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.

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).

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

Mammalian nuclei become licensed for DNA replication during late telophase.

Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase.