A Mathematical Modelling for the Cheliped Regeneration with Handedness in Fiddler Crab

An enormously developed giant cheliped with the other small one characterizes the adult male fiddler crab. Some experiments with artificial severances of cheliped indicate that such a handedness in the cheliped size is maintained even after the regeneration of severed cheliped. Other experimental researches give some results about an unknown physiological system which controls the emergence and the regeneration of the handedness in the cheliped size. In this paper, with two hypothesized factors relevant to the regeneration of a severed cheliped, we propose a simple mathematical model to describe the experimental result about the cheliped regeneration with a handedness after the cheliped severance for the fiddler crab. Our model gives a suggestion about an underlying system for the cheliped regeneration in the fiddler crab or some other crustacean species.     

Megalin-mediated endocytosis of cystatin C in proximal tubule cells

Serum levels of cystatin C, an endogenous cysteine proteinase inhibitor, are often used as an indicator of glomerular filtration rate. Although it is known that cystatin C is filtered by glomeruli and metabolized in proximal tubule cells (PTC), the precise molecular mechanism underlying this process is undetermined. Using quartz-crystal microbalance analyses, we demonstrate that cystatin C binds directly to megalin, an endocytic receptor in PTC, in a Ca(+)-dependent manner. We also find that cystatin C is endocytosed specifically via megalin in rat yolk sac epithelium-derived L2 cells which share a variety of characteristics with PTC. Finally, in vivo studies using kidney-specific megalin knockout mice provide evidence that megalin mediates proximal tubular uptake of cystatin C. We conclude that megalin is an endocytic receptor of cystatin C in PTC.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

Roles of RecA protein in spontaneous mutagenesis in Escherichia coli

To verify the extent of contribution of spontaneous DNA lesions to spontaneous mutagenesis, we have developed a new genetic system to examine simultaneously both forward mutations and recombination events occurring within about 600 base pairs of a transgenic rpsL target sequence located on Escherichia coli chromosome. In a wild-type strain, the recombination events were occurring at a frequency comparable to that of point mutations within the rpsL sequence. When the cells were UV-irradiated, the recombination events were induced much more sharply than point mutations. In a recA null mutant, no recombination event was observed. These data suggest that the blockage of DNA replication, probably caused by spontaneous DNA lesions, occurs often in normally growing E. coli cells and is mainly processed by cellular functions requiring the RecA protein. However, the recA mutant strain showed elevated frequencies of single-base frameshifts and large deletions, implying a novel mutator action of this strain. A similar mutator action of the recA mutant was also observed with a plasmid-based rpsL mutation assay. Therefore, if the recombinogenic problems in DNA replication are not properly processed by the RecA function, these would be a potential source for mutagenesis leading to single-base frameshift and large deletion in E. coli. Furthermore, the single-base frameshifts induced in the recA-deficient cells appeared to be efficiently suppressed by the mutS-dependent mismatch repair system. Thus, it seems likely that the single-base frameshifts are derived from slippage errors that are not directly caused by DNA lesions but made indirectly during some kind of error-prone DNA synthesis in the recA mutant cells.     

Homophilic Dscam interactions control complex dendrite morphogenesis

Alternative splicing of the Drosophila gene Dscam results in up to 38,016 different receptor isoforms proposed to interact by isoform-specific homophilic binding. We report that Dscam controls cell-intrinsic aspects of dendrite guidance in all four classes of dendrite arborization (da) neurons. Loss of Dscam in single neurons causes a strong increase in self-crossing. Restriction of dendritic fields of neighboring class III neurons appeared intact in mutant neurons, suggesting that dendritic self-avoidance, but not heteroneuronal tiling, may depend on Dscam. Overexpression of the same Dscam isoforms in two da neurons with overlapping dendritic fields forced a spatial segregation of the two fields, supporting the model that dendritic branches of da neurons use isoform-specific homophilic interactions to ensure minimal overlap. Homophilic binding of the highly diverse extracellular domains of Dscam may therefore limit the use of the same "core" repulsion mechanism to cell-intrinsic interactions without interfering with heteroneuronal interactions.     

Variation in Storage α-Polyglucans of Red Algae: Amylose and Semi-Amylopectin Types in <Emphasis Type="Italic">Porphyridium</Emphasis> and Glycogen Type in <Emphasis Type="Italic">Cyanidium</Emphasis>

