The Xenopus cdc2 protein is a component of MPF, a cytoplasmic regulator of mitosis

In Xenopus, a cytoplasmic agent known as MPF induces entry into mitosis. In fission yeast, genetic studies have shown that the cdc2 kinase regulates mitotic initiation. The 13 kd product of the suc1 gene interacts with the cdc2 kinase in yeast cells. We show that the yeast suc1 gene product (p13) is a potent inhibitor of MPF in cell-free extracts from Xenopus eggs. p13 appears to exert its antagonistic effect by binding directly to MPF. MPF activity is quantitatively depleted by chromatography on a p13 affinity column. Concomitantly, the Xenopus counterpart of the yeast cdc2 protein is adsorbed to the column. A 42 kd protein also binds specifically to the p13 affinity matrix. These findings suggest that the Xenopus cdc2 protein and the 42 kd protein are components of MPF.     

Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

RCC1, a regulator of mitosis, is essential for DNA replication.

Temperature-sensitive mutants in the RCC1 gene of BHK cells fail to maintain a correct temporal order of the cell cycle and will prematurely condense their chromosomes and enter mitosis at the restrictive temperature without having completed S phase. We have used Xenopus egg extracts to investigate the role that RCC1 plays in interphase nuclear functions and how this role might contribute to the known phenotype of temperature-sensitive RCC1 mutants. By immunodepleting RCC1 protein from egg extracts, we find that it is required for neither chromatin decondensation nor nuclear formation but that it is absolutely required for the replication of added sperm chromatin DNA. Our results further suggest that RCC1 does not participate enzymatically in replication but may be part of a structural complex which is required for the formation or maintenance of the replication machinery. By disrupting the replication complex, the loss of RCC1 might lead directly to disruption of the regulatory system which prevents the initiation of mitosis before the completion of DNA replication.     

Evidence that the G1-S and G2-M transitions are controlled by different cdc2 proteins in higher eukaryotes.

Xenopus eggs contain two distinct cdc2 homologs of 34 and 32 kd. We show that the 32 kd cdc2 protein, like the 34 kd protein, is a kinase. However, unlike the 34 kd homolog, the 32 kd cdc2 kinase activity does not decrease dramatically at the end of mitosis. The 32 kd protein does not associate with mitotic cyclins B1 and B2 but does associate with cyclin A and a novel doublet of proteins of 54 kd that may regulate its activity. We also show that depletion of the 32 kd cdc2 homolog from a Xenopus extract blocks DNA replication, but does not inhibit entry into mitosis. By contrast, depletion of the 34 kd cdc2 homolog or absence of mitotic cyclins from an extract does not inhibit replication, but does block entry into mitosis. Our results indicate that in higher eukaryotes, DNA replication (G1-S) and mitosis (G2-M) may be controlled by distinctly different cdc2 proteins.     

A Role for Cdk2 Kinase in Negatively Regulating DNA Replication during S Phase of the Cell Cycle

Using cell-free extracts made from Xenopus eggs, we show that cdk2-cyclin E and A kinases play an important role in negatively regulating DNA replication. Specifically, we demonstrate that the cdk2 kinase concentration surrounding chromatin in extracts increases 200-fold once the chromatin is assembled into nuclei. Further, we find that if the cdk2–cyclin E or A concentration in egg cytosol is increased 16-fold before the addition of sperm chromatin, the chromatin fails to initiate DNA replication once assembled into nuclei. This demonstrates that cdk2–cyclin E or A can negatively regulate DNA replication. With respect to how this negative regulation occurs, we show that high levels of cdk2–cyclin E do not block the association of the protein complex ORC with sperm chromatin but do prevent association of MCM3, a protein essential for replication. Importantly, we find that MCM3 that is prebound to chromatin does not dissociate when cdk2– cyclin E levels are increased. Taken together our results strongly suggest that during the embryonic cell cycle, the low concentrations of cdk2–cyclin E present in the cytosol after mitosis and before nuclear formation allow proteins essential for potentiating DNA replication to bind to chromatin, and that the high concentration of cdk2–cyclin E within nuclei prevents MCM from reassociating with chromatin after replication. This situation could serve, in part, to limit DNA replication to a single round per cell cycle.     

