The Role of Kupffer Cells as Mediators of Adipose Tissue Lipolysis.

Kupffer cells (KCs) are the resident macrophages of the liver, and they respond to and counteract metabolic stresses, such as those imposed by high-fat diet feeding in mouse models. However, little is known regarding the role of these cells in maintaining metabolic homeostasis under metabolically normal conditions. In this study, we found that depletion of KCs in vivo led to enhanced lipolysis in adipose tissue by increasing the expression of FGF21, a metabolic regulator, in hepatocytes. IL-1ß secreted from KCs contributed to the suppression of FGF21 expression in hepatocytes. FGF21 overexpression led to a lean phenotype and enhanced lipolysis in mice. KC depletion resulted in a lack of IL-1ß signaling in the liver, leading to elevated expression of FGF21 in hepatocytes. FGF21 promoted lipolysis in adipose tissue and led to hyperlipidemia and decreased body weight. The secretion of IL-1ß in KCs was mediated by bacterial products. Antibiotic treatment also led to enhanced lipolysis. Therefore, the current study identified a physiological role of KCs in the regulation of adipose lipolysis.

A practical approach to enrich intact tryptic N-glycopeptides through size exclusion chromatography and hydrophilicity (SELIC) using an acrylamide-agarose composite gel system.

Increasing researches proved that abnormal glycosylation is strongly correlated with many diseases. Specially, site-specific glycosylation and its associated heterogeneity are closely related to the function and activity of the glycoprotein. However, intact N-glycopeptide analysis still faces great challenges because the presence of highly abundant non-glycosylated peptides would suppress the ionization of lowly abundant glycopeptides. In the present study, we developed a practical intact tryptic N-glycopeptide enrichment method using acrylamide-agarose composite gel that combined the size exclusion chromatography and hydrophilic (named SELIC) effects, aimed to remove the detergent rapidly and effectively, as well as enrich intact N-glycopeptides while extracting peptides. This is a useful tool to facilitate the intact N-glycopeptides analysis of complex protein mixtures, particularly for samples that extracted from formalin-fixed and paraffin-embedded (FFPE) tissues by SDS. Using this method, we successfully identified 700 site-specific intact tryptic N-glycopeptides corresponding to 261 glycosylation sites on 191 glycoproteins from FFPE thymoma tissues.

Clinical features and genetic diagnosis of hereditary spinocerebellar ataxia 3.

Spinocerebellar ataxia type 3 (SCA3) is a rare inherited autosomal dominant progressive neurological disorder, which results from a CAG­repeat expansion in the gene encoding the deubiquitinating enzyme, ataxin­3. At present, no effective treatment is available for this fatal disorder; however, certain studies have suggested that reducing the levels of mutant ataxin­3 protein may reverse or halt the progression of disease in patients with SCA3. In the present study, clinical examinations were performed on a patient with SCA3 who exhibited disease features including coughing, expectoration and was bedridden with mobility limitation. CAG repetitions at SCA­associated genes were detected in the patient's family by performing standard polymerase chain reaction (PCR) and triple­repeat primed PCR. The numbers of CAG­repeats within the two alleles of the gene of interest in the patient were 15 and 78. Notably, the patient's brother, who harbored 76 CAG­repeats in one allele of the gene of interest, did not exhibit severe disease symptoms. These results suggest that the number of CAG­repeats is a critical for determination of SCA3 disease severity and time of onset. In addition, the defined phenotypic characteristics of the patient in the present study provide useful insight for more accurate clinical diagnosis and genotyping of future patients.

[Analysis of the association of human leukocyte antigen DQ gene polymorphisms with unexplained recurrent spontaneous abortion among ethnic Han Chinese from Wenzhou region].

OBJECTIVE: To assess the association of human leukocyte antigen DQ gene polymorphisms with unexplained recurrent spontaneous abortion (URSA) among ethnic Han Chinese from Wenzhou region. METHODS: Fifty couples with URSA (URSA group) and 66 couples with normal pregnancy history (control group) were recruited. The alleles of HLA-DQA1 and HLA-DQB1 were analyzed by polymerase chain reaction with specific sequence primers (PCR-SSP) in all subjects. The frequency distribution of HLA-DQ alleles, odds ratios (OR) between each group and sharing of HLA-DQ alleles were calculated. RESULTS: The frequency distribution of HLA-DQB1*03:03 allele in the females with URSA was significantly higher than that healthy females (21.00% vs. 9.85%, OR=2.433, 95%CI: 1.232-4.894, χ(2)=5.657, P<0.05). The HLA-DQB1*05:03 allele was present among the healthy females with a frequency of 3.03%, and was not detected among females with URSA. For both males and females, the HLA-DQB1*05:02 allele were only typed in control group with frequencies of 6.06% and 5.30%, respectively. The sharing of HLA-DQA1 alleles in couples with URSA was increased compared with the control group (70.27% vs. 44.64%, OR=2.931, 95%CI: 1.216-7.067, P<0.05). CONCLUSION: The increased sharing of HLA-DQA1 alleles may contribute to the susceptibility of URSA among ethnic Han Chinese from Wenzhou region. The allele of HLA-DQB1*03:03 in the females may be predisposing factor for URSA. However, the HLA-DQB1*05:02 allele in both gender and HLA-DQB1*05:03 allele in females may confer a protective effect.

Metallothionein prevents cardiac pathological changes in diabetes by modulating nitration and inactivation of cardiac ATP synthase.

Mitochondrial ATP production is the main energy source for the cell. Diabetes reduces the efficient generation of ATP, possibly due to the inactivation of ATP synthase. However, the exact mechanism by which diabetes induces inactivation of ATP synthase remains unknown, as well as whether such inactivation has a role in the development of pathological abnormalities of the diabetic heart. To address these issues, we used cardiac metallothionein-transgenic (MT-TG) and wild-type (WT) mice with streptozotocin-induced diabetes, since we have demonstrated previously that diabetes-induced cardiac damage and remodeling were found in WT diabetic mice, but not in MT-TG diabetic mice. Immunohistochemical and biochemical assays were used to compare pathological and biochemical changes of the heart between MT-TG and WT diabetic mice, and a proteomic assay to evaluate ATP synthase expression and tyrosine nitration, with its activity. LC/MS analysis revealed that diabetes increased tyrosine nitration of the ATP synthase α subunit at Tyr(271), Tyr(311), and Tyr(476), and the ß subunit at Tyr(269) and Tyr(508), and also significantly reduced ATP synthase activity by ~32%. These changes were not observed in MT-TG diabetic mice. Furthermore, parallel experiments with induced expression of cardiac MT by zinc supplementation in diabetic mice produced similar effects. These results suggest that MT can preserve ATP synthase activity in streptozotocin-induced diabetes, probably through the inhibition of ATP synthase nitration.

An improved protocol for better detection of protein using 8-anilino-1-naphthalenesulfonate.

This study describes the optimum conditions at the staining time and the signal intensity for using 8-anilino-1-naphthalenesulfonate (ANS) as a fluorescent probe to detect proteins in SDS-PAGE. Using the optimized protocol, protein can be easily detected by short time fixing (20 min) and washing (2 × 5 min), followed by 10 min of staining. As low as 1-2 ng of the protein band can be detected, approximately thirty-fold higher than that of the original protocol. Furthermore, the compatibility of the staining method with MS was also explored by comparing the peptide mass fingerprinting results data of serial dilutions of BSA and ovalbumin stained by ANS with SYPRO Ruby.

Metallothionein prevents diabetes-induced cardiac pathological changes, likely via the inhibition of succinyl-CoA:3-ketoacid coenzyme A transferase-1 nitration at Trp(374).

We previously demonstrated that metallothionein (MT)-mediated protection from diabetes-induced pathological changes in cardiac tissues is related to suppression of superoxide generation and protein nitration. The present study investigated which diabetes-nitrated protein(s) mediate the development of these pathological changes by identifying the panel of nitrated proteins present in diabetic hearts of wild-type (WT) mice and not in those of cardiac-specific MT-overexpressing transgenic (MT-TG) mice. At 2, 4, 8, and 16 wk after streptozotocin induction of diabetes, histopathological examination of the WT and MT-TG diabetic hearts revealed cardiac structure derangement and remodeling, significantly increased superoxide generation, and 3-nitrotyrosine accumulation. A nitrated protein of 58 kDa, succinyl-CoA:3-ketoacid CoA transferase-1 (SCOT), was identified by mass spectrometry. Although total SCOT expression was not significantly different between the two types of mice, the diabetic WT hearts showed significantly increased nitration content and dramatically decreased catalyzing activity of SCOT. Although SCOT nitration sites were identified at Tyr(76), Tyr(117), Tyr(135), Tyr(226), Tyr(368), and Trp(374), only Tyr(76) and Trp(374) were found to be located in the active site by three-dimensional structure modeling. However, only Trp(374) showed a significantly different nitration level between the WT and MT-TG diabetic hearts. These results suggest that MT prevention of diabetes-induced pathological changes in cardiac tissues is most likely mediated by suppression of SCOT nitration at Trp(374).

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user-friendly silver stain to meet the needs in high-efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde-based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user-friendly method could be a good choice for LPS visualization on polyacrylamide gels.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y.

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.