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.

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)

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.

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)

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.

Cosmetic surgery and criminal rehabilitation.

The contribution of physical appearance to criminal behavior has long been a matter of general interest. We investigated the influence of cosmetic surgery on recidivism rates in the Texas state prison system. The baseline prison population recidivism rates have been 14%, 32%, and 36% at one, two, and three years, respectively. The study group consisted of 253 inmates who had cosmetic procedures between 1982 and 1984 and who were released from prison between the time of operation and the end of 1986. The recidivism rates, calculated by life table analysis, were 8% at one year, 17% at two years, and 25% at three years. All were significantly less than baseline (P less than .01, P less than .001, P less than .01). The rates for those who had been incarcerated for violent crimes were 3.3%, 7.7%, and 15.0% (all P less than .001). We conclude that a positive relationship exists between cosmetic surgery and criminal rehabilitation, as measured by a decrease in recidivism.

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)

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.

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.

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.

Specific adsorption of a platelet membrane glycoprotein by human insoluble collagen.

A platelet membrane glycoprotein, 61 kDa, has been identified, which binds specifically to insoluble collagen. The detection of this protein was accomplished by incubating radiolabeled Triton-solubilized platelet supernatant with insoluble collagen, and, after washing the collagen pellet, extracting the 61-kDa glycoprotein from the pellet with sodium dodecyl sulfate buffer. The optimal conditions for specific binding were incubation of 120 micrograms of total platelet supernatant protein with 2 mg of collagen at 4 degrees C for 0.5 h in 0.5 ml of the incubating buffer (20 mM Tris, 150 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, and 0.2% Triton, pH 7.4). The 61-kDa glycoprotein is cleaved by trypsin into a major peptide (44 kDa) and a smaller peptide(s) linked together by disulfide bonds in a molecule which still binds to collagen. When intact platelets are treated first with trypsin and then with dithiothreitol, the 44-kDa peptide is released and was shown to bind to collagen. We conclude that the 61-kDa glycoprotein is a platelet membrane protein which specifically interacts through its extracellular domain with insoluble collagen, and, thus, must be considered as a possible component of the initial platelet-matrix adhesion process which leads to platelet aggregation in vivo.

Interaction of specific platelet membrane proteins with collagen: evidence from chemical cross-linking.

Two recently developed membrane-impermeant cross-linkers, 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP) and bis(sulfosuccinimidyl) suberate (BS3), have been used to examine the interaction of human platelets with collagen. Reaction of human platelets with either of the two cross-linking reagents at micromolar concentrations completely inhibited platelet aggregation in response to collagen but not in response to thrombin. Platelet adhesion to collagen was, however, not affected by these reagents. Inhibition of collagen-induced platelet aggregation by DTSSP or BS3 appears to be due to cross-linking and not simply to the chemical modification of membrane proteins, since the homologous monofunctional reagent sulfosuccinimidyl propionate had no effect on platelet aggregation. Inhibition of platelet aggregation by BS3 was accompanied by a decrease in the intensity of glycoprotein bands IIb, IIIa, and IV when analyzed on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels. In order to determine if collagen is directly interacting with a specific platelet membrane glycoprotein, 3H-labeled platelets were allowed to adhere to collagen and then cross-linked with various concentrations of DTSSP. Proteins which remain associated with collagen after lysis and washing were analyzed on NaDodSO4 gels. At concentrations of 16-50 microM DTSSP, glycoproteins IIb and IIIa appeared to be specifically cross-linked to collagen. These results suggest that the glycoprotein IIb-IIIa complex, which has previously been implicated as the fibrinogen receptor in activated platelets, may also be directly involved in collagen-induced platelet aggregation.

Adhesion of human platelets to collagen: evidence for two states of platelet activation.

Normal human platelets suspended in buffer were exposed to a suspension of bovine tendon collagen fibrils and the rate of adhesion observed over a period of 60 minutes. In fresh platelet preparations, approximately 15-20% of the platelets adhered within one minute, and the total percent adhesion, following pseudo first order kinetics after the first minute, approached 60% after 60 minutes. Pre-treatment with N-ethylmaleimide, dithiothreitol, and glutaraldehyde reduced adhesion at 1 minute to less than 5%, but only N-ethylmaleimide completely blocked adhesion at 60 minutes. Thus, freshly collected, washed human platelets contain two distinct subpopulations exhibiting greatly differing rates of adhesion, reflecting different levels of reactivity toward collagen.

Comparative analysis of glycopeptides derived from human platelet membrane glycoprotein Ib.

Although several purification procedures have been reported for platelet glycocalicin and macroglycopeptide, compositional data suggest that contamination with tightly associated peptide fragments is a continuing problem. This, together with the lack of a reliable estimate of molecular weights, has delayed a clear resolution of the relationship of intact platelet membrane glycoprotein Ib and these proteolytically derived glycopeptides. A new procedure was developed for purification of both macroglycopeptide and glycocalicin from human platelet plasma membranes. It consists of ion-exchange chromatography on diethylaminoethyl-Sephacel, lectin affinity chromatography on wheat germ agglutinin coupled to Sepharose, and gel filtration chromatography under denaturing conditions and avoids exposure of these sialylated glycoproteins to acidic conditions. Electrophoretic evidence for the purity of macroglycopeptide and glycocalicin prepared by this procedure was obtained by Laemmli (1970) [Laemmli, U.K. (1970) Nature (London) 227, 680-685] sodium dodecyl sulfate-polyacrylamide gel electrophoresis of samples radiolabeled by sequential sodium metaperiodate oxidation and borotritide reduction. Electrophoresis gave apparent molecular weights of 108 000 and 118 000 for macroglycopeptide and glycocalicin, respectively. However, sedimentation equilibrium centrifugation experiments using the meniscus-depletion method in 6 M guanidine hydrochloride established the weight-average molecular weights of macroglycopeptide and glycocalicin as 59 700 and 105 600, respectively. The molecular weight determinations are the first by a primary physical method for platelet macroglycopeptide and glycocalicin and, together with compositional analyses, permitted calculation of the compositions of the two glycopeptides in terms of residues per molecule, which is consistent with the derivation of macroglycopeptide from glycocalicin by proteolysis.