Evaluation of the non-clinical toxicity of an antiparasitic agent: diminazene aceturate.

The diminazene aceturate (C14H15N7.2C4H7NO3) is a chemotherapeutic agent with more than six decades of use, however more studies regarding its toxicity still need to be performed. Thus, the present study determined the acute toxicity (14 days) of diminazene acetate (DIZE) in male and female swiss mice by changes in body mass, food consumption, biochemical and hematological parameters, locomotor activity and motor coordination. DIZE was administered at a single dose (1000 and 2000 mg/kg) orally. In addition, in vitro antioxidant capacity, hemolytic activity, toxicity in Artemia salina and in silico evaluation were also performed. The results obtained include several signs of toxicity (hypoactivity, loss of the straightening reflex and tachycardia), reduction of behavioral activity (locomotor activity and motor coordination) and significant changes (p < 0.05) in biochemical and hematological parameters. According to the in silico study, the DIZE can be classified based on the mean lethal dose (LD50) in category 4 (300 mg/kg < LD50 ≤ 2000 mg/kg, ProTox-II) or 3 (50 mg/kg < LD50 ≤ 300 mg/kg, AdmetSAR 1.0). Additionally, DIZE (30.3-969.9 nM) was not toxic to A. salina in the first 48 hours of treatment and was not cytotoxic to rat red blood cells after induced hemolysis. In vitro results indicated low antioxidant capacity against DPPH• and ABTS•+ radicals. Therefore, DIZE induces several adverse effects with influence on the central nervous system, changes in hematological and biochemical parameters and even mortality at the highest dose. However, absence of toxicity was observed in A. salina and rats red blood cells.

Non-clinical toxicity of ß-caryophyllene, a dietary cannabinoid: Absence of adverse effects in female Swiss mice.

ß-caryophyllene is a food additive that is found in food plants and has broad pharmacological potential. However, little toxicological information has been reported and its use is based on the fact that this bicyclic sesquiterpene is daily consumed as a plant food in much larger quantities than as a food additive. Thus, this study evaluated acute (14-day) and repeated-dose (28 days) oral ß-caryophyllene toxicity in female Swiss mice analyzing changes in body weight, food intake, water intake, hematological and biochemical parameters, organ weight after necropsy, oxidative stress markers and histopathology of various tissues. Acute (300 and 2000 mg/kg) and repeated-dose (300 and 2000 mg/kg) toxicity studies were performed according to the Organization for Economic Cooperation and Development (OECD) guideline 423 and 407, respectively. There was absence of adverse clinical signs and mortality in any animal subjected to acute and repeated-dose toxicity study. In addition, no significant changes in body weight, food and water intake, oxidative stress biomarkers, hematological and biochemical parameters were observed when compared to control group from single-dose and repeated-dose toxicity study. Therefore, the results of this study provide an understanding of the toxicity profile of ß-caryophyllene which can be considered a compound with toxicity at doses higher than 2000 mg/kg body weight.

Absence of toxicity in Swiss mice following treatment with 7-acetoxy-4-aryl-3,4-dihydrocoumarin: Acute and repeated-dose toxicity study.

Neoflavonoids, which are classified as 4-arylcoumarin (neoflavone), 3,4-dihydro-4-arylcoumarin and neoflavene, have been the subject of a number of studies with respect to their therapeutic potential and, despite promising in vitro, ex vivo and in vivo pharmacological activities, there is a lack of studies demonstrating their toxicological properties. Therefore, this study aims to evaluate the acute (14 days) and repeated-dose (28 days) toxicity of synthetic neoflavonoid 7-acetoxy-4-aryl-3,4-dihydrocoumarin in Swiss mice through parameters related to changes in body weight, food and water intake, hematological and biochemical parameters. Toxicity studies using acute doses (300 and 2000 mg/kg) and repeated doses (250, 500 and 1000 mg/kg) orally were carried out as per Organization for Economic Co-operation and Development (OECD) guidelines 423 and 407, respectively. Based on the results of this study, treatment with 7-acetoxy-4-aryl-3,4-dihydrocoumarin was found to not cause clinical adverse symptoms and mortality in any animal used in the acute and repeated-dose toxicity study. In addition, no significant changes were observed in body weight and internal organs, food and water intake, hematological and biochemical parameters, compared to control group. Therefore, these results provide an initial understanding regarding the toxicity profile of 7-acetoxy-4-aryl-3,4-dihydrocoumarin, which can be considered a neoflavonoid with toxicity seen at doses higher than 2000 mg/kg in Swiss mice.

Comparison of different degrees of variability in tidal volume to prevent deterioration of respiratory system elastance in experimental acute lung inflammation.

