Cockroach allergen serine proteinases: Isolation, sequencing and signalling via proteinase-activated receptor-2.

BACKGROUND: Allergy to the German cockroach (Blattella germanica) is a significant asthma risk factor for inner-city communities. Cockroach, like other allergens, contains trypsin-like enzyme activity that contributes to allergenicity and airway inflammation by activating proteinase-activated receptors (PARs). To date, the enzymes responsible for the proteolytic activity of German cockroach allergen have not been characterized. OBJECTIVES: We aimed to identify, isolate and characterize the trypsin-like proteinases in German cockroach allergen extracts used for clinical skin tests. For each enzyme, we sought to determine (1) its substrate and inhibitor enzyme kinetics (Km and IC50), (2) its amino acid sequence and (3) its ability to activate calcium signalling and/or ERK1/2 phosphorylation via PAR2. METHODS: Using a trypsin-specific activity-based probe, we detected three distinct enzymes that were isolated using ion-exchange chromatography. Each enzyme was sequenced by mass spectometery (deconvoluted with an expressed sequence tag library), evaluated kinetically for its substrate/inhibitor profile and assessed for its ability to activate PAR2 signalling. FINDINGS: Each of the three serine proteinase activity-based probe-labelled enzymes isolated was biochemically distinct, with different enzyme kinetic profiles and primary amino acid sequences. The three enzymes showed a 57%-71% sequence identity with a proteinase previously cloned from the American cockroach (Per a 10). Each enzyme was found to activate both Ca++ and MAPK signalling via PAR2. CONCLUSIONS AND RELEVANCE: We have identified three different serine proteinases from the German cockroach that may, via PAR2 activation, play different roles for allergen sensitization in vivo and may represent attractive therapeutic targets for asthma.

Programmable quantum emitter formation in silicon.

Silicon-based quantum emitters are candidates for large-scale qubit integration due to their single-photon emission properties and potential for spin-photon interfaces with long spin coherence times. Here, we demonstrate local writing and erasing of selected light-emitting defects using femtosecond laser pulses in combination with hydrogen-based defect activation and passivation at a single center level. By choosing forming gas (N2/H2) during thermal annealing of carbon-implanted silicon, we can select the formation of a series of hydrogen and carbon-related quantum emitters, including T and Ci centers while passivating the more common G-centers. The Ci center is a telecom S-band emitter with promising optical and spin properties that consists of a single interstitial carbon atom in the silicon lattice. Density functional theory calculations show that the Ci center brightness is enhanced by several orders of magnitude in the presence of hydrogen. Fs-laser pulses locally affect the passivation or activation of quantum emitters with hydrogen for programmable formation of selected quantum emitters.

Proteinase-activated receptor-2 activation participates in allergic sensitization to house dust mite allergens in a murine model.

BACKGROUND: Many aeroallergens contain proteinase activity and are able to induce allergic sensitization when presented to mucosal surfaces. Some of these allergens activate proteinase-activated receptor-2 (PAR2 ). OBJECTIVE: To determine the role of PAR2 activation in a murine house dust mite (HDM) allergy model. METHODS: We sensitized and challenged PAR2 -deficient mice with HDM, and examined allergic outcomes compared to wild-type animals. To focus on the role of PAR2 in allergic sensitization, we administered a PAR2 blocking antibody to wild-type animals during the sensitization phase and examined the outcomes immediately after sensitization or following subsequent allergen challenge. RESULTS: We found PAR2 -deficient mice sensitized and challenged with HDM failed to develop airway inflammation, did not produce HDM-specific IgG1 and had less IL-4 mRNA in the lungs than wild-type animals. Prevention of PAR2 activation during sensitization in wild-type mice diminished the levels of Th2 mediators, including IL-4, IL-5 and IL-13, in the lungs. Blocking PAR2 during the sensitization phase also led to decreased manifestations of allergic disease, including airway hyperresponsiveness (AHR) and airway inflammation following subsequent allergen challenge. HDM-induced proliferation of splenocytes obtained from animals sensitized in the presence of PAR2 antibody was reduced relative to those that did not receive antibody. The effect of PAR2 blockade could be transferred to naïve mice through splenic CD4(+) T cells from sensitized mice. CONCLUSIONS AND CLINICAL RELEVANCE: PAR2 activation plays a key role during the sensitization phase of our HDM allergy model, leading to increased lung cytokine production and augmented lung reactivity. PAR2 activation is a common mechanism for sensitization to a wide variety of allergens and is therefore a potential pharmacological target to prevent allergy.

