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Sperm beta-acrosin activity is inhibited by suramin, a polysulfonated naphthylurea compound with therapeutic potential as a combined antifertility agent and microbicide. A kinetic analysis of enzyme inhibition suggests that three and four molecules of suramin bind to one molecule of ram and boar beta-acrosins respectively. Surface charge distribution models of boar beta-acrosin based on its crystal structure indicate several positively charged exosites that represent potential 'docking' regions for suramin. It is hypothesised that the spatial arrangement and distance between these exosites determines the capacity of beta-acrosin to bind suramin.

Trypsin has been isolated and purified from the digestive glands of the slipper lobster, Thenus orientalis. It is a glycoprotein with a molecular mass of approximately 35 kDa as judged by both SDS-PAGE and gel filtration. The N-terminal amino acid sequence has strong homology to crustacean trypsins. This is confirmed by the cross-reaction of crustacean trypsins with antibodies to the T. orientalis enzyme. Despite a 40% identity with the bovine trypsin N-terminal sequence, there was no cross-reaction with the mammalian serine proteases. The optimum kcat and kcat/Km values for N-alpha-benzoylarginine-p-nitroanalide were 0.91 s-1 and 9.7 x 10(3) M-1 s-1, respectively, with this specificity constant being lower than those reported for other crustacean trypsins. Inhibition studies indicated the presence of serine and histidine at the active site and pKa of the catalytic histidine residue was found to be 5.7 in the free enzyme and 4.7 in the Michaelis complex.

The mechanism for the inhibition of thrombin by the serpins antithrombin and protease nexin 1 has been investigated using several kinetic techniques at pH 7.9 and 37 degrees C with an ionic strength of 0.3 M. Rapid kinetic studies demonstrated that a two-step mechanism for the formation of the stable thrombin-serpin complex applied to both serpins. The inhibition constant for the initial thrombin-antithrombin complex was 265 microM, and the rate constant for the conversion of this complex to the final one was 3.9 s-1; the corresponding values for PN1 were 3.4 microM and 6.0 s-1. By using slow-binding kinetics, it was possible to obtain estimates of the second-order rate constants for the formation of the stable thrombin-serpin complexes (1.2 x 10(4) and 1.5 x 10(6) M-1 s-1 for antithrombin and protease nexin 1, respectively) and the dissociation constants for these complexes (< 1 nM for both serpins). The influence of viscosity on the reactions indicated that the rate of interaction of both serpins with thrombin was diffusion-controlled. Moreover, the results indicated that the initial complex reacted more rapidly to form the stable complex than it dissociated to free enzyme and inhibitor; i.e., the behavior of the serpins was analogous to that of "sticky" substrates. By using the results from slow-binding, viscosity, and rapid kinetic studies, it was possible to set values for all of the rate constants for the interactions of antithrombin and protease nexin 1 with thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)

The serpins antithrombin, protease nexin 1, and alpha 1-antitrypsin with a reactive-center arginine (Arg-alpha 1-antitrypsin) were found to inhibit the sperm protease acrosin with varying efficiency. The serpins were titrated against acrosin to determine their specific activity with respect to this enzyme. While antithrombin was fully active against acrosin, more than one molecule of Arg-alpha 1-antitrypsin and protease nexin 1 was required to inhibit one molecule of acrosin. In particular, only 2.7% of protease nexin 1 molecules interacting with acrosin formed stable complexes with the enzyme at 37 degrees C and this value decreased to 0.03% at 12 degrees C. N-terminal sequence analysis indicated that acrosin had cleaved protease nexin 1 at its reactive-center Arg-Ser bond. The results could be interpreted in terms of protease nexin 1 acting as a suicide substrate for acrosin; after the formation of an initial complex, the serpin partitioned between pathways yielding either inactivated (cleaved) serpin or a stable serpin-enzyme complex. The association rate constant (k(ass)) and inhibition constant (Ki) for the stable complexes were determined for each of the serpins by using slow-binding kinetics. The values of k(ass) were 2 x 10(5), 4 x 10(4), and 5 x 10(3) M-1 s-1 for Arg-alpha 1-antitrypsin, antithrombin, and protease nexin 1, respectively. The Ki values for the serpins were 1 nM or less. Heparin markedly accelerated the inhibition of acrosin by antithrombin and protease nexin 1; at the optimal concentration, the degree of heparin acceleration of the inhibition rate was 250- and 500-fold for antithrombin and protease nexin 1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparin was found to be an allosteric modulator of the amidolytic activity of the protease acrosin. In the presence of saturating concentrations of heparin, there was a 4.9-fold decrease in the value of the Michaelis constant for the substrate D-Ile-Pro-Arg-p-nitroanilide and the value of kcat was 2.5-fold lower. Analysis of the data yielded a dissociation constant of 0.22 +/- 0.04 microM for the heparin-acrosin complex. The presence of relatively high concentrations of protein C inhibitor in seminal plasma [Laurell, M., Christensson, A., Abrahamson, P., Stenflo, J., & Lilja, H. (1992) J. Clin. Invest. 89, 1094-1101] suggests that this serpin may be involved in the control of the activity of acrosin. Acrosin was found to be rapidly inhibited by protein C inhibitor with the association rate constant (kass) for the formation of the complex being (2.41 +/- 0.03) x 10(5) M-1 s-1. The value of kass showed a bell-shaped dependence on the concentration of heparin; it was maximal at concentrations of heparin between 0.08 and 3 microM and decreased at lower and higher concentrations. At the optimal heparin concentration, the value of kass for the acrosin-protein C inhibitor reaction was 230-fold higher ((5.6 +/- 0.1) x 10(7) M-1 s-1) than in the absence of heparin. The results suggest that protein C inhibitor may be important in the physiological control of acrosin activity, particularly where the presence of heparin-like glycosaminoglycans would stimulate the acrosin-protein C inhibitor reaction.

