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Integrins, Integrin Regulators, and the Extracellular Matrix
Published in Bruce S. Bochner, Adhesion Molecules in Allergic Disease, 2020
Stephen W. Hunt, Sirid-Aimée Kellermann, Yoji Shimizu
A genetic approach has also been employed to elucidate the mechanisms by which activation upregulates β1 integrin activity (91). Mutants of the Jurkat T cell line were isolated by γ-irradiation and selection for cells that were unable to bind to Fn-coated plates following stimulation with PMA or anti-CD3 mAbs. These mutant cell lines expressed normal levels of β1 integrins. Furthermore, the ability of these mutants to bind to Fn and VCAM-1 following direct stimulation of β1 integrins with an activating β1-specific mAb suggested that the mutation induced in these cells did not alter the structural integrity of the ligand-binding domain. Genetic complementation studies indicated that at least three genetically distinct mutant types were isolated. The defect in β1 integrin function in one of these mutant types was also associated with the expression of an altered form of the MAPK isoform ERK-1 and defective production of IL-2 following CD3 stimulation. While the precise nature of the defects in β 1 integrin function in these mutant cells remains to be elucidated, early signaling events that impact integrin functional activity, such as tyrosine kinase activity, PKC activity, and intracellular Ca2+ flux, appear normal.
Mitochondrial Dysfunction and Epilepsies
Published in Shamim I. Ahmad, Handbook of Mitochondrial Dysfunction, 2019
Bindu Parayil Sankaran, Arun B. Taly
Mitochondria are highly specialized subcellular organelles present in almost all eukaryotic cells whose primary function is generation of adenosine tri phosphate (ATP) through the oxidative phosphorylation [OXPHOS]. (Chinnery and Hudson 2013) Oxidative phosphorylation involves five enzymatic complexes situated in the inner mitochondrial membrane, the mitochondrial respiratory chain (MRC). (Hatefi 1985) The most important and elegant aspect of MRC is that it is the result of genetic complementation between nuclear DNA and mitochondrial DNA. (Chinnery and Hudson 2013) Mitochondrial biogenesis requires the concerted interaction of more than 1500 proteins derived from these two distinct genomes. (Raymond, Horvath, and Chinnery 2018). MtDNA is a circular, double-stranded, 16,569 base-pair molecule of DNA that encodes 37 genes, including 13 polypeptides essential for the formation and function of four of the five MRC complexes, namely complex I, III, IV and V, two ribosomal RNAs (12S and 16S rRNA), and 22 transfer RNAs (tRNA). (Anderson et al. 1981) All other OXPHOS-related proteins, including most of the MRC subunits or factors controlling their expression, assembly function, and turnover is specifically targeted, sorted and imported to their final mitochondrial location (Mokranjac and Neupert 2005). Consequently OXPHOS dysfunction results from mutations in either mitochondrial genes or mito-nuclear genes. (Smeitink, van Den Heuvel, and DiMauro 2001).
Diseased States in Man and Other Vertebrates
Published in D. B. Keech, J. C. Wallace, Pyruvate Carboxylase, 2018
The mechanism of inheritance of PCD has been investigated by measuring the activity of pyruvate carboxylase in cultured fibroblasts, lymphocytes, and leukocytes obtained from members of the families of patients with a deficiency of the enzyme. For two patients, PC (Portland) and PC (St. Louis), the activity of pyruvate carboxylase in cells obtained from the parents and a sister was decreased (Figure 3).32,230 The results were found to be consistent with inheritance of the defect in an autosomal recessive manner (Figure 3).32 The mutation which leads to inherited PCD has been designated pyc.273 Genetic complementation studies conducted with fibroblast cell lines derived from patients deficient in pyruvate carboxylase and from two patients with biotin-responsive multiple carboxylase deficiency have indicated that the locus pyc is separate from the loci of mutations for biotin-responsive multiple carboxylase deficiency (bio).273
Peptidase PepP is a novel virulence factor of Campylobacter jejuni contributing to murine campylobacteriosis
Published in Gut Microbes, 2020
Markus M. Heimesaat, Anna-Maria Schmidt, Soraya Mousavi, Ulrike Escher, Nicole Tegtmeyer, Silja Wessler, Gabriele Gadermaier, Peter Briza, Dirk Hofreuter, Stefan Bereswill, Steffen Backert