BACKGROUND: Variable ventilation improves respiratory function, but it is not known whether the amount of variability in tidal volume (VT) can be reduced in recruited lungs without a deterioration of respiratory system elastance. METHODS: Acute lung inflammation was induced by intratracheal instillation of lipopolysaccharide in 35 Wistar rats. Twenty-eight animals were anaesthetized and ventilated in volume-controlled mode. Lungs were recruited by random variation of VT (mean 6 ml kg(-1), coefficient of variation 30%, normal distribution) for 30 min. Animals were randomly assigned to different amounts of VT variability (n=7 for 90 min per group): 30, 15, 7.5, or 0%. Lung function, diffuse alveolar damage, and gene expression of biological markers associated with cell mechanical stress, inflammation, and fibrogenesis were assessed. Seven animals were not ventilated and served as controls for post-mortem analyses. RESULTS: A VT variability of 30%, but not 15, 7.5, or 0%, prevented deterioration of respiratory system elastance [Mean (SD) -7.5 (8.7%), P<0.05; 21.1 (9.6%), P<0.05; 43.3 (25.9), P<0.05; and 41.2 (16.4), P<0.05, respectively]. Diffuse alveolar damage was lower with a VT variability of 30% than with 0% and without ventilation, because of reduced oedema and haemorrhage. A VT variability of 30, 15, or 7.5% reduced the gene expression of amphiregulin, cytokine-induced neutrophil chemoattractant-1, and tumour necrosis factor α compared with a VT variability of 0%. CONCLUSIONS: In this model of acute lung inflammation, a VT variability of 30%, compared with 15 and 7.5%, was necessary to avoid deterioration of respiratory system elastance and was not associated with lung histological damage.

Ecology of phlebotomines (Diptera: Psychodidae) in a transitional area between the Amazon and the Cerrado in the State of Maranhão, Brazil.

The Amazon rainforest and the Brazilian Cerrado both possess high phlebotomine diversity. The fragmentation of these habitats has resulted in the appearance of human cases of cutaneous leishmaniasis. In one altered area of mixed primary vegetation (forest and Cerrado) and its adjacent settlement in the northeast state of Maranhão, Brazil, evidence exists for the active transmission of cutaneous leishmaniasis. Accordingly, an entomological investigation was performed in both the forest and the settlement to compare the phlebotomine vector faunain each environment. The study was conducted from September 2009 to August 2010 in the municipality of Itapecuru Mirim in the state of Maranhão, Brazil. The phlebotomine species were captured using 24 light Center for Disease Control and Prevention traps that were placed in the forest and the settlement (peridomicile and intradomicile). The similarity between the phlebotomine compositions in the forest and those in the settlement was determined using a Principal Coordinate Analysis based on a dissimilarity matrix that was calculated using the Bray-Curtis index (relative abundance) and the Jaccard index (presence and absence of species). In total, 29 Lutzomyia species and one Brumptomyia species were collected. The phlebotomines were diverse and abundant in both the forest fragment (27 species, 4,606 specimens) and the settlement (22 species, 753 specimens). The most abundant species were L. infraspinosa (25%), L. davisi (21%), L. antunesi (21%), L. longipalpis (9%), L. saulensis (6%), L. flaviscutellata (5%), and L. wellcomei (4%). Some species were found strictly in the forest, other species were exclusive to the anthropic environment, and some species colonized both of the studied environments. The phlebotomines adaptation to these modified environments explains the autochthonous outbreak of cutaneous leishmaniasis.

Anticonvulsant, Anxiolytic and Antidepressant Properties of the ß-caryophyllene in Swiss Mice: Involvement of Benzodiazepine-GABAAergic, Serotonergic and Nitrergic Systems.

BACKGROUND: Central nervous system disorders such as anxiety, depression and epilepsy are characterized by sharing several molecular mechanisms in common and the involvement of the L-arginine/NO pathway in neurobehavioral studies with ß-caryophyllene is still little discussed. OBJECTIVES: One of the objectives of the present study was to demonstrate the anxiolytic behavioral effect of ß-caryophyllene (ß-CBP) in female Swiss mice, as well as to investigate the molecular mechanisms underlying the results obtained. METHODS: This study evaluated the neurobehavioral effects of ß-CBP using the open field test, rota- rod test, elevated plus maze test, novelty suppressed feeding test, tail suspension test and forced swim test, as well as pilocarpine, pentylenetetrazole and isoniazid-induced epileptic seizure models. RESULTS: The results demonstrated that the neuropharmacological activities of ß-CBP may involve benzodiazepine/GABAergic receptors, since the pre-treatment of ß-CBP (200 mg/kg) associated with flumazenil (5 mg/kg, benzodiazepine receptor antagonist) and bicuculline (1 mg/kg, selective GABAA receptor antagonist) reestablished the anxiety parameters in the elevated plus-maze test, as well as the results of reduced latency to consume food in the novelty suppressed feeding test. In addition to benzodiazepine/GABAergic receptors, the neuropharmacological properties of ß-CBP may be related to inhibition of nitric oxide synthesis, since pre-treatment with L-arginine (500-750 mg/kg) reversed significantly the anxiolytic, antidepressant and anticonvulsant activities of ß-CBP. CONCLUSION: The results obtained provide additional support in understanding the neuromolecular mechanisms underlying the anxiolytic, antidepressant and anticonvulsive properties of ß-CBP in female Swiss mice.