Red algae are widely known to produce floridean starch but it remains unclear whether the molecular structure of this algal polyglucan is distinct from that of the starch synthesized by vascular plants and green algae. The present study shows that the unicellular species Porphyridium purpureum R-1 (order Porphyridiales, class Bangiophyceae) produces both amylopectin-type and amylose-type alpha-polyglucans. In contrast, Cyanidium caldarium (order Porphyridiales, class Bangiophyceae) synthesizes glycogen-type polyglucan, but not amylose. Detailed analysis of alpha-1,4-chain length distribution of P. purpureum polyglucan suggests that the branched polyglucan has a less ordered structure, referred to as semi-amylopectin, as compared with amylopectin of rice endosperm having a tandem-cluster structure. The P. purpureum linear amylose-type polyglucan, which has a lambda(max) of 630 nm typical of amylose-iodine complex and is resistant to Pseudomonas isoamylase digestion, accounts for less than 10% of the total polyglucans. We produced and isolated a cDNA encoding a granule-bound starch synthase (GBSS)-type protein of P. purpureum, which is probably the approximately 60-kDa protein bound tightly to the starch granules, resembling the amylose-synthesizing GBSS protein of green plants. The present investigation indicates that the class Bangiophyceae includes species producing both semi-amylopectin and amylose, and species producing glycogen alone.     

Selective photo-cross-linking of 2'-O-psoralen-conjugated oligonucleotide with RNAs having point mutations

It has been reported that point mutations in genes are responsible for various cancers and the selective regulation of the gene expression is an important issue to develop a new type of anticancer drugs. In this report, we present a new type of antisense molecule that photo-cross-links to an oligoribonucleotide having a point mutation site in a sequence specific manner. 2'-O-psoralen-conjugated adenosine was synthesized in four steps from adenosine and introduced in the middle of an oligodeoxyribonucleotide (2'-Ps-oligo). Compared with 5'-O-psoralen-conjugated oligodeoxyribonucleotide (5'-Ps-oligo), which has a psoralen at the 5'-end, 2'-Ps-oligo more selectively photo-cross-linked to a pyrimidine base of the site of alteration from purine to pyrimidine in the oligoribonucleotide.     

Arsonic acid-presenting superparamagnetic iron oxide for pH-responsive aggregation under slightly acidic conditions

We report the synthesis and characterization of the arsonic acid-presenting superparamagnetic iron oxide (SPIO). We used arsonoacetic acid as the ligand for SPIOs in aqueous media. The surface modification of the SPIOs was accomplished via the ligand exchange from undecanoic acid to the carboxyl moiety of arsonoacetic acid. Consequently, the well-dispersed arsonic acid-presenting SPIOs in water were obtained. We found that the dispersion state of the arsonic acid-presenting SPIOs can be sharply regulated by pH changes in the biological significant region. The well dispersion state of the arsonic acid-presenting SPIOs can be maintained at the neutral pH region. In contrast, the arsonic acid-presenting SPIOs can sensitively form the aggregation below pH 6.1. Moreover, these dispersion states can be controlled reversibly by the pH alteration in the narrow region.     

Induction of insulin-like growth factor 2 expression in a mesenchymal cell line co-cultured with an ameloblast cell line

Various growth factors have been implicated in the regulation of cell proliferation and differentiation during tooth development. It has been unclear if insulin-like growth factors (IGFs) participate in the epithelium–mesenchyme interactions of tooth development. We previously produced three-dimensional sandwich co-culture systems (SW) containing a collagen membrane that induce the differentiation of epithelial cells. In the present study, we used the SW system to analyze the expression of IGFs and IGFRs. We demonstrate that IGF2 expression in mesenchymal cells was increased by SW. IGF1R transduces a signal; however, IGF2R does not transduce a signal. Recombinant IGF2 induces IGF1R and IGF2R expression in epithelial cells. IGF1R expression is increased by SW; however, IGF2R expression did not increase by SW. Thus, IGF2 signaling works effectively in SW. These results suggest that IGF signaling acts through the collagen membrane on the interaction between the epithelium and mesenchyme. In SW, other cytokines may be suppressed to induce IGF2R induction. Our results suggest that IGF2 may play a role in tooth differentiation.     

Neonatal lethality in knockout mice expressing the kinase-dead form of the gefitinib target GAK is caused by pulmonary dysfunction

Gefitinib (Iressa) is an inhibitor of the epidermal growth factor receptor (EGFR) that has shown promising activity in the treatment of patients with non-small cell lung cancer (NSCLC). However, adverse side effects of gefitinib treatment, such as respiratory dysfunction, have limited the therapeutic benefit of this targeting strategy. The present results show that this adverse effect can be attributed to the inhibition of the novel gefitinib target GAK (Cyclin G-associated kinase), which is as potently inhibited by the drug as the tyrosine kinase activity of EGFR. Knockout mice expressing the kinase-dead form of GAK (GAK-kd) died within 30 min after birth primarily due to respiratory dysfunction. Immunohistochemical analysis revealed that surfactant protein A (SP-A) was abundant within alveolar spaces in GAK-kd(+/+) mice but not in GAK-kd(-/-) pups. E-cadherin and phosphorylated EGFR signals were also abnormal, suggesting the presence of flat alveolar cells with thin junctions. These results suggest that inhibition of GAK by gefitinib may cause pulmonary alveolar dysfunction, and the present study may help prevent side effects associated with gefitinib therapy in NSCLC patients.