• Applied Microscopy
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• v.31 no.1
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• pp.19-35
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• 2001

Outlines for plasma $estradiol-17\beta$, components, electrophoretic patterns, and ultrastructural changes were obtained in female rainbow trout (Oncorhynchus mykiss) during the seasonal reproductive cycles. Plasma $estradiol-17\beta$ under the natural conditions, exhibited distinct seasonal variation, peaking very late in vitellogenic season during September, decreasing gradually the halt of spawning in December, and ultimately falling during the early stages of seasonal ovarian recrudescence in February and March. This change in $estradiol-17\beta$ appeared to stimulate vitellogenin production as evidenced by increases in plasma calcium, phosphorus, glucose, albumin and total protein levels. The electrophoretic patterns of late maturing or spawning oocytes were stained more intensively than those of late perinucleolus oocytes (molecular weights of approximately 70,000 and 200,000). Two protein bands were found in the SDS-PAGE separation, coincident with the $estradiol-17\beta$ hormone peak. Gonadosomatic indices (GSI) significantly increased from October to January, and showed the highest peak in January, coinciding with the numerically abrupt increase of ripe ova in female. A positive correlation (r=0.701, p<0.01) was established between plasma $estradiol-17\beta$ levels and the gonadosomatic index during the prespawning. The highest level of hepatosomatic index (HSI) observed in December. During the breeding season (December), the gonadotropes were large and filled with GTH-containing inclusions such as granules and globules. The vitellogenic phase began as late perinurleolus oocytes became transformed into early maturing oocytes through the accumulation of yolk, and oocytes reached the late maturing stages as the ooplasm was completely packed with yolk. Marked ultrastructural changed in the granulosa cells during nuclear migration involve the dilation of the rough endoplasmic reticulum and the appearance of the rod-shaped mitochondria with tubular cristae. Microvilli (finger-like projections), from the zona radiata and from the oocyte grew, and made contact with each other in the pore canals of the zona radials during vitellogenesis, but were withdrawn as the zona radiata became more compact and devoid of pore canals during oocyte maturation. The zona radiata grew to a tripartite structure such as an outer thin homogeneous layer, and two inner thick helicoidal layers (zona radials interna and zona radiata externa). Under the normal conditions, the ovarian follicle influenced the histological development and periodical secretion of the hormones , sufficient for a oogenesis and gonadal steroid production.

• Korean Journal of Microbiology
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• v.52 no.2
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• pp.192-201
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• 2016

Recently, Psychrobacter sp. ArcL13 strain showing the extracellular lipase activity was isolated from the Chuckchi Sea of the Arctic Ocean. However, due to the low expression levels of the enzyme in the natural strain, the production of recombinant lipase is crucial for various applications. Identification of the gene for the enzyme is prerequisite for the production of the recombinant protein. Therefore, in the present study, a novel lipase gene (ArcL13-Lip) was isolated from Psychrobacter sp. ArcL13 strain by gene prospecting using PCR, and its complete nucleotide sequence was determined. Sequence analysis showed that ArcL13-Lip has high amino acid sequence similarity to lipases from bacteria of some Psychrobacter genus (84-90%) despite low nucleotide sequence similarity. The lipase gene was cloned into the bacterial expression plasmid and expressed in E. coli. SDS-PAGE analysis of the cells showed that ArcL13-Lip was expressed as inclusion bodies with a molecular mass of about 35 kDa. Refolding was achieved by diluting the unfolded protein into refolding buffers containing various additives, and the highest refolding efficiency was seen in the glucose-containing buffer. Refolded ArcL13-Lip showed high hydrolytic activity toward p-nitrophenyl caprylate and p-nitrophenyl decanoate among different p-nitrophenyl esters. Recombinant ArcL13-Lip displayed maximal activity at $40^{\circ}C$ and pH 8.0 with p-nitrophenyl caprylate as a substrate. Activity assays performed at various temperatures showed that ArcL13-Lip is a cold-active lipase with about 40% and 73% of enzymatic activity at $10^{\circ}C$ and $20^{\circ}C$, respectively, compared to its maximal activity at $40^{\circ}C$.