Effect of aging on neuroglobin expression in rodent brain

Neuroglobin (Ngb), a recently discovered O2-binding heme protein related to hemoglobin and myoglobin, protects neurons from hypoxic-ischemic injury in vitro and in vivo. In immunostained mouse brain sections, we found widespread expression of Ngb protein in neurons, but not astrocytes, of several brain regions that are prominently involved in age-related neurodegenerative disorders. Western blots from young adult (3 month), middle-aged (12 month), and aged (24 month) rats showed an age-related decline in Ngb expression in cerebral neocortex, hippocampus, caudate-putamen, and cerebellum. Loss of this neuroprotective protein may have a role in increasing susceptibility to age-related neurological disorders.     

The relative contribution of direct and indirect antigen recognition pathways to the alloresponse and graft rejection depends upon the nature of the transplant

In this study, we measured direct and indirect T-cell alloresponses mediated by CD4(+) and CD8(+) T cells in three mouse transplantation models: skin, cornea, and retina. We show that the contribution of direct and indirect antigen recognition pathways to the alloresponse to fully allogeneic grafts varies depending upon the nature of the tissue/organ transplanted. The implications of this finding for understanding the cellular mechanisms by which rejection is mediated in different transplant models are discussed.     

Total serum IgD is increased in atopic subjects

We have developed a sandwich-type ELISA system for measuring total IgD levels in the serum of atopics and non-atopic controls. In this ELISA system, affinity purified goat anti-human IgD was used for capture. Results were superior to those obtained with monoclonal anti-human IgD antibody. No cross-reactivity could be demonstrated to IgG, IgM, IgA or IgE. The assay showed minimal non-specific binding even with initial serum dilutions of 1:2. The results obtained were reproducible among replicates (Mean CV +/- SEM = 0.03 +/- 0.002; n = 251), between dilutions (CV = 0.08 +/- 0.006; n = 108), and between assays (CV = 0.05 +/- 0.12; n = 5). We used routine radioimmunoassay for measuring total serum IgE. Using these assays total serum IgD and IgE levels were measured in 75 atopic patients and 33 normal subjects. None of the atopics had recent immunotherapy. As expected, the geometric mean serum IgE in atopics (373 ng/ml) was significantly higher than that in normal subjects (49 ng/ml) (P less than 0.01). However, geometric mean serum IgD was also significantly higher in atopics (20.3 micrograms/ml) than that in normal subjects (8.4 micrograms/ml) (P less than 0.02). In both atopic and normal groups, mean serum IgD level did not differ significantly on the bases of age, sex or asthmatic status. Furthermore, total serum IgD was not significantly correlated with total serum IgE (r = 0.14; P = 0.14; n = 108), indicating that immunoregulatory control of the basal levels of the two isotypes is not linked.(ABSTRACT TRUNCATED AT 250 WORDS)     

Early atherosclerotic lesions in infancy: role of parental cigarette smoking

Cigarette smoking is associated with an increased incidence of atherosclerotic diseases. The aim of this study was to examine the progression of the preatherosclerotic lesions previously observed by us in coronary arteries of fetuses of smoker mothers and in infants with smoker parents. We examined the coronary arteries of 34 infants, aged 1–36 months, and the histological and biological [c-fos, proliferating cell nuclear antigen (PCNA), and apoptosis] features of the early atherosclerotic lesions. In 17 infants (50%), at least one parent smoked, generally more than five cigarettes a day. In 18 cases (53%), we observed variable thickening of the coronary walls from preatherosclerotic lesions to juvenile atherosclerotic plaques, related to parental smoking habit. This morphological progression of the lesions was accompanied by a sequence of biological changes in the smooth muscle cells of the tunica media. We suggest that the oxidants present in the gas phase of the parental cigarette smoke pass through the endothelium and induce at first the c-fos gene activation and subsequently the PCNA positivity, that is, a proliferative process.     

Memory for object and object-location after lesions to the ventromedial prefrontal cortex in humans

The aim of the present study was to investigate the effect of small unilateral lesions to the ventromedial portion of the prefrontal cortex on two memory functions: memory for objects and memory for object locations. Patients, who had undergone surgery of the anterior communicating artery aneurysm, and normal control subjects, participated in the study. The patients were subdivided into two groups: with and without unilateral resection of the gyrus rectus. Subjects were presented with two memory tests, that required remembering either simultaneously presented visual stimuli (object memory test; OMT) or locations of the stimuli (location memory test; LMT). In the OMT, patients with resection of the gyrus rectus were impaired in comparison to patients without resection and normal control subjects. In the LMT, the three groups did not differ from each other. Our results suggest that the ventromedial prefrontal cortex is specifically involved in memory for objects.     

