Molecular cloning and transcriptional regulation of two γ-carbonic anhydrase genes in the green macroalga Ulva prolifera.

Ulva prolifera O.F. Müller (Ulvophyceae, Chlorophyta) is well known as a typical green-tide forming macroalga which has caused the world's largest macroalgal blooms in the Yellow Sea of China. In this study, two full-length γ-carbonic anhydrase (γ-CA) genes (UpγCA1 and UpγCA2) were cloned from U. prolifera. UpγCA1 has three conserved histidine residues, which act as an active site for binding a zinc metal ion. In UpγCA2, two of the three histidine residues were replaced by serine and arginine, respectively. The two γ-CA genes are clustered together with other γ-CAs in Chlorophyta with strong support value (100% bootstrap) in maximum likelihood (ML) phylogenetic tree. Quantitative real-time PCR (qRT-PCR) analysis showed that stressful environmental conditions markedly inhibited transcription levels of these two γ-CA genes. Low pH value (pH 7.5) significantly increased transcription level of UpγCA2 not UpγCA1 at 12 h, whereas high pH value (pH 8.5) significantly inhibited the transcription of these two γ-CA genes at 6 h. These findings enhanced our understanding on transcriptional regulation of γ-CA genes in response to environmental factors in U. prolifera.

Automation of RNA-based biomarker extraction from dried blood spots for the detection of blood doping.

Aim: Transcriptomic biomarkers originating from reticulocytes measured in dried blood spots (DBSs) may be reliable indicators of blood doping. Methods/results: Here, we examined changes in the expression levels of the erythropoiesis-related ALAS2, CA1 and SLC4A1 genes in DBS samples from elite athletes and volunteers of clinical study with recombinant erythropoietin dose. Conclusion: By comparing the mean intraday coefficients of variation for ALAS2L, ALASLC, CA1 and SLC4A1 between manual and automated RNA extractions, an average improvement was observed, whereas the assessment of interday variability provided comparable results for both manual and automated approaches. Our results confirmed that RNA biomarkers on DBS support are efficient to detect blood doping.

Trichinella spiralis-induced mastocytosis and erythropoiesis are simultaneously supported by a bipotent mast cell/erythrocyte precursor cell.

Anti-helminth responses require robust type 2 cytokine production that simultaneously promotes worm expulsion and initiates the resolution of helminth-induced wounds and hemorrhaging. However, how infection-induced changes in hematopoiesis contribute to these seemingly distinct processes remains unknown. Recent studies have suggested the existence of a hematopoietic progenitor with dual mast cell-erythrocyte potential. Nonetheless, whether and how these progenitors contribute to host protection during an active infection remains to be defined. Here, we employed single cell RNA-sequencing and identified that the metabolic enzyme, carbonic anhydrase (Car) 1 marks a predefined bone marrow-resident hematopoietic progenitor cell (HPC) population. Next, we generated a Car1-reporter mouse model and found that Car1-GFP positive progenitors represent bipotent mast cell/erythrocyte precursors. Finally, we show that Car1-expressing HPCs simultaneously support mast cell and erythrocyte responses during Trichinella spiralis infection. Collectively, these data suggest that mast cell/erythrocyte precursors are mobilized to promote type 2 cytokine responses and alleviate helminth-induced blood loss, developmentally linking these processes. Collectively, these studies reveal unappreciated hematopoietic events initiated by the host to combat helminth parasites and provide insight into the evolutionary pressure that may have shaped the developmental relationship between mast cells and erythrocytes.

Comprehensive Analysis for Identifying Diagnostic and Prognostic Biomarkers in Colon Adenocarcinoma.

Colon adenocarcinoma (COAD) is a common noncutaneous carcinoma worldwide with high morbidity and mortality. Effective prevention methods are far from being met. Both diagnostic and prognostic models that can precisely and accurately predict the status and survival time of COAD are urgently needed. In the field of COAD, there have been limited studies on molecular biomarkers that can predict disease status and prognosis. Hence, an important task is to identify these biomarkers. We aimed to identify important risk genes that have the ability not only to diagnose tumors but also to predict overall survival. A comprehensive analysis was performed in this study. Finally, carbonic anhydrase 1 (CA1) and CA4 were identified as potential biomarkers due to their predictive roles in diagnosis and prognosis, and the results were further confirmed by a series of analyses. Overall, these findings are of great importance and may facilitate individualized treatment in diagnosis and prognosis.

