The effect of arginine (Arg) and Ornitargin^® (OT) [a compound containing the aminoacids Arg, citrulline (Cit) and ornithine (Orn)] administration upon growth hormone (GH) gene expression was studied both in vivo and in vitro (hemipituitaries and GH3 cells) by Northern blot analysis. For in vivo studies, adult male Wistar rats were anesthetized, subjected to i.v. infusion of 200 µl of 150 mM NaCl (control group), Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) at a rate of 20 µl/min, and killed 50 min thereafter. For the in vitro studies, hemipituitaries or GH3 cells were incubated in 1 ml of appropriate medium containing Arg (15 or 150 mg) or OT (15 mg of Arg, 1 mg of Cit and 4 mg of Orn) for 60 min. The pituitaries of the in vivo and in vitro studies and GH3 cells were subsequently processed for RNA extraction. Total RNA was subjected to electrophoresis in agarose (1%)/formaldehyde gel, transferred to a nylon membrane and subjected to hybridization with a rat GH ³²P-cDNA, and ³²P-18S rRNA probe to correct for the variability in RNA loading. After autoradiography of the membrane, the abundance of GH mRNA and 18S rRNA bands was quantified by densitometry. The in vivo study demonstrated that Arg and OT infusion induced a 2.3-fold increase in GH mRNA expression, which could result from the Arg-mediated inhibition of somatostatin release. In addition, in vitro Arg, but not OT, induced GH gene expression in hemipituitaries and GH3 cells, indicating that the aminoacid can act per se at the pituitary somatotrope level. In conclusion, our data show for the first time that arginine stimulates GH gene expression in parallel to its recognized GH-releasing activity.

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The amino acid arginine (Arg) is a recognized secretagogue of growth hormone (GH), and has been shown to induce GH gene expression. Arg is the natural precursor of nitric oxide (NO), which is known to mediate many of the effects of Arg, such as GH secretion. Arg was also shown to increase calcium influx in pituitary cells, which might contribute to its effects on GH secretion. Although the mechanisms involved in the effects of Arg on GH secretion are well established, little is known about them regarding the control of GH gene expression. We investigated whether the NO pathway and/or calcium are involved in the effects of Arg on GH gene expression in rat isolated pituitaries. To this end, pituitaries from approximately 170 male Wistar rats (∼250 g) were removed, divided into two halves, pooled (three hemi-pituitaries) and incubated or not with Arg, as well as with different pharmacological agents. Arg (71 mM), the NO donor sodium nitroprusside (SNP, 1 and 0.1 mM) and a cyclic guanosine monophosphate (cGMP) analogue (8-Br-cGMP, 1 mM) increased GH mRNA expression 60 min later. The NO acceptor hemoglobin (0.3 µM) blunted the effect of SNP, and the combined treatment with Arg and L-NAME (an NO synthase (NOS) inhibitor, 55 mM) abolished the stimulatory effect of Arg on GH gene expression. The calcium channel inhibitor nifedipine (3 µM) also abolished Arg-induced GH gene expression. The present study shows that Arg directly induces GH gene expression in hemi-pituitaries isolated from rats, excluding interference from somatostatinergic neurons, which are supposed to be inhibited by Arg. Moreover, the data demonstrate that the NOS/NO signaling pathway and calcium mediate the Arg effects on GH gene expression.

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Fifteen female canines with mammary tumors and 6 normal females were used to study mutations in exons 4 to 8 of the p53 gene. DNA samples from the tumors, respective adjacent normal mammary tissue and mammary glands from healthy animals were sequenced and analyzed for the presence of mutations. Mutations were found in 71.8% of the samples and the most frequent were missense mutations. The most attacked exons in the mammary tumor were 5, 7 and 8, with 23.4, 31.6 and 23.4% mutations, respectively. Canine mammary tumors are related to mutations in gene p53 and mutations mostly occur in the region of the protein that is linked to the DNA in the cell nucleus, which can change the functionality of the cell and propitiate tumor growth. Despite being macroscopically normal, the mammary tissue adjacent to the tumors has mutations that can lead to recurrence if not removed together with the tumor.

Artisan fresh cheese producing farms from six provinces of Cuba were studied to identify the presence of bacterial hazards and the results are presented in this research communication. The bacterial hazards identified in milk and cheese respectively were: Listeria spp. (9.5 and 18.9%), Bacillus cereus (23.2 and 24.2%), Escherichia coli O157 (12.6 and 13.7%), Salmonella spp. (10.5 and 17.9%), and Staphylococcus aureus (29.5 and 51.6%). Listeria monocytogenes was not detected. Nine Salmonella serotypes corresponding to Salmonella enterica subsp. enterica and Salmonella enterica subsp. arizonae were isolated, whereas Salmonella Anatum was present most often. Biofilm formation by the isolated species and enterotoxin production by S. aureus strains demonstrated the pathogenic potential of the identified bacterial hazards. Results proved the presence of bacterial hazards in the raw milk and cheeses analyzed, so that good manufacturing practices must be accomplished throughout the entire production process in order to avoid the occurrence of foodborne diseases in the population.

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Breast cancer is the most commonly diagnosed cancer in women under 60. Localized breast cancer is easily treated, resulting in high survival rates. However, treatment for advanced disease is inadequate, with a five-year survival rate of less than 24%. Thus, there is a great need for new therapies capable of increasing therapeutic efficacy. Synthetic mesoionic compounds, belonging to the 1,3-thiazolium-5-thiolate group, are recognized for their broad spectrum of biological activities including antibiotic, antiparasitic, antiviral, anticonvulsant, antidepressant, antioxidant, analgesic, anti-inflammatory, and more recently for their potential antitumor activity. These compounds have the ability to cross cell membranes; the characteristic of mesoionic structures having distinct regions of positive and negative charge associated with a poly-heterocyclic aromatic ring system, indicates the capability of strong interactions with biomolecules such as DNA and proteins. In this study, the cytotoxic effects of mesoionic compound MI H 2.4 alone and in combination with zinc was examined in breast cancer cell lines (4TI, BT-20, BT-549, MCF7, MDA-MB-231, MDA-MB-436, MM2MT, T-47D, and ZR-75-1) and normal breast cell lineages (HuMEC, MCF-10A, and MCF-12A) were evaluated. The effect of this agent on cell cycle was also investigated. Different concentrations of mesoionic compound MI H 2.4 (MI H 2.4 free) and in combination with zinc (MI H 2.4 Zinc) were added to the cultured cells and incubated for 24, 48, 72 and 96 h. Cell survival and cytotoxicity were evaluated using crystal violet and MTT assays. Cell cycle analysis was performed using MCF7 cells that were stained with propidium iodide and analyzed by flow cytometry. The cytotoxic effects of mesoionic compounds (MI H 2.4 free and MI H 2.4 Zinc) were highest at72 and 96 h. The MI H 2.4 free and MI H 2.4 Zinc showed a similar inhibitory effect on breast cancer cell growth in the μM range. In contrast, the normal breast cell lineages showed low cytotoxicity to treatment with the mesoionic compounds. Treatment of MCF7 cells cultured with MI H 2.4 free blocked cell cycle progression at the G2 phase of the cell cycle after 24 h of treatment. Mitochondrial function of MCF7 cells was determined using a Seahorse XF-24 Extracellular Flux Analyzer. Treatment with MI H 2.4 free and MI H 2.4 Zinc for 24 h resulted in a decreased basal and maximal mitochondrial respiration. In summary, mesoionic compound MI H 2.4 may offer a novel therapeutic strategy in the treatment of breast cancer, considering that it has significant antitumoral activity in breast cancer cell lines and low cytotoxicity in normal cells. Citation Format: Luciana Amaral de Mascena Costa, Filipe Cássio Silva de Lima, Rodrigo da Silva Viana, Silvany de Sousa Araujo, Aurea Wischral, Helivaldo Diógenes da Silva Souza, Petrônio Filgueiras de Athayde-Filhoa, Leandro Araújo de Azevedo, Severino Alves Júnior, Manoel Adrião, J. Michael Mathis. Antitumor activity of the mesoionic compound MI H 2.4 on breast cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5877.