Biomedical subjects

P L Clark

Publications and source records attributed to P L Clark.

12 recordsLinked to original sources

PubMed2002

Safety and efficacy of nitric oxide in chronic lung disease.

BACKGROUND: Therapies for neonatal chronic lung disease (CLD) of prematurity have had limited success. AIMS: To determine whether inhaled nitric oxide (INO) administered to very low birthweight infants with developing CLD might improve oxygenation without adverse effects. METHODS: Subjects were 10-30 days of age, birth weight < 1250 g, with developing or established CLD, and requiring mechanical ventilation with mean airway pressure > or = 7 cm H2O and FIO2 . or = 0.40. We monitored changes in oxygenation and FIO2 requirement during treatment with INO (initial dose 20 ppm). Tracheal aspirate samples obtained before, during, and after treatment were analysed for interleukin 1beta (IL-1beta), IL-8, 8-epi-prostaglandin F2alpha (8-epi-PGF2alpha), laminin, and endothelin 1 (ET-1) to assess any potential effects of INO on markers of inflammation peroxidation, basement membrane injury, or vasoactivity. RESULTS: Thirty three patients met entry criteria. Mean gestational age was 25 (SD 2) weeks; birth weight was 736 (190 g); age of study infants was 19 (6) days (range 9-29). Mean FIO2 decreased from baseline (0.75) to 0.58 at 72 hours. Duration of therapy was seven days. Tracheal aspirate concentrations of IL-1beta, IL-8, 8-epi-PGF2alpha, ET-1, and laminin were unchanged between baseline and 48 hours of INO, and 48 hours after discontinuation of INO. No new cases of, nor extension of, intraventricular haemorrhage occurred. Four infants died. CONCLUSION: INO (< or = 20 ppm) improved oxygenation in most infants with early CLD, without inducing changes in markers of inflammatory or oxidative injury.

Biomarkers↗

PubMed2001

A newly synthesized, ribosome-bound polypeptide chain adopts conformations dissimilar from early in vitro refolding intermediates.

Little is known about the conformations of newly synthesized polypeptide chains as they emerge from the large ribosomal subunit, or how these conformations compare with those populated immediately after dilution of polypeptide chains out of denaturant in vitro. Both in vivo and in vitro, partially folded intermediates of the tailspike protein from Salmonella typhimurium phage P22 can be trapped in the cold. A subset of monoclonal antibodies raised against tailspike recognize partially folded intermediates, whereas other antibodies recognize only later intermediates and/or the native state. We have used a pair of monoclonal antibodies to probe the conformational features of full-length, newly synthesized tailspike chains recovered on ribosomes from phage-infected cells. The antibody that recognizes early intermediates in vitro also recognizes the ribosome-bound intermediates. Surprisingly, the antibody that did not recognize early in vitro intermediates did recognize ribosome-bound tailspike chains translated in vivo. Thus, the newly synthesized, ribosome-bound tailspike chains display structured epitopes not detected upon dilution of tailspike chains from denaturant. As opposed to the random ensemble first populated when polypeptide chains are diluted out of denaturant, folding in vivo from the ribosome may begin with polypeptide conformations already directed toward the productive folding and assembly pathway.

Antibodies, Monoclonal↗

PubMed2001

Distinct cysteine sulfhydryl environments detected by analysis of Raman S-hh markers of Cys-->Ser mutant proteins.

Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.

Amino Acid Substitution↗

PubMed1998

Probing the folding pathway of a beta-clam protein with single-tryptophan constructs.

BACKGROUND: Cellular retinoic acid binding protein I (CRABPI) is a small, predominantly beta-sheet protein with a simple architecture and no disulfides or cofactors. Folding of mutants containing only one of the three native tryptophans has been examined using stopped-flow fluorescence and circular dichroism at multiple wavelengths. RESULTS: Within 10 ms, the tryptophan fluorescence of all three mutants shows a blue shift, and stopped-flow circular dichroism shows significant secondary structure content. The local environment of Trp7, a completely buried residue located near the intersection of the N and C termini, develops on a 100 ms time scale. Spectral signatures of the other two tryptophan residues (87 and 109) become native-like in a 1 s kinetic phase. CONCLUSIONS: Formation of the native beta structure of CRABPI is initiated by rapid hydrophobic collapse, during which local segments of chain adopt significant secondary structure. Subsequently, transient yet specific interactions of amino acid residues restrict the arrangement of the chain topology and initiate long-range associations such as the docking of the N and C termini. The development of native tertiary environments, including the specific packing of the beta-sheet sidechains, occurs in a final, highly cooperative step simultaneous with stable interstrand hydrogen bonding.

Circular Dichroism↗

PubMed1997

Cavity formation before stable hydrogen bonding in the folding of a beta-clam protein.

The time course of folding of a small beta-sheet protein reveals formation of a central ligand binding cavity before the consolidation of the native hydrogen bonding network. These results suggest that side chain interactions and not stable hydrogen bonding determine the beta-sheet architecture and play crucial roles in the overall chain topology.

Hydrogen Bonding↗

PubMed1996

Intrinsic tryptophans of CRABPI as probes of structure and folding.

The native state fluorescence and CD spectra of the predominantly beta-sheet cellular retinoic acid-binding protein I (CRABPI) include contributions from its three tryptophan residues and are influenced by the positions of these residues in the three-dimensional structure. Using a combination of spectroscopic approaches and single Trp-mutants of CRABPI, we have deconvoluted these spectra and uncovered several features that have aided in our analysis of the development of structure in the folding pathway of CRABPI. The emission spectrum of native CRABPI is dominated by Trp 7. Trp 109 is fluorescence-silent due to its interaction with the guanidino group of Arg 111. Although the far-UV CD spectrum of CRABPI is largely determined by the protein's secondary structure, aromatic clustering around Trp 87 and the aromatic-charge interaction between Arg 111 and Trp 109 give rise to a characteristic feature in the CD spectrum at 228 nm. The near-UV CD bands of CRABPI arise largely from additive contributions of the three tryptophan residues. Trp 7 and Trp 87 give a negative CD band at 275 nm. The near-UV CD band from Trp 109 is positive and shifted to longer wavelengths (to 302 nm) due to the charge-aromatic interaction between Arg 111 and Trp 109. Our deconvolution of the equilibrium spectra have been used to interpret kinetic folding experiments monitored by stopped-flow fluorescence. These dynamic experiments suggest the early evolution of a well-populated, hydrophobically collapsed intermediate, which undergoes global rearrangement to form the fully folded structure. The results presented here suggest several additional strategies for dissecting the folding pathway of CRABPI.

Circular Dichroism↗

PubMed1985

The Rh phenotype r'r' in Polynesians.

Since 1970, 19 persons with the r'r' phenotype have been detected at the Auckland Blood Transfusion Centre. All have been Maoris or Pacific Islanders. This study reports the detailed serological investigations on 11 r'r' samples. No evidence was found for the presence of the Du antigen, and there was no evidence that the Polynesian r' was related to the Negroid r'. It is postulated that the r' (Cde) of Polynesians arose from a mutation of CDe. No morphological abnormalities of r'r' red cells were found. Blood samples were also tested for various high- and low-frequency antigens, and one specimen was found to be r'r' Jk(a-b-).

Erythrocytes↗

PubMed1983