Overexpression of CA1 mRNA and the CA I Protein in Tumor Cells Does Not Change the Gene Expression of the ECM Proteins.

In our study, we performed retroviral transduction to overexpress codon-optimized variant of gene encoding human carbonic anhydrase I (optiCA1) in two tumor cell lines PC3 and MDA-MB-231, derived from human prostatic and breast carcinoma respectively. We achieved significantly enhanced and stable overexpression of exogenous optiCA1 gene. The expression of endogenous, wild CA1 gene was found to be normally low (Ct 28.6 for PC3 cells) or below to the detection limit (Ct 35.5 for MDA-MB-231 cells). No morphological changes and no decreasing viability of tumor cells were observed upon stable overexpression of the optiCA1 gene. In our study we have shown that the overexpression of the optimized human CA1 in engineered PC3 and MDA-MB-231 cells did not induce similar changes as we observed in tumor cells cultivated in the presence of human sera containing extensively high titers of anti-CA I autoantibodies from patients with complete remission of malignant disease. In both optiCA1transduced cell lines, the expression of selected genes responsible for basal lamina assembly, cytoskeleton, extracellular matrix proteins and proto-oncogenes (COL1A1, COL4A4, LAMC2, CTHRC1, and WNT7B) was not changed.

Pseudomonas aeruginosa ß-carbonic anhydrase, psCA1, is required for calcium deposition and contributes to virulence.

Calcification of soft tissue leads to serious diseases and has been associated with bacterial chronic infections. However, the origin and the molecular mechanisms of calcification remain unclear. Here we hypothesized that a human pathogen Pseudomonas aeruginosa deposits extracellular calcium, a process requiring carbonic anhydrases (CAs). Transmission electron microscopy confirmed the formation of 0.1-0.2 µm deposits by P. aeruginosa PAO1 growing at 5 mM CaCl2, and X-ray elemental analysis confirmed they contain calcium. Quantitative analysis of deposited calcium showed that PAO1 deposits 0.35 and 0.75 mM calcium/mg protein when grown at 5 mM and 10 mM CaCl2, correspondingly. Fluorescent microscopy indicated that deposition initiates at the cell surface. We have previously characterized three PAO1 ß-class CAs: psCA1, psCA2, and psCA3 that hydrate CO2 to HCO3-, among which psCA1 showed the highest catalytic activity (Lotlikar et. al. 2013). According to immunoblot and RT-qPCR, growth at elevated calcium levels increases the expression of psCA1. Analyses of the deletion mutants lacking one, two or all three psCA genes, determined that psCA1 plays a major role in calcium deposition and contributes to the pathogen's virulence. In-silico modeling of the PAO1 ß-class CAs identified four amino acids that differ in psCA1 compared to psCA2, and psCA3 (T59, A61A, A101, and A108), and these differences may play a role in catalytic rate and thus calcium deposition. A series of inhibitors were tested against the recombinant psCA1, among which aminobenzene sulfonamide (ABS) and acetazolamide (AAZ), which inhibited psCA1 catalytic activity with KIs of 19 nM and 37 nM, correspondingly. The addition of ABS and AAZ to growing PAO1 reduced calcium deposition by 41 and 78, respectively. Hence, for the first time, we showed that the ß-CA psCA1 in P. aeruginosa contributes to virulence likely by enabling calcium salt deposition, which can be partially controlled by inhibiting its catalytic activity.

Silencing of carbonic anhydrase I enhances the malignant potential of exosomes secreted by prostatic tumour cells.

We report results showing that the silencing of carbonic anhydrase I (siCA1) in prostatic (PC3) tumour cells has a significant impact on exosome formation. An increased diameter, concentration and diversity of the produced exosomes were noticed as a consequence of this knock-down. The protein composition of the exosomes' cargo was also altered. Liquid chromatography and mass spectrometry analyses identified 42 proteins significantly altered in PC3 siCA1 exosomes compared with controls. The affected proteins are mainly involved in metabolic processes, biogenesis, cell component organization and defense/immunity. Interestingly, almost all of them have been described as 'enhancers' of tumour development through the promotion of cell proliferation, migration and invasion. Thus, our results indicate that the reduced expression of the CA1 protein enhances the malignant potential of PC3 cells.

