Differentiated Approach to Pharmacotherapy of Autism Spectrum Disorders: Biochemical Aspects.

Autism Spectrum Disorders (ASD) are highly heterogeneous neurodevelopmental disorders caused by a complex interaction of numerous genetic and environmental factors and leading to deviations in the nervous system formation at the very early developmental stages. Currently, there are no accepted pharmacological treatments for the so-called core symptoms of ASD, such as social communication disorders and restricted and repetitive behavior patterns. Lack of knowledge about biological basis of ASD, absence of the clinically significant biochemical parameters reflecting abnormalities in the signaling cascades controlling the nervous system development and functioning, and lack of methods for selection of clinically and biologically homogeneous subgroups are considered as causes for the failure of clinical trials of ASD pharmacotherapy. This review considers the possibilities of applying differentiated clinical and biological approaches to the targeted search for ASD pharmacotherapy with emphasis on biochemical markers associated with ASD and attempts to stratify patients by biochemical parameters. The use of such approach as "the target-oriented therapy and assessment of the target status before and during the treatment to identify patients with a positive response to treatment" is discussed using the published results of clinical trials as examples. It is concluded that identification of biochemical parameters for selection of the distinct subgroups among the ASD patients requires research on large samples reflecting clinical and biological diversity of the patients with ASD, and use of unified approaches for such studies. An integrated approach, including clinical observation, clinical-psychological assessment of the patient behavior, study of medical history and description of individual molecular profiles should become a new strategy for stratifying patients with ASD for clinical pharmacotherapeutic trials, as well as for evaluating their efficiency.

Links of platelet glutamate and glutathione metabolism with attenuated positive and negative symptoms in depressed patients at clinical high risk for psychosis.

Aim of the study is to reveal clinical and biological correlations in patients with adolescent depression and attenuated psychotic symptoms. Activity of platelet enzymes involved in glutamate-, glutathione- and energy metabolism was evaluated in control group and in the patients, because these systems are suspected as related to pathogenesis of psychosis. Adolescents (78 men, 16-25 years old) hospitalized with the first acute depressive state composed two groups: with prevalence of attenuated psychotic positive or negative symptoms (Gr1 and Gr2, 48 and 30 patients, respectively). Control group comprised 20 mentally healthy men of 19-25 years old. Gr1 differed significantly from Gr2 in scores by the Scale of Prodromal Symptoms (SOPS) for positive symptoms, p < 0.001, for disorganization symptoms, p < 0.003, and for total SOPS score, p < 0.001, before the treatment started. When patients from either Gr1 or Gr2 were compared with the control group, significantly decreased baseline activities of platelet glutamate dehydrogenase (GDH), glutathione reductase (GR) and glutathione S-transferase (GST) were found (p < 0.0001). Different correlations were found between baseline enzymatic activities in Gr1 and Gr2: GDH activity correlated with GR activity in Gr1 (R = 0.37), and with GST activity in Gr2 (R = 0.70). Significant correlations were found only in Gr2 between the delta of scores by SOPS negative symptoms (SOPS-N) under treatment and baseline GDH, GST, and GR activities (R = - 0.36, R = - 0.60, and R = 0.38, respectively). The found correlations of the baseline enzymatic activity levels with the value of the decrease (delta) in SOPS-N scores under the treatment represent interest for the prediction of the pharmacotherapy efficiency.

Late onset psychosis treatment with adjunctive medicines.

Background: A number of studies have shown the feasibility of using adjunctive drugs in late onset psychosis (LOP). Aim: Testing hypothesis that among LOP people treated with antipsychotics and antidepressants, basing on certain clinical characteristics a subgroup of patients might be distinguished, for whom adjunctive therapy is advantageous. This subgroup might be identified by measurement of blood biochemical parameters. Methods: 59 in-patients with LOP, treated neuroleptics and antidepressants, were included, and followed in real clinical practice. Database containing demographic, clinical data (scores by PANSS, CDSS, CGI-S, HAMD-17), prescribed therapy, adverse effects of antipsychotic and antidepressant treatment, and blood biochemical parameters (enzymatic activities of glutamate- and glutathione metabolism enzymes in platelets and erythrocytes) at baseline and after the treatment course was created. Results: Three groups of patients (Gr1, Gr2, and Gr3), based on the adjunctive therapy usage were identified: Gr1 (n = 16) was without adjunctive therapy, two other groups (Gr2 and Gr3) were with adjunctive medicines, such as 2-ethyl-6-methyl-3-hydroxypyridine succinate (EMHS; Gr2, n = 20), or other drugs, such as citicoline, cerebrolysin, cortexin, actovegin, gliatilin (choline alfoscerate; Gr3, n = 23). The enzymatic activities were assessed also in the matched control group (n = 38). In all three patient groups, as compared with controls, activity of erythrocyte glutathione reductase was decreased at baseline and after the treatment course. In Gr2, unlike Gr1 or Gr3, there was a significant decrease in baseline glutamate dehydrogenase and glutathione-S-transferase activities. Certain clinical criteria were also elucidated for prescription of EMHS as adjunctive therapy for patients of Gr2. Glutamate dehydrogenase and glutathione-S-transferase activities returned closer to control levels after the treatment course in Gr2, unlike Gr1, where they declined yet more after psychotropic treatment without adjunctive medicine. Different significant links between biochemical parameters and scores by clinical scales were observed in Gr1, Gr2, and Gr3, some having predictive value for evaluation of antipsychotic treatment efficacy. Conclusion: We demonstrate the validity of adjunctive neuroprotective medicines' usage in addition to antipsychotic and antidepressant therapy in distinct subgroups of patients suffering with LOP, especially those who have prominent side effects accompanying their psychotropic treatment. Returning of biochemical parameters to control range following the treatment course observed in patients of the subgroup treated with adjunctive EMHS is evidence for their metabolism normalization.

Activity of energy, glutamate, and glutathione metabolism enzymes in blood cells of elderly patients with depression

Heterogeneity of depression in older adults is a challenge for the development of person-centered treatment. To address this, we studied glutamate and glutathione metabolism enzymes in blood cells in 53 older adult patients with depression and 20 controls. Patients with depression had decreased platelet glutathione-S-transferase and erythrocyte glutathione reductase. The biochemical and clinical data contributed to three clinical clusters that are not linked to the onset of depression or its duration but were related to anxiety, cerebrovascular and cardiovascular comorbidities, including parkinsonian features such as tremor, akathisia, and rigidity. These findings could aid person-centered diagnosis and outcomes and timely successful treatment(s) of depression in older adults. (AU)

DNA Methylation Profiles of GAD1 in Human Cerebral Organoids of Autism Indicate Disrupted Epigenetic Regulation during Early Development.

DNA methylation profiling has become a promising approach towards identifying biomarkers of neuropsychiatric disorders including autism spectrum disorder (ASD). Epigenetic markers capture genetic risk factors and diverse exogenous and endogenous factors, including environmental risk factors and complex disease pathologies. We analysed the differential methylation profile of a regulatory region of the GAD1 gene using cerebral organoids generated from induced pluripotent stem cells (iPSCs) from adults with a diagnosis of ASD and from age- and gender-matched healthy individuals. Both groups showed high levels of methylation across the majority of CpG sites within the profiled GAD1 region of interest. The ASD group exhibited a higher number of unique DNA methylation patterns compared to controls and an increased CpG-wise variance. We detected six differentially methylated CpG sites in ASD, three of which reside within a methylation-dependent transcription factor binding site. In ASD, GAD1 is subject to differential methylation patterns that may not only influence its expression, but may also indicate variable epigenetic regulation among cells.

Changes in systemic GDF15 across the adult lifespan and their impact on maximal muscle power: the Copenhagen Sarcopenia Study.

BACKGROUND: Although growth differentiation factor 15 (GDF15) is known to increase with disease and is associated with low physical performance, the role of GDF15 in normal ageing is still not fully understood. Specifically, the influence of circulating GDF15 on impairments in maximal muscle power (a major contributor to functional limitations) and the underlying components has not been investigated. METHODS: Data from 1305 healthy women and men aged 20 to 93 years from The Copenhagen Sarcopenia Study were analysed. Circulating levels of GDF15 and markers of inflammation (tumor necrosis factor-alpha, interleukin-6, and high-sensitivity C-reactive protein) were measured by ELISA (R&D Systems) and multiplex bead-based immunoassays (Bio-Rad). Relative (normalized to body mass), allometric (normalized to height squared), and specific (normalized to leg muscle mass) muscle power were assessed by the Nottingham power rig [leg extension power (LEP)] and the 30 s sit-to-stand (STS) muscle power test. Total body fat, visceral fat, and leg lean mass were assessed by dual energy X-ray absorptiometry. Leg skeletal muscle index was measured as leg lean mass normalized to body height squared. RESULTS: Systemic levels of GDF15 increased progressively as a function of age in women (1.1 ± 0.4 pg·mL-1 ·year-1 ) and men (3.3 ± 0.6 pg·mL-1 ·year-1 ) (both P < 0.05). Notably, GDF15 increased at a faster rate from the age of 65 years in women (11.5 ± 1.2 pg·mL-1 ·year-1 , P < 0.05) and 70 years in men (19.3 ± 2.3 pg·mL-1 ·year-1 , P < 0.05), resulting in higher GDF15 levels in men compared with women above the age of 65 years (P < 0.05). Independently of age and circulatory markers of inflammation, GDF15 was negatively correlated to relative STS power (P < 0.05) but not LEP, in both women and men. These findings were mainly explained by negative associations of GDF15 with specific STS power in women and men (both P < 0.05). CONCLUSIONS: A J-shaped relationship between age and systemic GDF15 was observed, with men at older age showing steeper increases and elevated GDF15 levels compared with women. Importantly, circulating GDF15 was independently and negatively associated with relative STS power, supporting the potential role of GDF15 as a sensitive biomarker of frailty in older people.

