A new MMPI-derived indicator of liability to develop schizophrenia: evidence from the New York High-Risk Project.

A large body of research indicates that the liability to develop schizophrenia is largely genetically mediated, although phenotypic expression requires environmental triggers/insults and/or epigenetic and/or stochastic factors. In an effort to identify the precise environmental factors that precipitate a predisposition to schizophrenia, researchers have implemented a high-risk model-the prospective study of offspring born to schizophrenic parents. As it is difficult to ascertain exactly which of the "high-risk" participants will actually develop the disorder, we examined the validity of an experimental MMPI scale, Schizophrenia Proneness (SzP), and the Moldin-Gottesman psychometric index to identify such individuals. Results suggest that the SzP scale can be an effective predictor of schizophrenia-related psychoses. A revised psychometric index is offered for further study.

Vascular effects of etomidate administered for electroencephalographic burst suppression in humans.

Although its status as a neuroprotectant is controversial, etomidate is often employed for pharmacologic cerebral protection in aneurysm surgery. One purported advantage of etomidate over thiopental is its hemodynamic stability. This study examined the cardiovascular effects of etomidate given for electroencephalographic (EEG) burst suppression during cerebral aneurysm clipping in humans and the direct effects of etomidate on arteries in vitro. The charts of intracranial aneurysm surgery patients were retrospectively reviewed to determine the dose of etomidate employed, the frequency of concurrent vaspressor administration, and whether hemodynamic changes were associated with etomidate use. Against a background of balanced anesthesia, the dose of etomidate to induce burst suppression was 0.73 +/- 0.49 mg/kg (mean +/- SD) and the maintenance dose was 48 +/- 30 microg/kg/min. Etomidate produced an immediate decrease in mean arterial pressure that was sustained in patients who did not receive vasopressor support. During etomidate administration, 48% of patients (10 of 21) received some form of vasopressor support such as phenylephrine or ephedrine, and 62% of patients (13 of 21) receiving isoflurane had the anesthetic discontinued or its inspired concentration decreased. Etomidate in vitro produced dose-dependent relaxation of human internal mammary arterial rings that had been preconstricted by potassium or norepinephrine. Etomidate, in EEG burst suppression doses, decreases mean arterial pressure in anesthetized patients undergoing cerebral aneurysm surgery. One mechanism of etomidate-induced hypotension may be direct relaxation of vascular smooth muscle, because etomidate directly dilates preconstricted human arteries in vitro.

Myristoylation of flagellar creatine kinase in the sperm phosphocreatine shuttle is linked to its membrane association properties.

TCK, the flagellar creatine kinase (ATP:creatine N-phosphotransferase) of sperm from the sea urchin Strongylocentrotus purpuratus is a membrane-associated lipophilic protein involved in energy transport. The cDNA derived protein sequence contains a consensus site sufficient for the covalent attachment of myristate. To examine whether TCK was myristoylated, mouse fibroblast Swiss 3T3 and baby hamster kidney cell lines were transfected with a cDNA encoding the entire TCK protein linked to a metallothionein promotor. TCK expression was induced by zinc and paralleled by incorporation of [3H]myristic acid derived label into the protein. 3H Label incorporated into TCK was resistant to hydroxylamine treatment. The 3H-labeled material released from TCK by acid methanolysis eluted from a C18 reverse phase high pressure liquid chromatography column at the positions of myristic acid and methylmyristate. Thus, TCK expressed in transfected mammalian cell lines contains authentic myristic acid, covalently attached through amide linkage. [3H]Myristoyl TCK comigrated on two-dimensional gels with the purified lipophilic isoform TCK II from sea urchins. Furthermore, like TCK II, [3H]myristoyl TCK associated with phospholipid liposomes, suggesting that myristoylation may mediate the observed membrane association of TCK. Myristoylation of sea urchin sperm flagellar creatine kinase may play a role in confining this enzyme to the flagellum during spermatogenesis.