Carbonic Anhydrase Inhibitors as Novel Drugs against Mycobacterial ß-Carbonic Anhydrases: An Update on In Vitro and In Vivo Studies.

Mycobacteria cause a variety of diseases, such as tuberculosis, leprosy, and opportunistic diseases in immunocompromised people. The treatment of these diseases is problematic, necessitating the development of novel treatment strategies. Recently, ß-carbonic anhydrases (ß-CAs) have emerged as potential drug targets in mycobacteria. The genomes of mycobacteria encode for three ß-CAs that have been cloned and characterized from Mycobacterium tuberculosis (Mtb) and the crystal structures of two of the enzymes have been determined. Different classes of inhibitor molecules against Mtb ß-CAs have subsequently been designed and have been shown to inhibit these mycobacterial enzymes in vitro. The inhibition of these centrally important mycobacterial enzymes leads to reduced growth of mycobacteria, lower virulence, and impaired biofilm formation. Thus, the inhibition of ß-CAs could be a novel approach for developing drugs against the severe diseases caused by pathogenic mycobacteria. In the present article, we review the data related to in vitro and in vivo inhibition studies in the field.

Transcriptomic biomarkers of altered erythropoiesis to detect autologous blood transfusion.

Autologous blood transfusion is a powerful means of improving performance and remains one of the most challenging methods to detect. Recent investigations have identified 3 candidate reticulocytes genes whose expression was significantly influenced by blood transfusion. Using quantitative reverse transcription polymerase chain reaction as an alternative quantitative method, the present study supports that delta-aminolevulinate synthase 2 (ALAS2), carbonic anhydrase (CA1), and solute carrier family 4 member 1 (SLC4A1) genes are down-regulated post-transfusion. The expression of these genes exhibited stronger correlation with immature reticulocyte fraction than with reticulocytes percentage. Moreover, the repression of reticulocytes' gene expression was more pronounced than the diminution of immature reticulocyte fraction and reticulocyte percentage following blood transfusion. It suggests that the 3 candidate genes are reliable predictors of bone marrow's response to blood transfusion and that they represent potential biomarkers for the detection of this method prohibited in sports.

Loss of the Chloroplast Transit Peptide from an Ancestral C3 Carbonic Anhydrase Is Associated with C4 Evolution in the Grass Genus Neurachne.

Neurachne is the only known grass lineage containing closely related C3, C3-C4 intermediate, and C4 species, making it an ideal taxon with which to study the evolution of C4 photosynthesis in the grasses. To begin dissecting the molecular changes that led to the evolution of C4 photosynthesis in this group, the complementary DNAs encoding four distinct ß-carbonic anhydrase (CA) isoforms were characterized from leaf tissue of Neurachne munroi (C4), Neurachne minor (C3-C4), and Neurachne alopecuroidea (C3). Two genes (CA1 and CA2) each encode two different isoforms: CA1a/CA1b and CA2a/CA2b. Transcript analyses found that CA1 messenger RNAs were significantly more abundant than transcripts from the CA2 gene in the leaves of each species examined, constituting ∼99% of all ß-CA transcripts measured. Localization experiments using green fluorescent protein fusion constructs showed that, while CA1b is a cytosolic CA in all three species, the CA1a proteins are differentially localized. The N. alopecuroidea and N. minor CA1a isoforms were imported into chloroplasts of Nicotiana benthamiana leaf cells, whereas N. munroi CA1a localized to the cytosol. Sequence analysis indicated an 11-amino acid deletion in the amino terminus of N. munroi CA1a relative to the C3 and C3-C4 proteins, suggesting that chloroplast targeting of CA1a is the ancestral state and that loss of a functional chloroplast transit peptide in N. munroi CA1a is associated with the evolution of C4 photosynthesis in Neurachne spp. Remarkably, this mechanism is homoplastic with the evolution of the C4-associated CA in the dicotyledonous genus Flaveria, although the actual mutations in the two lineages differ.

Comparative Proteomics Reveals that Phosphorylation of ß Carbonic Anhydrase 1 Might be Important for Adaptation to Drought Stress in Brassica napus.

