PubMed Health⌕ Search

Biomedical subjects

M Kirschner

Publications and source records attributed to M Kirschner.

At least 37 records · Page 2Linked to original sources
PubMed1991

mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2.

wee1 acts antagonistically to cdc25 in the tyrosine dephosphorylation and activation of cdc2, yet biochemical evidence suggests that wee1 is not required for tyrosine phosphorylation and its role is obscure. We show here that a related 66 kd kinase, called mik1, acts redundantly with wee1 in the negative regulation of cdc2 in S. pombe. A null allele of mik1 has no discernible phenotype, but a mik1 wee1 double mutant is hypermitotically lethal: all normal M phase checkpoints are bypassed, including the requirement for initiation of cell cycle "start," completion of S phase, and function of the cdc25+ mitotic activator. In the absence of mik1 and wee1 activity, cdc2 rapidly loses phosphate on tyrosine, both in strains undergoing mitotic lethality and in those that are viable owing to a compensating mutation within cdc2. The data suggest that mik1 and wee1 act cooperatively on cdc2, either directly as the inhibitory tyrosine kinase or as essential activators of that kinase.

Amino Acid Sequence↗
PubMed1991

Microtubule polymer assembly and transport during axonal elongation.

As axons elongate, tubulin, which is synthesized in the cell body, must be transported and assembled into new structures in the axon. The mechanism of transport and the location of assembly are presently unknown. We report here on the use of tubulin tagged with a photoactivatable fluorescent group to investigate these issues. Photoactivatable tubulin, microinjected into frog embryos at the two-cell stage, is incorporated into microtubules in neurons obtained from explants of the neural tube. When activated by light, a fluorescent mark is made on the microtubules in the axon, and transport and turnover can be visualized directly. We find that microtubules are generated in or near the cell body and continually transported distally as a coherent phase of polymer during axon elongation. This vectorial polymer movement was observed at all levels on the axon, even in the absence of axonal elongation. Measurements of the rate of polymer translocation at various places in the axon suggest that new polymer is formed by intercalary assembly along the axon and assembly at the growth cone in addition to transport of polymer from the cell body. Finally, polymer movement near the growth cone appeared to respond in a characteristic manner to growth cone behavior, while polymer proximally in the axon moved more consistently. These results suggest that microtubule translocation is the principal means of tubulin transport and that translocation plays an important role in generating new axon structure at the growth cone.

Animals↗
PubMed1991

Microtubule behavior during guidance of pioneer neuron growth cones in situ.

The growth of an axon toward its target results from the reorganization of the cytoskeleton in response to environmental guidance cues. Recently developed imaging technology makes it possible to address the effect of such cues on the neural cytoskeleton directly. Although high resolution studies can be carried out on neurons in vitro, these circumstances do not recreate the complexity of the natural environment. We report here on the arrangement and dynamics of microtubules in live neurons pathfinding in response to natural guidance cues in situ using the embryonic grasshopper limb fillet preparation. A rich microtubule network was present within the body of the growth cone and normally extended into the distal growth cone margin. Complex microtubule loops often formed transiently within the growth cone. Branches both with and without microtubules were regularly observed. Microtubules did not extend into filopodia. During growth cone steering events in response to identified guidance cues, microtubule behaviour could be monitored. In turns towards guidepost cells, microtubules selectively invaded branches derived from filopodia that had contacted the guidepost cell. At limb segment boundaries, microtubules displayed a variety of behaviors, including selective branch invasion, and also invasion of multiple branches followed by selective retention in branches oriented in the correct direction. Microtubule invasion of multiple branches also was seen in growth cones migrating on intrasegmental epithelium. Both selective invasion and selective retention generate asymmetrical microtubule arrangements within the growth cone, and may play a key role in growth cone steering events.

Animals↗
PubMed1991

The need for unity in the biomedical research community.

The author provides a perspective on how basic scientists view contemporary issues of biomedical research funding by discussing five topics of concern to the biomedical research community and the need for that community to form a new consensus as the basis for new approaches to legislative efforts in Congress and communication with the public. The topics are (1) the reasons scientific societies became active in legislative efforts in Congress, (2) the reasons there was initial friction between these societies and other institutions in the biomedical research community, (3) the history of the founding of a biomedical research community, (3) the history of the founding of a biomedical research caucus in the House of Representatives, (4) the effects of the Institute of Medicine's recent report on funding health sciences research, and (5) the weakness of the present institutional coalition. The author proposes specific steps to develop a more unified approach to achieve a common biomedical research agenda.

