Origin of the novel chemoreceptor Aesthetasc "Y" in Ostracoda: morphogenetical thresholds and evolutionary innovation

SUMMARY The morphology and developmental processes of the two types of ostracod chemoreceptors, the Aesthetasc "Y" and the "Grouped setae," were compared. Cypridoidea and Pontocypridoidea, belonging to Cypridocopina, have a large baseball bat-like seta as an autapomorphic character on the second antenna, whereas most ostracod taxa with plesiomorphic characters bear "Grouped setae" consisting of multiple setae on the second antenna. Their budding positions, morphology, and ontogenetic changes were compared, and our deduction is that the Aesthetasc "Y" originated from "Grouped setae-like" organ in the Paleozoic. The morphogenetic processes in the molting period of these chemoreceptors were compared at the cellular level. The observations suggest that the "Grouped setae" are formed by hypodermal cells and share sheath cells corresponding to those of the Aesthetasc "Y" as a common constraint in the molting process of setae. We conclude that modification of the morphogenetic processes in the molting period of the "Grouped setae" gave rise to the Aesthetasc "Y" as a novel organ in the evolutionary pathway of the Ostracoda.     

Colominic acid inhibits the proliferation of cultured bovine aortic endothelial cells and injures their monolayers: cell density-dependent effects prevented by sulfation

Colominic acid (CA), produced by Escherichia coli K1, is a polymer of sialic acid linked through alpha (2-->8) glycosidic linkages. Although there are several studies on the biological activities of chemically sulfated CA, the activity of CA has been incompletely understood. In the present study, we investigated the effects of CA, prepared as an alpha2,8-linked homopolymer of N-acetylneuraminic acid, on the proliferation and monolayer maintenance of bovine aortic endothelial cells in culture. The results indicate that CA potently inhibits the proliferation of sparse endothelial cells without nonspecific cell damage. The inhibitory effect of CA was markedly stronger than those of sodium spirulan and calcium spirulan, known polysaccharides that inhibit endothelial cell proliferation. On the other hand, in dense endothelial cells, CA induced nonspecific cell damage and markedly injured the monolayer. These results indicate that CA has two distinct effects on vascular endothelial cells: one is the inhibition of proliferation when the cell density is low, and the other is the nonspecific cytotoxicity when the cell density is high. Interestingly, these cell density-dependent effects of CA could be prevented by sulfation of the CA chains. Therefore, it is concluded that CA not only inhibits the proliferation of sparse endothelial cells without nonspecific cell damage but also injures dense cells in a monolayer by nonspecific cytotoxicity, which can be prevented by sulfation of the polysaccharide.     

Par6B and atypical PKC regulate mitotic spindle orientation during epithelial morphogenesis

Cdc42 plays an evolutionarily conserved role in promoting cell polarity and is indispensable during epithelial morphogenesis. To further investigate the role of Cdc42, we have used a three-dimensional matrigel model, in which single Caco-2 cells develop to form polarized cysts. Using this system, we previously reported that Cdc42 controls mitotic spindle orientation during cell division to correctly position the apical surface in a growing epithelial structure. In the present study, we have investigated the specific downstream effectors through which Cdc42 controls this process. Here, we report that Par6B and its binding partner, atypical protein kinase C (aPKC), are required to regulate Caco-2 morphogenesis. Depletion or inhibition of Par6B or aPKC phenocopies the loss of Cdc42, inducing misorientation of the mitotic spindle, mispositioning of the nascent apical surface, and ultimately, the formation of aberrant cysts with multiple lumens. Mechanistically, Par6B and aPKC function interdependently in this context. Par6B localizes to the apical surface of Caco-2 cysts and is required to recruit aPKC to this compartment. Conversely, aPKC protects Par6B from proteasomal degradation, in a kinase-independent manner. In addition, we report that depletion or inhibition of aPKC induces robust apoptotic cell death in Caco-2 cells, significantly reducing both cyst size and number. Cell survival and apical positioning depend upon different thresholds of aPKC expression, suggesting that they are controlled by distinct downstream pathways. We conclude that Par6B and aPKC control mitotic spindle orientation in polarized epithelia and, furthermore, that aPKC coordinately regulates multiple processes to promote morphogenesis.     

Destruction of amyloid fibrils of keratoepithelin peptides by laser irradiation coupled with amyloid-specific thioflavin T

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of β(2)-microglobulin K3 fragments and amyloid β by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.     

