Cell and Tissue Research最新文献

Toll-like receptors (TLRs) are a class of pattern-recognition receptors that recognize pathogen- and damage-associated molecular patterns and initiate immune responses. TLRs selectively recruit distinct adapter molecules such as the myeloid differentiation primary response protein 88 (MyD88) that mediates signaling downstream of all TLRs, with the exception of TLR3. To investigate TLR signaling pathways in fish, we engineered a knockout clonal epitheliod fish cell line, named MYD88C2, using CRISPR/Cas9-mediated genome editing to disrupt the myd88 gene. We characterized the phenotype of this cell line alongside a wild-type cell line through gene-expression profiling and reporter-gene analyses in the context of stimulation with heat-killed Vibrio anguillarum, heat-killed Escherichia coli, flagellin, zymosan, and inoculation with a panel of viruses. We demonstrate that the proinflammatory response to zymosan and flagellin, as measured through the induction of proinflammatory genes, was greatly reduced in the MYD88C2 cell line. The responsiveness to zymosan was found to be partially restored by transfecting the MYD88C2 cell line with a myd88-expression plasmid. In contrast, the loss of the myd88 gene had no impact on the cytopathic effect associated with the replication of viral haemorrhagic septicemia virus (VHSV), infectious haematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), spring viraemia of carp virus (SVCV) or infectious salmon anaemia virus (ISAV). These findings highlight the critical role of MyD88 in mediating specific proinflammatory responses to bacterial and fungal stimuli, while its absence has no detectable impact on viral replication or cytopathogenicity in epithelioid fish cells under the conditions tested.

Fibulin-4, an extracellular matrix protein, is indispensable for elastic fiber assembly. Fibulin-4 null mice show bilateral forelimb contracture and patients with EFEMP2/FBLN4 mutations demonstrate similar defects, besides joint laxity, vascular and pulmonary abnormalities. Here we report that limb tendons in fibulin-4 null mice developed normally until E17.5-18.5, but thereafter　thinner tendons showed abnormalities, suggesting that fibulin-4 maintains the integrity of certain tendons. Tendon/ligament specific conditional knockout mice of Efemp2/Fbln4 (ScxCre-H;Fbln4^flox/- mice) were generated in order to elucidate its role of collagen fibril organization, collagen cross-linking and mechanical features of tendons. Curiously, however, the conditional Fbln4 knockout mice did not show forelimb contractures or other obvious morphological defects. We could detect small amounts of fibulin-4 in tendon extracts, but isolated tenocytes from the conditional knockout mice did not secrete fibulin-4, confirming that the Efemp2/Fbln4 gene was properly deleted in tenocytes. Electron microscopic analyses revealed an enhanced proportion of thinner fibrils in tendons from the conditional knockout mice. Furthermore, mechanical stress tests of patellar tendons of the conditional knockout mice revealed strongly reduced strain resistance compared to the tendons of control mice, although cross-link formation and thermal stability of tendon collagen were not affected. These finding indicate that fibulin-4 has an important role in the organization and stability of collagen fibrils in tendons.

Catecholamines, including dopamine, are thought to play important roles in the nervous system of mollusks. In the brains of the terrestrial gastropods, the procerebrum is the higher olfactory center involved in olfactory associative learning. Dopamine is known to affect the activities of interneurons in the procerebrum. However, little is known about from where and how dopaminergic neurons project to the procerebrum. In the present study, we generated a specific antibody against tyrosine hydroxylase (TH) and visualized catecholaminergic neurons, including dopaminergic neurons, in the central and peripheral nervous systems of the terrestrial slug Limax valentianus. We found that (1) the number of the cell bodies of catecholaminergic neurons was larger in the ventral aspect than in the dorsal aspect of the brain; (2) most of the TH-immunoreactive putative sensory afferents in the superior tentacle projected to the brain along the axis as bundles in a regularly spaced manner while bypassing the procerebrum following entry into the brain; (3) TH-immunoreactive nerves in the procerebrum were derived from at least three distinct origins outside and within the procerebrum; and (4) dopamine upregulated the oscillatory frequency of the local field potential oscillation in the procerebrum, which was occluded by pre-incubation with sulpiride, a mammalian D₂/D₃ receptor antagonist. This is the first study to investigate the catecholaminergic system, with a special focus on the procerebrum, using a specific antibody against TH in terrestrial gastropods. The present study uncovered catecholaminergic regulation of procerebrum activity through innervation from both the outside and inside of the procerebrum.

Peroxisomes are the site of important metabolic pathways as they contain enzymes involved in the β-oxidation of fatty acids, the degradation of reactive oxygen species and the synthesis of ether-lipids and steroid hormone precursors. They are present in virtually all eukaryotic cells, yet they demonstrate considerable heterogeneity of number and protein composition. The presence of peroxisomes and concomitant activity of peroxisomal enzymes was previously documented in ovarian cells and it was suggested that peroxisomal oxidative stress management through catalase plays a pivotal role during oogenesis. In the present article, an exhaustive analysis is presented of the changes in peroxisome abundance during folliculogenesis in mouse oocytes. To this end, a quantitative assessment of the number of peroxisomes was conducted through morphometric analysis in the oocytes in follicular and oestrus cycle stages. Furthermore, a qualitative investigation was undertaken into the distribution of peroxisomal proteins involved in their biogenesis, membrane transport and lipid/hormone and reactive oxygen species metabolism in secondary and tertiary follicles. Given the metabolic connection between peroxisomes and mitochondria, the study was complemented with immunofluorescence stainings for reactive oxygen species and mitochondrial electron transport chain complexes and antioxidative enzymes. Our results confirmed the presence of peroxisomes in all follicular cells, including the oocyte. They further prove that peroxisomal abundance and protein composition are subject to variation depending on follicular and oestrus cycle stage and follicular cell type. Most strikingly, the study provided evidence of the near absence of catalase and of oxidative stress marker 8-oxo-2'-deoxyguanosine in the oocyte.

