Method to Improve Azo-Compound (AAPH)-Induced Hemolysis of Erythrocytes for Assessing Antioxidant Activity of Lipophilic Compounds.

We examined the method of oxidative hemolysis for assessment of antioxidant activity of various compounds, especially lipophilic compounds. 2,2'-Azobis(amidinopropane) dihydrochloride (AAPH) was used as the source of free radicals for the oxidative hemolysis of horse erythrocytes. We found that absorbance at 540 nm is not appropriate for monitoring AAPH-induced hemolysis. Instead, we should use absorbance at 523 nm (an isosbestic point), because AAPH oxidizes the oxygenated hemoglobin to methemoglobin and absorbance at 540 nm does not correctly reflect the amount of released hemoglobin by AAPH-induced hemolysis. The corrected method of AAPH-induced hemolysis was applicable to assess the antioxidant activity of various hydrophilic compounds such as ascorbic acid, (-)-epicatechin, and edaravone. For the assessment of antioxidant activity of lipophilic compounds, we need appropriate dispersing agents for these lipophilic compounds. Among several agents tested, 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC) liposome at a concentration of 0.34 mM was found to be useful. Exogenous α-tocopherol incorporated using DMPC liposome as a dispersing agent was shown to protect erythrocytes from AAPH-induced hemolysis in a concentration-dependent manner.

Intrinsic cell permeability of the GAGA zinc finger protein into HeLa cells.

We examined the intrinsic cell permeability of a GAGA zinc finger obtained from the Drosophila melanogaster transcription factor and analyzed its mechanism of cellular uptake using confocal microscopy and flow cytometry. HeLa cells were treated with the Cy5-labeld GAGA peptides (containing a fluorescent chromophore) to detect fluorescence signals from the fluorescent labeling peptides by confocal microscopy. The results clearly indicated that GAGA peptides possess intrinsic cell permeability for HeLa cells. Based on the results of the flow cytometry analysis and the theoretical net positive charge of the GAGA peptides, the efficiency of cellular uptake of the GAGA peptides was predicted to depend on the net positive charge of the GAGA peptide as well as the cationic component ratio of Arg residues to Lys residues.

Possible role of interferon tau on the bovine corpus luteum and neutrophils during the early pregnancy.

When pregnancy is established, interferon tau (IFNT), a well-known pregnancy recognition signal in ruminants, is secreted by embryonic trophoblast cells and acts within the uterus to prepare for pregnancy. IFNT acts as an endocrine factor on the corpus luteum (CL) to induce refractory ability against the luteolytic action of PGF2 α. Hypothesising that IFNT may influence not only the uterine environment but also the CL in cows via local or peripheral circulation, we investigated qualitative changes in the CL of pregnant cows during the maternal recognition period (day 16) and the CL of non-pregnant cows. The CL of pregnant animals had a higher number of neutrophils, and the expression of interleukin 8 (IL8) mRNA and its protein was higher as well as compared with the CL of non-pregnant animals. Although IFNT did not affect progesterone (P4) secretion and neutrophil migration directly, it stimulated IL8 mRNA expression on luteal cells (LCs), influencing the neutrophils, resulting in the increased migration of IFNT-activated neutrophils. Moreover, both IFNT-activated neutrophils and IL8 increased P4 secretion from LCs in vitro. Our novel finding was the increase in neutrophils and IL8 within the CL of pregnant cows, suggesting the involvement of IFNT function within the CL toward establishment of pregnancy in cows. The present results suggest that IFNT upregulates neutrophil numbers and function via IL8 on LCs in the CL of early pregnant cows and that both neutrophils and IL8, stimulated by IFNT, are associated with an increase in P4 concentrations during the maternal recognition period in cows.

Transcriptional regulation in the early ectodermal lineage of ascidian embryos.

