Actin filament organization of foot processes in vertebrate glomerular podocytes

We investigated the actin filament organization and immunolocalization of actin-binding proteins (α-actinin and cortactin) in the podocyte foot processes of eight vertebrate species (lamprey, carp, newt, frog, gecko, turtle, quail, and rat). Three types of actin cytoskeleton were found in these foot processes. (1) A cortical actin network with cortactin filling the space between the plasma membrane and the other actin cytoskeletons described below was found in all of the species examined here. The data indicated that the cortical actin network was the minimal essential actin cytoskeleton for the formation and maintenance of the foot processes in vertebrate podocytes. (2) An actin bundle with α-actinin existing along the longitudinal axis of foot process above the level of slit diaphragms was only observed in quail and rat. (3) An actin fascicle consisting of much fewer numbers of actin filaments than that of the actin bundle was observed in the species other than quail and rat, but at various frequencies. These findings suggest that the actin bundle is an additional actin cytoskeleton reflecting a functional state peculiar to quail and rat glomeruli. Considering the higher intraglomerular pressure and the extremely thin filtration barrier in birds and mammals, the foot processes probably mainly protect the thinner filtration barrier from the higher internal pressure occurring in quail and rat glomeruli. Therefore, we consider that the actin bundle plays a crucial role in the mechanical protection of the filtration barrier. Moreover, the actin fascicle may be a potential precursor of the actin bundle.     

Enzymatic oxidation of dipyridamole in homogeneous and micellar solutions in the horseradish peroxidase-hydrogen peroxide system

Enzymatic oxidation of dipyridamole (DIP) by horseradish peroxidase-hydrogen peroxide system (HRP-H2O2) in aqueous and micellar solutions was carried out. The reaction was monitored by optical absorption and fluorescence techniques. In aqueous solution at pH 7.0 and pH 9.0, the disappearance of the characteristic bands of DIP centered at 400 nm and 280 nm was observed. A new strong band at 260 nm is observed for the oxidation product(s) with shoulders at 322 nm and 390 nm. A non-fluorescent product is formed upon oxidation. In cationic cethyl trimethyl-1-ammonium chloride (CTAC) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonium) propane sulfonate (HPS) micellar solutions the same results are observed: three, well-defined, isosbestic points in the optical spectra suggest the transformation between two species. In anionic micellar sodium dodecylsulfate solution (SDS), the appearance of a new band centered around 506 nm was observed, associated to a solution color change from the usual yellow to deep blue/violet, characteristic of a radical species associated to the one-electron oxidation of DIP to its cation radical (DIP*+), observed previously in electrochemical oxidation. Experiments of radical decay kinetics monitoring the absorbance change at 506 nm were performed and analyzed in the frame of a kinetic model taking into account the species both in homogeneous and micellar media. The reaction medium is composed of bulk solution, SDS micelle/solution interface and enzyme catalytic site(s). The variation of DIP*+ concentration was analyzed assuming: (1) synthesis of DIP*+ by HRP through one-electron oxidation; (2) decomposition of DIP*+ by further one-electron oxidation; (3) direct two-electron oxidation of DIP by HRP; (4) bimolecular DIP*+ disproportionation. The main results of the analysis are as follows: (1) kinetic data can be divided in two phases, an HRP active phase and another phase which proceeds in the absence of enzyme activity due to consumption of all H2O2; (2) the reactions of DIP*+ formation, DIP*+ decomposition and DIP two-electron oxidation are HRP concentration dependent; (3) since DIP*+ formation constant seems to be overestimated, it is proposed that two-electron oxidation is another source of DIP*+, through the comproportionation reaction. Evidences for this reaction were also observed previously in electrochemical experiments; and (4) the kinetic analysis provides evidences that the bimolecular reaction of DIP*+ takes place mainly in the absence of active HRP and in this phase the combination of, at least, two second-order kinetic processes is needed to model the experimental data. Our data suggest that HRP oxidizes DIP in general by a two-electron process or that the cation radical is very unstable so that the one-electron process is only detected in the presence of anionic surfactant, which stabilizes significantly the DIP*+ intermediate.     

