Biomedical subjects

D Beach

Publications and source records attributed to D Beach.

At least 109 records · Page 6Linked to original sources

PubMed1990

Direct activation of cdc2 with phosphatase: identification of p13suc1-sensitive and insensitive steps.

In Xenopus oocytes, activation of MPF during prophase-metaphase transition is associated with the tyrosine dephosphorylation of the cdc2 protein. In vivo and in cell-free extracts kinase activation can be inhibited by excess p13suc1, a subunit of the protein kinase. Here we have demonstrated that affinity-purified cdc2 from Xenopus prophase oocytes may be activated in vitro by exposure to potato acid phosphatase. In vitro, excess p13 does not inhibit tyrosine dephosphorylation of prophase cdc2, but nonetheless binds and prevents the activation of the enzyme. By contrast, fully activated enzyme from metaphase Xenopus eggs is insensitive to excess p13. These observations define a p13-sensitive state in the activation of fully active cdc2 that follows tyrosine dephosphorylation.

Animals↗

PubMed1990

A versatile microtiter assay for the universal cdc2 cell cycle regulator.

A microassay for p34cdc2 based on the high affinity association between cdc2 and Schizosaccharomyces pombe p13suc1 has been developed. p13 purified from Escherichia coli was immobilized on microtiter plates and cellular lysate was incubated in the wells to allow the binding of cdc2 and its associated proteins. p34cdc2 was assayed either as a histone kinase or by immunological methods. The method was optimized for S. pombe cell extracts but can also be applied to other organisms such as Xenopus oocytes or HeLa cells. This rapid assay allows the specific determination of p34cdc2 histone H1 kinase activity in a very large number of samples.

Animals↗

PubMed1990

Imaging of kinked configurations of DNA molecules undergoing orthogonal field alternating gel electrophoresis by fluorescence microscopy.

The dynamics of individual DNA molecules undergoing orthogonal field alternating gel electrophoresis (OFAGE) have been studied by use of T2 DNA molecules labeled with a dye and visualized with a fluorescence microscope. The mechanism of reorientation used by a molecule to align itself in the direction of the new orthogonal field depends on the degree of extension of the chain immediately before the application of this field. The formation of kinks is promoted when time is allowed between the application of the two orthogonal fields so that the molecule attains a partially relaxed configuration. In this case, the chain appears bunched up in domains moving along the contour of the molecule. These regions are found to be the locations where the kinks are formed upon application of the second field perpendicular to the chain. The formation of kinks provides a significant retardation of the reorientation of the molecules, relative to molecules that do not form kinks, and appears to play an important role in the fractionation attained with OFAGE. A classification of various reorientation mechanisms observed in molecules that form kinks is presented.

DNA↗

PubMed1990

Fission yeast cdc25 is a cell-cycle regulated protein.

Fission yeast cell division is initiated by the cdc2/cdc13-cyclin protein kinase which in its catalytically active state comprises the mitotic inducer. During interphase the cdc2/cyclin complex is assembled in an inactive state that requires cdc25+ gene function for M-phase activation. The cdc25+ product, a 76 kd phosphoprotein, is shown to oscillate in abundance during the cell cycle, reaching a peak at G2/M, and to be sensitive to nitrogen starvation. The level of cdc25 is subject to feedback regulation involving both cdc25 and cdc2.

Blotting, Western↗

PubMed1990

High linoleic acid diets ameliorate diabetic nephropathy in rats.

The value of high polyunsaturated fatty acid (PUFA) diets in preventing diabetic nephropathy in rats was studied. Diabetes was induced by intravenous injection of streptozotocin (SZ), 65 mg/kg. Rats were divided in four groups fed diets containing 11% fat for 38 weeks. Dietary fat derived from four sources: beef tallow (BT; rich in saturated fatty acids), evening primrose oil (EPO; rich in gamma linolenic [GLA] and linoleic acids [LA]), safflower oil (SO; rich in LA), and fish oil (FO; rich in eicosapentaenoic [EPA] and docosahexaenoic [DHA] acids). Ultralente insulin was administered every other day to maintain the blood glucose levels between 11.1 and 22.2 mmol/L (200 and 400 mg/dL). The diets prepared with EPO and SO had a clear beneficial effect on proteinuria, glomerular sclerosis, and tubular abnormalities, as compared with BT. Both diets also increased the ratio of renal cortical production of 6-keto-PGF1 alpha to thromboxane B2 (TXB2), the stable metabolites of PGI2 and TXA2, respectively. They did not induce significant changes in plasma lipid composition. The FO diet did not have an effect on renal disease, but decreased plasma lipids and inhibited eicosanoid synthesis by platelets and kidney cortex. FO feeding was associated with a lowered 6-keto-PGF1 alpha/TXB2 ratio. It is concluded that high LA diets are protective in this model of diabetic nephropathy. The effect may be secondary to modifications of the eicosanoid balance. Diets containing FO have a beneficial effect on plasma lipids in this model.

Animals↗

PubMed1990

Human p53 is phosphorylated by p60-cdc2 and cyclin B-cdc2.

The human anti-oncoprotein p53 is shown to be a substrate of cdc2. The primary site of phosphorylation is serine-315. Serine-315 is phosphorylated by both p60-cdc2 and cyclin B-cdc2 enzymes. The phosphorylation of p53 is cell cycle-dependent. The abundance of p53 also oscillates during the cell cycle. The protein is largely absent from cells that have just completed division but accumulates in cells during G1 phase. Phosphorylation by cdc2 might regulate the antiproliferative activity of p53.

Amino Acid Sequence↗

PubMed1990

Activation of M-phase-specific histone H1 kinase by modification of the phosphorylation of its p34cdc2 and cyclin components.

An M-phase-specific histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types undergoing the G2/M transition in the cell division cycle. We have used p13suc1-Sepharose affinity chromatography to purify H1K to near homogeneity from matured starfish oocytes. A yield of 67% was obtained. Active H1K behaves as a 90- to 100-kD protein and appears to be constituted of equimolar amounts of cyclin and p34cdc2. The p34cdc2 subunit becomes tyrosine-dephosphorylated as the H1K is activated during entry of the oocytes into M phase, whereas the cyclin subunit is reciprocally phosphorylated. Acid phosphatase treatment of inactive p34cdc2/cyclin complex induces p34cdc2 dephosphorylation and three- to eightfold stimulation of the enzyme activity. These results suggest that active M-phase-specific H1K is constituted of both dephosphorylated p34cdc2 and phosphorylated cyclin.

Acid Phosphatase↗

PubMed1990

Cell cycle regulation of p34cdc2 kinase activity in Physarum polycephalum.

The regulation of the mitotic histone H1 kinase activity has been analyzed during the naturally synchronous cell cycle of Physarum polycephalum plasmodia. The universal binding property of the p13suc1 Schizosaccharomyces pombe gene product was used to precipitate and assay the cdc2 histone H1 kinase activity. The kinase activity peaks at the beginning of metaphase and its decline, which requires protein synthesis, appears to be an early event during the metaphase process. Microtubular poisons, temperature shifts and DNA synthesis inhibitors were used to perturb cell cycle regulatory pathways and characterize their effects on cdc2 kinase activation. Our results suggest that the full activation of the mitotic kinase requires at least two successive triggering signals involving microtubular components and DNA synthesis.

Aphidicolin↗

PubMed1989

Phosphorylation of large tumour antigen by cdc2 stimulates SV40 DNA replication.

Simian virus 40 large tumour antigen (T) is a replication origin binding protein required for viral DNA synthesis. Unphosphorylated T antigen is deficient in promoting DNA replication in vitro but can be activated by phosphorylation at residue threonine 124 by the cdc2 protein kinase. This observation demonstrates that T is regulated by phosphorylation and provides a model for cdc2 function in the control of DNA replication.

Antigens, Polyomavirus Transforming↗

PubMed1989

A 60 kd cdc2-associated polypeptide complexes with the E1A proteins in adenovirus-infected cells.

p60 is a cellular protein that binds to the adenovirus E1A protein complex in virally infected or transformed human cells. In both infected and uninfected cells, p60 was found in a complex with the cdc2 protein kinase. Immune complexes containing p60 and cdc2 display a cell cycle-dependent histone H1 kinase activity that is most active in interphase. The previously described cdc2-p62/cyclin complex also acts as a histone H1 kinase but is maximally active in mitotic metaphase. The shift in the timing of activation of different cdc2-containing complexes suggests that each might play a distinct role in regulation of the cell cycle.

Adenovirus Early Proteins↗

PubMed1989

Reversible tyrosine phosphorylation of cdc2: dephosphorylation accompanies activation during entry into mitosis.

Tyrosine phosphorylation of cdc2 is regulated in the cell cycle of mouse 3T3 fibroblasts. Phosphotyrosine in cdc2 is detectable at the onset of DNA synthesis and becomes maximal in the G2 phase of the cell cycle. Quantitative tyrosine dephosphorylation of cdc2 occurs during entry into mitosis and no phosphotyrosine is detected during the G1 phase of the cell cycle. While increasing tyrosine phosphorylation of cdc2 correlates with the formation of a cdc2/p62 complex, the tyrosine phosphorylated cdc2 is inactive as a histone H1 kinase. cdc2 is fully dephosphorylated in its most active mitotic form, yet specific tyrosine dephosphorylation of interphase cdc2 in vitro is insufficient to activate the kinase. In vivo inhibition of tyrosine dephosphorylation by exposure of cells to a phosphatase inhibitor is associated with G2 arrest, which is reversible upon the removal of the phosphatase inhibitor. Tyrosine dephosphorylation of cdc2 may be one of a number of obligatory steps in the mitotic activation of the kinase.

Animals↗

PubMed1989

Involvement of a type 1 protein phosphatase encoded by bws1+ in fission yeast mitotic control.

Fission yeast cdc25+ and wee1+ interact genetically with cdc2+ in the regulation of cell division, respectively as a mitotic activator and inhibitor. cdc25+ is normally essential for mitosis, but this requirement is alleviated in a loss-of-function wee1 mutant background. A plasmid-borne sequence, other than wee1+, that causes a cdc25ts wee1- double mutant to revert to a temperature-sensitive cdc phenotype has been isolated. The gene carried by this plasmid is called bws1+ (for bypass of wee suppression). bws1+ also bypasses the ability of alleles of cdc2 that confer a wee phenotype (cdc2w) to suppress loss-of-function cdc25 mutants. The nucleotide sequence of bws1+ shows that the predicted protein shares 81% amino acid identity with the catalytic subunit of mammalian type 1 protein phosphatase. Thus a genetic screen that might have yielded a protein kinase (wee1+) uncovered a phosphatase that also appears to be involved in the pathway of mitotic control.

Alleles↗

PubMed1989

The cdc2 kinase is a nuclear protein that is essential for mitosis in mammalian cells.

A homolog of the fission yeast cdc2-encoded protein kinase (p34) is a component of M phase promoting factor in Xenopus oocytes. The homologous kinase in human HeLa cells is maximally active during mitosis, suggesting a mitotic role in mammalian somatic cells. This has been directly investigated by microinjection of anti-p34 antibodies into serum-stimulated rat fibroblasts. DNA synthesis was unaffected but cell division was quantitatively blocked in injected cells. Injection of antibodies against p13suc1, a component of the p34 kinase complex, did not block mitosis but caused mitotic abnormalities resulting in cells containing multiple micronuclei in the subsequent interphase. p34 localized in the nucleus during interphase. During mitosis, a fraction tightly associated with centrosomes. p13 was more evenly distributed between the nucleus and cytoplasm. These observations demonstrate that cdc2 is a nuclear and centrosomal protein that is required for mitosis in mammalian cells.

Animals↗

PubMed1989

Cdc2 protein kinase is complexed with both cyclin A and B: evidence for proteolytic inactivation of MPF.

In the clam, Spisula, two previously described proteins known as cyclin A and B display the unusual property of selective proteolytic degradation at the end of each mitosis. We show here that clam oocytes and embryos contain a cdc2 protein kinase. This protein kinase is a component of the M phase promoting factor (MPF) in frog eggs and the M phase-specific histone H1 kinase in starfish. Clam cdc2 is found in association with both cyclin A and B, probably not as a trimolecular association, but as separate cdc2/cyclin A and cdc2/cyclin B complexes. Clam cdc2 and the associated cyclins bind to p13suc1-Sepharose. The p13-bound complex, and also anti-cyclin A or B immunoprecipitates, each display cell cycle-dependent histone H1 kinase activity. We suggest that in addition to the cdc2 protein kinase, the cyclins are further components of the M phase promoting factor and that cyclin proteolysis provides the mechanism of MPF inactivation and thus exit from mitosis.

Animals↗

PubMed1989

Cyclin is a component of the sea urchin egg M-phase specific histone H1 kinase.