Membrane association of flagellar creatine kinase in the sperm phosphocreatine shuttle.

The flagellar creatine kinase (TCK) of Strongylocentrotus purpuratus sperm is both a principal component of sperm tail membrane preparations and a cytosolic enzyme. An improved purification scheme identified three pools of TCK, termed TCK I, TCK II, and TCK III. TCK I and II were essentially homogeneous protein preparations, while TCK III was heavily contaminated with other flagellar proteins, predominantly guanylate cyclase, and alpha- and beta-tubulin. The three TCK species are roughly present in a 1:10:1 ratio as assessed by activity measurements. TCK I and II are similar proteins as shown by two-dimensional gel electrophoresis, partial proteolytic fragmentation, and cellulose polyacetate electrophoresis and have the same pH-dependent specific activity. However, they are functionally distinct with respect to their capacity to associate with lipids. TCK II associated readily with phospholipid liposomes and detergent micelles, while TCK I did not. Association of TCK II was as a protein monomer with an apparent Kd of approximately 1-2 mM at a 10(4):1 lipid or detergent to protein ratio. Whereas the Kd estimates were pH independent, the rate of association increased 2-3-fold between pH 6.5 and 8. The data are consistent with membrane-association of TCK II being a two-step process, involving a pH-dependent, intramolecular, TCK-specific step and a charge-facilitated, but pH-independent, membrane association step. Membrane association of TCK may, together with microtubule association (Tombes, R.M., Farr, A., and Shapiro, B.M. (1988) Exp. Cell Res. 178, 307-317) represent a mechanism required for specific accumulation of the enzyme within the flagellum.

Functional domains of proteoliaisin, the adhesive protein that orchestrates fertilization envelope assembly.

Ovoperoxidase, the enzyme that hardens the sea urchin fertilization envelope, is inserted into the assembling extracellular matrix through the action of an intermediary protein, proteoliaisin (PLN). The domain structure of PLN, a large, rod-shaped protein that binds to ovoperoxidase and the vitelline layer, was examined by limited proteolytic cleavage. Purified proteolytic fragments of PLN were tested for their ability to bind ovoperoxidase, inhibit the binding of 125I-PLN to the vitelline layer, or act as substrates for the hardening reaction. Based on these results, the vitelline layer-binding domain can be placed near the amino terminus, followed by the binding site for ovoperoxidase; the distal two-thirds of the protein contain sites for ovoperoxidase-catalyzed dityrosine formation. The pentapeptide GRGDS (but not RGD) inhibited PLN-vitelline layer binding half-maximally at 0.2 mM. Moreover, PLN promoted adhesion of bovine aortic endothelial cells to plastic dishes, a process inhibited by GRGDS. Thus PLN is a new member of the adhesive protein family, the function of which is to coordinate the morphogenesis of a specific, rapidly assembled extracellular matrix.

The control of oxidant stress at fertilization.

Metazoan eggs alter their coats after fertilization to protect the early embryo. In sea urchins, this modification consists of a rapid, coordinated set of noncovalent macromolecular assembly steps that are stabilized by protein cross-linking. The sea urchin egg uses an oxidative cross-linking reaction that requires hydrogen peroxide and a secreted peroxidase and thus faces the challenge of oxidant stress at the beginning of its development. Protection from the deleterious effects of this oxidative mechanism is afforded by regulation of the production and utilization of oxidizing species. This regulation requires a specific protein kinase C-activated oxidase and ovothiol, an intracellular antioxidant.

Protein kinase C activates the respiratory burst of fertilization, but not cortical granule exocytosis, in ionophore-stimulated sea urchin eggs.

