Investigating the Transepithelial Transport and Enzymatic Stability of Lactononadecapeptide (NIPPLTQTPVVVPPFLQPE), a 19-Amino Acid Casein-Derived Peptide in Caco-2 Cells.

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE), a casein-derived peptide comprising 19 residues, is known for its capacity to enhance cognitive function. This study aimed to explore the transepithelial transport and stability of LNDP. Results showed that LNDP retained over 90% stability after 2 h of treatment with gastrointestinal enzymes. The stability of LNDP on Caco-2 cell monolayers ranged from 93.4% ± 0.9% to 101.1% ± 1.2% over a period of 15-60 min, with no significant differences at each time point. The permeability of LNDP across an artificial lipid membrane was very low with the effective permeability of 3.6 × 10-11 cm/s. The Caco-2 assay demonstrated that LNDP could traverse the intestinal epithelium, with an apparent permeability of 1.22 × 10-6 cm/s. Its transport was significantly inhibited to 67.9% ± 5.0% of the control by Gly-Pro, a competitor of peptide transporter 1 (PEPT1). Furthermore, PEPT1 knockdown using siRNA significantly inhibited LNDP transport by 77.6% ± 1.9% in Caco-2 cell monolayers. The LNDP uptake in PEPT1-expressing HEK293 cells was significantly higher (54.5% ± 14.6%) than that in mock cells. These findings suggest that PEPT1 plays a crucial role in LNDP transport, and LNDP exhibits good resistance to gastrointestinal enzymes.

Condensed but liquid-like domain organization of active chromatin regions in living human cells.

In eukaryotes, higher-order chromatin organization is spatiotemporally regulated as domains, for various cellular functions. However, their physical nature in living cells remains unclear (e.g., condensed domains or extended fiber loops; liquid-like or solid-like). Using novel approaches combining genomics, single-nucleosome imaging, and computational modeling, we investigated the physical organization and behavior of early DNA replicated regions in human cells, which correspond to Hi-C contact domains with active chromatin marks. Motion correlation analysis of two neighbor nucleosomes shows that nucleosomes form physically condensed domains with ~150-nm diameters, even in active chromatin regions. The mean-square displacement analysis between two neighbor nucleosomes demonstrates that nucleosomes behave like a liquid in the condensed domain on the ~150 nm/~0.5 s spatiotemporal scale, which facilitates chromatin accessibility. Beyond the micrometers/minutes scale, chromatin seems solid-like, which may contribute to maintaining genome integrity. Our study reveals the viscoelastic principle of the chromatin polymer; chromatin is locally dynamic and reactive but globally stable.

Effects of Prebiotic Yeast Mannan on Gut Health and Sleep Quality in Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Study.

Human gut health is closely related to sleep. We aimed to evaluate the efficacy of yeast mannan (YM) in improving bowel habits and sleep quality, along with metabolomics in fecal samples. A total of 40 healthy adults (age range, 22-64 years) with discomfort in defecation were enrolled and randomly allocated to receive either YM (n = 20; 1.1 g/day) or placebo (n = 20) for four weeks. Participants recorded their defecation habits throughout the test periods. Sleep electroencephalogram (EEG) recording using an EEG device and fecal sampling were performed pre- and post-treatment. The YM group significantly increased defecation frequency and stool volumes compared to the placebo group. After 4 weeks of treatment, the non-REM sleep stage 3 (N3) duration in the YM group was significantly higher than that in the placebo group. YM ingestion significantly lengthened total time in bed (TIB) and significantly shortened N3 latency compared to placebo intake during the trial. The metabolomics analysis found a total of 20 metabolite differences between the YM and placebo groups. As a result of stepwise linear regression, changes in fecal propionate and gamma-aminobutyric acid (GABA) levels were identified as the primary factors explaining changes in TIB and N3 latency, respectively. Our findings suggest that the prebiotic YM could be beneficial to gut health and sleep quality.

Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm.

