Nutritional diagnosis of phytoplankton in Lake Baikal

To diagnose the nutritional status of phytoplankton in Lake Baikal, surveys for the determination of concentrations of particulate carbon (PC), nitrogen (PN) and phosphorus (PP) and their ratios were conducted at six stations in March, June, August and October 1999. The concentrations of PC and PN were lower than, and those of PP were similar to, those in another mesotrophic lake except at the station near the mouth of the largest input river, Selenga River, of Lake Baikal. The PC : PN : PP ratio was 102 : 13 : 1, considerably close to the Redfield ratio. The ratio was constant against spatiotemporal changes. These indicate that phytoplankton in Lake Baikal were exposed to no deficiency in nitrogen nor phosphorus. From a viewpoint of the nutritional status of phytoplankton, Lake Baikal might be viewed as an ocean rather than as a lake.     

Alzheimer's β-secretase cleaves a glycosyltransferase as a physiological substrate

Alzheimer's beta-secretase (BACE1) is a membrane-bound protease that cleaves the amyloid precursor protein (APP) in the trans-Golgi network, an initial step in the pathogenesis of Alzheimer's disease. Although BACE1 is distributed among various tissues including brain, its physiological substrate other than APP have not been identified. We have recently found that when BACE1 was overexpressed in COS cells together with α2,6-sialyltransferase (ST6Gal I), the secretion of ST6Gal I markedly increased, suggesting that BACE1 cleaves ST6Gal I as a physiological substrate. Thus BACE1 is the first identified protease that is responsible for the cleavage and secretion of glycosyltransferases. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Growth history and migration of the threespine stickleback <Emphasis Type="Italic">Gasterosteus aculeatus</Emphasis> in Otsuchi Bay,northeastern Japan

 Both growth and migratory history of the Pacific Ocean forms of the threespine stickleback Gasterosteus aculeatus collected in Otsuchi Bay, northeastern Japan, were examined using otolith microstructure and analysis of strontium (Sr) and calcium (Ca) concentrations with wavelength dispersive X-ray spectrometry by an electron microprobe. Age of the juveniles (21.6–25.9 mm in total length) examined ranged from 101 to 128 days (115 ± 8.5 days; mean ± SD), hatching being estimated as having occurred between March and April 2001. The Sr : Ca ratios in the otoliths changed with both ontogenic development and salinity of the habitat. The otolith Sr : Ca ratios increased gradually from 4.1 × 10⁻³ around the core to 7.5 × 10⁻³ around the edge of the otolith. The fluctuation pattern of otolith Sr : Ca ratios was different from those observed in both freshwater resident and anadromous forms in previous studies. These results suggested that the fish sampled spend their lives in the estuarine and sea environment without freshwater life after hatching. Received: June 5, 2002 / Revised: September 11, 2002 / Accepted: September 24, 2002 Acknowledgments We thank Mr. K. Morita and crews of the Otsuchi Marine Research Center, Ocean Research Institute, The University of Tokyo 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     

High-level expression and bulk crystallization of recombinant l-methionine γ-lyase, an anticancer agent

l-Methionine γ-lyase is a pyridoxal 5′-phosphate-dependent enzyme which has tumor selective anticancer activity. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. The plasmid was optimized with a promoter and the region of the ribosome-binding site. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-l fermentor. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-l crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.     

The Tottori (D7N) and English (H6R) familial Alzheimer disease mutations accelerate Abeta fibril formation without increasing protofibril formation

A subset of Alzheimer disease cases is caused by autosomal dominant mutations in genes encoding the amyloid beta-protein precursor or presenilins. Whereas some amyloid beta-protein precursor mutations alter its metabolism through effects on Abeta production, the pathogenic effects of those that alter amino acid residues within the Abeta sequence are not fully understood. Here we examined the biophysical effects of two recently described intra-Abeta mutations linked to early-onset familial Alzheimer disease, the D7N Tottori-Japanese and H6R English mutations. Although these mutations do not affect Abeta production, synthetic Abeta(1-42) peptides carrying D7N or H6R substitutions show enhanced fibril formation. In vitro analysis using Abeta(1-40)-based mutant peptides reveal that D7N or H6R mutations do not accelerate the nucleation phase but selectively promote the elongation phase of amyloid fibril formation. Notably, the levels of protofibrils generated from D7N or H6R Abeta were markedly inhibited despite enhanced fibril formation. These N-terminal Abeta mutations may accelerate amyloid fibril formation by a unique mechanism causing structural changes of Abeta peptides, specifically promoting the elongation process of amyloid fibrils without increasing metastable intermediates.     