In a "respiratory burst", fertilized sea urchin eggs consume oxygen to produce H2O2 as an extracellular oxidant to crosslink their protective surface envelopes. The egg generates H2O2 via a NADPH-specific oxidase that requires protein kinase C for activation. To further study the physiological regulation of the respiratory burst and cortical granule exocytosis, we have measured azide-insensitive oxygen uptake and fertilization envelope assembly in ionophore-stimulated eggs. Procaine, trifluoperazine, staurosporine, and H-7, which inhibit protein kinase C by different mechanisms, suppressed egg oxygen consumption without affecting fertilization envelope assembly. In contrast, both exocytosis and oxygen uptake were blocked in N-ethylmaleimide-treated eggs. When the eggs were stimulated with ionophore in Na-free artificial seawater, which prevents the increase in pHi, oxidase activity was inhibited. This effect was reversed by elevation of cytoplasmic pH with the membrane-permeant base NH4Cl. We conclude that protein kinase C was not involved in the events downstream from the ionophore-dependent elevation of Ca2+ which induced cortical granule exocytosis. However, the respiratory burst was inhibited despite the increase in Ca2+ that triggered exocytosis. The likely target for inhibition of the burst was protein kinase C. Cytoplasmic alkalinization was necessary for optimal rates of H2O2 synthesis, further implicating pHi as a regulator of the egg oxidase.

The phosphocreatine shuttle of sea urchin sperm: flagellar creatine kinase resulted from a gene triplication.

TCK, the creatine kinase (ATP:creatine N-phosphotransferase) from sperm flagella of the sea urchin Strongylocentrotus purpuratus, is a Mr 145,000 axonemal protein that is employed in energy transport. Its amino acid sequence was obtained by analysis of fragments from cyanogen bromide digestion and by sequencing cDNA clones from two sea urchin testis libraries. TCK contains three complete but nonidentical creatine kinase segments joined by regions of sequence that are not creatine kinase-like and flanked by unique amino and carboxyl termini. Each creatine kinase segment is homologous to vertebrate creatine kinases of both muscle and brain types, and all three repeats contain the essential active-site cysteine. The sequence differences among repeats suggest an ancient gene triplication, around the time of the chordate-echinoderm divergence. The echinoderm, with a unique creatine kinase in sperm, arginine kinase in eggs, and both phosphagen kinases in somatic cells, may represent a preserved branch point in evolution, and TCK may be a relic of this event.

Superoxide peroxidase activity of ovoperoxidase, the cross-linking enzyme of fertilization.

Ovoperoxidase, an enzyme secreted by the eggs of the sea urchin Stronglycocentrotus purpuratus upon activation, catalyzes the formation of dityrosine residues in the fertilization envelope. This cross-linking reaction requires extracellular H2O2, which is produced by the egg during the cyanide-insensitive "respiratory burst" of fertilization. While investigating the possibility that the sea urchin oxidase might generate O2- as a precursor to H2O2, we discovered that ovoperoxidase possessed O2- degrading activity. Ovoperoxidase catalyzed the breakdown of O2- in a reaction that was sensitive to inhibition by catalase, indicating a requirement for H2O2. High concentrations of either O2- or H2O2 inhibited the O2- degrading activity of ovoperoxidase, as did the peroxidase inhibitors aminotriazole, azide, and phenylhydrazine. When ovoperoxidase was heated at 56 degrees C, it lost O2- degrading activity in parallel with peroxidase activity. In contrast, the copper-chelating agent diethyldithiocarbamate, which completely inactivated CuZn superoxide dismutase, failed to affect ovoperoxidase. The requirement for H2O2 and the inhibition by aminotriazole, azide, and phenylhydrazine support the hypothesis that ovoperoxidase catalyzes the breakdown of O2- by a peroxidative mechanism. Ovoperoxidase may play a role in protecting the developing embryo from oxidants derived from O2-.

A specific requirement for protein kinase C in activation of the respiratory burst oxidase of fertilization.