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. Elucidating this temperature compensation is essential for understanding the KaiABC circadian clock, but its mechanism has remained a mystery. We analyzed the KaiABC temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model explained the experimentally observed responses of the oscillation phase to the temperature shift or the ADP-concentration change and suggested that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.

Landscape-Based View on the Stepping Movement of Myosin VI.

Myosin VI dimer walks toward the minus end of the actin filament with a large and variable step size of 25-36 nm. Two competing models have been put forward to explain this large step size. The Spudich model assumes that the myosin VI dimer associates at a distal tail near the cargo-binding domain, which makes two full-length single α-helix (SAH) domains serve as long legs. In contrast, the Houdusse-Sweeney model assumes that the association occurs in the middle (between residues 913 and 940) of the SAH domain and that the three-helix bundles unfold to ensure the large step size. Their consistency with the observation of stepping motion with a large and variable step size has not been examined in detail. To compare the two proposed models of myosin VI, we computationally characterized the free energy landscape experienced by the leading head during the stepping movement along the actin filament using the elastic network model of two heads and an implicit model of the SAH domains. Our results showed that the Spudich model is more consistent with the 25-36 nm step size than the Houdusse-Sweeney model. The unfolding of the three-helix bundles gives rise to the free energy bias toward a shorter distance between two heads. Besides, the stiffness of the SAH domain is a key factor for giving strong energetic bias toward the longer distance of stepping. Free energy analysis of the stepping motion complements the visual inspection of static structures and enables a deeper understanding of underlying mechanisms of molecular motors.

The Structural Rule Distinguishing a Superfold: A Case Study of Ferredoxin Fold and the Reverse Ferredoxin Fold.

Superfolds are folds commonly observed among evolutionarily unrelated multiple superfamilies of proteins. Since discovering superfolds almost two decades ago, structural rules distinguishing superfolds from the other ordinary folds have been explored but remained elusive. Here, we analyzed a typical superfold, the ferredoxin fold, and the fold which reverses the N to C terminus direction from the ferredoxin fold as a case study to find the rule to distinguish superfolds from the other folds. Though all the known structural characteristics for superfolds apply to both the ferredoxin fold and the reverse ferredoxin fold, the reverse fold has been found only in a single superfamily. The database analyses in the present study revealed the structural preferences of αß- and ßα-units; the preferences separate two α-helices in the ferredoxin fold, preventing their collision and stabilizing the fold. In contrast, in the reverse ferredoxin fold, the preferences bring two helices near each other, inducing structural conflict. The Rosetta folding simulations suggested that the ferredoxin fold is physically much more realizable than the reverse ferredoxin fold. Therefore, we propose that minimal structural conflict or minimal frustration among secondary structures is the rule to distinguish a superfold from ordinary folds. Intriguingly, the database analyses revealed that a most stringent structural rule in proteins, the right-handedness of the ßαß-unit, is broken in a set of structures to prevent the frustration, suggesting the proposed rule of minimum frustration among secondary structural units is comparably strong as the right-handedness rule of the ßαß-unit.

Generation of dynamic three-dimensional genome structure through phase separation of chromatin.

Three-dimensional genome structure and dynamics play critical roles in regulating DNA functions. Flexible chromatin structure and movements suggested that the genome is dynamically phase separated to form A (active) and B (inactive) compartments in interphase nuclei. Here, we examine this hypothesis by developing a polymer model of the whole genome of human cells and assessing the impact of phase separation on genome structure. Upon entry to the G1 phase, the simulated genome expanded according to heterogeneous repulsion among chromatin chains, which moved chromatin heterogeneously, inducing phase separation of chromatin. This repulsion-driven phase separation quantitatively reproduces the experimentally observed chromatin domains, A/B compartments, lamina-associated domains, and nucleolus-associated domains, consistently explaining nuclei of different human cells and predicting their dynamic fluctuations. We propose that phase separation induced by heterogeneous repulsive interactions among chromatin chains largely determines dynamic genome organization.

