Allocleaning behavior by the sentinel crab Macrophthalmus banzai: a case of mutual cooperation

The macrophthalmid crab Macrophthalmus banzai performs allocleaning behavior, in which an individual feeds off the carapace or walking legs of a conspecific male or female. Cleaning continues until the recipient (‘cleanee’) rejects the cleaning or the cleaner ceases cleaning. In cases where the recipient did not reject the cleaning, the cleanee often retreated to its own burrow site after the cleaner ceased cleaning, allowing the cleaner to feed on its territory. We suggest that this is mutual cooperation, in which the cleaner assists the recipient to clean the body surface, and the recipient in return provides a feeding ground for the cleaner. Using field observation, we explored the factors responsible for the retreat of the cleanee in response to the cleaning behavior. Factors influencing the retreat of the cleanee were the time of the cleaning incident relative to low tide, the cleanee’s position relative to its own burrow, and the duration of the cleaning bout. The third factor showed that retreat was more common when the cleaning was thorough, which suggests that allocleaning is a form of mutual cooperation in M. banzai.     

The utility of Apc-mutant rats in modeling human colon cancer

Prior to the advent of genetic engineering in the mouse, the rat was the model of choice for investigating the etiology of cancer. Now, recent advances in the manipulation of the rat genome, combined with a growing recognition of the physiological differences between mice and rats, have reignited interest in the rat as a model of human cancer. Two recently developed rat models, the polyposis in the rat colon (Pirc) and Kyoto Apc Delta (KAD) strains, each carry mutations in the intestinal-cancer-associated adenomatous polyposis coli (Apc) gene. In contrast to mouse models carrying Apc mutations, in which cancers develop mainly in the small intestine rather than in the colon and there is no gender bias, these rat models exhibit colonic predisposition and gender-specific susceptibility, as seen in human colon cancer. The rat also provides other experimental resources as a model organism that are not provided by the mouse: the structure of its chromosomes facilitates the analysis of genomic events, the size of its colon permits longitudinal analysis of tumor growth, and the size of biological samples from the animal facilitates multiplexed molecular analyses of the tumor and its host. Thus, the underlying biology and experimental resources of these rat models provide important avenues for investigation. We anticipate that advances in disease modeling in the rat will synergize with resources that are being developed in the mouse to provide a deeper understanding of human colon cancer.KEY WORDS: APC, Allelic series, Animal models, Colorectal cancer     

JNK- and IkappaB-dependent pathways regulate MCP-1 but not adiponectin release from artificially hypertrophied 3T3-L1 adipocytes preloaded with palmitate in vitro

Obese conditions increase the expression of adipocytokine monocyte chemoattractant protein-1 (MCP-1) in adipose tissue as well as MCP-1 plasma levels. To investigate the mechanism behind increased MCP-1, we used a model in which 3T3-L1 adipocytes were artificially hypertrophied by preloading with palmitate in vitro. As observed in obesity, under our model conditions, palmitate-preloaded cells showed significantly increased oxidative stress and increased MCP-1 expression relative to control cells. This increased MCP-1 expression was enhanced by adding exogenous tumor necrosis factor-alpha (TNF-alpha; 17.8-fold vs. control cells, P < 0.01) rather than interleukin-1beta (IL-1beta; 2.6-fold vs. control cells, P < 0.01). However, endogenous TNF-alpha and IL-1beta release was not affected in hypertrophied cells, suggesting that these endogenous cytokines do not mediate hypertrophy-induced increase in MCP-1. MCP-1 secretion from hypertrophied cells was significantly decreased by treatment with antioxidant N-acetyl-cysteine, JNK inhibitors SP600125 and JIP-1 peptide, and IkappaB phosphorylation inhibitors BAY 11-7085 and BMS-345541 (P < 0.01). MCP-1 secretion was not affected by peroxisome proliferator-activated receptor-gamma (PPARgamma) antagonists assayed. Adiponectin, another adipocytokine studied in parallel, also showed increased release in hypertrophy relative to control cells. But in contrast to MCP-1, adiponectin release was significantly suppressed by both exogenous TNF-alpha and IL-1beta as well as by PPARgamma antagonists bisphenol A diglycidyl ether and T0070907 (P < 0.01). JNK inhibitors and IkappaB phosphorylation inhibitors showed no significant effect on adiponectin. We conclude that adipocyte hypertrophy through palmitate loading causes oxidative stress, which in turn increases MCP-1 expression and secretion through JNK and IkappaB signaling. In contrast, the parallel increase in adiponectin expression appears to be related to the PPARgamma ligand properties of palmitate.     