A so-called 'growth-associated' or 'M-phase specific' histone H1 kinase (H1K) has been described in a wide variety of eukaryotic cell types; p34cdc2 has previously been shown to be a catalytic subunit of this protein kinase. In fertilized sea urchin eggs the activity of H1K oscillates during the cell division cycle and there is a striking temporal correlation between H1K activation and the accumulation of a phosphorylated form of cyclin. H1K activity declines in parallel with proteolytic cyclin destruction of the end of the first cell cycle. By virtue of the high affinity of the fission yeast p13suc1 for the p34cdc2 protein, H1K strongly binds to p13-Sepharose beads. Cyclin, p34cdc2 and H1K co-purify on this affinity reagent as well as through several conventional chromatographic procedures. Anticyclin antibodies immunoprecipitate the M-phase specific H1K in crude extracts or in purified fractions. Sea urchin eggs appear to contain much less cyclin than p34cdc2, suggesting that p34cdc2 may interact with other proteins. These results demonstrate that cyclin and p34cdc2 are major components of the M-phase specific H1K.

Animals↗

PubMed1989

Effect of decidua angiogenic factors on experimental dermis allografts.

Meshed human dermis allografts containing human uterine angiogenic factor were implanted over full thickness surgical skin wounds in rats. Enhanced angiogenesis of the wound bed, three times more than that observed in control grafts, was found in experimental grafts at 3 days postimplantation. This was accompanied by a greater amount of granulation tissue formation, enhanced granulation tissue penetration into the dermal grafts and accelerated incorporation of the grafts. Angiogenesis of the wound bed regressed to control levels at 7 days postimplantation. The growth and penetration of the granulation tissue and the accelerated incorporation of the dermis grafts, however, continued ahead in the experimental grafts while necrosis appeared in the control grafts. The relevance of these results for acceleration of wound healing and improvement of the wound bed for subsequent application of cultured epidermal grafts is discussed. This method may be extended to the treatment of ulcers and burns.

Angiogenesis Inducing Agents↗

PubMed1989

Uterine angiogenic factor induces vascularization of collagen sponges in guinea-pigs.

The need for an adequate blood supply is of prime importance in successful skin grafting and in the take of keratinocyte cultures. Thus, the human uterine angiogenic factor (HUAF) extract, which induces neovascularization of the chorioallantoic membrane (CAM), was employed. The bioassay of HUAF was performed on an in vivo model of subdermally implanted collagen sponges and on sponges implanted into full skin thickness burn wounds in guinea-pigs. The HUAF extract was injected into the sponges every other day for 10 days. Each injection contained 10 micrograms decidual proteins with a total of 50 micrograms/sponge. The animals were sacrificed and the sponge together with the surrounding structures were extirpated, examined macro- and microscopically and by histological techniques. HUAF induced growth of blood vessels from the surrounding vascular bed into the implanted sponges. The angiogenesis was characterized by dense tortuous vessels with centripetal orientation. The control sponges exhibited only sporadic growth of blood vessels. This phenomenon repeated itself in the animals which were inflicted with burn wounds. The present study demonstrates that HUAF extract is also active on the in vivo model of experimental burns and wounds.

Angiogenesis Inducing Agents↗

PubMed1989

Activation of human CDC2 protein as a histone H1 kinase is associated with complex formation with the p62 subunit.

p34 kinase, the product of the CDC2 gene, is a cell-cycle regulated protein kinase that is most active during mitosis. In HeLa cells, p34 kinase has previously been shown to exist in both a low- and a high-molecular-mass form, the latter of which is only found in cells in the G2/M phase of the cell cycle and contains a 62-kDa subunit. Here we show that although each form of the kinase phosphorylates casein in vitro, only the high-molecular-mass form uses histone H1 as substrate. The high-molecular-mass form of p34 kinase from nocodazole-treated HeLa cells was purified 6700-fold. The apparent molecular mass of the mitotic CDC2-encoded protein kinase complex was 220 kDa. The purified enzyme phosphorylated not only its endogenous 62-kDa subunit but also phosphorylated histone H1 with a Km of 3 microM and used ATP 40 times more efficiently than GTP (Km 54 microM and 2 mM, respectively). The enzyme activity was unaffected by cAMP, calcium/calmodulin, or by the heat-stable inhibitor of cAMP-dependent protein kinase. These characteristics are typical of growth-associated histone H1 kinase from different organisms. These results suggest that CDC2 protein may be activated as an M-phase-specific protein kinase in part by its association with the p62 subunit.

CDC2 Protein Kinase↗