Adipose tissue-organotypic culture system as a promising model for studying adipose tissue biology and regeneration

Adipose tissue consists of mature adipocytes, preadipocytes and mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. We have recently developed “adipose tissue-organotypic culture system” that maintains unilocular structure, proliferative ability and functions of mature adipocytes for a long term, using three-dimensional collagen gel culture of the tissue fragments. In this system, both preadipocytes and MSCs regenerate actively at the peripheral zone of the fragments. Our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome. Thus, it seems to be a promising model for investigating adipose tissue biology and regeneration. In this article, we introduce adipose tissue-organotypic culture, and propose two theories regarding the mechanism of tissue regeneration that occurs specifically at peripheral zone of tissue fragments in vitro.Key words: adipose tissue-organotypic culture, three-dimensional, tissue fragments, peripheral zone, central zone, mature adipocytes, preadipocytes (immature adipocytes), mesenchymal stem cells, adipokines, tissue regeneration     

Leucine Dehydrogenase from Corynebacterium pseudodiphtheriticum: Purification and Characterization

Leucine dehydrogenase [EC 1.4.1.9] was purified to homogeneity from Corynebacterium pseudodiphtheriticum ICR 2210. The enzyme consisted of a single polypeptide with a molecular weight of about 34,000. Stepwise Edman degradation provided the N-terminal sequence of the first 24 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 2 amino acids. Although the enzyme catalyzed the reversible deamination of various branched-chain l-amino acids, l-valine was the best substrate for oxidative deamination at pH 10.9 and the saturated concentration. The enzyme, however, had higher reactivity for l-leucine, and the k_cat/Km value for l-leucine was higher than that for l-valine. The enzyme required NAD⁺ as a natural coenzyme. The NAD⁺ analogs 3-acetylpyridine-NAD⁺ and deamino-NAD⁺ were much better coenzymes than NAD ⁺. The enzyme activity was significantly reduced by sulfhydryl reagents and pyridoxal 5′-phosphate. d-Enantiomers of the substrate amino acids competitively inhibited the oxidation of l-valine.     

Reduced Renal α-Klotho Expression in CKD Patients and Its Effect on Renal Phosphate Handling and Vitamin D Metabolism

Renal α-Klotho (α-KL) plays a fundamental role as a co-receptor for fibroblast growth factor 23 (FGF23), a phosphaturic hormone and regulator of 1,25(OH)₂ vitamin D₃ (1,25VitD₃). Disruption of FGF23-α-KL signaling is thought to be an early hallmark of chronic kidney disease (CKD) involving reduced renal α-KL expression and a reciprocal rise in serum FGF23. It remains unclear, however, whether the rise in FGF23 is related to the loss of renal α-KL. We evaluated α-KL expression in renal biopsy samples and measured levels of several parameters of mineral metabolism, as well as soluble α-KL (sKL), in serum and urinary samples from CKD patients (n = 236). We found that although renal α-KL levels were significantly reduced and serum FGF23 levels were significantly elevated in early and intermediate CKD, serum phosphate levels remained within the normal range. Multiple regression analysis showed that the increases in FGF23 were significantly associated with reduced renal function and elevated serum phosphate, but were not associated with loss of renal α-KL. Moreover, despite falling renal α-KL levels, the increase in FGF23 enhanced urinary fractional excretion of phosphate and reduced serum 1,25VitD₃ levels in early and intermediate CKD, though not in advanced CKD. Serum sKL levels also fell significantly over the course of CKD, and renal α-KL was a significant independent determinant of sKL. These results demonstrate that FGF23 levels rise to compensate for renal failure-related phosphate retention in early and intermediate CKD. This enables FGF23-α-KL signaling and a neutral phosphate balance to be maintained despite the reduction in α-KL. In advanced CKD, however, renal α-KL declines further. This disrupts FGF23 signaling, and serum phosphate levels significantly increase, stimulating greater FGF23 secretion. Our results also suggest the serum sKL concentration may be a useful marker of renal α-KL expression levels.     

DNAX-activating Protein 10 (DAP10) Membrane Adaptor Associates with Receptor for Advanced Glycation End Products (RAGE) and Modulates the RAGE-triggered Signaling Pathway in Human Keratinocytes

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes.     

A radioimmunoassay for human pro-luteinizing hormone-releasing factor [pro-LRF(14-69)OH]

A radioimmunoassay (RIA) for human pro-LRF(14-69)OH was developed with an antiserum, generated in a rabbit, to [Tyr67]pro-LRF(47-67)NH2 conjugated to BSA. This antiserum bound 28-32% of [125I]pro-LRF(14-69)OH at a final dilution of 1:2500 and the binding was inhibited by pro-LRF(14-69)OH in a dose-dependent manner. The sensitivity of the RIA was 31.2-62.5 pg and the dose that inhibited 50% of the binding to the tracer was 280-320 pg. Intra- and inter-assay coefficients of variation at 50% inhibition were 8 and 12%, respectively. Neither LRF nor pro-LRF(14-37)OH was recognized by the antiserum. The dilution curve generated with human hypothalamic extract was parallel to that of pro-LRF(14-69)OH. In addition the extract yielded a major immunoreactive peak emerging in elution volumes concordant with [125I]pro-LRF(14-69)OH on Sephadex G-50 chromatography.     

Genetic divergence between and within two color morphotypes of <Emphasis Type="Italic">Parapercis sexfasciata</Emphasis> (Perciformes: Pinguipedidae) from Tosa Bay,southern Japan

The anterior half of the mitochondrial DNA control region (mtCR) sequence (ca. 400 base pairs) was compared between two color morphotypes (A, B) of Parapercis sexfasciata from Tosa Bay, southern Japan, using 16 and 21 specimens, respectively. Intramorphotypic mtCR divergences were only 0.0–0.5% and 1.0–2.5% for morphotypes A and B, respectively. In contrast, intermorphotypic mtCR divergence was much greater, 12.7–14.0%. Furthermore, phylogenetic analysis using a neighbor-joining algorithm, with P. multifasciata as an outgroup, showed that each morphotype was reciprocally monophyletic. These results and the distinct coloration and overlapping distribution indicate that the two color morphotypes of P. sexfasciata represent two distinct species. Mismatch distribution analysis suggested that both morphotypes had undergone population expansion; however, estimates of initial population sizes and mutational timescales suggested that morphotype B comprises historically larger and older populations than morphotype A.