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Biomedical subjects

L W Cunningham

Publications and source records attributed to L W Cunningham.

At least 19 recordsLinked to original sources
PubMed1991

Pulmonary infiltrates associated with Vibrio vulnificus septicemia.

In the case described, fatal Vibrio vulnificus primary septicemia was complicated by development of diffuse pulmonary infiltrates. Clinical features of this recently discovered infectious disease are discussed. Various aspects of diagnoses of pulmonary infiltrates in patients with V vulnificus septicemia are also presented. Vibriosis should be suspected in patients with chronic, underlying, predisposing diseases, recent ingestion of raw seafood, and characteristic skin lesions.

Humans↗
PubMed1990

Separation and localization of the four cysteine-949 residues in human alpha 2-macroglobulin using fluorescence energy transfer.

By use of the intermediate form (I-form) [Gettins, Crews, & Cunningham (1989) Biochemistry 28, 5613-5618], alpha 2-macroglobulin can be specifically labeled with fluorescent probes in a manner that allows the determination of the topology of the four thiol ester derived Cys949 residues within this large tetrameric protease inhibitor. Freshly prepared I-form alpha 2-macroglobulin was reacted with 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonate (1,5-I-AEDANS) to produce alpha 2-macroglobulin specifically and stoichiometrically labeled with 1,5-AEDANS (donor) at the two Cys949 SH groups in the first protease interaction site. Upon subsequent reaction of this labeled species with chymotrypsin, the remaining two bait regions and thiol ester linkages were opened, generating two free SH groups on the two Cys949 residues in the second protease interaction site. These SH groups were specifically and stiochiometrically labeled with 5-(iodoacetamido)fluorescein (acceptor). Fluorescence energy transfer from donor to acceptor results in 82% loss of AEDANS fluorescence intensity. By use of an R0(2/3) value of 43.5 A, calculated from the spectral parameters of this system, an R(2/3) separation between donor and acceptor of 33.9 A was calculated. From fluorescence anisotropy measurements of both donor and acceptor attached to alpha 2-macroglobulin, upper and lower limits on the separation of 43.4 and 26.1 A, respectively, were calculated. These separations, small in the context of the alpha 2-macroglobulin tetramer, which has approximate dimensions of 190 x 90 x 90 A, severely restrict the possible locations of the four Cys949 residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1990

Inhibition and partial reversal of the methylamine-induced conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin by modification of the thiols.

It has been shown previously [Van Leuven, F., Marynen, P., Cassiman, J. J., & Van den Berghe, H. (1982) Biochem. J. 203, 405-411] that 2,4-dinitrophenyl thiocyanate (DNPSCN) can block the conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin (alpha 2M) normally resulting from reaction of alpha 2M with methylamine. The kinetics of reaction of DNPSCN with alpha 2M in the presence of methylamine are examined here and shown to approximate pseudo first order, reflecting the rate-limiting reaction of alpha 2M with methylamine [Larsson, L. J., & Björk, I. (1984) Biochemistry 23, 2802-2807]. One mole of DNPS is liberated per mole of free thiol in alpha 2M, consistent with cyanylation of the thiol liberated upon scission of the internal thiol esters by methylamine. I3(-) can also react with the methylamine-generated thiol groups of alpha 2M with a stoichiometry consistent with conversion of the thiol to a sulfenyl iodide. Reaction of the thiol groups with either DNPSCN or I3(-) inhibits the conversion of alpha 2M from the "slow" to the "fast" electrophoretic form. Furthermore, DNPSCN added after the conformational change can partially reverse the change. A similar reversal can be effected by cyanylation, with NaCN, of methylamine-treated alpha 2M in which the liberated thiols have first been converted to mixed disulfides by reaction with dithiobis(nitrobenzoic acid). Differential scanning calorimetry shows nearly identical properties for the methylamine-treated "fast" form and the cyanylated "slow" form of alpha 2M.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1990

Cholecystopleural fistula with cholelithiasis presenting as a right pleural effusion.

