The Impact of Stressor Exposure and Glucocorticoids on Anxiety and Fear.

Anxiety disorders and trauma- and stressor-related disorders, such as posttraumatic stress disorder (PTSD), are common and are associated with significant economic and social burdens. Although trauma and stressor exposure are recognized as a risk factors for development of anxiety disorders and trauma or stressor exposure is recognized as essential for diagnosis of PTSD, the mechanisms through which trauma and stressor exposure lead to these disorders are not well characterized. An improved understanding of the mechanisms through which trauma or stressor exposure leads to development and persistence of anxiety disorders or PTSD may result in novel therapeutic approaches for the treatment of these disorders. Here, we review the current state-of-the-art theories, with respect to mechanisms through which stressor exposure leads to acute or chronic exaggeration of avoidance or anxiety-like defensive behavioral responses and fear, endophenotypes in both anxiety disorders and trauma- and stressor-related psychiatric disorders. In this chapter, we will explore physiological responses and neural circuits involved in the development of acute and chronic exaggeration of anxiety-like defensive behavioral responses and fear states, focusing on the role of the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid hormones.

Serum amyloid P component: its role in platelet activation stimulated by sphingomyelinase D purified from the venom of the brown recluse spider (Loxosceles reclusa).

Serum amyloid P component or serum amyloid protein is a ubiquitous, highly conserved glycoprotein whose function is unknown. Although the related pentraxin, C-reactive protein, is an acute phase reactant in man, there is no direct evidence that human serum amyloid protein is involved in an inflammatory response. Here we show that serum amyloid protein is required by sphingomyelinase D, the principal necrotic agent of the venom of Loxosceles reclusa, for the in vitro-activation of human platelets. Furthermore, this platelet activation is dependent upon the presence of only serum amyloid protein; no other plasma components are necessary. Secretion of [3H]-serotonin and aggregation of platelets are nearly maximal following incubation of the platelets with purified sphingomyelinase D (0.3 micrograms/ml) and 5 micrograms/ml pure serum amyloid protein in the presence of calcium. Since the platelets are no longer activated when this 10% physiologic amount of serum amyloid protein is omitted, serum amyloid protein is likely to have a role in the necrosis caused by brown recluse spider venom.

Kinetic distinction between rapid-equilibrium random and abortive ordered enzymatic mechanisms using alternative substrates or kinetic isotope effects.

Alternative substrates, such as those isotopically-labeled, which differ in their rate constants of catalysis but not in their rate constants of binding, generate identical values of V/Ka in ordered kinetic mechanisms of bireactant enzymes. This is shown to be true even for the rapid-equilibrium ordered mechanism in which an abortive complex between free enzyme and the second substrate is formed. In contrast, rapid-equilibrium random mechanisms have non-identical values for V/Ka. Consequently, the effect of alternative substrates or isotope effects on V/Ka provides a means to distinguish between these nearly identical kinetic mechanisms.

Alternative substrate and inhibition kinetics of aminoglycoside nucleotidyltransferase 2''-I in support of a Theorell-Chance kinetic mechanism.

Aminoglycoside nucleotidyltransferase 2''-I conveys multiple antibiotic resistance to Gram-negative bacteria because the enzyme adenylylates a broad range of aminoglycoside antibiotics as substrates [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (preceding paper in this issue)]. The enzyme also catalyzes the transfer of a variety of nucleotides [Van Pelt, J. E., & Northrop, D. B. (1984) Arch. Biochem. Biophys. 230, 250-263]. This doubly broad substrate specificity makes it an excellent candidate for application of the alternative substrate diagnostic [Radika, K., & Northrop, D. B. (1984) Anal. Biochem. 141, 413-417] as a means to determine its kinetic mechanism. The kinetic patterns presented here are composed of one set of intersecting lines and one coincident line and are consistent with a Theorell-Chance kinetic mechanism in which nucleotide binding precedes aminoglycosides, pyrophosphate is released prior to the nucleotidylated aminoglycoside (Q), and turnover is controlled by the rate-limiting release of the final product. Substrate inhibition by tobramycin (B) is partial and uncompetitive versus Mg-ATP, indicating that B binds to the EQ complex, but not in the usual dead-end fashion common to an ordered sequential release of products; instead, Q may escape from the abortive EQB complex at a finite rate. Dead-end inhibition by neomycin C (I) is also partial and uncompetitive versus Mg-ATP but is slope-linear, intercept-hyperbolic, partial noncompetitive versus gentamicin A; both kinetic patterns signify the formation of a partial abortive EQI complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate specificities and structure-activity relationships for the nucleotidylation of antibiotics catalyzed by aminoglycoside nucleotidyltransferase 2''-I.

Aminoglycoside nucleotidyltransferase 2''-I (formerly gentamicin adenylyltransferase) conveys antibiotic resistance to Gram-negative bacteria by transfer of AMP to the 2''-hydroxyl group of 4,6-substituted deoxystreptamine-containing aminoglycosides. The kinetics constants of thirteen aminoglycoside antibiotics and the magnesium chelates of eight nucleotide triphosphates were determined with purified enzyme. Eleven of the antibiotics exhibit substrate inhibition attributed to secondary binding of the aminoglycoside to an enzyme-AMP-aminoglycoside complex. Maximal velocities vary by only 4-fold, versus variation of values of Vmax/Km for the aminoglycosides of nearly 4000-fold, consistent with a Theorell-Chance kinetic mechanism as proposed for this enzyme [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)] with the added specification that the binding of aminoglycosides is in rapid equilibrium. Under these conditions, Vmax/Km becomes kcat/Kd, where kcat is the net rate constant for catalysis (but not turnover) and Kd is the dissociation constant of aminoglycosides from a complex with enzyme and nucleotide. Values of kcat fall closely together into three distinct sets, with the 3',4'-dideoxygentamicins greater than gentamicins greater than kanamycins. These sets reflect unusual structure-activity correlations which are specific for catalysis but have nothing to do with the maximal velocity of this enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Determination of the rate-limiting segment of aminoglycoside nucleotidyltransferase 2''-I by pH and viscosity-dependent kinetics.

Aminoglycoside nucleotidyltransferase 2''-I follows a Theorell-Chance kinetic mechanism in which turnover is controlled by the rate-limiting release of the final product (Q), a nucleotidylated aminoglycoside [Gates, C. A., & Northrop, D. B. (1988) Biochemistry (second of three papers in this issue)]. The effects of viscosity on the kinetic constants of netilmicin, gentamicin C1, and sisomicin aminoglycoside substrates are as follows: no change in the substrate inhibition constants of all three antibiotics, a small but significant and highly unusual increase in Vmax/Km for netilmicin but large, normal decreases for gentamicin C1 and sisomicin, and marked decreases in the maximal velocities for all three. The lack of effect on substrate inhibition provides essential control experiments, signifying that glycerol does not interfere with binding of aminoglycosides to EQ and that the steady-state distribution of EQ does not increase as the release of Q is slowed by a viscosogen. The decrease in the Vmax/Km of better substrates indicates dominance by a diffusion-controlled component in the catalytic segment, attributed to the release of pyrophosphate. The presence of an increase in the Vmax/Km of the poor substrate, however, is inexplicable in terms of either single or multiple diffusion-controlled steps. Instead, it is here attributed to an equilibrium between conformers of the enzyme-nucleotide complex in which glycerol favors the conformation necessary for binding of aminoglycosides. The decrease in Vmax is consistent with the diffusion-controlled release of the final product determining enzymatic turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

FLP recombinase is an enzyme.

The FLP protein of the yeast 2-microns plasmid catalyzes intermolecular site-specific recombination with a turnover number of approximately equal to 0.12 min-1 (per FLP monomer) for relaxed DNA substrates. Under conditions that enhance its stability, the protein can be used in catalytic rather than stoichiometric amounts. The reaction rate exhibits a strong dependence on FLP protein concentration even when the protein is present in excess relative to available recombination sites.

