Superoxide dismutase isozymes in cerebral organoids from autism spectrum disorder patients.

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.

Androgens increase excitatory neurogenic potential in human brain organoids.

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 individuals1,2, but a mechanistic understanding of how this difference arises remains unknown. Here we use brain organoids3 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.

An isoform of Dicer protects mammalian stem cells against multiple RNA viruses.

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.

Integrated multi-omics analyses on patient-derived CRC organoids highlight altered molecular pathways in colorectal cancer progression involving PTEN.

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.

Evaluation of Tissue Stem Cell-Derived Human Intestinal Organoids, a Physiologically Relevant Model to Evaluate Cytochrome P450 Induction in Gut.

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 EC50 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.

Microbiota-derived metabolite promotes HDAC3 activity in the gut.

The coevolution of mammalian hosts and their beneficial commensal microbes has led to development of symbiotic host-microbiota relationships1. Epigenetic machinery permits mammalian cells to integrate environmental signals2; 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 (InsP3). Both intestinal exposure to InsP3 and phytate ingestion promoted recovery following intestinal damage. Of note, InsP3 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 InsP3 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.

Genetic Manipulation of Human Intestinal Enteroids Demonstrates the Necessity of a Functional Fucosyltransferase 2 Gene for Secretor-Dependent Human Norovirus Infection.

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.

ROCK inhibitor increases proacinar cells in adult salivary gland organoids.

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 Mist1CreERT2; R26TdTomato 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.

DNA Damage Activates TGF-ß Signaling via ATM-c-Cbl-Mediated Stabilization of the Type II Receptor TßRII.

Activation of both the DNA damage response (DDR) and transforming growth factor ß (TGF-ß) signaling induces growth arrest of most cell types. However, it is unclear whether the DDR activates TGF-ß 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-ß type II receptor (TßRII) and thus enhancement of TGF-ß signaling. Mechanistically, ATM phosphorylates and stabilizes c-Cbl, which promotes TßRII neddylation and prevents its ubiquitination-dependent degradation. Consistently, DNA damage enhances the interaction among ATM, c-Cbl, and TßRII. The ATM-c-Cbl-TßRII 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-ß signaling by stabilizing TßRII. 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.

Aß oligomers promote oligodendrocyte differentiation and maturation via integrin ß1 and Fyn kinase signaling.

Alzheimer´s disease (AD) is characterized by a progressive cognitive decline that correlates with the levels of amyloid ß-peptide (Aß) oligomers. Strong evidences connect changes of oligodendrocyte function with the onset of neurodegeneration in AD. However, the mechanisms controlling oligodendrocyte responses to Aß are still elusive. Here, we tested the role of Aß in oligodendrocyte differentiation, maturation, and survival in isolated oligodendrocytes and in organotypic cerebellar slices. We found that Aß 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. Aß oligomers required integrin ß1 receptor, Src-family kinase Fyn and Ca2+/CaMKII as effectors to modulate MBP protein expression. The pharmacological inhibition of Fyn kinase also attenuated oligodendrocyte differentiation and survival induced by Aß oligomers. Similarly, using ex vivo organotypic cerebellar slices Aß promoted MBP upregulation through Fyn kinase, and modulated oligodendrocyte population dynamics by inducing cell proliferation and differentiation. Importantly, application of Aß to cerebellar organotypic slices enhanced remyelination and oligodendrocyte lineage recovery in lysolecithin (LPC)-induced demyelination. These data reveal an important role of Aß in oligodendrocyte lineage function and maturation, which may be relevant to AD pathogenesis.

Vitamin D Regulation of the Uridine Phosphorylase 1 Gene and Uridine-Induced DNA Damage in Colon in African Americans and European Americans.

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)2D3 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)2D3 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)2D3 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)2D3 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)2D3 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)2D3 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)2D3 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)2D3. Differences in expression of UPP1 in response to vitamin D could contribute to the increased risk of CRC in African Americans.

Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells.

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.

Oncogenic ß-catenin and PIK3CA instruct network states and cancer phenotypes in intestinal organoids.

Colorectal cancer is driven by cooperating oncogenic mutations. In this study, we use organotypic cultures derived from transgenic mice inducibly expressing oncogenic ß-catenin and/or PIK3CAH1047R to follow sequential changes in cancer-related signaling networks, intestinal cell metabolism, and physiology in a three-dimensional environment mimicking tissue architecture. Activation of ß-catenin alone results in the formation of highly clonogenic cells that are nonmotile and prone to undergo apoptosis. In contrast, coexpression of stabilized ß-catenin and PIK3CAH1047R 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 ß-catenin and PIK3CAH1047R 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.

RIPK1 ensures intestinal homeostasis by protecting the epithelium against apoptosis.

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.

A three-dimensional in vitro model system to study the adaptation of craniofacial skeletal muscle following mechanostimulation.

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.

Expression of cytochrome P450 enzymes in hepatic organoid reconstructed by rat small hepatocytes.

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.

Alterations in the expression and localization of protein kinase C isoforms during mammary gland differentiation.

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.

Butyrylcholinesterase regulates laminar retinogenesis of the chick embryo in vitro.

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.

Melanogenic regulatory factors in coated vesicles from melanoma cells.

Coated vesicles have been found to contain much higher tyrosinase and gamma-glutamyl transpeptidase activities than premelanosomes. This indicates that similar to tyrosinase, gamma-glutamyl transpeptidase, an enzyme responsible for pheomelanogenesis, is highly concentrated in coated vesicles after its maturation in Golgi associated endoplasmic reticulum (GERL). Furthermore, in the pre- and post-dopaquinone melanogenic pathway, coated vesicles convert dopachrome to colorless indole compounds more quickly than in premelanosomes because of their higher dopachrome conversion factor activity. Melanosomes have been found to exhibit indole conversion factor activity, while coated vesicles show indole blocking factor activity. In moderately tyrosinase-rich premelanosomes, the levels of dopachrome conversion factor and indole blocking factor are lower than in coated vesicles or melanosomes. High levels of indole blocking factor in coated vesicles may indicate why melanin polymer formation does not occur there in vivo despite their high tyrosinase activity.

[An ultracytochemical study of the hydrogenosomes of Trichomonas vaginalis]. / Ul'tratsitokhimicheskoe izuchenie gidrogenosom Trichomonas vaginalis.

The ultracytochemical study of cultural T. vaginalis has been carried out. The activity of magnesium-dependent ATPase, transport (sodium-potassium-dependent) ATPase and adenylate cyclase was detected in dense corpuscles, located on the membranes of hydrogenosomes having a rounded form. The authors suggest that the dense corpuscles are the zones of the active transport of ions through the membranes of hydrogenosomes.