Title: Glyoxal fixation: An approach to solve immunohistochemical problem in neuroscience research Konno K, Yamasaki M, Miyazaki T, Watanabe M Ref: Sci Adv, 9:eadf7084, 2023 : PubMed
The gold-standard fixative for immunohistochemistry is 4% formaldehyde; however, it limits antibody access to target molecules that are buried within specialized neuronal components, such as ionotropic receptors at the postsynapse and voltage-gated ion channels at the axon initial segment, often requiring additional antigen-exposing techniques to detect their authentic signals. To solve this problem, we used glyoxal, a two-carbon atom di-aldehyde. We found that glyoxal fixation greatly improved antibody penetration and immunoreactivity, uncovering signals for buried molecules by conventional immunohistochemical procedures at light and electron microscopic levels. It also enhanced immunosignals of most other molecules, which are known to be detectable in formaldehyde-fixed sections. Furthermore, we unearthed several specific primary antibodies that were once judged to be unusable in formaldehyde-fixed tissues, allowing us to successfully localize so far controversial synaptic adhesion molecule Neuroligin 1. Thus, glyoxal is a highly effective fixative for immunostaining, and a side-by-side comparison of glyoxal and formaldehyde fixation is recommended for routine immunostaining in neuroscience research.
RATIONALE: Only few cases of myasthenia gravis (MG) associated with small-cell lung cancer (SCLC) have been reported, and cases positive for acetylcholine receptor antibody (AChR-ab) are even rarer. The efficacy of standard MG treatment, such as cholinesterase inhibitor therapy, immunosuppressive therapy using steroids and immunosuppressive drugs, plasma exchange, and intravenous immune globulin (IVIg), for these cases is unclear. PATIENT CONCERNS AND DIAGNOSES: A 71-year-old man complained of bilateral eyelid ptosis. He also presented with dysphagia and masticatory muscle fatigue after chewing. The edrophonium test was positive, and the serum AChR-ab level was increased; therefore, the patient was diagnosed with MG. Computed tomography scan showed a nodule on the left upper lobe of the lung and mediastinal lymphadenopathy. Further examination revealed the lesion as SCLC. Finally, he was diagnosed with AChR-ab-positive MG associated with SCLC. INTERVENTIONS AND OUTCOMES: Oral pyridostigmine and tacrolimus were administered to treat MG; however, his symptoms worsened. Therefore, methylprednisolone and IVIg were administrated, which temporarily improved his symptoms. However, they remained uncontrolled. Meanwhile, chemotherapy with carboplatin and etoposide was administered to treat his SCLC. The lesions shrunk, and the MG symptoms and serum AChR-ab level also improved. LESSONS: AChR-ab-positive MG may develop as a comorbidity of SCLC. In such cases, management might require treatment for SCLC in addition to the standard MG treatment to stabilize the MG symptoms.
The majority of systemic sclerosis (SSc) patients have gastrointestinal tract involvement, but therapies of prokinetic agents are usually unsatisfactory. Patients are often compromised by the use of steroid; therefore, a surgical indication including fundoplication has been controversial. There is no report that advanced SSc with severe gastroesophageal reflux disease (GERD) is successfully treated with acotiamide, which is the acetylcholinesterase (AChE) inhibitor designed for functional dyspepsia (FD). We report a 44-year-old woman of SSc with severe GERD successfully treated with acotiamide. She had received medical treatment in our hospital since 2003. She had been aware of the significant gastroesophageal reflux symptoms (e.g., heartburn, chest pain, and dysphagia) due to the development of esophageal hardening associated with SSc since 2014. As a result of upper gastrointestinal series, upper gastrointestinal endoscopy, and 24-h pH monitoring and frequency scale for the symptoms of the GERD (FSSG) scoring, she has been diagnosed with GERD associated with SSc. First of all, she started to take prokinetic agents Rikkunshito and mosapride and proton pump inhibitor; there was no change in reflux symptoms. So, we started to prescribe her the acotiamide.After oral administration started, reflux symptoms have been improved. Five months after oral administration, FSSG score, a questionnaire for evaluation of the symptoms of GERD, was improved. Since its introduction of acotiamide, the patient has kept free from symptoms for 6 months.