Inhibition of steroid C17(20) lyase with C-17-heteroaryl steroids.

Steroids bearing a heteroaromatic substituent at C-17 were designed as inhibitors of C17(20) lyase. The thiazoles, furans, and thiophenes appended to the steroid nucleus were positioned on the alpha-face and the beta-face of the steroid, and conjugated with a 16,17-olefin, to test their ability to coordinate the heme iron of the P450 enzyme complex. The position of the heterocycle with respect to the steroid skeleton was determined to be important for optimum affinity and, in general, compounds with the heterocycle attached to a trigonal center at C-17, had the best affinity for C17(20) lyase. Simple molecular models were used to compare the three types of heterocyclic-substituted steroids.

Simultaneous PCR detection of Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus types 1 and 2 from genital ulcers.

A multiplex PCR (M-PCR) assay with colorimetric detection was devised for the simultaneous amplification of DNA targets from Haemophilus ducreyi, Treponema pallidum, and herpes simplex virus (HSV) types 1 and 2. By using target-specific oligonucleotides in a microwell format, 298 genital ulcer swab specimens collected in New Orleans during three intervals from 1992 through 1994 were evaluated. The results of the M-PCR assay were compared with the results of dark-field microscopy and H. ducreyi culture on two different culture media. HSV culture results were available for 99 specimens collected during the third interval. Confirmatory PCR assays targeting different gene sequences for each of the three organisms were used to validate the M-PCR results. Specimens were resolved as positive for the determination of sensitivity if the reference diagnostic test was positive or if the results of both the M-PCR and the confirmatory PCR were positive. The resolved sensitivities of M-PCR for HSV, H. ducreyi, and T. pallidum were 100, 98.4, and 91%, respectively. The resolved sensitivities of HSV culture, H. ducreyi culture, and dark-field microscopy were 71.8, 74.2, and 81%, respectively. These results indicate that the M-PCR assay is more sensitive than standard diagnostic tests for the detection of HSV, H. ducreyi, and T. pallidum from genital ulcers.

Purification of the FLP site-specific recombinase by affinity chromatography and re-examination of basic properties of the system.

The FLP protein, a site-specific recombinase encoded by the 2 micron plasmid of yeast, has been purified to near homogeneity from extracts of E. coli cells in which the protein has been expressed. The purification is a three column procedure, the final step employing affinity chromatography. The affinity ligand consists of a DNA polymer with multiple FLP protein binding sites arranged in tandem repeats. This protocol yields 2 mg of FLP protein which is 85% pure. The purified protein is highly active, stable for several months at -70 degrees C and free of detectable nucleases. The molecular weight and N-terminal sequence are identical to that predicted for the FLP protein by the DNA sequence of the gene. Purified FLP protein primarily, but not exclusively, promotes intramolecular recombination. Intermolecular recombination becomes the dominant reaction when E. coli extracts containing no FLP protein are added to the reaction mixture. These extracts are not specifically required for recombination, but demonstrate that some properties previously attributed to FLP protein can be assigned to contaminating proteins present in E. coli.

Acceleration of onset of collagen-induced arthritis by intra-articular injection of tumour necrosis factor or transforming growth factor-beta.

We examined whether tumour necrosis factor (TNF) or transforming growth factor-beta 1 (TGF-beta 1) could alter the course of collagen-induced arthritis (CIA). Injection of 100 ng TNF or 500 ng TGF-beta 1 into ankle joints of normal rats induced a very limited inflammatory response, observable only upon histological analysis. However, when injected into ankle joints of rats 9 days after immunization with bovine type II collagen (CII), identical doses of TNF or TGF-beta 1 induced a sustained, clinically obvious inflammation and oedema that began within 8 h on average, as compared to 90 h in CII-immunized control rats given no injections or intra-articular injections of buffer. The incidence of arthritis at 2 weeks post-immunization was 100% for TNF-injected hindpaws, compared with 55% for the control groups, a statistically significant difference. In rats passively immunized with a subarthritic dose of affinity purified antibody to rat-CII, intra-articular injection of 100 ng TNF or 500 ng of TGF-beta 1 also induced intense, though transient arthritis. The rapid proinflammatory effects in CIA described in this study and the synergy demonstrated between anti-CII IgG and either cytokine, suggest that these cytokines can participate locally in the pathogenesis of arthritis.

