Blood-based analysis of 84 microRNAs identifies molecules deregulated in individuals with type-2 diabetes, risk factors for the disease or metabolic syndrome.

AIM: Micro-RNAs (miRNAs) are implicated in insulin-signaling and the development of type-2 diabetes (T2D). Their deregulated expression is mostly described in the pancreas, liver, skeletal muscle, or adipose tissue of diabetic animals. Relevant studies in humans are limited due to difficulties in accessing tissue-biopsies. Though, circulating miRNAs are indicators of organ-specific pathophysiological events and could potentially serve as disease biomarkers. We explored the profile of 84 T2D-related miRNAs in peripheral blood of subjects with or without the disease. METHODS: An RT-qPCR array screening 84 T2D-related miRNAs was applied in samples of T2D (n = 6) versus non-T2D (n = 6) subjects. The deregulated miRNAs were thereafter analyzed in peripheral blood samples of a validation cohort of 40 T2D and 37 non-T2D individuals [16 controls and 21 subjects with metabolic syndrome (Met-S) and/or T2D risk factors (T2D-RF)], using specific RT-qPCR assays. Correlations with clinicopathological parameters and risk factors were evaluated. RESULTS: Subjects with the disease displayed decreased levels of miR-214-3p, miR-24-3p and let-7f-5p, compared to those without. MiRNA levels correlated with serum insulin and HbA1c levels in individuals with T2D or Met-S/T2D-RF, and with higher BMI, dyslipidemia and family history in controls. CONCLUSIONS: Blood levels of miR-214-3p, miR-24-3p and let-7f-5p are down-regulated in T2D- and Met-S/T2D-RF subjects. Future studies are needed to evaluate their potential as disease biomarkers and elucidate the associated tissue-specific pathogenetic mechanisms.

Decreased expression of microRNAs targeting type-2 diabetes susceptibility genes in peripheral blood of patients and predisposed individuals.

AIM: Certain microRNA molecules (miRNAs) that target genes involved in beta-cell growth and insulin resistance are found deregulated in patients with type-2 diabetes mellitus (T2D) and correlate with its complications. However, the expression profile of miRNAs that regulate genes bearing T2D-related single-nucleotide polymorphisms has been hardly studied. We recently reported that the mRNA patterns of specific T2D-susceptibility genes are impaired in patients, and associate with disease parameters and risk factors. The aim of this study was to explore the levels of miRNAs that target those genes, in peripheral blood of patients versus controls. METHODS: A panel of 14 miRNAs validated to target the CDKN2A, CDK5, IGF2BP2, KCNQ1, and TSPAN8 genes, was developed upon combined search throughout the DIANNA TarBase v7.0, miRTarBase, miRSearch v3.0-Exiqon, miRGator v3.0, and miRTarget Link Human algorithms. Specifically developed poly(A)polyadenylation(PAP)-reverse transcription(RT)-qPCR protocols were applied in peripheral blood RNA samples from patients and controls. Possible correlations with the disease, clinicopathological parameters and/or risk factors were evaluated. RESULTS: T2D patients expressed decreased levels of let-7b-5p, miR-1-3p, miR-24-3p, miR-34a-5p, miR-98-5p, and miR-133a-3p, compared with controls. Moreover, these levels correlated with certain disease features including insulin and % HbA1c levels in patients, as well as BMI, triglycerides' levels and family history in controls. CONCLUSIONS: A T2D-specific expression profile of miRNAs that target disease-susceptibility genes is for the first time described. Future studies are needed to elucidate the associated transcription-regulatory mechanisms, perchance involved in T2D pathogenesis, and to evaluate the potential of these molecules as possible biomarkers for this disorder.

Blood-based analysis of type-2 diabetes mellitus susceptibility genes identifies specific transcript variants with deregulated expression and association with disease risk.

Despite significant progress by genome-wide association studies, the ability of genetic variants to conduce to the prediction or prognosis of type-2 diabetes (T2D) is weak. Expression analysis of the corresponding genes may suggest possible links between single-nucleotide polymorphisms and T2D phenotype and/or risk. Herein, we investigated the expression patterns of 24 T2D-susceptibility genes, and their individual transcript variants (tv), in peripheral blood of T2D patients and controls (CTs), applying RNA-seq and real-time qPCR methodologies, and explore possible associations with disease features. Our data revealed the deregulation of certain transcripts in T2D patients. Among them, the down-regulation of CAPN10 tv3 was confirmed as an independent predictor for T2D. In patients, increased expression of CDK5 tv2, CDKN2A tv3 or THADA tv5 correlated positively with serum insulin levels, of CDK5 tv1 positively with % HbA1c levels, while in controls, elevated levels of TSPAN8 were associated positively with the presence of T2D family history. Herein, a T2D-specific expression profile of specific transcripts of disease-susceptibility genes is for the first time described in human peripheral blood. Large-scale studies are needed to evaluate the potential of these molecules to serve as disease biomarkers.