At autopsy, multiple gallstones were recovered from the right pleural space of an elderly patient who presented with a massive right pleural effusion and septic shock. The mechanisms of gallstone migration and fistula formation between the gallbladder and right pleural space are described. Despite atypical presentations, gallbladder disease remains an important differential consideration of right pleural effusion in the elderly.

Aged↗
PubMed1989

Preparation and initial characterization of an intermediate, half-cleaved form of human alpha 2-macroglobulin.

A form of human alpha 2-macroglobulin (alpha 2M) has been prepared that has properties intermediate to those of native alpha 2-macroglobulin and 2:1 protease-alpha 2 M ternary complex by using Sepharose-linked chymotrypsin. The intermediate form has mobility on native polyacrylamide gels between the fast and slow forms of alpha 2M and migrates as a diffuse band. Two bait regions and two thiol esters per alpha 2M tetramer are cleaved, although no chymotrypsin is detectable in the modified alpha 2-macroglobulin species. The remaining bait regions and thiol esters can be cleaved by further reaction with other proteases. Intermediate-form alpha 2M can trap 1.18 mol of chymotrypsin, 0.85 mol of trypsin, and 0.65 mol of thrombin. Although both thrombin and methylamine react with intermediate-form alpha 2M at rates not distinguishable within experimental error from those of their reactions with native alpha 2M, chymotrypsin-Sepharose reacts much more slowly with the intermediate form than with native alpha 2 M, indicating a nonequivalence of the two reactive sites on alpha 2M. This nonequivalence may be present initially or be induced by reaction at the first site. Comparison of ESR results obtained from spin-labeling methylamine-treated or protease-reacted alpha 2M with those from spin-labeling of the free SH groups in intermediate-form alpha 2M shows that trapped protease influences the mobility of the attached nitroxide either through direct contact or by producing a different conformation from that present in methylamine-treated or intermediate-form alpha 2M.

Binding Sites↗
PubMed1988

Proximity of thiol esters and bait region in human alpha 2-macroglobulin: paramagnetic mapping.

The two key structural features of alpha 2-macroglobulin (alpha 2M) involved in inhibitory caging of proteases are the thiol ester and the bait region. This paper examines the environment of the hydrolyzed thiol ester in methylamine-treated human alpha 2M and the separation between the bait region and the thiol ester and between the four thiol esters in the tetramer to try to further our understanding of how bait region proteolysis triggers thiol ester cleavage. The sulfhydryl groups of Cys-949, formed upon cleavage of the thiol ester by methylamine, were specifically labeled with the nitroxide spin-labels 3-(2-iodoacetamido)-PROXYL (iodo-I) (PROXYL = 2,2,5,5-tetramethylpyrrolidine-1-oxyl), 3-[2-(2-iodoacetamido)acetamido]-PROXYL (iodo-II), and 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidine-1-oxyl (iodo-III). ESR spectra of these alpha 2M derivatives showed that label I is firmly held and label II has limited freedom of rotation consistent with location of the cysteine residue in a narrow cavity. Label III has much greater motional freedom. From the absence of dipole-dipole splittings in the ESR spectra, it is concluded that the four nitroxide groups in the tetramer are more than 20 A apart for both label I and label II. Label I broadens 1H NMR signals from one phenylalanyl, one tyrosyl, and four histidyl residues in the bait region. Separations of 11-17 A are estimated between the nitroxide of label I and these residues. Label II is further away and only broadens resonances from one of the histidines.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1987

In support of the trap hypothesis. Chymotrypsin is not rigidly held in its complex with human alpha 2-macroglobulin.

