CGM2, a Member of the Carcinoembryonic Antigen Gene Family is Down- Regulated in Colorectal Carcinomas

We have determined the precise chromosomal location, the exon structure, and the expression pattern of CGM2, a member of the carcinoembryonic antigen (CEA) gene family. CGM2 cDNA was amplified by reverse transcription-polymerase chain reaction (RT/PCR) from the colon adenocarcinoma cell line, LS174T. A defective exon is missing from this cDNA clone, leading to a novel domain organization for the human CEA family with two immunoglobulin-like domains. The derived C-terminal domain predicts that the CGM2 protein is membrane-bound through a glycosyl phosphatidylinositol anchor. RT/PCR analyses identified CGM2 transcripts in mucinous ovarian and colonic adenocarcinomas as well as in adjacent colonic tissue, but not in other tumors including leukocytes from six chronic myeloid leukemia patients. Thus, unlike several other family members, CGM2 is not expressed in granulocytes but reveals a more CEA-like expression pattern. Northern blot analyses identified a 2.5-kilobase CGM2 mRNA that is strongly down-regulated in colonic adenocarcinomas compared with adjacent colonic mucosa, suggesting a possible tumor suppressor function. In addition, a 3.2- kilobase transcript was observed in a number of colon tumors that is not detectable in normal colonic tissue. This mRNA species could represent a tumor-specific CGM2 splice variant

Learning induced neuronal activation pattern measured by c-fos expression in murine hippocampus and nucleus accumbens

Neuronale Aktivierungen, induziert durch räumliches Lernen, wurden anhand der c-fos Genexpression in Mäusen untersucht. Das Immediate-Early Gene c-fos, wichtig zur Konsolidierung von Langzeit-Gedächtnis, ermöglicht mit ubiquitärer und transienter Expression die nicht-invasive Analyse neuronaler Aktivierung verschiedener Gehirn-Areale mit hoher zeitlich-räumlicher Auflösung auf zellulärem Niveau. Ein Circular-Maze-Task wurde etabliert, um hippocampus-spezifisches räumliches Lernen unter optimierten Bedingungen für die c-fos Analyse zu erzielen. Die c-fos Expressionsmuster verdeutlichten die funktionelle Untergliederung des Hippocampus und Nucleus Accumbens in räumlichen Lernprozessen anhand einer starken spezifischen Aktivierung im gyrus dentatus und der CA1- im Gegensatz zur CA3-Region des Hippocampus und einer schwachen Involvierung der Shell-Region des Nucleus Accumbens. Ein reiner Novelty-Effekt wurde ausgeschlossen, da ein Novelty-Task keine Veränderung der c-fos Expression ergab

Cloning of the Complete Gene for Carcinoembryonic Antigen

Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region

Efficient production of the Nylon 12 monomer ω-aminododecanoic acid methyl ester from renewable dodecanoic acid methyl ester with engineered Escherichia coli

The expansion of microbial substrate and product scopes will be an important brick promoting future bioeconomy. In this study, an orthogonal pathway running in parallel to native metabolism and converting renewable dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to 12-aminododecanoic acid methyl ester (ADAME), a building block for the high-performance polymer Nylon 12, was engineered in Escherichia coli and optimized regarding substrate uptake, substrate requirements, host strain choice, flux, and product yield. Efficient DAME uptake was achieved by means of the hydrophobic outer membrane porin AlkL increasing maximum oxygenation and transamination activities 8.3 and 7.6-fold, respectively. An optimized coupling to the pyruvate node via a heterologous alanine dehydrogenase enabled efficient intracellular L-alanine supply, a prerequisite for self-sufficient whole-cell transaminase catalysis. Finally, the introduction of a respiratory chain-linked alcohol dehydrogenase enabled an increase in pathway flux, the minimization of undesired overoxidation to the respective carboxylic acid, and thus the efficient formation of ADAME as main product. The completely synthetic orthogonal pathway presented in this study sets the stage for Nylon 12 production from renewables. Its effective operation achieved via fine tuning the connectivity to native cell functionalities emphasizes the potential of this concept to expand microbial substrate and product scopes

Combined bezafibrate and medroxyprogesterone acetate: potential novel therapy for acute myeloid leukaemia

Background: The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis. Principal Findings: Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D2 (PGD2) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Δ12,14 PGJ2 (15d-PGJ2). BEZ increased PGD2 synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ2 by inhibiting the PGD2 11β -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD2 to 9α11β-PGF2α. B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ2. Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors. Significance: Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ2. These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML

Glial-Specific Deletion of Med12 Results in Rapid Hearing Loss via Degradation of the Stria Vascularis

Mediator protein complex subunit 12 (Med12) is a core component of the basal transcriptional apparatus and plays a critical role in the development of many tissues. Mutations in Med12 are associated with X-linked intellectual disability syndromes and hearing loss; however, its role in nervous system function remains undefined. Here, we show that temporal conditional deletion of Med12 in astrocytes in the adult central nervous system results in region specific alterations in astrocyte morphology. Surprisingly, behavioral studies revealed rapid hearing loss after adult deletion of Med12 that was confirmed by a complete abrogation of auditory brainstem responses. Cellular analysis of the cochlea revealed degeneration of the stria vascularis, in conjunction with disorganization of basal cells adjacent to the spiral ligament and downregulation of key cell adhesion proteins. Physiological analysis revealed early changes in endocochlear potential, consistent with strial-specific defects. Together, our studies reveal that Med12 regulates auditory function in the adult by preserving the structural integrity of the stria vascularis