Partially reduced oxygen species are toxic, yet activated sea urchin eggs produce H2O2, suggesting that the control of oxidant stress might be critical for early embryonic development. We show that the Ca2(+)-stimulated NADPH oxidase that generates H2O2 in the "respiratory burst" of fertilization is activated by a protein kinase, apparently to regulate the synthesis of this potentially lethal oxidant. The NADPH oxidase was separated into membrane and soluble fractions that were both required for H2O2 synthesis. The soluble fraction was further purified by anion exchange chromatography. The factor in the soluble fraction that activated the membrane-associated oxidase was demonstrated to be protein kinase C (PKC) by several criteria, including its Ca2+/phophatidylserine/diacyl-glycerol-stimulated histone kinase activity, its response to phorbol ester, its inhibition by a PKC pseudosubstrate peptide, and its replacement by purified mammalian PKC. Neither calmodulin-dependent kinase II, the catalytic subunit of cyclic AMP-dependent protein kinase, casein kinase II, nor myosin light chain kinase activated the oxidase. Although the PKC family has been ubiquitously implicated in cellular regulation, enzymes that require PKC for activation have not been identified; the respiratory burst oxidase is one such enzyme.

Molecular mechanisms of sea-urchin sperm activation before fertilization.

Several mechanisms are used to control the behaviour of sea urchin spermatozoa while fertilizing eggs. These include discrete regulatory steps that modulate the sperm activation sequence from spawning to gamete membrane fusion. After release from the testis, sperm motility is instantaneously activated, by using intracellular pH as a throttle mechanism to control the rate of the dynein motor that catalyses axonemal bending. To support motility, energy is transported from the mitochondrion to the tail, by using a shuttle mechanism involving phosphocreatine diffusion. This shuttle employs a novel, endotriplicated, creatine kinase of Mr 140,000 in the flagellar axoneme as its terminus. The steering mechanism that determines where the spermatozoon swims is unknown, but may involve an egg peptide-induced guanylate cyclase activation, mediated by a cGMP-dependent Ca2+ channel, and attenuated by a plasma membrane cGMP phosphodiesterase. Upon arriving at the egg, which is identified by virtue of its proteoglycan coat (egg jelly), the spermatozoon undergoes a univesicular secretion that prepares it to fuse with the egg. This acrosome reaction involves several altered ionic fluxes in its mechanism, terminating in a massive Ca2+ uptake. If the spermatozoon is fortunate enough to fuse with an egg, a new member of the species is generated; if the acrosome reaction occurs without gamete fusion, the spermatozoon rapidly dies. All of these activation processes involve changes in the intracellular ionic milieu that are co-ordinated with altered enzyme activities, often in a causal fashion. Even with our current imperfect understanding of the process, a few of the steps in sperm activation may be defined by biochemical pathways that include specific modulatory control points.

Phosphagen kinase evolution. Expression in echinoderms.

Arginine kinase and creatine kinase that catalyze the transfer of a phosphate group between ATP and arginine and creatine, respectively, play an important role in cellular energetics. In contrast to most animals which exhibit a single phosphagen kinase activity (creatine kinase in chordates and arginine kinase in protostomians), echinoderms exhibit both arginine kinase and creatine kinase activities, sometimes in the same tissue. In contrast to chordates in which creatine kinases are dimers (consisting of two subunits of 40 kDa) and protostomians in which arginine kinases are usually monomers (40 kDa), echinoids contain specific phosphagen kinases: a dimeric arginine kinase (consisting of two subunits of 42 kDa) in eggs and a monomeric creatine kinase (145 kDa) in sperm. We have examined echinoderms from the five existing classes (echinoids, asteroids, ophiuroids, holothurians and crinoids) for the expression of these specific phosphagen kinases in different tissues. Gel filtration was used to determine the molecular masses of the native enzymes. Antibodies specific for arginine kinase or for creatine kinase were used to characterize the subunit composition of arginine kinase and creatine kinase after SDS/PAGE and transfer. In all echinoderms analyzed, arginine kinase always occurred as an enzyme of about 81 kDa consisting of two subunits of 42 kDa and creatine kinase as a monomeric enzyme of 140-155 kDa. The occurrence in echinoderms of both phosphagen kinases with distinct specificities and specific molecular structures is discussed from both a developmental and evolutionary point of view.

