Endogenous neurotoxic dopamine derivative covalently binds to Parkinson's disease‐associated ubiquitin C‐terminal hydrolase L1 and alters its structure and function

Parkinson's disease (PD) is a common neurodegenerative disease, but its pathogenesis remains elusive. A mutation in ubiquitin C‐terminal hydrolase L1 (UCH‐L1) is responsible for a form of genetic PD which strongly resembles the idiopathic PD. We previously showed that 1‐(3′,4′‐dihydroxybenzyl)‐1,2,3,4‐tetrahydroisoquinoline (3′,4′DHBnTIQ) is an endogenous parkinsonism‐inducing dopamine derivative. Here, we investigated the interaction between 3′,4′DHBnTIQ and UCH‐L1 and its possible role in the pathogenesis of idiopathic PD. Our results indicate that 3′,4′DHBnTIQ binds to UCH‐L1 specifically at Cys152 in vitro. In addition, 3′,4′DHBnTIQ treatment increased the amount of UCH‐L1 in the insoluble fraction of SH‐SY5Y cells and inhibited its hydrolase activity to 60%, reducing the level of ubiquitin in the soluble fraction of SH‐SY5Y cells. Catechol‐modified UCH‐L1 as well as insoluble UCH‐L1 were detected in the midbrain of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐treated PD model mice. Structurally as well as functionally altered UCH‐L1 have been detected in the brains of patients with idiopathic PD. We suggest that conjugation of UCH‐L1 by neurotoxic endogenous compounds such as 3′,4′DHBnTIQ might play a key role in onset and progression of idiopathic PD.

     

Production and Accumulation of Xylooligosaccharides with Long Chains by Growing Culture and Xylanase of a Mutant Strain of Bacillus pumilus X-6-19

Bacillus pumilus X-6-9 isolated from soil and subsequently identified, produced xylooligosacchatides with long chainsfrom xylan and accumulated them in the culture. By improving the culture conditions and mutating the bacterium, a 3.2-fold increasein the production of the xylooligosaccharides was established, when compared to the original culture conditions of B. pumilus X-6-19.The addition of D-glucose to the culture of the mutant swain U-3 of B. pumilus X-6-9 repressed the synthesis of β-xylosidase, but notxylanase. Thus, it was revealed that strain U-3 was a good organism for the production and accumulation of xylooligosaccharideswith long chains from xylan by a microbial culture. Xylanase produced by strain U-3 was purified to homogeneity and characterized.The hydrolyzates generated by the purified xylanase contained xylobiose, xylotrinse, xylotewaose, and xylopentaose, but not xylose.     

Isolation and characterization of thermotolerant bacterium utilizing ammonium and nitrate ions under aerobic conditions

A thermotolerant bacterium, identified as Bacillus licheniformis, completely utilized 0.1% (w/v) NH₄NO₃ at 30 and 50°C under aerobic condition. The addition of 0.5 mM Fe²⁺ to the NH₄NO₃ medium markedly promoted the utilization of NH₄⁺ and NO₃⁻. At 50°C, of total nitrogen originally provided, 24% was taken up into the cells and 20% remained in the culture supernatant. Residual nitrogen (56%) was probably removed into the atmosphere. The cell extracts contained enzymes involved in denitrification. GC-MS demonstrated that NH₄ ¹⁵NO₃ had been converted to ¹⁵N₂O. These results indicate that the strain has denitrification ability under aerobic condition.     

In vitro phosphorylation of the tumor suppressor gene RB protein by mitosis-specific histone H1 kinase

The major components of the mitosis-specific histone H1 kinase are CDC2 kinase and cyclin and the consensus amino acid sequence for phosphorylation by this enzyme has been proposed. We have noted the presence of such sequences in six sites of the tumor suppressor gene RB protein and determined whether or not RB protein is in fact phosphorylated by this kinase. Highly purified enzyme was used for this purpose. HeLa cell extracts immunoprecipitated with anti-RB antiserum as well as RB proteins expressed in E. coli cells were shown to be phosphorylated by this kinase in vitro. Synthetic peptides for the six expected sites were also phosphorylated. These results suggest the possibility that the function of RB protein is regulated by CDC2 kinase.     

Progression of Oral Squamous Cell Carcinoma Accompanied with Reduced E-Cadherin Expression but Not Cadherin Switch

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.     

Suprainguinal ectopic scrota of TS inbred rats

A new rat line (TS inbred rats) showing uni- or bilateral ectopic scrota in about 70% of the males was established. Ectopic scrota were elongated pouches of the thin muscle layer under the suprainguinal body skin, in which hypoplastic testes and epididymides were seen. Genetic analysis revealed that the ectopic scrotum was controlled by multiple genes. Groups of 5 normal, uni- and bilaterally affected rats were killed at 25 weeks of age. Histologically, ectopic testes of uni- and bilaterally affected rats showed arrest of spermatogenesis at the primary spermatocyte stage. Contralateral testes of unilaterally affected rats were normally descended and showed normal spermatogenesis. No abnormality was seen in the urinary system. Plasma concentrations of FSH were significantly elevated in bilaterally affected rats but plasma concentrations of testosterone and LH were unchanged in uni- and bilaterally affected rats. Pituitary contents of LH and FSH were significantly elevated in bilaterally affected rats. The endocrinological status of TS inbred rats was therefore similar to that of experimentally induced cryptorchid rats.     

Localization of synapsin I in normal fibers and regenerating axonal sprouts of the rat sciatic nerve

The localization of synapsin I, a synaptic vesicle-associated protein, was investigated immunocytochemically in normal nerve fibers and regenerating axonal sprouts following crush-injuries to the rat sciatic nerve. In normal myelinated axons, weak synapsin I immunoreactivity was found in the axoplasmic/smooth endoplasmic domains, but not in the cytoskeletal domains comprising neurofilaments and microtubules. In non-myelinated axons without dense cytoskeletal structures, moderate immunoreactivity was distributed diffusely throughout the axoplasm. In the crush-injured nerves, intense synapsin I immunoreactivity was demonstrated by light microscopy in early regenerating sprouts emerging from nodes of Ranvier. These nodal sprouts subsequently elongated as regenerating axons through the space between the basal lamina and the myelin sheath (or Schwann cell plasma membrane). Intense synapsin I immunoreactivity was also found in the growth cones of such long regenerating axons. Electron microscopy revealed that synapsin I immunoreactivity was associated mainly with vesicular organelles in the nodal sprouts and growth cones of regenerating axons. Long regenerating axons exhibited no synapsin I immunoreactivity in the shaft, which contained an abundance of neurofilaments. However, vesicle accumulations remaining in the periphery of the shaft still exhibited intense synapsin I immunoreactivity. Thus, it can be concluded that synapsin I is localized at especially high density in the domains comprising vesicular organelles, which are characteristic of early nodal sprouts, as well as in growth cones of regenerating axons. These findings, together with the proposed functions of synapsin I investigated in other studies, suggest that synapsin I may play important roles in vesicular dynamics including the translocation of vesicles to the plasma membrane in sprouts and growth cones of regenerating axons.     

Use of a surface plasmon resonance biosensor for the determination of binding constants of transiently expressed recombinant antibody to its antigen

Summary By using a commercially available surface plasmon resonance (SPR) biosensor, the values of the association rate constant (kass), dissociation rate constant (kdiss), and association constant (KA = kass / kdiss) for binding to the antigens were determined. They were almost the same for the recombinant antibody expressed in COS cells, CHO cells, and mouse hybridoma cells. The system of transient expression of the recombinant antibody (Ab) in COS cells and SPR analysis of the supernatant should be useful for rapid expression and evaluation of the binding ability of large numbers of engineered Abs.     

Visceral Fat Accumulation,Insulin Resistance,and Elevated Depressive Symptoms in Middle-Aged Japanese Men

Objective

To investigate visceral fat accumulation and markers of insulin resistance in relation to elevated depressive symptoms (EDS).

Methods

Participants were 4,333 male employees (mean age, 49.3 years) who underwent abdominal computed tomography scanning, measured fasting insulin, and did not self-report diabetes and mental disorders under treatment and history of cancer, myocardial infarction, and stroke. Multivariable logistic regression was used to assess the association of EDS with abdominal fat deposition and markers of insulin resistance.

Results

Visceral fat area (VFA) and fasting insulin were significantly, positively associated with EDS. Multivariable-adjusted odds ratios (95% confidence interval) of high VFA for the lowest through highest quartile of depression score were 1 (reference), 1.18 (0.97–1.42), 1.25 (1.02–1.54), 1.23 (1.01–1.51), respectively, and corresponding figures for high fasting insulin were 1 (reference), 0.98 (0.80–1.19), 1.12 (0.91–1.38), and 1.29 (1.06–1.57), respectively. Subcutaneous fat area was not associated with EDS.

Conclusions

Results suggest that EDS is related to visceral, but not subcutaneous, fat accumulation and insulin resistance in middle-aged Japanese men.