A concerted structural transition in the plasminogen activator inhibitor-1 mechanism of inhibition

The inhibition mechanism of serpins requires a change in structure to entrap the target proteinase as a stable acyl-enzyme complex. Although it has generally been assumed that reactive center loop insertion and associated conformational change proceeds in a concerted manner, this has not been demonstrated directly. Through the substitution of tryptophan with 7-azatryptophan and an analysis of transient reaction kinetics, we have described the formation of an inhibited serpin-proteinase complex as a single concerted transition of the serpin structure. Replacement of the four tryptophans of plasminogen activator inhibitor type-1 (PAI-1) with the spectrally unique analogue 7-azatryptophan permitted observations of conformational changes in the serpin but not those of the proteinase. Formation of covalent acyl-enzyme complexes, but not noncovalent Michaelis complexes, with tissue-type plasminogen activator (t-PA) or urokinase (u-PA) resulted in rapid decreases of fluorescence coinciding with insertion of the reactive center loop and expansion of beta-sheet A. Insertion of an octapeptide consisting of the P14-P7 residues of the reactive center loop into beta-sheet A produced the same conformational change in serpin structure measured by 7-azatryptophan fluorescence, suggesting that introduction of the proximal loop residues induces the structural rearrangement of the serpin molecule. The atom specific modification of the tryptophan indole rings through analogue substitution produced a proteinase specific effect on function. The reduced inhibitory activity of PAI-1 against t-PA but not u-PA suggested that the mechanism of loop insertion is sensitive to the intramolecular interactions of one or more tryptophan residues.     

Absence of anxiolytic response to chlordiazepoxide in two common background strains exposed to the elevated plus-maze: importance and implications of behavioural baseline

Although genetic background is acknowledged as a potentially important determinant of mutant phenotypes, publications on genetically modified mice far outnumber those on progenitor strains. We have recently reported major differences in basal anxiety levels (elevated plus-maze & light/dark exploration) among three strains (C57BL/6JOlaHsd, 129/SvEv and 129S2/SvHsd) employed as progenitor stock in European laboratories (Rodgers et al. in press). Furthermore, the phenotypes of these inbred strains differed significantly from that of an outbred strain (Swiss-Webster) commonly used in behavioural pharmacology. In view of these findings, the present study assessed possible differences in the anxiolytic efficacy of chlordiazepoxide (0, 7.5 & 15.0 mg/kg, IP) in three of these strains (Swiss-Webster (SW), C57BL/6JOIaHsd (C57) & 129S2/SvHsd (129)). Experimentally naive mice were exposed to the elevated plus-maze, sessions were videotaped and behaviour analysed using ethological software. The performance of control subjects confirmed significant strain differences in basal levels of activity (SW > C57 > 129) and anxiety-related behaviours (129 = SW > C57), with hypolocomotion dominating the 129 profile. SW mice displayed an anxioselective response to both doses of chlordiazepoxide (CDP), with significant reductions in open arm avoidance and risk assessment observed in the absence of any change in general activity. In direct contrast, the lower dose of CDP (7.5 mg/kg) was without effect in either inbred strain, whereas treatment with 15.0 mg/kg induced a profile indicative of muscle relaxation/mild sedation in C57 mice and virtually abolished all behavioural activity in 129 mice. Although the absence of an anxiolytic response to CDP in C57 mice may be attributed to their low basal anxiety levels, the profile of 129 mice strongly suggests an abnormality in benzodiazepine/GABAA receptor function. The implications of these findings for research on mutant mice are discussed.     

Paternally biased cpDNA inheritance in Turnera ulmifolia (Turneraceae)

We end-labeled Hin fI restriction digests of a PCR-amplified plastid encoded gene, the large subunit of ribulose bisphosphate carboxylase, to investigate patterns of cpDNA inheritance in Turnera ulmifolia. A total of 70 progeny from crosses among plants taken from ten populations revealed varying patterns of inheritance. A majority of progeny inherited the paternal cpDNA (64%), while 19% exhibited maternal and 17% biparental inheritance. Eight variegated progeny showed biparental inheritance and were analyzed in greater detail. We extracted and analyzed the cpDNA content of light- vs. dark- green leaf sectors from these plants. The results showed that vegetative segregation of cpDNA had occurred for seven of the eight plants.     