Pooled protein tagging,cellular imaging,and in situ sequencing for monitoring drug action in real time

The levels and subcellular localizations of proteins regulate critical aspects of many cellular processes and can become targets of therapeutic intervention. However, high-throughput methods for the discovery of proteins that change localization either by shuttling between compartments, by binding larger complexes, or by localizing to distinct membraneless organelles are not available. Here we describe a scalable strategy to characterize effects on protein localizations and levels in response to different perturbations. We use CRISPR-Cas9-based intron tagging to generate cell pools expressing hundreds of GFP-fusion proteins from their endogenous promoters and monitor localization changes by time-lapse microscopy followed by clone identification using in situ sequencing. We show that this strategy can characterize cellular responses to drug treatment and thus identify nonclassical effects such as modulation of protein–protein interactions, condensate formation, and chemical degradation.

Currently available mass-spectrometry methods (Rix and Superti-Furga 2009; Martinez Molina et al. 2013; Savitski et al. 2014; Huber et al. 2015; Drewes and Knapp 2018) for monitoring the effects of cellular perturbations on proteomes cannot be scaled efficiently to monitor time-dependent effects in high throughput. A different approach to study drug action is live-cell imaging of protein dynamics in cells expressing a protein of interest fused to a fluorescent tag. Traditionally, such reporter cells are generated either by overexpression to nonphysiologic levels, by oligonucleotide-directed homologous recombination in yeast, or by using CRISPR-Cas9 and homology-directed repair (HDR) to endogenously tag proteins in human cells (Ghaemmaghami et al. 2003; Huh et al. 2003; Chong et al. 2015; Leonetti et al. 2016). In addition to those targeted approaches, “gene trapping” or “CD-tagging” strategies, which rely on the random, viral integration of fluorescent tags as synthetic exons, have been used for analyzing dynamic changes in response to drugs (Jarvik et al. 1996; Morin et al. 2001; Cohen et al. 2008; Kang et al. 2016), but they are limited by integration site biases and require the isolation and characterization of clones before using them in an arrayed format. Recently, a strategy combining genome engineering and gene trapping using homology-independent CRISPR-Cas9 editing to place a fluorescent tag as a synthetic exon into introns of individual target genes has been described (Serebrenik et al. 2019). The strategy relies on a generic sgRNA excising a fluorescent tag flanked by splice acceptor and donor sites from a generic donor plasmid, which is coexpressed with a gene-specific intron-targeting sgRNA specifying the integration site. Here we show the scalability of that strategy to enable pooled protein tagging of more than 900 metabolic enzymes and epigenetic modifiers. Exposing the GFP-tagged cells to compounds allows us to monitor drug effects on the localization and levels of hundreds of proteins in real time in a pooled format, followed by identification of responding clones by in situ sequencing of the expressed intron-targeting sgRNA that corresponds to the tagged protein (Fig. 1A).Open in a separate windowFigure 1.Pooled GFP intron-tagging of metabolic enzymes. (A) Schematic outline of the approach. (B) Identification of targetable introns within metabolic genes. (C) FACS sorting of clones with successful GFP-tagging by signal enrichment over background mCherry intensity used as control for autofluorescence. (D) Representative image of sorted GFP-tagged cell pool. Scale bar, 25 µm. (E) Comparison of RNA-seq expression in HAP1 cells between genes for which GFP-tagged cells could be isolated and genes that were targeted in the sgRNA library but did not result in successful clone isolation.     

Kinetics and mechanism of formation and properties of polymers composed of paired macromolecules

The influence of formation conditions on structure and properties of reaction products from two different macromolecules (paired polymers) such as polystyrene and poly(1,1,2-trichlorobutadiene) or polystyrene and poly(vinyl chloride) is investigated. Mechanical properties, molecular mobility, heat resistance, thermostability, and fire resistance are shown to be regulated over a wide range by changing the molecular weight of the initial polymers, their ratio in the reaction mixture, etc. The interaction of different macromolecules in solution to form paired polymers is analyzed theoretically and experimentally. An analysis of structure and properties of the resulting products by refractometry, viscometry, sedimentation velocity, statistical analysis, and others shows that paired polymers are systems of the “coil-in-coil” type held together by chemical bonds in the zones of mutual penetration.     

Multiple mechanisms govern the dynamics of depression at neocortical synapses of young rats

Synaptic transmission between pairs of excitatory neurones in layers V ( N = 38) or IV ( N = 6) of somatosensory cortex was examined in a parasagittal slice preparation obtained from young Wistar rats (14–18 days old). A combined experimental and theoretical approach reveals two characteristics of short-term synaptic depression. Firstly, as well as a release-dependent depression, there is a release-independent component that is evident in smaller postsynaptic responses even following failure to release transmitter. Secondly, recovery from depression is activity dependent and is faster at higher input frequencies. Frequency-dependent recovery is a Ca²⁺-dependent process and does not reflect an underlying augmentation. Frequency-dependent recovery and release-independent depression are correlated, such that at those connections with a large amount of release-independent depression, recovery from depression is faster. In addition, both are more pronounced in experiments performed at physiological temperatures. Simulations demonstrate that these homeostatic properties allow the transfer of rate information at all frequencies, essentially linearizing synaptic responses at high input frequencies.