Free amino acids in brain, liver, and skeletal muscle tissue of voles infected with Trypanosoma brucei gambiense.

The concentrations of several acidic and neutral amino acids of brain, liver, and skeletal muscle were determined in field voles, Microtus montanus, and compared to values obtained from voles harboring a chronic infection of Trypanosoma brucei gambiense. All of the amino acids examined were found at comparable levels in brain tissue from both groups of animals with the exception of tyrosine, which was reduced by approximately 45% in the infected voles. Similarly, the only difference noted in liver tissue was 32% decrease of free tyrosine in the infected animals. With respect to muscle tissue, in addition to a 45% reduction of free tyrosine in the infected voles, decreases of a smaller magnitude were also noted for threonine, glutamate, and valine. The relatively specific alteration of free tyrosine concentrations in the investigated tissues of trypanosome-infected animals suggests an alteration in host metabolism of this amino acid and/or parasite utilization.     

The cell cycle dependence of protein synthesis during Xenopus laevis development.

The regulation of early embryonic development in the amphibian Xenopus laevis depends largely upon translational and post-translational regulatory mechanisms to direct the complex cytodifferentiations that take place during early cleavage and blastula formation. The cell cycle dependence of protein synthesis was examined in developing Xenopus embryos as well as in cycling cell-free lysates from Xenopus eggs. In both cases M-phase and the activation of the M-phase kinase were found to be correlated with an inhibition of translation. Translation in both the rough endoplasmic reticulum and cytosolic-free ribosomes were affected by this inhibition. Since elongation was found to be unaffected by M-phase, shifts in the polysome profiles during M-phase indicated that the inhibition affected initiation processes. The activity of the M-phase kinase may inhibit initiation through the modification of initiation factors or some other component during this process. The cell cycle dependence of translation may affect developmental mechanisms controlled by the titration of regulatory proteins.     

Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components

At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins.     

The influence of cell elastic modulus on inertial positions in Poiseuille microflows

Microchannels are used as a transportation highway for suspended cells both in vivo and ex vivo. Lymphatic and cardiovascular systems transfer suspended cells through microchannels within the body, and microfluidic techniques such as lab-on-a-chip devices, flow cytometry, and CAR T-cell therapy utilize microchannels of similar sizes to analyze or separate suspended cells ex vivo. Understanding the forces that cells are subject to while traveling through these channels are important because certain applications exploit these cell properties for cell separation. This study investigated the influence that cytoskeletal impairment has on the inertial positions of circulating cells in laminar pipe flow. Two representative cancer cell lines were treated using cytochalasin D, and their inertial positions were investigated using particle streak imaging and compared between benign and metastatic cell lines. This resulted in a shift in inertial positions between benign and metastatic as well as treated and untreated cells. To determine and quantify the physical changes in the cells that resulted in this migration, staining and nanoindentation techniques were then used to determine the cells’ size, circularity, and elastic modulus. It was found that the cells’ exposure to cytochalasin D resulted in decreased elastic moduli of cells, with benign and metastatic cells showing decreases of 135 ± 91 and 130 ± 60 Pa, respectively, with no change in either size or shape. This caused benign, stiffer cancer cells to be more evenly distributed across the channel width than metastatic, deformable cancer cells; additionally, a decrease in the elastic moduli of both cell lines resulted in increased migration toward the channel center. These results indicate that the elastic modulus may play more of a part in the inertial migration of such cells than previously thought.     

Polymorphism in NOD2, Crohn's disease, and susceptibility to pulmonary tuberculosis

The nucleotide oligomerization binding domain 2 gene (NOD2) encodes an intracellular receptor for bacterial components, which is expressed in monocytes and is associated with Crohn's Disease (CD). This finding, along with epidemiological evidence, supports a role for infection in the pathogenesis of CD. Speculation that mycobacteria are involved in CD led us to investigate NOD2 in susceptibility to tuberculosis (TB), a global public health problem caused by Mycobacterium tuberculosis. CD-associated NOD2 variants were absent in a case-control study of 640 Gambians, where CD is rare. Novel NOD2 promoter polymorphisms were identified but showed no association with TB in this African population sample.