Humans↗
PubMed1990

[3 new antibody detection tests. A comparative study].

Erythrocyte antibodies are of clinical importance since they may cause decreased red cell survival as the result of hemolytic transfusion reactions, hemolytic disease of the newborn or autoimmune hemolytic anemia. A variety of in vitro antibody screening tests are employed to detect their presence in donor and patient sera. Three new methods now available for pretransfusion testing--two solid phase antiglobulin tests and a gel centrifugation test--are compared in parallel with a conventional tube test.

Blood Group Incompatibility↗
PubMed1989

Contractures in patients with Williams syndrome.

Williams syndrome is a multisystem disorder, including a characteristic facies and habitus. Intellectual, neurologic, and cardiovascular dysfunction occur; vascular disease may be progressive. Joint contractures affected 10 of a group of 20 children and young adults. In 3 of 10 patients, the contractures were severe enough to interfere with daily activities; in 3 the effect was only moderate. The contractures developed in early childhood and usually did not improve or worsen with time. Large and small joints were affected and usually were symmetrical. Their occurrence did not correlate with vascular disease. The contractures could not be attributed to a neurologic cause.

Adolescent↗
PubMed1988

New features of microtubule behaviour observed in vivo.

The microtubule cytoskeleton is thought to be intimately involved in generating and maintaining cell polarity and can generate many different morphological structures from a few structural elements. The mechanism by which these structures are generated has been partially elucidated from studies of microtubule polymerization both in vitro and in vivo. Microtubules in vitro exist in growing (polymerizing) and shrinking (depolymerizing) populations that interconvert infrequently. This behaviour, termed dynamic instability, permits microtubules in the cell rapidly to explore different arrangements and allows selective stabilization of specific morphologies. To investigate the regulation of these processes, we have implemented techniques for direct observation of fluorescently labelled microtubules and developed them to observe the dynamic behaviour of individual microtubules in single living cells. Sammak and Borisy recently used this technique to show that the dynamics of microtubules in fibroblasts is explained by dynamic instability. Although we also conclude here that dynamic instability explains much of microtubule behaviour in vivo, we find significant deviations from the properties of tubulin in vitro. These results suggest that local cytoplasmic factors strongly influence microtubule dynamics; such control has important implications for cellular morphogenesis.

Animals↗
PubMed1988

The primary structure and heterogeneity of tau protein from mouse brain.

Tau protein is a family of microtubule binding proteins, heterogeneous in molecular weight, that are induced during neurite outgrowth and are found prominently in neurofibrillary tangles in Alzheimer's disease. The predicted amino acid sequences of two forms of tau protein from mouse brain were determined from complementary DNA clones. These forms are identical in their amino-terminal sequences but differ in their carboxyl-terminal domains. Both proteins contain repeated sequences that may be tubulin binding sites. The sequence suggests that tau is an elongated molecule with no extensive alpha-helical or beta-sheet domains. These complementary DNAs should enable the study of various functional domains of tau and the study of tau expression in normal and pathological states.

Amino Acid Sequence↗
PubMed1988

Regulation of microtubule protein levels during cellular morphogenesis in nerve growth factor-treated PC12 cells.

Nerve growth factor induces neurite process formation in pheochromacytoma (PC12) cells and causes the parallel increase in levels of the microtubule-associated proteins, tau and MAP1, as well as increases in tubulin levels. Mechanisms to insure balanced accumulation of microtubule proteins and make their levels highly responsive to nerve growth factor were investigated. The effects on tau, MAP1, and tubulin are due to changes in protein synthesis rates, which for tau and tubulin we could show are due in part to changes in the mRNA levels. Whereas tubulin shows feedback regulation to modulate synthesis up or down, tau protein synthesis is not affected in a straightforward way by microtubule polymerization and depolymerization. The degradation of tau, MAP1, and both tubulin polypeptides, however, are stimulated by microtubule depolymerization caused by colchicine, or nerve growth factor removal. Combined feedback on synthesis and stability make tubulin levels highly responsive to assembly states. In addition, the linkage of tau and MAP1 turnover with the state of microtubule polymerization amplifies any change in their rate of synthesis, since tau and MAP1 promote microtubule polymerization. This linkage lends itself to rapid changes in the state of the system in response to nerve growth factor.

Animals↗
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↗