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Biomedical subjects

M Kirschner

Publications and source records attributed to M Kirschner.

At least 55 records · Page 3Linked to original sources
PubMed1987

Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo.

The primary patterning event in early vertebrate development is the formation of the mesoderm and its subsequent induction of the neural tube. Classic experiments suggest that the vegetal region signals the animal hemisphere to diverge from the pathway of forming ectoderm to form mesoderm such as muscle. Here we show that bovine basic FGF has a limited capacity to induce muscle actin expression in animal hemisphere cells. This level of expression can be raised to levels normally induced in the embryo by another mammalian growth factor, TGF-beta, which by itself will not induce actin expression. We show that the Xenopus embryo contains an mRNA encoding a protein highly homologous to basic FGF. These results together with the identification of a maternal mRNA with strong homology to TGF-beta, suggest that molecules closely related to FGF and TGF-beta are the natural inducers of mesoderm in vertebrate development.

Actins↗
PubMed1987

The events of the midblastula transition in Xenopus are regulated by changes in the cell cycle.

The midblastula transition (MBT) in Xenopus can be initiated prematurely by blocking the fundamental cell-cycle oscillator with cycloheximide, in which case motility and transcription are quickly initiated. Using various inhibitors of specific events of the cell cycle that do not inhibit the autonomous oscillator, we have shown that transcription is activated when DNA synthesis is interrupted and motility is activated when cell cleavage is inhibited. Furthermore, very low levels of transcription are found to occur before the MBT. These results demonstrate that the pre-MBT egg is fully competent for transcription and motility and suggest that different features of the rapid early cell cycle normally suppress these events.

Animals↗
PubMed1987

Phosphorylation changes associated with the early cell cycle in Xenopus eggs.

Enucleated and nondividing amphibian eggs undergo cyclic changes in cell morphology and in the level of maturation promoting factor (MPF) with a period similar to the early cleavage cycle. We show here that there is a corresponding phosphorylation and dephosphorylation of specific proteins associated with this fundamental cell cycle. M-phase is associated with a general increase in phosphatase activity and specific phosphorylation of a small set of M-phase proteins, reflected in an increased stochiometry of phosphate and increased turnover. At the end of metaphase and correlated with a drop in MPF the phosphoproteins are rapidly lost. By microinjecting M-phase phosphoproteins into arrested interphase and metaphase eggs we could show that the specific M-phase phosphorylation was not due to specificity in phosphatase action. The ability to segregate synthesis from phosphorylation demonstrates that regulation is not on the level of synthesis of the M-phase proteins. Taken together these data suggest that regulation of kinase activity in M-phase in the face of general rapid phosphate turnover in the egg plays an important role in the regulation of the fundamental mitotic cycle.

Adenosine Triphosphate↗
PubMed1987

Dynamic and stable populations of microtubules in cells.

Using a new immunocytochemical technique, we have visualized the spatial arrangement of those microtubules in cells that are stable to biotin-tubulin incorporation after microinjection. Cells fixed at various periods of time after injection were exposed to antibody to biotinylated tubulin and several layers of secondary antibodies; these layers prevented reaction of biotin-containing microtubules with antitubulin antibodies. The microtubules that had not incorporated biotin-tubulin could then be stained with anti-tubulin and a fluorescent secondary antibody. In BSC1 cells, most microtubules in the cell exchange with a half-time of 10 min. A separate population of microtubules can be detected, using the above techniques, that are stable to exchange for 1 h or more; these have a characteristic pericentrosomal spatial arrangement as compared to the majority of dynamic microtubules. Unlike the dynamic microtubules, most of the stable microtubules are nongrowing. The average BSC-1 cell contains approximately 700 microtubules: approximately 500 growing at 4 micron min-1, 100 shrinking at approximately 20 micron min-1, and approximately 100 that are relatively more stable to exchange. The potential significance of these stable microtubules is discussed.

Animals↗
PubMed1987

Posttranslational modification and microtubule stability.

We have probed the relationship between tubulin posttranslational modification and microtubule stability, using a variation of the antibody-blocking technique. In human retinoblastoma cells we find that acetylated and detyrosinated microtubules represent congruent subsets of the cells' total microtubules. We also find that stable microtubules defined as those that had not undergone polymerization within 1 h after injection of biotin-tubulin were all posttranslationally modified; furthermore dynamic microtubules were all unmodified. We therefore conclude that in these cells the stable, acetylated, and detyrosinated microtubules represent the same subset of the cells' total network. Posttranslational modification, however, is not a prerequisite for microtubule stability and vice versa. Potorous tridactylis kidney cells have no detectable acetylated microtubules but do have a sizable subset of stable ones, and chick embryo fibroblast cells are extensively modified but have few stable microtubules. We conclude that different cell types can create specific microtubule subsets by modulating the relative rates of posttranslational modification and microtubule turnover.

Animals↗
PubMed1986

Direct radioimmunoassay for human atrial natriuretic peptide (hANP) and its clinical evaluation.

A direct radioimmunoassay for the rapid and accurate detection of human ANP from unextracted plasma is described. The sensitivity was approximately 50 pg/ml, respectively 2.5 pg/tube, the intra-assay variation 4%, and the inter-assay variation less than 12%. Rat ANP (1-28, 5-25, 5-27 and 5-28), oxydized and reduced hANP as well as plasma samples from various patients run in parallel to the 1-28 hANP standard curve. These findings imply, that the antibody primarily recognizes the mid-region (amino acids 6-25) of the intact ANP, that the C-terminal portion further increases the immunoreactivity, and that circulating plasma hANP is reliably measured. Plasma hANP ranged from 50-166 pg/ml (mean +/- SD: 98.3 +/- 44.6) in healthy individuals, there was no significant difference between samples were drawn in upright or lying position, the apparent half-life of injected hANP was 5.65 minutes. Patients with liver cirrhosis revealed significantly higher hANP levels of 244.5 +/- 173.5 pg/ml. Patients with various forms of cardiac disease had hANP concentrations ranging from 50 to 1744 pg/ml, depending at least partially on the right atrial pressure. No difference was observed if the samples were drawn from either right or left intracardial locations. Our findings with this system demonstrate that hANP is reliably measured even without prior extraction.

Antibody Specificity↗
PubMed1986

Sites of microtubule assembly and disassembly in the mitotic spindle.

We have microinjected biotinylated tubulin into mitotic fibroblast cells to identify the sites in the spindle at which new subunits are incorporated into microtubules (MTs). Labeled subunits were visualized in the electron microscope using an antibody to biotin followed by a secondary antibody coupled to colloidal gold. Astral MTs incorporate labeled subunits very rapidly by elongation of existing MTs and by new nucleation from the centrosome. At a slower rate, kinetochore MTs incorporate subunits at the kinetochore progressively during metaphase, suggesting a slow poleward flux of subunits in the kinetochore fiber. When cells injected in metaphase were examined in anaphase, a significant fraction of kinetochore MTs was unlabeled, suggesting that depolymerization had occurred at the kinetochore concomitant with chromosome to pole movement. The existence of opposite fluxes at the kinetochore during metaphase and anaphase suggests that two separate forces are responsible for chromosome congression and anaphase movement.

Anaphase↗
PubMed1986

Direct observation of steady-state microtubule dynamics.

Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.

Alkanesulfonates↗
PubMed1986

Microtubule dynamics in interphase cells.

The sites of microtubule growth and the kinetics of elongation have been studied in vivo by microinjection of biotin-labeled tubulin and subsequent visualization with immunocytochemical probes. Immunofluorescence and immunoelectron microscopy demonstrate that injected biotin-labeled subunits are incorporated into new segments of growth which are contiguous with unlabeled microtubules. Rapid incorporation occurs by elongation of existing microtubules and new nucleation off the centrosome. The growth rate is 3.6 micron/min and is independent of the concentration of injected labeled tubulin. This rate of incorporation together with turnover of existing microtubules leads to approximately 80% exchange in 15 min. The observed kinetics and pattern of microtubule turnover allow for an evaluation of the relevance of several in vitro models for steady-state dynamics to the in vivo situation. We have also observed a substantial population of quasi-stable microtubules that does not exchange subunits as rapidly as the majority of microtubules and may have specialized functions in the cell.

Animals↗
PubMed1986

Morphogenesis and the control of microtubule dynamics in cells.

