Structural study of the sugar chains of alpha-amylases produced ectopically in tumors

About thirty percent of two alpha-amylases produced from a serous papillary cystadenocarcinoma of the ovarium (case 1) and a bronchioloalveolar adenocarcinoma of the lung (case 2) was glycoproteins containing 1 mol of asparagine-linked sugar chain, respectively. The structures of the sugar moieties were found by sequential enzymatic degradation and methylation analysis to be as follows: [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6(3)][GlcNAc beta 1 leads to 2Man alpha 1 leads to 3(6)]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc and [(Gal beta 1 leads to 4)0 or 1GlcNAc beta 1 leads to 2Man alpha 1 leads to 6][NeuAc alpha 2 leads to 6Gal beta 1 leads to 4GlcNAc beta 1 leads to 2Man alpha 1 leads to 3]Man beta 1 leads to 4GlcNAc beta 1 leads to 4(Fuc alpha 1 leads to 6)GlcNAc. Structures of asparagine-linked sugar chains were the same in the tumors of cases 1 and 2 and were incomplete in comparison with those of the parotid amylase.     

Oligosaccharides of human milk. Isolation and characterization of three new disialyfucosyl hexasaccharides.

Renal mitochondrial 25-hydroxyvitamin D₃-1-hydroxylase (1-hydroxylase) is sensitive to inhibition by 2 × 10^?5m calcium and 5 × 10^?3m phosphate when hydroxylation is supported by either malate or NADPH. This sensitivity to ion inhibition is observed in mitochondria from both vitamin D-deficient and repleted chicks and remains when mitochondria are frozen and thawed or are incubated in a hypotonic medium. The ionophore A23187 inhibits the 1-hydroxylase but partially reverses the inhibition exerted by 2, 5, or 7.5 × 10^?5m calcium. Addition of a kidney soluble cell fraction (37,000g supernatant) to isolated mitochondria did not enhance the 1-hydroxylase activity under conditions of varied substrate concentration, osmolarity of the incubation medium, or mitochondrial washes. It is concluded that a soluble cellular component is not involved in the regulation of the 1-hydroxylase but that intramitochondrial calcium and phosphate may well play a role in its regulation.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Diacylglycerol kinase alpha regulates globular adiponectin-induced reactive oxygen species

It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.     

A novel serum chitinase that is expressed in bovine liver

Chitinases are ubiquitous chitin-fragmenting hydrolases. They are synthesized by a vast array of organisms, including those not composed of chitin. Here, we describe a novel serum chitinase (chitin-binding protein b04, CBPb04), which is expressed in bovine liver. Although CBPb04 is secreted as an endocrine chitinase, it shows higher homology with human gastrointestinal tract exocrine chitinase (AMCase) than with macrophage endocrine chitinase (human chitotriosidase). This suggests that cows have a specific defense against chitin-containing microorganisms. CBPb04 mRNA is expressed in hepatocytes. This is the first report of a hepatogenic mammalian chitinase.     

Fusion of bone marrow-derived cells with renal tubules contributes to renal dysfunction in diabetic nephropathy

Although diabetic nephropathy (DN) is a major cause of end-stage renal disease, the mechanism of dysfunction has not yet been clarified. We previously reported that in diabetes proinsulin-producing bone marrow-derived cells (BMDCs) fuse with hepatocytes and neurons. Fusion cells are polyploidy and produce tumor necrosis factor (TNF)-α, ultimately causing diabetic complications. In this study, we assessed whether the same mechanism is involved in DN. We performed bone marrow transplantation from male GFP-Tg mice to female C57BL/6J mice and produced diabetes by streptozotocin (STZ) or a high-fat diet. In diabetic kidneys, massive infiltration of BMDCs and tubulointerstitial injury were prominent. BMDCs and damaged tubular epithelial cells were positively stained with proinsulin and TNF-α. Cell fusion between BMDCs and renal tubules was confirmed by the presence of Y chromosome. Of tubular epithelial cells, 15.4% contain Y chromosomes in STZ-diabetic mice, 8.6% in HFD-diabetic mice, but only 1.5% in nondiabetic mice. Fusion cells primarily expressed TNF-α and caspase-3 in diabetic kidney. These in vivo findings were confirmed by in vitro coculture experiments between isolated renal tubular cells and BMDCs. It was concluded that cell fusion between BMDCs and renal tubular epithelial cells plays a crucial role in DN.     

Axotomy induces axonogenesis in hippocampal neurons through STAT3

After axotomy of embryonic hippocampal neurons in vitro, some of the axotomized axons lose their identity, and new axons arise and grow. This axotomy-induced axonogenesis requires importin, suggesting that some injury-induced signals are transported via axons to elicit axonogenesis after axotomy. In this study, we show that STAT3 is activated in response to axotomy. Because STAT3 was co-immunoprecipitated with importin β in the axotomized neurons, we suggest that STAT3 is retrogradely transported as molecular cargo of importin α/β heterodimers. Indeed, inhibition of importin α binding with STAT3 resulted in the attenuation of axonogenesis. Silencing STAT3 blocked the axonogenesis, demonstrating that STAT3 is necessary for axotomy-induced axonogenesis. Furthermore, the overexpression of STAT3 enhanced axotomy-induced axonogenesis. Taken together, these results demonstrate that activation and retrograde transport of STAT3 in injured axons have key roles in the axotomy-induced axonogenesis of hippocampal neurons.     

Quantifying sequence and structural features of protein–RNA interactions

Increasing awareness of the importance of protein–RNA interactions has motivated many approaches to predict residue-level RNA binding sites in proteins based on sequence or structural characteristics. Sequence-based predictors are usually high in sensitivity but low in specificity; conversely structure-based predictors tend to have high specificity, but lower sensitivity. Here we quantified the contribution of both sequence- and structure-based features as indicators of RNA-binding propensity using a machine-learning approach. In order to capture structural information for proteins without a known structure, we used homology modeling to extract the relevant structural features. Several novel and modified features enhanced the accuracy of residue-level RNA-binding propensity beyond what has been reported previously, including by meta-prediction servers. These features include: hidden Markov model-based evolutionary conservation, surface deformations based on the Laplacian norm formalism, and relative solvent accessibility partitioned into backbone and side chain contributions. We constructed a web server called aaRNA that implements the proposed method and demonstrate its use in identifying putative RNA binding sites.