Vittorio Colantuoni

Impaired retinal function and vitamin A availability in mice lacking retinol‐binding protein

Retinol‐binding protein (RBP) is the sole specific transport protein for retinol (vitamin A) in the circulation, and its single known function is to deliver retinol to tissues. Within tissues, retinol is activated to retinoic acid, which binds to nuclear receptors to regulate transcription of >300 diverse target genes. In the eye, retinol is also activated to 11‐cis‐retinal, the visual chromophore. We generated RBP knockout mice (RBP−/−) by gene targeting. These mice have several phenotypes. Although viable and fertile, they have reduced blood retinol levels and markedly impaired retinal function during the first months of life. The impairment is not due to developmental retinal defect. Given a vitamin A‐sufficient diet, the RBP−/− mice acquire normal vision by 5 months of age even though blood retinol levels remain low. Deprived of dietary vitamin A, vision remains abnormal and blood retinol declines to undetectable levels. Another striking phenotype of the mutant mice is their abnormal retinol metabolism. The RBP−/− mice can acquire hepatic retinol stores, but these cannot be mobilized. Thus, their vitamin A status is extremely tenuous and dependent on a regular vitamin A intake. Unlike wild‐type mice, serum retinol levels in adult RBP−/− animals become undetectable after only a week on a vitamin A‐deficient diet and their retinal function rapidly deteriorates. Thus RBP is needed for normal vision in young animals and for retinol mobilization in times of insufficient dietary intake, but is otherwise dispensable for the delivery of retinol to tissues.

Fenofibrate prevents and reduces body weight gain and adiposity in diet-induced obese rats

Fibrates are hypolipidemic drugs that activate the peroxisome proliferator‐activated receptors. Since fibrates may also increase energy expenditure, we investigated whether fenofibrate (FF) had this effect in diet‐induced obese rats. A 2‐month administration of a high‐fat palatable diet to adult rats increased body weight by 25% and white adipose mass by 163% compared with a standard diet. These effects were prevented by FF, both when administered for the 2 months of high‐fat feeding and when given for only the second month. Consequently, FF‐treated rats had a final body weight and white adipose tissue mass similar to untreated animals on the standard diet. FF also increased resting metabolic rate, hepatic peroxisomal and mitochondrial palmitoyl‐dependent oxygen uptake and mRNA levels of acyl‐CoA oxidase and lipoprotein lipase. Finally, FF lowered mRNA levels of uncoupling protein‐2 and did not affect mitochondrial respiration in skeletal muscle. Therefore, FF seems to act as a weight‐stabilizer mainly through its effect on liver metabolism.

Structure and cell-specific expression of a cloned human retinol binding protein gene: the 5'-flanking region contains hepatoma specific transcriptional signals.

Human plasma retinol binding protein (RBP) is coded by a single gene and is specifically synthesized in the liver. We have characterized a lambda clone, from a human DNA library, carrying the gene coding for plasma RBP. Southern blot analysis and DNA sequencing show that the gene is composed of six exons and five introns. Primer elongation and S1 mapping experiments allowed the definition of the initiation of transcription and the identification of the putative promoter. The 5′‐flanking region of the RBP gene was fused upstream to the coding sequence of the bacterial enzyme chloramphenicol acetyl transferase (CAT): the chimeric gene was introduced, by calcium phosphate precipitation, into the human hepatoma cell line Hep G2 and into HeLa cells. Efficient expression of CAT was obtained only in Hep G2. Primer elongation analysis of the RNA extracted from transfected Hep G2 showed that initiation of transcription of the transfected chimeric gene occurs at a position identical to that of the natural gene. Transcriptional analysis of Bal31 deletions from the 3′ end of the RBP 5′‐flanking DNA allowed the identification of the RBP gene promoter.

De novo expression of uncoupling protein 3 is associated to enhanced mitochondrial thioesterase‐1 expression and fatty acid metabolism in liver of fenofibrate‐treated rats

Uncoupling protein 3 (UCP3) is a member of the mitochondrial carrier superfamily, preferentially expressed in skeletal muscle. Its function is not fully understood and it is debated whether it uncouples oxidative phosphorylation as does UCP1 in brown adipose tissue. Recent evidences suggest a role for UCP3 in the flux of fatty acids in and out mitochondria and their utilization in concert with mitochondrial thioesterase‐1 (MTE‐1). In fact, mice overexpressing muscle UCP3 also show high levels of MTE‐1. Fenofibrate is a hypolipidemic drug that prevents body weight gain in diet‐induced obese rats and enhances lipid metabolism by activating peroxisome proliferator‐activated receptors (PPARs). Because fatty acids and fenofibrate stimulate PPARs and in turn UCP3, we investigated whether UCP3 expression might be induced ‘de novo’ in situations of increased hepatic mitochondrial fatty acid utilization caused by a combined effect of a high‐fat diet and fenofibrate treatment. We also investigated whether Mte‐1 expression and β‐oxidation were affected. We show here that Ucp3 is induced in liver of fenofibrate‐treated rats at the mRNA and protein level. Expression was restricted to hepatocytes and was unevenly distributed in the liver. No increase in cell proliferation, inflammatory or fibrotic responses was found. Mte‐1 expression and mitochondrial β‐oxidation were upregulated. Thus, Ucp3 can be transactivated in tissues where it is normally silent and fenofibrate can attain this effect in liver. The data demonstrate that UCP3 is involved in fatty acid utilization and support the notion that UCP3 and MTE‐1 are linked within the same metabolic pathway.

In Vivo and In Vitro Studies of Cytosolic Phospholipase A2 Expression in Helicobacter pylori Infection

ABSTRACT Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with differentH. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2activity in the gastric mucosae of patients with H. pyloriinfection and no change in epithelial cell lines exposed to H. pylori.

