Increased systemic inflammatory interleukin-1ß and interleukin-6 during agitation as predictors of Alzheimer's disease.

OBJECTS: Identification of biomarkers for Alzheimer's disease (AD) is important for its early diagnosis and prevention and a key in advancing our understanding of its pathophysiology. The aim of this study was to determine whether systemic inflammatory interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) as well as hypertension (HT), diabetes mellitus (DM), and body mass index (BMI) are predictors of AD. METHODS: We performed a 10-year follow-up study on 133 elderly who were institutionalized in a nursing home. The associations of IL-1ß and IL-6 at both rest and agitation, as well as HT, DM, and BMI at baseline, were analyzed with the incidences of vascular dementia (VD) and AD during a 10-year follow-up period. RESULTS: The Kaplan-Meier method with log-rank test and Cox regression analyses for the total of 133 subjects showed significantly higher incidences of both VD and AD in subjects with DM or HT at baseline. Resting IL-1ß or IL-6 value, or agitation score, was not significantly associated with the subsequent development of VD or AD. The analyses of 40 subjects who had shown agitation at least once in the previous 3 months demonstrated that IL-1ß and IL-6 values at the agitation stage were significantly associated with AD, but not with VD. CONCLUSION: Our results indicate that systemic inflammatory IL-1ß and IL-6 at the agitation stage are risk factors for the development of AD, but not VD. Inflammatory mechanisms for AD seem to be causal and specific to the development of AD.

Clinical benefits of precision medicine in treating solid cancers: European Society of Medical Oncology-Magnitude of Clinical Benefit Scale score-based analysis.

BACKGROUND: Precision and matched cancer medicine has the potential to complement the existing biomarker approaches in cancer treatment. However, despite their promising potential, certain negative results have highlighted their limitations in molecular biology-driven treatment strategies. This study aimed to evaluate the clinical benefits of precision therapies. MATERIALS AND METHODS: Three reviewers independently identified and assessed precision and matched cancer treatment studies published between January 2015 and December 2020. Clinical benefits of the treatments included in our cohort were assessed using two established frameworks; the European Society of Medical Oncology-Magnitude of Clinical Benefit Scale version 1.1 (ESMO-MCBS) and the American Society of Clinical Oncology Value Framework. RESULTS: Of the 290 eligible studies, 130 were for lung cancer, 51 for solid tumors, 24 for melanoma, and 24 for breast cancer. The common targets were: epidermal growth factor receptor (N = 66), serine/threonine-protein kinase B-Raf (N = 40), anaplastic lymphoma kinase (ALK) (N = 34), breast cancer protein (N = 26), phosphatidylinositol-3 kinase/protein kinase B/phosphatase and tensin homolog (PI3K/AKT/PTEN) pathway (N = 19), receptor tyrosine-protein kinase erbB-2 (HER2) (N = 19), mitogen-activated protein kinase (RAS/RAF/MAPK) pathway (N = 18), programmed death-ligand 1 (N = 12), fibroblast growth factor receptor (N = 8), and others (N = 43). The ESMO-MCBS scales ranged from 0 to 4. Based on the clinical benefit values, tumor mutational burden/mismatch repair-deficient/microsatellite instability-high for immunotherapy, anaplastic lymphoma kinase, and neurotrophic receptor tyrosine kinase therapeutic targets were considered high, whereas RAS/RAF/MAPK and PI3K/AKT/PTEN were considered low. Additionally, we found a significant difference between each average score (P < 0.001). CONCLUSIONS: This study showed that precision and matched cancer therapies require further improvement. This is consistent with the views of the tumor board and of clinicians that precision strategies need to be revised to improve their therapeutic effects.

Association between altered systemic inflammatory interleukin-1beta and natural killer cell activity and subsequently agitation in patients with Alzheimer disease.

OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disorder that is the most common cause of dementia in the elderly and is frequently accompanied by emotional disorder, including agitation. Although evidence of neuroendocrine immune and inflammatory functions during emotional changes has been accumulated, the pathogenic mechanisms in the development of agitation accompanied by AD remain to be elucidated. METHODS: To clarify the involvement of neuroendocrine and immune and inflammatory systems in agitation in AD, we examined agitation levels, circadian rhythms of behavior, cortisol, interleukin-1beta (IL-1beta), and natural killer cell activity (NKCA) in controls without dementia and 16 AD patients who were recognized to be easily agitated in their nursing homes. These behavioral and blood indicators were assessed according to the progress of the stage of agitation in 16 AD patients (stable, pre-agitation, and agitation stages). RESULTS: Elevations in night behavior and blood cortisol, IL-1beta and an reduced blood NKCA level in the evening were observed not only in the agitation stage, but also when stable in AD patients as compared to the control. Increased IL-1beta and decreased NKCA occurred in both the morning and evening in pre-agitation and agitation stages in AD. CONCLUSIONS: The increased IL-1beta and decreased NKCA with the progress of agitation in AD suggest that inflammation produces agitation and aggravates AD.

Simple and low-cost tabletop NMR system for chemical-shift-resolution spectra measurements.

We have been working on developing a low-cost tabletop NMR system. We reported that a field homogeneity as high as 50 ppm was achieved with a simple NMR magnet by employing two facing ferrite magnets with iron disks in between (Chonlathep et al., 2017). In this paper, we report two improvements added to our previous system: (1) an FPGA based signal processing unit to improve the S/N ratio and (2) a simple shimming mechanism to improve the field homogeneity. We obtained as high as 1 ppm field homogeneity in the best case. The signals from hydrogen nuclear spins in a methyl and carboxy group in acetic acid were resolved in NMR spectra.

Purification and properties of a magnesium-dependent endodeoxyribonuclease endogenous to rat-liver nuclei.

An Mg2+-dependent endonuclease has been purified from a 0.6 M NaCl extract of rat-liver nuclei by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) was shown to have a pI value of 7.1 and to migrate as a single band to a molecular-weight position of 36,500 on SDS-polyacrylamide gel. The IEF fraction was subjected to a sedimentation analysis. In a hypotonic buffer (10 mM Tris), the nuclease activity sedimented to have an S value of 4.1 S. However, in an isotonic buffer (0.15 M NaCl), this fraction exhibited two activity peaks of 2.8 and 4.3 S. In a hypertonic buffer (0.3 M NaCl), almost all of the nuclease activity sedimented at 2.7-2.8 S. In this connection, values of 2.8 and 4.3 S were determined to correspond to molecular weights of about 36,000 and 70,000, respectively. Thus, an Mg2+-dependent endonuclease, endogenous to rat-liver nuclei, has been inferred to exist in the reversible form of a monomer/homodimer as its native state. Moreover, the IEF fraction formed single-strand nicks more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyguanosine 5'-monophosphate termini in the DNA at an early incubation time. In addition, RNAase activity was not detected in this fraction.

Purification and properties of an endonuclease endogenous to rat-liver nuclei.

An endonuclease endogenous to rat-liver nuclei has been purified by a series of chromatographic procedures and finally by isoelectric focusing (IEF) electrophoresis. The nuclease fraction prepared by the IEF electrophoresis (IEF fraction) showed a pI value of 5.7 and migrated as a single band to a molecular weight position of 46,000 on an SDS-polyacrylamide gel. The activity for single-stranded DNA was enhanced by 10 mM MgCl2 and/or by 5-15 mM MgCl2 in the presence of 2 mM CaCl2 (an optimum pH, 7.0), but was lowered by CaCl2 alone and inhibited strongly by ZnCl2 or MnCl2. The activity for duplex DNA was rather low, although an optimum condition was 10 mM MgCl2. In fact, even under this condition, the activity was about 40% lower than that for single-stranded DNA. Moreover, the IEF fraction formed single-strand nicks much more rapidly than double-strand cuts in pBR322 DNA, and preferentially produced deoxyadenosine 5'-monophosphate termini in the DNA. In addition, RNAase activity was also detected in this fraction.

A nuclease from rat-liver nuclei with endo- and exonucleolytic activity.

An Mg2(+)-dependent endonuclease endogenous to rat-liver nuclei had an exonuclease activity for single-stranded DNA, but not for duplex DNA. The activity was about twice as high in the 3'----5' direction as in the 5'----3' direction. The products by 3'----5' activity were mononucleotides alone. The 5'----3' activity released mononucleotides as main products and small amounts of di-, tri-, tetra- and oligonucleotides. Another major endonuclease endogenous to the nuclei, a Ca2+/Mg2(+)-dependent endonuclease, did not have such exonuclease activities.

