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Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic citron rho-interacting serine/threonine kinase (CIT) missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it did not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lost cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupted the polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

Autism spectrum disorder is a pervasive neurodevelopmental disorder with a substantial contribution to the global disease burden. Despite intensive research efforts, the aetiopathogenesis remains unclear. The Janus-faced antioxidant enzymes superoxide dismutase 1-3 have been implicated in initiating oxidative stress and as such may constitute a potential therapeutic target. However, no measurement has been taken in human autistic brain samples. The aim of this study is to measure superoxide dismutase 1-3 in autistic cerebral organoids as an in vitro model of human foetal neurodevelopment. Whole brain organoids were created from induced pluripotent stem cells from healthy individuals (n = 5) and individuals suffering from autism (n = 4). Using Pierce bicinchoninic acid and enzyme-linked immunosorbent assays, the protein and superoxide dismutase 1, 2, and 3 concentrations were quantified in the cerebral organoids at days 22, 32, and 42. Measurements were normalized to the protein concentration. Results represented using medians and interquartile ranges. Using Wilcoxon matched-pairs signed-rank test, an abrupt rise in the superoxide dismutase concentration was observed at day 32 and onwards. Using Wilcoxon rank-sum test, no differences were observed between healthy (SOD1: 35.56 ng/mL ± 3.46; SOD2: 2435.80 ng/mL ± 1327.00; SOD3: 1854.88 ng/mL ± 867.94) and autistic (SOD1: 32.85 ng/mL ± 5.26; SOD2: 2717.80 ng/mL ± 1889.10; SOD3: 1690.18 ng/mL ± 615.49) organoids. Cerebral organoids recapitulate many aspects of human neurodevelopment, but the diffusion restriction may render efforts in modelling differences in oxidative stress futile due to the intrinsic hypoxia and central necrosis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

The biological basis of male-female brain differences has been difficult to elucidate in humans. The most notable morphological difference is size, with male individuals having on average a larger brain than female individuals 1,2 , but a mechanistic understanding of how this difference arises remains unknown. Here we use brain organoids 3 to show that although sex chromosomal complement has no observable effect on neurogenesis, sex steroids-namely androgens-lead to increased proliferation of cortical progenitors and an increased neurogenic pool. Transcriptomic analysis and functional studies demonstrate downstream effects on histone deacetylase activity and the mTOR pathway. Finally, we show that androgens specifically increase the neurogenic output of excitatory neuronal progenitors, whereas inhibitory neuronal progenitors are not increased. These findings reveal a role for androgens in regulating the number of excitatory neurons and represent a step towards understanding the origin of sex-related brain differences in humans., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

In mammals, early resistance to viruses relies on interferons, which protect differentiated cells but not stem cells from viral replication. Many other organisms rely instead on RNA interference (RNAi) mediated by a specialized Dicer protein that cleaves viral double-stranded RNA. Whether RNAi also contributes to mammalian antiviral immunity remains controversial. We identified an isoform of Dicer, named antiviral Dicer (aviD), that protects tissue stem cells from RNA viruses-including Zika virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-by dicing viral double-stranded RNA to orchestrate antiviral RNAi. Our work sheds light on the molecular regulation of antiviral RNAi in mammalian innate immunity, in which different cell-intrinsic antiviral pathways can be tailored to the differentiation status of cells., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Colorectal cancer (CRC) represents the fourth leading cause of cancer-related deaths. The heterogeneity of CRC identity limits the usage of cell lines to study this type of tumor because of the limited representation of multiple features of the original malignancy. Patient-derived colon organoids (PDCOs) are a promising 3D-cell model to study tumor identity for personalized medicine, although this approach still lacks detailed characterization regarding molecular stability during culturing conditions. Correlation analysis that considers genomic, transcriptomic, and proteomic data, as well as thawing, timing, and culturing conditions, is missing., Methods: Through integrated multi-omics strategies, we characterized PDCOs under different growing and timing conditions, to define their ability to recapitulate the original tumor., Results: Whole Exome Sequencing allowed detecting temporal acquisition of somatic variants, in a patient-specific manner, having deleterious effects on driver genes CRC-associated. Moreover, the targeted NGS approach confirmed that organoids faithfully recapitulated patients' tumor tissue. Using RNA-seq experiments, we identified 5125 differentially expressed transcripts in tumor versus normal organoids at different time points, in which the PTEN pathway resulted of particular interest, as also confirmed by further phospho-proteomics analysis. Interestingly, we identified the PTEN c.806_817dup (NM_000314) mutation, which has never been reported previously and is predicted to be deleterious according to the American College of Medical Genetics and Genomics (ACMG) classification., Conclusion: The crosstalk of genomic, transcriptomic and phosphoproteomic data allowed to observe that PDCOs recapitulate, at the molecular level, the tumor of origin, accumulating mutations over time that potentially mimic the evolution of the patient's tumor, underlining relevant potentialities of this 3D model.

Induction of cytochrome P450 can cause drug-drug interactions and efficacy failure. Induction risk in liver and gut is typically inferred from experiments with plated hepatocytes. Organoids are physiologically relevant, multicellular structures originating from stem cells. Intestinal stem cell-derived organoids retain traits of normal gut physiology, such as an epithelial barrier and cellular diversity. Matched human enteroid and colonoid lines, generated from ileal and colon biopsies from two donors, were cultured in extracellular matrix for 3 days, followed by a single 48-hour treatment with rifampin, omeprazole, CITCO, and phenytoin at concentrations that induce target genes in hepatocytes. After treatment, mRNA was analyzed for induction of target genes. Rifampin induced CYP3A4; estimated EC 50 and maximal fold induction were 3.75 µM and 8.96-fold, respectively, for ileal organoids and 1.40 µM and 11.3-fold, respectively, for colon organoids. Ileal, but not colon, organoids exhibited nifedipine oxidase activity, which was induced by rifampin up to 14-fold. The test compounds did not increase mRNA expression of CYP1A2, CYP2B6, multidrug resistance transporter 1 (P-glycoprotein), breast cancer resistance protein, and UDP-glucuronosyltransferase 1A1 in ileal organoids. Whereas omeprazole induced CYP3A4 (up to 5.3-fold, geometric mean, n = 4 experiments), constitutive androstane receptor activators phenytoin and CITCO did not. Omeprazole failed to induce CYP1A2 mRNA but did induce CYP1A1 mRNA (up to 7.7-fold and 15-fold in ileal and colon organoids, respectively, n = 4 experiments). Despite relatively high intra- and interexperimental variability, data suggest that the model yields induction responses that are distinct from hepatocytes and holds promise to enable evaluation of CYP1A1 and CYP3A4 induction in gut. SIGNIFICANCE STATEMENT: An adult intestinal stem cell-derived organoid model to test P450 induction in gut was evaluated. Testing several prototypical inducers for mRNA induction of P450 isoforms, UDP-glucuronosyltransferase 1A1, P-glycoprotein, and breast cancer resistance protein with both human colon and ileal organoids resulted in a range of responses, often distinct from those found in hepatocytes, indicating the potential for further development of this model as a physiologically relevant gut induction test system., Competing Interests: All authors are employees of AbbVie and may own AbbVie stock. AbbVie sponsored and funded the study; contributed to the design; participated in the collection, analysis, and interpretation of data; and participated in writing, reviewing, and approval of the final publication., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

The coevolution of mammalian hosts and their beneficial commensal microbes has led to development of symbiotic host-microbiota relationships 1 . Epigenetic machinery permits mammalian cells to integrate environmental signals 2 ; however, how these pathways are fine-tuned by diverse cues from commensal bacteria is not well understood. Here we reveal a highly selective pathway through which microbiota-derived inositol phosphate regulates histone deacetylase 3 (HDAC3) activity in the intestine. Despite the abundant presence of HDAC inhibitors such as butyrate in the intestine, we found that HDAC3 activity was sharply increased in intestinal epithelial cells of microbiota-replete mice compared with germ-free mice. This divergence was reconciled by the finding that commensal bacteria, including Escherichia coli, stimulated HDAC activity through metabolism of phytate and production of inositol-1,4,5-trisphosphate (InsP 3 ). Both intestinal exposure to InsP 3 and phytate ingestion promoted recovery following intestinal damage. Of note, InsP 3 also induced growth of intestinal organoids derived from human tissue, stimulated HDAC3-dependent proliferation and countered butyrate inhibition of colonic growth. Collectively, these results show that InsP 3 is a microbiota-derived metabolite that activates a mammalian histone deacetylase to promote epithelial repair. Thus, HDAC3 represents a convergent epigenetic sensor of distinct metabolites that calibrates host responses to diverse microbial signals.

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene ( FUT2 ) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme, while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals, while HIEs from secretors are permissive to infection. However, whether FUT2 expression alone is critical for infection remains unproven, since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication, we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies, these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status. IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however, previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains., (Copyright © 2020 Haga et al.)