BACKGROUND: A significant problem to be solved for patients after liver transplantation (LT) is malnutrition with anorexia in the early posttransplant period. We hypothesized that this problem was due to the change in ghrelin metabolism during LT. The aim of this study was to examine the balance of acyl ghrelin (AG) and desacyl ghrelin and the dependence of the regulation mechanism on hepatic-related enzymes in patients during LT. MATERIALS AND METHODS: AG, desacyl ghrelin, and acyl/total ghrelin (A/T) concentrations in blood samples were measured in 15 patients with liver failure (LF), 15 patients after LT, and 10 controls. The correlations between the participants' ghrelin profiles and hepatic function-related data, including liver enzymes, were evaluated. In vitro assays using synthetic AG for assessment of deacylation activity in serum were performed. RESULTS: AG and A/T ratio were significantly higher in the LF patients than the patients after LT and controls (AG: 25.9 +/- 12.6 versus 16.4 +/- 12.6 and 9.8 +/- 7.6 fmol/mL, P < 0.05; A/T ratio: 17.4 +/- 4.1 versus 12.2 +/- 5.5 and 11.8% +/- 5.9%, P < 0.05). The serum cholinesterase level was inversely correlated with AG and A/T ratio (P < 0.01). In vitro assays showed that deacylation activity was significantly lower in patients with LF than controls (10.5% versus 42.4%, 90 min; P < 0.01). Degradation of AG was partially suppressed by a cholinesterase inhibitor. CONCLUSIONS: Deacylation activity was lower in LF patients, which could cause elevation of AG levels. Serum cholinesterase may be responsible for deacylation in humans.
Title: Lack of molecular-anatomical evidence for GABAergic influence on axon initial segment of cerebellar Purkinje cells by the pinceau formation Iwakura A, Uchigashima M, Miyazaki T, Yamasaki M, Watanabe M Ref: Journal of Neuroscience, 32:9438, 2012 : PubMed
The axon initial segment (AIS) of cerebellar Purkinje cells (PCs) is embraced by ramified axons of GABAergic basket cells (BCs) called the pinceau formation. This unique structure has been assumed to be a device for the modulation of PC outputs through electrical and/or GABAergic inhibition. Electrical inhibition is supported by enriched potassium channels, absence of sodium channels, and developed septate-like junctions between BC axons. The neurochemical basis for GABAergic inhibition, however, has not been well investigated. Here we addressed this issue using C56BL/6 mice. First, we confirmed previous observations that typical synaptic contacts were rare and confined to proximal axonal portions, with the remaining portions being mostly covered by astrocytic processes. Then we examined the expression of molecules involved in GABAergic signaling, including GABA synthetic enzyme glutamic acid decarboxylase (GAD), vesicular GABA transporter vesicular inhibitory amino acid transporter (VIAAT), cytomatrix active zone protein bassoon, GABA receptor GABA(A)Ralpha1, and cell adhesion molecule neuroligin-2. These molecules were recruited to form a functional assembly at perisomatic BC-PC synapses and along the AIS of hippocampal and neocortical pyramidal cells. GAD and VIAAT immunogold labeling was five times lower in the pinceau formation compared with perisomatic BC terminals and showed no accumulation toward the AIS. Moreover, bassoon, neuroligin-2, and GABA(A)Ralpha1 formed no detectable clusters along the ankyrin-G-positive AIS proper. These findings indicate that GABAergic signaling machinery is organized loosely and even incompletely in the pinceau formation. Together, BCs do not appear to exert GABAergic synaptic inhibition on the AIS, although the mode of action of the pinceau formation remains to be explored.
BACKGROUND: A single-nucleotide polymorphism (SNP), G994T, in the lipoprotein-associated phospholipase A(2) (Lp-PLA(2)) gene is known to have a potent influence on the activity of the enzyme. As this enzyme hydrolyzes oxidized low-density lipoprotein (oxLDL), which is an important player in atherogenesis, the present study evaluated effects of the G994T genotype on the oxLDL level as well as on intima media thickness (IMT) in vivo. METHODS: Participants of a health examination (1,307 in total) were recruited from two rural communities in Shimane, Japan. Genotyping was performed by an allele-specific PCR and the TaqMan method. The oxLDL level was determined by an enzyme immunoassay. RESULTS: The minor allele (994T) frequency (0.19) in the studied populations was consistent with previous reports on Japanese. The 994T allele increased the plasma oxLDL/LDL ratio in a recessive manner, whereas 994T had a codominant effect on the Lp-PLA(2) activity. A multivariate analysis revealed that age and the G994T genotype had independent effects on the oxLDL/LDL level. By contrast, the G994T genotype was not associated with IMT. All of these results were reproducible in the two independent populations studied. CONCLUSIONS: G994T influenced plasma LDL oxidation. Further studies on the effect of this polymorphism in cardiovascular diseases are warranted.
Title: Cloning and physical mapping of the EcoRI fragments of the giant linear plasmid SCP1. Redenbach M, Ikeda K, Yamasaki M, Kinashi H Ref: Journal of Bacteriology, 180:2796, 1998 : PubMed
Title: Molecular cloning of a gene, DHS1, which complements a drug- hypersensitive mutation of the yeast Saccharomyces cerevisiae. Lee YS, Shimizu J, Yoda K, Yamasaki M Ref: Biosci Biotechnol Biochem, 58:391, 1994 : PubMed
