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At least 19 recordsLinked to original sources
PubMed2025

MCM5 UFMylation regulates replication origin firing and fork progression.

Modification with UFM1 (UFMylation) is essential for cell proliferation, but its precise mechanism of action is unclear. Furthermore, the UFMylation pathway has been associated with microcephalic primordial dwarfism (MPD) disorders, and mutations causative for MPD are also identified in genes encoding components of the replicative DNA helicase complex, including the MCM hexamer. Here, we reveal that UFMylation regulates DNA replication, and that all MPD-associated mutations in UFMylation enzymes impair replication. Mechanistically, the UFM1 E3 ligase UFL1 catalyzes Lys583 UFMylation of MCM5, a critical component of the CMG replicative DNA helicase complex. Mutation of Lys583 blocking this UFMylation event destabilizes the helicase complex, delaying origin firing and slowing replication fork progression. We conclude that MCM5 UFMylation is essential for efficient origin firing and replication fork progression, both of which ensure accurate DNA replication, cell proliferation, and prevention of MPD disorders.

DNA Replication↗
PubMed2026

CDC7 and APC/CCdh1 gate distinct routes to initiate DNA replication.

Precise temporal control of DNA replication initiation is essential for faithful cell division, yet there are conflicting prevailing models for how cells trigger origin firing. Here, we demonstrate that human origins are not fired at fixed thresholds of cell cycle regulators E2F, APC/CCdh1, CDK2/1, and CDC7. Instead, origin firing is triggered at a tunable CDK2/1 threshold that is gated by either CDC7 or APC/CCdh1 activity. CDC7 phosphorylates MCM helicase, enabling Cyclin E-CDK2 to trigger origin firing at low CDK2/1 activity, independent of APC/CCdh1 inactivation. In contrast, APC/CCdh1 inactivation enables Cyclin A-CDK1 to phosphorylate MCM at distinct CDK2/1-specific sites and trigger origin firing, independent of CDC7 activity. Strikingly, this requires much higher CDK2/1 activity, which CDK2 cannot normally reach alone. Thus, having two distinct routes necessitates blocking both routes to prevent S phase: either by inhibiting CDC7 or CDK2 while also preventing APC/CCdh1 inactivation or inhibiting CDK1.

Humans↗
PubMed1992

The development of a new small-scale smoke toxicity test method and its comparison with real-scale fire tests.

A comprehensive methodology has been developed for obtaining and using smoke toxicity data for fire hazard analysis. This bench-scale method can simulate diverse fire conditions and identify extremely toxic smoke under both pre- and post-flashover conditions. However, incidence data show that most of the fire deaths in the U.S. occur outside the room of fire origin from smoke and toxic gases that are generated from a fire under post-flashover conditions. Therefore, the most relevant real-scale combustion conditions to simulate in the bench-scale apparatus would be the post-flashover conditions which are achieved by using radiant heat, a high heat flux, and correcting the bench-scale carbon monoxide (CO) results to agree with CO yields observed in real-scale post-flashover fires. The number of test animals (Fischer 344 male rats) is minimized by using the N-Gas Model to estimate the LC50 value from the chemical analysis of the smoke. The current N-Gas Model predicts the toxicity of complex fire gas mixtures based on a large data base of experimental results of individual and mixed gases that include CO, CO2, reduced O2, HCN, HCl, HBr, and NOx. The prediction is checked with a small number of animal tests and an approximate LC50 value is determined. The bench-scale results have been validated with full-scale room wall burns of a limited number of materials of widely differing characteristics chosen to challenge the system. The toxic potency values are assessed to determine if the smoke from a material or product is unusually or extremely toxic and can then be used in computations of fire hazard.

Animals↗
PubMed1975

Piribedil and apomorphine: pre- and postsynaptic effects on dopamine synthesis and neuronal activity.

Piribedil and apomorphine can cause a complete but temporary inhibition in firing of the DA-containing neurons. The cells recover to approximately 30 to 40% of the original firing rate, and with subsequent administration of the drug become more and more resistant to further inhibition. At this point they are also resistant to inhibition by other direct- or indirect-acting DA agonists that are normally able to inhibit the firing of DA cells. This effect may be due to an interaction of the DA agonists with the postsynaptic DA receptors, but apomorphine at least is able to inhibit the firing of DA cells directly when iontophoresed onto the DA cell body (Aghajanian and Bunney, 1973). These drugs can block the increase in DA synthesis observed in the absence of impulse flow, indicating a presynaptic site of acttion. Taken together, these results suggest that in interpreting the effects of drugs interacting with the DA systems several points of interaction should be considered.

Animals↗
PubMed1992

[Ca2+ channels and the abnormal electrical activity of demyelinated nerve].