Synergistic Transcriptional Changes in AMPA and GABAA Receptor Genes Support Compensatory Plasticity Following Unilateral Hearing Loss.

Debilitating perceptual disorders including tinnitus, hyperacusis, phantom limb pain and visual release hallucinations may reflect aberrant patterns of neural activity in central sensory pathways following a loss of peripheral sensory input. Here, we explore short- and long-term changes in gene expression that may contribute to hyperexcitability following a sudden, profound loss of auditory input from one ear. We used fluorescence in situ hybridization to quantify mRNA levels for genes encoding AMPA and GABAA receptor subunits (Gria2 and Gabra1, respectively) in single neurons from the inferior colliculus (IC) and auditory cortex (ACtx). Thirty days after unilateral hearing loss, Gria2 levels were significantly increased while Gabra1 levels were significantly decreased. Transcriptional rebalancing was more pronounced in ACtx than IC and bore no obvious relationship to the degree of hearing loss. By contrast to the opposing, synergistic shifts in Gria2 and Gabra1 observed 30 days after hearing loss, we found that transcription levels for both genes were equivalently reduced after 5 days of hearing loss, producing no net change in the excitatory/inhibitory transcriptional balance. Opposing transcriptional shifts in AMPA and GABA receptor genes that emerge several weeks after a peripheral insult could promote both sensitization and disinhibition to support a homeostatic recovery of neural activity following auditory deprivation. Imprecise transcriptional changes could also drive the system toward perceptual hypersensitivity, degraded temporal processing and the irrepressible perception of non-existent environmental stimuli, a trio of perceptual impairments that often accompany chronic sensory deprivation.

Two directions of plasticity in the sensory-deprived adult cortex.

Damage or deprivation of a localized region of the skin surface has been shown to induce a selective expansion of adjacent skin surface representations in the adult somatosensory cortex. Here, we use repeated optical imaging in conjunction with single unit recordings to assess the plasticity of a single whisker's functional representation in the adult rat. We observed a large-scale expansion of a single whisker's functional representation following innocuous removal of all neighboring whiskers. Surprisingly, the same manipulation can also induce a large-scale contraction of the representation if the animal is removed from its home cage and given a brief opportunity to use its whiskers for active exploration of a different environment. Both the expansion and contraction reverse upon regrowth of the deprived whiskers. Thus, allowing the animal to use its deprived receptor organ in active exploration can determine the direction of plasticity in the adult cortex.

Unbalanced synaptic inhibition can create intensity-tuned auditory cortex neurons.

Intensity-tuned auditory cortex neurons have spike rates that are nonmonotonic functions of sound intensity: their spike rate initially increases and peaks as sound intensity is increased, then decreases as sound intensity is further increased. They are either "unbalanced," receiving disproportionally large synaptic inhibition at high sound intensities; or "balanced," receiving intensity-tuned synaptic excitation and identically tuned synaptic inhibition which neither creates enhances nor creates intensity-tuning. It has remained unknown if the synaptic inhibition received by unbalanced neurons enhances intensity-tuning already present in the synaptic excitation, or if it creates intensity-tuning that is not present in the synaptic excitation. Here we show, using in vivo whole cell recordings in pentobarbital-anesthetized rats, that in some unbalanced intensity-tuned auditory cortex neurons synaptic inhibition enhances the intensity-tuning; while in others it actually creates the intensity-tuning. The lack of balance between synaptic excitation and inhibition was not always apparent in their peak amplitudes, but could sometimes be revealed only by considering their relative timing. Since synaptic inhibition is essentially cortical in origin, the unbalanced neurons in which inhibition creates intensity-tuning provide examples of auditory feature-selectivity arising de novo at the auditory cortex.

Visualizing and quantifying evoked cortical activity assessed with intrinsic signal imaging.

Intrinsic signal imaging (ISI) measures changes in light reflectance from the illuminated cortex (intrinsic signals or IS) attributed to various vascular and metabolic sources that, when using illumination in the 600 nm range, appear to co-localize with neuronal activity. Given the multiple sources contributing to the collected IS, the common practice of averaging across an extended post-stimulus time epoch before dividing by baseline data typically visualizes evoked IS overlying both the cortical tissue and the large surface blood vessels. In rat PMBSF, the contribution from these vessels are problematic as they do not co-localize with known PMBSF function. Determining a means for quantifying the evoked IS area poses an additional challenge. Here, we describe how exploiting IS collected shortly after stimulus onset (within 1.5 s), which coincides with fast oxygen consumption of active neurons, visualizes evoked IS overlying the cortical tissue without the large surface vessels. We also describe how the use of absolute thresholds combined with a baseline determined from data collected immediately prior to stimulus onset (within 1 s) targets most precisely a specific evoked IS amplitude, a method that should be especially useful when evoked areas are expected to occupy a substantial portion of the total imaged area and/or when peak activity is expected to differ between subjects.

Opiate disruption of maternal behavior: morphine reduces, and naloxone restores, c-fos activity in the medial preoptic area of lactating rats.

Morphine significantly impairs maternal behavior; naloxone, an opiate antagonist, restores it. Maternal behavior is associated with c-fos expression, an immediate early gene product, in the medial preoptic area (mPOA) of females. In two experiments, the effects of morphine-alone and morphine plus naloxone on the expression of c-fos were examined. On postpartum day 5, females were injected with morphine or saline (experiment 1), and morphine + naloxone or morphine + saline (experiment 2) and placed back in the home-cage, separated from their pups by a wire-mesh partition. A separate group in experiment 1 was injected but not exposed to pups. Processing for c-fos immunohistochemistry commenced, and c-fos positive cells in a proscribed portion of mPOA were counted. Morphine-treated females had fewer c-fos cells in mPOA compared to saline-treated females, and the presence of pups accounted for a significant increase in c-fos-expressing neurons, whereas in females not exposed to pups, morphine treatment did not significantly reduce baseline c-fos expression (experiment 1). Furthermore, naloxone mitigated the effect as morphine + naloxone-treated females expressed more c-fos cells compared to morphine + saline females (experiment 2). Morphine-treated females, therefore, may exhibit reductions in maternal behavior because of relative opiate-induced inactivation of areas of the brain devoted to the regulation of maternal behavior.

The efficacy of a prophylactic knee brace to reduce knee injuries in football. A prospective, randomized study at West Point.

The purpose of this prospective, randomized study was to determine the efficacy of a prophylactic knee brace to reduce the frequency and severity of acute knee injuries in football in an athletic environment in which the athletic shoe, playing surface, athlete-exposure, knee injury history, and brace assignment were either statistically or experimentally controlled. The participants in the study were 1396 cadets at the United States Military Academy, West Point, New York, who experienced a total of 21,570 athlete-exposures in the 1986 and 1987 fall intramural tackle football seasons. The use of prophylactic knee braces significantly reduced the frequency of knee injuries, both in the total number of subjects injured and in the total number of medial collateral ligament injuries incurred. However, the reduction in the frequency of knee injuries (total and medial collateral ligament) was dependent on player position. Defensive players who wore prophylactic knee braces had statistically fewer knee injuries than players who served as controls. This was not true of offensive players who served as controls; they had statistically no difference in the number of knee injuries from players who wore prophylactic knee braces. The severity of medical collateral ligament and anterior cruciate ligament knee injuries was not significantly reduced with the use of prophylactic knee braces.

Biased signalling and proteinase-activated receptors (PARs): targeting inflammatory disease.

Although it has been known since the 1960s that trypsin and chymotrypsin can mimic hormone action in tissues, it took until the 1990s to discover that serine proteinases can regulate cells by cleaving and activating a unique four-member family of GPCRs known as proteinase-activated receptors (PARs). PAR activation involves the proteolytic exposure of its N-terminal receptor sequence that folds back to function as a 'tethered' receptor-activating ligand (TL). A key N-terminal arginine in each of PARs 1 to 4 has been singled out as a target for cleavage by thrombin (PARs 1, 3 and 4), trypsin (PARs 2 and 4) or other proteases to unmask the TL that activates signalling via Gq , Gi or G12 /13 . Similarly, synthetic receptor-activating peptides, corresponding to the exposed 'TL sequences' (e.g. SFLLRN-, for PAR1 or SLIGRL- for PAR2) can, like proteinase activation, also drive signalling via Gq , Gi and G12 /13 , without requiring receptor cleavage. Recent data show, however, that distinct proteinase-revealed 'non-canonical' PAR tethered-ligand sequences and PAR-activating agonist and antagonist peptide analogues can induce 'biased' PAR signalling, for example, via G12 /13 -MAPKinase instead of Gq -calcium. This overview summarizes implications of this 'biased' signalling by PAR agonists and antagonists for the recognized roles the PARs play in inflammatory settings.

Specific and rapid effects of acoustic stimulation on the tonotopic distribution of Kv3.1b potassium channels in the adult rat.

Recent studies have demonstrated that total cellular levels of voltage-gated potassium channel subunits can change on a time scale of minutes in acute slices and cultured neurons, raising the possibility that rapid changes in the abundance of channel proteins contribute to experience-dependent plasticity in vivo. In order to investigate this possibility, we took advantage of the medial nucleus of the trapezoid body (MNTB) sound localization circuit, which contains neurons that precisely phase-lock their action potentials to rapid temporal fluctuations in the acoustic waveform. Previous work has demonstrated that the ability of these neurons to follow high-frequency stimuli depends critically upon whether they express adequate amounts of the potassium channel subunit Kv3.1. To test the hypothesis that net amounts of Kv3.1 protein would be rapidly upregulated when animals are exposed to sounds that require high frequency firing for accurate encoding, we briefly exposed adult rats to acoustic environments that varied according to carrier frequency and amplitude modulation (AM) rate. Using an antibody directed at the cytoplasmic C-terminus of Kv3.1b (the adult splice isoform of Kv3.1), we found that total cellular levels of Kv3.1b protein-as well as the tonotopic distribution of Kv3.1b-labeled cells-was significantly altered following 30 min of exposure to rapidly modulated (400 Hz) sounds relative to slowly modulated (0-40 Hz, 60 Hz) sounds. These results provide direct evidence that net amounts of Kv3.1b protein can change on a time scale of minutes in response to stimulus-driven synaptic activity, permitting auditory neurons to actively adapt their complement of ion channels to changes in the acoustic environment.

Malignant melanoma arising intradermally in a small congenital nevus of an adult.

The occurrence of a malignant melanoma in a small congenital nevus is a rare event. A malignant melanoma arising intradermally in a small congenital nevus in an adult has not been previously reported. The clinical implications and known information about the prognosis of intradermal malignant melanomas are discussed.

Varying the degree of single-whisker stimulation differentially affects phases of intrinsic signals in rat barrel cortex.

Using intrinsic signal optical imaging (ISI), we have shown previously that the point spread of evoked activity in the rat barrel cortex in response to single-whisker stimulation encompasses a surprisingly large area. Given that our typical stimulation consists of five deflections at 5 Hz, the large area of evoked activity might have resulted from repetitive stimulation. Thus in the present study, we use ISI through the thinned skull to determine whether decreasing the degree of single-whisker stimulation decreases the area of the cortical point spread. We additionally outline a protocol to quantify stimulus-related differences in the temporal characteristics of intrinsic signals at a fine spatial scale. In 10 adult rats, whisker C2 was stimulated randomly with either one or five deflections delivered in a rostral-to-caudal fashion. Each deflection consisted of a 0.5-mm displacement of the whisker as measured at the point of contact, 15 mm from the snout. The number of whisker deflections did not affect the area or peak magnitude of the cortical point spread based on the intrinsic signal activity occurring from 0.5 up to 1.5 s poststimulus onset. In contrast, the magnitude and time course of intrinsic signal activity collected after 1.5-s poststimulus onset did reflect the difference in the degree of stimulation. Thus decreasing the degree of stimulation differentially affected the early and late phases of the evoked intrinsic signal response. The implications of the present results are discussed in respect to probable differences in the signal source underlying the early versus later phases of evoked intrinsic signals.