In addition to the neurotropic role of brain-derived neurotrophic factor (BNDF) in cortical circuit plasticity, there is a good positive correlation between the cortical expression level of BDNF and developmental changes in visual acuity. Here, the authors find that directly impairing BDNF signaling using transgenic methods causes visual impairment by affecting the systems level control on contrast gain. During development and aging and in amblyopia, visual acuity is far below the limitations set by the retina. Expression of brain-derived neurotrophic factor (BDNF) in the visual cortex is reduced in these situations. We asked whether neurotrophic tyrosine kinase receptor, type 2 (TrkB) regulates cortical visual acuity in adult mice. We found that genetically interfering with TrkB/BDNF signaling in pyramidal cells in the mature visual cortex reduced synaptic strength and resulted in a loss of neural responses to high spatial-frequency stimuli. Responses to low spatial-frequency stimuli were unaffected. This selective loss was not accompanied by a change in receptive field sizes or plasticity, but apparent contrast was reduced. Our results indicate that a dependence on spatial frequency in the Heeger normalization model explains this selective effect of contrast reduction on high-resolution vision and suggest that it involves contrast gain control operating in the visual cortex.

How the visual cortex responds to specific stimuli is strongly influenced by visual experience during development. Monocular deprivation, for example, changes the likelihood of neurons in the visual cortex to respond to input from the deprived eye and reduces its visual acuity. Because these functional changes are accompanied by extensive reorganization of neurite morphology and dendritic spine turnover, genes regulating neuronal morphology are likely to be involved in visual plasticity. In recent years, Notch1 has been shown to mediate contact inhibition of neurite outgrowth in postmitotic neurons and implicated in the pathogenesis of various degenerative diseases of the CNS. Here, we provide the first evidence for the involvement of neuronal Notch1 signaling in synaptic morphology and plasticity in the visual cortex. By making use of the Cre/Lox system, we expressed an active form of Notch1 in cortical pyramidal neurons several weeks after birth. We show that neuronal Notch1 signals reduce dendritic spine and filopodia densities in a cell-autonomous manner and limit long-term potentiation in the visual cortex. After monocular deprivation, these effects of Notch1 activity predominantly affect responses to visual stimuli with higher spatial frequencies. This results in an enhanced effect of monocular deprivation on visual acuity. Copyright © 2008 Society for Neuroscience.

The introduction of forward genetic screens in the mouse asks for techniques that make rapid screening of visual function possible. Transcranial imaging of intrinsic signal is suitable for this purpose and could detect the effects of retinal degeneration, and the increased predominance of the contralateral eye in albino animals. We quantified visual response properties of the cortex by introducing a normalization method to reduce the impact of biological noise. In addition, the presentation of a 'reset'-stimulus shortly after the probing stimulus at a different visual location could reduce the interstimulus time necessary for the decay of the response. Applying these novel methods, we found that acuity of C57Bl/6J mice rises from 0.35 cycles per degree (cpd) at postnatal day 25 to 0.56 cpd in adults. Temporal resolution was lower in adults than in juvenile animals. There was no patchy organization of spatial or temporal frequency preference at the intrinsic signal resolution. Monocular deprivation, a model for amblyopia and critical period plasticity, led to a loss in acuity and a shift towards the nondeprived eye in juvenile animals. Short deprivation did not lead to increased acuity of the nondeprived eye. In adults, a small ocular dominance shift was detectable with urethane anaesthesia. This was not observed when the combination of the opiate fentanyl, fluanisone with a benzodiazepine was used, adding evidence to the hypothesis that enhancing GABA(A)-receptor function masks an adult shift. Together, these novel applications confirm that noninvasive screening of many functional properties of the visual cortex is possible.