Peripheral nerve demyelination was produced in adult rats by placing loosely-constrictive ligatures around the common sciatic nerve. The postoperative behavior of these rats indicated that hyperalgesia, allodynia and possible spontaneous pain were produced. In the meantime, abnormal spontaneous afferent activities (ectopic firings)originating from the demyelinated region were recorded. Evidence showed that the application of Ca2+ and Ca2+ channel blockers modulated the abnormal activity of the injured nerve. Ca2+ facilitation was dependent on its concentration (in the range of 10-20 mol/L), while 40mol/L Ca2+ always abolished firing. Verapamil, as well as La3+, applied locally or i. v. (for verapamil) not only strongly inhibited the spontaneous ectopic firings, but also blocked discharges elicited by tetraethylammonium. It is suggested that newly formed Ca2+ channels on the naked axolemma are largely responsible for the abnormal afferent activities following demyelination of the nerve.

Animals↗
PubMed1992

Initiation of replication in the Chinese hamster dihydrofolate reductase domain.

Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a approximately 50 kb "initiation locus" (although the level of initiation detected at any given site within this region was relatively low). This result contrasts markedly with data from an in vitro strand switching assay suggesting that > 80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiation per se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of 3H-thymidine moves through the structures observed on 2-D gels with the kinetics expected of bonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.

Animals↗
PubMed2026

Lamin-ating the genome: quantitative gatekeeping of replication initiation.

Discovered in the 1970s, nuclear lamins control chromatin organization and are linked to many diseases. Zhang et al. now find that lamin A/C quantitatively constrains DNA replication initiation by limiting chromatin accessibility and sequestering proliferating cell nuclear antigen, extending lamin's long-known role in replication to the control of origin firing.

Journal Article↗
PubMed2026

Direct visualization of MCM helicase activation and replisome coupling in situ.

Deciphering the spatial organization of molecular machines that copy the genome remains a fundamental challenge in biology. Essential for eukaryotic DNA replication, Mini-Chromosome Maintenance (MCM2-7) helicases are loaded during G1 as double hexamers (DHs) to license replication origins. Upon activation in S phase, each DH is thought to split into two single hexamers (SHs) that form the active CMG helicases and travel bidirectionally. However, the field has long been divided: biochemical and structural studies define CMG helicases as autonomous, independent motors, while genomic and cellular imaging assays suggest sister replisomes remain physically coupled within replication factories. Here, we use MINFLUX nanoscopy to localize individual MCM complexes down to nanometer precision in situ, directly resolving DHs in human cells and capturing their separation into SHs upon origin firing. We find that the resulting sister replisomes do not diffuse apart: they remain coupled at a characteristic distance of ~40 nm throughout S phase. Depletion experiments identify two distinct contributions to this coupling: local, protein-mediated tethering by the AND1 scaffold, and higher-order spatial confinement dependent on cohesin, which is dispensable for MCM loading in G1 but required to maintain coupling in S phase. By linking the nanometer-scale architecture of the replisome to the genome-wide topology of replication fountains, these findings provide direct spatial evidence that sister forks are coupled during DNA synthesis and define the molecular forces that organize replisomes within their native nuclear context.

DNA replication↗
PubMed1979

Acceleration of idioventricular rhythms by histamine in guinea pig heart: mediation by H2 receptors.

To evaluate the ability of histamine to induce ventricular arrhythmias, we studied the effects of histamine on ventricular rhythmicity in the isolated guinea pig heart with complete atrioventricular conduction block. As a function of dose (0.1-30 microgram), histamine enhanced the idioventricular rate by increasing the rate of firing of the original pacemaker and also by causing the sudden appearance of faster idioventricular rhythms that coincided with changes in pacemaker site. Anaphylaxis in the isolated guinea pig heart with complete atrioventricular conduction block caused histamine release and acceleration of idioventricular rate. The effects of histamine on idioventricular rhythmicity were not attenuated by the histamine H1 receptor antagonist chlorpheniramine, but were antagonized by the H2 receptor antagonist cimetidine. Moreover, the selective H2 agonist 4-methylhistamine (4MeH) accelerated the idioventricular rate, whereas 2-(2-thiazolyl) ethylamine (ThEA), at doses selective for H1 receptor activation, did not. The effects of histamine on idioventricular rhythmicity were not modified by the beta-adrenergic blocker pindolol. The mechanism by which histamine increases idioventricular rate probably involves two components: (1) an enhancement in automaticity of the original pacemaker, and (2) the induction of faster rhythms via reentry and/or afterdepolarizations. Whatever the mechanism, both components of the ventricular chronotropic action of histamine appear to involve exclusively histamine receptors of the H2 type. Thus, our results suggest that H2 receptor antagonists may have a role as specific antiarrhythmic agents in the treatment of cardiac dysfunctions caused by histamine release.