In ascidian embryos, ectodermal tissues derive from blastomeres in the animal hemisphere. The animal hemisphere-specific gene expression is observed as early as the 16-cell stage. Here, we characterized animal hemisphere-specific enhancers of three genes, Ci-ephrin-Ad, Ci-TGFß-NA1 and Ci-Fz4. Deletion analyses identified minimal essential elements. Although these elements contained multiple GATA sequences, electrophoretic mobility shift assays revealed that only some of them were strong binding sites for the transcription factor Ci-GATAa. On the other hand, the motif-searching software MEME identified an octamer, GA (T/G) AAGGG, shared by these enhancers. In Ci-ephrin-Ad and Ci-TGFß-NA1, the octamer was GATAAGGG, which strongly bound Ci-GATAa. The 397-bp upstream region of Ci-ephrin-Ad contained two strong Ci-GATAa-binding sites, one of which was the octamer motif. Mutation in the octamer motif, but not the other Ci-GATAa-binding site, severely affected the enhancer activity. The 204-bp upstream region of Ci-TGFß-NA1 contained four strong Ci-GATAa-binding sites, including the octamer motif. Mutation only in the octamer motif, leaving the other three Ci-GATAa-binding sites intact, abolished the enhancer activity. These results suggest a crucial role for the octamer motif.

Clinical Features of Syphilis Patients with Ocular Symptoms as the Initial Manifestation.

Purpose: This study aimed to investigate the clinical features of patients with syphilis having ocular symptoms as the initial manifestation. Patients and Methods: Eleven patients diagnosed with ocular syphilis at Dokkyo Medical University Hospital and Jichi Medical University Hospital between November 2018 and April 2022 were studied retrospectively. Results: Six patients were diagnosed with secondary, three were latent, and one was tertiary stage syphilis. Ten out of 11 patients underwent cerebrospinal fluid analysis, and 1 refused. Nine out of 10 patients tested positive, of which 4 presented with neurological symptoms and the others were asymptomatic. Nine out of 11 patients tested negative for human immunodeficiency virus. Antiluetic therapy was administered to 10 out of 11 patients, which improved or maintained visual acuity at -0.1 logMAR in 9 patients. One patient achieved the best-corrected visual acuity -0.1 logMAR in one eye, whereas the other showed no improvement due to severe chorioretinal degeneration. Conclusion: Ocular syphilis presents with various clinical findings and has no significant ocular manifestations without acute syphilitic posterior placoid chorioretinitis. Patients diagnosed with syphilis based on ocular symptoms should undergo cerebrospinal fluid analysis.

Upregulation of interferon-stimulated genes and interleukin-10 in peripheral blood immune cells during early pregnancy in dairy cows.

In cows, interferon-tau (IFNT) regulates maternal recognition around days 15-19 after artificial insemination (AI). The present study hypothesized that if key target genes of IFNT are clearly upregulated in earlier stages of pregnancy, these genes could be use as indices of future pregnancy in cows. Therefore, we determined the expression of these genes in peripheral blood mononuclear leukocytes (PBMCs) and polymorphonuclear granulocytes (PMNs) during the maternal recognition period (MRP). Twenty multiparous Holstein cows were subjected to AI on day 0 and categorized into the following groups: pregnancy (Preg, n = 9), embryonic death (ED, n = 5) and non-pregnancy (NP, n = 6). Progesterone levels in the Preg group were higher than those in the NP group on days 12-21. ISG15 and OAS-1 (IFN-stimulated genes: ISGs) mRNA in PBMCs on day 8 was higher in the Preg group than in the NP group, and these mRNAs in PMNs was higher in the Preg group on day 5 than in the NP and ED groups. Interleukin-10 (IL-10, Th2 cytokine) mRNA expression increased on day 8 in the PBMCs of pregnant cows. Tumor necrosis factor α (TNFα, Th1 cytokine) mRNA expression was stable in all groups. In an in vitro cell culture experiment, IFNT stimulated mRNA expression of ISGs in both PBMCs and PMNs. IFNT stimulated IL-10 mRNA expression in PBMCs, whereas IFNT increased TNFα mRNA levels in PBMCs in vitro. The results suggest that ISGs and IL-10 could be responsive to IFNT before the MRP in peripheral blood immune cells and may be useful target genes for reliable indices of pregnancy before the MRP.

Combination of delirium and coma predicts psychiatric symptoms at twelve months in critically ill patients: A longitudinal cohort study.

