From oocyte maturation to the in vitro cell cycle: the history of discoveries of Maturation-Promoting Factor (MPF) and Cytostatic Factor (CSF)

This article briefly reviews the classical cell cycle studies using oocytes and zygotes of mainly amphibians in the past century. The discussions are focused on the investigations into the cytoplasmic factors that regulate meiosis during oocyte maturation and the initiation of mitosis during fertilisation, which were carried out in the author's lab between 1967 and 1987. This chronicle traces the development of the problems and the direction in which their solutions were attempted in the course of these investigations. The author tries to answer the following questions: why he decided to study oocyte maturation, how he discovered progesterone as a maturation-inducing hormone, how he discovered and characterised the cytoplasmic regulators of the cell cycle, Maturation-Promoting Factor (MPF) and Cyto-Static Factor (CSF), and how he invented the method of observing cell cycle processes in a cytoplasmic extract in vitro.     

Extensin Secreted into the Culture Medium by Tobacco Cells I. Purification and Some Properties

The medium of 12-day-old culturs of tobacco cells (Nicotianatabacum L., var Xanthi; line XD-6S) contain c.a. 160mg/literof protein, of which 14% of the constituent ami no acids werefound to be hydroxyproline. By sequential column chromatographiesand CsCl density-gradient centrifugation, a basic hydroxyproline-richglycoprotein was purified from the medium and found to havean amino acid composition typical of extensin; with a high levelof hydroxyproline (33mole%), tyrosine (13%), and lysine (14%).The glycoprotein contained 42% (w/w) of sugars, among whicharabinose was the major component (85%). The proportion of thisextensin in the proteins in the culture medium was estimatedto be much higher than that of arabino-galactan protein (about5 times higher) on a protein basis, with extensin comprisingbetween 25% and 41%, and probably about 37% of the proteinsin the medium. (Received September 19, 1988; Accepted December 26, 1988)     

Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Growth of mouse plasmacytoma cells in serum-free, hormone-supplemented medium: procedure for the determination of hormone and growth factor requirements for cell growth

The procedure of analyzing hormone and growth factor requirements for the growth of MPC-11 cells and of developing a serum-free medium for this cell line has been described. In this medium, MPC-11 cells grow as fast as in serum-supplemented medium, up to 50 generation. MPC-11 cells grown in serum-supplemented medium secrete IgG_2b and K light chain into the medium as they do in serum-containing medium.     

Amino acid substitution of mating factor of Saccharomyces cerevisiae structure-activity relationship.

Analogs of the mating factor of $\text{Saccharomyces cerevisiae}$, Trp¹- Trp³-Leu-Gln-Leu⁶-Lys⁷-Pro⁸-Gly-Gln-Pro¹¹-Met¹²-Tyr¹³, from which amino acids were eliminated or substituted for other amino acid, were synthesized. These analogs showed lower biological activity than the natural mating factor if assayed after 6 hours incubation with $\text{a}$-mating type cells of $\text{S. cerevisiae}$. However, if assayed after 24 or 48 hours incubation, the situation changed, i.e. the analogs in which Leu⁶ or Lys⁷ were replaced by the corresponding D-isomer, showed higher mating factor activity than the unsubstituted mating factor. The same result was obtained with the analogs in which Met was replaced by norleucine.     

Incorporation of [14C]glucose from UDP[14C]glucose into a phosphoglycolipid by cell-free particulate systems of a moderately halophilic gram-negative bacterium, Pseudomonas halosaccharolytica ATCC 29423.

A glucosyl group from uridine diphosphate [U-14C]glucose is incorporated into a phosphoglycolipid, probably a glucosylphosphatidylglycerol, by a disrupted membrane enzyme preparation from a gram-negative, moderately halophilic bacterium, Pseudomonas halosaccharolytica ATCC 29423. The conversion of [14C]phosphatidylglycerol into phosphoglycolipid by the particulate preparation was also enhanced in the presence of non-labelled UDP-glucose. A chemical degradation study of labelled phosphoglycolipid showed the bulk of the radioactivity from UDP[U-14C]glucose to be associated with the glucose moiety, which also appeared to be attached to the hydroxyl group of a second glycerol.     

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.     

灵井许昌人遗址第5层细石核工艺

本文研究的细石核出自灵井"许昌人"遗址第5层,该层为桔黄色粉细砂,2008-2013年发掘,出土细石核82件,其他还有与之相关的材料。该层碳十四年龄为13402±406BP。细石核素材一般为燧石质的石片、小砾石等。根据其毛坯形状、细石叶剥离进度等的差异,形成了角锥形、柱形、圆锥形等各种形状。在剥离石叶过程中,曾运用过台面修理、工作面上端(细石叶头部)修正、台面更新、台面转移等调整手法。灵井石器工业的细石叶工艺是一种以角锥形(型)细石核为主的技术。通过对比,灵井与华北各地的同类细石核大小尺寸接近,与本省临近的大岗、李家沟等细石器遗址应属相同或相近技术传统的细石叶工艺。