Clinical evaluation of microRNA-145 expression in renal cell carcinoma: a promising molecular marker for discriminating and staging the clear cell histological subtype.

The vast majority of malignancies detected in renal parenchyma are diagnosed as renal cell carcinoma (RCC), whose subtype discrimination and determination of prognosis may contribute to the selection of the adequate therapy. Recently, a new class of small non-coding RNAs, known as microRNAs, has proven to be among the most promising biomarkers for providing this information. Herein, we sought to add up to this knowledge by evaluating the expression levels of microRNA-145 (miR-145) in RCC. For that purpose, total RNA from 58 cancerous and 44 adjacent non-cancerous renal tissues was firstly extracted and then polyadenylated and reverse transcribed to cDNA. MiR-145 levels were finally analyzed by developing and applying a highly sensitive real-time PCR protocol, while their clinical significance was determined via comprehensive statistical analysis. Our data showed that miR-145 was significantly downregulated in cancerous samples and could discriminate between clear cell and non-clear cell subtypes. Moreover, miR-145 expression was found to be correlated with primary tumor staging of cancerous samples, something also noticed in the clear cell RCC subset, in which miR-145 levels were negatively correlated with tumor size as well. Overall, these results indicate that miR-145 might constitute a promising molecular marker for RCC classification and staging.

Uncovering the clinical utility of miR-143, miR-145 and miR-224 for predicting the survival of bladder cancer patients following treatment.

Accurate prognosis is a key factor in establishing optimal therapeutic decisions; yet in the case of bladder cancer (BlCa) current prognostic indicators cannot ensure optimal disease management. Here, we aimed to evaluate the previously unexplored clinical potential of the urological cancer-related miR-145, miR-143 and miR-224 in BlCa. A total of 279 bladder tissue specimens were included in this study (133 BlCa, 107 adjacent normal and 39 healthy samples). Total RNA was extracted from tissues, it was polyadenylated and reverse transcribed to cDNA. The expression of target molecules was measured via quantitative real-time PCR. The expression levels of both miR-143 and miR-145 were significantly decreased, whereas those of miR-224 were increased in BlCa. Receiver operating characteristic curve analysis indicated a significant discriminatory capacity for miR-143/miR-145 levels. Important associations with disease aggressiveness were observed for all three microRNAs; elevated levels were observed in tumors of higher stage and grade, as well as in 'high-risk' TaT1 patients. More importantly, high miR-143/145 levels could effectively prognose inferior overall survival for muscle-invasive patients and could independently predict the progression of superficial tumors. Finally, the combination of miR-143/145 overexpression with the widely used prognostic markers of European Organization for Research and Treatment of Cancer-risk groups or recurrence at the first follow-up cystoscopy resulted to a superior positive prediction of non-muscle-invasive bladder cancer short-term progression compared with the use of the abovementioned markers alone. The cancer-related miR-143, miR-145 and miR-224 were investigated for the first time in the clinical setting of BlCa, and miR-143/145 cluster constitutes a novel marker helpful for providing an enhanced prediction of oncologic outcome for BlCa patients.

L-DOPA decarboxylase mRNA levels provide high diagnostic accuracy and discrimination between clear cell and non-clear cell subtypes in renal cell carcinoma.