Salisphere-derived adult epithelial cells have been used to improve saliva production of irradiated mouse salivary glands. Importantly, optimization of the cellular composition of salispheres could improve their regenerative capabilities. The Rho Kinase (ROCK) inhibitor, Y27632, has been used to increase the proliferation and reduce apoptosis of progenitor cells grown in vitro. In this study, we investigated whether Y27632 could be used to improve expansion of adult submandibular salivary epithelial progenitor cells or to affect their differentiation potential in different media contexts. Application of Y27632 in medium used previously to grow salispheres promoted expansion of Kit + and Mist1 + cells, while in simple serum-containing medium Y27632 increased the number of cells that expressed the K5 basal progenitor marker. Salispheres derived from Mist1 CreERT2 ; R26 TdTomato mice grown in salisphere media with Y27632 included Mist1-derived cells. When these salispheres were incorporated into 3D organoids, inclusion of Y27632 in the salisphere stage increased the contribution of Mist1-derived cells expressing the proacinar/acinar marker, Aquaporin 5 (AQP5), in response to FGF2-dependent mesenchymal signals. Optimization of the cellular composition of salispheres and organoids can be used to improve the application of adult salivary progenitor cells in regenerative medicine strategies., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Activation of both the DNA damage response (DDR) and transforming growth factor β (TGF-β) signaling induces growth arrest of most cell types. However, it is unclear whether the DDR activates TGF-β signaling that in turn contributes to cell growth arrest. Here, we show that in response to DNA damage, ataxia telangiectasia mutated (ATM) stabilizes the TGF-β type II receptor (TβRII) and thus enhancement of TGF-β signaling. Mechanistically, ATM phosphorylates and stabilizes c-Cbl, which promotes TβRII neddylation and prevents its ubiquitination-dependent degradation. Consistently, DNA damage enhances the interaction among ATM, c-Cbl, and TβRII. The ATM-c-Cbl-TβRII axis plays a pivotal role in intestinal regeneration after X-ray-induced DNA damage in mouse models. Therefore, ATM not only mediates the canonical DDR pathway but also activates TGF-β signaling by stabilizing TβRII. The double brake system ensures full cell-cycle arrest, allowing efficient DNA damage repair and avoiding passage of the damaged genome to the daughter cells., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Alzheimer´s disease (AD) is characterized by a progressive cognitive decline that correlates with the levels of amyloid β-peptide (Aβ) oligomers. Strong evidences connect changes of oligodendrocyte function with the onset of neurodegeneration in AD. However, the mechanisms controlling oligodendrocyte responses to Aβ are still elusive. Here, we tested the role of Aβ in oligodendrocyte differentiation, maturation, and survival in isolated oligodendrocytes and in organotypic cerebellar slices. We found that Aβ peptides specifically induced local translation of 18.5-kDa myelin basic protein (MBP) isoform in distal cell processes concomitant with an increase of process complexity of MBP-expressing oligodendrocytes. Aβ oligomers required integrin β1 receptor, Src-family kinase Fyn and Ca 2+ /CaMKII as effectors to modulate MBP protein expression. The pharmacological inhibition of Fyn kinase also attenuated oligodendrocyte differentiation and survival induced by Aβ oligomers. Similarly, using ex vivo organotypic cerebellar slices Aβ promoted MBP upregulation through Fyn kinase, and modulated oligodendrocyte population dynamics by inducing cell proliferation and differentiation. Importantly, application of Aβ to cerebellar organotypic slices enhanced remyelination and oligodendrocyte lineage recovery in lysolecithin (LPC)-induced demyelination. These data reveal an important role of Aβ in oligodendrocyte lineage function and maturation, which may be relevant to AD pathogenesis.

Background & Aims: African Americans have the greatest colorectal cancer (CRC) burden in the United States; interethnic differences in protective effects of vitamin D might contribute to disparities. 1α,25(OH) 2 D 3 vitamin D (the active form of vitamin D) induces transcription of the uridine phosphorylase gene (UPP1) in colon tissues of European Americans but to a lesser extent in colon tissues of African Americans. UPP1-knockout mice have increased intestinal concentrations of uridine and Deoxyuridine triphosphate (dUTP), have increased uridine-induced DNA damage, and develop colon tumors. We studied 1α,25(OH) 2 D 3 regulation of UPP1 and uridine-induced DNA damage in the colon and differences in these processes between African and European Americans., Methods: We quantified expression and activity of UPP1 in response to 1α,25(OH) 2 D 3 in young adult mouse colonic cells, human CRC cells (LS174T), and organoids (derived from rectosigmoid biopsy samples of healthy individuals undergoing colonoscopies) using quantitative polymerase chain reaction, immunoblot, and immunocytochemistry assays. Binding of the vitamin D receptor to UPP1 was tested by chromatin immunoprecipitation. Uridine-induced DNA damage was measured by fragment-length analysis in repair enzyme assays. Allele-specific 1α,25(OH) 2 D 3 responses were tested using luciferase assays., Results: Vitamin D increased levels of UPP1 mRNA, protein, and enzymatic activity and increased vitamin D receptor binding to the UPP1 promoter in young adult mouse colonic cells, LS174T cells, and organoids. 1α,25(OH) 2 D 3 significantly reduced levels of uridine and uridine-induced DNA damage in these cells, which required UPP1 expression. Organoids derived from colon tissues of African Americans expressed lower levels of UPP1 after exposure to 1α,25(OH) 2 D 3 and had increased uridine-induced DNA damage compared with organoids derived from tissues of European Americans. Luciferase assays with the T allele of single nucleotide polymorphism rs28605337 near UPP1, which is found more frequently in African Americans than European Americans, expressed lower levels of UPP1 after exposure to 1α,25(OH) 2 D 3 than assays without this variant., Conclusions: We found vitamin D to increase expression of UPP1, leading to reduce uridine-induced DNA damage, in colon cells and organoids. A polymorphism in UPP1 found more frequently in African Americans than European Americans reduced UPP1 expression upon cell exposure to 1α,25(OH) 2 D 3 . Differences in expression of UPP1 in response to vitamin D could contribute to the increased risk of CRC in African Americans., (Copyright © 2018 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: ( i ) continuous PKC inhibition and ( ii ) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin., Competing Interests: The authors declare no conflict of interest.