Parathyroid hormone-responsive Smad3-related factor, Tmem119, promotes osteoblast differentiation and interacts with the bone morphogenetic protein-Runx2 pathway

The mechanisms whereby the parathyroid hormone (PTH) exerts its anabolic action on bone are incompletely understood. We previously showed that inhibition of ERK1/2 enhanced Smad3-induced bone anabolic action in osteoblasts. These findings suggested the hypothesis that changes in gene expression associated with the altered Smad3-induced signaling brought about by an ERK1/2 inhibitor would identify novel bone anabolic factors in osteoblasts. We therefore performed a comparative DNA microarray analysis between empty vector-transfected mouse osteoblastic MC3T3-E1 cells and PD98059-treated stable Smad3-overexpressing MC3T3-E1 cells. Among the novel factors, Tmem119 was selected on the basis of its rapid induction by PTH independent of later increases in endogenous TGF-β. The levels of Tmem119 increased with time in cultures of MC3T3-E1 cells and mouse mesenchymal ST-2 cells committed to the osteoblast lineage by BMP-2. PTH stimulated Tmem119 levels within 1 h as determined by Western blot analysis and immunocytochemistry in MC3T3-E1 cells. MC3T3-E1 cells stably overexpressing Tmem119 exhibited elevated levels of Runx2, osteocalcin, alkaline phosphatase, and β-catenin, whereas Tmem119 augmented BMP-2-induced Runx2 levels in mesenchymal cells. Tmem119 interacted with Runx2, Smad1, and Smad5 in C2C12 cells. In conclusion, we identified a Smad3-related factor, Tmem119, that is induced by PTH and promotes differentiation in mouse osteoblastic cells. Tmem119 is an important molecule in the pathway downstream of PTH and Smad3 signaling in osteoblasts.     

Prophylactic Effects of the Glucagon-Like Peptide-1 Analog Liraglutide on Hyperglycemia in a Rat Model of Type 2 Diabetes Mellitus Associated with Chronic Pancreatitis and Obesity

The objective of this study was to investigate the effects of liraglutide, an analog of human glucagon-like peptide 1 (GLP1), on WBN/Kob-Lepr^fa (fa/fa) rats, which spontaneously develop type 2 diabetes mellitus with pancreatic disorder and obesity. Male fa/fa rats (age, 7 wk) were allocated into 4 groups and received liraglutide (37.5, 75, 150 μg/kg SC) or saline (control group) once daily for 4 wk. All rats in the control group became overweight and developed hyperglycemia as they aged. Although the rats given liraglutide showed a dose-dependent reduction in food intake, no significant effects on body weight or fat content occurred. In the liraglutide groups, the development of hyperglycemia was suppressed, even as plasma insulin concentrations increased in a dose-dependent manner. Intravenous glucose tolerance testing of the liraglutide-treated rats confirmed improvement of glucose tolerance and enhanced insulin secretion. Histologic examination revealed increased numbers of pancreatic β-cell type islet cells and increased proliferation of epithelial cells of the small ducts in the liraglutide-treated groups. Although our study did not reveal a significant decrease in obesity after liraglutide administration, the results suggest a marked antidiabetic effect characterized by increased insulin secretion in fa/fa rats with pancreatic disorders.Abbreviations: GLP1, glucagon-like peptide-1; IVGTT, intravenous glucose tolerance testing; T2DM, type 2 diabetes mellitusThe number of patients diagnosed with diabetes has more than doubled over the last 30 y, and diabetes has become an important public health concern worldwide.⁶ Approximately 90% of patients with diabetes are diagnosed with type 2 diabetes mellitus (T2DM).³¹ The onset of T2DM is determined by multiple factors that lead to reduced insulin secretion or insulin resistance, including genetic predisposition and lifestyle-associated habits such as lack of exercise, overeating, and obesity. Many drugs are already used clinically to treat T2DM;^9,11 nevertheless, the search and development of more efficient and safe drugs is currently underway. In this regard, incretin has recently gained attention as a member of a class of drugs used to treat T2DM.^9,11Enteroendocrine cells secret incretin hormones, which augment glucose-induced insulin secretion in response to food ingestion, in a glucose-dependent manner. Currently, the 2 confirmed incretins are glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 (GLP1). Research has shown that GLP1 derivatives have functions in addition to the promotion of insulin secretion, including facilitation of β-cell proliferation,²⁸ suppression of β-cell apoptosis,¹² and promotion of β-cell differentiation or de novo formation of β cells.^29,30 GLP1 derivatives have been reported to have multiple nonpancreatic functions, including suppression of appetite and body weight,^7,13 suppression of gastric secretions,¹⁹ reduction of lipid accumulation in the liver,¹⁷ and promotion of sensitivity to insulin in adipose cells and skeletal muscle.^8,22WBN/Kob-type male rats are a relevant animal model for diabetes without obesity. These rats typically show disease conditions including chronic pancreatitis and pancreatic endocrine disorders.^18,26 A new model rat for diabetes was established recently by crossing rats carrying the Lepr^fa obesity gene with wild-type WBN/Kob rats, yielding a fa/fa congenic strain.¹ The obesity gene (Lepr^fa) is a spontaneous mutation derived from Zucker-fatty rats that leads to dysfunction of the leptin receptor. Rats homozygous for this gene are obese, resistant to insulin, and hyperinsulinemic.^4,16,32 Male WBN/Kob-Lepr^fa rats (hereafter referred to as fa/fa rats) represent a new animal model in which the animals spontaneously develop diabetes in addition to endogenous insulin resistance. Compared with the parental strains, fa/fa rats are characterized by an earlier onset of diabetes and more severe pancreatic complications.^1,2 Our previous investigations have revealed that fa/fa rats present with hyperinsulinemia at a prediabetic stage as a compensatory response to insulin resistance, but these rats show high blood glucose levels because of a difficulty in maintaining blood insulin concentrations as a consequence of declining pancreatic β-cell function associated with advancing age.¹⁴In the current study, to further validate fa/fa rats as a T2DM model, we investigated the effects of a GLP1 analog in fa/fa rats with hyperglycemia (age, 7 to 11 wk). We used liraglutide, a human GLP1 analog, which has been shown to be clinically effective in T2DM patients.^9,11     

