Thromboxane A2 synthase. Modification during "suicide" inactivation.

Thromboxane synthase is a ferrihemoprotein which undergoes mechanism-based inactivation during catalysis. This "suicide" process may be an important factor for limiting thromboxane A2 biosynthesis in cells. Although the kinetics have been characterized for purified enzyme and platelets, the chemical basis for inactivation has remained unclear. Protein modification or alteration of the heme prosthetic group is each compatible with the irreversible nature of suicide inactivation of thromboxane synthase. We have investigated these two possibilities using enzyme purified to homogeneity. Our data show that the Soret absorbance spectrum of thromboxane synthase is unaltered by additions of prostaglandin endoperoxide H2 which cause enzymatic inactivation. Using a coupled cyclooxygenase/thromboxane synthase system and polyacrylamide gel electrophoresis we have demonstrated that the enzyme retains radiolabel under nondenaturing gel conditions. Label incorporation is reduced by the competitive thromboxane synthase inhibitor U63557, an agent that also protects the enzyme from inactivation. Under denaturing conditions the radiolabel localizes with the released heme prosthetic group. In addition, interaction of the heme prosthetic group with cyanide was prevented by inactivating the enzyme with prostaglandin H2. In similar experiments, the lipid hydroperoxide 15(S)-hydroperoxyeicosatetraenoic acid inactivated thromboxane synthase with concurrent bleaching of the Soret spectrum. Labeling studies with a coupled soybean lipoxygenase/thromboxane synthase system indicate that, in this case, the apoenzyme is modified. These results suggest that the mechanism of thromboxane synthase inactivation during thromboxane A2 biosynthesis involves a tight, nondestructive association of substrate or product with the prosthetic heme group. Inactivation by hydroperoxides, however, appears to result from apoenzyme modification. These reactions may have important implications for cellular physiology and pathophysiology of thrombosis.     

Bacillary angiomatosis. A "new" disease with a broadening clinicopathologic spectrum.

Bacillary angiomatosis (BA) is a reactive vasoproliferative lesion occurring almost exclusively in immunocompromised individuals in response to infection by a bacillus closely related to Rochalimaea quintana. The commonest site of involvement is the skin, in the form of multiple erythematous nodules, but bacillary angiomatosis can also present in a wide variety of sites such as soft tissues, bone, lymph node, liver and spleen. Some patients may present with persistent fever and bacteraemia. Bacillary angiomatosis is characterized histologically by proliferation of blood vessels lined by plump endothelium, associated with an interstitial eosinophilic or amphophilic material formed by aggregated bacilli, best demonstrated by the Warthin-Starry stain. A heavy infiltrate of neutrophils is frequently, but not invariably, present. In the liver and spleen, there may be in addition features of peliosis. It is important to be able to diagnose bacillary angiomatosis correctly because prompt treatment with antibiotics is potentially life-saving.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. II. A study of the "inside-out" configuration

The single K-channel current reported in a previous note was also studied in "outside-out" conditions. The electrode filling solutions used for the "cell-attached" experiments faced in this case the intracellular side of the membrane patches, the extracellular side facing the bath saline, i.e. Ringer standard. The most significant observations were obtained with filling solutions with varying proportions in K/Na concentrations solutions. In the absence of Na+ ([K+] = 110 mM), the elementary conductance was still around 90 pS and the I/V diagram was again somewhat bell shaped, though the distinctive reduction of the elementary conductance began at more positive potentials (+110 mV). No inward current could be detected upon membrane repolarization also in this case. The rectification became less evident and conductance increased with increasing Na+ concentration in the filling solution, until the I/V curve became a linear one and conductance was 270 pS with standard Ringer. Distinct inward elementary currents were evident upon repolarization in these conditions. Thus a complex interaction between Na+ and K+ takes place for conduction through the outward K channel in the frog oocyte, both cations probably competing for at least one active site inside. Another interesting observation concerns the process of gating of the OPC: the open times of the elementary currents were in fact much greater in outside out experiments as compared to cell-attached experiments, probably due to the presence of Ca++ in contact with the inner membrane side. Even increasing Na+ concentration prolonged the open time duration. The gating of the OPC in the membrane was not only voltage dependent, but also Ca++ and Na+ dependent.     