Purification and characterization of nuclear scaffold proteins which bind to a highly repetitive bent DNA from rat liver.

Our previous work (Hibino et al. (1992) Biochem. Biophys. Res. Commun. 184, 853-858) has shown that the binding affinities of a highly repetitive DNA component for rat nuclear scaffold proteins, P123 and P130, depend on the degree of sequence-directed bending of the helix axis. In the present experiment, these proteins have been purified and finally isolated by DNA-Sepharose column chromatography. The pI values of P123 and P130 were 7.2 and 8.1, respectively. The southwestern blotting revealed that a highly repetitive bent DNA (370-bp XmmI fragment) from rat liver binds readily to the isolated proteins under a hypotonic condition (50 mM NaCl) and that the level of the binding affinity for each protein was lowered with increasing NaCl concentration. The sedimentation analysis predicted that direct interaction between the XmnI fragment and P123 or P130 results in the formation of a complex which consists of two of the fragments and one molecule of the protein, alternatively, one of the fragment and three molecules of the proteins. Distamycin A, an antibiotic which binds specifically to AT-rich DNA, removed the bend in the XmnI fragment and inhibited binding of the fragment to P123 or P130, whereas neither removal of the bend nor binding inhibition was observed with chromomycin A3, an antibiotic specific for GC-rich sites in DNA. These results imply that AT-rich regions in a highly repetitive DNA component cause bending of the helix axis to be recognized by some of nuclear scaffold proteins.

Involvement of phosphorylation in binding of nuclear scaffold proteins from rat liver to a highly repetitive DNA component.

The results of our previous work [Hibino et al., Biochim. Biophys. Acta 1174 (1993) 162-170] suggested that a highly repetitive DNA component facilitates bending of the helix axis to be recognized by the nuclear scaffold proteins from rat liver, P123 and P130. In the present experiment, it was shown that binding of these proteins to such a repetitive DNA component from rat liver nuclei (370-bp XmnI fragment) is based on a cooperative mode of interaction, although the binding activity of P130 is much higher than that of P123. The immunoblot analysis with anti-phosphoamino acid antibodies suggested that phosphorylation of serine and threonine residues occurs on P123 and P130, but also of tyrosine residue(s) on P130. The phosphatase assay showed that phosphoryl groups on these proteins may be involved in altering the DNA binding activities of the proteins. Thus, the results in the present study imply that phosphorylation of a nuclear scaffold protein in addition to the degree of bending of the DNA helix axis plays an important role in anchoring chromatin to the nuclear scaffold and in construction of a higher-order chromatin structure.

Cloning and nucleotide sequencing of phenylalanine dehydrogenase gene of Bacillus sphaericus.

The gene coding for phenylalanine dehydrogenase [PDH; L-phenylalanine: NAD+ oxidoreductase (deaminating); EC 1.4.1.-] from Bacillus sphaericus SCRC-79a was cloned onto plasmid pUC9, and the nucleotide sequence of the 2-kb DNA region of the insert was determined. A 1143-bp open reading frame consisting of 381 codons was identified as a pdh gene coding for PDH.

An endodeoxyribonuclease activity in nuclei from rat-ascites hepatoma.

The DNA in nuclei from rat-ascites hepatoma (AH) was rather resistant to endogenous endonucleolytic attack (autodigestion), compared with that in nuclei from normal rat liver (RL). In contrast, by micrococcal nuclease, the DNA in AH nuclei was cleaved in the same manner as in RL nuclei. A 0.6 M NaCl extract was prepared from RL or AH nuclei and subjected to Sephadex G-100 filtration. The resulting-nuclease fraction was separated further into two nuclease fractions, I and II, by CM-Sephadex column chromatography. The activity ratio of II to I was 7.1 for the RL and 2.0 for the AH nuclei. Moreover, the activity of fraction II from the AH nuclei was rather low, compared with that from the RL nuclei. Regenerating-liver nuclei from the normal rat were also assayed in the same way. The results obtained were very similar to those from the AH nuclei. In addition, each of fractions, I and II, cleaved pBR322 DNA of superhelical form; in other words, each had endonucleolytic ability.

Binding affinities of highly repetitive DNA components for a nuclear scaffold protein from rat-ascites hepatoma cells.

