Bruno Varriale

Cell Biology of the Harderian Gland

The harderian gland is an orbital gland of the majority of land vertebrates. It is the only orbital gland in anuran amphibians since the lacrimal gland develops later during phylogenesis in some reptilian species. Perhaps because it is not found in man, little interest was paid to this gland until about four decades ago. In recent years, however, the scientific community has shown new interest in analyzing the ontogenetic and morphofunctional aspects of the harderian gland, particularly in rodents, which are the preferred experimental model for physiologists and pathologists. One of the main characteristics of the gland is the extreme variety not only in its morphology, but also in its biochemical properties. This most likely reflects the versatility of functions related to different adaptations of the species considered. The complexity of the harderian gland is further shown in its control by many exogenous and endogenous factors, which vary from species to species. The information gained so far points to the following functions for the gland: (1) lubrication of the eye and nictitating membrane, (2) a site of immune response, particularly in birds, (3) a source of pheromones, (4) a source of saliva in some chelonians, (5) osmoregulation in some reptiles, (6) photoreception in rodents, (7) thermoregulation in some rodents, and (8) a source of growth factors.

Role of orexin system in obesity

Obesity is a public health disease and its incidence is steadily increasing both in adults and in children especially in the Western World. It is important to understand the underlying mechanisms of obesity and possible treatments as the orexin system with its receptors, which are involved in different physiological processes. In fact, the aim of this mini-review is to consider the importance of the orexin system and the role that orexin plays in the regulation of obesity and physical activity. Furthermore to demonstrate how the orexin and its receptors fit within a network distributed in multiple brain areas, each with specific actions, whose activation and interconnection has been seen to lead to a lower propensity for increase of fat mass, it could thus constitute an important future target for prevention and treatment of obesity

Regulation of androgen receptor mRNA expression in primary culture of Harderian gland cells: cross-talk between steroid hormones

The androgen receptor (AR) must be considered a transcription factor belonging to the steroid-thyroid hormones receptor superfamily. Previous results gained from the Harderian gland, a tubulo-alveolar gland located in the orbital cavity of the golden hamster, indicate that Harderian gland cells express mRNAs encoding for androgen, glucocorticoid, thyroid hormone (T(3)), and estrogen receptors, respectively. Since in other systems, these receptors have been related to the expression of the androgen receptor, we have studied the regulation of AR expression in primary cultures of the male hamster Harderian gland. Our in vitro experiments show that androgen, and thyroid hormones increase the expression of AR. Retinoic acids also show a positive effect on AR expression, while exposure to glucocorticoid or estrogen blocks AR expression. Since these steroids differently modulate AR expression, our results must be considered in the context of multi-hormonal control of gene expression that could act through cross-talk between members of the steroid-thyroid hormones.

The hamster androgen receptor promoter : A molecular analysis

The steroid/thyroid hormone receptors are members of a very large family of nuclear-activated transcription factors. These receptors play a crucial role in most biological function, including regulation of development, metabolism, behaviour and reproduction. Among androgen receptor (AR), we have recently demonstrated that its expression in the Harderian gland (HG) of the male hamster is under a well-co-ordinated cross-talk between various steroid hormone receptors. Here, are presented data on the sequence of hamster AR promoter region (5UTR) and the molecular tools of its regulation. The 5UTR is 1585 bp. The promoter region shows various responsive elements. Two putative CREM elements are present at -71 and -1576 bp. A putative retinoic acid responsive element is present at -1476 bp. An androgen/glucocorticoid responsive element is present at -473 bp. A putative thyroid hormone-responsive element at -381 bp and an estrogen responsive element at -230 bp. Also, a homopurinic stretch is evident between -1199 and -1118. Furthermore, Sp1 sites are also spread along the sequence. As well as for human, mouse, rat and pig, the hamster lacks the canonical promoter TATA and CCAAT boxes. Gel retardation experiments confirm the presence of active responsive elements for AR, estrogen receptor, glucocorticoid receptor and thyroid hormone receptor. Previous data on the regulation of expression of AR by other members of steroid/thyroid hormone receptors well correlate with sequence analysis and gel retardation experiments. Thus, androgens, thyroid hormone, stimulate the AR transcription, while synthetic glucocorticoid (Dex) and estrogen are potent inhibitors of AR expression. The comparison of hamster AR promoter sequence with other AR promoter shows an 89, 82, 84 and 84% identity with human, rat, mouse and pig AR promoter, respectively. These results, in the light of the extreme plasticity of hamster HG, suggest that the comparative study of expression and regulation of AR gene in the HG of the hamster offers a useful tool to approach the normal and pathological phenotype in human.

FMO3 allelic variants in Sicilian and Sardinian populations: trimethylaminuria and absence of fish-like body odor.

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria. The aim of this study was to determine the allelic and genotypic distributions of these four FMO3 variants in 528 healthy individuals collected from the Sicilian and Sardinian populations together with haplotype and linkage analyses. Finally, we present data on the genotype-phenotype correlation by ESI-MS/MS TMA/TMAO urinary determination in 158KK/308EG individuals. Variant 158K shows the same frequency in Sicilian and Sardinian populations while variant 257M was not observed in the Sardinian sampling. No significant differences were found for 308G and 475D variants among two populations. Cis-linkage between 158K and 308G was confirmed with the compound variant (158K-308G) being found in a proportion of 0.9% and 0.3% of Sicilian subjects, and 0.01% and 0.5% in Sardinian population. Urinary determination of TMA/TMAO ratio in 158KK/308EG individuals showed a considerable reduction in FMO3 activity although they do not show the classical features of trimethylaminuria as a strong body odor and breath. Our data support the conclusion that trimethylaminuria is not always accompanied by a fish-like odor, despite the coexistence in the same individual of the two variants 158K and 308G, and other factors account for the expression of that phenotype.

Synergistic Interplay between Curcumin and Polyphenol-Rich Foods in the Mediterranean Diet: Therapeutic Prospects for Neurofibromatosis 1 Patients

Neurofibromas are the hallmark lesions in Neurofibromatosis 1 (NF1); these tumors are classified as cutaneous, subcutaneous and plexiform. In contrast to cutaneous and subcutaneous neurofibromas, plexiform neurofibromas can grow quickly and progress to malignancy. Curcumin, a turmeric-derived polyphenol, has been shown to interact with several molecular targets implicated in carcinogenesis. Here, we describe the impact of different dietary patterns, namely Mediterranean diet (MedDiet) compared to the Western diet (WesDiet), both with or without curcumin, on NF1 patients’ health. After six months, patients adopting a traditional MedDiet enriched with 1200 mg curcumin per day (MedDietCurcumin) presented a significant reduction in the number and volume of cutaneous neurofibromas; these results were confirmed in subsequent evaluations. Notably, in one patient, a large cranial plexiform neurofibroma exhibited a reduction in volume (28%) confirmed by Magnetic Resonance Imaging. Conversely, neither unenriched MedDiet nor WesDiet enriched with curcumin exhibited any significant positive effect. We hypothesize that the combination of a polyphenol-rich Mediterranean diet and curcumin was responsible for the beneficial effect observed on NF1. This is, to the best of our knowledge, the first experience with curcumin supplementation in NF1 patients. Our report suggests that an integrated nutritional approach may effectively aid in the management of NF1.

A novel diagnostic method to detect truncated neurofibromin in Neurofibromatosis 1

Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic condition caused by dominant loss‐of‐function mutations of the tumor suppressor gene NF1 that encodes neurofibromin, a negative regulator of RAS activity. Mutation analysis of NF1 located at 17q11.2 has been hampered by the large size of the gene, the high rate of new mutations, the lack of mutational clustering, and the presence of several homologous loci. To date, about 80% of the reported NF1 mutations are predicted to result in protein truncation, but very few studies have correlated the causative NF1 mutation with its effect at the protein level. We evaluated a novel diagnostic method to detect truncated forms of neurofibromin in a large cohort of unrelated subjects suspected of having NF1, according to the NIH consensus criteria. Western blot analysis was carried out on protein extracts from patients leukocytes to highlight the possible presence of altered neurofibromin as a result of mutations in NF1. Truncated neurofibromin was identified in 274/336 patients (81%), confirming the usefulness and reproducibility of the proposed diagnostic approach. Our methodology can be routinely applied in the diagnostic setting, thanks to its simplicity and reliability. Combined with molecular approaches, it may increase the accuracy and efficiency of NF1 genetic testing.