A knockout mutant of pepP was constructed by homologous gene recombination using a construct in which the gene was interrupted by a kanamycin gene. The knockout mutant was checked by PCR and subsequent amplicon sequence analysis. Genetic complementation of the ∆pepP mutant with the WT pepP gene was performed as described previously.24 Presence or absence of PepP expression and proteolytical activity was confirmed by Western blotting using generated α-M24 (PepP) antibodies (Figure 1d) and casein zymography (Figure 1e), respectively. No significant growth differences were observed between the deletion mutant and its parental WT strain when grown on rich media (Supplementary Figure S3). Next, the knockout mutant and the isogenic WT strain were tested in the IL-10−/- mouse infection model.28 Therefore, microbiota-depleted IL10−/- mice were perorally infected with approximately 109 CFU of the C. jejuni ΔpepP, the pepP complemented or the WT strain on d 0 and 1 by gavage. Daily cultural analysis of fecal samples revealed high median bacterial loads of approximately 109 colony forming units (CFU) per g from d 2 until d 6 p.i., without differences between the three tested strains (Supplemental Figure S4). Likewise, the postmortem luminal loads on d 6 in the stomach, duodenum, and colon were comparable between respective strains (n.s.; Figure 2), whereas ileal bacterial numbers were slightly, but significantly lower in mice that had received the ΔpepP mutant as compared to the WT or pepP complemented strains (p < .05) (Figure 2). Hence, inactivation of the pepP gene did not compromise the colonization properties of C. jejuni in the colon, duodenum, and stomach, but decreased this ability in the ileum following peroral infection of microbiota-depleted IL-10−/- mice.
Cell cycle kinase CHEK2 in macrophages alleviates the inflammatory response to Staphylococcus aureus-induced pneumonia
Published in Experimental Lung Research, 2022
Fei Xie, Ruidong Chen, Jie Zhao, Chunyan Xu, Chunxiang Zan, Bin Yue, Wenqiu Tian, Wenxia Yi
There are some limitation should be indicated here. Only the protective effect of Chek2 in the acute phase of pneumonia is investigated in this research, while the treatment benefit of Chek2 in prolonged pneumonia is needed. The genetic complementation experiment testifies that the inhibition role of Chek2 to NF-κB can be attributed to the kinase activity of Chek2, while a more detailed and testified mechanism is further required.
Implications of NAD metabolism in pathophysiology and therapeutics for neurodegenerative diseases
Published in Nutritional Neuroscience, 2021
Keisuke Hikosaka, Keisuke Yaku, Keisuke Okabe, Takashi Nakagawa
The relationship between NAD metabolism and axonal degeneration has been intensively investigated in Wallerian degeneration, a process of removing injured distal axons (Table 2). At first, a strain of mice, C57BL/6/Ola, was discovered that exhibited a very slow Wallerian degeneration [22]. Subsequently, the locus affecting this phenotype was distally mapped on the mouse chromosome 4, and the mutant gene was designated as Wallerian degeneration slow (WldS) [23]. Further studies identified the WldS gene as a Ube4b/Nmnat1 chimeric gene, which encoded an N-terminal fragment of ubiquitination factor E4B (Ube4b) fused to full-length of Nmnat1 gene. In these mice, the chimeric protein WldS protected axons in a dose-dependent manner [24]. Furthermore, a transgenic mouse line, in which cytoplasm-targeted Nmnat1 (cytNmnat1) was overexpressed in the nervous system, showed a similar protective effect against Wallerian degeneration [25]. Overexpression of Nmnat3 was also reported to have protective effects against axonal degeneration in both in vivo and in vitro models [26–28]. In addition, overexpression of Nmnat1 or Nmnat3 exhibited a protective role in models of neonatal cerebral hypoxia-ischemia [29,30]. Depletion of endogenous Nmnat2 was adequate to induce Wallerian-like degeneration of uninjured axons [31]. The loss of Nmnat2 in mice led to embryonic lethality because of peripheral and central nervous system defects, and Nmnat2 compound heterozygous (Nmnat2gtBay/gtE) mice, which have one silenced and one partially silenced Nmnat2 allele and showed lower expression levels of Nmnat2, exhibited early and age-dependent peripheral nerve axon defects [32]. The genetic complementation of WldS in Nmnat2-deficient mice rescued the perinatal lethality by correcting the neuronal defect [33,34]. Thus, all Nmnat isozymes are considered to be important factors in axon growth and maintenance, both under physiological and pathological conditions.