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At least 19 recordsLinked to original sources
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↗
PubMed2017

Rapid DNA replication origin licensing protects stem cell pluripotency.

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.

Cell Cycle↗
PubMed1992

Propionate metabolism in cultured human cells after overexpression of recombinant methylmalonyl CoA mutase: implications for somatic gene therapy.

Strategies for somatic gene therapy must consider the metabolic consequences of expressing the recombinant gene product in addition to methods for gene transfer and expression. We describe studies of propionate metabolism in cultured cells transfected with methylmalonyl CoA mutase (MCM), the enzyme deficient in mut methylmalonic acidemia. Transfection of MCM into mut fibroblasts restores propionate metabolism to normal levels in a dose-dependent manner. Overexpression of MCM, or the addition of excess propionate, carnitine, or cobalamin, does not increase propionate metabolism in normal human fibroblasts, lymphoblasts, or hepatoma cells, although hepatic cells exhibit > 10-fold higher levels of propionate metabolism. Significantly, the restoration of propionate metabolism in mut fibroblasts is disproportionately greater than the efficiency of transfection, suggesting the presence of a cooperative phenomenon between cells. Intercellular participation in propionate metabolism is evident in cocultures of MCM-deficient and propionyl CoA carboxylase-deficient cells. We conclude that the liver is the preferred target for gene therapy of MCM deficiency because of its greater capacity for propionate metabolism and that cooperation between cells could enhance the biological effect of a subpopulation of cells transformed with recombinant MCM.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

Interleukin-1, platelet derived growth factor, free radicals and monocyte aryl hydrocarbon hydroxylase activity in liver disease. Role of cell communication.

Monocytes were isolated from blood of human origin and cultured in supplemented Leibovitz (L-15) medium for 24 hr. The medium was then decanted and filtered, and all subsequent tests were done on monocyte conditioned medium (MCM). The monocytes of patients with liver disease spontaneously secrete temperature-sensitive arylhydrocarbon hydroxylase (AHH) inhibitory factors detectable in the MCM. Anti-interleukin-1 antibody (IL-1Ab) reduced the AHH inhibitory activity of the MCM, suggesting that part of the AHH inhibitory activity was due to interleukin-1 (IL-1). Platelet derived growth factor did not affect AHH activity. Interleukin-1 beta was detectable in MCM but did not differ significantly between patients and normal volunteers. A time course experiment indicated that interleukin-1 beta inhibited hepatocyte AHH activity after only 2 hr of incubation. Catalase partially blocked the AHH inhibitory activity of MCM suggesting that activated oxygen intermediates are partially involved in the AHH inhibitory activity of the MCM. Simultaneous incubation of interleukin-1 beta and catalase did not prevent or augment the inhibitory action of IL-1 on AHH activity. IL-1 stimulates collagen synthesis and elevates serum procollagen type 3 peptide (P-III-P). Results indicated that serum P-III-P was elevated in blood sources producing temperature-sensitive AHH inhibitory factor.

Antibodies↗
PubMed1977

Clinical and biochemical assessment of a modified evaporated milk for infant feeding.

A clinical and biochemical evaluation has been made of a new milk formula, Modified Carnation milk (MCM), based on cows' milk but with the mineral content and concentration of caloric nutrients altered to make it correspond more closely to human milk. MCM produced higher plasma calcium and magnesium concentrations in 6-day-old infants than those produced by unmodified evaporated and dried milks, achieving concentrations closer to those of breast milk. Plasma free amino acid concentrations in MCM-fed infants are nearer breast-fed values than those in unmodified milk-fed infants where higher individual plasma amino acid concentrations persist during the first 3 months. MCM-fed infants had low plasma urea concentrations and lower urine osmolalities at 6 days, 3 weeks, 6 weeks, 3 months, and 6 months than infants fed on the evaporated and dried milks, and similar plasma urea and urine osmolalities to those of breast-fed infants. MCM is likely to be superior to unmodified evaporated and dried milks in preventing convulsions of the hypocalcaemic/hypomagnesaemic/hyperphosphataemic type, and seems less likely to cause hypertonic dehydration. MCM is easily prepared, readily accepted by babies, and appears to be nutritionally adequate for the feeding of term infants.

Amino Acids↗
PubMed1977

[Genetic syndromes of multiple congenital developmental defects].