PURPOSE: We aimed to determine any associations between delirium and comas during intensive care unit (ICU) stay, and long-term psychiatric symptoms and disability affecting activity of daily living (ADL). MATERIALS AND METHODS: In this prospective observational study, we enrolled critically ill adult patients that were emergently admitted to an ICU. We assessed psychiatric symptoms and disability affecting ADL at three and twelve months after ICU discharge. RESULTS: Among the 81 and the 47 patients that responded to the questionnaires at three and twelve months, 22 (27%) and 13 (28%) patients experienced delirium, respectively. During their ICU stay, 28 (35%) and 21 (45%) had been in comas, respectively. At three and twelve months, 51 (63%) and 23 (49%) of patients experienced composite psychiatric symptoms or disability affecting ADL, respectively. After adjusting predefined confounders, the combination of delirium and comas was an independent risk factor for the presence of composite psychiatric symptoms or disability affecting ADL (adjusted odds ratio [aOR] 3.38; 1.10-10.38 at three months; aOR 8.28; 1.48-46.46 at twelve months). CONCLUSIONS: In critically ill adults, combination of delirium and comas during ICU stay is a predictor of psychiatric symptoms or ADL disability. TRIAL REGISTRATION: UMIN Clinical Trial Registry no. UMIN000023743, September 1, 2016.

Development of enzyme immunoassay for chromogranin A (CgA) and profiling of plasma CgA concentrations in the cow.

The objectives of this study were to establish and characterize a homologous immunoassay for bovine chromogranin A (bCgA) and to profile plasma bCgA concentrations during early pregnancies. We synthesized oligopeptide corresponding to the amino acid sequence 341-355 of bCgA for immunizing rabbits and peptide corresponding to the amino acid sequence 336-365 of bCgA for both a biotinylated tracer and reference standards. Recombinant bCgA protein was also generated in Escherichia coli lysate. Dose-dependent displacement curves were obtained from 1 to 1,000 nM of the reference standards. The displacement curves showed a good relationship between the reference standards of the synthetic peptide and the serially diluted plasma sample or recombinant bCgA protein generated in the present study. The assay sensitivity defined as the value of two standard deviations below the zero standard was calculated as 0.46 nM. The intraassay and interassay coefficients of variation were 6.48% and 13.4%, respectively. Changes in the plasma bCgA concentrations in early pregnancies undulated in nonpregnant animals. The results of the present study suggest that assaying plasma bCgA concentrations could be utilized as measures to evaluate the physiological status of cattle.

The effect of standard and high dose of rikkunshito on achievement of enteral nutrition target in critically ill patients: a pilot randomized controlled trial.

AIM: Rikkunshito is a traditional Japanese medicine used for delayed gastric emptying in intensive care units in Japan. This study aimed to investigate whether standard- or high-dose rikkunshito can improve the achievement of enteral calorie target among critically ill adults. METHODS: This open-label, single-center, pilot randomized controlled trial was carried out from March 2018 until December 2018 and enrolled critically ill adult patients requiring enteral nutrition by gastric tube for at least 5 days. Patients were randomized into the control group, the standard-dose rikkunshito group (2.5 g three times daily), and the high-dose rikkunshito group (5 g three times daily). Intervention was given for 5 days. The primary outcome measure was the percentage of enteral calorie intake achieved in the target at the fifth day after randomization. RESULTS: The cohort comprised 26 patients; of these, 9, 8, and 9 were included in the control group, the standard-dose group, and the high-dose group, respectively. Twenty-one patients (81%) were included in the primary analysis. The percentage of enteral calorie intake achieved in the target at the fifth day was 59% (interquartile range [IQR], 39-63%), 40% (IQR, 26-61%), and 62% (IQR, 17-83%) in the control, the standard-dose, and the high-dose groups, respectively (P = 0.42). The number of adverse events did not differ significantly between the groups (control group, 4 [44%]; standard-dose group, 3 [38%]; and high-dose group, 4 [44%], P = 1.00). CONCLUSIONS: Standard- or high-dose rikkunshito did not improve the achievement of enteral calorie target in critically ill adults.

