Imaging data on characterization of retinal autofluorescent lesions in a mouse model of juvenile neuronal ceroid lipofuscinosis (CLN3 disease).

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease), a lethal pediatric neurodegenerative disease without cure, often presents with vision impairment and characteristic ophthalmoscopic features including focal areas of hyper-autofluorescence. In the associated research article "Loss of CLN3, the gene mutated in juvenile neuronal ceroid lipofuscinosis, leads to metabolic impairment and autophagy induction in retinal pigment epithelium" (Zhong et al., 2020) [1], we reported ophthalmoscopic observations of focal autofluorescent lesions or puncta in the Cln3Δex7/8 mouse retina at as young as 8 month old. In this data article, we performed differential interference contrast and confocal imaging analyses in all retinal layers to localize and characterize these autofluorescent lesions, including their spectral characteristics and morphology. We further studied colocalization of these autofluorescent lesions with the JNCL marker mitochondrial ATP synthase F0 sub-complex subunit C and various established retinal cell type markers.

Loss of CLN3, the gene mutated in juvenile neuronal ceroid lipofuscinosis, leads to metabolic impairment and autophagy induction in retinal pigment epithelium.

Juvenile neuronal ceroid lipofuscinosis (JNCL, aka. juvenile Batten disease or CLN3 disease) is a lysosomal storage disease characterized by progressive blindness, seizures, cognitive and motor failures, and premature death. JNCL is caused by mutations in the Ceroid Lipofuscinosis, Neuronal 3 (CLN3) gene, whose function is unclear. Although traditionally considered a neurodegenerative disease, CLN3 disease displays eye-specific effects: Vision loss not only is often one of the earliest symptoms of JNCL, but also has been reported in non-syndromic CLN3 disease. Here we described the roles of CLN3 protein in maintaining healthy retinal pigment epithelium (RPE) and normal vision. Using electroretinogram, fundoscopy and microscopy, we showed impaired visual function, retinal autofluorescent lesions, and RPE disintegration and metaplasia/hyperplasia in a Cln3 ~ 1 kb-deletion mouse model [1] on C57BL/6J background. Utilizing a combination of biochemical analyses, RNA-Seq, Seahorse XF bioenergetic analysis, and Stable Isotope Resolved Metabolomics (SIRM), we further demonstrated that loss of CLN3 increased autophagic flux, suppressed mTORC1 and Akt activities, enhanced AMPK activity, and up-regulated gene expression of the autophagy-lysosomal system in RPE-1 cells, suggesting autophagy induction. This CLN3 deficiency induced autophagy induction coincided with decreased mitochondrial oxygen consumption, glycolysis, the tricarboxylic acid (TCA) cycle, and ATP production. We also reported for the first time that loss of CLN3 led to glycogen accumulation despite of impaired glycogen synthesis. Our comprehensive analyses shed light on how loss of CLN3 affect autophagy and metabolism. This work suggests possible links among metabolic impairment, autophagy induction and lysosomal storage, as well as between RPE atrophy/degeneration and vision loss in JNCL.

Percutaneous transhepatic choledochoscopic lithotomy as a rescue therapy for removal of bile duct stones in Billroth II gastrectomy patients who are difficult to perform ERCP.

BACKGROUND: Endoscopic retrograde cholangiopancreatography (ERCP) is more difficult and dangerous in patients with Billroth II (B II) gastrectomy than those with normal anatomy. OBJECTIVES: To evaluate the clinical efficacy of percutaneous transhepatic choledochoscopic lithotomy (PTCSL) for removing common bile duct stones in B II gastrectomy patients who are difficult to perform ERCP. METHODS: This study was designed as prospectively uncontrolled in a large tertiary referral center. In 20 patients, mostly men, with bile duct stones and an earlier B II gastrectomy, PTCSL was tried because of failed ERCP and high risk. The PTCSL was performed using electrohydraulic lithotripsy or papillary balloon dilation. Successful stone removal and complications were measured. RESULTS: Stone removal was achieved in all 20 patients. The mean number of procedures and session time were 4.5 and 45 min, respectively. Minor PTCSL-related complications, such as fever, hemobilia, hyperamylasemia, and wound pain, occurred in five patients (25%). There were no major procedure-related complications, including perforation or mortality. CONCLUSION: The PTCSL procedure is an effective and safe rescue therapy for common bile duct stones in B II gastrectomy patients with failed ERCP and high risk.

Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p.

We present an example of expression and purification of a biologically active G-protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR alpha-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5% n-dodecyl maltoside (DM). Approximately 120 microg of purified alpha-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (K(d)) of the purified alpha-factor receptor in DM micelles was 28 nM as compared to K(d)=12.7 nM for Ste2p in cell membranes, and approximately 40% of the purified receptor was correctly folded as judged by ligand saturation binding. About 50% of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the alpha-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

The coffee diterpene kahweol suppress the inducible nitric oxide synthase expression in macrophages.