Analysis of developmental defects in 615 children who had died in hospitals in Minsk during the 5-year period (1971--1975)showed that in 274 children defects were multiple. This number includes 78 children with chromosomal diseases and 37 children with genic syndromes of multiple congenital malformations (MCM). The total number of genic syndromes of MCM is extremely great, at present over 250 of them are known. Analysis of the available literature and the authors' own findings made it possible to develop criteria of morphological diagnosis of genic syndromes of MCM with due account for different diagnostic significance of various defects. Genealogical studies and estimation of population incidence of individual malformations are also necessary for diagnosis of genic syndromes of MCM and identification of new syndromes. An etiological classification of MCM and that of genic syndromes of MCM are considered on the basis of isolation of similar clinical forms, which are of important for differential diagnosis.

Abnormalities, Multiple↗
PubMed1992

Optimized formulation of magnetic chitosan microspheres containing the anticancer agent, oxantrazole.

A combined emulsion/polymer cross-linking/solvent evaporation technique was used to prepare magnetic chitosan microspheres (MCM) containing the anticancer drug, oxantrazole. A central composite experimental design was used to simultaneously evaluate a variety of formulation factors on a number of response variables, such as the percentage of oxantrazole entrapped in the MCM. In association with the study design, statistical optimization procedures indicated the factors that significantly influence MCM preparation and what levels of the factors are needed to produce optimum MCM. Entrapment of anticancer agents into biodegradable microspheres is difficult because of low aqueous drug solubility and porosity of the particles. The latter effect was circumvented by a chitosan cross-linking step that resulted in approximately 3% (w/w) oxantrazole entrapment in the MCM via the optimization procedures. The combined formulation and statistical optimization strategy provide a basis to develop other microparticulate systems and led to a dosage form that can be used for future in vivo investigations.

Anthraquinones↗
PubMed1992

Optimal protocol and trajectory visualization for conformational searches of peptides and proteins.

Conformational searches by molecular dynamics and different types of Monte Carlo or build-up methods usually aim to find the lowest-energy conformation. However, this is often misleading, as the energy functions used in conformational calculations are imprecise. For instance, though positions of local minima defined by the repulsive part of the Lennard-Jones potential are usually altered only slightly by functional modification, the relative depths of the minima could change significantly. Thus, the purpose of conformational searches and, correspondingly, performance criteria should be reformulated and appropriate methods found to extract different local minima from the search trajectory and allow visualization in the search space. Attempts at convergence to the lowest-energy structure should be replaced with efforts to visit a maximum number of different local energy minima with energies within a certain range. We use this quantitative criterion consistently to evaluate performances of different search procedures. To utilize information generated in the course of simulation, a "stack" of low energy conformations is created and stored. It keeps track of variables and visit numbers for the best representatives of different conformational families. To visualize the search, projection of multidimensional walks onto a principal plane defined by a set of reference structures is used. With Met-enkephalin as a structural example and a Monte Carlo procedure combined with energy minimization (MCM) as a basic search method, we analyzed the influence on search efficiency of different characteristics as temperature schedules, the step size for variable modification, constrained random step and response mechanisms to search difficulties. Simulated annealing MCM had comparable efficiency with MCM at constant and elevated temperature (about 600 K). Constraining the randomized choice of side-chain chi angles to optimal values (rotamers) on every MCM step did not improve, but rather worsened, the search efficiency. Two low-energy Met-enkephalin conformations with parallel Tyr1 and Phe4 rings, a gamma-turn around the Gly2 residue, and Phe4 and Met5 side-chains forming together a compact hydrophobic cluster were found and are suggested as possible structural candidates for interaction with a receptor or a membrane.

Amino Acid Sequence↗
PubMed1992

Mood-congruent memory in depression: emotional priming or elaboration?

We investigated whether mood-congruent memory (MCM) bias in depression is a function of implicit or explicit memory. Implicit memory is taken as a measure of ease of activation, whereas explicit memory also taps elaboration. As expected, MCM bias was found in the explicit memory task but not in the implicit memory task. We believe this finding supports the involvement of elaborative mechanisms in MCM. In addition, memory bias was found with words related to depression but not with words denoting physical threat. Thus, the MCM bias in explicit memory was found to be specific to information that was congruent with depression rather than to all negative information.

Adult↗
PubMed1992

Influence of rat testicular macrophages on Leydig cell function in vitro.

