Transblot and cytochemical identification of avidin-interacting proteins in mitochondria of cultured cells

Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.     

Protective Effect of Oren-gedoku-to Against Induction of Neuronal Death by Transient Cerebral Ischemia in the C57BL/6 Mouse

We examined the neuroprotective effects of oren-gedoku-to (TJ15), a herbal medicine, after transient forebrain ischemia. Transient forebrain ischemia was induced by occlusion of both common carotid arteries for 15 min in C57BL/6 mice treated with TJ15. In the control ischemic group without TJ15 treatment, histologic examination of brain tissue collected seven days after reperfusion showed death of pyramidal cells in CA2-3 area of the hippocampus, unilaterally or bilaterally. In mice treated with oral TJ15 (845 mg/kg/day) for five weeks, the frequency of ischemic neuronal death was significantly lower. Immunohistochemistry for Cu/Zn-superoxide dismutase (Cu/Zn-SOD) showed strongly reactive astrocytes in the hippocampus of ischemic mice treated with TJ15. Damage to nerve cells by free radicals plays an important role in the induction of neuronal death by ischemia-reperfusion injury. Our results suggest that TJ15 protects against ischemic neuronal death by increasing the expression of Cu/Zn-SOD and suggest that oren-gedoku-to reduces the exposure of hippocampal neurons to oxidative stress.     

Dorsal specification in blastoderm at the blastula stage in the goldfish, Carassius auratus

The teleost dorsoventral axis cannot be morphologically distinguished before gastrulation. Previous studies by the current authors have shown that localized dorsalizing activity in the yolk cell (YC) induces the dorsal tissues in the overlying blastoderm. In order to examine whether or not dorsal blastomeres are committed to their dorsal fate before the gastrula stage, a variety of transplant operations were performed in goldfish blastoderms at the mid- to late-blastula stages. When the blastoderm was cut from the YC, rotated horizontally at 180°, and recombined with the YC, the blastoderm frequently developed two axes, indicating that dorsal blastomeres of the blastula had already acquired the ability to differentiate into the organizer in the absence of dorsalizing signals from the YC. This result was further confirmed by experiments using ventralized embryos in which no dorsal structures formed: the axis formation was frequently observed in the normal blastoderm combined with the ventralized YC at the blastula stage. However, the axes formed in the absence of dorsal information from the YC exhibited a lower dorso-anterior index. Furthermore, the dorsal specification was not stably maintained when the dorsal cells were located far from the YC. These results suggest that the inductive and permissive influence of the YC may be required for the blastoderm to undergo full dorsal differentiation.     

Molecular Dissection of the Rho-associated Protein Kinase (p160ROCK)-regulated Neurite Remodeling in Neuroblastoma N1E-115 Cells

A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho–ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.     

Multiple mating and lifetime reproductive output in female dobsonflies that receive nuptial gifts

In mating of the dobsonfly, Protohermes grandis (Thunberg), the male attaches the spermatophore externally to the female genitalia. The spermatophore includes a large gelatinous mass which the female detaches and feeds on after mating. While the female consumes this nuptial food gift, sperm is evacuated from the remaining portion of the spermatophore (sperm package) into her reproductive tract. Under laboratory conditions, mated females maintained receptivity throughout their lifetime, and they remated even on the day following copulation. A single insemination may supply enough sperm, as females mated only once deposited fertile eggs throughout life and, when dissected after death, all females had sperm in the spermatheca. There was a positive correlation between longevity and the number of matings. Lifetime fecundity also increased as mating multiplied. However, the size of eggs and hatchlings was not influenced by the number of matings. It seems that large spermatophore consumption by female P. grandis provides nutrients that increase fitness not in offspring quality, but in their quantity.     

Fascin, an Actin-bundling Protein, Induces Membrane Protrusions and Increases Cell Motility of Epithelial Cells

Fascin is an actin-bundling protein that is found in membrane ruffles, microspikes, and stress fibers. The expression of fascin is greatly increased in many transformed cells, as well as in specialized normal cells including neuronal cells and antigen-presenting dendritic cells. A morphological characteristic common to these cells expressing high levels of fascin is the development of many membrane protrusions in which fascin is predominantly present. To examine whether fascin contributes to the alterations in microfilament organization at the cell periphery, we have expressed fascin in LLC-PK1 epithelial cells to levels as high as those found in transformed cells and in specialized normal cells. Expression of fascin results in large changes in morphology, the actin cytoskeleton, and cell motility: fascin-transfected cells form an increased number of longer and thicker microvilli on apical surfaces, extend lamellipodia-like structures at basolateral surfaces, and show disorganization of cell–cell contacts. Cell migration activity is increased by 8–17 times when assayed by modified Boyden chamber. Microinjection of a fascin protein into LLC-PK1 cells causes similar morphological alterations including the induction of lamellipodia at basolateral surfaces and formation of an increased number of microvilli on apical surfaces. Furthermore, microinjection of fascin into REF-52 cells, normal fibroblasts, induces the formation of many lamellipodia at all regions of cell periphery. These results together suggest that fascin is directly responsible for membrane protrusions through reorganization of the microfilament cytoskeleton at the cell periphery.     