OBJECTIVES: Renal cell carcinoma (RCC) is the most frequent type of kidney cancer. RCC patients frequently present with arterial hypertension due to various causes, including intrarenal dopamine deficiency. L-DOPA decarboxylase (DDC) is the gene encoding the enzyme that catalyzes the biosynthesis of dopamine in humans. Several studies have shown that the expression levels of DDC are significantly deregulated in cancer. Thus, we herein sought to analyze the mRNA levels of DDC and evaluate their clinical significance in RCC. DESIGN AND METHODS: DDC levels were analyzed in 58 surgically resected RCC tumors and 44 adjacent non-cancerous renal tissue specimens via real-time PCR. Relative levels of DDC were estimated by applying the 2(-ΔΔC)T method, while their diagnostic accuracy and correlation with the clinicopathological features of RCC tumors were assessed by comprehensive statistical analysis. RESULTS: DDC mRNA levels were found to be dramatically downregulated (p<0.001) in RCC tumors, exhibiting remarkable diagnostic accuracy as assessed by ROC curve analysis (AUC: 0.910; p<0.001) and logistic regression (OR: 0.678; p=0.001). Likewise, DDC was found to be differentially expressed between clear cell RCC and the group of non-clear cell subtypes (p=0.001) consisted of papillary and chromophobe RCC specimens. Furthermore, a statistically significant inverse correlation was also observed when the mRNA levels of DDC were analyzed in relation to tumor grade (p=0.049). CONCLUSIONS: Our data showed that DDC constitutes a highly promising molecular marker for RCC, exhibiting remarkable diagnostic accuracy and potential to discriminate between clear cell and non-clear cell histological subtypes of RCC.

Human L-DOPA decarboxylase mRNA is a target of miR-145: A prediction to validation workflow.

l-DOPA decarboxylase (DDC) is a multiply-regulated gene which encodes the enzyme that catalyzes the biosynthesis of dopamine in humans. MicroRNAs comprise a novel class of endogenously transcribed small RNAs that can post-transcriptionally regulate the expression of various genes. Given that the mechanism of microRNA target recognition remains elusive, several genes, including DDC, have not yet been identified as microRNA targets. Nevertheless, a number of specifically designed bioinformatic algorithms provide candidate miRNAs for almost every gene, but still their results exhibit moderate accuracy and should be experimentally validated. Motivated by the above, we herein sought to discover a microRNA that regulates DDC expression. By using the current algorithms according to bibliographic recommendations we found that miR-145 could be predicted with high specificity as a candidate regulatory microRNA for DDC expression. Thus, a validation experiment followed by firstly transfecting an appropriate cell culture system with a synthetic miR-145 sequence and sequentially assessing the mRNA and protein levels of DDC via real-time PCR and Western blotting, respectively. Our analysis revealed that miR-145 had no significant impact on protein levels of DDC but managed to dramatically downregulate its mRNA expression. Overall, the experimental and bioinformatic analysis conducted herein indicate that miR-145 has the ability to regulate DDC mRNA expression and potentially this occurs by recognizing its mRNA as a target.

Effect of cytostatic drugs on the mRNA expression levels of ribonuclease κ in breast and ovarian cancer cells.

Breast and ovarian cancers remain a major public health issue, constituting a major cause of female morbidity and mortality worldwide. Even though systemic chemotherapy remains the main course of action for both types of cancer, current research studies aim at the discovery of novel therapeutic targets. Ribonucleases, due to their apparent implication in gene expression regulation, may serve as potential anticancer drugs. Human RNase κ is an endoribonuclease belonging in a recently identified family of proteins. Recent data from expression microarray studies reveal that the RNase κ gene is found either up- or downregulated in a number of human cancers, indicating a possible diagnostic and/or prognostic utility.The aim of this study was to investigate modulations in the expression levels of RNase κ gene, in breast and ovarian cancer cells, as a response to treatment with different chemotherapeutic agents. BT-20 breast cancer cells and SKOV-3 ovarian cancer cells were treated with the cytotoxic drugs paclitaxel, docetaxel, cisplatin, carboplatin, epirubicin and vinorelbine. Gene expression analysis was performed by the comparative CT method also known as 2(-ΔΔCT) method. The results revealed a distinct increase in the expression levels of RNase κ mRNA (up to 9-fold) after treatment with the antineoplastic agent paclitaxel in both cell lines, while treatment with the remaining anticancer drugs did not alter drastically the mRNA levels of RNase κ. Based on the fact that paclitaxel exerts its cytotoxic action by inducing apoptosis, the results could be indicative of a potential implication of RNase κ in apoptosis-related pathways.

Quantification and study of the L-DOPA decarboxylase expression in gastric adenocarcinoma cells treated with chemotherapeutic substances.