A study of the "outward potassium channel" (OPC) in the frog oocyte. I. A study of the "cell-attached" configuration

A single channel current was studied in the membrane of the immature oocyte of the european frog (Rana esculenta) by using the "patch clamp" technique in the "cell attached" configuration. Single channel activity appeared as short outward currents when membrane potential was made positive inside; full activation required seconds to be complete, no inactivation being appreciable. Deactivation (or current block) upon membrane repolarization was so fast that no inward current could be detected in any case. The reversal potential, estimated by interpolating the I/V diagrams, was -30 mV using standard Ringer as electrode filling solution, and the elementary conductance was 95 pS. Neither reversal potential nor elementary conductance were affected by removal of external Ca2+ (Mg2+ or Ba2+ substitution) or external Cl- (methanesulphonate substitution). The reversal potential moved towards positive potentials by substituting external Na+ with K+, the magnitude of the shifts being consistent with a ratio PK/PNa = 6.4. A distinctive property of the current/voltage relation for this K-current is its anomalous bell-shape, the outward current displaying a maximum at membrane potentials around 75 mV with standard Ringer as electrode filling solution and tending to zero with more positive potentials.     

Characterization of the Mlsf system. I. A novel "polymorphism" of endogenous superantigens.

In addition to Mlsa (Mls-1a) and Mlsc (Mls-2a, Mls-3a), we and others have recently described a third set of stimulatory minor lymphocyte stimulating (Mls) determinants, which are ligands for "I-E related" V beta, V beta 5, V beta 11, and V beta 12. Although all V beta associated with the recognition of the conventional Mls determinants are, in general, uniformly deleted in those animals expressing relevant Mls, expression of Mlsf-related V beta reveals various deletion patterns among different strains. Here we describe extensive genetic studies to evaluate the relationship among the self-Ag responsible for clonal deletion of T cells bearing Mlsf-related V beta by using antibodies specific for TCR V beta chain. In addition, a panel of T cell clones specific for the Mlsf determinant were generated and employed to analyze the determinant specificity, which is recognized by Mlsf-reactive T cells in vitro as well as the role of class II molecules in T cell recognition of the Mlsf determinants. The results of these two independent approaches provide evidence that the Mlsf system is composed of a set of gene products that reveal a unique polymorphism in the induction of clonal deletion in vivo and in T cell activation in vitro. One of these gene products causes almost complete deletion of the self-Mlsf reactive T cell repertoire in vivo and elicits a strong proliferative response to Mlsf-specific T cell clones. Expression of the other gene products results in the clonal deletion of only part of the Mlsf-reactive T cell repertoire. Furthermore, the response pattern of Mlsf-specific clones to intra-MHC recombinant inbred strains and the inhibition pattern of these clones by anti-class II antibody suggested that although expression of the I-E molecule is essential for T cell recognition of Mlsf determinants, the A beta gene may also contribute to the efficient presentation of Mlsf determinants by forming unique class II E alpha A beta molecules.     

Characterization of the "microprotease" from Bacillus cereus. A zinc neutral endoprotease.

The neutral protease isolated from Bacillus cereus (BRL-70) has been purified by affinity chromatography and characterized. The enzyme exhibits a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, has a molecular weight of 34 000 by ultracentrifugation, and contains one enzymatically essential zinc atom per 34 000 g. These data together with the amino acid composition, response to metal substitution, chemical modification, and substrate specificity all indicate that this protease is monomeric and is a typical bacterial neutral metalloprotease.     

"Nonclassic" cytologic signs of cervical condyloma. A case-control study

The association between "nonclassic" cytologic signs of condyloma and human papillomavirus (HPV) infection in women with negative Papanicolaou smears was analyzed via a case-control study. The cytologic signs considered were mild koilocytosis, mild dyskeratosis, binucleation or multinucleation, cleared cytoplasm and nuclear hyperchromatism. The Papanicolaou smears of 166 cases that showed colposcopic and histologic evidence of HPV infection (but whose smears lacked the classic cytologic signs of condyloma) and 166 controls that were negative colposcopically were randomly admixed and blindly reviewed by a panel of cytologists. A significant association to HPV infection was observed for all of the nonclassic signs studied, but multivariate analysis showed a weakly independent association only for mild koilocytosis. The sensitivity (0.46) and the specificity (0.87) of these nonclassic signs were not satisfactory. The utility of selecting women with negative Papanicolaou smears for colposcopy on the basis of these signs is discussed.     

A functional protein pore with a "retro" transmembrane domain.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.