Separate samples of a self-ligated tandem dimer of a highly repetitive DNA component (369-bp HindIII fragment) from rat-ascites hepatoma nuclei were digested with different restriction enzymes that cleave only once in the monomer. The resulting 369-bp sequence-permuted monomers showed anomalously slow gel electrophoretic mobility. Of them, the XmnI fragment had the slowest mobility. This suggests that bending of the helix axis is the strongest in this fragment. Our previous work has shown that such a repetitive bent DNA has selective affinities for two nuclear scaffold proteins from rat liver that have molecular weights of 123,000 and 130,000 Hibino et al. (1992) Biochem. Biophys. Res. Commun., 184, 853-858; Hibino et al. (1993) Biochim. Biophys. Acta, 1174, 162-170). In the present experiment, it has been found that the nuclear scaffold fraction from rat-ascites hepatoma cells does not contain these proteins, but does have a repetitive bent DNA-binding protein that has a molecular weight of about 230,000. These results imply that there is some difference in the structure of nuclear DNA attachment region between rat liver and the hepatoma.

Anticarcinogenic actions of water-soluble and alcohol-insoluble fractions from culture medium of Lentinus edodes mycelia.

The water-soluble (LEM) and alcohol-insoluble (LAP and LAP1) fractions were prepared from the culture medium of Lentinus edodes mycelia which was composed of bagasse and rice bran. LEM suppressed rat hepatocarcinogenesis and its cell proliferation of rat-ascites hepatoma to about 50% or less of each control group. LAP also suppressed cell proliferation at almost the same rate. LAP1 induced many small cells in the ascites and significantly raised the survival rate of hepatoma-bearing rats. Thus, anticarcinogenic action was revealed in LAP or LAP1 fractions which were mainly composed of xylose-containing polysaccharide and protein.

Base sequences of highly repetitive components in nuclear DNAs from rat liver and rat-ascites hepatoma.

A 370-bp highly repetitive component in each of the nuclear DNAs from rat liver (RL) and rat-ascites hepatoma (AH) was isolated by HindIII digestion and cloned in pUC9. Ten of the resulting clones were arbitrarily selected and sequenced. Heterogeneity of size was found in 7 of the RL clones (366-369 bp), but in only 2 of the AH clones (369 bp). The sequence homology was 64.6% among the RL clones; 80.3% among the AH clones. The base compositions were AT-rich, ranging from 61.1% to 64.7%. Many A and/or T runs consisting of 2-5 bases were interspersed throughout each sequence. The restriction sites reported previously, EcoRI, HaeIII, HindIII, HinfI and HphI sites, were confirmed in almost all of the clones. In the present experiment, 12 kinds of the sites were further found in both RL and AH clones.

Anticarcinogenic action of an alcohol-insoluble fraction (LAP1) from culture medium of Lentinus edodes mycelia.

From the culture medium of Lentinus edodes mycelia, water-soluble material (LEM) was prepared and further fractionated by alcohol precipitation and gel filtration on Sepharose 6B. The resulting fraction of xylose-rich proteoglycan at the void volume was designated as LAP1. The 25% and 50% survival rates of hepatoma-bearing rats were raised by intraperitoneal (i.p.) administration of LAP1 at doses of 3-10 mg/kg (an optimum dose, 3 mg/kg). This fraction did not suppress in vitro cell proliferation of the hepatoma. Moreover, the i.p. administration of LAP1 significantly augmented the activity of macrophage-migration inhibition of the splenic cells from hepatoma-bearing rats in the early stage after transplantation. Thus, the anticarcinogenic action of LAP1 would partly be interpreted by host-dependent immunomodulation.

Enhancement of excision repair of cisplatin-DNA adducts by cell-free extract from a cisplatin-resistant rat cell line.

To characterize the enhanced repair synthesis of defined DNA lesions, oligodeoxyribonucleotides were synthesized and inserted into plasmid DNA. The inserted plasmid DNA was treated with cis-diamminedichloroplatinum(II) (cisplatin) and subjected to in vitro DNA repair assay with soluble extract from the rat liver cell line Ac2F. All cisplatin adducts tested stimulated DNA repair synthesis. Moreover, two cisplatin-resistant cell lines, Ac2F-CR4 and Ac2F-CR10, were established by stepwise exposure of Ac2F cells to this drug. The DNA repair synthesis was enhanced 3- to 4-fold in the extract from cisplatin-resistant Ac2F cells relative to that from Ac2F cells. Such repair synthesis was suppressed by the specific DNA polymerase inhibitor aphidicolin. The results of the present study suggested that the enhanced repair activity induced by a cisplatin adduct can be detected by in vitro DNA repair assay with soluble cell extract.