Protein Phosphorylation Signaling Cascades in Autism: The Role of mTOR Pathway.

The mammalian target of rapamycin (mTOR) signaling pathway is a central regulator of cell metabolism, growth, and survival in response to hormones, growth factors, nutrients, and stress-induced signals. In this review, we analyzed the studies on the molecular abnormalities of the mTOR-associated signaling cascades in autism spectrum disorders (ASDs) and outlined the prospects for the pathogenicity-targeting pharmacotherapeutic approaches to ASDs, in particular syndromic ASDs. Based on available experimental and clinical data, we suggest that very early detection of molecular abnormalities in the ASD risk groups can be facilitated by using peripheral blood platelets. Also, identification of the time window of critical dysregulations in the described pathways in the ASD risk groups might suggest further research directions leading to more efficacious pharmacotherapeutic interventions in ASDs.

Skeletal Muscle Microvascular Changes in Response to Short-Term Blood Flow Restricted Training-Exercise-Induced Adaptations and Signs of Perivascular Stress.

Aim: Previous reports suggest that low-load muscle exercise performed under blood flow restriction (BFR) may lead to endurance adaptations. However, only few and conflicting results exist on the magnitude and timing of microvascular adaptations, overall indicating a lack of angiogenesis with BFR training. The present study, therefore, aimed to examine the effect of short-term high-frequency BFR training on human skeletal muscle vascularization. Methods: Participants completed 3 weeks of high-frequency (one to two daily sessions) training consisting of either BFR exercise [(BFRE) n = 10, 22.8 ± 2.3 years; 20% one-repetition maximum (1RM), 100 mmHg] performed to concentric failure or work-matched free-flow exercise [(CON) n = 8, 21.9 ± 3.0 years; 20% 1RM]. Muscle biopsies [vastus lateralis (VL)] were obtained at baseline, 8 days into the intervention, and 3 and 10 days after cessation of the intervention to examine capillary and perivascular adaptations, as well as angiogenesis-related protein signaling and gene expression. Results: Capillary per myofiber and capillary area (CA) increased 21-24 and 25-34%, respectively, in response to BFRE (P < 0.05-0.01), while capillary density (CD) remained unchanged. Overall, these adaptations led to a consistent elevation (15-16%) in the capillary-to-muscle area ratio following BFRE (P < 0.05-0.01). In addition, evaluation of perivascular properties indicated thickening of the perivascular basal membrane following BFRE. No or only minor changes were observed in CON. Conclusion: This study is the first to show that short-term high-frequency, low-load BFRE can lead to microvascular adaptations (i.e., capillary neoformation and changes in morphology), which may contribute to the endurance effects previously documented with BFR training. The observation of perivascular membrane thickening suggests that high-frequency BFRE may be associated with significant vascular stress.

Synthesis and biological evaluation of 2-acylbenzofuranes as novel α-glucosidase inhibitors with hypoglycemic activity.

A series of benzofuran derivatives was synthesized as analogues of known natural α-glucosidase inhibitors. Their activity was evaluated in enzymatic assay and in rat model of diabetes mellitus. Newly identified inhibitors demonstrate significant potency with IC50 values ranging from 6.50 to 722.2 µm, as well as hypoglycemic activity exceeding the reference drug acarbose. Docking simulations provided insight into structure-activity relationships to direct further development of these novel hypoglycemic agents.

Blood flow restricted training leads to myocellular macrophage infiltration and upregulation of heat shock proteins, but no apparent muscle damage.