Colorectal cancer is driven by cooperating oncogenic mutations. In this study, we use organotypic cultures derived from transgenic mice inducibly expressing oncogenic β-catenin and/or PIK3CA H1047R to follow sequential changes in cancer-related signaling networks, intestinal cell metabolism, and physiology in a three-dimensional environment mimicking tissue architecture. Activation of β-catenin alone results in the formation of highly clonogenic cells that are nonmotile and prone to undergo apoptosis. In contrast, coexpression of stabilized β-catenin and PIK3CA H1047R gives rise to intestinal cells that are apoptosis-resistant, proliferative, stem cell-like, and motile. Systematic inhibitor treatments of organoids followed by quantitative phenotyping and phosphoprotein analyses uncover key changes in the signaling network topology of intestinal cells after induction of stabilized β-catenin and PIK3CA H1047R We find that survival and motility of organoid cells are associated with 4EBP1 and AKT phosphorylation, respectively. Our work defines phenotypes, signaling network states, and vulnerabilities of transgenic intestinal organoids as a novel approach to understanding oncogene activities and guiding the development of targeted therapies., (© 2017 Riemer et al.)

Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.

The aim of this study was to investigate the in vitro response of human craniofacial muscle-derived myotubes (primitive/nascent muscle fibres), in three-dimensional constructs, to strain in vitro to mimic clinical scenarios, using expression of the mechanoresponsive gene gelatinase-A/matrix metalloproteinase-2 (MMP-2) as a marker of remodelling of muscle extracellular matrix. Three-dimensional (3D) constructs of cells derived from explants of human masseter muscle (human craniofacial muscle-derived cells; hCMDC) in collagen sponges were subjected to mechanical, uniaxial strain using the Bio-Stretch system. 3D myotube constructs were exposed to the strain regimes of rapid ramp stretch (RRS) or cyclical ramp strain (CRS) with 7.5% and 15% strain. The activity of MMP-2 was assessed by zymography of construct-conditioned medium, whilst lysates of the constructs were used to measure creatine phosphokinase (CPK) activity to confirm the presence of myotubes in the strained constructs. Scanning electron microscopy of the collagen sponges and the CPK assays confirmed the presence of myotubes. MMP-2 was expressed by all the samples and controls, but expression was found to be significantly higher in those cultures strained continuously (RRS), compared to cyclical strain (CRS), and in those strained at 15% compared to 7.5%. Thus, MMP-2 expression, and hence extracellular matrix remodelling, is up-regulated in response to strain and is dependent upon the amount and type of strain to which the muscle is subjected., ((c) Eur J Oral Sci, 2005)

Background and Aims: Small hepatocytes (SH), which are hepatic progenitor cells, were isolated from an adult rat liver. SH in a colony sometimes change their shape from small to large and from flat to rising/piled-up. The morphological changes of SH may be correlated with hepatic maturation. Cytochrome P450s (CYP) are drug-metabolizing enzymes and the expression is one of hepatic differentiated functions. However, it is well known that the re-expression and maintenance of CYP activity are very difficult in cultured hepatocytes. We investigated the expression of CYP and the enzymatic activities in long-term cultured SH., Methods: SH were isolated from adult rat livers and SH colonies were collected, replated on new dishes, and then cultured. CYP1A1/2, CYP2B1, CYP3A2, CYP4A1, and CYP2E1 were induced by the addition of 3-methylcholanthrene, phenobarbital, pregnenolone-16alpha-carbonitrile, clofibric acid, and ethanol, respectively. Immunocytochemistry, immunoblots, and enzyme activities were examined., Results: SH could differentiate into mature hepatocytes by the addition of Matrigel and re-express constitutive CYPs. The expression of CYP1A1/2, CYP2B1, CYP3A2, and CYP4A1 dose-dependently increased and the amounts gradually increased with time in culture, especially in the cells treated with Matrigel. Activities of CYP1A, CYP2B, CYP3A and CYP2E in SH treated with Matrigel induced by each of the inducers were approximately 120-fold, 2.8-fold, 6.4-fold and 0.8-fold higher than in the control., Conclusion: The matured SH could re-express the constitutive CYP and recover inducibility, not only of protein expression but also of enzyme activities., ((c) 2005 Blackwell Publishing Asia Pty Ltd.)