Role of tachykinins in airway responses to ozone in rats

Previousstudies that used neonatal capsaicin (Cap) treatment toablate C fibers indicate that C fibers act to inhibit lung damage andairway hyperresponsiveness after ozone(O₃) exposure in rats. Thepurpose of this study was to determine1) the role of tachykinins in theseprotective effects and 2) whetherdifferences in minute ventilation (E)during O₃ exposure might accountfor the effect of Cap. In the first study, male Sprague-Dawley rats were exposed to 1 part/million O₃or air for 3 h. Four hours later, a bronchoalveolar lavage (BAL) wasperformed or airway responsiveness was measured. Rats were treated withCP-99994 and SR-48968, selective neurokinin-1- and -2-receptorantagonists, respectively, or with vehicle (Veh).O₃ caused an increase in thenumber of neutrophils recovered from BAL fluid in both the Veh-treatedand tachykinin-receptor antagonist (TKRA)-treated rats, but the numberof neutrophils was approximately twofold greater in the TKRA-treatedrats. In contrast, TKRA treatment had no effect on baseline pulmonarymechanics or airway responsiveness. AfterO₃ exposure, the number ofneutrophils in BAL fluid was also greater in Cap- than in Veh-treatedrats. O₃ reducedE in both Veh- and Cap-treated rats,but the response was greater (reduction of 44.7 ± 3.7 vs. 27.8 ± 6.8%) and occurred earlier (10 vs. 70 min) in Cap- than inVeh-treated rats (P < 0.02). Theseresults suggest that tachykinins mediate protective effects of C fibersagainst O₃-induced lunginflammation. The results also indicate that the more pronounced effectof O₃ on BAL neutrophils inCap-treated rats is not the result of a greater inhaled dose ofO₃ resulting from greaterE.

     

The effect of rate of eruption on periodontal ligament glycosylaminoglycan content and enamel formation in the rat incisor

The rate of eruption of rat mandibular incisors was either increased by cutting one tooth out of occlusion or eliminated by means of pinning. The effects of such changes in eruption rate on the sulphated glycosylaminoglycan content of the periodontal ligaments was analysed. The length of the enamel secretory zone and the composition of the developing enamel matrix protein was also compared. Sulphated glycosylaminoglycan content of the periodontal ligament increased fourfold (P<0.001) during accelerated eruption but decreased to a corresponding extent (P<0.001) in the absence of eruption, when compared with controls. The length of the enamel secretory zone was also significantly reduced in the immobilised teeth, although the protein content was similar compared with controls. The results demonstrate the differential response to varied eruption rates of the periodontal ligament and enamel, particularly in respect of the extracellular matrix. The data are consistent with the view that the ground substance of the periodontal ligament plays a role in the generation of the eruptive force.     

Self-organization of the Earth's biosphere-geochemical or geophysiological?

We explore the implications of indicating the biosphere's self-organization by the trend over time of the net entropic flow from the Earth's surface, the actual physical boundary of virtually all biotic mass. This flow, derived from the radiative surface entropy budget, is approximately inversely related to the surface temperature when the solar incident flux remains constant. In the geophysiological (gaian) interpretation, biospheric self-organization has increased with the progressive colonization of the continents and evolutionary developments in the land biota, as a result of surface cooling arising from biotic enhancement of weathering. The key site for this self-organization is at the interface between land and atmosphere, the soil, where carbon is sequestered by its reaction (as carbonic and organic acids) with calcium magnesium silicates. Along with disequilibrium (steady-state) levels of carbon dioxide in the atmosphere, the occurrence of differentiated soil is the critical material evidence for biospheric self-organization, whether it be geophysiological or geochemical (ie., purely result of inorganic reactions). The computed equilibrium levels of carbon dioxide and corresponding equilibrium temperatures in the past are dramatically different from the steady-state levels. With future solar luminosity increase, the biospheric capacity for climatic regulation will decrease, leading to the ending of self-organization some two billion years from now. The Earth's surface will then approach chemical equilibrium with respect to the carbonate-silicate cycle.     