Mechanism of autonomous synchronization of the circadian KaiABC rhythm.

The cyanobacterial circadian clock can be reconstituted by mixing three proteins, KaiA, KaiB, and KaiC, in vitro. In this protein mixture, oscillations of the phosphorylation level of KaiC molecules are synchronized to show the coherent oscillations of the ensemble of many molecules. However, the molecular mechanism of this synchronization has not yet been fully elucidated. In this paper, we explain a theoretical model that considers the multifold feedback relations among the structure and reactions of KaiC. The simulated KaiC hexamers show stochastic switch-like transitions at the level of single molecules, which are synchronized in the ensemble through the sequestration of KaiA into the KaiC-KaiB-KaiA complexes. The proposed mechanism quantitatively reproduces the synchronization that was observed by mixing two solutions oscillating in different phases. The model results suggest that biochemical assays with varying concentrations of KaiA or KaiB can be used to test this hypothesis.

Effects of a single dose of tablets containing lactononadecapeptide on cognitive function in healthy adults: a randomized, double-blind, cross-over, placebo-controlled trial.

Lactononadecapeptide (LNDP; NIPPLTQTPVVVPPFLQPE) is a memory-improving peptide. The current study aimed to determine the effects of a single dose of tablets containing LNDP on cognitive function in healthy Japanese men aged 30-59 years. A randomized, double-blind, cross-over, placebo-controlled trial was conducted in participants randomly assigned to receive LNDP or placebo tablets. The Uchida-Kraepelin test was used to induce cognitive load in participants as a model of work load. Cognitive function was evaluated using the Japanese version of the CNS Vital Signs. Composite memory and verbal memory were significantly higher following consumption of LNDP than placebo tablets. Carryover effects were observed in attention and concentration domains so that period 1 data was analyzed. LNDP consumption led to higher processing speed, executive function, and cognitive flexibility than placebo. Thus, supplementation with a single dose of LNDP tablets may improve cognitive functions including memory, attention, concentration, and information processing in daily life.

Stochastic epigenetic dynamics of gene switching.

Epigenetic modifications of histones crucially affect eukaryotic gene activity, while the epigenetic histone state is largely determined by the binding of specific factors such as the transcription factors (TFs) to DNA. Here, the way in which the TFs and the histone state are dynamically correlated is not obvious when the TF synthesis is regulated by the histone state. This type of feedback regulatory relation is ubiquitous in gene networks to determine cell fate in differentiation and other cell transformations. To gain insights into such dynamical feedback regulations, we theoretically analyze a model of epigenetic gene switching by extending the Doi-Peliti operator formalism of reaction kinetics to the problem of coupled molecular processes. Spin-1 and spin-1/2 coherent-state representations are introduced to describe stochastic reactions of histones and binding or unbinding of TFs in a unified way, which provides a concise view of the effects of timescale difference among these molecular processes; even in the case that binding or unbinding of TFs to or from DNA is adiabatically fast, the slow nonadiabatic histone dynamics gives rise to a distinct circular flow of the probability flux around basins in the landscape of the gene state distribution, which leads to hysteresis in gene switching. In contrast to the general belief that the change in the amount of TF precedes the histone state change, flux drives histones to be modified prior to the change in the amount of TF in self-regulating circuits. Flux-landscape analyses shed light on the nonlinear nonadiabatic mechanism of epigenetic cell fate decision making.

A milk-derived pentapeptide reduces blood pressure in advanced hypertension in a CCK system-dependent manner.

