Disassembly of in vitro formed lamin head-to-tail polymers by CDC2 kinase.

The nuclear lamina is an intermediate filament-type network underlying the inner nuclear membrane. At the onset of mitosis it depolymerizes, presumably in response to phosphorylation of the lamin proteins. Recently, cdc2 kinase, a major regulator of the eukaryotic cell cycle, was shown to induce lamina depolymerization when incubated with isolated nuclei. Here, we have analysed the structural consequences of lamin phosphorylation by cdc2 kinase using lamin head-to-tail polymers reconstituted in vitro from bacterially expressed chicken lamin B2 protein as a substrate. The effects of phosphorylation were monitored by both a pelleting assay and electron microscopy. We show that lamin B2 head-to-tail polymers disassemble in response to phosphorylation of specific sites that are phosphorylated also during mitosis in vivo. These sites are located within SP/TP motifs N- and C-terminal to the central alpha-helical rod domain of lamin proteins. Subsequent dephosphorylation of these sites by purified phosphatase 1 allows reformation of lamin head-to-tail polymers. The relative importance of N- and C-terminal phosphorylation sites for controlling the assembly state of nuclear lamins was assessed by mutational analysis. Polymers formed of lamin proteins carrying mutations in the C-terminal phosphoacceptor motif could still be disassembled by cdc2 kinase. In contrast, a single point mutation in the N-terminal site (Ser16----Ala) rendered head-to-tail polymers resistant to disassembly. These results emphasize the importance of the N-terminal end domain for lamin head-to-tail polymerization in vitro, and they demonstrate that phosphorylation-dephosphorylation is sufficient to control the longitudinal assembly of lamin B2 dimers.     

Differential phosphorylation of vertebrate p34cdc2 kinase at the G1/S and G2/M transitions of the cell cycle: identification of major phosphorylation sites.

The cdc2 kinase is a key regulator of the eukaryotic cell cycle. The activity of its catalytic subunit, p34cdc2, is controlled by cell cycle dependent interactions with other proteins as well as by phosphorylation--dephosphorylation reactions. In this paper, we examine the phosphorylation state of chicken p34cdc2 at various stages of the cell cycle. By peptide mapping, we detect four major phosphopeptides in chicken p34cdc2; three phosphorylation sites are identified as threonine (Thr) 14, tyrosine (Tyr) 15 and serine (Ser) 277. Analysis of synchronized cells demonstrates that phosphorylation of all four sites is cell cycle regulated. Thr 14 and Tyr 15 are phosphorylated maximally during G2 phase but dephosphorylated abruptly at the G2/M transition, concomitant with activation of p34cdc2 kinase. This result suggests that phosphorylation of Thr 14 and/or Tyr 15 inhibits p34cdc2 kinase activity, in line with the location of these residues within the putative ATP binding site of the kinase. During M phase, p34cdc2 is also phosphorylated, but phosphorylation occurs on a threonine residue distinct from Thr 14. Finally, phosphorylation of Ser 277 peaks during G1 phase and drops markedly as cells progress through S phase, raising the possibility that this modification may contribute to control the proposed G1/S function of the vertebrate p34cdc2 kinase.     

Cyclic-AMP-dependent protein kinase type II is associated with the Golgi complex and with centrosomes

The subcellular distribution of the type II enzyme of cAMP-dependent protein kinase (cAMP-dPK II) in epithelial and fibroblastic cells was determined by indirect immunofluorescence microscopy. In interphase cells both regulatory (R II) and catalytic (C) subunits were concentrated in a perinuclear area. By comparison of the R II distribution with the location of a bona fide Golgi membrane constituent, this area was identified as the Golgi complex. The cytochemical localization of R II was confirmed by subcellular fractionation. In addition, cAMP-dPK II was associated with microtubule-organizing centers, in particular with mitotic spindle poles. These distributions of cAMP-dPK II probably represent important factors in mediating the effects of cAMP on basic cellular activities ranging from secretion and proliferation to cell shape and motility.     

A technique for normalizing centre of pressure paths

Centre of pressure paths may be expected to provide useful information in the analysis of gait. In this paper, a technique for normalizing and averaging centre of pressure paths is presented. It utilizes force data obtained from force platform measurements together with information about the placement of the foot on the force platform. This placement is described by specifying the location of the 'centre' and 'midline' of the foot which are defined for a footprint. The coordinate system associated with a footprint is defined and centre of pressure locations are determined in relation to this system. A comparison of the centre of pressure paths obtained using this method and the method proposed by Cavanagh [J. Biomechanics 11, 487-491 (1978)] shows significant differences. It is concluded that the selection of an appropriate technique to obtain centre of pressure paths is important.     

Drosophila Cdk8, a kinase partner of cyclin C that interacts with the large subunit of RNA polymerase II.