3,4-Dihydroxy-L-phenylalanine decarboxylase (DDC) is an enzyme implicated in the biosynthetic pathways of the neurotransmitters dopamine and probably serotonin. DDC gene expression has been studied in numerous malignancies and the corresponding data have shown remarkable alterations in the mRNA and/or protein levels encoded by the gene. The aim of this study was to examine any modulations in the DDC mRNA levels in gastric cancer cells after their treatment with the chemotherapeutic agents 5-fluorouracil, leucovorin, irinotecan, etoposide, cisplatin, and taxol. The sensitivity of the AGS gastric adenocarcinoma cells to the antineoplastic drugs was evaluated using the MTT assay. Total RNA was extracted and reverse transcribed into cDNA. A highly sensitive quantitative real-time PCR methodology was developed for the quantification of DDC mRNA. GAPDH was used as a housekeeping gene. Relative quantification analysis was carried out using the comparative C T method ((Equation is included in full-text article.)). The treatment of AGS cells with several concentrations of various broadly used anticancer drugs resulted in significant modulations of the DDC mRNA levels compared with those in the untreated cells in a time-specific and drug-specific manner. Generally, DDC expression levels appeared to decrease after three time periods of exposure to the selected chemotherapeutic agents, suggesting a characteristic DDC mRNA expression profile that is possibly related to the mechanism of each drug. Our experimental data show that the DDC gene might serve as a new potential molecular biomarker predicting treatment response in gastric cancer cells.

L-Dopa decarboxylase (DDC) constitutes an emerging biomarker in predicting patients' survival with stomach adenocarcinomas.

PURPOSE: Stomach adenocarcinoma represents a major health problem and is regarded as the second commonest cause of cancer-associated mortality, universally, since it is still difficult to be perceived at a curable stage. Several lines of evidence have pointed out that the expression of L-Dopa decarboxylase (DDC) gene and/or protein becomes distinctively modulated in several human neuroendocrine neoplasms as well as adenocarcinomas. METHODS: In order to elucidate the clinical role of DDC on primary gastric adenocarcinomas, we determined qualitatively and quantitatively the mRNA levels of the gene with regular PCR and real-time PCR by using the comparative threshold cycle method, correspondingly, and detected the expression of DDC protein by immunoblotting in cancerous and normal stomach tissue specimens. RESULTS: A statistically significant association was disclosed between DDC expression and gastric intestinal histotype as well as tumor localization at the distal third part of the stomach (p = 0.025 and p = 0.029, respectively). Univariate and multivariate analyses highlighted the powerful prognostic importance of DDC in relation to disease-free survival and overall survival of gastric cancer patients. According to Kaplan-Meier curves, the relative risk of relapse was found to be decreased in DDC-positive (p = 0.031) patients who, also, exhibited higher overall survival rates (p = 0.016) than those with DDC-negative tumors. CONCLUSIONS: This work is the first to shed light on the potential clinical usefulness of DDC, as an efficient tumor biomarker in gastric cancer. The provided evidence underlines the propitious predictive value of DDC expression in the survival of stomach adenocarcinoma patients.

L-DOPA decarboxylase mRNA expression is associated with tumor stage and size in head and neck squamous cell carcinoma: a retrospective cohort study.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) represents one of the most commonly diagnosed malignancies worldwide. The DDC gene encodes L-DOPA decarboxylase, an enzyme catalyzing the decarboxylation of L-DOPA to dopamine. We have recently shown that DDC mRNA is a significant predictor of patients' prognosis in colorectal adenocarcinoma and prostate cancer. The aim of the current study was to analyze the DDC mRNA expression in HNSCC patients. METHODS: 53 malignant tumors were resected from the larynx, pharynx, tongue, buccal mucosa, parotid glands, and nasal cavity, as well as from 34 adjacent non-cancerous tissues of HNSCC patients, and were homogenized. Total RNA was isolated and converted into first-strand cDNA. An ultrasensitive real-time PCR method based on the SYBR Green chemistry was used for DDC mRNA quantification in head and neck tissue specimens. Relative quantification was performed using the comparative Ct (2-ddCt) method. RESULTS: DDC mRNA levels were lower in squamous cell carcinomas (SCCs) of the larynx and tongue than in adjacent non-cancerous tissue specimens. Furthermore, low DDC mRNA expression was noticed in laryngeal and tongue tumors of advanced TNM stage or bigger size, compared to early-stage or smaller tumors, respectively. No statistically significant differences were observed between SCCs resected from pharynx, buccal mucosa, or nasal cavity, and their normal counterparts. CONCLUSION: This is the first study examining the DDC mRNA expression in HNSCC. According to our results, DDC mRNA expression may constitute a potential prognostic biomarker in tongue and/or larynx SCCs, which principally represent the overwhelming majority of HNSCC cases.

l-DOPA Decarboxylase (DDC) Expression Status as a Novel Molecular Tumor Marker for Diagnostic and Prognostic Purposes in Laryngeal Cancer.