Pannexin 1a (Panx1a), a neuronal ATP-release channel, is increasingly recognized for its role in neurodevelopment, yet its contribution to synaptic homeostasis under metabolic stress remains poorly defined. We demonstrate that Panx1a coordinates synaptic and metabolic processes supporting neural circuit stability in the developing zebrafish brain. Using a genetic Panx1a knockout model and pharmacological induction of oxidative stress via MPTP, we reveal that Panx1a loss exacerbates metabolic alterations, reduces extracellular ATP availability, and triggers transcriptional activation of AMPK-mTORC1 signaling, autophagy, and apoptosis. These molecular changes coincide with impaired synaptic gene expression and increased neuronal cell death, particularly in the tectum and pallium. Electrophysiological recordings further show that Panx1a may function as a regulator for preserving local field potential coherence and phase-amplitude coupling, with knockout larvae displaying aberrant oscillatory activity and reduced network adaptability. Our findings identify Panx1a as a regulator of the metabolic-synaptic interface during a vulnerable developmental window and suggest that its ablation could contribute to pathophysiological mechanisms underlying neurodevelopmental disorders.

Age-related loss of volume and strength of the external urethral sphincter (EUS) is involved in the pathogenesis of stress urinary incontinence. Here, we explored if aged EUS undergoes capillary rarefaction, a characteristic of sarcopenia. Ca²⁺ dynamics in pericytes of EUS capillaries were also investigated to understand their roles in regulating EUS blood supply. Florescence immunohistochemistry was employed to compare the density of striated muscle fibres, fibroblasts, capillaries and pericytes between young and aged female mouse EUS. Electrical filed stimulation (EFS)-induced Ca²⁺ dynamics in EUS capillary pericytes were investigated using young NG2-GCaMP6 mice. In comparison to young EUS, aged EUS displayed a reduced density of striated muscle fibres, an increased density of PDGFRα⁺ fibroblasts, as well as a reduced density of capillaries and pericytes. EFS evoked prolonged Ca²⁺ transients and transient Ca²⁺ reductions in α-smooth muscle actin negative pericytes. The EFS-induced Ca²⁺ transients were prevented by either guanethidine (10 µM) or phentolamine (1 µM), despite the lack of pericapillary tyrosine hydroxylase-positive sympathetic fibres. The EFS-induced Ca²⁺ reductions were prevented by L-nitroarginine (10 µM). Pericytes were not innervated by neuronal NOS (nNOS)-positive nerve fibres, while EUS muscle fibres express sarcolemmal nNOS immunoreactivity. Thus, capillary rarefaction may contribute to the age-related EUS loss associated fibrosis. The Ca²⁺ reductions in pericytes appear to be mediated by NO released from EUS muscle fibres. Since pericytes express ANO1 Ca²⁺-activated chloride channels, the Ca²⁺ reductions would cause hyperpolarisation that spreads to the upstream resulting in arteriolar dilatation to increase blood supply to contracting EUS fibres.

Bitter taste receptors (TAS2Rs), known for their role in oral taste perception, are also expressed in extra-oral tissues, suggesting broader physiological functions. This study examined TAS2R expression in porcine tissues and their potential link to detoxification via cytochrome P450 (CYP) enzymes. Using transcriptome profiling complemented by PCR validation, we examined 14 TAS2Rs and detected 8 receptors across multiple tissues, including tongue, liver, jejunum, duodenum, kidney, colon, ileum, and white adipose. Distinct tissue-specific expression patterns were observed, with the highest diversity and abundance in metabolically active organs. Correlation analysis revealed that TAS2R expression levels were not only strongly associated across tissues but also closely aligned with hepatic CYP content, suggesting a functional relationship. These findings provide novel insights into the extra-oral roles of TAS2Rs in pigs, highlighting their potential involvement in xenobiotic sensing, detoxification, and metabolic regulation.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer, with existing therapies largely ineffective for advanced stages. Ankyrin 3 (ANK3), a protein involved in various cellular processes, has been associated with poor prognosis and immune infiltration in ccRCC. However, its role in ccRCC development and progression remains poorly understood. In the present study, ANK3-knockdown 786-O cells were generated using a shRNA approach. Cellular assays revealed that ANK3 knockdown significantly enhanced cell proliferation, migration, and invasion compared to shControl cells. ANK3-knockdown cells also displayed increased nuclear size and accumulation of G2/M phase of the cell cycle. Proteomic analysis identified 59 significantly altered proteins in ANK3-knockdown cells, primarily involved in cell adhesion and structural integrity. Among these, 10 proteins were significantly associated with overall survival in ccRCC patients. Morphological analysis showed that ANK3-knockdown cells exhibited a more spindle-like shape and increased cellular protrusions (filopodia). Western blot analysis further demonstrated altered expression levels of junctional and cytoskeleton proteins, including decreased E-cadherin and increased ZO-1, ICAM-1, vimentin, and β-actin. Immunofluorescence analysis revealed the accumulation of F-actin in the cytoplasm and filopodia in ANK3-knockdown cells. Additionally, ANK3 knockdown enhanced cell adhesion and aggregation capabilities. Finally, a decrease in ANK3 expression was confirmed in ccRCC tissues (grades I and II) compared to adjacent normal tissues. These findings suggest that ANK3 acts as a potential modulator of cell adhesion and cytoskeleton remodeling in ccRCC and may represent a promising therapeutic target for this malignancy.