Little is known about the mechanism of drought tolerance in rapeseed (Brassica napus L.). In this study, different morphological and physiological responses to drought stress were studied in three rapeseed cultivars. For the cultivar 2AF009 with high drought tolerance, comparative proteomic analyses were conducted to determine the molecular mechanism behind. Approximately 138 differentially abundant proteins (DAPs) and 1232 phosphoproteins containing 4469 phosphopeptides were identified. Furthermore, 337 phosphoproteins containing 547 phosphorylation sites demonstrated significant changes. These drought-responsive DAPs and phosphoproteins were mainly involved in signal transduction, photosynthesis, and glutathione-ascorbate metabolism. Notably, 9 DAPs were also identified as drought-responsive phosphoproteins, especially beta carbonic anhydrase 1 (ßCA1), which was represented by eight distinct protein spots with different abundant levels during drought stress. Tyr207 phosphorylated site of ßCA1 was down-regulated at the phosphorylation level during drought stress, which was also located in the substrate-binding active region of three-dimensional (3D) structure. Moreover, drought stress inhibited CA activity. We concluded that Tyr207 was the most likely phosphorylation target affecting the enzyme activity, and phosphorylation of ßCA1 might be important for the response to drought stress in rapeseed. The study provided a new clue for the drought tolerance mechanism in B.napus.

Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4 Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

Antibodies reacting to carbonic anhydrase isozymes (I and II) and albumin in sera from dogs.

IgGs to carbonic anhydrase isozymes (CA-I and CA-II) and albumin were identified in dog serum. IgG titers were determined in the sera of asymptomatic dogs, and in dogs with atopic dermatitis, diarrhea and/or vomiting, diabetes and/or pancreatitis, kidney disease, hepatic disease, and thyroid gland disease, using ELISA. Low titres of IgG-reactive CA-I, CA-II, BSA, and CSA were found in the sera of healthy beagles. Compared with healthy beagles, there was a significant difference in the titers of antibodies against CA-I in asymptomatic dogs, dogs with diabetes and/or pancreatitis, or thyroid gland disease, or hepatic disease. Compared with healthy beagles, there was a significant difference in the antibody titer of anti-CA-II IgG in asymptomatic dogs and in those with hepatic disease. There was a significant difference in the antibody titer of anti-BSA IgG between healthy beagles and dogs with hepatic disease.

CA1 contributes to microcalcification and tumourigenesis in breast cancer.

BACKGROUND: Although mammary microcalcification is frequently observed and has been associated with poor survival in patients with breast cancer, the genesis of calcification remains unclear. Carbonic anhydrase I (CA1) has been shown to promote calcification by catalysing the hydration of CO2. This study aimed to determine whether CA1 was correlated with microcalcification and with other processes that are involved in breast cancer tumourigenesis. METHODS: CA1 expression in breast cancer tissues and blood samples was detected using western blotting, real-time PCR, immunohistochemistry and ELISA. Calcification was induced in the cultured 4T1 cell line originating from mouse breast tumours, using ascorbic acid and ß-glycerophosphate. Acetazolamide, a chemical inhibitor of CA1, was also added to the culture to determine the role of CA1 in calcification. The MCF-7 human breast cancer cell line was treated with anti-CA1 siRNA and was assessed using a CCK-8 cell proliferation assay, an annexin V cell apoptosis assay, transwell migration assay and a human breast cancer PCR array. The tag SNP rs725605, which is located in the CA1 locus, was genotyped using TaqMan® genotyping. RESULTS: Increased CA1 expression was detected in samples of breast carcinoma tissues and blood obtained from patients with breast cancer. A total of 15.3 % of these blood samples exhibited a 2.1-fold or higher level of CA1 expression, compared to the average level of CA1 expression in samples from healthy controls. Following the induction of calcification of 4T1 cells, both the number of calcium-rich deposits and the expression of CA1 increased, whereas the calcification and CA1 expression were significantly supressed in the presence of acetazolamide. Increased migration and apoptosis were observed in MCF-7 cells that were treated with anti-CA1 siRNA. The PCR array detected up-regulation of the androgen receptor (AR) and down-regulation of X-box binding protein 1 (XBP1) in the treated MCF-7 cells. Significant differences in the allele and genotype frequencies of rs725605 were detected in the cohort of patients with breast cancer but not in other tumours. CONCLUSION: The results of this study suggested that CA1 is a potential oncogene and that it contributes to abnormal cell calcification, apoptosis and migration in breast cancer.

[Association of gene polymorphisms with the susceptibility of schizophrenia in Han Chinese population].