l-DOPA decarboxylase (DDC) plays an essential role in the enzymatic synthesis of dopamine and alterations in its gene expression have been reported in several malignancies. Our objective was to analyze DDC messenger RNA (mRNA) and protein expression in laryngeal tissues and to evaluate the clinical implication of this molecule in laryngeal cancer. In this study, total RNA was isolated from 157 tissue samples surgically removed from 100 laryngeal cancer patients. A highly sensitive real-time polymerase chain reaction methodology based on SYBR Green I fluorescent dye was developed for the quantification of DDC mRNA levels. In addition, Western blot analysis was performed for the detection of DDC protein. DDC mRNA expression was revealed to be significantly downregulated in primary laryngeal cancer samples compared with their nonmalignant counterparts (P = .001). A significant negative association was also disclosed between DDC mRNA levels and TNM staging (P = .034). Univariate analysis showed that patients bearing DDC-positive tumors had a significantly decreased risk of death (hazard ratio = 0.23, P = .012) and local recurrence (hazard ratio = 0.32, P =.006), whereas DDC expression retained its favorable prognostic significance in the multivariate analysis. Kaplan-Meier curves further demonstrated that DDC-positive patients experienced longer overall and disease-free survival periods (P = .006 and P = .004, respectively). Moreover, DDC protein was detected in both neoplastic and noncancerous tissues. Therefore, our results suggest that DDC expression status could qualify as a promising biomarker for the future clinical management of laryngeal cancer patients.

Essential cysteine residues for human RNase κ catalytic activity.

Human RNase κ is an endoribonuclease expressed in almost all tissues and organs and belongs to a highly conserved protein family bearing representatives in all metazoans. To gain insight into the role of cysteine residues in the enzyme activity or structure, a recombinant active form of human RNase κ expressed in Pichia pastoris was treated with alkylating agents and dithiothreitol (DTT). Our results showed that the human enzyme is inactivated by DDT, while it remains fully active in the presence of alkylating agents. The unreduced recombinant protein migrates on SDS/PAGE faster than the reduced form. This observation in combination with the above findings indicated that human RNase κ does not form homodimers through disulfide bridges, and cysteine residues are not implicated in RNA catalysis but participate in the formation of intramolecular disulfide bond(s) essential for its ribonucleolytic activity. The role of the cysteine residues was further investigated by expression and study of Cys variants. Ribonucleolytic activity experiments and SDS/PAGE analysis of the wild-type and mutant proteins under reducing and non-reducing conditions demonstrated that Cys7, Cys14 and Cys85 are not essential for RNase activity. On the other hand, replacement of Cys6 or Cys69 with serine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of human RNase κ by forming a disulfide bond. Due to the absolute conservation of these cysteine residues, the Cys6-Cys69 disulfide bond is likely to exist in all RNase κ family members.

Efficient cloning of alternatively polyadenylated transcripts via hybridization capture PCR.

Cloning of alternatively polyadenylated transcripts is crucial for studying gene expression and function. Recent transcriptome analysis has mainly focused on large EST clone collections. However, EST sequencing techniques in many cases are incapable of isolating rare transcripts or address transcript variability. In most cases, 3' RACE is applied for the experimental identification of alternatively polyadenylated transcripts. However, its application may result in nonspecific amplification and false positive products due to the usage of a single gene specific primer. Additionally, internal poly(A) stretches primed by oligo(dT) primer in mRNAs with AU-rich 3'UTR may generate truncated cDNAs. To overcome these limitations, we have developed a simple and rapid approach combining SMART technology for the construction of a full length cDNA library and hybrid capture PCR for the selection and amplification of target cDNAs. Our strategy is characterized by enhanced specificity compared to other conventional RT-PCR and 3' RACE procedures.

Release of membrane-associated L-dopa decarboxylase from human cells.

L-Dopa Decarboxylase is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of L-Dopa to dopamine. In this study, we investigated the cellular topology of the active human enzyme. Fractionation of membranes from human cell lines, of neural and non-neural origin, by temperature-induced phase separation in Triton X-114 resulted in the detection of DDC molecules in all separation phases. Solubilization of membrane-associated DDC was observed in a pH and time-dependent manner and was affected by divalent cations and protease inhibitors, suggesting the involvement of a possible release mechanism. The study of the biological properties and function of the solubilization phenomenon described here, as well as, the study of the membrane-associated enzyme could provide us with new information about the participation of the human L-Dopa decarboxylase in physiological and aberrant processes.

L-Dopa decarboxylase expression profile in human cancer cells.

