Molecular design of a highly selective and strong protein inhibitor against matrix metalloproteinase-2 (MMP-2).

Synthetic inhibitors of matrix metalloproteinases (MMPs), designed previously, as well as tissue inhibitors of metalloproteinases (TIMPs) lack enzyme selectivity, which has been a major obstacle for developing inhibitors into safe and effective MMP-targeted drugs. Here we designed a fusion protein named APP-IP-TIMP-2, in which the ten amino acid residue sequence of APP-derived MMP-2 selective inhibitory peptide (APP-IP) is added to the N terminus of TIMP-2. The APP-IP and TIMP-2 regions of the fusion protein are designed to interact with the active site and the hemopexin-like domain of MMP-2, respectively. The reactive site of the TIMP-2 region, which has broad specificity against MMPs, is blocked by the APP-IP adduct. The recombinant APP-IP-TIMP-2 showed strong inhibitory activity toward MMP-2 (Ki(app) = 0.68 pm), whereas its inhibitory activity toward MMP-1, MMP-3, MMP-7, MMP-8, MMP-9, or MT1-MMP was six orders of magnitude or more weaker (IC50 > 1 µm). The fusion protein inhibited the activation of pro-MMP-2 in the concanavalin A-stimulated HT1080 cells, degradation of type IV collagen by the cells, and the migration of stimulated cells. Compared with the decapeptide APP-IP (t½ = 30 min), APP-IP-TIMP-2 (t½ ≫ 96 h) showed a much longer half-life in cultured tumor cells. Therefore, the fusion protein may be a useful tool to evaluate contributions of proteolytic activity of MMP-2 in various pathophysiological processes. It may also be developed as an effective anti-tumor drug with restricted side effects.

Structural basis for matrix metalloproteinase-2 (MMP-2)-selective inhibitory action of ß-amyloid precursor protein-derived inhibitor.

Unlike other synthetic or physiological inhibitors for matrix metalloproteinases (MMPs), the ß-amyloid precursor protein-derived inhibitory peptide (APP-IP) having an ISYGNDALMP sequence has a high selectivity toward MMP-2. Our previous study identified amino acid residues of MMP-2 essential for its selective inhibition by APP-IP and demonstrated that the N to C direction of the decapeptide inhibitor relative to the substrate-binding cleft of MMP-2 is opposite that of substrate. However, detailed interactions between the two molecules remained to be clarified. Here, we determined the crystal structure of the catalytic domain of MMP-2 in complex with APP-IP. We found that APP-IP in the complex is indeed embedded into the substrate-binding cleft of the catalytic domain in the N to C direction opposite that of substrate. With the crystal structure, it was first clarified that the aromatic side chain of Tyr(3) of the inhibitor is accommodated into the S1' pocket of the protease, and the carboxylate group of Asp(6) of APP-IP coordinates bidentately to the catalytic zinc of the enzyme. The Ala(7) to Pro(10) and Tyr(3) to Ile(1) strands of the inhibitor extend into the nonprime and the prime sides of the cleft, respectively. Therefore, the decapeptide inhibitor has long range contact with the substrate-binding cleft of the protease. This mode of interaction is probably essential for the high MMP-2 selectivity of the inhibitor because MMPs share a common architecture in the vicinity of the catalytic center, but whole structures of their substrate-binding clefts have sufficient variety for the inhibitor to distinguish MMP-2 from other MMPs.

Partial REM-sleep deprivation increases the dream-like quality of mentation from REM sleep and sleep onset.

STUDY OBJECTIVES: Sleep onset (SO) is cognitively and physiologically similar to rapid eye movement (REM) sleep, supporting the notion that REM sleep-related processes are 'covertly' active at this time. The objective was to determine if SO mentation is sensitive to REM sleep deprivation. DESIGN: Two-group cross-sectional design; sleep recordings for 3 nights. SETTING: Standard sleep laboratory with 24-channel polysomnography recording. PARTICIPANTS: Fourteen female, 13 male healthy volunteers (18-41 yrs, mean=24.8 +/- 6.07). INTERVENTIONS: On Night 2, half were and half were not partially REM sleep-deprived (REMD), recalled REM mentation, and rated it for dream-like quality (DLQ), sleepiness, and sensory attributes. On Night 3, all were awakened from SO substages 4 and 5 for mentation reports and further ratings. REMD measures were derived from scored sleep tracings. MEASUREMENTS AND RESULTS: REMD produced increases in DLQ for both REM and SO reports (P < .05); DLQ scores were higher for REM than for SO mentation (P < .001). Covarying sleepiness preserved the (REMD) effect but abolished the REM/SO difference. Whereas 2 sensory attributes (presence of self, visual intensity) tended to distinguish the REM-mentation reports of REMD and control subjects, only 1, self-movement, distinguished their SO mentation reports (P < .06). Multiple regression revealed that increased DLQ of both REM and SO mentation was associated with increased sleepiness and decreased REM sleep time on Night 2. CONCLUSIONS: SO mentation responds to REMD much like REM mentation does, a finding consistent with other work supporting the notion of covert REM-sleep processes at SO. DLQ may be mediated by both increases in REM-sleep propensity and a circadian process indexed by sleepiness ratings.

Detection of rapid-eye movements in sleep studies.