Complexes (2:1) of chymotrypsin with human alpha 2-macroglobulin have been prepared in the presence of 200 mM methylamine such that 90% of the chymotrypsin remains noncovalently bound to the alpha 2-macroglobulin. Reaction of this complex with the active-site-directed spin-labeling reagent 4-[(ethoxyfluorophosphinyl)oxy]-2,2,6,6-tetramethylpiperidinyl+ ++-1-oxy results in nitroxide labeling of the active-site serine residue of the complexed chymotrypsin. Electron spin resonance (ESR) spectra of this complex were recorded at 275 K in buffer and at 263 K in 50% glycerol. At 263 K in 50% glycerol the spectrum is that expected for a rigid glass, whereas at room temperature the ESR spectrum shows that the chymotrypsin is only slightly immobilized compared with free spin-labeled chymotrypsin. These findings are discussed in relation to possible models of inhibition of protease activity by alpha 2-macroglobulin. It is concluded that the trap mechanism of Barrett and Starkey [Barrett, A. J., & Starkey, P. M. (1973) Biochem. J. 133, 709-724] is the only model currently considered that can account for the present findings.

Chymotrypsin↗
PubMed1986

A unique pair of zinc binding sites in the human alpha 2-macroglobulin tetramer. A 35Cl and 37Cl NMR study.

35Cl NMR has been used to demonstrate that human alpha 2-macroglobulin tetramer possesses a unique pair of zinc binding sites. Zinc bound at these sites does not affect the 35Cl NMR line width of free Cl-. Additional lower affinity zinc sites exist that bind chloride weakly and cause broadening of the free chloride resonance through fast exchange with bound chloride. Using both 35Cl and 37Cl relaxation measurements it has been shown that chloride bound at these sites has an internal correlation time of 5.1 ns and a quadrupolar interaction, chi, of 4.2 MHz with zinc. Manganese binds to apo-alpha 2-macroglobulin analogously to zinc. alpha 2-macroglobulin that has been reacted with methylamine still possesses two classes of zinc sites per tetramer, but their relative affinities differ more than for unreacted alpha 2-macroglobulin. These data are discussed with respect to possible models for the subunit arrangement in the tetramer.

Binding Sites↗
PubMed1986

Identification of 1H resonances from the bait region of human alpha 2-macroglobulin and effects of proteases and methylamine.

The 1H NMR spectrum of human alpha 2-macroglobulin, Mr 716,000, consists of predominantly extremely broad unresolved resonances but also has nine relatively sharp (delta nu 1/2 less than 25 Hz) resonances from aromatic residues. By treatment of alpha 2-macroglobulin with methylamine, chymotrypsin, and subtilisin, it has been shown that eight of these resonances arise from bait region residues. More specifically, assignment has been made of resonances at 6.80 and 7.11 ppm to the ortho and meta protons, respectively, of tyrosine-685 and tentative assignment of a resonance at 7.29 ppm to the aromatic protons of phenylalanine-684. C2 proton resonances from five histidine residues are also visible. Four of these are attributed to residues in the bait region or immediately adjacent to this, at positions 675, 694, 699, and 704. The sharpness of resonances from bait region residues demonstrates the great flexibility of this region of the polypeptide. It is proposed that the flexible region extends from residue 675 to residue 710. These resonances are all affected by proteolytic cleavage in the bait region but are not influenced by the subsequent conformational rearrangement of the whole protein tetramer. The significance of these findings is discussed in relation to the current structural models of alpha 2-macroglobulin.

Humans↗
PubMed1979

Cross-linking experiments with the adenosine triphosphatase of sarcoplasmic reticulum.

The proteins of sarcoplasmic reticulum were cross-linked by rapid oxidation of thiol groups with I2. About two-thirds of the thiols were oxidized without any significant cross-linking, implying an extensive formation of intramolecular disulphide bonds. When the thiols were completely oxidized at room temperature a series of oligomers containing up to five molecules were observed, as well as large aggregates which were excluded from the gels. Complete oxidation at -10 degrees C left most of the ATPase (adenosine triphosphatase) as monomer. Similar results were obtained when copper-phenanthroline complexes or dimethyl suberimidate were used as cross-linking reagents. We conclude that most of the cross-linked species arise by linking of randomly colliding ATPase molecules which are present in the membrane at very high concentration.

Adenosine Triphosphatases↗
PubMed1979

Fibronectin and the multiple interaction model for platelet-collagen adhesion.

