We seek alterations in protein patterns at the earliest possible step on the path to cancer, namely, in cells of the target tissue from normal persons versus the corresponding normally appearing cells from persons who are heterozygous for mutation in a tumor suppressor gene that predisposes strongly to carcinoma in that tissue. To begin a systematic comparison of the proteomes of cells from normal and from neoplastic colons, we have undertaken the isolation of human colon crypts that are derived from the normal-appearing mucosa of left (descending) colon of patients with sporadic colorectal cancer. Two-dimensional (2D) gel electrophoresis is a proteomic approach that excels in the resolution of protein isoforms. Here, we document the practicality of this approach with human samples using gels of three overlapping pH ranges. For the first time, about 800 nonredundant proteins and 900 isoforms from purified human colonic crypts were identified, permitting an assessment of the contributions of protein isoforms. These interactive, searchable, hyperlink-enabled proteome maps and gene ontology analyses will facilitate future studies to discover the earliest markers and intervention targets during progression to colon cancer.
SUMMARYEpidermal growth factor (EGF) is mitogenic to gastric mucosal cells, protects against gastric mucosal injury induced by ulcerogenic agents, and stimulates cell migration in many systems. Restitution, the rapid re-establishment of mucosal integrity following damage, involves epithelial cell migration and can be monitored by measuring the transmucosal potential difference (PD) of tissue mounted in an Ussing chamber. The role of EGF in restitution is poorly characterized. To assess the effect of exogenous EGF on restitution, samples of rat gastric mucosa were mounted in an Ussing chamber with a solution of 10 nM EGF bathing either the mucosal or serosal surface. Matched control tissues from the same animals were untreated. Mucosal damage was induced by exposure of tissues to hyperosmolar sodium chloride. Restitution following injury was monitored by measuring the recovery of transmucosal PD, which reflects the degree of mucosal integrity. Undamaged mucosa maintained a PD of approximately -30 mV for several hours (mucosa negative with respect to serosa). Mucosal exposure to 1-2 M NaCl for 2 min reduced the PD to near 0 mV. Thereafter, the PD returned in approximately 60 min to plateau at a value representing maximal recovery. Tissues mucosally treated with 10 nM EGF recovered following damage to a maximum of 602 + 5.1 % of pre-exposure PD, while matched controls recovered to only 46.2 + 5-8 %, a significant difference. Tissues serosally treated with EGF recovered to 61.0 + 3.2 % and matched controls recovered to 51-1 + 2-9 %, also a significant difference. These results suggest that both serosally and mucosally applied EGF stimulates the restitution of rat gastric mucosa in vitro.
Background: Neurodevelopmental disorder (NDD) risk genes have pleiotropic biological functions, such as control over both developmental and non-developmental processes that influence disease-related phenotypes. Currently, it remains unclear how developmental versus non-developmental processes influence the duration and/or effectiveness of permissive treatment windows for NDDs. SYNGAP1 haploinsufficiency causes an NDD defined by autistic traits, cognitive impairment, and epilepsy. Syngap1 heterozygosity in mice disrupts a developmental critical period, and, consistent with this, certain behavioral abnormalities are resistant to gene therapy initiated in adulthood. However, the Syngap1 endophenotype is extensive and this protein has diverse cell biological functions. Therefore, SynGAP pleiotropy may influence the permissive treatment window for previously untested disease-relevant phenotypes.Methods: A whole-body gene restoration technique was used to determine how restoration of SynGAP protein in adult heterozygous mice impacted previously untested phenotypes, such as memory, seizure susceptibility, systems-level cortical hyperexcitability, and hippocampal oscillations linked to mnemonic processes.Results: Adult restoration of SynGAP protein in haploinsufficient mice reversed long-term contextual memory deficits and behavioral measures of seizure susceptibility. Moreover, SynGAP re-expression in adult mice eliminated brain state-dependent, patient-linked paroxysmal interictal spiking and increased the amplitude of hippocampal theta oscillations.Conclusions: SynGAP protein in the mature brain dynamically regulates neural circuit function and influences disease-relevant phenotypes. The impact of these findings is that treatments targeting certain debilitating aspects of SYNGAP1-related disorders may be effective throughout life. Moreover, the efficacy of experimental treatments for SYNGAP1 patients may be quantifiable through changes in species-conserved, state-dependent pathological electroencephalogram signals.
30SYNGAP1 is a major genetic risk factor for global developmental delay, autism spectrum 31 disorder, and epileptic encephalopathy. De novo loss-of-function variants in this gene cause a 32 neurodevelopmental disorder defined by cognitive impairment, social-communication disorder, 33 and early-onset seizures. Cell biological studies in mouse and rat neurons have shown that 34 Syngap1 regulates developing excitatory synapse structure and function, with loss-of-function 35 variants driving formation of larger dendritic spines and stronger glutamatergic transmission. 36However, studies to date have been limited to mouse and rat neurons. Therefore, it remains 37 unknown how SYNGAP1 loss-of-function impacts the development and function of human 38 neurons. To address this, we employed CRISPR/Cas9 technology to ablate SYNGAP1 protein 39 expression in neurons derived from a human induced pluripotent stem cell line (hiPSC). 40Reducing SynGAP protein expression in developing hiPSC-derived neurons enhanced dendritic 41 morphogenesis, leading to larger neurons compared to those derived from isogenic controls. 42Consistent with larger dendritic fields, we also observed a greater number of morphologically 43 defined excitatory synapses in cultures containing these neurons. Moreover, neurons with 44 reduced SynGAP protein had stronger excitatory synapses and expressed synaptic activity 45 earlier in development. Finally, distributed network spiking activity appeared earlier, was 46 substantially elevated, and exhibited greater bursting behavior in SYNGAP1 null neurons. We 47 conclude that SYNGAP1 regulates the postmitotic maturation of human neurons made from 48 hiPSCs, which influences how activity develops within nascent neural networks. Alterations to 49 this fundamental neurodevelopmental process may contribute to the etiology of SYNGAP1-50 related disorders. 51 52 53Pathogenic loss-of-function variants in the SYNGAP1 gene are causally-linked to a range of 55 neuropsychiatric disorders, including global developmental delay (GDD)/intellectual disability 56
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