The heme environment of ovoperoxidase as determined by optical spectroscopy.

Native ovoperoxidase exhibited an optical absorption spectrum with certain similarities to lactoperoxidase, but not horseradish peroxidase, over the pH range 4.5-11.5. Ovoperoxidase had three distinct spectral forms dependent on pH, with transitions at apparent pKa values of 6.6 and 3.0. Complexes of ovoperoxidase with CN-, N3-, F-, or when reduced and ligated to carbon monoxide, CN-, or pyridine, were distinct from other peroxidases. Ovoperoxidase formed two specific and different spectral derivatives at pH 6.0 and 8.0, either in the native state, or when combined with CN-, when reduced, or when reduced and ligated to CN-. The position of the Soret band when mixed with near-stoichiometric amounts of H2O2. This cycling was inhibited by phenylhydrazine, 3-amino-1,2,4-triazole, or low pH (less than or equal to 6). Compound II was formed when ovoperoxidase was mixed with ethyl hydrogen peroxide in a 1:3 ratio, but not with H2O2. With a great excess of H2O2, Compound III was formed at pH 8.0; at pH 6.0 or below, the Soret band shifted slightly with excess of H2O2, but Compound III was never formed. Even when ovoperoxidase was bound to proteoliaisin (Weidman, P. J., and Shapiro, B. M. (1987) J. Cell Biol. 105, 561-567), ovoperoxidase exhibited spectral characteristics of the free enzyme.

Energy transport and cell polarity: relationship of phosphagen kinase activity to sperm function.

The energy required for motility of sea urchin sperm is transported from the mitochondrion to the flagellum by a phosphocreatine shuttle involving diffusion of phosphocreatine (PCr) between isozymes of creatine kinase (CrK) localized at the two sites (Tombes and Shapiro, Cell, 41:325, '85; Tombes et al., Biophys. J., 52:75, '87). The present studies demonstrate that high sperm CrK (various echinoderms; sea squirt, bristle worm, salmon) or arginine kinase (molusc, barnacle, moth) activity is seen in several species with sperm of a primitive morphology (mitochondrion at the base of the head, relatively long flagellum). In contrast, CrK activity is 10-100-fold less abundant in sperm of other species (frog, mouse, rooster, rabbit, bull, and human) that either possess a modified morphology (mitochondria that extend along the flagellum) and/or utilize glycolytic metabolism. We interpret these findings as support for the use of phosphagen kinase-dependent energy transport in cells in which the production of adenosine triphosphate (ATP) by the mitochondrion is distant from its utilization, leading to a form of metabolic polarization. Two other cell types, frog photoreceptors and rabbit oviduct cells, whose morphology and function also suggest that they exhibit metabolic polarization, contain relatively high CrK activity. The presence of high phosphagen kinase activity in metabolically polarized gametes and somatic cells further substantiates the role of such enzymes in facilitating energy transport.

Respiratory burst oxidase of fertilization.

Partially reduced oxygen species are toxic, yet sea urchin eggs synthesize H2O2 in a "respiratory burst" at fertilization, as an extracellular oxidant to crosslink their protective surface envelopes. To study the biochemical mechanism for H2O2 production, we have isolated an NADPH-specific oxidase fraction from homogenates of unfertilized Strongylocentrotus purpuratus eggs that produces H2O2 when stimulated with Ca2+ and MgATP2-. Concentrations of free Ca2+ previously implicated in regulation of egg activation modulate the activity of the oxidase. Inhibitors were used to test the relevance of this oxidase to the respiratory burst of fertilization. Procaine, two phenothiazines, and N-ethylmaleimide (but not iodoacetamide) inhibited H2O2 production by the oxidase fraction and oxygen consumption by activated eggs. The ATP requirement suggested that protein kinase activity might regulate the respiratory burst of fertilization; consonant with this hypothesis, H-7 and staurosporine were inhibitory. The respiratory burst oxidase of fertilization is an NADPH:O2 oxidoreductase that appears to be regulated by a protein kinase; although it bears a remarkable resemblance to the neutrophil oxidase, unlike the latter it does not form O2- as its initial product.

