling the diagnostic criteria for vasomotor rhinitis and the results were compared with 75 sex- and age-matched control subjects. Patients with vasomotor rhinitis had significant abnormalities of their sudomotor, cardiovagal, and adrenergic subscores. Their composite autonomic scale score was significantly impaired at 2.43, as compared with 0.11 for controls (P < .005). CONCLUSION: Autonomic nervous system dysfunction is significant in patients with vasomotor rhinitis. Possible factors that trigger this dysfunction including nasal trauma and extraesophageal manifestations of gastroesophageal reflux are discussed.

Adult↗
PubMedDate not supplied

Nature vs. nurture: two brothers with schizophrenia.

TOPIC: The nature vs. nurture argument as it pertains to two brothers. PURPOSE: To explore the synergistic effects of heritability and environment in the cases of two brothers with schizophrenia. SOURCES: Review of the literature and the authors' clinical experience. CONCLUSIONS: The nature vs. nurture dichotomy may not be as relevant as looking at the interaction between these two forces.

Environment↗