Age-related changes of element contents in the human meniscus

The relative contents (RCs) of elements in the human menisci from 23 subjects in the age range between 65 and 93 yr were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of sulfur, calcium, and phosphorus in menisci increased progressively until the 80s, being the highest in the 80s, and thereafter decreased. The RCs of magnesium in menisci increased progressively until the 90s. Regarding the medial and lateral menisci, higher RCs of magnesium and iron, and a lower RC of phosphorus were found in lateral menisci in comparison with those in medial menisci. There were sexual differences in the RCs of calcium and phosphorus of medial and lateral menisci. The RCs of calcium and phosphorus were about 50% higher in women’s menisci than in men’s. Histological examinations showed that structureless mucoid masses were observed in the menisci, with very high RCs of calcium and phosphorus being detected.     

Brown Adipose Tissue in Cetacean Blubber

Brown adipose tissue (BAT) plays an important role in thermoregulation in species living in cold environments, given heat can be generated from its chemical energy reserves. Here we investigate the existence of BAT in blubber in four species of delphinoid cetacean, the Pacific white-sided and bottlenose dolphins, Lagenorhynchus obliquidens and Tursiops truncates, and Dall’s and harbour porpoises, Phocoenoides dalli and Phocoena phocoena. Histology revealed adipocytes with small unilocular fat droplets and a large eosinophilic cytoplasm intermingled with connective tissue in the innermost layers of blubber. Chemistry revealed a brown adipocyte-specific mitochondrial protein, uncoupling protein 1 (UCP1), within these same adipocytes, but not those distributed elsewhere throughout the blubber. Western blot analysis of extracts from the inner blubber layer confirmed that the immunohistochemical positive reaction was specific to UCP1 and that this adipose tissue was BAT. To better understand the distribution of BAT throughout the entire cetacean body, cadavers were subjected to computed tomography (CT) scanning. Resulting imagery, coupled with histological corroboration of fine tissue structure, revealed adipocytes intermingled with connective tissue in the lowest layer of blubber were distributed within a thin, highly dense layer that extended the length of the body, with the exception of the rostrum, fin and fluke regions. As such, we describe BAT effectively enveloping the cetacean body. Our results suggest that delphinoid blubber could serve a role additional to those frequently attributed to it: simple insulation blanket, energy storage, hydrodynamic streamlining or contributor to positive buoyancy. We believe delphinoid BAT might also function like an electric blanket, enabling animals to frequent waters cooler than blubber as an insulator alone might otherwise allow an animal to withstand, or allow animals to maintain body temperature in cool waters during sustained periods of physical inactivity.     

Chemosystematics of tea trees based on tea leaf polyphenols as phenetic markers

This study examined the polyphenols of tea leaves as chemotaxonomic markers to investigate the phenetic relationship between 89 wild (the small-leaved C. sinensis var. sinensis and large-leaved C. sinensis var. assamica), hybrid, and cultivated tea trees from China and Japan. (?)-Epigallocatechin 3-O-gallate, EGCG (1); (?)-epigallocatechin, EGC (2); (?)-epicatechin 3-O-gallate, ECG (3); (?)-epicatechin, EC (4); (+)-catechin, CA (5); strictinin, STR (6); and gallic acid, GA (7) were used as polyphenolic markers. Of the 13 polyphenol patterns observed, Principal Component Analysis (PCA) indicated that the structure-types of the flavonoid B-rings, such as the pyrogallol-(EGCG (1) and EGC (2)) and catechol-(ECG (3) and EC (4)) types, greatly influenced the classification. Ward’s minimum-variance cluster analysis was used to produce a dendrogram that consisted of three sub-clusters. One sub-cluster (A) was composed of old tea trees ‘Gushu’ cha (C. sinensis var. assamica) and cv ‘Taidi’ cha, suggesting that relatively primitive tea trees contain greater amounts of compounds 3 and 4 and lower amounts of compounds 1 and 2. The other two sub-clusters B and C, made up of Chinese hybrids (sub-cluster B) and Japanese and Taiwanese tea trees (sub-cluster C), had lower contents of 3 and 4 than sub-cluster A. Therefore, PCA and cluster analysis indicated that the greater the amounts of 1 and 2 (and the lower of 3 and 4), the more recent the origin of the tea line. Based on morphological characteristics, geographical information, and the historical information on tea trees, these results show good agreement with the current theory of tea tree origins, and this suggests that the Xishuangbanna district and Puer City are among the original sites of the tea tree species.