OBJECTIVE: To investigate the potential association of carbonic anhydrase I (CA1) anterior pharynx-defective 1A ( APH1A), neurodevelopment protein 1-like 1 (NDEL1) and serine racemase (SRR) gene polymorphisms with the susceptibility of schizophrenia (SZ). METHODS: A case-control study was performed to identify polymorphisms of the CA1, APH1A, NDEL1 and SRR gene that may confer susceptibility to SZ in the Han Chinese population. Five single nucleotide polymorphisms (SNPs) were genotyped in 516 paranoid SZ patients and 516 control subjects by real time quantitative polymerase chain reaction. The association between genotypes and positive and negative symptoms scale was also explored. RESULTS: No significant differences in genotype or allele frequencies of five SNPs were observed between schizophrenic patients and healthy controls (P = 0.163, 0.322, 0.494, 0.338, 0.545; 0.259, 0.149, 0.417, 0.527, 0.720; respectively). However, the frequency of CA haplotypes in SZ group was higher than control group (P = 0.041). The scores of depression/anxiety, positive and excited/hostile factors in SZ patients with genotype of rs2298161 (AG), rs4523957 (CC) and rs8081273 (GG) were higher than other genotypes (P = 0.008, 0.001, 0.000, respectively). CONCLUSIONS: These data suggests that the CA1, APH1A, NDEL1 and SRR gene may not be association with susceptibility to SZ in the Han Chinese population. However, the haplotype of CA may be the susceptible factor of SZ. Rs2298161, rs4523957 and rs8081273 may be associated with some phenotypes of SZ.

Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas.

Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.

Carbonic anhydrase activation is associated with worsened pathological remodeling in human ischemic diabetic cardiomyopathy.

BACKGROUND: Diabetes mellitus (DM) has multifactorial detrimental effects on myocardial tissue. Recently, carbonic anhydrases (CAs) have been shown to play a major role in diabetic microangiopathy but their role in the diabetic cardiomyopathy is still unknown. METHODS AND RESULTS: We obtained left ventricular samples from patients with DM type 2 (DM-T2) and nondiabetic (NDM) patients with postinfarct heart failure who were undergoing surgical coronary revascularization. Myocardial levels of CA-I and CA-II were 6- and 11-fold higher, respectively, in DM-T2 versus NDM patients. Elevated CA-I expression was mainly localized in the cardiac interstitium and endothelial cells. CA-I induced by high glucose levels hampers endothelial cell permeability and determines endothelial cell apoptosis in vitro. Accordingly, capillary density was significantly lower in the DM-T2 myocardial samples (mean±SE=2152±146 versus 4545±211/mm(2)). On the other hand, CA-II was mainly upregulated in cardiomyocytes. The latter was associated with sodium-hydrogen exchanger-1 hyperphosphorylation, exaggerated myocyte hypertrophy (cross-sectional area 565±34 versus 412±27 µm(2)), and apoptotic death (830±54 versus 470±34 per 10(6) myocytes) in DM-T2 versus NDM patients. CA-II is activated by high glucose levels and directly induces cardiomyocyte hypertrophy and death in vitro, which are prevented by sodium-hydrogen exchanger-1 inhibition. CA-II was shown to be a direct target for repression by microRNA-23b, which was downregulated in myocardial samples from DM-T2 patients. MicroRNA-23b is regulated by p38 mitogen-activated protein kinase, and it modulates high-glucose CA-II-dependent effects on cardiomyocyte survival in vitro. CONCLUSIONS: Myocardial CA activation is significantly elevated in human diabetic ischemic cardiomyopathy. These data may open new avenues for targeted treatment of diabetic heart failure.

Transgenic mice over-expressing carbonic anhydrase I showed aggravated joint inflammation and tissue destruction.

