1,242,466 research outputs found
Influence of Bcl-2 on cell death during the cultivation of a Chinese hamster ovary cell line expressing a chimeric antibody
No full textThe influence of Bcl-2 expression on the robustness of a CHO cell line (22H11) developed for the industrial production of a chimeric antibody was evaluated. Western blot analysis following transfection with the expression vector unexpectedly revealed upregulation of endogenous Bcl-2 expression in the control (Neo) cell line in response to exposure to the selection drug G418. This indicated that geneticin may function by inducing apoptosis in cells not carrying the control plasmid or expressing very low levels of survival genes. Thus, exposure to the drug enriched the culture for a population of cells which expressed enhanced levels of endogenous Bcl-2. In batch cultures, ectopic bcl-2 expression resulted in a 75% increase in maximum viable cell density over control cultures. Moreover, the rate of decrease in viability in the Bcl-2 cultures was significantly lower than that in the control cultures. After 18 days, the Bcl-2 viability was around 90%, compared to 20% in the control cultures. Evaluation of the mechanism of cell death revealed very few cells with classical apoptotic morphology. Around 10% were clearly necrotic, but the majority of dead cells were seen as chromatin free but otherwise relatively intact structures. Because of the relatively low rate of cell death in both cell lines, few cells were observed in the transitional, easily identifiable early stages of apoptosis. However, DNA gel electrophoresis revealed a clear ladder-pattern, but only in the control cultures, thus confirming high levels of apoptotic death. Antibody concentrations during both sets of cultures were very similar, both during the growth and death phases, with a maximum titer of around 40 mu g/ml. Analysis of Bcl-2 expression by flow cytometry revealed that the cultures contained two populations of cells: a large population which expressed high levels of Bcl-2 and a relatively smaller low-expressing population. During the course of the batch, the smaller, low-expressing population declined in frequency, suggesting that these cells were more sensitive to cell death. In addition, the mean level of Bcl-2 expression in the overexpressing population also declined significantly, presumably reflecting the exhaustion of precursors for protein synthesis following nutrient depletion. Importantly, when cells were taken from day 40 of the significantly extended Bcl-2 batch cultures, they immediately proliferated, confirming that they had retained their replicative potential. Cultivation of the cells in basal medium lacking (individually) serum, all amino acids, glutamate/asparagine, and, finally, glucose, resulted in relatively lower viable cell numbers and viability in the control cell line compared to the Bcl-2 cell line. Exposure of cells to ammonia toxicity also revealed the relative robustness of the bcl-2 transfected cells. When growth was arrested by treatment with 4 mM thymidine, Bcl-2 overexpressing cells exhibit a viability of over 80% after 5 days in culture, compared to only 40% in the control cell line. However, under growth-arrested conditions, there was no major difference in antibody titer between the two cell lines. (C) 2000 John Wiley & Sons, Inc
Bcl-2 mediated suppression of apoptosis in myeloma NS0 cultures
No full textThe influence of Bcl-2 expression on the suppression of apoptosis during the cultivation of an NS0 cell line expressing a chimeric antibody was investigated. Following selection of transfectants in medium containing G418, Western analysis revealed evidence of some up-regulation of endogenous Bcl-2 expression even in the control vector transfectants. Cultivation of the two cell lines in suspension batch cultures clearly demonstrated the enhanced robustness of the bcl-2 vector transfected cells. Suppression of apoptosis resulted in an approximately 20% increase in maximum viable cell number, and a doubling in culture duration compared to the control transfected cells. However, despite the significant affect on viability, Bcl-2 expression did not result in an increase in final antibody titre in comparison with the control cell line. Exposure of cells to various nutrient limited conditions further emphasised the influence of Bcl-2 on cell survival. After 3 days of exposure to serum, glucose, glutamate and asparagine deprivation, the viable cell number and viability were significantly higher in the bcl-2 transfected cell line. When control cells were deprived of all amino acids, there was a complete loss of viability and viable cell number within 3 days. By contrast, the bcl-2 transfected cell line retained greater than 75% of the initial viable cell number and about 70% viability. In response to exposure to 8 mM thymidine (a cytostatic agent) the control cell line underwent complete loss of viability and viable cell number after 6 days. This compared with 18 days for complete loss of viability in the bcl-2 transfected cell line. As under batch culture conditions, there was no difference between the two cell lines in final antibody titre, which indicated that MAb synthesis is limited by nutrient availability during the latter stages of culture in both cases. When fed batch cultures were carried out using a concentrated essential amino acid feed, the bcl-2 cell line exhibited a 60% increase in maximum viable cell number and a 50% increase in culture duration, when compared to the control cell line. Moreover, the bcl-2 cell line exhibited a greater than 40% increase in maximum antibody titre. (C) 2000 Elsevier Science B.V. All rights reserved
The unfolded protein response
No full textProtein folding is the rate-limiting step for secretion of recombinant proteins in nearly all expression systems. Overexpression of recombinant proteins easily exhausts the cellular protein folding machinery in the endoplasmic reticulum (ER). In this ER stress situation a signal transduction network, called the unfolded protein response (UPR), is activated. The UPR coordinates adaptive responses to this stress situation and, presumably in response to prolonged ER stress, induces apoptosis. A molecular understanding of the UPR will enable the engineering of expression systems with the aim to increase cellular productivities and to improve the cost effectiveness of these expression systems. In this paper we review our current molecular understanding of the UPR and its implications for the engineering of recombinant protein expression systems
PEMIKIRAN KH. MUHADJIRIN AMSAR AL-DARY (1924-2003) DALAM KITAB MISHB?H AL-DHAL?M: SYARH BUL?GH AL-MAR?M MIN ADILLAH AL-AHK?M
No full textThis paper examines the thoughts of KH. Muhadjirin Amsar Al-Dary in Mishb?h al-Dhal?m: Syarh Bul?gh al-Mar?m min Adillah al-Ahk?m. The book is one of the results of a phenomenal writing, when he studied in Makkah and Medina. This study uses the approach of Philology and Phenomenology, which are used as the analytical tool to obtain information from a text through the work of KH. Muhadjirin Amsar Ad-Dary (1924-2003) and to reveal facts in a certain time span based on the views of a group of people or someone who is considered representative. This study shows that the main characteristics and identity of KH. Muhadjirin’s work viewed from the systematic of Mishb?h al-Dhal?m: Syarh Bul?gh al-Mar?m min Adillah al-Ahk?m, very clearly visible. In the book, he presents several studies which describe the differences among several schools, especially the popular school of jurisprudence.