The influence of co-cultures of rat testicular macrophages and Leydig cells (LC) on LC morphology and steroidogenesis was investigated with and without macrophage stimulation by a bacterial lipopolysaccharide (LPS). LC showed an elongated form in the presence of stimulated testicular macrophages. In the presence of non-stimulated testicular macrophages a significant inhibition of testosterone production was observed (decrease of 33%) from 48 h in co-culture while an increase of 16% was obtained at the same culture time, after stimulation of macrophages by LPS. When LC were treated with testicular macrophage-conditioned media (MCM) obtained from LPS-treated macrophages, they became fusiform and there was stimulation (78%) of steroid production. After human FSH stimulation (1-1000 mIU ml-1), MCM from testicular macrophages was no more effective in enhancing testosterone production by LC than was media from untreated LC. Similar experiments with LPS were conducted with macrophages of peritoneal origin. Peritoneal macrophages stimulated or not by LPS in co-cultures with LC or peritoneal MCM did not significantly modify testosterone production. However, these cells were able to modify LC morphology when LPS-MCM was added to LC-culture medium. The present results suggest strongly that testicular macrophage-LC interactions could be important in the control of LC steroidogenesis.

Animals↗
PubMedDate not supplied

[Morphology and growth cycle of Hyphomicrobium with a screw-like prostheca].

Hyphomicrobium with a screw-like prostheca was isolated from a mixed culture of soil bacteria. Its morphology and growth cycle were studied by electron microscopy. The adult organisms are 1.6-1.8 mcm long and 0.8 mcm thick. The diameter of the prostheca is 0.2-0.3 mcm and sometimes up to 10-12 mcm. It has a peculiar screw-like structure of the cell wall surface and forms branches at whose ends daughter organisms develop. The bacterium multiplies not only by vegetative growth but also by conjugation.

Bacteria↗
PubMed1978

[Characteristics of the lymphoid cells of rabbits].

By means of scanning electron microscopy it has been stated that the thymus gland, spleen, paratracheal lymph node, Peyer's patches and appendix in an intact rabbit of "Shinshilla" strain have T- and B-lymphocytes in different proportion. T-lymphocytes are seen as spherical cells with rough surface, with a few microprocesses, have 3.0 +/- 0.1 mcm--4.7 +/- 0.2 mcm in diameter depending on their location. B-lymphocytes are seen as spherical cells with a considerable amount of microvilli on their surface, have 2.9 +/- 0.1 mcm--4.4 +/- 0.1 mcm in diameter depending also on their location. In the thymus gland, cells with rough surface, a few microprocesses and crater-like hollows prevail. In the spleen and lymph node, most of the cells are of similar diameter and have microvilli on the surface. Lymphoid population in the Peyer's patch and appendix is represented by large cells with microprocesses.

Animals↗
PubMedDate not supplied

[Study of the sequential stages of Proteus vulgaris L-transformation by scanning electron microscopy].

Consecutive stages of L-transformation caused by penicillin in Proteus vulgaris were studied, from spheroplasts and unstable, easily reversing L-forms to stable L-forms which did not reverse. To maintain natural topography of the culture on the growth medium, millipore filters were placed on the surface of the medium and then were fixed with the culture. All the main elements of L-cultures were found at the stage of spheroplast formation, before the first passage onto the medium for cultivating L-forms, i.e. spherical cells having a diameter from 3.5 to 1 mcm, elementary bodies 1.0-0.6 mcm large, filamentous structures with a diameter to 4 mcm, large bodies having dimensions of 20 mcm and more, and "cell-free" masses. The composition of the structural elements and their reproduction are supposed to be almost the same in the bacterial culture and L-forms at the stage of spheroplast formation.

L Forms↗
PubMed1978

Surface modification of guinea pig sperm during in vitro capacitation: an assessment using lectin-induced agglutination of living sperm.

Plant lectins have been used to advantage to study carbohydrate-containing cell surface receptors in numerous systems. In this study, a simple, reliable assay was developed to quantitate lectin-induced agglutinability of sperm. This assay was used successfully to compare some of the surface properties of uncapacitated and capacitated guinea pig sperm. Capacitation was induced by incubating sperm in minimum capacitation medium (MCM) or modified Tyrodes solution (T-PL). Control incubations were done in Ham's F-10 or Hank's balanced salt solution which do not support capacitation. At timed intervals during incubation, sperm samples were assessed for pattern and degree of lectin-induced agglutination. Results establish that: (1) soybean agglutinin (SBA) and to a lesser extent concanavalin A (Con A) induced agglutinability of guinea pig sperm increase during in vitro capacitation in MCM; (2) a similar increase in SBA induced agglutinability occurs during capacitation in T-PL, but not in the non-capacitating media; and (3) for sperm incubated in MCM or T-PL, there is a significant increase in tail to tail agglutination after capacitation. The results with SBA demonstrate that D-galactose and/or N-acetyl-D-galactosamine containing receptor sites or the guinea pig sperm surface are affected by capacitation, and this effect occurs, at least in part, in the sperm tail. Possible explanations for the observed increase in agglutinability are discussed. The agglutination assay may prove useful as a direct test for the occurrence of capacitation and may be especially valuable for species having a small acrosome or limited number of eggs.