Fibrodysplasia Ossificans Progressiva-related Activated Activin-like Kinase Signaling Enhances Osteoclast Formation during Heterotopic Ossification in Muscle Tissues

Fibrodysplasia ossificans progressiva is characterized by extensive ossification within muscle tissues, and its molecular pathogenesis is responsible for the constitutively activating mutation (R206H) of the bone morphogenetic protein type 1 receptor, activin-like kinase 2 (ALK2). In this study, we investigated the effects of implanting ALK2 (R206H)-transfected myoblastic C2C12 cells into nude mice on osteoclast formation during heterotopic ossification in muscle and subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells with BMP-2 in nude mice induced robust heterotopic ossification with an increase in the formation of osteoclasts in muscle tissues but not in subcutaneous tissues. The implantation of ALK2 (R206H)-transfected C2C12 cells in muscle induced heterotopic ossification more effectively than that of empty vector-transfected cells. A co-culture of ALK2 (R206H)-transfected C2C12 cells as well as the conditioned medium from ALK2 (R206H)-transfected C2C12 cells enhanced osteoclast formation in Raw264.7 cells more effectively than those with empty vector-transfected cells. The transfection of ALK2 (R206H) into C2C12 cells elevated the expression of transforming growth factor (TGF)-β, whereas the inhibition of TGF-β signaling suppressed the enhanced formation of osteoclasts in the co-culture with ALK2 (R206H)-transfected C2C12 cells and their conditioned medium. In conclusion, this study demonstrated that the causal mutation transfection of fibrodysplasia ossificans progressiva in myoblasts enhanced the formation of osteoclasts from its precursor through TGF-β in muscle tissues.     

In vitro processing of amyloid precursor protein by cathepsin D

The formation of beta A4 amyloid in the brains of individuals with Alzheimer's disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimer's disease.     

Release of ribosome-bound ribosome recycling factor by elongation factor G

Elongation factor G (EF-G) and ribosome recycling factor (RRF) disassemble post-termination complexes of ribosome, mRNA, and tRNA. RRF forms stable complexes with 70 S ribosomes and 50 S ribosomal subunits. Here, we show that EF-G releases RRF from 70 S ribosomal and model post-termination complexes but not from 50 S ribosomal subunit complexes. The release of bound RRF by EF-G is stimulated by GTP analogues. The EF-G-dependent release occurs in the presence of fusidic acid and viomycin. However, thiostrepton inhibits the release. RRF was shown to bind to EF-G-ribosome complexes in the presence of GTP with much weaker affinity, suggesting that EF-G may move RRF to this position during the release of RRF. On the other hand, RRF did not bind to EF-G-ribosome complexes with fusidic acid, suggesting that EF-G stabilized by fusidic acid does not represent the natural post-termination complex. In contrast, the complexes of ribosome, EF-G and thiostrepton could bind RRF, although with lower affinity. These results suggest that thiostrepton traps an intermediate complex having RRF on a position that clashes with the P/E site bound tRNA. Mutants of EF-G that are impaired for translocation fail to disassemble post-termination complexes and exhibit lower activity in releasing RRF. We propose that the release of ribosome-bound RRF by EF-G is required for post-termination complex disassembly. Before release from the ribosome, the position of RRF on the ribosome will change from the original A/P site to a new location that clashes with tRNA on the P/E site.     

Crystal structure of inorganic pyrophosphatase from Thermus thermophilus.

The 3-dimensional structure of inorganic pyrophosphatase from Thermus thermophilus (T-PPase) has been determined by X-ray diffraction at 2.0 A resolution and refined to R = 15.3%. The structure consists of an antiparallel closed beta-sheet and 2 alpha-helices and resembles that of the yeast enzyme in spite of the large difference in size (174 and 286 residues, respectively), little sequence similarity beyond the active center (about 20%), and different oligomeric organization (hexameric and dimeric, respectively). The similarity of the polypeptide folding in the 2 PPases provides a very strong argument in favor of an evolutionary relationship between the yeast and bacterial enzymes. The same Greek-key topology of the 5-stranded beta-barrel was found in the OB-fold proteins, the bacteriophage gene-5 DNA-binding protein, toxic-shock syndrome toxin-1, and the major cold-shock protein of Bacillus subtilis. Moreover, all known nucleotide-binding sites in these proteins are located on the same side of the beta-barrel as the active center in T-PPase. Analysis of the active center of T-PPase revealed 17 residues of potential functional importance, 16 of which are strictly conserved in all sequences of soluble PPases. Their possible role in the catalytic mechanism is discussed on the basis of the present crystal structure and with respect to site-directed mutagenesis studies on the Escherichia coli enzyme. The observed oligomeric organization of T-PPase allows us to suggest a possible mechanism for the allosteric regulation of hexameric PPases.