Localization and developmental fate of ovoperoxidase and proteoliaisin, two proteins involved in fertilization envelope assembly.

Fertilization of the sea urchin egg leads to the assembly of an extracellular matrix, the fertilization envelope. Ovoperoxidase, the enzyme implicated in hardening the fertilization envelope, is inserted into the assembling structure via a Ca2+-dependent interaction with the protein proteoliasin (P. Weidman and B. M. Shapiro, 1987, J. Cell Biol. 105, 561-567). In the present report, polyclonal antisera were raised to ovoperoxidase and proteoliasin (purified from eggs of Strongylocentrotus purpuratus) and characterized by Western blot analysis and an enzyme-linked immunoabsorbent assay (ELISA). By indirect immunofluorescence microscopy all cortical granules of unfertilized eggs, as well as the fertilization envelope, contained both proteoliasin and ovoperoxidase. At the ultrastructural level both proteins are localized to the electron-dense spiral lamellae of the cortical granules. Western blot analysis revealed that ovoperoxidase and proteoliasin persist in early embryos until hatching, but are absent from later developmental stages. Homogenates of eggs of several other echinoderm species (Strongylocentrotus droebachiensis, Strongylocentrotus franciscanus, Pisaster ochraceus, Dendraster excentricus, and Lytechinus pictus) also contain proteins antigenically similar to ovoperoxidase and proteoliaisin, indicating that many echinoderms utilize a similar strategy for assembly of the fertilization envelope. The results underline the need for postsecretory controls in the extracellular matrix modifications that accompany the cortical reaction.

Hierarchies of protein cross-linking in the extracellular matrix: involvement of an egg surface transglutaminase in early stages of fertilization envelope assembly.

The involvement of transglutaminase activity in fertilization envelope (FE) formation was investigated using eggs from the sea urchin, Strongylocentrotus purpuratus. Eggs fertilized in the presence of the transglutaminase inhibitors, putrescine and cadaverine, had disorganized and expanded FEs with inhibition of the characteristic I-T transition. The permeability of the FE was increased by these agents, as revealed by the loss of proteins from the perivitelline space and the appearance of ovoperoxidase activity in supernates from putrescine-treated eggs. [3H]putrescine was incorporated into the FE during fertilization in a reaction catalyzed by an egg surface transglutaminase that could also use dimethylcasein as a substrate in vitelline layer-denuded eggs. Egg secretory products alone had no transglutaminase activity. The cell surface transglutaminase activity was transient and maximal within 4 min of activation. The enzyme was Ca2+ dependent and was inhibited by Zn2+. We conclude that sea urchin egg surface transglutaminase catalyzes an early step in a hierarchy of cross-linking events during FE assembly, one that occurs before ovoperoxidase-mediated dityrosine formation (Foerder, C. A., and B. M. Shapiro. 1977. Proc. Natl. Acad. Sci. USA. 74:4214-4218). Thus it provides a graphic example of the physiological function of a cell surface transglutaminase.

Ovothiol replaces glutathione peroxidase as a hydrogen peroxide scavenger in sea urchin eggs.

Despite its potential toxicity, H2O2 is used as an extracellular oxidant by Stronglylocentrotus purpuratus eggs to cross-link their fertilization envelopes. These eggs contain 5 mM 1-methyl-N alpha,N alpha-dimethyl-4-mercaptohistidine (ovothiol C), which reacts with H2O2. In consuming H2O2 and being reduced by glutathione, ovothiol acts as a glutathione peroxidase and replaces the function of the enzyme in eggs. The ovothiol system is more effective than egg catalase in destroying H2O2 at concentrations produced during fertilization and constitutes a principal mechanism for preventing oxidative damage at fertilization.