Development of the Timed Re-Insemination (TRI-synch) program re-inseminating 24 days after the initial service in dairy cows.

For the timed re-insemination at the minimal interbreeding interval, cows were treated with a progesterone (P4 )-releasing intravaginal device from Days 13-15 to 21 post-insemination (Day 0 = estrus), followed by plasma P4 assay on Day 23 and then subjected to the Experiments 1 and 2. In Experiment 1, of 18 cows, 6 cows were determined as luteolysis with low (<1 ng/ml) plasma P4 concentrations on Day 23 and ovulated on Days 24 (3 cows), 25 (1 cow), and 26 (1 cow) except a cow affected by ovarian quiescence. In Experiment 2, all cows were treated with GnRH on Day 23. Cows with low (<1 ng/ml) plasma P4 concentrations on Day 23 were diagnosed as non-pregnant and subjected to the re-insemination in the morning of Day 24 irrespective of estrous signs. Of 36 cows, 15 cows were diagnosed as being non-pregnant on Day 23. Fourteen cows of the non-pregnant animals were re-inseminated in the morning of Day 24 irrespective of estrous signs and the pregnancy rate of re-insemination was 36%. The conception rates of initial and re-inseminations were 50% (18/36) and 36% (5/14), respectively. The overall pregnancy rate by adding the rates of initial and re-inseminations was 64% (23/36).

Adolescent rats find repeated Delta(9)-THC less aversive than adult rats but display greater residual cognitive deficits and changes in hippocampal protein expression following exposure.

The current study examined whether adolescent rats are more vulnerable than adult rats to the lasting adverse effects of cannabinoid exposure on brain and behavior. Male Wistar rats were repeatedly exposed to Delta-9-tetrahydrocannabinol (Delta(9)-THC, 5 mg/kg i.p.) in a place-conditioning paradigm during either the adolescent (post-natal day 28+) or adult (post-natal day 60+) developmental stages. Adult rats avoided a Delta(9)-THC-paired environment after either four or eight pairings and this avoidance persisted for at least 16 days following the final Delta(9)-THC injection. In contrast, adolescent rats showed no significant place aversion. Adult Delta(9)-THC-treated rats produced more vocalizations than adolescent rats when handled during the intoxicated state, also suggesting greater drug-induced aversion. After a 10-15 day washout, both adult and adolescent Delta(9)-THC pretreated rats showed decreased social interaction, while only Delta(9)-THC pretreated adolescent rats showed significantly impaired object recognition memory. Seventeen days following their last Delta(9)-THC injection, rats were euthanased and hippocampal tissue processed using two-dimensional gel electrophoresis proteomics. There was no evidence of residual Delta(9)-THC being present in blood at this time. Proteomic analysis uncovered 27 proteins, many involved in regulating oxidative stress/mitochondrial functioning and cytoarchitecture, which were differentially expressed in adolescent Delta(9)-THC pretreated rats relative to adolescent controls. In adults, only 10 hippocampal proteins were differentially expressed in Delta(9)-THC compared to vehicle-pretreated controls. Overall these findings suggest that adolescent rats find repeated Delta(9)-THC exposure less aversive than adults, but that cannabinoid exposure causes greater lasting memory deficits and hippocampal alterations in adolescent than adult rats.

Alcoholism: protein expression profiles in a human hippocampal model.

It is well known that chronic, excessive consumption of alcohol can cause brain damage/structural changes in the regions important for neurocognitive function. Some of the damages are permanent, while others are reversible. Molecular mechanisms underlying alcohol-induced and/or -related brain damage are largely unknown, although it is generally believed that three factors (ethanol, nutritious and hepatic factors) play important roles. Recently, we have been employing a high-throughput proteomics technology to investigate several alcohol-sensitive brain regions from uncomplicated and hepatic cirrhosis-complicated alcoholics to understand the mechanisms of alcohol effects on the CNS at the level of protein expression. The changes of protein expression profiles in the hippocampus of alcoholic subjects were firstly demonstrated using 2D gel electrophoresis-based proteomics. Protein expression profiles identified in the hippocampus of alcoholic subjects were significantly different from those previously identified by our group in other brain regions of the same alcoholic cases, possibly indicating that these different brain regions react differently to chronic alcohol ingestion at the level of protein expression. Identified changes of protein expression associated with astrocyte and oxidative stress may indicate the possibility that increased levels of CNS ammonia and reactive oxygen species induced by alcoholic mild hepatic damage/dysfunction could cause selective damage in astrocytes of the hippocampus. Although our data did not demonstrate any evidence of direct alcohol effects to induce the alteration of protein expression in association with brain damage, high-throughput neuroproteomics approaches have proved to have the potential to dissect the mechanisms of complex brain disorders. Proteomics studies on human hippocampus, an important region for neurocognitive function and psychiatric illnesses (e.g., Alzheimer's disease, alcoholism and schizophrenia) are still sparse, and further investigation is warranted to understand the underlying mechanisms.

