A role for cyclin D3 in the endomitotic cell cycle.

Platelets, essential for thrombosis and hemostasis, develop from polyploid megakaryocytes which undergo endomitosis. During this cell cycle, cells experience abrogated mitosis and reenter a phase of DNA synthesis, thus leading to endomitosis. In the search for regulators of the endomitotic cell cycle, we have identified cyclin D3 as an important regulatory factor. Of the D-type cyclins, cyclin D3 is present at high levels in megakaryocytes undergoing endomitosis and is markedly upregulated following exposure to the proliferation-, maturation-, and ploidy-promoting factor, Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the platelet lineage display a striking increase in endomitosis, similar to changes seen following Mpl ligand administration to normal mice. Electron microscopy analysis revealed that unlike such treated mice, however, D3 transgenic mice show a poor development of demarcation membranes, from which platelets are believed to fragment, and no increase in platelets. Thus, while our model supports a key role for cyclin D3 in the endomitotic cell cycle, it also points to the unique role of Mpl ligand in priming megakaryocytes towards platelet fragmentation. The role of cyclin D3 in promoting endomitosis in other lineages programmed to abrogate mitosis will need further exploration.     

Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.     

Characterization of 2'':3''-Cyclic Nucleotide 3''-Phosphodiesterase: Rapid Isolation, Native Enzyme Analysis, Identification of a Serum-Soluble Activity, and Kinetics

The enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was isolated from bovine brain white matter by a rapid (72 h) procedure. The minimum molecular weight (MW) of the enzyme was approximately 52,500 as estimated by sucrose density gradient analysis. When this isolated enzyme was stimulated with bovine serum albumin (BSA), the peak of activity was shifted to approximately 90,000 MW. Prior treatment by trypsin blocked the expression of the higher MW form of CNPase, but not the BSA activation of the enzyme. If the trypsin digestion was allowed to progress, the MW was gradually lowered to a broad peak sedimenting between 20,000 and 50,000 MW. An apparently soluble form of CNPase found in serum is described. Kinetic and MW comparisons between the serum soluble enzyme and CNPase isolated from bovine brain, as well as an analysis of substrate specificity, were made and it was concluded that the two enzymes were identical.     

Characterization of 2'':3''-Cyclic Nucleotide 3''-Phosphodiesterase: Limited Proteolytic Digestion, Plant Lectin Affinity Chromatography, and Immunological Identification

Purified bovine brain 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) migrates as a protein double band in SDS-polyacrylamide gel electrophoresis. The positions of the two protein bands correspond to approximate molecular weights (MW) of 56,000 and 53,000. Limited protease treatment of isolated CNPase leads to subsequent degradation of the enzyme into smaller polypeptides having MWs of approximately 40,000, 30,000, and 20,000. During proteolytic digestion CNPase remains enzymatically active. Binding studies with several immobilized plant lectins as well as periodic acid-Schiff reagent (PAS) staining of SDS gels indicate that CNPase is a glycoprotein. An antiserum against purified CNPase, prepared in rabbits, was used to confirm the immunological identity of various CNPase preparations obtained in our laboratory.     

Ultrastructural analysis of acute immune thrombocytopenia in mice: dissociation between alterations in megakaryocytes and platelets

Thrombopoiesis was studied in mice after the induction of acute immune thrombocytopenia with platelet antiserum (PAS). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 4, 8, 12, 24, 48, 72, and 120 hr after administration of PAS. Four to 24 hr after injection of PAS, the majority of bone marrow MK were normal in size and organelle distribution. The demarcation membrane system (DMS) extended normally throughout the mature cell cytoplasm at these times. However, approximately 50% of MK observed 48 hr and 72 hr after injection of PAS were significantly larger than normal, and often had demarcation membranes confined to an area between a peripheral organelle-deficient zone and a central nuclear zone. The median values for sectional areas of platelets obtained 8-72 hr after administration of PAS were significantly greater than the median value for sectional areas of platelets in a pooled control sample. The proportion of cytoplasm to surface-connected canalicular system appeared greater than normal in most large platelets from the PAS samples; and increased numbers of profiles of Golgi complex and endoplasmic reticulum were observed. By 48 hr post-injection of PAS (at which time the modal ploidy class of MK has shifted from 16N to 32N; Corash et al., Blood 70:177, 1987), most platelets were normal in size and cytoplasmic appearance. At 120 hr post-injection of PAS, virtually all platelets exhibited a normal size and complement of organelles, and MK also had returned to normal. Our data indicate that in response to acute thrombocytopenia, MK prematurely release platelets which differ from normal platelets in size and cytoplasmic appearance. There was a marked dissociation between alterations in platelets and MK, since a statistically significant increase in platelet sectional area occurred 40 hr before the shift in modal ploidy class of MK, and platelet size subsequently decreased toward normal during the period that has been shown to be associated with the maximum shift in MK ploidy. These results strongly suggest that the characteristics of platelet release do not depend on the ploidy or cytoplasmic characteristics of MK.     

