SKGQA, a Peptide Derived from the ANA/BTG3 Protein, Cleaves Amyloid-ß with Proteolytic Activity.

Despite the extensive research conducted on Alzheimer's disease (AD) over the years, no effective drug for AD treatment has been found. Therefore, the development of new drugs for the treatment of AD is of the utmost importance. We recently reported the proteolytic activities of JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMA), synthetic peptides of nine amino acids each, derived from the Box A region of Tob1 and ANA/BTG3 proteins, respectively. Furthermore, two components of ANA-TA9, ANA-YA4 (YRMI) at the C-terminus end and ANA-SA5 (SKGQA) at the N-terminus end of ANA-TA9, exhibited proteolytic activity against amyloid-ß (Aß) fragment peptides. In this study, we identified the active center of ANA-SA5 using AEBSF, a serine protease inhibitor, and a peptide in which the Ser residue of ANA-SA5 was replaced with Leu. In addition, we demonstrate the proteolytic activity of ANA-SA5 against the soluble form Aß42 (a-Aß42) and solid insoluble form s-Aß42. Furthermore, ANA-SA5 was not cytotoxic to A549 cells. These results indicate that ANA-SA5 is a promising Catalytide and a potential candidate for the development of new peptide drugs targeting Aß42 for AD treatment.

Effect of an Outbound Scheduling Team on the Timeliness of Scheduling Referrals to Pediatric Otolaryngology.

OBJECTIVE: Families preferring to receive care in a language-other-than-English have disparities in access to care. We studied the effect of implementing an ambulatory outbound scheduling team on the timeliness of scheduling referrals to pediatric otolaryngology. We hypothesized this intervention could increase access to care. STUDY DESIGN: Retrospective cohort analysis. SETTING: Tertiary care academic center. METHODS: Data were abstracted from the hospital's enterprise database for patients referred to Otolaryngology over 3 years (October 2019-August 2022; 7675 referrals). An outbound scheduling team was created April 2021 and tasked with calling out to schedule referrals within one business day of receipt. Referral lag was compared across patient cohorts before and after the scheduling intervention. Log-transformed linear regression models were used to assess the impact of the scheduling intervention on referral lag for language cohorts. RESULTS: The median preintervention referral lag was 6 days (interquartile range [IQR] 2-18), which was reduced to 1 day postintervention (IQR 0-5; P < .001). Preintervention language-other-than-English families had a median referral lag of 8 days (IQR 2-23), which was 1.27 times higher than for patients speaking English (P < .001). With implementation of the scheduling intervention, language-other-than-English families were scheduled in a median of 1 day (IQR 0-6), and the disparity in timeliness of scheduling was eliminated (P = .131). Postintervention, referral lag was reduced by 58% in the English and 64% in the language other than English cohorts. CONCLUSION: Implementation of an outbound ambulatory scheduling process reduces referral lag for all patients and eliminated a disparity in referral lag for language-other-than-English families.

[Surveillance of the Naturally Derived Benzoic Acid Levels in Fruits and Fruit Products, and Comparison of Analytical Methods].

Benzoic acid (BA) is typically found in natural food; therefore, naturally occurring BA must be distinguished from added BA preservatives. In this study, we investigated BA levels in 100 samples of fruit products and their fresh fruits as raw materials using dialysis and steam distillation approaches. BA was detected in the range (minimum-maximum) of 2.1-1380 µg/g and 2.2-1950 µg/g in dialysis and steam distillation, respectively. Steam distillation indicated higher BA levels than dialysis.

Five-mer peptides prevent short-term spatial memory deficits in Aß25-35-induced Alzheimer's model mouse by suppressing Aß25-35 aggregation and resolving its aggregate form.