Structure and characteristics of reassembled fluorescent protein, a new insight into the reassembly mechanisms

Bimolecular fluorescence complementation (BiFC) assay has been used widely to visualize protein-protein interactions in cells. However, there is a problem that fluorescent protein fragments have an ability to associate with each other independent of an interaction between proteins fused to the fragments. To facilitate the BiFC assay, we have attempted to determine the structure and characteristics of reassembled fluorescent protein, Venus. The anion-exchange chromatography showed an oligomer and a monomer of reassembled Venus. Our results suggested that the oligomer was formed by β-strands swapping without any serious steric clashes and was converted to the monomer. Crystal structure of reassembled Venus had an 11-stranded β-barrel fold, typical of GFP-derived fluorescent proteins. Based on the structural features, we have mutated to β-strand 7 and measured T_m values. The results have revealed that the mutation influences the thermal stability of reassembled fluorescent complex.     

Stability of DNA/Td3717 complexes for gene transfection,defined by the size and polydispersity of the complex

The amphiphilic α-helical peptide, Td3717, is a bi-functional synthetic peptide that acts as both a polycation for DNA binding and a ligand for targeted delivery to tumor cells. Td3717 forms a stable complex with plasmid DNA, and the complex maintained high transfection efficiency after storage at 4 °C for six months and after four freeze/thaw cycles. During the storage and freeze/thaw cycling, the particle size of the DNA/Td3717 complex remained less than 100 nm. The size of the complex is an important factor for its internalization into cells via the endocytosis pathway; therefore, the stability of the particles will strongly contribute to high transfection efficiencies after storage and repeated freezing/thawing.     

Utility of NucleoCounter for the chondrocyte count in the collagenase digest of human native cartilage

In cartilage tissue engineering, viable cell numbers should be correctly counted in the collagenase digest of the biopsied cartilage. However, this is a difficult task due to the presence of matrix debris, cell ghosts and their aggregates. To search for the correct cell counting method in this situation, we evaluated the utility of an automatic cell counting device, the NucleoCounter, and compared it with conventional staining using the LIVE/DEAD® kit. We first measured the cell numbers of a standard chondrocyte sample by the NucleoCounter, which showed a high accuracy (R² = 0.9999) and reproducibility (%CV: 2.00–8.66). We then calculated the cell numbers and viability in some collagenase digests of native cartilage using either the NucleoCounter or LIVE/DEAD® kit, revealing that the total cell numbers, viable ones and viability were highly correlated between them (R² = 0.9601, 0.9638 and 0.917, respectively). However, both the intrapersonal and interpersonal variabilities in the NucleoCounter was significantly decreased to about 1/20–1/5, compared to that of the LIVE/DEAD® kit. The NucleoCounter was regarded as a useful tool for simple, rapid, and highly reproducible cell counts, which may not only provide constant experimental data in a certain laboratory, but also contribute to the high reproducibility of the clinical results of cartilage tissue engineering among multiple institutions.     

Augmentation of antitumor resistance by a strain of unicellular green algae,Chlorella vulgaris

Summary Growth of Meth-A tumor in CDF1 mice was inhibited significantly by injection of a hot water extract of a strain of Chlorella vulgaris (CE) into the tumor or into the subcutaneous tissue near the tumor. The augmentation of resistance by CE may require the participation of T cells and macrophages, since it was abolished or reduced in athymic nude mice or mice treated with carrageenan, a macrophage blocker. Mice treated with CE exhibited antigen-specific augmented resistance against rechallenge with tumor. Moreover, the antitumor effect of CE was comparable with that of Corynebacterium parvum, but its mechanism of effect might be different.