Microtubules show unusual dynamic properties at steady state in vitro. While overall the polymer mass remains stable, individual polymers in the population are either growing or shrinking. This phenomenon called dynamic instability is best explained by the known coupling of polymerization to GTP hydrolysis, and the hypothesis that the stability or instability of the whole polymer is determined by whether GTP or GDP is bound to the terminal subunit. Similar unusual dynamics have now also been found in vivo. By visualizing new subunit assembly after injection of tubulin modified with biotin into living fibroblast cells, we can visualize new growth on individual microtubules with antibody to biotin. Microtubules grow in vivo at about 4 microns min-1 and after rapid and precessive depolymerization old microtubules are replaced by new growth from the centrosome. Some microtubules turn over much more slowly and these stable microtubules have a different spatial distribution from the majority of dynamic ones. The existence of both stable and dynamic microtubules in the same cell suggests a model for morphogenesis of the microtubule cytoskeleton. The rapid turnover of microtubules in the cell provides a complex population upon which selective factors can act. Stability can be generated at the end of the polymer and affects the entire microtubule. This model of selective stabilization at the microtubule ends is discussed in terms of recent experiments on the establishment of kinetochore-pole microtubules during mitosis.

Animals↗
PubMed1986

Human atrial natriuretic peptide (ANP) for the treatment of patients with liver cirrhosis and ascites.

The acute effects of human atrial natriuretic peptide (ANP) were investigated in 10 patients with liver cirrhosis and ascites. In all patients, diuresis and natriuresis were stimulated with a wide individual variation (50 to 500%) in response to a bolus injection of 30 micrograms ANP. No side effects of treatment were observed. Continuous infusion of ANP (300 micrograms/10 h/d) in a patient with liver cirrhosis and ascites, resistant to conservative forms of diuretic therapy, resulted in an initial increase of diuresis and natriuresis which subsequently returned to pretreatment levels. After initiation of pulsatile nocturnal treatment (5 pulses of 30 micrograms ANP every 3 h), diuresis increased, leading to a persistent normalization of sodium and chloride excretion. The patient lost 8 kg of weight during 16 days of treatment. Out of 3 additional patients on the same therapeutic regime, only one experienced a weight loss of 5 kg due to increased natriuresis and chloruresis. The remaining 2 patients did not respond during 5 resp. 7 days of therapy.

Adult↗
PubMed1985

Influence of the centrosome on the structure of nucleated microtubules.

The capacity of the centrosome to influence the lattice structure of nucleated microtubules was studied in vitro. Brain microtubules self-assembled to give predominantly (98%) 14-protofilament microtubules. However, under exactly the same conditions of assembly they grew off of purified centrosomes from neuroblastoma cells to give mostly (82%) 13-protofilament microtubules. Thus, the nucleation sites on the centrosome constrained the microtubule lattice to yield the number of protofilaments usually found in vivo.

Animals↗
PubMed1985

M-phase promoting factors from eggs of Xenopus laevis.

When an M-phase promoting factor (MPF) is injected into Xenopus oocytes, which are naturally arrested at the G2/prophase boundary, it induces rapid entry of the cells into M-phase. MPF is present in late G2 and in M-phase of a variety of cell types, such as Xenopus eggs (naturally arrested in M), cleaving embryos, yeast, HeLa, and CHO cultures. MPF has been purified approximately 50-fold from eggs. It is stabilized by gamma-thio-ATP and by phosphoprotein phosphatase inhibitors. It runs as a protein of approximately 100 kd size on gel filtration. Oocytes contain a precursor of MPF, which is activated by post-translational means when a small amount of purified MPF is injected into the cell. Thus, MPF appears to be an auto-activating cytoplasmic trigger of M-phase. At anaphase of the cell cycle, MPF is inactivated due to the appearance of an 'anti-MPF' activity. Monoclonal antibodies have been prepared to partially purified MPF stabilized by gamma-thio-ATP, and several preparations which inactivate MPF were obtained. The antibodies are directed against thio-phosphate groups carried by a set of proteins including MPF. This indicates that MPF is present in our active preparations as a thio-phosphoprotein. These and other data suggest that MPF is normally activated in the cell cycle by a phosphorylation reaction.

Animals↗
PubMed1984

Temporal and spatial regulation of fibronectin in early Xenopus development.

Pattern formation and temporal control of gene expression in Xenopus development were investigated using fibronectin as a biochemical marker. We determined the spatial localization of fibronectin in the embryo by immunofluorescence and the temporal program of its expression by biosynthesis studies and Western blotting techniques. At the start of gastrulation, fibronectin is localized on the roof of the blastocoel which serves as the surface upon which mesodermal cells will migrate. However, since we find fibronectin secreted by all parts of the embryo, localization is probably achieved through spatially localized receptors that bind secreted fibronectin. Fibronectin levels and fibronectin synthesis rates increase following the midblastula stage. This increase is independent of transcription and therefore involves activation of maternal RNA for fibronectin. Since this message mobilization also occurs in activated but unfertilized eggs, this event must be regulated separately from the midblastula transition.

Animals↗