The restrictive proteolysis of α-fodrin to a 120 kDa fragment is not catalyzed by calpains during thymic apoptosis

The -subunit (240 kDa) of fodrin was found to be digested selectively to a 120 kDa fragment during apoptosis of rat thymocytes in vivo and in vitro. This fragment was detected by an antibody (Ab) against full length -fodrin, but not by the anti-N-terminal sequence (GMMPR) of the -calpain-generated 150 kDa fragment Ab or the anti-PEST sequence of -fodrin Ab. On the other hand, basal levels of the 150 kDa fragment were constantly recognized by these three antibodies during apoptosis. The production of the 120 kDa fragment during apoptosis was not affected by the addition of calpain inhibitors such as Ac-LLLnal and E-64d, despite inhibition of the generation of the 150 kDa fragment. When x-irradiated thymocytes were incubated in the presence of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), both production of the 120 kDa fragment and apoptosis were suppressed. Purified - and m-calpain did not catalyze the formation of the 120 kDa fragment from purified -fodrin in vitro. These results suggest that a protease different from calpains is involved in the major process of -fodrin proteolysis to a 120 kDa fragment during thymic apoptosis.     

Nucleotide sequence of a rice cDNA similar to a maize NADP-dependent malic enzyme

We have isolated a rice cDNA clone that is homologous to the gene for the maize NADP-dependent malic enzyme (EC 1.1.1.40; NADP-ME). The deduced amino acid sequence coded for by the cDNA indicates a high level of homology to chloroplast type NADP-ME, including a transit peptide with pronounced hydrophobic properties at the amino terminus. Northern blot analysis indicates that the expression of this gene is regulated by external stress such as submergence.     

Biosynthesis and in vitro enzymatic synthesis of the isoleucine conjugate of 12-oxo-phytodienoic acid from the isoleucine conjugate of α-linolenic acid

The isoleucine conjugate of 12-oxo-phytodienoic acid (OPDA-Ile), a new member of the jasmonate family, was recently identified in Arabidopsis thaliana and might be a signaling molecule in plants. However, the biosynthesis and function of OPDA-Ile remains elusive. This study reports an in vitro enzymatic method for synthesizing OPDA-Ile, which is catalyzed by reactions of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) using isoleucine conjugates of α -linolenic acid (LA-Ile) as the substrate. A. thaliana fed LA-Ile exhibited a marked increase in the OPDA-Ile concentration. LA-Ile was also detected in A. thaliana. Furthermore, stable isotope labelled LA-Ile was incorporated into OPDA-Ile. Thus, OPDA-Ile is biosynthesized via the cyclization of LA-Ile in A. thaliana.     

How have spawning ground investigations of the Japanese eel Anguilla japonica contributed to the stock enhancement?

After Schmidt’s discovery of the spawning area of the Atlantic eels Anguilla anguilla and A. rostrata, the search for the Japanese eel A. japonica began in the Pacific Ocean. In 1991, the spawning area of the Japanese eel was determined to be the western North Pacific. Because of enthusiastic research, eggs and maturing eels have been collected in the Japanese eel. These findings are the first for one of the 19 freshwater eels. The population sizes of the Japanese and Atlantic eels are linearly decreasing. Thus, these eel population sizes are considered outside of safe biological limits, and the current fisheries are not sustainable. Artificial propagation has not yet succeeded for the freshwater eels. Stock assessment and management of the European eel have received increasing attention; however, such assessments and management of the Japanese eel have not yet been seriously considered. This paper is an overview of the results of intensive spawning ground investigations of the Japanese eel and describes how the outcomes of these studies have contributed not only to biological interests but also to stock enhancement. During the past 20 years of expeditions, noticeable findings have only been collected for wild eggs and mature adult specimens in spite of the expenditure of large research grants and the large amounts of time invested. The outcomes throughout an expedition do not necessarily contribute to the development and improvement of artificial breeding techniques and stock enhancement. Thus, eel research should be more focused on the studies related to eel stock management.     

Emi1 Maintains Genomic Integrity during Zebrafish Embryogenesis and Cooperates with p53 in Tumor Suppression

A growing body of evidence indicates that early mitotic inhibitor 1 (Emi1) is essential for genomic stability, but how this function relates to embryonic development and cancer pathogenesis remains unclear. We have identified a zebrafish mutant line in which deficient emi1 gene expression results in multilineage hematopoietic defects and widespread developmental defects that are p53 independent. Cell cycle analyses of Emi1-depleted zebrafish or human cells showed chromosomal rereplication, and metaphase preparations from mutant zebrafish embryos revealed rereplicated, unsegregated chromosomes and polyploidy. Furthermore, EMI1-depleted mammalian cells relied on topoisomerase IIα-dependent mitotic decatenation to progress through metaphase. Interestingly, the loss of a single emi1 allele in the absence of p53 enhanced the susceptibility of adult fish to neural sheath tumorigenesis. Our results cast Emi1 as a critical regulator of genomic fidelity during embryogenesis and suggest that the factor may act as a tumor suppressor.Successful cell division requires faithful replication of the genome, and defects in this process can contribute to genomic instability and subsequent malignant transformation (23). A key regulator of the normal cell cycle is the early mitotic inhibitor 1 (EMI1/FBXO5), a zinc finger protein expressed by a variety of adult tissues and especially in proliferating Ki-67-positive cells (39). Studies of the mammalian and Xenopus homologues of EMI1 have shown that it inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase complex that targets cell cycle-regulated proteins, such as the S- and M-phase cyclins (A and B), securin, and geminin (13, 25, 31). Depletion of EMI1 by small interfering RNA (siRNA) knockdown in cell lines or immunodepletion in cycling Xenopus extracts results in the untimely degradation of APC/C substrates, delaying G₁/S- and M-phase progression and inducing rereplication (6, 21, 25, 31). Such rereplication is a consequence of decreased levels of the APC/C substrates cyclin A and geminin, which are regulators of replication licensing (6, 21). The result of EMI1 depletion in some cell lines is senescence (39).Despite these insights into the molecular underpinnings of EMI1 function, little is known about the role of this protein in development. Knockout of murine Emi1 results in an embryonic-lethal phenotype prior to implantation, while a deficiency of Emi1 in cultured pronuclear zygotes leads to multipolar and tangled spindle structures, orphan chromosomes, large nuclei, and apoptosis by the 16-cell stage (17). Otherwise, the dynamic influence of EMI1 on early vertebrate development remains undefined. We sought to close this gap by taking advantage of the zebrafish model system. Zebrafish embryos harboring homozygous mutations of emi1 (emi1^m/m) develop beyond the onset of circulation, providing a unique opportunity to examine the developmental roles of Emi1 in vivo. The zebrafish emi1 mutant (hi2648) line was originally identified by a proviral insertional mutagenesis screen designed to identify genes that are necessary for normal morphological development in embryos (1, 8, 9). Subsequent studies showed that the insertion was located between the first and second exons of the emi1 gene (2). The morphological defects in emi1^m/m embryos at 2 days postfertilization (p.f.) are described in the public access zebrafish model organism database (http://zfin.org). Briefly, abnormalities in emi1^m/m embryos can be identified as early as 20 h p.f. and include slightly smaller heads and a lack of ventral curving of the posterior presomitic mesoderm. By 25 h p.f., the tail is more ventrally curved than in normal embryos, and increased cell death is observed throughout the central nervous system. Mutant embryos have circulating blood cells, although the onset of circulation is delayed. We became interested in this mutant because it harbors defects in the numbers and morphology of granulocytes, an important myeloid cell type within the innate immune system.There is evidence that EMI1 may function in cancer pathogenesis, and a variety of human tumors express this factor very highly, although in some cases this may be a consequence of elevated proliferation rates (11, 18). In fact, the human homologue of emi1 resides within chromosome 6q25, a region often deleted in leukemia, which, together with the cell cycle-regulatory role of EMI1, suggested that this factor may also function as a tumor suppressor whose loss of function could promote genetic instability. Thus, in addition to investigating the role of zebrafish emi1 in zebrafish development, with particular emphasis on hematopoiesis, we examined mammalian cells to identify mechanisms that may be important in EMI1-related malignant transformation and explored a putative tumor suppressor role for this cell cycle-regulatory protein.     

Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.     

Differences in the Trace Element Deposition in Otoliths Between Marine- and Freshwater-resident Japanese Eels, Anguilla japonica, as Determined by Laser Ablation ICPMS

Synopsis In order to determine whether the trace element composition in otolith of the Japanese eel Anguilla japonica could be used to determine its habitat use, we used laser ablation inductivity coupled plasma mass spectrometry (LA-ICPMS) to assay sectioned otoliths of both marine-resident (sea eels) and freshwater-resident (river eels) eels. A close linear relationship in the Sr:Ca ratios between EPMA (X-ray analysis with an electron microprobe) and LA-ICPMS analyses was found, suggesting that the latter technique could be used to separate the marine and freshwater life phases. Elemental signatures in the otolith outside the elver mark showed significant differences in Cr:Ca, Mn:Ca, and Ba:Ca ratios as well as Sr:Ca ratios between sea and river eels. These results indicate that the elemental compositions may reflect environmental variability between marine and fresh water masses. Thus, those elemental ratios determined by LA-ICPMS analysis seem to have the potential to help distinguish the habitat of the eel.     

A part of the Japan Sea form of the threespine stickleback, Gasterosteus aculeatus, spawns in the seawater tidal pools of western Hokkaido Island, Japan

The Japan sea form of the threespine stickleback, Gasterosteus aculeatus, was found to spawn in the seawater tidal pool of the Cape Benkei, western Hokkaido Island, Japan, in 2001 and 2003. Nest-guarding males, adult females and juveniles of this form were also observed in four tidal pools of the Capes Benkei, Kabuto and Kamui, in 2001–2003. Otolith Sr:Ca ratios in sticklebacks collected from these tidal pools were relative high from the core to the edge, suggesting they lived in seawater environments throughout its life cycle. These findings suggest that a part of the Japan Sea formȁ9s individuals in western Hokkaido Island have the potential to breed in the coastal sea and the life history style has evolved in dependence on the seawater environment.     

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.     

Differential roles of two major brain structures, mushroom bodies and central complex, for Drosophila male courtship behavior

Drosophila male courtship is a complex and robust behavior, the potential for which is genetically built into specific neural circuits in the central nervous system. Previous studies using male-female mosaics and the flies with defects in particular brain structures implicated the critical central regions involved in male courtship behavior. However, their acute physiological roles in courtship regulation still largely remain unknown. Using the temperature-sensitive Dynamin mutation, shibire(ts1), here we demonstrate the significance of two major brain structures, the mushroom bodies and the central complex, in experience-independent aspects of male courtship. We show that blocking of synaptic transmission in the mushroom body intrinsic neurons significantly delays courtship initiation and reduces the courtship activity by shortening the courtship bout length when virgin females are used as a sexual target. Interestingly, however, the same treatment affects neither initiation nor maintenance of courtship toward young males that release courtship-stimulating pheromones different from those of virgin females. In contrast, blocking of synaptic transmission in a central complex substructure, the fan-shaped body, slightly but significantly reduces courtship activity toward both virgin females and young males with little effect on courtship initiation. Taken together, our results indicate that the neuronal activity in the mushroom bodies plays an important role in responding to female-specific sex pheromones that stimulate initiation and maintenance of male courtship behavior, whereas the fan-shaped body neurons are involved in maintenance of male courtship regardless of the nature of courtship-stimulating cues.