Protein kinase C (PKC) is involved in signaling that modulates the proliferation and differentiation of many cell types, including mammary epithelial cells. In addition, changes in PKC expression or activity have been observed during mammary carcinogenesis. In order to examine the involvement of specific PKC isoforms during normal mammary gland development, the expression and localization of PKCs alpha, delta, epsilon and zeta were examined during puberty, pregnancy, lactation, and involution. By immunoblot analysis, expression of PKC alpha, delta, epsilon and zeta proteins was increased in mammary epithelial organoids during the transition from puberty to pregnancy. In mammary gland frozen sections, PKCs alpha, delta, epsilon and zeta were stained in the luminal epithelium and myoepithelium, in varying isoform-and developmental stage-specific locations. PKC alpha was found in a punctate apical localization in the luminal epithelium during pregnancy. During lactation, PKC epsilon was present in the nucleus, and PKC zeta was concentrated in the subapical region of the luminal epithelium. Additionally, marked staining for PKCs alpha, delta, epsilon, and zeta was observed in the myoepithelial cells at the base of ducts and alveoli. This basal ductal and alveolar staining differed in intensity in a developmentally-specific fashion. During most time points (virgin, pregnant, lactating, and early involution), myoepithelial cells of the duct were more intensely stained than those lining the alveoli for PKCs alpha, delta, epsilon and zeta. During late involution (days 9-12), the preferential staining of ducts was lost or reversed, and the myoepithelial cells lining the regressing alveolar structures stained equally (PKCs epsilon and zeta) or more intensely (PKCs alpha and delta), coincident with the thickening of the myoepithelial cells surrounding the regressing alveoli. The increased PKC isoform staining at the base of alveoli during involution suggests that alveolar regression may be influenced by alterations in signaling in the alveolar myoepithelium.

During chicken neurogenesis, the sequential expression of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) between final cell proliferation and differentiation is functionally not understood. Recently, cholinesterases have been shown to regulate neurite growth in vitro. Here, we investigated the effects of inhibition of BChE on laminar histogenesis in retinospheroids that arise from dissociated embryonic chicken retinal cells in rotation culture. In the presence of the BChE inhibitor iso-OMPA (tetraisopropyl pyrophosphoramide), the number of spheroids/dish is increased, and their diameter is decreased by about 20%, corresponding to about 50% volume size. As a corollary, the course of histotypical differentiation is dramatically accelerated. Thus as a consequence of BChE inhibition both, organization of nuclear cell layers and of plexiform-like (neuropile) areas, as detected by an antibody to the fiber fasciculation protein F11, is temporally advanced by at least two days. Moreover, AChE is almost fully diminished in these areas. The results further demonstrate novel roles of cholinesterases during laminar histogenesis of coherent neural networks in vitro.

A low-speed supernatant from dog liver was prepared and subjected to analytical subcellular fractionation using either preformed Percoll or reorientating sucrose density gradient centrifugation. In Percoll the following organelles, with marker enzymes and modal densities between brackets, were characterised: plasma membrane (alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, 1.039); endoplasmic reticulum (neutral alpha-glucosidase, 1.039); lysosomes (N-acetyl-beta-glucosaminidase, 1.087; alpha-mannosidase, 1.081) peroxisomes (catalase, 1.045) and mitochondria (succinate dehydrogenase, 1.081). In sucrose alkaline phosphatase had a bimodal particulate distribution (1.120, 1.187) distinct from that of 5' nucleotidase (1.160) and of gamma-glutamyl transferase (1.173). Other modal densities were: endoplasmic reticulum (1.187), lysosomes (1.227, 1.200), peroxisomes (1.213) and mitochondria (1.187). Further resolution was achieved by homogenisation in digitonin which disrupted lysosomes and, in sucrose, selectively increased the densities of the plasma membrane components. Both procedures therefore achieved distinct but quite different resolutions of organelles and should prove valuable for investigating subcellular pathology.

The Ca2+-ATPase activity in basal bodies of Tetrahymena pyriformis was determined by cytochemical and biochemical methods. It was found that the sites of the Ca2+-ATPase activity are associated with the basal body microtubules. A method for the isolation of the basal bodies in a purified form in amounts for biochemical assay has been developed. Some properties of basal body ATPase were examined. It was established that the enzyme activity is specific for the hydrolysis of ATP and deoxy-ATP. The characteristic behaviour of the ATPase activity in response to divalent cations was assessed: the enzyme is stimulated by Ca2+ and inhibited by Mg2+. Basal body ATPase has a pH optimum of 7.5. Electrophoretic analysis of the protein composition of basal bodies revealed proteins with molecular weights of 56 and 43 kDa, corresponding to those of tubulin and actin respectively. It is suggested that the basal body ATPase may be involved in beating of the cilia, participating in the mechanochemical processes needed for motility of Tetrahymena pyriformis.

Various procedures which reduce or deplete the kallikrein content of the cat's submandibular gland correspondingly reduce the number of apical granules in the striated duct cells. The kallikrein content is greatly reduced after chronic parasympathetic but not after sympathetic nerve section which suggests that the parasympathetic innervation is required for synthesis or storage of this enzyme.

Male Wistar rats were fed a diet with or without di(2-ethylhexyl)phthalate (DEHP), a peroxisome proliferator, for 2 weeks. The increases in the individual enzymes of the hepatic peroxisomal beta-oxidation system after administration of DEHP were 31- to 33-fold. It was found by in vivo experiments using L-[4,5-3H]leucine and the immunoprecipitation technique that the rates of synthesis of the enzymes were 16- to 20-fold higher and those of degradation were 1.7- to 1.9-fold lower in the DEHP group. The translation rates of these enzymes in vitro with liver RNA in the reticulocyte-lysate system were 12- to 14-fold higher in the DEHP group. Short-term kinetic labeling experiments on acyl-CoA oxidase consisting of three subunits were conducted in vivo to explore the biogenesis of peroxisomes. The label was found in the biggest subunit of the enzyme in the supernatant fraction shortly after the label injection, but was distributed to the smaller subunits later. The labeling in the smaller subunits in the peroxisomal fraction was greater than that of the supernatant. The distribution of the label among the subunits in these subcellular fractions was the same as that of the protein amounts 1 day after the label injection. This paper reports that the increase in the quantities of peroxisomal enzymes upon administration of DEHP is mainly due to the increase in their synthesis rates caused by the increase in amounts of mRNA coding for these enzymes.