Diet Therapy of Obesity: Observations on the Usefulness of Weekly Supervision in the Improvement of Weight Loss

Background and aims: Obesity, a condition born in the rich countries of the West, is rising in the rest of the world. The developing countries are adopting the eating habits and the lifestyle of the rich countries. The purpose of this study was to evaluate, on obese subjects, the effect of a diet therapy with weekly control with respect to the effect of the same monthly check, as by traditional protocol. Methods: Forty patients with first degree of obesity were treated with hypocaloric diet therapy for 6 months with weekly checks, while other 40 patients were treated with the same diet therapy but with monthly control. Examination has allowed to assess nutritional status, to estimate body composition and provide guidance for the calculation of individual nutrient needs and to evaluate the effectiveness of nutritional therapy. The analysis of the parameters was carried out mainly through the survey of the body data changes as per protocol. Moreover, taking advantage of the BIA (Bio-impedance) we were able to follow the trend of the effectiveness of the diet regimen in both groups. Results: The slimming of the weekly control group resulted 63% greater than the monthly control group. In both men and women the weekly check produces significant differences by increasing the effects of diet therapy treatment. Reaching statistical significance not only for the decrease in body weight but also for the majority of BIA parameters considered in our analysis. Conclusion: The results, thus obtained, indicate that the weekly monitoring can improve the “compliance” of the patient in diet therapy.

Ontogenesis of the sexual dimorphism in the Harderian gland of the domestic fowl (Gallus domesticus)

AdultGallus domesticus shows a sexual dimorphism of the electrophoretic pattern of Harderian gland (HG) proteins. The female protein pattern is characterized by two fractions whose size is about 210 and 80kDa respectively. The male pattern shows two proteic fractions of about 180 and 50kDa respectively. The estrogen receptor mRNA has been detected only in the female. Androgen receptor mR-NA has not been found in either sexes. Sexual dimorphism of the electrophoretic pattern of HG proteins appears at 45 days after hatching.RiassuntoGallus domesticus mostra un dimorfismo sessuale nel pattern elettroforetico delie proteine delia ghiandola di Harder. II pattern della femmina adulta è caratterizzato da due frazioni proteiche di circa 210 e 80kDa, mentre quello del maschio adulto da due frazioni di circa 180 e 50kDa. L’mRNA per il recettore degli estrogeni è presente solo nella ghiandola della femmina, mentre manca completamente 1’mRNA per il recettore degli androgeni sia nel maschio che nella femmina. Le differenze sessuali nel pattern elettroforetico appaiono nei pulcini di 45 giorni.

Sex steroid binding proteins in the Harderian gland of nonmammalian tetrapods

The presence of sex hormone receptors in the Harderian gland (HG) of nonmammalian tetrapods has been investigated. In the male and female green frog,Rana esculenta, androgen receptors were found in both nuclear and cytosolic extracts (Kd = 1.8 ± 1.2 nM); their seasonal variations correlate with plasmatic androgen profiles as well as with the cyclic secretory activity of the gland. These findings suggest a direct participation of intracellular androgens in the seromucoidal secretion of frog Harderian gland. No estradiol specific binding was detectable. In the toad,Bufo viridis, an androgen-estrogen binding protein (Kd = 1.8 ±0.7 nM) was evidenced only in the cytosolic fraction of the both sexes. Androgen and/or estrogen binding proteins were undetectable in the lizardPodarcis s. sicula and the chicken.Riassuntoè stata studiata la presenza di recettori per gli ormoni sessuali nella ghiandola di Harder di alcuni tetrapodi non mammiferi. InRana esculenta sono stati messi in evidenza recettori per gli androgeni sia negli estratti nucleari che in quelli citoplasmatici sia del maschio che della femmina (Kd = 1.8 ± 1.2 nM); le loro variazioni stagionali sono correlabili sia con i profill degli androgeni plasmatici che con il ciclo dell’attività secretoria della ghiandola. Questi dati suggeriscono una diretta partecipazione degli androgeni intracellulari alia secrezione sieromucosa della ghiandola di Harder, mentre non è stato messo in evidenza alcun legame specirico per l’estradiolo. Nella ghiandola di Harder di entrambi i sessi diBufo viridis è stata evidenziata, nella sola frazione citoplasmatica, una proteina che lega sia gli androgeni che gli estrogeni (Kd= 1.8 ±0.7 nM), mentre in quella della lucertola,Podarcis s. sicula, e del polio, non è stato evidenziato alcun legame specirico per gli androgeni e/o per gli estrogeni.