Mouse Lefty2 and zebrafish antivin are feedback inhibitors of nodal signaling during vertebrate gastrulation.

Mammalian lefty and zebrafish antivin form a subgroup of the TGF beta superfamily. We report that mouse mutants for lefty2 have an expanded primitive streak and form excess mesoderm, a phenotype opposite to that of mutants for the TGF beta gene nodal. Analogously, overexpression of Antivin or Lefty2 in zebrafish embryos blocks head and trunk mesoderm formation, a phenotype identical to that of mutants caused by loss of Nodal signaling. The lefty2 mutant phenotype is partially suppressed by heterozygosity for nodal. Similarly, the effects of Antivin and Lefty2 can be suppressed by overexpression of the nodal-related genes cyclops and squint or the extracellular domain of ActRIIB. Expression of antivin is dependent on Nodal signaling, revealing a feedback loop wherein Nodal signals induce their antagonists Lefty2 and Antivin to restrict Nodal signaling during gastrulation.     

Expression of trypsin in human cancer cell lines and cancer tissues and its tight binding to soluble form of Alzheimer amyloid precursor protein in culture.

It was recently found that overexpression of the trypsin gene in tumor cells stimulates their growth in culture and in nude mice. In the present study, expression of trypsin in various human cancer cell lines and tissues was studied by gelatin zymography and immunoblotting before and after enterokinase treatment and by immunohistochemistry. The analyses showed that many stomach, colon, and breast cancer cell lines secreted trypsinogens-1 and/or -2, as well as an unidentified serine proteinase of about 70 kDa, into culture medium. Lung cancer cell lines secreted 18- and 19-kDa unidentified trypsin-like proteins. Stomach cancer cell lines frequently secreted active trypsin, suggesting that they produced an endogenous activator of trypsinogen, most likely enterokinase. Active trypsin formed a complex with a soluble form of Alzheimer amyloid precursor protein (sAPP), a Kunitz-type trypsin inhibitor, which was secreted by all cell lines tested. This indicated that sAPP is a primary inhibitor of secreted trypsin. Immunohistochemical analysis showed that trypsin(ogen) was frequently expressed at high levels in stomach and colon cancers, but scarcely in breast cancers. In the stomach cancers, the trypsin immunoreactivity was higher in the malignant, non-cohesive type than in the cohesive type. These results support the hypothesis that tumor-derived trypsin is involved in the malignant growth of tumor cells, especially stomach cancer cells.     

Mechanism of Regulatory Target Selection by the SOX High-Mobility-Group Domain Proteins as Revealed by Comparison of SOX1/2/3 and SOX9

SOX proteins bind similar DNA motifs through their high-mobility-group (HMG) domains, but their action is highly specific with respect to target genes and cell type. We investigated the mechanism of target selection by comparing SOX1/2/3, which activate δ-crystallin minimal enhancer DC5, with SOX9, which activates Col2a1 minimal enhancer COL2C2. These enhancers depend on both the SOX binding site and the binding site of a putative partner factor. The DC5 site was equally bound and bent by the HMG domains of SOX1/2 and SOX9. The activation domains of these SOX proteins mapped at the distal portions of the C-terminal domains were not cell specific and were independent of the partner factor. Chimeric proteins produced between SOX1 and SOX9 showed that to activate the DC5 enhancer, the C-terminal domain must be that of SOX1, although the HMG domains were replaceable. The SOX2-VP16 fusion protein, in which the activation domain of SOX2 was replaced by that of VP16, activated the DC5 enhancer still in a partner factor-dependent manner. The results argue that the proximal portion of the C-terminal domain of SOX1/2 specifically interacts with the partner factor, and this interaction determines the specificity of the SOX1/2 action. Essentially the same results were obtained in the converse experiments in which COL2C2 activation by SOX9 was analyzed, except that specificity of SOX9-partner factor interaction also involved the SOX9 HMG domain. The highly selective SOX-partner factor interactions presumably stabilize the DNA binding of the SOX proteins and provide the mechanism for regulatory target selection.     