The effect of ionic strength and pH on the release of some enzymes of the matrix of peroxisomes in rat's liver was studied. Catalase, L ALpha-hydroxy acid oxidase, isocitrate dehydrogenase, glycerophosphate dehydrogenase and lactate dehydrogenase were easily released from the particles during their lysis and treatment with 0.16 M KCl, whereas urate oxidase, NADH cytochrome c reductase and D-amino acid oxidase were not solubilized. After the solubilization of peroxisomal membrane by 0.2% Triton X-100, the remaining core contained about 50% amino acid oxidase activity, and had 1.28--1.30 g/cm3 density. These results suggest that D-amino acid oxidase associates with urate oxidase in the peroxisomal core.

We have localized a fraction of the enzyme, purine nucleoside phosphorylase (PNP), to the centrioles and basal bodies of mammalian, avian, and protozoan cells. Two completely independent methods were used, one based on the ultrastructural cytochemistry of the enzyme activity and one based on immunofluorescence microscopy using an antibody raised in rabbit against purified human PNP. PNP catalyzes the reversible conversion of purine nucleosides and inorganic phosphate to the corresponding purine bases and ribose-1-phosphate. Its partial localization to centrioles and basal bodies raises the possibility that purine compounds are involved in centriole replication and/or in the regulation of microtubule assembly in vivo. No centriolar PNP could be detected in primary skin fibroblast from two infants with severe immunodeficiency disease associated with the absence of soluble PNP. This raises the possibility that defects in centriole function may contribute to the impaired division and maturation of T lymphoid precursor in this inherited disorder. Initially, the immunofluorescence analyses were complicated by a residual centriole-binding antibody that persisted in immunoglobulins from immune animals after complete removal of anti-PNP by affinity chromatography. Binding was abolished by exposure of cells to sodium periodate, indicating that this (and possibly other) "spontaneous" anticentriole antibodies in rabbit serum may be directed against carbohydrates.

The orotate phosphoribosyltransferase of the epimastigote form of Trypanosoma cruzi was studied in its particulate state in preparations derived from glycosomes. Maximum activity was observed at pH 9. There was little activity in the absence of Mg2+; optimum [Mg2+] was related to [5'-phosphoribosyl-alpha-1-pyrophosphate]; Mn2+ could partially substitute for it. Kinetic analyses ruled out a substituted mechanism and suggested instead that the mechanism may be sequential. The apparent Km orotate was 2 microM; that for 5'-phosphoribosyl-alpha-1-pyrophosphate was 8 microM. The enzyme could not use uracil as substrate and was apparently not regulated by naturally-occurring nucleotides. It was, however, sensitive to inhibition by a wide range of pyrimidine analogues, the most active of which was 5-fluoroorotate. These inhibitors were as effective against the enzyme activity of intact glycosomes as broken preparations. This observation, when considered with an apparent lack of latency, suggests that the enzyme is located on the outside of the glycosome. The product of its reaction, orotidine 5'-phosphate, did not exchange readily with exogenous orotidine 5'-phosphate, suggesting that it is channeled directly to orotidine 5'-phosphate decarboxylase, the next enzyme in the pathway.

1. Enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase copurified when extracts from cotyledons of 5-day-old cucumber seedlings were fractionated by acetone precipitation, ion-exchange chromatography on CM-cellulose and affinity chromatography on blue-dextran-Sepharose. The protein was purified 600-fold with 16% recovery. 2. Bifunctionality of the protein was substantiated by exactly coinciding activity profiles upon chromatography on hydroxyapatite, CM-cellulose, or blue-dextran-Sepharose respectively. Analysis of the purified protein using isoelectric focusing as well as native electrophoresis confirmed the presence of a bifunctional protein. A monospecific antiserum could be raised against the homogeneous protein. 3. Further characterization of the protein revealed that the two enzyme activities are contained in a single peptide chain of Mr 75000. An extremely alkaline isoelectric point of pH 9.8 was determined. 4. The bifunctional protein was localized in glyoxysomes which were shown to be the exclusive site of beta-oxidation at this stage of germination. At least 10% of the enzyme were attributable to the organelle's membrane.