Anaphylaxis↗
PubMed1975

Neural regulation of respiration.

The main respiratory muscles are under both voluntary and involuntary (automatic) control. These two control systems come from separate sites in the CNS and have separate descending pathways; the final integration of these outputs occurs at segmental levels in the cord. Voluntary control arises from the motor and premotor cortex and descends in the cord in the corticospinal tract. Involuntary control is mediated by both rhythmic and nonrhythmic systems located in the brainstem. Recent studies have associated the classic respiratory centers with specific nuclei in the brainstem. The pneumotaxic center is located in the nucleus parabrachialis, and the medullary respiratory centers are located in the vicinity of the nucleus of the solitary tract (dorsal respiratory group) and the nuclei ambiguus and retroambigualis (ventral respiratory group). Most axons from the medullary nuclei cross in the medulla and descend in the ventral and lateral columns to segmental levels. The classic medullary respiratory centers described by Pitts have been shown to be the site of origin of tonically firing long reticulospinal axons that descend in the ventral and lateral columns. This system is thought to provide a nonrhythmic involuntary biasing of the membrane potential of respiratory motoneurons in the cord. The site of generation of eupnic breathing and the mechanism of rhythm generation remain unknown. However, recent studies indicate that reciprocal inhibition between populations of inspiratory and expiratory cells (bistable oscillator model) does not occur in the medulla; rather we suggest that inhibitory phasing of inspiratory cells generates inspiratory rhythm, and periodic inhibition of tonically active expiratory neurons results in respiratory rhythm. We suggest that the inhibitory phasing occurs in the dorsal respiratory group, which is also the site of integration of respiratory afferents. The main site of integration of the voluntary and involuntary control systems is the cord where the respiratory motoneuron output is determined by descending information from these systems, as well as with intrasegmental and intersegmental reflexes. Separation of the voluntary and involuntary control systems also occurs in man and discrete lesions may interrupt one system without significant alteration of the other.

Animals↗
PubMed1978

II. Effect of CO2 on afferent vagal endings in the canine lung.

We have attempted to identify the afferent endings responsible for the pulmonary-CO2 ventilatory reflex. We recorded afferent vagal impulses arising from the left lung in anesthetized dogs with separately ventilated lungs. When the left pulmonary artery was occluded, left lung PCO2 fell to 3 mm Hg and slowly-adapting pulmonary stretch receptor activity increased 46%. Firing declined to its original intensity when left lung PCO2 was raised in steps by administration of CO2, firing decreasing most between 2 and 19 mm Hg, and least between 30 and 50 mm Hg. Irritant receptor activity also increased (from 2.8 to 7.4 impulses/sec) after pulmonary arterial occlusion, the effect being reversed by administration of CO2. These procedures caused trivial changes in pulmonary and bronchial C-fiber activity. Effects on both slowly-adapting stretch receptors and irritant receptors appeared to result from a direct action of CO2 on the endings themselves, rather than from mechanical changes in the lung. Changes in slowly-adapting stretch receptor activity provide an adequate explanation for the pulmonary-CO2 ventilatory reflex, the relationship between impulse frequency and lung PCO2 suggesting that these afferents may have a role in limiting CO2 loss under conditions causing hypocapnia, but be less effective in stimulating breathing during hypercapnia.

Afferent Pathways↗
PubMed1976

Circadian and other rhythmic activity of neurones in the ventromedial nuclei and lateral hypothalamic area.

1. The frequency of firing was simultaneously recorded from single neurones of the ventromedial nuclei (VMN) and the lateral hypothalamic area (LHA) in urethane anaesthetized rats for many hours. 2. There were circadian changes of VMN and LHA neurone activity. The pattern of this circadian rhythm is as follows: throughout the day LHA neurones show higher activity than that of VMN, as indicated by higher frequency and more fluctuations in their rates of firing. In late afternoon the discharge rate of LHA neurones increases further, showing oscillations of short duration. In the early evening hours LHA neurone activity gradually goes down, as the VMN neurones become active. Throughout the night, VMN neurones are more active than those of LHA, just the opposite of the day period. In early morning hours VMN neurones gradually become quiet, while LHA neurones begin to show activity. 3. Superimposed on the circadian rhythm, at certain periods of the day, VMN and LHA neurones showed short duration oscillations in rate of firing, roughly every 7-15 sec and every 3-5 min. 4. Activities in neurones of the VMN and LHA were reciprocally related; a decrease in firing rate of one was associated with an increase in the other. This phenomenon was shown clearly by analysis of auto- and cross-correlation functions of firing patterns of VMN and LHA neurones. 5. The effects of stimulations of the prefrontal cortex and splanchnic afferents on VMN and LHA neurones depended on the basic firing frequency, thus they varied with the time of day. Definite relationships exist between basic firing frequency of a cell and the magnitude of changes evoked by these stimuli. Reactions of VMN and LHA neurones were the opposite in most instances. Septal stimulations (at more than 10/sec) always produced inhibition of LHA neurone activity. 6. Intravenous injection of glucose inhibited LHA neurones and accelerated firing of VMN cells. This was true during the day period as well as at night when background activities of VMN and LHA neurones were different from that of the day. 7. Stimulation of the septal area with subthreshold pulses at a low rate (1-0.3/sec) suppressed or altered oscillations in firing frequency of LHA neurones. Severance of connection between LHA and structures caudal thereto had no effect on LHA neurone firing rates or rhythms. Sections between the septal area and LHA, however, abolished or greatly altered the oscillatory rhythms of LHA cell activity, although spontaneous discharges continued at a somewhat lower rate for periods of hours. 8. Stimulation of suprachiasmatic nuclei with weak intensity and low frequency also changed oscillatory fluctuations in firing of LHA neurones. 9. Possible origins of circadian rhythm and oscillations of short duration in firing pattern of VMN and LHA neurones were discussed.

Action Potentials↗
PubMed1992

Different firing patterns generated in dendrites and somata of CA1 pyramidal neurones in guinea-pig hippocampus.

1. Intracellular recordings, taken from CA1 pyramidal cells in guinea-pig hippocampal slices, were used to examine the origins of repetitive and burst firing in these cells. Single action potentials were elicited by depolarizing current injection at somatic recording sites. In contrast, current injection during intradendritic recordings initiated burst firing in the dendrites. Burst firing could be elicited in the soma by direct depolarization of distal apical dendrites (> 150 microns from the cell body layer) with large extracellular polarizing electrodes. 2. Intracellular recordings were taken simultaneously from the apical dendrites and pyramidal cell somata with the intention of impaling the same neurone with both electrodes. Paired dendrite-soma recordings confirmed that rhythmic single action potentials were generated at the cell soma, whereas bursts of action potentials were initiated in the distal apical dendrites (> 150 microns from the cell body layer). Fast spikes in the dendrite often triggered fast spikes in the soma, but not all fast spikes in the dendritic burst were 'relayed' to the soma. 3. In paired recordings, when a dendritic action potential failed to elicit a full somatic action potential, a 'd-spike' was commonly recorded in the soma. Somatic d-spikes were uniform all-or-none responses that could be shown, in some cases, to trigger the full somatic action potentials. 4. Attenuated spikes could be recorded in the dendrites, triggered by action potentials initiated at the cell soma. Dendritic responses to somatic stimulation sometimes varied in amplitude, but always showed a direct correspondence with somatic action potentials. 5. Dendritic recordings taken closer to the pyramidal cell bodies (< 150 microns from the cell body layer) showed a 'transitional' region where single action potentials rather than burst discharges could be evoked. After-potentials of these single spikes differed from those associated with somatic spikes in that proximal dendritic spikes had depolarizing after-potentials. The observed shift from after-hyperpolarization to depolarizing after-potentials in intradendritic recordings taken progressively further from the cell body corresponds to the change from repetitive to burst firing. 6. The results indicate that activity of the CA1 pyramidal cell soma, presumably a reflection of its output, can be either burst or repetitive firing. Somatic 'bursts,' unlike the burst discharges seen in the apical dendrites or the burst discharges reported in CA3 cells, are not initiated locally. Rather, they appear to be simply a rapid spike-for-spike response by the soma to the fast spikes that form part of the apical dendritic burst.(ABSTRACT TRUNCATED AT 400 WORDS)

Action Potentials↗
PubMed2025

Regulation of replication timing in Saccharomyces cerevisiae.

In order to maintain genomic integrity, DNA replication must be highly coordinated. Disruptions in this process can cause replication stress which is aberrant in many pathologies including cancer. Despite this, little is known about the mechanisms governing the temporal regulation of DNA replication initiation, thought to be related to the limited copy number of firing factors. Here, we present a high (1-kilobase) resolution stochastic model of Saccharomyces cerevisiae whole-genome replication in which origins compete to associate with limited firing factors. After developing an algorithm to fit this model to replication timing data, we validated the model by reproducing experimental inter-origin distances, origin efficiencies, and replication fork directionality. This suggests the model accurately simulates the aspects of DNA replication most important for determining its dynamics. We also use the model to predict measures of DNA replication dynamics which are yet to be determined experimentally and investigate the potential impacts of variations in firing factor concentrations on DNA replication.

Saccharomyces cerevisiae↗