Medullary thyroid cancer, papillary thyroid microcarcinoma and Graves' disease: an unusual clinical coexistence.

We describe the unusual case of a Caucasian woman who had a diagnosis of medullary thyroid cancer and papillary microcarcinoma 5 years after a diagnosis of Graves’ disease. The patient came to our observation for recurrence of hyperthyroidism. An ultrasound scan revealed diffuse thyroid enlargement with a nodule, recently increased in size. The serum CT and carcinoembrional antigen were elevated, and the fine-needle aspiration cytology with immunocytochemical analysis for CT was suggestive for medullary thyroid carcinoma. The nodular lesion showed intense 111In-pentetreotide uptake, whereas total body scintigraphy with the same tracer and with Thallium- 201, 99mTc (V) dimercaptosuccinic acid was negative for lymph node and distant metastasis. The histological examination of thyroidectomy specimens confirmed the diagnosis of medullary thyroid cancer, showing a lymphocytic intratumoral infiltration. The histological analysis of the controlateral lobe showed an occult papillary microcarcinoma. Medullary thyroid carcinoma and papillary microcarcinoma showed intense staining with policlonal anti-RET antibodies, although genetic analysis was negative for RET mutations most frequently involved in familial and sporadic medullary thyroid carcinomas. Possible implications about the coexistence of the 3 thyroid diseases are discussed.

A novel germline mutation in Peroxisome Proliferator-Activated Receptor γ gene associated with large intestine polyp formation and dyslipidemia

We report a novel PPARG germline mutation in a patient affected by colorectal cancer that replaces serine 289 with cysteine in the mature protein (S289C). The mutant has impaired transactivation potential and acts as dominant negative to the wild type receptor. In addition, it no longer restrains cell proliferation both in vitro and in vivo. Interestingly, the S289C mutant poorly activates target genes and interferes with the inflammatory pathway in tumor tissues and proximal normal mucosa. Consistently, only mutation carriers exhibit colonic lesions that can evolve to dysplastic polyps. The proband presented also dyslipidemia, hypertension and overweight, not associated to type 2 diabetes; of note, family members tested positive for the mutation and display only a dyslipidemic profile at variable penetrance with other biochemical parameters in the normal range. Finally, superimposing the mutation to the crystal structure of the ligand binding domain, the new Cys289 becomes so closely positioned to Cys285 to form an S-S bridge. This would reduce the depth of the ligand binding pocket and impede agonist positioning, explaining the biological effects and subcellular distribution of the mutant protein. This is the first PPARG germline mutation associated with dyslipidemia and colonic polyp formation that can progress to full-blown adenocarcinoma.

Genetics of medullary thyroid cancer: An overview

Medullary thyroid carcinoma (MTC) represents 3-5% of thyroid cancers. 75% is sporadic and 25% is the dominant component of the hereditary multiple endocrine neoplasia (MEN) type 2 syndromes. Three different subtypes of MEN2, such as MEN2A, MEN2B, and Familial MTC (FMTC) have been defined, based on presence or absence of hyperparathyroidism, pheocromocytoma and characteristic clinical features. Mutations of the RET proto-oncogene are implicated in the pathogenesis of MTC, but there are many other mutational patterns involved. In MEN2A, Codon 634 in exon 11 (Cys634Arg), corresponding to a cysteine in the extracellular cysteine-rich domain, is the most commonly altered codon. Many other mutations include codons 611, 618, 620. In the genetical testing of RET mutations in MTCs, Next-Generation Sequencing (NGS) is taking an increasingly important role. One of the most important benefit is the comprehensive analysis of molecular alterations in MTC, which allows rapidly to select patients with different risk levels. There is a difference in miRNA expression pathway between sporadic and hereditary MTCs. Among sporadic cases, expression of miR-127 was significantly lower in those who harbor somatic RET mutations than those with wild-type RET. CDKN1B mutations are associated with many clinical pictures of cancers, such as MEN4. V109G polymorphism is associated with sporadic MTCs negative for RET mutations, and might influence the clinical course of the patients affected by MTC. Although surgery (i.e. total thyroidectomy with neck lymph node dissection) is the elective treatment for MTCs, about 80% of patients have distant metastases at diagnosis and in this cases surgery is not enough and an additional treatment is needed. Interesting results come from two large phase III clinical trials with two targeted tyrosine kinase inhibitors (TKIs), vandetanib and cabozantinib. CONCLUSIONS New genetical testings and therapeutical approaches open new perspectives in MTC management.

Fractionation of Escherichia coli isoaccepting tRNA species by Sepharose 4B Column chromatography

Abstract We have determined the elution profile on Sepharose 4B chromatographic column ofthe tRNA isoaccepting species of all 20 amino acids from Escherichia coli MRE 600. Further chromatography on a reversed phase column (RPC-5) is sufficient, in some cases, for a complete purification.

Seventeen-year-long follow-up of a family affected by type 2A Multiple Endocrine Neoplasia (MEN 2A)

This paper reports the results of a 17-year-long follow-up covering 17 members of a family affected by multiple endocrine neoplasia (MEN) type 2A, first diagnosed in 1980. This family is enrolled in our screening program. The thyroid, parathyroid and adrenal glands of the family members were investigated using the most sophisticated and sensitive techniques which have become available during this period, and their DNA was genetically tested for detecting RET mutations. Thanks to the combination of these two approaches it was possible to confirm the diagnosis in the members concerned from the genetic point of view, and to achieve an early diagnosis in the young members of the last generation before the clinical onset of the disease. The detection of a RET mutation also prompted a prophylactic thyroidectomy in a four year-old boy, in a pre-tumoral stage of the disease. Lastly, evidence is provided that genetic analysis of the DNA of the chorionic villi can be carried out as a prenatal test during routine amniocentesis.