Male Wistar rats were given a diet containing 0.05% (w/w) LK-903 (alpha-methyl-p-myristyroxycinnamic acid 1-monoglyceride) for 2 weeks. The activities of four hepatic peroxisomal enzymes involved in the fatty acyl-CoA beta-oxidizing system were determined. The activities of fatty acyl-CoA oxidase, crotonase, beta-hydroxybutyryl-CoA dehydrogenase and thiolase were all increased about three times by administration of LK-903. The intraparticulate localizations of the four enzymes were then investigated by treatment of the purified peroxisomes with Triton X-100, by sonication, and by sucrose-density-gradient centrifugation after Triton X-100 treatment. The results suggest that thiolase is localized in the matrix of peroxisomes, that crotonase and beta-hydroxybutyryl-CoA dehydrogenase are located in the core, and that all or at least part of fatty acyl-CoA oxidase is associated with the core, though its association is weak.

Endogenous peroxidase activity was demonstrated by cytochemistry in mucous cells of the submucosal glands and tracheobronchial epithelium of guinea pigs. It is localized in the nuclear envelope, in cisternae of rough endoplasmic reticulum, and in secretory granules. It was not seen in Golgi saccules or in the airway lumen. By contrast, all epithelial cells within the lung including mucous (goblet) cells lack activity. Reaction product is also absent from alveolar macrophages and mast cells. The appearance of peroxidase in mucous cells is age-related. No activity was seen at 1.5 ms of age. A few mucous cells were positive at 2.5 and 3 ms while the proportion of positive cells increased substantially up to 7 ms. Thus, the age of guinea pigs in HRP transport studies must receive careful consideration in order to avoid misinterpretation of results. The function of mucous cell peroxidase is unknown. The results of this study suggest that it is secreted. Whether it plays a significant role in lung defense through its well documented anti-infectious properties remains to be determined.

Using trans-stilbene oxide as substrate, the subcellular distribution of epoxide hydrolase was investigated in livers from DBA/2 mice. The highest specific activities were found in cytosolic and light mitochondrial fractions. Isopycnic subfractionation of the light mitochondrial fraction showed that the organelle-bound trans-stilbene oxide hydrolase is localized in peroxisomes.

A peroxisome fraction was isolated from rat liver tissue using hypotonic treatment of mitochondria and lysosomes. In the fraction obtained activity of marker enzymes was studied in various subcellular structures. Content of mitochondria, lysosomes and microsomes in the peroxisome fraction was about 15-20% of the total protein in the fraction. The results of hypotonic treatment of lysosomes and mitochondria are considered. Flotation activity of lysosomes was decreased to 1.16-1.20 g/cm3 after osmotic lysis and elimination of matrix enzymes from these organelles.

The localization of cyclic 3', 5'-nucleotide phosphodiesterase activity at the neuromuscular junction and in skeletal muscle of the newt was studied cytochemically. In the axon terminal, activity was localized to a site on the membranes of synaptic vesicles. In skeletal muscle, activity occurred in the sarcoplasmic reticulum but was restricted to the longitudinal tubules. Junctional folds of the motor end plate were unreactive. These findings are suggestive of a role of cyclic AMP in the functions of synaptic vesicles and sarcoplasmic reticulum and indicate its effects are terminated at these sites.

Rectal biopsy specimens from control subjects, patients with either active or quiescent ulcerative colitis, and patients with Crohn's colitis were examined histologically and assayed for marker enzymes associated with tissue organelles. They were catalase (peroxisome); neutral alpha-glucosidase (endoplasmic reticulum); alkaline phosphatase (plasma membrane); malate dehydrogenase (mitochondria); lactate dehydrogenase (cytosol). There was no significant change in these enzyme activities in patient samples compared with controls. Activities of three acid hydrolases (lysosomal enzymes), beta-glucuronidase, acid phosphatase, and N-acetyl-beta-glucosaminidase, were also assayed in the biopsy samples. Decreased activities of all three enzymes were noted in ulcerative colitis, particularly in active disease. Normal values were obtained in Crohn's colitis. Measurement of lysosomal integrity by assays of latent N-acetyl-beta-glucosaminidase activity revealed similar results in control and colitic subjects. It is suggested that the lysosomal changes reflect a specific tissue release of enzyme and may be implicated in the pathogenesis of the tissue damage.

The activities of five peroxisomal enzymes (fatty acyl-CoA oxidase, FAO; uricase, U; L-hydroxyacid oxidase, LHAO; D-aminoacid oxidase, DAO; and catalase, C and the triglyceride content were determined in the regenerating liver of rats after partial hepatectomy. The FAO activity was significantly increased on day 3. Thus, its rise followed the increase of the triglyceride content and preceded the disappearance of steatosis. For U and LHAO a similar progressive increase in activities was obtained on days 3, 5, and 7 of regeneration. DAO exhibited a highly significant increase on day 1. C increased on day 1 and then again after day 3. It is concluded that peroxisomal functions are not restored at parallel rates during liver regeneration.