BACKGROUND: The development of drugs for Alzheimer's disease (AD), which is related to the misfolding and aggregation of amyloid-ß (Aß), is high in demand due to the growing number of AD patients. In this study, we screened 22 kinds of 5-mer synthetic peptides derived from the Box A region of Tob1 protein to find a peptide effective against Aß aggregation. METHODS: A Thioflavin T (ThT) assay was performed to evaluate aggregation and screen aggregation inhibitors. Male ICR mice (6 weeks old) were administered saline, 9 nmol Aß25-35, or a mixture of 9 nmol Aß25-35 and 9 nmol GSGFK in the right lateral ventricle. Short-term spatial memory was assessed through Y-maze. Microglia cells (BV-)2 cells were plated on 24-well plates (4 × 104 cells/well) and incubated for 48 h, and then, the cells were treated with 0.01, 0.05, 0.1, 0.2, or 0.5 mM GSGFK. After incubation for 24 h, bead uptake was evaluated using a laser confocal microscope and Cytation 5. RESULTS: We found two kinds of peptides, GSGNR and GSGFK, that were not only suppressed by aggregation of Aß25-35 but also resolved the aggregated Aß25-35. Results obtained from the Y-maze test on an Aß25-35-induced AD model mouse indicated that GSGFK prevents the deficits in short-term memory induced by Aß25-35. The effect of GSGFK on phagocytosis in BV-2 cells proved that GSGFK activates the phagocytic ability of microglia. CONCLUSIONS: In conclusion, 5-mer peptides prevent short-term memory deficit in Aß25-35 induced AD model mouse by reducing the aggregated Aß25-35. They may also upregulate the phagocytic ability of microglia, which makes 5-mer peptides suitable candidates as therapeutic drugs against AD.

Structure-Activity Relationship of 5-mer Catalytides, GSGYR and RYGSG.

We recently discovered JAL-TA9 (YKGSGFRMI), a short hydrolytic peptide that we termed a Catalytide. The catalytic center of JAL-TA9 was modeled using MM2 and MMFF94 parameters and identified as GSGFR. Additionally, a structure-activity relationship study showed that GSGYR cleaved Aß11-29. Here, we developed a novel Catalytide in silico. Molecular dynamics simulations of GSGYR and RYGSG using MM2 and MMFF94 parameters suggested that both peptides may form catalytic triads and oxyanion holes. The hydrolytic potency of RYGSG was five times higher than that of GSGYR. Moreover, both peptides showed three common cleavage positions for Aß11-29; namely, L17-V18, V18-F19, and E22-D23. The aggregation ratio analyzed by the thioflavin-T assay correlated well with proteolytic activity, suggesting that the aggregation of Aß11-29 was suppressed by the cleavage reaction. Docking simulations with the carbonyl carbon of L17 or the carbonyl carbon of E22 in Aß11-29 were conducted using the secondary structures of GSGYR and RYGSG. The distance between the hydroxyl group of serine and the carbonyl carbon of the two cleavage sites proved that RYGSG was closer to Aß11-29 than to GSGYR. This study demonstrated that Catalytides are useful for understanding structure-activity relationships.

Direct Delivery of ANA-TA9, a Peptide Capable of Aß Hydrolysis, to the Brain by Intranasal Administration.

We have recently reported Catalytides (Catalytic peptides) JAL-TA9 (YKGSGFRMI) and ANA-TA9 (SKGQAYRMI), which are the first Catalytides found to cleave Aß42. Although the Catalytides must be delivered to the brain parenchyma to treat Alzheimer's disease, the blood-brain barrier (BBB) limits their entry into the brain from the systemic circulation. To avoid the BBB, the direct route from the nasal cavity to the brain was used in this study. The animal studies using rats and mice clarified that the plasma clearance of ANA-TA9 was more rapid than in vitro degradation in the plasma, whole blood, and the cerebrospinal fluid (CSF). The brain concentrations of ANA-TA9 were higher after nasal administration than those after intraperitoneal administration, despite a much lower plasma concentration after nasal administration, suggesting the direct delivery of ANA-TA9 to the brain from the nasal cavity. Similar findings were observed for its transport to CSF after nasal and intravenous administration. The concentration of ANA-TA9 in the olfactory bulb reached the peak at 5 min, whereas those in the frontal and occipital brains was 30 min, suggesting the sequential backward translocation of ANA-TA9 in the brain. In conclusion, ANA-TA9 was efficiently delivered to the brain by nasal application, as compared to other routes.

Amyloid beta cleavage by ANA-TA9, a synthetic peptide from the ANA/BTG3 Box A region.

INTRODUCTION: We recently discovered a short synthetic peptide derived from the ANA/BTG3 protein Box A region called ANA-TA9 (SKGQAYRMI), which possesses catalytic activity. Herein we demonstrated the proteolytic activity of ANA-TA9 against amyloid beta 42 (Aß42). METHODS: The proteolytic activity of ANA-TA9 against both the authentic soluble form Aß42 (a-Aß42) and the solid insoluble form Aß42 (s-Aß42) was analyzed by high-performance liquid chromatography and mass spectrometry. Plasma clearance, brain uptake, and cell viability were examined. RESULTS: ANA-TA9 cleaved not only a-Aß42 but also s-Aß42. Proteolytic activity was partially inhibited by 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, a serine protease inhibitor. Plasma clearance was very rapid, and the brain concentration indicated efficient brain delivery of ANA-TA9 via nasal application. Cell viability analysis indicated that ANA-TA9 did not display toxicity. DISCUSSION: ANA-TA9 is an attractive potential candidate for the development of novel peptide drugs in Alzheimer's disease treatment.

Zinc-aggravated M1 microglia regulate astrocytic engulfment via P2×7 receptors.

BACKGROUND: Glial cells such as astrocytes and microglia play an important role in the central nervous system via communication between these glial cells. Activated microglia can exhibit either the inflammatory M1 phenotype or the anti-inflammatory M2 phenotype, which influences astrocytic neuroprotective functions, including engulfment of cell debris. Recently, extracellular zinc has been shown to promote the inflammatory M1 phenotype in microglia through intracellular zinc accumulation and reactive oxygen species (ROS) generation. PURPOSE: Here, we investigated whether the zinc-enhanced inflammatory M1 phenotype of microglia affects the astrocytic engulfing activity. METHODS: Engulfing activity was assessed in astrocytes treated with microglial-conditioned medium (MCM) from lipopolysaccharide (LPS)-activated or from ZnCl2-pretreated LPS-activated M1 microglia. The effect of zinc on microglia phenotype was also validated using the zinc chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) and the ROS scavenger Trolox. RESULTS: Although treatment of astrocytes with LPS showed no significant effect on the engulfing activity, MCM from LPS-induced M1 microglia increased the beads uptake by astrocytes. This increased uptake activity was suppressed when MCM from LPS-induced M1 microglia pretreated with ZnCl2 was applied to astrocytes, which was further abolished by the intracellular zinc chelator TPEN and the ROS scavenger Trolox. In addition, expression of P2×7 receptors (P2×7R) was increased in astrocytes treated with MCM derived from M1 microglia but not in the M1 microglia pretreated with ZnCl2. CONCLUSION: These findings suggest that zinc pre-treatment abolishes the ability of LPS-induced M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2×7R.

Catalytides derived from the Box A region in the ANA/BTG3 protein cleave amyloid-ß fragment peptide.

We have recently reported about shorter proteolytic peptides termed Catalytide as general name. JAL-TA9 (YKGSGFRMI), a fragment peptide derived from Box A region of Tob1 protein, is the first Catalytide and cleaves Aß42 and its fragment peptides. Herein, we demonstrate the enzymatic properties of ANA-TA9 corresponding region to JAL-TA9 in ANA/BTG3 protein. ANA-TA9 showed the auto-proteolytic activity and cleaved 3 kinds of synthetic fragment peptides derived from Aß42, especially on the central region of Aß42 with a serine protease like activity. Interestingly, 2 kinds of components, ANA-SA5 (SKGQA) and ANA-YA4 (YRMI), also showed similar proteolytic activity. These results indicate that ANA-TA9 is composed of two different Catalytides.

Development of a novel class of peroxisome proliferator-activated receptor (PPAR) gamma ligands as an anticancer agent with a unique binding mode based on a non-thiazolidinedione scaffold.

We previously identified dibenzooxepine derivative 1 as a potent PPARγ ligand with a unique binding mode owing to its non-thiazolidinedione scaffold. However, while 1 showed remarkably potent MKN-45 gastric cancer cell aggregation activity, an indicator of cancer differentiation-inducing activity induced by PPARγ activation, we recognized that 1 was metabolically unstable. In the present study, we identified a metabolically soft spot, and successfully discovered 3-fluoro dibenzooxepine derivative 9 with better metabolic stability. Further optimization provided imidazo[1,2-a]pyridine derivative 17, which showed potent MKN-45 gastric cancer cell aggregation activity and excellent PK profiles compared with 9. Compound 17 exerted a growth inhibitory effect on AsPC-1/AG1 pancreatic tumor in mice. Furthermore, the decrease in the hematocrit (an indicator of localized edema, a serious adverse effect of PPARγ ligands) was tolerable even with oral administration at 200 mg/kg in healthy mice.

Effects of Cu2+ on conformational change and aggregation of hPrP180-192 with a V180I mutation of the prion protein.

Prion diseases are neurodegenerative disorders caused by misfolding of the prion protein (PrP) from a normal cellular protein (PrPC) to a protease-resistant isoform (PrPSc). However, the aggregation mechanism is not entirely understood because of the physical properties of PrP, such as its solubility or aggregation in vitro and conformational or mutation diversity. Recently, we reported the physical and physiological properties of a synthetic fragment peptide. In the present study, we assessed the importance of a point mutation at the C-terminal region of PrP in structural conversion and aggregation and evaluated the physical and physiological properties of the point-mutated human-PrP180-192 V180I (hPrP180-192 V180I) using circular dichroism spectra, high-performance liquid chromatography, Affinix QNµ, and thioflavin-T staining, including the effects of Cu2+. The secondary structure of hPrP180-192 V180I changed from a random coil to a ß-sheet in Cu2+ free buffer. In addition, we observed molecular interactions in hPrP180-192 V180I and aggregation with itself, which were inhibited by Cu2+. We conclude that the point mutation in the C-terminal region of PrP, including hPrP180-192 V180I, and Cu2+ may play an important role in the conversion of PrPC to PrPSc.

The discovery of shorter synthetic proteolytic peptides derived from Tob1 protein.

We screened nearly 1000 synthetic peptides and found that JAL-AK22 (KYEGHWYPEKPYKGSGFRCIHI), which is derived from the BoxA domain in the Tob1 protein, activates both unfolded and folded proMMP-7. Interestingly, the smaller derivative of JAL-AK22, termed JAL-TA9 (YKGSGFRMI) possessed auto-proteolytic activity and cleaved three synthetic peptides fragment (MMP18-33, MMP18-40, and Aß11-29) under physiological conditions. The kcat of JAL-TA9 was 4.58 × 10-4 min-1 against MMP18-33 and 6.5 × 10-4 min-1 against MMP18-40. These kinetic parameters are lower than those of general proteinases like trypsin, for which the kcat is 247.2 × 105 min-1 against benzoyl-l-arginine ethyl ester. In addition, a 5-mer peptide derived from JAL-TA9, GSGFR also cleaved Aß11-29. These proteolytic activities were inhibited by AEBSF (4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride), a serine protease inhibitor. Our results demonstrate that some small synthetic peptides have protease activity. Thus, we propose calling small peptides possessing with protease activity Catalytides (catalytic peptides). We expect that our findings will stimulate the development of novel Catalytides and related applications such as the development of strategic peptide drugs.

Information exchange using a prescribed form and involvement of occupational health nurses promotes occupational physicians to collaborate with attending physicians for supporting workers with illness in Japan.

The maintenance of a balance between work and disease treatment is an important issue in Japan. This study explored factors that affect collaboration between occupational physicians (OPs) and attending physicians (APs). A questionnaire was mailed to 1,102 OPs. The questionnaire assessed the demographic characteristics of OPs; their opinions and behaviors related to collaboration, including the exchange of medical information with APs; and the occupational health service system at their establishments. In total, 275 OPs completed the questionnaire (25.0% response rate). Over 80% of respondents believed OPs should collaborate with APs. After adjusting for company size, collaboration ≥10 times/yr (with regard to both returning to work following sick leave and annual health check-ups for employees) was significantly associated with environmental factors, such as the presence of occupational health nurses (odds ratio (OR): 5.56 and 5.01, respectively, p<0.05) and the use of prescribed forms for information exchange (OR: 4.21 and 3.63, respectively, p<0.05) but not with the demographic characteristics of the OPs (p>0.05). The majority of OPs believed that collaboration with APs is important for supporting workers with illnesses. Support systems including prescribed forms of information exchange and occupational health nurses, play pivotal roles in promoting this collaboration.

Attenuation of zinc-enhanced inflammatory M1 phenotype of microglia by peridinin protects against short-term spatial-memory impairment following cerebral ischemia in mice.