Acrosome↗
PubMed1978

Aerobic work capacity in chronically undernourished adult males.

Maximal O2 consumption (VO2max) and maximal endurance time at 80% Vo2max (T80) were determined in 49 subjects with varying degrees of nutritional compromise. Vo2max was lover by 21% and 52% in subjects with moderate and severe undernutrition, respectively, when compared to men with mild nutritional compromise. Most (80%) of the change in Vo2max could be related to the estimated differences in muscle cell mass (MCM). A significant reduction (-18%) in the Vo2max per unit of MCM was found in the severely undernourished subjects, indicating basic physiological changes. Total body hemoglobin showed the highest correlation with Vo2max (r = 0.709) and at constant MCM (partial r = 0.796). A multiple regression equation was derived to estimate the Vo2max of populations on the basis of simple anthropometry and blood hemoglobin levels. T80 (93-103 min) and estimated maximum sustained 8-h percent effort (32%) in mild, intermediate, or severe undernutrition were not significantly different; by contrast, endurance at a given absolute work load was markedly lower in severe undernutrition (8 vs. 1.5 h) due presumably to the difference in Vo2max.

Adipose Tissue↗
PubMed1992

Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria.

Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts. One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin. We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae. Three novel base changes were observed. Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute [14C]propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of [14C]propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal. These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.

Amino Acid Sequence↗
PubMed1992

Human osteoblasts in culture synthesize collagenase and other matrix metalloproteinases in response to osteotropic hormones and cytokines.

Collagenase production by rodent osteoblasts in response to calciotropic hormones has led to the hypothesis that bone cells play a major role in bone resorption by degrading the surface osteoid layer, thereby exposing the underlying mineralized matrix to osteoclastic action. Many studies suggest, however, that this model might not apply to bone resorption in the human. Human osteoblasts have been shown to produce gelatinase-A (72 kDa) and TIMP-1 (tissue inhibitor of metalloproteinases), but previous investigators have been unable to demonstrate the synthesis of collagenase by human osteoblasts either constitutively or in response to bone resorptive agents. In the present study the ability of human osteoblasts to produce the matrix metalloproteinases (MMPs) collagenase, gelatinase and stromelysin, and their specific inhibitors TIMPs-1 and 2, was examined using highly sensitive and specific antisera and by zymography. Semi-quantitative histomorphometric data showed that cells cultured on either glass or a type I collagen substratum constitutively synthesized gelatinase-A and TIMP-1. On type I collagen, however, a small proportion of unstimulated cells produce both collagenase (7%) and gelatinase-B (95 kDa; 3%). Treatment of cells with either parathyroid hormone (PTH), 1,25-dihydroxy-vitamin D3 (1,25(OH)2D3), or partially purified mononuclear cell conditioned medium (MCM), stimulated the synthesis of collagenase, gelatinase-B and stromelysin; MCM was 2- to 3-fold more potent than either PTH or 1,25(OH)2D3. Zymography using SDS/PAGE on conditioned media from cells cultured on type I collagen films revealed the presence of active gelatinase-A and that MCM stimulated progelatinase-B synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Effects of lemakalim and thalium on early afterdepolarization in mouse atrial fibers.

The effects of lemakalim (BRL 38227) and thallium ion (Tl) on action potential (AP) and on early afterdepolarization (EAD) were studied in mouse atrial fibers. Under treatment with lemakalim (25 mcM) or Tl (0.2 mM), the AP was not significantly changed except for the reduction of duration of AP (APD). EAD induced by 3.0 mM K superfusion was abolished by 50 mcM lemakalim and only partially inhibited or was not effected under 25 mcM lemakalim. However, EAD was abolished completely and rapidly by Tl treatment. With addition of K concentration in superfusate to 5.0 mM the EAD could only be partially inhibited. By further increasing the K concentration to 7.0 mM the EAD could be completely abolished. It is suggested that the activator of outward current could inhibit EAD as well as the blockers of inward currents.

Action Potentials↗