We recently found that a peptide that activates the cholecystokinin (CCK) system effectively reduces blood pressure in spontaneously hypertensive rats (SHR) after the development of hypertension, after which hypotensive drugs are sometimes less effective. In this study, we investigated the vasorelaxation and antihypertensive effects of a peptide derived from a milk protein in SHR with advanced hypertension. The vasorelaxing activity was measured using the mesenteric artery isolated from SHR and the systemic blood pressure was measured by the tail-cuff method. KFWGK was released from bovine serum albumin (BSA) and the model peptide after subtilisin digestion. KFWGK relaxed the mesenteric artery and this vasorelaxation activity was inhibited by lorglumide, an antagonist of the CCK1 receptor. KFWGK more potently relaxed the artery from advanced-stage SHR than that from early-stage SHR. Orally administered KFWGK exhibited potent and long-lasting antihypertensive effects in SHR after the development of hypertension (the minimum effective dose was 5 µg kg-1). The KFWGK-induced antihypertensive effects were also blocked by a CCK antagonist, suggesting that it activates the CCK system. In conclusion, KFWGK, a CCK-dependent vasorelaxant peptide, exhibited potent antihypertensive effects in SHR after the development of hypertension.

Heterogeneous fluid-like movements of chromatin and their implications to transcription.

Eukaryotic chromatin is a complex of genome DNA and associated proteins, and its structure and dynamics play a crucial role in regulating DNA functions. Chromatin takes rather irregular structures in the nucleus and exhibits heterogeneous sub-diffusive movements as polymers fluctuating in a fluid state. Using genome-wide single-nucleosome tracking data, heterogeneity of movements was statistically analyzed, which categorized chromatin into two types: slow chromatin that moves under structurally constrained environments and fast chromatin that moves with less constraints. Interactions of chromatin to various protein factors determine the motional constraints. For example, loss of the cohesin complex that bundles the chromatin chains reduces the motional constraints and increases the population of fast chromatin. Another example is the transcriptional machinery. While it was previously thought that the transcriptional activity is associated with more open and dynamic chromatin structure, recent studies suggested a more nuanced role of transcription in chromatin dynamics: dynamic association/dissociation of active RNA polymerase II (RNAPII) and other transcription factors and Mediators (TF-Meds) transiently bridges transcriptionally active DNA regions, which forms a loose network of chromatin and constrains chromatin movement, enhancing the slow chromatin population. This new view on the dynamical effects of transcription urges a reflection on the traditional model of transcription factories and invites the more recent models of condensates/phase-separated liquid droplets of RNAPII, transcription factors, and Mediators. The combined procedure of genome-wide single-nucleosome tracking and its statistical analysis would unveil heterogeneity in the chromatin movement, which should provide a key to understanding the relations among chromatin dynamics, structure, and function.

Ser-Tyr and Asn-Ala, vasorelaxing dipeptides found by comprehensive screening, reduce blood pressure via different age-dependent mechanisms.

To understand the changes in physiological responses due to aging, a number of bioactive probes based on different signal transduction pathways are necessary. In this study, we comprehensively and systematically investigated changes in blood vessel function with age using a 336-dipeptide library. In the early stage of hypertension, the most potent vasorelaxant dipeptide was Ser-Tyr (SY) in the mesenteric artery isolated from spontaneously hypertensive rats (SHR). SY-induced vasorelaxation and anti-hypertensive effects were blocked by L-NAME, an inhibitor of nitric oxide synthase (NOS), suggesting that SY activates the NO system. On the other hand, the patterns of dipeptides with vasorelaxation activity in early and advanced stages of hypertension were different. In the advanced stage, the most potent vasorelaxing dipeptide was Asn-Ala (NA). Orally administered NA (1.5 mg/kg) reduced the blood pressure in the advanced stage, at which drugs were sometimes less effective, and the anti-hypertensive effects lasted for 6 hr. The NA-induced vasorelaxation and anti-hypertensive activity was blocked by lorglumide, an antagonist of the cholecystokinin CCK1 receptor, suggesting that NA activated the CCK system. Taken together, in the early and advanced stages of hypertension, SY and NA exhibited vasorelaxing and anti-hypertensive effects via the NO and CCK systems, respectively.

Organization of fast and slow chromatin revealed by single-nucleosome dynamics.