KEY POINTS: Muscular contractions performed using a combination of low external loads and partial restriction of limb blood flow appear to induce substantial gains in muscle strength and muscle mass. This exercise regime may initially induce muscular stress and damage; however, the effects of a period of blood flow restricted training on these parameters remain largely unknown. The present study shows that short-term, high-frequency, low-load muscle training performed with partial blood flow restriction does not induce significant muscular damage. However, signs of myocellular stress and inflammation that were observed in the early phase of training and after the training intervention, respectively, may be facilitating the previously reported gains in myogenic satellite cell content and muscle hypertrophy. The present results improve our current knowledge about the physiological effects of low-load muscular contractions performed under blood flow restriction and may provide important information of relevance for future therapeutic treatment of muscular atrophy. ABSTRACT: Previous studies indicate that low-load muscle contractions performed under local blood flow restriction (BFR) may initially induce muscle damage and stress. However, whether these factors are evoked with longitudinal BFR training remains unexplored at the myocellular level. Two distinct study protocols were conducted, covering 3 weeks (3 wk) or one week (1 wk). Subjects performed BFR exercise (100 mmHg, 20% 1RM) to concentric failure (BFRE) (3 wk/1 wk), while controls performed work-matched (LLE) (3 wk) or high-load (HLE; 70% 1RM) (1 wk) free-flow exercise. Muscle biopsies (3 wk) were obtained at baseline (Pre), 8 days into the intervention (Mid8), and 3 and 10 days after training cessation (Post3, Post10) to examine macrophage (M1/M2) content as well as heat shock protein (HSP27/70) and tenascin-C expression. Blood samples (1 wk) were collected before and after (0.1-24 h) the first and last training session to examine markers of muscle damage (creatine kinase), oxidative stress (total antibody capacity, glutathione) and inflammation (monocyte chemotactic protein-1, interleukin-6, tumour necrosis factor α). M1-macrophage content increased 108-165% with BFRE and LLE at Post3 (P < 0.05), while M2-macrophages increased (163%) with BFRE only (P < 0.01). Membrane and intracellular HSP27 expression increased 60-132% at Mid8 with BFRE (P < 0.05-0.01). No or only minor changes were observed in circulating markers of muscle damage, oxidative stress and inflammation. The amplitude, timing and localization of the above changes indicate that only limited muscle damage was evoked with BFRE. This study is the first to show that a period of high-frequency, low-load BFR training does not appear to induce general myocellular damage. However, signs of tissue inflammation and focal myocellular membrane stress and/or reorganization were observed that may be involved in the adaptation processes evoked by BFR muscle exercise.

Delayed Effect of Blood Flow-restricted Resistance Training on Rapid Force Capacity.

PURPOSE: The aim of the present study was to investigate the effect and time course of high-frequent low-load blood flow-restricted (BFR) resistance training on rapid force capacity (i.e., rate of torque development [RTD]). MATERIALS AND METHODS: Ten male subjects (22.8 ± 2.3 yr) performed four sets of knee extensor exercise (20% one-repetition maximum) to concentric failure during concurrent BFR of the thigh (100 mm Hg), and eight work-matched controls (21.9 ± 3.0 yr) trained without BFR (CON). Twenty-three training sessions were performed within 19 d. Maximal slow and fast knee joint velocity muscle strength and rapid force capacity (e.g., RTD) and evoked twitch contractile parameters were assessed before (Pre) and 5 and 12 d after (Post5 and Post12) training. Muscle biopsies were obtained Pre, after 8 d (Mid8), and 3 and 10 d after (Post3 and Post10) training to examine changes in myofiber area and expression of myocellular proteins known to be modified by cellular stress (CaMKII, annexin A6, SNO-CYS). RESULTS: RTD remained unchanged after BFR training at Post5, while increasing 15%-20% Post12 (P < 0.01). Evoked muscle twitch parameters showed a general decline Post5 (P < 0.01) while returning to baseline levels at Post12. All contractile parameters essentially remained unchanged in CON. Elevated CaMKII was observed with BFR training at Post3 (57%) and Post10 (71%) (P < 0.05), whereas SNO-CYS increased in CON at Mid8 (P < 0.05). CONCLUSION: This study is the first to show that low-load resistance exercise performed with BFR leads to marked increases in rapid force capacity (RTD). However, a general delayed adaptive response was observed for voluntary contractile parameters (including RTD) in parallel with a decline and subsequent recovery in evoked contractile properties, suggesting the delayed gain in rapid force capacity mainly have a peripheral origin.