A number of cyclins have been described, most of which act together with their catalytic partners, the cyclin-dependent kinases (Cdks), to regulate events in the eukaryotic cell cycle. Cyclin C was originally identified by a genetic screen for human and Drosophila cDNAs that complement a triple knock-out of the CLN genes in Saccharomyces cerevisiae. Unlike other cyclins identified in this complementation screen, there has been no evidence that cyclin C has a cell-cycle role in the cognate organism. Here we report that cyclin C is a nuclear protein present in a multiprotein complex. It interacts both in vitro and in vivo with Cdk8, a novel protein-kinase of the Cdk family, structurally related to the yeast Srb10 kinase. We also show that Cdk8 can interact in vivo with the large subunit of RNA polymerase II and that a kinase activity that phosphorylates the RNA polymerase II large subunit is present in Cdk8 immunoprecipitates. Based on these observations and sequence similarity to the kinase/cyclin pair Srb10/Srb11 in S. cerevisiae, we suggest that cyclin C and Cdk8 control RNA polymerase II function.     

Immunological characterization of avian MAP kinases: evidence for nuclear localization.

The subcellular distribution and regulation of MAP kinase isoforms in chicken hepatoma DU249 cells was investigated with antibodies directed against peptides patterned after sequences in the mitogen-activated protein (MAP) kinases, sea star p44mpk, and rat p44erk1. MonoQ chromatography of cytosol from these cells afforded the resolution of at least four peaks of myelin basic protein (MBP) phosphotransferase activity, but only one of these (peak II) was stimulated in extracts from phorbol ester-treated cells. A 40- to 41-kDa (p41) doublet on Western blots detected with three different MAP kinase antibodies was coincident with peak II, and it probably corresponded to the avian homolog of p42mapk/erk2. Immunofluorescent studies with DU249 cells and chicken embryo fibroblasts revealed that most of the cross-reactive protein with at least two different MAP kinase antibodies was distributed in the nucleus. Subcellular fractionation studies confirmed a predominantly nuclear localization for p41 MAP kinase. Nocodazole arrest of DU249 cells was exploited for the detection of an M-phase-activated MBP kinase that was resolved from p41 MAP kinase by phenyl-Superose chromatography. Western blotting analysis with antibodies for the cdc2-encoded protein kinase and p13suc1-agarose binding studies allowed positive identification of this MBP kinase as p34cdc2.     

High-pressure liquid-solid chromatography of the ecdysones--insect molting hormones

A rapid, high-pressure liquid-solid Chromatographie technique was developed for the separation, detection and analysis of ecdy-steroids. The advantages in using this system for the identification of ecdysteroids in combination with NMR and mass spectrometry and its potential application in analyzing material from biological and synthetic, systems are presented.     

Pattern classification of kinematic and kinetic running data to distinguish gender,shod/barefoot and injury groups with feature ranking

The identification of differences between groups is often important in biomechanics. This paper presents group classification tasks using kinetic and kinematic data from a prospective running injury study. Groups composed of gender, of shod/barefoot running and of runners who developed patellofemoral pain syndrome (PFPS) during the study, and asymptotic runners were classified.

The features computed from the biomechanical data were deliberately chosen to be generic. Therefore, they were suited for different biomechanical measurements and classification tasks without adaptation to the input signals. Feature ranking was applied to reveal the relevance of each feature to the classification task.

Data from 80 runners were analysed for gender and shod/barefoot classification, while 12 runners were investigated in the injury classification task. Gender groups could be differentiated with 84.7%, shod/barefoot running with 98.3%, and PFPS with 100% classification rate. For the latter group, one single variable could be identified that alone allowed discrimination.     

Biodistribution of sodium borocaptate (BSH) for boron neutron capture therapy (BNCT) in an oral cancer model

Boron neutron capture therapy (BNCT) is based on selective accumulation of ¹⁰B carriers in tumor followed by neutron irradiation. We previously proved the therapeutic success of BNCT mediated by the boron compounds boronophenylalanine and sodium decahydrodecaborate (GB-10) in the hamster cheek pouch oral cancer model. Based on the clinical relevance of the boron carrier sodium borocaptate (BSH) and the knowledge that the most effective way to optimize BNCT is to improve tumor boron targeting, the specific aim of this study was to perform biodistribution studies of BSH in the hamster cheek pouch oral cancer model and evaluate the feasibility of BNCT mediated by BSH at nuclear reactor RA-3. The general aim of these studies is to contribute to the knowledge of BNCT radiobiology and optimize BNCT for head and neck cancer. Sodium borocaptate (50 mg ¹⁰B/kg) was administered to tumor-bearing hamsters. Groups of 3–5 animals were killed humanely at nine time-points, 3–12 h post-administration. Samples of blood, tumor, precancerous pouch tissue, normal pouch tissue and other clinically relevant normal tissues were processed for boron measurement by optic emission spectroscopy. Tumor boron concentration peaked to therapeutically useful boron concentration values of 24–35 ppm. The boron concentration ratio tumor/normal pouch tissue ranged from 1.1 to 1.8. Pharmacokinetic curves showed that the optimum interval between BSH administration and neutron irradiation was 7–11 h. It is concluded that BNCT mediated by BSH at nuclear reactor RA-3 would be feasible.