One of the key features of rapid-eye movement (REM) sleep is the presence of bursts of REMs. Sleep studies routinely use REMs to classify sleep stages. Moreover, REM count or density has been used in studies involving learning and various psychiatric disorders. Most of these studies have been based on the visual identification of REMs, which is generally a very time-consuming task. This and the varying definitions of REMs across scorers have warranted the development of automatic REM detection methodologies. In this paper, we present a new detection scheme that combines many of the intrinsic properties of REMs and requires minimal parameter adjustments. In the proposed method, a single parameter can be used to control the REM detection sensitivity and specificity tradeoff. Manually scored training data are used to develop the method. We assess the performance of the method against manual scoring of individual REM events and present validation results using a separate data set. The ability of the method to discriminate fast horizontal ocular movement in REM sleep from other types of events is highlighted. A key advantage of the presented method is the minimal a priori information requirement. The results of training data (recordings from five subjects) show an overall sensitivity of 78.8% and specificity of 81.6%. The performance on the testing data (recording from five subjects different from the training data) showed overall sensitivity of 67.2% and specificity of 77.5%.

EEG activities during elicited sleep onset REM and NREM periods reflect different mechanisms of dream generation. Electroencephalograms. Rapid eye movement.

OBJECTIVE: To be the first to compare EEG power spectra during sleep onset REM periods (SOREMP) and sleep onset NREM periods (NREMP) in normal individuals and relate this to dream appearance processes underlying these different types of sleep periods. METHODS: Eight healthy undergraduates spent 7 consecutive nights in the sleep lab including 4 nights for SOREMP elicitation using the Sleep Interruption Technique. This enabled us to control preceding sleep processes between SOREMP and NREMP. EEG power spectra when participants did and did not report 'dreams' were compared between both types of sleep. Sleep stages, subjective measurements including dream property scores, sleepiness, mood, and tiredness after awakenings were also examined to determine their consistency with EEG findings. RESULTS: Increased alpha EEG activities (11.72-13.67 Hz) observed mainly in the central area were related to the absence of SOREMP dreams and appearance of NREMP dreams. Analyses of sleep stages combining two studies (16 participants) also supported the Fast Fourier Transform findings, showing that when dreams were reported there were decreased amounts of stage 2 and increased stage REM in SOREMP and increased stage W in NREMP. SOREMP dreams were more bizarre than NREMP dreams. Participants felt more tired after SOREMP with dreams than without dreams, while the opposite was observed after NREMP episodes. CONCLUSIONS: EEG power spectra patterns reflected different physiological mechanisms underlying generation of SOREMP and NREMP dreams. The same relationships were also reflected by sleep stage analyses as well as subjective measurements including dream properties and tiredness obtained after awakenings. This study not only supports the hypothesized relationships between REM mechanisms and REM dreams as well as arousal processes and NREM dreams, it also provides a new perspective to dream research due to its unique techniques to awaken participants and collect REM dreams during experimentally induced SOREMP.

Factors related to the occurrence of isolated sleep paralysis elicited during a multi-phasic sleep-wake schedule.

STUDY OBJECTIVES: To further investigate mechanisms of isolated sleep paralysis (ISP) in normal individuals, we experimentally elicited ISPs by facilitating sleep onset REM periods (SOREMP), a prerequisite of ISPs, and examined behavioral and psychological measurements relating to ISP appearances. DESIGN: The multi-phasic sleep/wake schedule (MPS) began at approximately midnight and ended when net sleep reached 7.5 hours. Participants were awakened after every 5 min of REM sleep to obtain a maximum number of SOREMPs. Upon each awakening, mentation reports and subjective measurements were collected. Performance tests were then assigned. SETTING: Sleep lab, Tokyo Metropolitan Institute for Neurosciences, Japan. PARTICIPANTS: Thirteen healthy Japanese students (10 males) with high self-reported frequencies of ISPs but no other narcolepsy-related symptoms. MEASUREMENTS AND RESULTS: From 184 sleep interruptions, 8 ISP episodes were obtained. In within participant comparisons between episodes with and without ISPs, the vigilance task (VT) reaction times were elevated before SOREMPs with ISPs. In between analyses (ISP vs non-ISP), the ISP group showed poorer performance, more complaints of physical, mental, and neurotic symptoms, increased subjective fatigue and increased stage 1 throughout the entire schedule. VT hit rates remained constant in the non-ISP group, but dropped in the later part of schedule in the ISP group. Subjective sleepiness dropped over time in the non-ISP group while it slightly increased in the ISP group. CONCLUSIONS: ISP is likely to appear as a phenotype of REM dissociation during SOREMP when participants with low tolerance for disrupted sleep-wake rhythms are placed in this type of schedule.

Elicitation of sleep-onset REM periods in normal individuals using the sleep interruption technique (SIT).

Use of the sleep interruption technique (SIT) to elicit sleep onset REM periods (SOREMPs) in normal individuals is introduced along with its theoretical bases, empirical findings, and potential applications. Capitalizing upon the circadian and ultradian nature of REM sleep, the SIT has been developed to examine various psychophysiological characteristics related to REM sleep. The SIT allows us to: (1) obtain SOREMPs at the discretion of the researcher; (2) avoid the contaminating effects of preceding non-REM (NREM)-REM stage ordering on subsequent target sleep episodes; and (3) obtain many REM episodes in a short time by repeating the sleep interruptions. The SIT has been applied in several studies, such as examination of physiological precursors to REM periods, correlates of dream mechanisms, and induction of sleep paralysis in normal individuals. Guidelines for eliciting SOREMP using the SIT, including the parameters to be manipulated, are provided, e.g. NREM duration before sleep interruption, time of night of awakenings, duration of sleep interruption and tasks employed. Directions for further research such as determining optimal type of task to promote SOREMP occurrences, generalization of SOREMP as usual REM periods, and forms of SOREMP occurrences under different conditions in normal individuals and clinical patients are discussed. Finally, possible future uses of the SIT, including combining this technique with new technologies, are also suggested.