Separate developmental programs for HLA-A and -B cell surface expression during differentiation from embryonic stem cells to lymphocytes, adipocytes and osteoblasts.

A major problem of allogeneic stem cell therapy is immunologically mediated graft rejection. HLA class I A, B, and Cw antigens are crucial factors, but little is known of their respective expression on stem cells and their progenies. We have recently shown that locus-specific expression (HLA-A, but not -B) is seen on some multipotent stem cells, and this raises the question how this is in other stem cells and how it changes during differentiation. In this study, we have used flow cytometry to investigate the cell surface expression of HLA-A and -B on human embryonic stem cells (hESC), human hematopoietic stem cells (hHSC), human mesenchymal stem cells (hMSC) and their fully-differentiated progenies such as lymphocytes, adipocytes and osteoblasts. hESC showed extremely low levels of HLA-A and no -B. In contrast, multipotent hMSC and hHSC generally expressed higher levels of HLA-A and clearly HLA-B though at lower levels. IFNγ induced HLA-A to very high levels on both hESC and hMSC and HLA-B on hMSC. Even on hESC, a low expression of HLA-B was achieved. Differentiation of hMSC to osteoblasts downregulated HLA-A expression (P = 0.017). Interestingly HLA class I on T lymphocytes differed between different compartments. Mature bone marrow CD4(+) and CD8(+) T cells expressed similar HLA-A and -B levels as hHSC, while in the peripheral blood they expressed significantly more HLA-B7 (P = 0.0007 and P = 0.004 for CD4(+) and CD8(+) T cells, respectively). Thus different HLA loci are differentially regulated during differentiation of stem cells.

Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis.

The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we provide a detailed method for creating SILAC media suitable for use in hESC experiments, additionally we describe methods for the isolation of membrane fractions and cytosolic and nuclear/membrane fractions.

System-wide temporal characterization of the proteome and phosphoproteome of human embryonic stem cell differentiation.

To elucidate cellular events underlying the pluripotency of human embryonic stem cells (hESCs), we performed parallel quantitative proteomic and phosphoproteomic analyses of hESCs during differentiation initiated by a diacylglycerol analog or transfer to media that had not been conditioned by feeder cells. We profiled 6521 proteins and 23,522 phosphorylation sites, of which almost 50% displayed dynamic changes in phosphorylation status during 24 hours of differentiation. These data are a resource for studies of the events associated with the maintenance of hESC pluripotency and those accompanying their differentiation. From these data, we identified a core hESC phosphoproteome of sites with similar robust changes in response to the two distinct treatments. These sites exhibited distinct dynamic phosphorylation patterns, which were linked to known or predicted kinases on the basis of the matching sequence motif. In addition to identifying previously unknown phosphorylation sites on factors associated with differentiation, such as kinases and transcription factors, we observed dynamic phosphorylation of DNA methyltransferases (DNMTs). We found a specific interaction of DNMTs during early differentiation with the PAF1 (polymerase-associated factor 1) transcriptional elongation complex, which binds to promoters of the pluripotency and known DNMT target genes encoding OCT4 and NANOG, thereby providing a possible molecular link for the silencing of these genes during differentiation.

Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells.

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research mainly because of the complex nature of hESC culture conditions. Here we describe complete SILAC labeling of hESCs with fully preserved pluripotency, self-renewal capabilities, and overall proteome status that was quantitatively analyzed to a depth of 1556 proteins and 527 phosphorylation events. SILAC-labeled hESCs appear to be perfectly suitable for functional studies, and we exploited a SILAC-based proteomics strategy for discovery of hESC-specific surface markers. We determined and quantitatively compared the membrane proteomes of the self-renewing versus differentiating cells of two distinct human embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell populations.

Teratoma formation by human embryonic stem cells is site dependent and enhanced by the presence of Matrigel.

When implanted into immunodeficient mice, human embryonic stem cells (hESCs) give rise to teratoma, tumor-like formations containing tissues belonging to all three germ layers. The ability to form teratoma is a sine qua non characteristic of pluripotent stem cells. However, limited data are available regarding the effects of implantation site and the methods employed for implantation on the success rate of teratoma formation. In this study, the rate of teratoma formation in immunodeficient mice was site dependent: subcutaneous (25-100%), intratesticular (60%), intramuscular (12.5%), and under the kidney capsule (100%). Co-injecting the hESCs with Matrigel increased subcutaneous teratoma formation efficiency from 25-40% to 80-100%. We did not observe site-specific differences in the teratoma composition at the histological level. However, subcutaneous teratomas were quite distinct, easy to remove, and caused minimal discomfort to the mice. Also, subcutaneous teratomas displayed larger proportion of solid tissues as opposed to cyst formation that dominated the teratomas formed at the other sites. Interestingly, a chromosomally abnormal hESCs with trisomy 20 formed teratomas where the ratio of differentiated to undifferentiated tissues was significantly decreased suggesting defective pluripotency of the cells. In conclusion, subcutaneous implantation of hESCs in presence of Matrigel appears to be the most efficient, reproducible, and the easiest approach for teratoma formation by hESCs. Also, teratoma formation can be employed to study the development defects exhibited by the chromosomally abnormal hESC lines.