Molecular characterization of mitocalcin, a novel mitochondrial Ca2+-binding protein with EF-hand and coiled-coil domains

Here we have identified and characterized a novel mitochondrial Ca2+-binding protein, mitocalcin. Western blot analysis demonstrated that mitocalcin was widely expressed in mouse tissues. The expression in brain was increased during post-natal to adult development. Further analyses were carried out in newly established neural cell lines. The protein was expressed specifically in neurons but not in glial cells. Double-labeling studies revealed that mitocalcin was colocalized with mitochondria in neurons differentiated from 2Y-3t cells. In addition, mitocalcin was enriched in the mitochondrial fraction purified from the cells. Immunohistochemical studies on mouse cerebellum revealed that the expression pattern of mitocalcin in glomeruli of the internal granular and molecular layers was well overlapped by the distribution pattern of mitochondria. Immunogold electron microscopy showed that mitocalcin was associated with mitochondrial inner membrane. Overexpression of mitocalcin in 2Y-3t cells resulted in neurite extension. Inhibition of the expression in 2Y-3t cells caused suppression of neurite outgrowth and then cell death. These findings suggest that mitocalcin may play roles in neuronal differentiation and function through the control of mitochondrial function.     

Quantitative screening of insect cell transformants stably expressing GFPuv-ß 1,3-N-acetylglucosaminyltransferase 2 fusion protein

Insect cell transformants, stably expressing human ß1,3-N-acetylglucosaminyltransferase 2 (ß3GnT2) as the green fluorescent protein (GFP_uv)-fused protein, were efficiently isolated on Western blot by the quantification of the densitometric intensity of the fusion protein. From almost 150 transformants containing the fusion gene linked to three different types of signal sequence, two transformants, Tn-pXme4a and-pX28a, were successfully selected, showing 8.3 and 8.6 mU/mL ß3GnT activity, respectively. This method requires a screening time almost one-half that required in the isolation of stably transformed cells with high expression levels, and at the same time allows the handling a large number of transformants.     

Individual radiation exposure dose due to support activities at safe shelters in Fukushima Prefecture

Immediately after the accidents in the nuclear power stations in Fukushima on March 11, the Japanese Government ordered the evacuation of the residents within a 20-km radius from the station on March 12, and asked various institutions to monitor the contamination levels of the residents. Hirosaki University, which is located 355 km north of Fukushima City, decided to send support staff to Fukushima. This report summarizes the results of the exposure of 13 individual teams from March 15 to June 20. The support teams surveyed more than 5,000 people during this period. Almost all subjects had external contamination levels of less than 13 kcpm on Geiger-Müller (GM) survey meter, which is categorized as "no contamination level." The 1(st) team showed the highest external exposure dose, but the 4(th) team onward showed no significant change. Subsequently, the internal radiation exposure was measured using a whole body counter that indicated undetectable levels in all staff members. Although the measured external radiation exposure dose cannot have serious biological effects on the health of an individual, a follow-up study of the residents in Fukushima and other regions where the radioactive material has spread will be required for a long time.     

Multicolor-based discrimination of 21 short tandem repeats and amelogenin using four fluorescent universal primers

The aim of this study was to develop a cost-effective genotyping method using high-quality DNA for human identification. A total of 21 short tandem repeats (STRs) and amelogenin were selected, and fluorescent fragments at 22 loci were simultaneously amplified in a single-tube reaction using locus-specific primers with 24-base universal tails and four fluorescent universal primers. Several nucleotide substitutions in universal tails and fluorescent universal primers enabled the detection of specific fluorescent fragments from the 22 loci. Multiplex polymerase chain reaction (PCR) produced intense FAM-, VIC-, NED-, and PET-labeled fragments ranging from 90 to 400 bp, and these fragments were discriminated using standard capillary electrophoretic analysis. The selected 22 loci were also analyzed using two commercial kits (the AmpFLSTR Identifiler Kit and the PowerPlex ESX 17 System), and results for two loci (D19S433 and D16S539) were discordant between these kits due to mutations at the primer binding sites. All genotypes from the 100 samples were determined using 2.5 ng of DNA by our method, and the expected alleles were completely recovered. Multiplex 22-locus genotyping using four fluorescent universal primers effectively reduces the costs to less than 20% of genotyping using commercial kits, and our method would be useful to detect silent alleles from commercial kit analysis.     