Indeterminate human immunodeficiency virus type 1 western blots: seroconversion risk, specificity of supplemental tests, and an algorithm for evaluation.

The human immunodeficiency virus type 1 (HIV-1) Western blot is indeterminate in 10%-20% of sera reactive by EIA. Eighty-nine individuals with prior repeatedly reactive EIA and indeterminate Western blots were followed prospectively to study the risk of seroconversion and specificity of supplemental tests. Four high-risk cases seroconverted within 10 months after enrollment (seroconversion risk, 4.5%, 95% confidence interval, 1.2%-11.1%). Among cases with p24 bands initially, 4 (18.2%) of 22 high-risk individuals seroconverted compared with 0 of 33 low-risk cases (P = .03). Specificities of HIV-1 culture, serum p24 antigen, polymerase chain reaction, and recombinant ENV 9 EIA were 100%, 100%, 98.6%, and 94.4%, respectively. An expedited evaluation protocol is proposed. Low-risk individuals with nonreactive EIAs upon repeat testing do not need further follow-up; high-risk individuals should be followed serologically for at least 6 months, especially those with p24 bands on Western blot.

Endocrine and antiprostatic effects of raloxifene (LY156758) in the male rat.

The benzothiophene anti-estrogen, raloxifene [LY156758; (6-hydroxy-2-(4-hydroxyphenyl) benzo(b)thien-3-yl)(4-(2-1-piperidinyl)ethoxy)phenyl methanone hydrochloride] has selective estrogen pharmacological antagonist activity in female rats. The present studies were done in the male rat to assess activity of raloxifene related to inhibition of prostatic growth and effects on the hypothalamic-pituitary-gonadal axis. Raloxifene did not compete for binding of the androgen, [3H]-methyltrienolone (R1881) in cytosolic extracts of ventral prostate. Similarly, the compound did not inhibit prostatic 5 alpha-reductase or testicular 17 alpha-hydroxy/C17,20-lyase activities. Raloxifene had no effect on the ventral prostatic uptake of [3H]-R1881 in vivo. Administration of estradiol to castrated male rats stimulated fourfold increases of in vitro ventral prostatic binding of [3H]-R1881. Raloxifene was devoid of agonist activity in castrated animals, because the compound had no stimulatory effect on prostatic androgen receptor binding activity. When raloxifene was coadministered with estradiol, the compound markedly antagonized the estrogen-induced increase of prostatic [3H]-R1881 binding, confirming its antiestrogenic properties in male rats. Serum prolactin was also elevated significantly (P < 0.05) with a single injection of raloxifene (20.0 mg/kg). In these same animals, serum FSH was significantly (P < 0.05) decreased by one dose (10.0 mg/kg) of the compound. Luteinizing hormone levels in castrated male rats were unaffected by raloxifene administration. Raloxifene treatment of castrated males significantly (P < 0.05) antagonized the stimulatory response of the ventral prostate (VP) to exogenous androgens in a dose-dependent manner. Raloxifene treatment of intact male rats for 14 and 28 days produced significant (P < 0.05) dose-dependent regression of the VP and seminal vesicles (SV). The VP regressive responses to raloxifene were associated with a decline in serum testosterone levels. Histological analysis of the VPs in raloxifene-treated rats was consistent with an androgen-deprived state. These findings support the contention that raloxifene is a pure estrogen antagonist and a physiological antagonist of androgen action in male rats. These pharmacological properties provide support for further structure-activity and mechanistic investigations with benzothiophenes in the medical management of prostatic neoplasia.