Proteolytic activation of protein kinase C delta and epsilon by caspase-3 in U937 cells during chemotherapeutic agent-induced apoptosis

Protein kinase C (PKC) family members play pivotal roles in cellular signal transduction and nPKCdelta and theta are known to be subjected to restrictive proteolysis during apoptosis. Here we show that nPKCepsilon was specifically cleaved and generates 43-kDa and 36-kDa C-terminal fragments during chemotherapeutic drug-induced apoptosis. The proteolytic cleavage of nPKCdelta and epsilon was completely inhibited by pretreatment with Ac-DEVD-cho, a specific inhibitor of caspase-3 family enzymes. Furthermore, nPKCepsilon in non-treated U937 cell lysates was cleaved by purified recombinant caspase-3 to generate the 43-kDa fragment, identical in size to the fragment observed in vivo. This cleavage was prevented by the addition of Ac-DEVD-cho. These results suggest that caspase-3 specifically cleaves nPKCepsilon. These findings suggest the possibility that nPKC subfamily members are generally involved in the execution of apoptosis but they are regulated diversely depending on the different apoptotic stimuli.     

APP mouse models for Alzheimer's disease preclinical studies

Animal models of human diseases that accurately recapitulate clinical pathology are indispensable for understanding molecular mechanisms and advancing preclinical studies. The Alzheimer's disease (AD) research community has historically used first‐generation transgenic (Tg) mouse models that overexpress proteins linked to familial AD (FAD), mutant amyloid precursor protein (APP), or APP and presenilin (PS). These mice exhibit AD pathology, but the overexpression paradigm may cause additional phenotypes unrelated to AD. Second‐generation mouse models contain humanized sequences and clinical mutations in the endogenous mouse App gene. These mice show Aβ accumulation without phenotypes related to overexpression but are not yet a clinical recapitulation of human AD. In this review, we evaluate different APP mouse models of AD, and review recent studies using the second‐generation mice. We advise AD researchers to consider the comparative strengths and limitations of each model against the scientific and therapeutic goal of a prospective preclinical study.     

Fecundity of the tropical catadromous eels <Emphasis Type="Italic">Anguilla bicolor bicolor</Emphasis>, <Emphasis Type="Italic">A. bengalensis bengalensis</Emphasis> and <Emphasis Type="Italic">A. marmorata</Emphasis>

Maturation is one of the most important ontogenetic transitions in an individual’s life. However, the reproductive ecology of the tropical anguillid eel genus Anguilla at the onset of oceanic spawning migration is poorly understood. To understand the reproductive ecology, the fecundity of the tropical eels Anguilla bicolor bicolor, A. bengalensis bengalensis and A. marmorata was examined using advanced migrating silver eels (Stage IV and V). A close linear relationship was found between total length and fecundity in A. bengalensis bengalensis. The fecundities of A. bicolor bicolor (0.55 to 4.96 × 10⁶), A. bengalensis bengalensis (0.33–1.72 × 10⁶) and A. marmorata (0.99 × 10⁶) were within the range of those observed in temperate eels.     

Thimet oligopeptidase cleaves the full-length Alzheimer amyloid precursor protein at a beta-secretase cleavage site in COS cells.

We developed an assay method using a novel quenched fluorescent substrate (QFS) flanking the beta-cleavage site of amyloid precursor protein (APP), and purified a candidate beta-secretase from bovine brain. N-terminal amino acid analysis showed the candidate to be thimet oligopeptidase (TOP). The cDNA for human TOP was cloned from a human brain cDNA library and expressed in COS cells. The enzyme was further purified on a Ni2+-agarose column. TOP cleaved the Swedish Alzheimer's substrate (SEVNLDAEFR) as well as the normal substrate (SEVKMDAEFR). We then coexpressed TOP with APP695 in COS cells, collected transfected cells and conditioned media, and analyzed them by immunoblotting. The antibody against the specific secreted APP cleaved by beta-secretase (sAPPbeta) detected the secretion of sAPPbeta only from APP/hTOP-overexpressing cells, and not from cells overexpressing of antisense hTOP cDNA. Finally, we analyzed the immunolocalization of overexpressed hTOP in COS cells. Most hTOP was localized in the nuclei, but a small amount was localized in the Golgi or other organelles around the nuclei. These results suggest that TOP has a beta-secretase-like activity responsible for the processing of APP.     

Calpastatin is up-regulated in response to hypoxia and is a suicide substrate to calpain after neonatal cerebral hypoxia-ischemia.

In a model of cerebral hypoxia-ischemia in the immature rat, widespread brain injury is produced in the ipsilateral hemisphere, whereas the contralateral hemisphere is left undamaged. Previously, we found that calpains were equally translocated to cellular membranes (a prerequisite for protease activation) in the ipsilateral and contralateral hemispheres. However, activation, as judged by degradation of fodrin, occurred only in the ipsilateral hemisphere. In this study we demonstrate that calpastatin, the specific, endogenous inhibitor protein to calpain, is up-regulated in response to hypoxia and may be responsible for the halted calpain activation in the contralateral hemisphere. Concomitantly, extensive degradation of calpastatin occurred in the ipsilateral hemisphere, as demonstrated by the appearance of a membrane-bound 50-kDa calpastatin breakdown product. The calpastatin breakdown product accumulated in the synaptosomal fraction, displaying a peak 24 h post-insult, but was not detectable in the cytosolic fraction. The degradation of calpastatin was blocked by administration of CX295, a calpain inhibitor, indicating that calpastatin acts as a suicide substrate to calpain during hypoxia-ischemia. In summary, calpastatin was up-regulated in areas that remain undamaged and degraded in areas where excessive activation of calpains and infarction occurs.     