BACKGROUND: Studies have demonstrated that carbonic anhydrase I (CA1) stimulates calcium salt precipitation and cell calcification, which is an essential step in new bone formation. Our study had reported that CA1 encoding gene has a strong association with rheumatoid arthritis (RA) and ankylosing spondylitis (AS), two rheumatic diseases with abnormal new bone formation and bone resorption in joints. This study investigated the effect of CA1 on joint inflammation and tissue destruction in transgenic mice that over-express CA1 (CA1-Tg). METHODS: CA1-Tg was generated with C57BL/6J mice by conventional methods. CA1-Tg was treated with collagen-II to induce arthritis (CIA). Wild-type mice, CA1-Tg treated with bovine serum albumin (BSA) and transgenic mice over-expressing PADI4 (PADI4-Tg), a gene known to be involved in rheumatoid arthritis, were used as controls. Histochemistry and X-ray radiographic assay were used to examine joint destruction. Western blotting and real time-PCR were used to examine CA1 expression. RESULTS: CIA was observed in 60% of CA1-Tg, 20% of PADI4-Tg and 20% of wild-type mice after collagen injections. No CIA was found in CA1-Tg mice that received injections of BSA. The arthritic score was 5.5 ± 0.84 in the CA1-Tgs but the score was less than 2 in the injected wild-type mice and the PADI4-Tgs. The thickness of the hind paws in the CA1-Tgs was 3.46 ± 0.11 mm, which was thicker than that of PADI4-Tgs (2.23 ± 0.08 mm), wild-type mice (2.08 ± 0.06 mm) and BSA-treated CA1-Tgs (2.04 ± 0.07 mm). Histochemistry showed obvious inflammation, synovial hyperplasia and bone destruction in the joints of CA1-Tg that was not detected in PADI4-Tgs or wild-type mice. X-ray assays showed bone fusion in the paws and spines of CA1-Tg mice. CONCLUSION: Over-expression of CA1 may aggravate joint inflammation and tissue destruction in the transgenic mice.

Ldb1 regulates carbonic anhydrase 1 during erythroid differentiation.

Carbonic anhydrase 1 (Car1), an early specific marker of the erythroid differentiation, has been used to distinguish fetal and adult erythroid cells since its production closely follows the γ- to ß-globin transition, but the molecular mechanism underlying transcriptional regulation of Car1 is unclear. Here, we show that Car1 mRNA decreases significantly when erythroid differentiation is induced in MEL cells. The Ldb1 protein complex including GATA1/SCL/LMO2 binds to the Car1 promoter in uninduced cells and reduced enrichment of the complex during differentiation correlates with loss of Car1 expression. Knockdown of Ldb1 results in a reduction of Ser2 phosphorylated RNA Pol II and Cdk9 at the Car1 promoter region, suggesting that Ldb1 is required for recruitment of Pol II as well as the transcription regulator P-TEFb to enhance elongation of Car1 transcripts. Taken together, these data show that Ldb1 forms a regulatory complex to maintain Car1 expression in erythroid cells.

Proteomic analysis shows down-regulations of cytoplasmic carbonic anhydrases, CAI and CAII, are early events of colorectal carcinogenesis but are not correlated with lymph node metastasis.

AIM: The aim of the study was to screen the markedly down-regulated proteins in colorectal cancer and analyze their relationship to carcinogenesis, cancer progression and pathological aspects. METHODS: Proteomic analysis was preformed on six fresh colorectal cancer tissues and paired normal colorectal mucosa by two-dimensional differential gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Two markedly down-regulated proteins among the proteins, of which the expressions were significantly decreased in colorectal cancer compared to normal mucosa, were confirmed by Western Blot in 12 colorectal cancers. Their relationship to carcinogenesis, cancer progression and pathological aspects of colorectal cancer were analyzed in 64 colorectal cancer and paired normal mucosa, 27 benign polyps, and 20 lymph node metastases by immunohistochemistry. RESULTS: Two-dimensional differential gel electrophoresis analysis showed there were 2 protein spots, of which the average abundances decreased 3.62 and 3.76 fold in colorectal cancer compared to normal mucosa, respectively. They were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry as carbonic anhydrase I and II (CAI and CAII). Validation by Western Blot in 12 colorectal cancers showed there were significantly different expressions of CAI and CAII between colorectal cancer and normal mucosa ( P = 0.002 and 0.027, respectively). Immunohistochemistry analysis indicated the expression of CAI and CAII was decreased from normal mucosa to benign polyps, and to colorectal cancer stepwise significantly (P <0.05). However, there were no differences in their expressions between lymph node metastasis and colorectal cancer (P >0.05). There were decreasing trends of CAI and CAII expressions from well to poor differentiation and from stage I or II to stage III or IV, but they were not statistically significant (P >0.05). CONCLUSIONS: CAI and CAII are necessary enzymes of the colorectum for their normal function. Down-regulations of CAI and CAII are early events of colorectum carcinogenesis. They have no correlations with lymph node metastasis.