Combined effects of social pressure from the inside and outside of fishing communities promote cooperation for stock management

We analyze a coupled socioeconomic and bioeconomic dynamics model for fisheries management in a local community. In the model, fishermen choose to cooperate with the fisheries management rule or not at each time step, depending on the utilities of each option. The social dynamics is coupled with the dynamics of the stock biomass. Fishermen’s utility basically depends on their catch, and the utility function of the noncooperative option is penalized by a psychological punishment, “social pressure”, which is affected by both the fraction of cooperative players and stock abundance. We classify social pressure into two categories: pressure originating from the inside and from the outside of the community. We develop a utility function with these social pressures and analyze the coupled dynamics. Our results show that all fishermen can end up cooperating with the management rule if both sources of internal and external social pressure have sufficient effect. The results imply that psychological aspects on fishermen can have an important role for successful management of local fisheries.     

Identification and synthesis of a heptapeptide in uremic fluid

A heptapeptide isolated from uremic fluid was synthesized by conventional method. The total amino acid sequence of this peptide was deduced as follows: H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH. Structural similarity was soon realized between this peptide and heptapeptde moiety corresponding to position 13 through 19 of β₂-microglobulin.     

The production-to-respiration ratio and its implication in Lake Biwa,Japan

Production-to-respiration (P:R) ratio was estimated at an offshore site of Lake Biwa in order to examine whether the plankton and benthic community is subsidized with allochthonous organic carbon, and to clarify the role of this lake as potential source or sink of carbon dioxide. The respiration rate of protozoan and metazoan plankton was calculated from their biomass and empirical equations of oxygen consumption rates, and that of bacterioplankton was derived from their production rate and growth efficiency. In addition, the carbon mineralization rate in the lake sediments was estimated from the accumulation rate of organic carbon, which was determined using a ²¹⁰Pb dating technique. On an annual basis, the sum of respiration rates of heterotrophic plankton was comparable to net primary production rate measured by the ¹³C method. However, when the mineralization rate in the lake sediments was included, the areal P:R ratio was 0.89, suggesting that Lake Biwa is net heterotrophic at the offshore site with the community being subsidized with allochthonous organic carbon. Such a view was supported by the surface water pCO₂ that was on average higher than that of the atmosphere. However, the estimate of net CO₂ release rate was close to that of carbon burial rate in the sediments. The result suggests that the role of Lake Biwa in relation to atmospheric carbon is almost null at the offshore site, although the community is supported partially by organic carbon released from the surrounding areas.     

Distinct mechanistic roles of calpain and caspase activation in neurodegeneration as revealed in mice overexpressing their specific inhibitors

Enzymatic proteolysis has been implicated in diverse neuropathological conditions, including acute/subacute ischemic brain injuries and chronic neurodegeneration such as Alzheimer disease and Parkinson disease. Calcium-dependent proteases, calpains, have been intensively analyzed in relation to these pathological conditions, but in vivo experiments have been hampered by the lack of appropriate experimental systems for a selective regulation of the calpain activity in animals. Here we have generated transgenic (Tg) mice that overexpress human calpastatin, a specific and the only natural inhibitor of calpains. In order to clarify the distinct roles of these cell death-associated cysteine proteases, we dissected neurodegenerative changes in these mice together with Tg mice overexpressing a viral inhibitor of caspases after intrahippocampal injection of kainic acid (KA), an inducer of neuronal excitotoxicity. Immunohistochemical analyses using endo-specific antibodies against calpain- and caspase-cleaved cytoskeletal components revealed that preclusion of KA-induced calpain activation can rescue the hippocampal neurons from disruption of the neuritic cytoskeletons, whereas caspase suppression has no overt effect on the neuritic pathologies. In addition, progressive neuronal loss between the acute and subacute phases of KA-induced injury was largely halted only in human calpastatin Tg mice. The animal models and experimental paradigm employed here unequivocally demonstrate their usefulness for clarifying the distinct contribution of calpain and caspase systems to molecular mechanisms governing neurodegeneration in adult brains, and our results indicate the potentials of specific calpain inhibitors in ameliorating excitotoxic neuronal damages.     

Calpain mediates excitotoxic DNA fragmentation via mitochondrial pathways in adult brains: evidence from calpastatin mutant mice

Calpain has been implicated in excitotoxic neurode-generation, but its mechanism of action particularly in adult brains remains unclear. We generated mutant mice lacking or overexpressing calpastatin, the only solely calpain-specific inhibitor ever identified or synthesized. Modulation of calpastatin expression caused no defect in the mice under normal conditions, indicating that calpastatin functions as a negative regulator of calpain only under pathological conditions. Kainate-evoked excitotoxicity in hippocampus resulted in proteolytic activation of a proapoptotic Bcl-2 subfamily member (Bid), nuclear translocation of mitochondria-derived DNA fragmentation factors (apoptosis-inducing factor and endonuclease G), DNA fragmentation, and nuclear condensation in pyramidal neurons. These apoptotic responses were significantly augmented by calpastatin deficiency. Consistently calpastatin overexpression suppressed them. No evidence of caspase-3 activation was detected. Our results demonstrated that calpain mediates excitotoxic signals through mobilization of proapoptotic factors in a caspase-independent manner. These mutant mice will serve as useful tools for investigating calpain involvement in various diseases.