A rapid, sensitive, and reproducible assay to determine the adhesion of platelets to collagen has been developed. Collagen fibers and adherent platelets are retained on polycarbonate membrane filters. Chemical modification of collagen by acetylation and of platelets by treatment with chymotrypsin markedly reduces adhesion. The role of fibronectin in the collagen-platelet interaction has been examined. Treatment of platelets with purified antibody or Fab' fragments to fibronectin only slightly reduces adhesion. Preincubation of platelets with high concentrations of gelatin reduces adhesion by only 22% but fails to inhibit aggregation. Thus, fibronectin has only a limited role in the adhesion of platelets to collagen and is either not involved in the adhesion that leads to aggregation or is only one of several adhesion mechanisms, any of which alone can initiate aggregation.

Animals↗
PubMed1977

Circular dichroism of glycopeptide fractions from alpha1-acid glycoprotein, thyroglobulin, and ovalbumin.

Using sequential pronase digestions, glycopeptide fractions were prepared from human alpha1-acid glycoprotein, hen egg ovalbumin, and bovine thyroglobulin, two types of glycopeptides being obtained from the latter. The fractions were characterized on the basis of hexose, hexosamine, sialic acid, and peptide content. The glycopeptide fraction from alpha1-acid glycoprotein is complex (i.e., the carbohydrate moiety contains mannose, galactose, N-acetylglucosamine, and sailic acid), as is one of the glycopeptide fractions from thyroglobulin (type I). The glycopeptide fraction from ovalbumin and the type II glycopeptide fractions from thyroglobulin are simple (i.e., the carbohydrate moiety contains only mannose and N-acetylglucosamine). The circular dichroic spectra of the two complex glycopeptide fractions and the ovalbumin glycopeptide fraction were similar and were characterized by a negative extremum between 207.5 nm and 211 nm with magnitudes in the range of --6400 deg-cm2-dmol-1 to --7200 deg-cm2-dmol-1 (referred to the molar concentration of N-acetylated sugars). The thyroglubulin type II glycopeptide fraction exhibited a circular dichroic spectrum with an extremum of --29 200 deg-cm2-dmol-1 at 205 nm. Removal of sialic acid from the complex glycopeptide fractions greatly increased the (negative) magnitude of ellipticity at the extremum. The circular dichroic spectra of the complex of glycopeptide fractions were reasonably additive using the spectra of monomeric sialic acid and the asialo-derivatives. This demonstrates that the contributions of sialic acid to the circular dichroic spectrum are nearly additive. The implications of this observation are that covalent attachment of these terminal residues to the oligosaccharides does not lead to strong interactions with other chromophores nor to positioning in particularly asymmetric environment. In contrast, the magnitudes of the observed ellipticity extrema in the circular dichroic spectra of the asialo-derivatives, in which N-acetylglucosamine is the major chromophore, are much greater than can be accounted for on the basis of monomeric contributions (i.e., free N-acetylglucosamine). This finding shows that the optical activity of N-acetyl-glucosamine is greatly influenced by the formation of the carbohydrate core in glycoproteins and suggests the possible formation of secondary structure in the carbohydrate moiety.

Amino Acids↗
PubMed1977

The synthesis of macromolecular 3-hydroxyproline by attaching and confluent cultures of human fibroblasts.

The synthesis of collagen has been studied during the attachment of freshly trypsinized human fibroblasts to culture vessels by measurement of the incorporation of radioactive proline into macromolecular hydroxyproline. Collagenous protein(s) was found to be a component of a substrate-attached material ('microexudate carpet') synthesized rapidly during cell attachment in the absence of serum. The ratio of 3-hydroxyproline/4-hydroxyproline in the collagenous proteins synthesized during cell attachment was found to be 4-5 fold higher than that of normal type I collagen. The synthesis of 3-hydroxyproline by confluent cultures was diminished by serum deprivation, and was shown to require higher concentrations of ascorbate than the synthesis of the 4-hydroxy isomer.

Ascorbic Acid↗