The BRCA1/BARD1 heterodimer modulates ran-dependent mitotic spindle assembly.

The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM--a known TPX2 partner--and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.

DNA replication of mitotic chromatin in Xenopus egg extracts.

Prereplication complexes are assembled at eukaryotic origins of DNA replication in the G1 phase of the cell cycle, and they are activated in S phase by cyclin-dependent kinase (Cdk)2/cyclin E and Cdk2/cyclin A. Previous experiments using Xenopus nuclear assembly egg extracts suggested that Cdk1/cyclin A, which is normally active in early mitosis, can replace the function of Cdk2 in driving DNA replication, whereas Cdk1/cyclin B, which functions later in mitosis, cannot. Here, we use a completely soluble replication system derived from Xenopus egg extracts to show that Cdk1/cyclin B also can support DNA replication. The ability of mitotic Cdks to drive DNA replication raises the question of whether DNA replication is possible in mitosis. To address this question, chromatin containing prereplication complexes was driven into mitosis with Cdk1/cyclin B. Strikingly, upon addition of a replication extract, the chromatin underwent a complete round of DNA replication. Replicating mitotic chromosomes became visibly decondensed, and, after DNA replication was complete, they recondensed. Our results indicate that there is extensive overlap in the substrate specificity of the major metazoan Cdk/cyclin complexes and that mitosis is not fundamentally incompatible with DNA replication. The results suggest that origins that fail to initiate DNA replication in S phase might still be able to do so in mitosis.

MCM2-7 complexes bind chromatin in a distributed pattern surrounding the origin recognition complex in Xenopus egg extracts.

The MCM2-7 complex is believed to function as the eukaryotic replicative DNA helicase. It is recruited to chromatin by the origin recognition complex (ORC), Cdc6, and Cdt1, and it is activated at the G(1)/S transition by Cdc45 and the protein kinases Cdc7 and Cdk2. Paradoxically, the number of chromatin-bound MCM complexes greatly exceeds the number of bound ORC complexes. To understand how the high MCM2-7:ORC ratio comes about, we examined the binding of these proteins to immobilized linear DNA fragments in Xenopus egg extracts. The minimum length of DNA required to recruit ORC and MCM2-7 was approximately 80 bp, and the MCM2-7:ORC ratio on this fragment was approximately 1:1. With longer DNA fragments, the MCM2-7:ORC ratio increased dramatically, indicating that MCM complexes normally become distributed over a large region of DNA surrounding ORC. Only a small subset of the chromatin-bound MCM2-7 complexes recruited Cdc45 at the onset of DNA replication, and unlike Cdc45, MCM2-7 was not limiting for DNA replication. However, all the chromatin-bound MCM complexes may be functional, because they were phosphorylated in a Cdc7-dependent fashion, and because they could be induced to support Cdk2-dependent Cdc45 loading. The data suggest that in Xenopus egg extracts, origins of replication contain multiple, distributed, initiation-competent MCM2-7 complexes.

Xenopus Mcm10 binds to origins of DNA replication after Mcm2-7 and stimulates origin binding of Cdc45.

Current models suggest that the replication initiation factor Mcm10 is required for association of Mcm2-7 with origins of replication to generate the prereplicative complex (pre-RC). Here we report that Xenopus Mcm10 (XMcm10) is not required for origin binding of XMcm2-7. Instead, the chromatin binding of XMcm10 at the onset of DNA replication requires chromatin-bound XMcm2-7, and it is independent of Cdk2 and Cdc7. In the absence of XMcm10, XCdc45 binding, XRPA binding, and initiation-dependent plasmid supercoiling are blocked. Therefore, XMcm10 performs its function after pre-RC assembly and before origin unwinding. As one of the earliest known pre-RC activation steps, chromatin binding of XMcm10 is an attractive target for regulation by cell cycle checkpoints.