L-Dopa decarboxylase (DDC) catalyses the decarboxylation of L-Dopa. It has been shown that the DDC gene undergoes alternative splicing within its 5'-untranslated region (UTR), in a tissue-specific manner, generating identical protein products. The employment of two alternative 5'UTRs is thought to be responsible for tissue-specific expression of the human DDC mRNA. In this study, we focused on the investigation of the nature of the mRNA expression in human cell lines of neural and non-neural origin. Our results show the expression of a neural-type DDC mRNA splice variant, lacking exon 3 in all cell lines studied. Co-expression of the full length non-neural DDC mRNA and the neural-type DDC splice variant lacking exon 3 was detected in all cell lines. The alternative DDC protein isoform, Alt-DDC, was detected in SH-SY5Y and HeLa cells. Our findings suggest that the human DDC gene undergoes complex processing, leading to the formation of multiple mRNA isoforms. The study of the significance of this phenomenon of multiple DDC mRNA isoforms could provide us with new information leading to the elucidation of the complex biological pathways that the human enzyme is involved in.

The U937 macrophage cell line expresses enzymatically active L-Dopa decarboxylase.

In the present study we demonstrate that enzymatically active l-Dopa decarboxylase (DDC) is expressed in U937 cells, an important model of human macrophage function. The mRNA coding for the enzyme was found to be of the neural-type. DDC was detected in both the soluble and membrane fractions. The active molecule is released from the membrane fraction in the absence of detergent. Furthermore, exposure of U937 cells to carbidopa and dopamine considerably affected cell viability. The expression of enzymatically active DDC shows the endogenous production of dopamine in U937 cells and raises new questions about the enzyme's involvement in immune responses.

A hybrid-specific, polymerase chain reaction-based amplification approach for chromosomal walking.

Here we report the development of an accurate and efficient genome walking approach based on ligation-mediated polymerase chain reaction (PCR) and magnetic capture hybrid selection technique. This approach overcomes the nonspecific amplification products that often occur in similar PCR-based methods. Our strategy was successfully applied for the cloning of the promoter region of the Cc RNase gene. This rapid, cost-effective, and sensitive protocol can easily be extended for use in the isolation and amplification of any target sequences for which only partial information is known such as identification of the position of transposable elements and integrated viral DNA sequences.

Detection, purification and identification of an endogenous inhibitor of L-Dopa decarboxylase activity from human placenta.

An endogenous inhibitor of L-Dopa decarboxylase activity was identified and purified from human placenta. The endogenous inhibitor of L-Dopa decarboxylase (Ddc) was localized in the membrane fraction of placental tissue. Treatment of membranes with phosphatidylinositol-specific phospholipase C or proteinase K did not affect membrane-associated Ddc inhibitory activity, suggesting that a population of the inhibitor is embedded within membranes. Purification was achieved by extraction from a nondenaturing polyacrylamide gel. The purification scheme resulted in the isolation of a single 35 kDa band, bearing L-Dopa decarboxylase inhibitory activity. The purified inhibitor was identified as Annexin V. The elucidation of the biological importance of the presence of an L-Dopa decarboxylase activity inhibitor in normal human tissues could provide us with new information leading to the better understanding of the biological pathways that Ddc is involved in.

Expression of enzymatically active L-DOPA decarboxylase in human peripheral leukocytes.

L-DOPA decarboxylase (DDC) is a pyridoxal 5-phosphate (PLP)-dependent enzyme that catalyses the decarboxylation of L-DOPA to dopamine. Dopamine (DA) has been found to be a regulating factor of the proliferation and differentiation of different leukocyte subtypes. In the present study, we report the expression of the gene that codes for the L-DOPA decarboxylase in human peripheral leukocytes and in T-lymphocytes, as well as the simultaneous detection of both neural and non-neural type DDC mRNA in the cellular components of this specialized connective tissue type. Furthermore, we have detected the neural type DDC transcript which lacks exon 3 and the alternative 37 kD alt-DDC protein isoform which lacks exons 10-15 but includes an alternative exon 10 in human peripheral leukocytes. Treatment of white blood cells with Triton X-114 resulted in the recovery of DDC in the detergent enriched and highly hydrophobic phases, suggesting association of DDC molecules with membranes in the studied cells. Enzymatic activity experiments revealed that DDC is active towards the decarboxylation of L-DOPA. The expression of enzymatically active DDC in human leukocytes could indicate a cross-talk between the nervous and the immune systems and raises new questions about the regulatory role of DDC in immune responses.