The native tonoplast and the mitochondrial H+-ATPase from oat roots were compared to determine whether the two enzymes have similar mechanisms. H+ pumping in low-density microsomal vesicles reflected activity from the tonoplast-type ATPase, as ATPase activity and ATP-dependent H+ pumping (quinacrine fluorescence quenching) showed similar sensitivities to inhibition by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4,4'-diisothiocyano-2,2'-stilbene disulfonate, nitrate, quercetin, or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. The tonoplast-type ATPase was stimulated by C1-,Br- greater than HCO3- whereas the mitochondrial ATPase was stimulated by HCO3- much greater than C1-,Br-. Both enzymes hydrolyzed ATP preferentially and were inhibited competitively by AMP or ADP. Apart from resistance to azide, the tonoplast-type ATPase was strikingly similar in its inhibitor sensitivities to the mitochondrial ATPase. The insensitivity to vanadate of both enzymes suggests the reaction mechanisms do not involve a covalent phosphoenzyme. Inhibition by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole and N-ethylmaleimide and protection by ATP suggests tyrosine and cysteine residues are in the catalytic site of the tonoplast ATPase. The mitochondrial ATPase was 100 times more sensitive to N,N'-dicyclohexyl-carbodiimide inhibition than the tonoplast H+-ATPase. These results suggest the tonoplast and the mitochondrial H+-ATPases share common steps in their catalytic and vectorial reaction mechanisms, yet sufficient differences exist to indicate they are two distinct ATPases.

The appearance of the characteristic crystalloid core of rat liver peroxisomes is emulated by the electron microscopic (EM) appearance of highly purified urate oxidase prepared from the same tissue. The purity of the enzyme preparation was established by gel electrophoresis under various conditions and the specific enzyme activity was at least as high as any previously reported. The amino acid composition of urate oxidase was determined. As additional evidence for close association of the peroxisomal core with urate oxidase, it was demonstrated that the biphasic changes in rat liver urate oxidase activity in response to prolonged starvation were paralleled by changes in the EM appearance of peroxisomes. Under comparable conditions catalase, another peroxisomal enzyme, did not show the same changes in activity as did urate oxidase. Evidence for the possible identity of urate oxidase with the peroxisomal crystalloid of rat liver has been presented, all materials having been obtained from, and experiments performed with, the rat.

Lutoids (vacuo-lysosomal particles) were isolated from the latex of Hevea brasiliensis. Using flow dialysis with 14C-methylamine uptake as a delta pH probe and 86Rb rubidium + valinomycin distribution for estimations of transmembrane electrical potential, intact lutoids exhibited a delta pH of 1 unit (interior more acid) and a delta psi of -70 mV (interior negative), when suspended in an isotonic medium at physiological concentrations of potassium (30 mM) and pH 7.0, in the absence of ATP. In most cases, the Donnan potential was shown to fully account for delta pH in nonenergized lutoids. The addition of MG-ATP (5 mM) resulted in a marked acidification of the lutoidic internal space (0.7 to 1 pH unit) depending on the composition of the medium, and in a membrane depolarization by 60 mV (interior becoming less negative). The resulting electrochemical potential of protons (delta approximately microH) increased by a hundred millivolts when lutoids were energized by ATP. These data strongly support an inward electrogenic proton translocating function for the ATPase of the vacuo-lysosomal membrane of lutoids. Results are discussed in terms of the in vivo maintenance of large "lutoids/cytoplasm" proton gradients, and of the rôle of these vacuo-lysosomes in the homeostasis of the cytoplasmic metabolism.

Protoplasts of Saccharomyces cerevisiae synthesized and degraded trehalose when they were incubated in a medium containing traces of glucose and acetate. Such protoplasts were gently lyzed by the polybase method and a particulate and soluble fraction was prepared. Trehalose was found in the soluble fraction and the trehalase activity mostly in the particulate fraction which also contained the vacuoles besides other cell organelles. Upon purification of the vacuoles, by density gradient centrifugation, the specific activity of trehalase increased parallel to the specific content of vacuolar markers. This indicates that trehalose is located in the cytosol and trehalase in the vacuole. It is suggested that trehalose, in addition to its role as a reserve may also function as a protective agent to maintain the cytosolic structure under conditions of stress.

A phosphatase, hydrolyzing pyridoxal-5-phosphate (P5P), a physiologically active component of the vitamin B6 complex and an essential co-enzyme in the synthesis of neurotransmitters, has been localized cytochemically in the perikarya of neurons in the peripheral, autonomic and central nervous systems of the rat. Neurons in dorsal root ganglia, sympathetic ganglia and ventral horn of spinal cord were studied by light and electron microscopy, while Purkinje cells, neurons in the dentate nucleus of the cerebellum, thalamus, and hypothalamus were studied by light microscopy only. Optimal conditions for demonstrating this activity in aldehyde-fixed tissue were determined with dorsal root ganglia. At the optimal pH of 5.0, neurons in these ganglia and in all other neurons studied show pyridoxal-5-phosphatase (P5Pase) activity in GERL. Small neurons in dorsal root ganglia also display enzyme activity in the endoplasmic reticulum (ER); activities in GERL and ER are also appreciably high at neutral pH. Small and large neurons in these ganglia, and neurons of sympathetic ganglia, show variable P5Pase activity in the Golgi apparatus. These localizations differ from the usual sites of both acid phosphatase and alkaline phosphatase activities. The P5Pase activity, demonstrated cytochemically, is a new acid hydrolase activity in GERL.