Continuous treatment with nicotine increases diazepam binding inhibitor (DBI) and its mRNA in the mouse brain.

Effect of chronic treatment with nicotine on DBI and its mRNA in mouse cerebral cortex were examined. Continuous treatment of mice with nicotine significantly increased DBI content and its mRNA expression, which was completely abolished by simultaneous administration of mecamylamine (1 mg/kg, i.p.). These results indicate that chronic functional interaction between nicotine and nicotinic acetylcholine receptors has a critical role in increases in DBI content and its mRNA expression.

Removal of hydroxyl radical facilitates Ca2+-dependent [3H]GABA release by peroxynitrite.

We investigated mechanisms for enhancement of peroxynitrite (OONO-; 5 microM)-evoked [3H] gamma-aminobutyric acid (GABA) release. Hydroxyl radical scavengers such as N,N'-dimethylthiourea (DMTU), mannitol, and uric acid, significantly increased OONO--evoked [3H]GABA release, whereas urea showed no effects on the release. Removal of Ca2+ from incubation buffer abolished the enhancement of the release by DMTU, although DMTU showed no effects on the basal release with and without Ca2+ in extracellular space. These results indicate that hydroxyl radical scavengers facilitate OONO--evoked [3H]GABA release dependent on Ca2+.

Functional involvement of benzodiazepine receptors in ethanol-induced increases of diazepam binding inhibitor (DBI) and its mRNA in the mouse brain.

We have attempted to clarify the mechanisms for alcohol (EtOH)-induced elevation of diazepam binding inhibitor (DBI) mRNA and to investigate whether the increase in DBI mRNA is paralleled with that in DBI using EtOH-treated mice and primary cultured neurons. Both the DBI content and the expression of DBI mRNA were elevated in the cerebral cortex of EtOH-inhaled and -withdrawn mice. Simultaneous administration of flunitrazepam (FLN) and Ro15-1788 with EtOH vapor completely abolished the EtOH-induced elevation of DBI mRNA. In addition, the exposure of the neurons for 3 days significantly elevated the expression of DBI mRNA, which was completely inhibited by concomitant exposure of FLN, Ro15-4513 and Ro-15-1788 with EtOH, while muscimol and bicuculline showed no effects on the EtOH-induced increase of DBI mRNA expression. These results indicate that functional interaction between EtOH and benzodiazepine (BDZ) receptors is a critical role in the increased expression of DBI mRNA.

Multiple actions of nitric oxide on voltage-dependent Ca2+ channels in mouse cerebral cortical neurons.

We investigated the effects of nitric oxide (NO) on voltage-dependent Ca2+ channels (VDCCs) by examining [45Ca2+]influx into mouse cerebral cortical neurons. S-nitroso-N-acetylpenicillamine (SNAP) induced a dose-dependent increase in [45Ca2+]influx, which was completely abolished by hemoglobin, tetrodotoxin and dibucaine. The NO-induced [45Ca2+influx was significantly inhibited by verapamil and omega-agatoxin VIA (omega-AGX), whereas omega-conotoxin GVIA (omega-CTX) had no effects on the NO-induced [45Ca2+]influx. KCl (30 mM) stimulated [45Ca2+]influx, and verapamil, omega-CTX and omega-AGX reduced the KCl-induced [45Ca2+]influx by about 40, 26 and 34%, respectively, indicating that the neurons used here possess L-, N- and P-typed VDCCs. SNAP itself reduced KCl-induced [45Ca2+]influx by about 28.5%. In the presence of both KCl and SNAP, omega-CTX showed no effects on the influx, while verapamil and omega-AGX significantly inhibited the influx and the concomitant presence of verapamil and omega-AGX completely abolished the influx. These results indicate that NO induces [45Ca2+] influx via the opening of L- and P-typed VDCCs subsequent to neuronal membrane depolarization and that NO itself inhibited the function of N-typed VDCC in the cerebral cortical neurons.