Transamidase kinetics. Amide formation in the enzymic reactions of thiol esters with amines.

1. Beta-Phenylpropionylthiocholine and N-(5-aminopentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylcadaverine) serve as a pair of water-soluble (pH7.5) model substrates for transamidating enzymes. Amide formation could be followed directly through fluorescence measurements by monitoring the continuous extraction of the water-soluble coupling product, N-(beta-phenylpropionyl)dansylcadaverine, into n-heptane. By this procedure, the steady-state kinetics of glutamine-lysine endo-gamma-glutamyltransferase from human plasma (fibrinoligase, thrombin- and Ca2+-activated blood coagulation Factor XII) and from guinea-pig liver (liver transglutaminase) were investigated at 25 degrees C. 2. With beta-phenylpropionylthiocholine as the varied substrate, Lineweaver-Burk plots with various concentrations of dansylcadaverine intercept on the horizontal axis, suggesting that formation of the acyl-enzyme is rate limiting. 3. On the basis of functional normality of active sites, kcat. values of 1.8 s(-1) and 0.9 s(-1) were obtained for the plasma and liver gamma-glutamyltransferase respectively. The two enzymes show identical affinities for the first substrate, beta-phenylpropionylthiocholine, with Ka 4 times 10(-4) M. 4. Utilization of the second substrate, dansylcadaverine, appears to be an order of magnitude more efficient with the liver enzyme. 5. N-(5-Amino-3-thiapentyl)-5-dimethylaminonaphthalene-1-sulphonamide (dansylthiacadaverine) could be used instead of dansylcadaverine in the fluorescent kinetic system. 6. Competitive inhibition by a non-fluorescent amine substrate histamine was also evaluated.     

KinMutBase, a database of human disease-causing protein kinase mutations.

KinMutBase (http://www.uta.fi/laitokset/imt/KinMut Base.html) is a registry of mutations in human protein kinases related to disorders. Kinases are essential cellular signalling molecules, in which mutations can lead into diseases including, e.g., immunodeficiencies, cancers and endocrine disorders. The first release of KinMutBase contains information for nine protein tyrosine kinases. There are altogether 170 entries representing 273 families and 403 patients. Mutations appear both in conserved hallmark residues of the kinases as well as in non-homologous sites. The KinMutBase WWW pages provide plenty of information, namely mutation statistics and display, clickable sequences with mutations, restriction enzyme patterns and online submission.     

Asynchronous population dynamics of Siberian lemmings across the Palaearctic tundra

The synchrony of Siberian lemming (Lemmus sibiricus L.) population dynamics was investigated during a ship-borne expedition along the Palaearctic tundra coast in the summer of 1994. On 12 sites along the coast from the Kola Peninsula to Wrangel Island, relative densities of lemmings were recorded using a standardised snap-trapping programme. The phase position of the lemming cycle in each of the studied populations was determined based on current density estimates, signs of previous density and the age profile of each population (ageing based on eye lens mass). In addition, dendrochronological methods were used to determine when the last peak in the density of microtine populations occurred at each site. The examined lemming populations were in different phases of the lemming cycle. Some populations were in the peak phase, as indicated by high current densities, an age profile in which older individuals were well represented, and signs of high previous density (abundant old lemming faeces). Other populations were in the decline phase, as reflected in a moderate current density, a predominance of older individuals and signs of high previous density. Populations in the low phase had an extremely low current density and showed signs of high previous density, while populations in the increase phase had a moderate current density, a predominance of younger individuals and showed signs of low previous density. The results of phase determinations based on dendrochronological methods support the findings based on lemming demography. Recent Russian studies carried out on some of the sites also agreed with our phase determination results. Thus, on a regional scale (across the whole Palaearctic tundra), the population dynamics of Siberian lemmings can be considered asynchronous. However, sites situated adjacent to each other were often phase synchronous, suggesting a more fine-grained pattern of dynamics with synchrony over distances as long as 1000 km or so, e.g. the Yamal and Taymyr Peninsulas. Received: 19 August 1998 / Accepted: 1 March 1999