Transient kinetic studies of substrate inhibition in the horse liver alcohol dehydrogenase reaction

The rate-limiting step of ethanol oxidation by alcohol dehydrogenase (E) at substrate inhibitory conditions (greater than 500 mM ethanol) is shown to be the dissociation rate of NADH from the abortive E-ethanol-NADH complex. The dissociation rate constant of NADH decreased hyperbolically from 5.2 to 1.4 s-1 in the presence of ethanol causing a decrease in the Kd of NADH binding from 0.3 microM for the binary complex to 0.1 microM for the abortive complex. Correspondingly, ethanol binding to E-NADH (Kd = 37 mM) was tighter than to enzyme (Kd = 109 mM). The binding rate of NAD+ (7 X 10(5) M-1s-1) to enzyme was not affected by the presence of ethanol, further substantiating that substrate inhibition is totally due to a decrease in the dissociation rate constant of NADH from the abortive complex. Substrate inhibition was also observed with the coenzyme analog, APAD+, but a single transient was not found to be rate limiting. Nevertheless, the presence of substrate inhibition with APAD+ is ascribed to a decrease in the dissociation rate of APADH from 120 to 22 s-1 for the abortive complex. Studies to discern the additional limiting transient(s) in turnover with APAD+ and NAD+ were unsuccessful but showed that any isomerization of the enzyme-reduced coenzyme-aldehyde complex is not rate limiting. Chloride increases the rate of ethanol oxidation by hyperbolically increasing the dissociation rate constant of NADH from enzyme and the abortive complex to 12 and 2.8 s-1, respectively. The chloride effect is attributed to the binding of chloride to these complexes, destabilizing the binding of NADH while not affecting the binding of ethanol.     

E-rosette receptors induced by phytohemagglutinin on human K cells expressing T-cell surface antigens.

Killer cells (K cells) enriched from human blood mononuclear cells which mediate antibody-dependent cellular cytotoxicity (ADCC) were examined for surface markers. Sixty-seven percent of the E-rosette-negative, sIg-negative cells reacted with anti-T cell serum (AMT) previously shown to react with immunochemically defined T-cell antigens. Phytohemagglutinin induced 25% of K cells to express an E-rosette receptor. When these induced cells were isolated, greater than 98% reacted with AMT and 17% expressed the Fc receptor for IgG. Furthermore, they retained their functional capacity in ADCC. These findings demonstrate that an E-rosette receptor can be induced on human K cells. The data suggest the K-cell fraction included a population of thymus-dependent lymphocytes which can function as effector cells in ADCC.     

The mononuclear cell in human blood which mediates antibody-dependent cellular cytotoxicity to virus-infected target cells. I. Identification of the population of effector cells.

Mononuclear cells (MC) from human blood were fractionated by a variety of physical and immunologic techniques, and the cellular subpopulations generated were assessed for their capacity to lyse herpes simplex virus (HSV)-infected target cells in the presence of IgG antibody to HSV. Latex phagocytosis and surface marker studies were performed in parallel in order to identify the major effector cells by their phagocytic properties and their possession of surface immunoglobulin and receptors for either sheep erythrocytes, C3, or the Fc fragment of IgG. Cytotoxic effector cell activity was unaffected or slightly enhanced after the removal of plastic-adherent or carbonyl iron-adherent MC, indicating that the major effector cell is not a classical monocyte. Similar results were obtained after removal of more than 90% of the T cells by depletion of rosette-forming cells. Likewise, effector cell activity was generally unchanged when more than 95% of the B cells were removed by filtering MC on nylon wool columns. Effector cell function was also found to be normal in three patients with B cell-deficient X-linked agammaglobulinemia. These observations strongly suggest that the effector cells are not T cells or B cells. A 4- to 5-fold enrichment in effector cells, however, was consistently found in a subpopulation, consisting of 5% of the unfractionated MC, that was dramatically enriched both for nonphagocytic cells with only Fc receptor (K cells) and for nonphagocytic cells with no detectable surface markers (null cells). Since, as is demonstrated in the accompanying report, effector surface Fc receptors play a critical role in the mediation of antibody-dependent cellular cytotoxicity directed at HSV-infected target cells, the major mononuclear effector cell in human blood is a K cell.