ROLE OF ENCYSTMENT AND EXCYSTMENT OF PERIDINIUM BIPES F. OCCULATUM (DINOPHYCEAE) IN FRESH WATER RED TIDES IN LAKE KIZAKI,JAPAN1

The encystment flux of Peridinium bipes f. occulatum (Dinophyceae) was investigated with sediment traps from 1968 to 1990 in Lake Kazki. Cysts of P. bipes were formed throughout the blooms, Encystment flux of P. bipes in the pelagic zone was usually lower than those at shallow sites, and the density of P. bipes cysts in lake sediment was higher in the shallow region than in the pelagic zone. However, in the shallower region, The concentration of P. bipes cysts varied widely, possibly due to high rates of encystment and excystment. Peridinium bipes encystment occurred between 15° and 25° C in the laboratory, with very little cyst formation below 10°C. Though cyst formation was observed in continous darkness, the rate increased with irradiance. Under continuous darkness, no excystment was observed at any temperature from 5° to 25° C. Eighty-one percent of the cysts illuminated at 105 μE m^?2 s^?1 excysted after 13 days incubation at 15° C, and lower irradiances decreased germination success. Results from laboratory experiments suggest that light is a critical factor in the germination of P. bipes cysts. Bottom depth thus can have a significant effect on germination because cysts only can excyst from depths where light is sufficient. The shallow region of the lake is thus very important as a seed bed for P. bipes during early spring. Cyst deposited in deeper waters may not ever germinate unless they are resuspended and transported to shallow areas where light reaches the bottom.     

Behavior of cell aggregate of Carthamus tinctorius L. cultured cells and correlation with red pigment formation

The formation processes of Carthamus tinctorius cell aggregates in a growth medium and the correlation of red pigment formation with cell aggregate sizes were investigated. About 80% of cell aggregates in the growth medium were > 1.00 mm in size. The growth rate of large cell aggregates was more rapid than that of small cell aggregates. Most cell aggregates > 0.50 mm in size became larger or smaller than their original sizes during the culture. A high level of red pigment formation was observed when cell aggregates obtained by the preculture using cell aggregates < 1.00 mm in size were cultured in the production medium.     

Reduced albumin reabsorption in the proximal tubule of early-stage diabetic rats

The aim of this study is to investigate the role of the proximal tubule in microalbuminuria in the early stage of diabetic nephropathy. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (50 mg/kg, i.v.). After 2 weeks, albumin delivery in the proximal tubule was measured using micropuncture and the endocytosis process of FITC-labeled albumin was evaluated with immunoelectron microscopy. Albumin was significantly reabsorbed in the proximal convoluted tubule (PCT) of controls (0.39+/-0.05 ng/min at early PCT to 0.17+/-0.08 at late PCT, P<0.05), whereas albumin reabsorption was inhibited in diabetic rats (0.27+/-0.05 to 0.21+/-0.08). Immunogold study revealed that FITC-albumin was significantly less reabsorbed in endosomes and lysosomes of S1 segments in diabetic rats than in controls (endosome: 1.20+/-0.10 vs 2.16+/-0.15 microm-1, P<0.0001; lysosome: 0.26+/-0.03 vs 0.83+/-0.07, P<0.0001). The expression of megalin, an endocytosis receptor, was decreased at the apical membrane of PCT in diabetic rats. The lipid peroxidation production in the proximal tubule was significantly increased in diabetic rats. In conclusion, albuminuria in early-stage diabetic rats can be partly explained by a decreased albumin endocytosis with reduced megalin expression and with increased lipid peroxidation in the proximal tubule.