Understanding chromatin organization and dynamics is important, since they crucially affect DNA functions. In this study, we investigate chromatin dynamics by statistically analyzing single-nucleosome movement in living human cells. Bimodal nature of the mean square displacement distribution of nucleosomes allows for a natural categorization of the nucleosomes as fast and slow. Analyses of the nucleosome-nucleosome correlation functions within these categories along with the density of vibrational modes show that the nucleosomes form dynamically correlated fluid regions (i.e., dynamic domains of fast and slow nucleosomes). Perturbed nucleosome dynamics by global histone acetylation or cohesin inactivation indicate that nucleosome-nucleosome interactions along with tethering of chromatin chains organize nucleosomes into fast and slow dynamic domains. A simple polymer model is introduced, which shows the consistency of this dynamic domain picture. Statistical analyses of single-nucleosome movement provide rich information on how chromatin is dynamically organized in a fluid manner in living cells.

Single nucleosome imaging reveals loose genome chromatin networks via active RNA polymerase II.

Although chromatin organization and dynamics play a critical role in gene transcription, how they interplay remains unclear. To approach this issue, we investigated genome-wide chromatin behavior under various transcriptional conditions in living human cells using single-nucleosome imaging. While transcription by RNA polymerase II (RNAPII) is generally thought to need more open and dynamic chromatin, surprisingly, we found that active RNAPII globally constrains chromatin movements. RNAPII inhibition or its rapid depletion released the chromatin constraints and increased chromatin dynamics. Perturbation experiments of P-TEFb clusters, which are associated with active RNAPII, had similar results. Furthermore, chromatin mobility also increased in resting G0 cells and UV-irradiated cells, which are transcriptionally less active. Our results demonstrated that chromatin is globally stabilized by loose connections through active RNAPII, which is compatible with models of classical transcription factories or liquid droplet formation of transcription-related factors. Together with our computational modeling, we propose the existence of loose chromatin domain networks for various intra-/interchromosomal contacts via active RNAPII clusters/droplets.

Effects of Stochastic Single-Molecule Reactions on Coherent Ensemble Oscillations in the KaiABC Circadian Clock.

How do many constituent molecules in a biochemical system synchronize, giving rise to coherent system-level oscillations? One system that is particularly suitable for use in studying this problem is a mixture solution of three cyanobacterial proteins, KaiA, KaiB, and KaiC: the phosphorylation level of KaiC shows stable oscillations with a period of approximately 24 h when these three Kai proteins are incubated with ATP in vitro. Here, we analyze the mechanism behind synchronization in the KaiABC system theoretically by enhancing a model previously developed by the present author. Our simulation results suggest that positive feedback between stochastic ATP hydrolysis and the allosteric structural transitions in KaiC molecules drives oscillations of individual molecules and promotes synchronization of oscillations of many KaiC molecules. Our simulations also show that the ATPase activity of KaiC is correlated with the oscillation frequency of an ensemble of KaiC molecules. These results suggest that stochastic ATP hydrolysis in each KaiC molecule plays an important role in regulating the coherent system-level oscillations. This property is robust against changes in the binding and unbinding rate constants for KaiA to/from KaiC or KaiB, but the oscillations are sensitive to the rate constants of the KaiC phosphorylation and dephosphorylation reactions.

Single-molecular and ensemble-level oscillations of cyanobacterial circadian clock.

When three cyanobacterial proteins, KaiA, KaiB, and KaiC, are incubated with ATP in vitro, the phosphorylation level of KaiC hexamers shows stable oscillation with approximately 24 h period. In order to understand this KaiABC clockwork, we need to analyze both the macroscopic synchronization of a large number of KaiC hexamers and the microscopic reactions and structural changes in individual KaiC molecules. In the present paper, we explain two coarse-grained theoretical models, the many-molecule (MM) model and the single-molecule (SM) model, to bridge the gap between macroscopic and microscopic understandings. In the simulation results with these models, ATP hydrolysis in the CI domain of KaiC hexamers drives oscillation of individual KaiC hexamers and the ATP hydrolysis is necessary for synchronizing oscillations of a large number of KaiC hexamers. Sensitive temperature dependence of the lifetime of the ADP bound state in the CI domain makes the oscillation period temperature insensitive. ATPase activity is correlated to the frequency of phosphorylation oscillation in the single molecule of KaiC hexamer, which should be the origin of the observed ensemble-level correlation between the ATPase activity and the frequency of phosphorylation oscillation. Thus, the simulation results with the MM and SM models suggest that ATP hydrolysis stochastically occurring in each CI domain of individual KaiC hexamers is a key process for oscillatory behaviors of the ensemble of many KaiC hexamers.

