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61.
This review outlines approaches to the cloning and expression of proteins in Escherichia coli. The expression vectors described here (pIN-III derivatives) utilize the strong lipoprotein promoter, which is controlled
by the lac-UV5 promoter-operator. These vectors provide the means for targeting a protein to any of the four subcellular compartments
of the bacterial cell: cytoplasm, cytoplasmic membrane, periplasm, and outer membrane. Of particular importance is that secretion
of proteins into the E. coli periplasm (using the OmpA signal peptide) is applicable for the production of both prokaryotic and eukaryotic proteins thereby
enhancing protein activity and stability. 相似文献
62.
We report a method for taking saliva samples from unrestrained, captive marmosets (Callithrix jacchus) to assess levels of free cortisol. Saliva samples can be obtained reliably, without any habituation, by encouraging the marmosets to lick and chew a cotton-wool bud coated in banana. Saliva is thus left on the bud. We also tested sweetened fruit-drink crystals and a number of other substances, but none of these attracted all of the marmosets, and even flavors that were effective once soon lost their attraction. The presence of banana in the samples collected was found to lower the measured concentration of cortisol; however, as shown in samples taken with and without the banana coating on the bud, it did so in a linear and consistent way, and did not vary significantly among subjects. Therefore, a simple conversion factor could be applied to correct for the presence of banana. A first experiment showed that the marmosets exhibited a rise in salivary cortisol levels in response to social isolation. A second experiment showed elevation of cortisol during a period when the marmosets were disturbed by increased human activity and noise levels in the building in which they were housed. Hence, this method of saliva sampling is a convenient, noninvasive means of assessing cortisol levels in marmosets. 相似文献
63.
In eukaryotes, mitosis is initiated by M phase promoting factor (MPF), composed of B-type cyclins and their partner protein kinase, CDK1. In animal cells, MPF is cytoplasmic in interphase and is translocated into the nucleus after mitosis has begun, after which it associates with the mitotic apparatus until the cyclins are degraded in anaphase. We have used a fusion protein between human cyclin B1 and green fluorescent protein (GFP) to study this dynamic behaviour in real time, in living cells. We found that when we injected cyclin B1-GFP, or cyclin B1-GFP bound to CDK1 (i.e. MPF), into interphase nuclei it is rapidly exported into the cytoplasm. Cyclin B1 nuclear export is blocked by leptomycin B, an inhibitor of the recently identified export factor, exportin 1 (CRM1). The nuclear export of MPF is mediated by a nuclear export sequence in cyclin B1, and an export-defective cyclin B1 accumulates in interphase nuclei. Therefore, during interphase MPF constantly shuttles between the nucleus and the cytoplasm, but the bulk of MPF is retained in the cytoplasm by rapid nuclear export. We found that a cyclin mutant with a defective nuclear export signal does not enhance the premature mitosis caused by interfering with the regulatory phosphorylation of CDK1, but is more sensitive to inhibition by the Wee1 kinase. 相似文献
64.
Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain 'locked' onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores. 相似文献
65.
Gur Pines 《FEBS letters》2010,584(12):2699-7079
The EGF-receptor is frequently mutated in a large variety of tumors. Here we review the most frequent mutations and conclude that they commonly enhance the intrinsic tyrosine kinase activity, or they represent loss-of-function of suppressive regulatory domains. Interestingly, the constitutive activity of mutant receptors translates to downstream pathways, which are subtly different from those stimulated by the wild-type receptor. Cancer drugs intercepting EGFR signaling have already entered clinical application. Both kinase inhibitors specific to EGFR, and monoclonal antibodies to the receptor are described, along with experimental approaches targeting the HSP90 chaperone. Deeper understanding of signaling pathways downstream to mutant receptors will likely improve the outcome of current EGFR-targeted therapies, as well as help develop new drugs and combinations. 相似文献
66.
In yeasts, the replication protein Cdc6/Cdc18 is required for the initiation of DNA replication and also for coupling S phase with the following mitosis. In metazoans a role for Cdc6 has only been shown in S phase entry. Here we provide evidence that human Cdc6 (HuCdc6) also regulates the onset of mitosis, as overexpression of HuCdc6 in G(2) phase cells prevents entry into mitosis. This block is abolished when HuCdc6 is expressed together with a constitutively active Cyclin B/CDK1 complex or with Cdc25B or Cdc25C. An inhibitor of Chk1 kinase activity, UCN-01, overcomes the HuCdc6 mediated G(2) arrest indicating that HuCdc6 blocks cells in G(2) phase via a checkpoint pathway involving Chk1. When HuCdc6 is overexpressed in G(2), we detected phosphorylation of Chk1. Thus, HuCdc6 can trigger a checkpoint response, which could ensure that all DNA is replicated before mitotic entry. We also present evidence that the ability of HuCdc6 to block mitosis may be regulated by its phosphorylation. 相似文献
67.
Garry G Sedgwick Daniel G Hayward Barbara Di Fiore Mercedes Pardo Lu Yu Jonathon Pines Jakob Nilsson 《The EMBO journal》2013,32(2):303-314
The Anaphase Promoting Complex/Cyclosome (APC/C) in complex with its co‐activator Cdc20 is responsible for targeting proteins for ubiquitin‐mediated degradation during mitosis. The activity of APC/C–Cdc20 is inhibited during prometaphase by the Spindle Assembly Checkpoint (SAC) yet certain substrates escape this inhibition. Nek2A degradation during prometaphase depends on direct binding of Nek2A to the APC/C via a C‐terminal MR dipeptide but whether this motif alone is sufficient is not clear. Here, we identify Kif18A as a novel APC/C–Cdc20 substrate and show that Kif18A degradation depends on a C‐terminal LR motif. However in contrast to Nek2A, Kif18A is not degraded until anaphase showing that additional mechanisms contribute to Nek2A degradation. We find that dimerization via the leucine zipper, in combination with the MR motif, is required for stable Nek2A binding to and ubiquitination by the APC/C. Nek2A and the mitotic checkpoint complex (MCC) have an overlap in APC/C subunit requirements for binding and we propose that Nek2A binds with high affinity to apo‐APC/C and is degraded by the pool of Cdc20 that avoids inhibition by the SAC. 相似文献
68.
O. Pines S. Shemesh E. Battat I. Goldberg 《Applied microbiology and biotechnology》1997,48(2):248-255
Saccharomyces cerevisiae accumulates l-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate
dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression
of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GAL10 or the constitutive PGK promoter causes a
6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in l-malic acid accumulation in the production medium. The high apparent K
m of MDH2 for l-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function
of the enzyme and differs from the previously published K
m of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be
a limiting factor, thus providing a system for further metabolic engineering of l-malic acid production. The overexpression of MDH2 activity also causes an elevation in the accumulation of fumaric acid and
citric acid. Accumulation of fumaric acid is presumably caused by high intracellular l-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing
possibility that cytosolic l-malic acid is a direct precursor of citric acid in yeast.
Received: 22 January 1997 / Received revision: 14 April 1997 / Accepted: 19 April 1997 相似文献
69.
The spindle assembly checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells from entering anaphase by inhibiting the ubiquitylation of cyclin B1 and securin by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator Cdc20. It is unclear how the SAC inactivates Cdc20 but most current models suggest that Cdc20 forms a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC, the APC/C ubiquitylates Cdc20 to target it for degradation. Thus, ubiquitylation of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest. 相似文献
70.