Excessive nitric oxide production by inducible nitric oxide synthase (iNOS) in stimulated inflammatory cells is thought to be a causative factor of cellular injury in cases of inflammation. In recent studies, it has been shown that kahweol, coffee-specific diterpene, exhibit chemoprotective effects. In this study, we investigated the effects of kahweol on the production of and the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. The nitrite production induced by LPS was markedly reduced in a dose-dependent manner. In addition, kahweol suppressed the expression of iNOS protein and iNOS mRNA. Since iNOS transcription has been shown to be under the control of the transcription factor, NF-kappaB, the effects of kahweol on NF-kappaB activation were examined. Transient transfection experiments showed that kahweol inhibited NF-kappaB-dependent transcriptional activity. Moreover, electrophoretic mobility shift assay experiments indicated that kahweol blocked the LPS-induced activation of NF-kappaB. The results of these studies suggest that the suppression of the transcriptional activation of iNOS by kahweol might be mediated through the inhibition of NF-kappaB activation. Taken together, the results of our study provide evidence that kahweol possess an anti-inflammatory potential, which constitutes a previously unrecognized biologic activity, and which may provide new insights into the inflammatory process.

Suppressive effects of the kahweol and cafestol on cyclooxygenase-2 expression in macrophages.

Inducible cyclooxygenase-2 (COX-2) has been suggested to play a role in the processes of inflammation and carcinogenesis. Recent studies have shown the chemoprotective effects of kahweol and cafestol, which are coffee-specific diterpenes. This study investigated the effects of kahweol and cafestol on the expression of COX-2 in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Kahweol and cafestol significantly suppressed the LPS-induced production of prostaglandin E(2), COX-2 protein and mRNA expression, and COX-2 promoter activity in a dose-dependent manner. Furthermore, kahweol blocked the LPS-induced activation of NF-kappaB by preventing IkappaB degradation and inhibiting IkappaB kinase activity. These results will provide new insights into the anti-inflammatory and anti-carcinogenic properties of kahweol and cafestol.

Induction of inducible nitric oxide synthase and proinflammatory cytokines expression by o,p'-DDT in macrophages.

Macrophages are crucial for the inflammatory response process because they release a number of proinflammatory mediators. 1,1,1 -Trichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl)ethane (o,p'-DDT) has been reported to possess immunomodulatory properties activity. However, its influence on cytokine production or the functions of the macrophages remains unclear. This study investigated the effects of o,p'-DDT on the production of nitric oxide (NO) and proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha) and analyzed the molecular mechanism in mouse macrophages. The addition of o,p'-DDT to macrophages induced NO and proinflammatory cytokines production in a dose-dependent manner. o,p'-DDT also increased inducible nitric oxide synthase (iNOS) and proinflammatory cytokines expression levels in the cells. NF-kappaB sites were identified in the promoter of the iNOS and proinflammatory cytokines genes. Pretreating the cells with NF-kappaB pathway inhibitors suppressed the iNOS and proinflammatory cytokines expression induced by o,p'-DDT. The transient expression and electrophoretic mobility shift assays with the NF-kappaB binding sites revealed that the o,p'-DDT-induced increase in the iNOS and proinflammatory cytokines expression level were mediated by the NF-kappaB transcription factor. However, pretreating the cells with o,p'-DDT and the NF-kappaB pathway inhibitors suppressed DDT-induced NF-kappaB activation. These results demonstrate that o,p'-DDT stimulates the production of NO and proinflammatory cytokines and can up-regulate the gene expression levels via NF-kappaB transactivation. Overall, the results of this study suggest evidence that o,p'-DDT might possess an inflammatory potential that is previously unrecognized immunomodulating activity of o,p'-DDT.

Suppressive effects of Platycodon grandiflorum on the progress of carbon tetrachloride-induced hepatic fibrosis.

The suppressive effects of Platycodi Radix (Changkil: CK), the root of Platycodon grandiflorum A. DC (Campanulaceae), on the progress of acute carbon tetrachloride (CCl4)-induced hepatic fibrosis were investigated in the rat. CK significantly suppressed CCl4-induced hepatic necrosis and inflammation, as determined by the serum enzymatic activities of alanine and aspartate aminotransferase and serum tumor necrosis factor-alpha levels, in dose-dependent manners. In addition, the increased hepatic fibrosis after acute CCl4 treatment was suppressed by the administration of CK. CK also significantly prevented the elevation of hepatic alpha1 (I) procollagen (type I collagen) mRNA and alpha-smooth muscle actin (alpha-SMA) expressions in the liver of CCl4-intoxicated rats and also suppressed the induction of alpha-SMA and type I collagen in cultured hepatic stellate cells, in dose-dependent manners. These results suggest that the suppressive effects of CK against the progress of acute CCl4-induced hepatic fibrosis possibly involve mechanisms related to its ability to block both hepatic inflammation and the activation of hepatic stellate cells.