CNS proteomes in alcohol and drug abuse and dependence.

Drugs of abuse, including alcohol, can induce dependency formation and/or brain damage in brain regions important for cognition. 'High-throughput' approaches, such as cDNA microarray and proteomics, allow the analysis of global expression profiles of genes and proteins. These technologies have recently been applied to human brain tissue from patients with psychiatric illnesses, including substance abuse/dependence and appropriate animal models to help understand the causes and secondary effects of these complex disorders. Although these types of studies have been limited in number and by proteomics techniques that are still in their infancy, several interesting hypotheses have been proposed. Focusing on CNS proteomics, we aim to review and update current knowledge in this rapidly advancing area.

Myostatin inhibits differentiation of bovine preadipocyte.

We investigated the effect of myostatin on the differentiation of bovine preadipocyte. Stromal-vascular cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, the differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin, and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with myostatin during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. The treatment with myostatin throughout the differentiation period severely suppressed the induction of all differentiation markers. The treatment with myostatin in the early phase of differentiation also suppressed the induction of terminal differentiation markers but three-fold higher dose of myostatin was required for the suppression compared with its treatment throughout the differentiation period. Myostatin treatment reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma mRNA and interfered with the induction of CCAAT/enhancer binding protein (C/EBP) alpha mRNA. We also observed that follistatin stimulates preadipocyte differentiation in the presence of myostatin. These results suggest that myostatin inhibits bovine preadiopocyte differentiation through suppressing PPARgamma and C/EBPalpha mRNA expressions and that follistatin counteracts the suppressive effect of myostatin.

Follistatin rescues the inhibitory effect of activin A on the differentiation of bovine preadipocyte.

We investigated the effect of activin A and follistatin on the differentiation of bovine preadipocytes. Stromal-vascular (SV) cells containing preadipocytes were prepared from perirenal adipose tissue of approximately 30-month-old Japanese Black steers. After confluence, differentiation was induced by 1-methyl-3-isobutyl-xanthine, dexamethasone, insulin and troglitasone for 2 days, and then subsequently cultured for 6 days. The cells were treated with activin A during the induction of differentiation (the early phase of differentiation) or throughout the differentiation period. We measured the terminal differentiation markers such as glycerol-3-phosphate dehydrogenase (GPDH) activity, lipid accumulation, and the expression of adipocyte fatty acid-binding protein mRNA at the end of cultures. Activin A suppressed the induction of all differentiation markers regardless of the duration of treatment. The treatment with activin A also reduced the expression of peroxisome proliferator-activated receptor (PPAR) gamma and CCAAT/enhancer binding protein (C/EBP) alpha mRNAs without affecting the expression of C/EBPbeta mRNA. We also observed that follistatin completely rescued the inhibitory effect of activin A on bovine preadipocyte differentiation. Furthermore, the higher doses of follistatin increased GPDH activity even in the presence of activin A compared with the cells treated with neither activin A nor follistatin. Additionally, the SV cells expressed activin A and myostatin mRNAs. These results suggest that activin A inhibits bovine preadiopocyte differentiation via affecting transcriptional cascade upstream of PPARgamma and C/EBPalpha expressions, and that follistatin suppresses the inhibitory effect of activin A on bovine preadipocyte differentiation. Endogenous activin A and/or myostatin possibly inhibit the differentiation of bovine preadipocytes.