Loss of the mammal-specific tectorial membrane component CEA cell adhesion molecule 16 (CEACAM16) leads to hearing impairment at low and high frequencies

The vertebrate-restricted carcinoembryonic antigen gene family evolves extremely rapidly. Among their widely expressed members, the mammal-specific, secreted CEACAM16 is exceptionally well conserved and specifically expressed in the inner ear. To elucidate a potential auditory function we inactivated murine Ceacam16 by homologous recombination. In young Ceacam16-/- mice the hearing threshold for frequencies below 10 kHz and above 22 kHz was raised. This hearing impairment progressed with age. A similar phenotype is observed in hearing-impaired members of Family 1070 with non-syndromic autosomal dominant hearing loss (DFNA4) who carry a missense mutation in CEACAM16. CEACAM16 was found in interdental and Deiters cells and was deposited in the tectorial membrane of the cochlea between postnatal day 12 and 15, when hearing starts in mice. In cochlear sections of Ceacam16-/- mice tectorial membranes were significantly more often stretched out as compared to wild-type mice where they were mostly contracted and detached from the outer hair cells. Homotypic cell sorting observed after ectopic cell surface expression of the carboxy-terminal immunoglobulin variable-like N2 domain of CEACAM16 indicated that CEACAM16 can interact in trans. Furthermore, Western blot analyses of membrane-bound CEACAM16 under reducing and non-reducing conditions demonstrated oligomerization via unpaired cysteines. Taken together, CEACAM16 probably can form higher order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin and influences the physical properties of the tectorial membrane. Evolution of CEACAM16 might have been an important step for the specialization of the mammalian cochlea allowing hearing over an extended frequency range

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

In response to cell swelling, volume-regulated anion channels (VRACs) participate in a process known as regulatory volume decrease (RVD). Only recently, first insight into the molecular identity of mammalian VRACs was obtained by the discovery of the leucine-rich repeats containing 8A (LRRC8A) gene. Here, we show that bestrophin 1 (BEST1) but not LRRC8A is crucial for volume regulation in human retinal pigment epithelium (RPE) cells. Whole-cell patch-clamp recordings in RPE derived from human-induced pluripotent stem cells (hiPSC) exhibit an outwardly rectifying chloride current with characteristic functional properties of VRACs. This current is severely reduced in hiPSC-RPE cells derived from macular dystrophy patients with pathologic BEST1 mutations. Disruption of the orthologous mouse gene (Best1−/−) does not result in obvious retinal pathology but leads to a severe subfertility phenotype in agreement with minor endogenous expression of Best1 in murine RPE but highly abundant expression in mouse testis. Sperm from Best1−/− mice showed reduced motility and abnormal sperm morphology, indicating an inability in RVD. Together, our data suggest that the molecular identity of VRACs is more complex—that is, instead of a single ubiquitous channel, VRACs could be formed by cell type- or tissue-specific subunit composition. Our findings provide the basis to further examine VRAC diversity in normal and diseased cell physiology, which is key to exploring novel therapeutic approaches in VRAC-associated pathologies

Investigation of sex-specific effects of apolipoprotein E on severity of EAE and MS

Background Despite pleiotropic immunomodulatory effects of apolipoprotein E (apoE) in vitro, its effects on the clinical course of experimental autoimmune encephalomyelitis (EAE) and multiple sclerosis (MS) are still controversial. As sex hormones modify immunomodulatory apoE functions, they may explain contentious findings. This study aimed to investigate sex-specific effects of apoE on disease course of EAE and MS. Methods MOG35-55 induced EAE in female and male apoE-deficient mice was assessed clinically and histopathologically. apoE expression was investigated by qPCR. The association of the MS severity score (MSSS) and APOE rs429358 and rs7412 was assessed across 3237 MS patients using linear regression analyses. Results EAE disease course was slightly attenuated in male apoE-deficient (apoE −/− ) mice compared to wildtype mice (cumulative median score: apoE −/−  = 2 [IQR 0.0–4.5]; wildtype = 4 [IQR 1.0–5.0]; n = 10 each group, p = 0.0002). In contrast, EAE was more severe in female apoE −/− mice compared to wildtype mice (cumulative median score: apoE −/−  = 3 [IQR 2.0–4.5]; wildtype = 3 [IQR 0.0–4.0]; n = 10, p = 0.003). In wildtype animals, apoE expression during the chronic EAE phase was increased in both females and males (in comparison to naïve animals; p < 0.001). However, in MS, we did not observe a significant association between MSSS and rs429358 or rs7412, neither in the overall analyses nor upon stratification for sex. Conclusions apoE exerts moderate sex-specific effects on EAE severity. However, the results in the apoE knock-out model are not comparable to effects of polymorphic variants in the human APOE gene, thus pinpointing the challenge of translating findings from the EAE model to the human disease

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy