The sea urchin major yolk protein is synthesized mainly in the gut inner epithelium and the gonadal nutritive phagocytes before and during gametogenesis

Major yolk protein (MYP), the predominant component of yolk granules in sea urchin eggs, is also contained in the coelomic fluid and nutritive phagocytes of the gonad in both sexes. MYP is stored in ovarian and testicular nutritive phagocytes prior to gametogenesis and is used during gametogenesis as material for synthesizing proteins and other components necessary for eggs and sperm. To reveal the expression profile and the main production site of MYP, we analyzed MYP mRNA expression in immature and maturing Pseudocentrotus depressus. Real‐time reverse‐transcribed polymerase chain reaction analysis showed that MYP mRNA was expressed predominantly in the digestive tract (stomach, intestine and rectum) and the gonad of both sexes. The total amounts of MYP mRNA in the whole digestive tract and in the whole gonad were at similar levels in both immature and maturing sea urchins. MYP mRNA was also detected in white morula cells and vibratile cells separated from the coelomic fluid by density gradient centrifugation, but the expression levels in these cells were very low compared with those in the digestive tract and the gonad. Using in situ hybridization analysis, MYP mRNA was detected in the inner epithelium of the digestive tract and in nutritive phagocytes of the ovary and testis, but was not detected in the germ cells. We conclude that the adult sea urchin has two predominant production sites for MYP regardless of sex and reproductive stage: the inner epithelium of the digestive tract and the nutritive phagocytes of the gonad. Mol. Reprod. Dev. 77: 59–68, 2010. © 2009 Wiley‐Liss, Inc.     

Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

Induction of cell size vesicles from human lymphoma cell lines and their application to drug carriers

Sodium butyrate (NaB) induced the membrane enclosed cell size vesicles from several IgM producing cell lines. We considered the application of the cell-derived vesicles (CDVs) to drug delivery system (DDS) using the lung cancer specific IgM producing AE6 cell line. Microscopic observation showed that the DiI fluorescence labeled AE6 vesicles were incorporated into the lung cancer cell line A549. The anticancer drug, actinomycin D (actD), contained in AE6 and Ramos vesicles decreased the A549 cell viability to 46 and 62% of control without actD, respectively. The cytotoxic effect in AE6 vesicles was superior to that in the Ramos vesicles that have the lung cancer non-specific IgM on their surfaces. However, the result of the Ramos vesicles suggests that the surface molecules other than IgM may interact with the A549 cells. In our method for vesicle production, more specific and abundant antibodies mounted vesicles can be generated by transfection of their genes into cells followed by NaB treatment. These suggest that the CDVs may be useful for the development of a drug carrier for DDS.     

Influence of physicochemical properties of rice flour on oil uptake of tempura frying batter

The physicochemical properties of rice flour and wheat flour influenced the oil uptake of tempura frying batter. Rice flour was better than wheat flour in the overall quality and crispness of the fried tempura batter. Rice flour resisted oil absorption more than wheat flour, and a higher level of apparent starch amylose and higher consistency/breakdown ratio of the pasting properties led to a lower oil uptake of the batter. Super hard EM10 rice showed the highest apparent amylose content and higher consistency/breakdown ratio than the other flour samples, the batter from EM10 revealing the lowest oil content after frying among all the batters examined. The apparent amylose content, consistency/breakdown ratio and oil absorption index are proposed as useful guides for oil absorption when frying from among the physicochemical properties that influence the oil content of fried batter. Our proposal for the "oil absorption index" could be a simple, although not perfect method for estimating the oil content of batter flour.     

Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma

H. Ohsaki, E. Hirakawa, K. Kagawa, M. Nakamura, H. Kiyomoto and R. Haba Value of computer‐assisted quantitative nuclear morphometry for differentiation of reactive renal tubular cells from low‐grade urothelial carcinoma Objective: To assess whether computer‐assisted quantitative morphological parameters can be an effective tool for objectively distinguishing reactive renal tubular cells from low‐grade urothelial carcinoma cells (LG‐UCs) in voided urine. Methods: Nuclear morphometry was performed by a computer‐assisted image analyser system on Papanicolaou‐stained cytological specimens. The circumference of reactive renal tubular cells (n = 40) or LG‐UC (n = 20) nuclei were manually traced, and the following nuclear morphometric parameters were analysed: (i) area, (ii) perimeter, (iii) roundness factor, (iv) maximum length, and (v) linear factor. For each nuclear measurement, we calculated the maximum, minimum, mean and standard deviation. Results: The mean nuclear area and nuclear perimeter were higher in reactive renal tubular cells compared to the LG‐UCs. The mean of roundness and linear factors (reflecting a tendency for the nuclear outline to be regular and oval, respectively) were higher in LG‐UCs compared with reactive renal tubular cells. Among nuclear areas, the nuclear perimeter, roundness factors and maximum length did not show any significant differences between reactive renal tubular cells and LG‐UCs. On the other hand, the linear factor showed a mean higher value among LG‐UCs than reactive renal tubular cells (P = 0.023). Conclusions: Of five quantitative nuclear morphological parameters, only linear factor was statistically significant in differentiating reactive renal tubular cells in renal disease from LG‐UCs.     

Fluorescent analysis of excess electron transfer through DNA

The DNA base stack provides unique features for the efficient long-range charge transfer. For the purpose of investigating excess electron transfer process through DNA, we developed a new method for fluorescence analysis of excess electron transfer based on reductive cleavage of a disulfide bond and a thiol-specific fluorescent probe. Excess electron transfer was detected by monitoring the fluorescence of emissive pyrene monomer generated by the reaction of pyrene maleimides with the cleaved disulfide bond (thiols). Mechanism of reductive cleavage of disulfides through excess electron transfer and subsequent reaction with the fluorescent probes were discussed. This facile and sensitive detection by fluorescence method can be applied for mechanistic study of excess electron transfer.     

Preparation and characterization of novel 4-bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide and the analogous phosphorus heterocycles or phospha sugars

4-Bromo-3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (3c) was first synthesized from 3,4-dimethyl-1-phenyl-2-phospholene 1-oxide (2c) by a bromo-radical substitution reaction occurred at C-4 position by N-bromosuccinimide and 2,2′-azobisisobutyronitrile. The novel phospha sugar analogue 3c exerted high anti-proliferative effect on U937 cells evaluated by MTT in vitro methods and was much more efficient than that of Gleevec®, which is known as a molecule targeting chemotherapeutical agent. The substitution of 2-phospholenes at C-3 and C-4 position with methyl groups as well as 4-bromo substituent suggests a good anti-proliferative effect.     

S-Nitrosylation of Drp1 links excessive mitochondrial fission to neuronal injury in neurodegeneration

Neurons are known to use large amounts of energy for their normal function and activity. In order to meet this demand, mitochondrial fission, fusion, and movement events (mitochondrial dynamics) control mitochondrial morphology, facilitating biogenesis and proper distribution of mitochondria within neurons. In contrast, dysfunction in mitochondrial dynamics results in reduced cell bioenergetics and thus contributes to neuronal injury and death in many neurodegenerative disorders, including Alzheimer’s disease (AD), Parkinson’s disease, and Huntington’s disease. We recently reported that amyloid-β peptide, thought to be a key mediator of AD pathogenesis, engenders S-nitrosylation and thus hyperactivation of the mitochondrial fission protein Drp1. This activation leads to excessive mitochondrial fragmentation, bioenergetic compromise, and synaptic damage in models of AD. Here, we provide an extended commentary on our findings of nitric oxide-mediated abnormal mitochondrial dynamics.