Orally active anti-hypertensive peptides found based on enteroendocrine cell responses to a dipeptide library.

We previously reported that an orally administered dipeptide, Arg-Phe (RF), which causes enteroendocrine cell responses, lowered blood pressure in spontaneously hypertensive rats (SHRs). In this study, we found that Phe-Trp (FW), induced the most potent enteroendocrine cell responses out of total 338 dipeptides. An FW analogue, Phe-Trp-Gly-Lys (FWGK), which was effectively produced by tryptic digestion of bovine serum albumin, decreased blood pressure after oral administration. The minimum effective dose of FWGK (50 µg/kg) was 1/300 of that of RF (15 mg/kg). FWGK stimulated cholecystokinin (CCK) secretion in the enteroendocrine cells and exhibited vasorelaxing and antihypertensive effects via the CCK1 system.

Role of ATP Hydrolysis in Cyanobacterial Circadian Oscillator.

A cyanobacterial protein KaiC shows a stable oscillation in its phosphorylation level with approximately one day period when three proteins, KaiA, KaiB, and KaiC, are incubated in the presence of ATP in vitro. During this oscillation, KaiC hydrolyzes more ATP molecules than required for phosphorylation. Here, in this report, a theoretical model of the KaiABC oscillator is developed to elucidate the role of this ATP consumption by assuming multifold feedback relations among reactions and structural transition in each KaiC molecule and the structure-dependent binding reactions among Kai proteins. Results of numerical simulation showed that ATP hydrolysis is a driving mechanism of the phosphorylation oscillation in the present model, and that the frequency of ATP hydrolysis in individual KaiC molecules is correlated to the frequency of oscillation in the ensemble of many Kai molecules, which indicates that the coherent oscillation is generated through the coupled microscopic intramolecular and ensemble-level many-molecular regulations.

Heterogeneous Spatial Distribution of Transcriptional Activity in Budding Yeast Nuclei.

Recent microscopic and simulation studies have shown that the genome structure fluctuates dynamically in the nuclei of budding yeast Saccharomyces cerevisiae. This genome-wide movement should lead to the fluctuations of individual genes in their territorial regions. This raises an intriguing question of whether the resulting distribution of genes is correlated to their transcriptional activity. An effective method for examining this correlation is to analyze how the spatial distribution of genes and their transcriptional activity are modified by mutation. In this study, we analyzed the modification observed in a budding yeast mutant in which genes necessary for anchoring telomeres to the nuclear envelope, yku70 and esc1, are silenced. Taddei et al. reported that 60 genes are clearly misregulated by this mutation, with 28 and 32 genes downregulated and upregulated, respectively. We calculated the probability density maps of the misregulated genes using a model of dynamical movement of the yeast genome in both wild-type (WT) and yku70 esc1 mutant and showed that the density of downregulated genes is larger near the nucleolus, whereas the density of upregulated genes is larger at the opposite side of the nucleus. By comparing these genes with those highly (top 200 of transcriptome) and lowly (bottom 200) expressed, we showed that the simulated distribution of 28 downregulated (12 out of 32 upregulated) genes has a distinctly larger overlap with the distribution of lowly (highly) expressed genes in the mutant than in the WT. The remaining 20 upregulated genes are localized near the nuclear envelope both in the WT and in the mutant. These results showed that the transcriptional level of genes is affected by their spatial distribution, thus highlighting the importance of the structural regulation in the yeast genome.