Activated microglia exhibit two opposite activation states, the inflammatory M1 and the anti-inflammatory M2 activation states. In the mammalian brain, ischemia elicits a massive release of zinc from hippocampal neurons, and the extracellular zinc primes M1 microglia-by inducing reactive oxygen species (ROS) generation-to enhance their production of proinflammatory cytokines, which ultimately results in short-term spatial memory impairment. Here, we examined how peridinin, a carotenoid in dinoflagellates, affects the zinc-enhanced inflammatory M1 phenotype of microglia. Treatment of microglia with 30-300 ng/mL peridinin caused a dose-dependent attenuation of zinc-enhanced interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α (TNFα) secretion when M1 activation was induced by lipopolysaccharide exposure. Moreover, peridinin inhibited the increase in ROS levels in zinc-treated microglia without directly interacting with zinc. Notably, when mice were administrated peridinin (20-200 ng/animal) intracerebroventricularly 5 min before cerebral ischemia-reperfusion, the peridinin treatment not only suppressed the increase in expression of IL-1ß, IL-6, TNFα, and the microglial M1 surface marker CD16/32, but also protected the mice against ischemia-induced short-term spatial-memory impairment. Our findings suggest that peridinin prevents extracellular zinc-enhanced proinflammatory cytokine secretion from M1 microglia by inhibiting the increase in microglial ROS levels, and that this anti-inflammatory effect of peridinin might result in protection against deficits in short-term spatial memory.

MMP-7 cleaves amyloid ß fragment peptides and copper ion inhibits the degradation.

The extracellular deposition of amyloid ß (Aß) is known to be the fundamental cause of Alzheimer's disease (AD). Aß1-42, generated by ß-secretases from the amyloid precursor protein (APP), is the main component of neuritic plaque, and the aggregation of this protein is shown to be dependent to an extent on metal ions such as copper and zinc. However, the mechanism by which Cu2+ affects the physicochemical properties of Aß1-42 or the central nervous system is still under debate. A recent series of studies have demonstrated that both the soluble-type matrix metalloproteinases (MMP-2 and MMP-9) and the membrane-type matrix metalloproteinase (MT1-MMP) are capable of degrading Aß peptides. MMP-7, one of the soluble-type matrix metalloproteinases, is expressed in hippocampal tissue; however, less information is available concerning the pathophysiological roles of this enzyme in the process and/or progress of Alzheimer's disease. In this study, we examined the degradation activity of MMP-7 against various Aß1-42's fragment peptides and the effect of Cu2+. Although Aß22-40 was degraded by MMP-7 regardless of Cu2+, Cu2+ inhibited the degradation of Aß1-19, Aß11-20, and Aß11-29 by MMP-7. These results indicate that MMP-7 is capable of degrading Aß1-42, and that Aß1-42 acquired resistance against MMP-7 cleavage through Cu2+-binding and structure changes. Our results demonstrate that MMP-7 may play an important role in the defensive mechanism against the aggregation of Aß1-42, which gives rise to the pathology of AD.

Severe pulmonary emphysema in a girl with interstitial deletion of 2q24.2q24.3 including ITGB6.

Owing to the large size of chromosome 2, partial monosomy of the long arm of this chromosome gives rise to many specific phenotypes. We report on a 2-month-old girl with an interstitial deletion of 2q24.2q24.3, which was confirmed by microarray-based comparative genomic hybridization analysis. The patient showed delayed growth and mental retardation, early myoclonic seizures, and characteristic dysmorphic features including thick arched eyebrows, upslanting palpebral fissures, long eyelashes, depressed nasal bridge, short nose, long philtrum, small mouth, micrognathia, and low set ears. Her early myoclonic seizures were likely due to haploinsufficiency of SCN1A and SCN2A, which are included in the deletion region. When she experienced acute bronchopneumonia, she showed severe pulmonary emphysema. The deletion region of 2q24.2 includes the integrin beta6 gene (ITGB6), which may prevent acute lung injury and pulmonary emphysema. Many previously reported patients with deletions of 2q24.2 showed poor outcomes because of respiratory failure. These observations suggest the possibility of a strong relationship between haploinsufficiency of ITGB6 and pulmonary dysfunction.