Keywords: syarh, kitab, KH. Muhadjirin
Uqūd al-zawāj عقود الزواج
No full textYaḥtawī ʻalá Khamsīn waraqah min nimrat 1 bi-tārīkh 22 Shaʻbān sanat 1309 li-nimrat 50 bi-tārīkh 24 Muḥarram 1310.
يحتوي على خمسين ورقة من نمرة 1 بتاريخ 22 شعبان سنة 1309 لنمرة 50 بتاريخ 24 محرم 1310.;
"" Maḥkamah al-Sharʻīyah -- Daftar zawāj Fum al-Maḥmūdaiyah -- nimrat al-daftar 59 -- Nimrat al-maḥfaẓah 8."" Cover title.
"" محكمة الشرعية دفتر زواج فم المحمودية نمرة الدفتر 59 نمرة المحفظة 8."";
The contracts has black an white color with stamps. All contracts are handwrittenMarriage contracts containing 50 papers from 1901-1903 in Egypt, Beheira Governorate, Ezab al-Takiyah. All contracts were completed at the Beheira Governorate Damanhur center
Uqūd al-zawāj عقود الزواج
No full textYaḥtawī ʻalá Khamsīn waraqah min nimrat 1 bi-tārīkh 22 Shaʻbān sanat 1309 li-nimrat 50 bi-tārīkh 24 Muḥarram 1310.
يحتوي على خمسين ورقة من نمرة 1 بتاريخ 22 شعبان سنة 1309 لنمرة 50 بتاريخ 24 محرم 1310.;
"" Maḥkamah al-Sharʻīyah -- Daftar zawāj Fum al-Maḥmūdaiyah -- nimrat al-daftar 59 -- Nimrat al-maḥfaẓah 8."" Cover title.
"" محكمة الشرعية دفتر زواج فم المحمودية نمرة الدفتر 59 نمرة المحفظة 8."";
The contracts has black an white color with stamps. All contracts are handwrittenMarriage contracts containing 50 papers from 1901-1903 in Egypt, Beheira Governorate, Ezab al-Takiyah. All contracts were completed at the Beheira Governorate Damanhur center
Uqūd al-zawāj عقود الزواج
No full textYaḥtawī ʻalá Khamsīn waraqah min nimrat 1 bi-tārīkh 22 Shaʻbān sanat 1309 li-nimrat 50 bi-tārīkh 24 Muḥarram 1310.
يحتوي على خمسين ورقة من نمرة 1 بتاريخ 22 شعبان سنة 1309 لنمرة 50 بتاريخ 24 محرم 1310.;
"" Maḥkamah al-Sharʻīyah -- Daftar zawāj Fum al-Maḥmūdaiyah -- nimrat al-daftar 59 -- Nimrat al-maḥfaẓah 8."" Cover title.
"" محكمة الشرعية دفتر زواج فم المحمودية نمرة الدفتر 59 نمرة المحفظة 8."";
The contracts has black an white color with stamps. All contracts are handwrittenMarriage contracts containing 50 papers from 1901-1903 in Egypt, Beheira Governorate, Ezab al-Takiyah. All contracts were completed at the Beheira Governorate Damanhur center
Uqūd al-zawāj عقود الزواج
No full textYaḥtawī ʻalá Khamsīn waraqah min nimrat 1 bi-tārīkh 22 Shaʻbān sanat 1309 li-nimrat 50 bi-tārīkh 24 Muḥarram 1310.
يحتوي على خمسين ورقة من نمرة 1 بتاريخ 22 شعبان سنة 1309 لنمرة 50 بتاريخ 24 محرم 1310.;
"" Maḥkamah al-Sharʻīyah -- Daftar zawāj Fum al-Maḥmūdaiyah -- nimrat al-daftar 59 -- Nimrat al-maḥfaẓah 8."" Cover title.
"" محكمة الشرعية دفتر زواج فم المحمودية نمرة الدفتر 59 نمرة المحفظة 8."";
The contracts has black an white color with stamps. All contracts are handwrittenMarriage contracts containing 50 papers from 1901-1903 in Egypt, Beheira Governorate, Ezab al-Takiyah. All contracts were completed at the Beheira Governorate Damanhur center
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