Changes in microtubule structures during the first cell cycle of physiologically polyspermic newt eggs

The unfertilized egg of the newt, Cynops pyrrhogaster, has a second meiotic spindle at the animal pole and numerous cortical cytasters. After physiologically polyspermic fertilization, all sperm nuclei incorporated into the egg develop sperm asters, and the cortical cytasters change into bundles of cortical microtubules. The size of the sperm asters in the animal hemisphere is ∼5.6-fold larger than that in the vegetal hemisphere. Only one sperm nucleus moves toward the center of the animal hemisphere to form a zygote nucleus with the egg nucleus. This movement is inhibited by nocodazole, but not by cytochalasin B. The centrosome in the zygote nucleus divides into two parts to form a bipolar spindle for the first cleavage synchronously with the nuclear cycle, but centrosomes of accessory sperm nuclei in the vegetal hemisphere remained to form monopolar interphase asters and subsequently degenerate around the first cleavage stage. The size of sperm asters in monospermically fertilized Xenopus eggs was ∼37-fold larger than those in Cynops eggs. Since sperm asters that formed in polyspermically fertilized Xenopus eggs exclude each other, the formation of a zygote nucleus is inhibited. Cynops sperm nuclei form larger asters in Xenopus eggs, whereas Xenopus sperm nuclei form smaller asters in Cynops eggs compared with those in homologous eggs. Since there was no significant difference in the concentration of monomeric tubulin between those eggs, the size of sperm asters is probably regulated by a component(s) in egg cytoplasm. Smaller asters in physiologically polyspermic newt eggs might be useful for selecting only one sperm nucleus to move toward the egg nucleus. Mol. Reprod. Dev. 47:210–221, 1997. © 1997 Wiley-Liss, Inc.     

Domain architecture of vasohibins required for their chaperone‐dependent unconventional extracellular release

Vasohibins (VASH1 and VASH2) are recently identified regulators of angiogenesis and cancer cell functions. They are secreted proteins without any classical secretion signal sequences, and are thought to be secreted instead via an unconventional protein secretion (UPS) pathway in a small vasohibin‐binding protein (SVBP)‐dependent manner. However, the precise mechanism of SVBP‐dependent UPS is poorly understood. In this study, we identified a novel UPS regulatory system in which essential domain architecture (VASH‐PS) of VASHs, comprising regions VASH1_91–180 and VASH2_80–169, regulate the cytosolic punctate structure formation in the absence of SVBP. We also demonstrate that SVBP form a complex with VASH1 through the VASH1_274–282 (SIa), VASH1_139‐144 (SIb), and VASH1_133–137 (SIc), leading to the dispersion in the cytosol and extracellular release of VASH1. The amino acid sequences of VASH‐SIa and VASH‐PS, containing SIb and SIc, are highly conserved among VASH family members in vertebrates, suggesting that SVBP‐dependent UPS may be common within the VASH family. This novel UPS regulatory system may open up new avenues for understanding fundamental protein secretion in vertebrates.     

Isolation, purification, characterization and glycan-binding profile of a d-galactoside specific lectin from the marine sponge, Halichondria okadai

A lectin recognizing both Galbeta1-3GlcNAc and Galbeta1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The pI value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl-d-galactopyranoside, N-acetyl-d-galactosamine, methyl-N-acetyl-d-galactosaminide, lactose, melibiose, and asialofetuin. The K(d) of the lectin against p-nitrophenyl-beta-lactoside was determined to be 2.76x10(-5) M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium.     

Food-chain length and adaptive foraging

Food-chain length, the number of feeding links from the basal species to the top predator, is a key characteristic of biological communities. However, the determinants of food-chain length still remain controversial. While classical theory predicts that food-chain length should increase with increasing resource availability, empirical supports of this prediction are limited to those from simple, artificial microcosms. A positive resource availability–chain length relationship has seldom been observed in natural ecosystems. Here, using a theoretical model, we show that those correlations, or no relationships, may be explained by considering the dynamic food-web reconstruction induced by predator''s adaptive foraging. More specifically, with foraging adaptation, the food-chain length becomes relatively invariant, or even decreases with increasing resource availability, in contrast to a non-adaptive counterpart where chain length increases with increasing resource availability; and that maximum chain length more sharply decreases with resource availability either when species richness is higher or potential link number is larger. The interactive effects of resource availability, adaptability and community complexity may explain the contradictory effects of resource availability in simple microcosms and larger ecosystems. The model also explains the recently reported positive effect of habitat size on food-chain length as a result of increased species richness and/or decreased connectance owing to interspecific spatial segregation.