Effects of Volatile Anesthetics on Glutamate Transporter, Excitatory Amino Acid Transporter Type 3: The Role of Protein Kinase C

Background: Glutamate transporters play an important role in maintaining extracellular glutamate homeostasis. The authors studied the effects of volatile anesthetics on one type of glutamate transporters, excitatory amino acid transporter type 3 (EAAT3), and the role of protein kinase C in mediating these effects.

Methods: Excitatory amino acid transporter type 3 was expressed in Xenopus oocytes by injection of EAAT3 mRNA. Using two-electrode voltage clamp, membrane currents were recorded before, during, and after application of l-glutamate. Responses were quantified by integrating the current trace and are reported as microcoulombs. Data are mean +/- SEM.

Results: l-Glutamate-induced responses were increased gradually with the increased concentrations of isoflurane, a volatile anesthetic. At 0.52 and 0.70 mm isoflurane, the inward current was significantly increased compared with control. Isoflurane (0.70 mm) significantly increased Vmax (maximum velocity) (3.6 +/- 0.4 to 5.1 +/- 0.4 [mu]C;P < 0.05) but not Km (Michoelis-Menten Constant) (55.4 +/- 17.0 vs. 61.7 +/- 13.6 [mu]m;P > 0.05) of EAAT3 for glutamate compared with control. Treatment of the oocytes with phorbol-12-myrisate-13-acetate, a protein kinase C activator, caused a significant increase in transporter current (1.7 +/- 0.2 to 2.5 +/- 0.2 [mu]C;P < 0.05). Responses in the presence of the combination of phorbol-12-myrisate-13-acetate and volatile anesthetics (isoflurane, halothane, or sevoflurane) were not greater than those when volatile anesthetic was present alone. Oocytes pretreated with any of the three protein kinase C inhibitors alone (chelerythrine, staurosporine, or calphostin C) did not affect basal transporter current. Although chelerythrine did not change the anesthetic effects on the activity of EAAT3, staurosporine or calphostin C abolished the anesthetic-induced increase of EAAT3 activity.     

Angio-immunoblastic lymphadenopathy: a clinical, immunological and molecular study

Twenty four patients with angio-immunoblastic lymphadenopathy (AILD) presenting between 1974 and 1985 have been reviewed. Clinical features at presentation included rash, fever, lymphadenopathy and hepatosplenomegaly in 75% of patients. Polyclonal hypergammaglobulinaemia was seen in 19/20 patients; 5 had normal immunoglobulin levels. Combination chemotherapy with MVPP was the optimal treatment with 6/7 patients achieving complete remission. Duration of remission ranged from 9 months to 4 years and was significantly longer in patients achieving complete as opposed to partial remission. In 6 patients phenotype studies were performed on single cell suspensions and immunoperoxidase studies on frozen sections of 7 lymph nodes. There was a reversal of the normal T suppressor/helper cell ratio with a predominance of T suppressor cells. Loss of normal B follicles was observed histologically in all except one lymph node. Germline configuration of the beta B-chain of the T cell receptor was observed in lymph nodes of 4 patients with AILD, and a rearranged T cell receptor was observed in 1 patient in whom a second lymph node biopsy had shown alteration of the histological picture to that of T-zone lymphoma. Frozen sera of 15 patients were screened for antibodies to HTLV I and III and were found to be negative.     

Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.     

Novel porous aortic elastin and collagen scaffolds for tissue engineering

Decellularized vascular matrices are used as scaffolds in cardiovascular tissue engineering because they retain their natural biological composition and three-dimensional (3-D) architecture suitable for cell adhesion and proliferation. However, cell infiltration and subsequent repopulation of these scaffolds was shown to be unsatisfactory due to their dense collagen and elastic fiber networks. In an attempt to create more porous structures for cell repopulation, we selectively removed matrix components from decellularized porcine aorta to obtain two types of scaffolds, namely elastin and collagen scaffolds. Histology and scanning electron microscopy examination of the two scaffolds revealed a well-oriented porous decellularized structure that maintained natural architecture of the aorta. Quantitative DNA analysis confirmed that both scaffolds were completely decellularized. Stress-strain analysis demonstrated adequate mechanical properties for both elastin and collagen scaffolds. In vitro enzyme digestion of the scaffolds suggested that they were highly biodegradable. Furthermore, the biodegradability of collagen scaffolds could be controlled by crosslinking with carbodiimides. Cell culture studies showed that fibroblasts adhered to and proliferated on the scaffold surfaces with excellent cell viability. Fibroblasts infiltrated about 120 microm into elastin scaffolds and about 40 microm into collagen scaffolds after 4 weeks of rotary cell culture. These results indicated that our novel aortic elastin and collagen matrices have the potential to serve as scaffolds for cardiovascular tissue engineering.     

The role of conditioned taste aversion in the suppression of food intake by estradiol

In three experiments, ovariectomized rats were given a familiar or novel diet prior to treatment with a brain-enhanced estradiol-chemical delivery system (E2-CDS, 1 mg/kg). Experiment 1 showed that food intake was suppressed in subjects receiving either diet, but animals given a novel diet initially showed a profound anorexia which eventually recovered to the moderate suppression of animals given the familiar diet. In Experiment 2, rats showed an aversion to a novel diet paired with the E2-CDS in a two-choice preference test given on Day 2 after the injection, indicating that the initial large reduction in intake was mediated by a conditioned taste aversion. However, no aversion was observed seven days after the E2-CDS, suggesting that the residual intake suppression was mediated by unconditioned aversion or appetite suppression. Experiment 3 showed that lengthening the postovariectomy time resulted in a taste aversion that persisted for a longer duration.     

IL-4–BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy

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