Occurrence of sea eels of <Emphasis Type="Italic">Anguilla japonica</Emphasis> along the Sanriku Coast of Japan

 The age and migratory history of the Japanese eel, Anguilla japonica, collected along the Sanriku Coast of Japan, were examined using otolith microstructure and analysis of strontium (Sr) and calcium (Ca) concentrations. The mean Sr : Ca ratios from the elver mark to the otolith edge indicated that there were eels with several general categories of migratory history, including sea eels that never entered freshwater and others which had entered freshwater for brief periods but returned to the estuary or bay. This first evidence of the occurrence of sea eels in this northern area indicates that Japanese eels of the Sanriku Coast do not necessarily migrate into freshwater rivers. Received: May 15, 2002 / Revised: August 4, 2002 / Accepted: August 15, 2002 Acknowledgments We thank Messrs. S. Yamane and K. Morita, and crews of the Otsuchi Marine Research Center, Ocean Research Institute, The University of Tokyo, for their assistance in collecting the eels. This work was supported in part by Grant-in-Aid No. 13760138 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:Takaomi Arai     

Migratory history and habitat use by New Zealand freshwater eels <Emphasis Type="Italic">Anguilla dieffenbachii</Emphasis> and <Emphasis Type="Italic">A. australis</Emphasis>, as revealed by otolith microchemistry

 The migratory history of Anguilla dieffenbachii and A. australis, collected from a coastal lake of New Zealand, was examined using analysis of strontium (Sr) and calcium (Ca) concentrations. Line analysis of Sr : Ca ratios along the life history transect of each otolith showed a peak (Ca. 16–20 × 10⁻³) between the core and elver mark, which corresponded to the period of their leptocephalus and early glass eel stages in the ocean. The mean Sr : Ca ratios from the elver mark to the otolith edge indicated that eels had different migratory histories, which included freshwater residency in some eels (average Sr : Ca ratios, 1.7 × 10⁻³–2.4 × 10⁻³) but not in others (average Sr : Ca ratios, 3.1 × 10⁻³–6.5 × 10⁻³). These findings suggest that New Zealand freshwater eels have a flexible migration strategy and an ability to adapt to various habitats and salinities. Received: November 25, 2002 / Revised: January 17, 2003 / Accepted: January 17, 2003     

Identifying sea-run brown trout, Salmo trutta, using Sr : Ca ratios of otolith

 The migratory history of the brown trout, Salmo trutta, collected from Japanese rivers, was examined in terms of strontium (Sr) and calcium (Ca) uptake in the otolith, by means of wavelength dispersive X-ray spectrometry on an electron microprobe. Sea-run (anadromous) and freshwater-resident (nonanadromous) types of S. trutta were found to occur sympatrically. Otolith Sr concentration or Sr : Ca ratios of anadromous S. trutta fluctuated strongly along the life history transect in accordance with the migration (habitat) pattern from sea to freshwater. In contrast, the Sr concentration or the Sr : Ca ratios of nonanadromous fish remained at consistently low levels throughout the otolith. The higher ratios in anadromous S. trutta, in the otolith region from the core to 1500 μm, corresponded to the initial seagoing period, probably reflecting the ambient salinity or the seawater–freshwater gradient in Sr concentration. The findings clearly indicated that otolith Sr : Ca ratios reflected individual life histories, enabling the sea-run S. trutta to be distinguished from the freshwater-resident brown trout. Received: March 18, 2002 / Revised: May 15, 2002 / Accepted: June 5, 2002 Acknowledgments The authors are grateful to Messrs. T. Ikeda, S. Kudo, Y. Miyakoshi, M. Nagata, K. Shimoda, T. Takami, K. Takeuchi, and M. Ueda for their assistance in collecting specimens. This work was supported in part by Grant-in-Aid No. 13760138 from the Ministry of Education, Culture, Sports, Science and Technology, Japan. Correspondence to:Takaomi Arai     

Variations in the migratory history of the tropical catadromous eels Anguilla bicolor bicolor and A. bicolor pacifica in south-east Asian waters

Fish movements between aquatic habitats of different salinity ranges (fresh, estuarine, marine) by the tropical catadromous eels Anguilla bicolor bicolor and A. bicolor pacifica were examined by analysing the otolith strontium and calcium concentrations of yellow (immature) and silver (mature) stage eels collected in south-east Asian (Indonesia, Malaysia and Vietnam) waters. The ratios suggest that all migratory-type eels, including freshwater, brackish water and marine residents, pass the river mouth. However, the habitat preference was different among the sites (countries). In Indonesia and Vietnam, most A. bicolor bicolor and A. bicolor pacifica were either marine or brackish water residents in this study. Alternatively, most A. bicolor bicolor were freshwater residents in Malaysia; such a typical catadromous migration pattern in these eels has not been found in previous studies. The wide range of otolith Sr:Ca in both subspecies indicates that the habitat use of these tropical eels was opportunistic among fresh, brackish and marine waters during their growth phases following recruitment to coastal areas. The geographical variability of migratory histories suggests that habitat use might be determined by the inter and intraspecific competition and environmental conditions at each site.