Insight into Anopheles (Nyssorhynchus) (Diptera: Culicidae) species from Brazil.

Anopheles (Nyssorhynchus) benarrochi s.l., Anopheles (Nyssorhynchus) oswaldoi s.l., and Anopheles (Nyssorhynchus) konderi s.l. collected in Acrelandia, state of Acre, Brazil, were identified based on morphological characters of the male genitalia, fourth-instar larvae, and pupae. Morphological variation was observed in the male genitalia of these species in comparison with specimens from other localities in Brazil. DNA sequence from the nuclear ribosomal second internal transcribed spacer of individuals identified as An. benarrochi s.l. by using male genitalia characteristics showed that the various morphological forms are conspecific but are distinct from An. benarrochi B from Colombia. Anopheles konderi s.l. and An. oswaldoi s.l. both misidentified as An. oswaldoi s.s. (Peryassti) throughout Brazil, may actually comprise at least two undescribed species. Diagnostic morphological characteristics of the male genitalia are provided to distinguish Anopheles benarrochi s.l., Anopheles oswaldoi s.l., and Anopheles konderi s.l. from morphologically similar species. Incrimination of An. oswaldoi s.s. in malaria transmission in Brazil needs further investigation because other undescribed species from Acre may have been confounded with this taxon.

Cyclooxygenase-2 inhibition increases gastric tone and delays gastric emptying in rats.

We evaluated the effects of cyclooxygenase-2 (COX-2) selective inhibitors, COX-1 selective inhibitor, or COX non-selective inhibitor on gastric emptying and intestinal transit of liquids, and evaluated the effect of a COX-2 selective inhibitor on gastric tonus (GT). Male Wistar rats were treated per os with saline (control), rofecoxib, celecoxib, ketorolac, rofecoxib + ketorolac, celecoxib + ketorolac, or indomethacin. After 1 h, rats were gavage-fed (1.5 mL) with the test meal (5% glucose solution with 0.05 g mL(-1) phenol red) and killed 10, 20 or 30 min later. Gastric, proximal, medial or distal small intestine dye recovery (GDR and IDR, respectively) were measured by spectrophotometry. The animals of the other group were treated with i.v. valdecoxib or saline, and GT was continuously observed for 120 min using a pletismomether system. Compared with the control group, treatment with COX-2 inhibitors, alone or with ketocolac, as well as with indomethacin increased GDR (P < 0.05) at 10-, 20- or 30-min postprandial intervals. Ketorolac alone did not change the GDR, but increased the proximal IDR (P < 0.05) at 10 min, and decreased medial IDR (P < 0.05) at 10 and 20 min. Valdecoxib increased (P < 0.01) GT 60, 80 and 100 min after administration. In conclusion, COX-2 inhibition delayed the gastric emptying of liquids and increased GT in rats.

Photoperiod Differences in Sand Fly (Diptera: Psychodidae) Species Richness and Abundance in Caves in Minas Gerais State, Brazil.

Caves are unique habitats that are inhabited by a diverse and singular biota. Among these inhabitants are sand flies, which are of great epidemiological interest in the Neotropical region because they are vectors of Leishmania The period of activity of these insects is usually crepuscular and nocturnal, but there are reports of diurnal activity of sand flies in caves. Thus, the aim of this study was to evaluate the periodicity of daily activity of sand flies in cave environments in the municipality of Pains, Minas Gerais. Sand flies were collected with light traps, which were operated for 5 consecutive days in the rainy season and in the dry season. Samples were collected every 12 h and separated between photophase and scotophase periods. In total, 1,777 sand flies of 23 species were collected. The most abundant species was Lutzomyia renei (Martins, Falcão, and Silva) (44%), followed by Lutzomyia longipalpis (Lutz and Neiva) (15%), Evandromyia edwardsi (Mangabeira) (11%), and Micropygomyia quinquefer (Costa Lima) (6%). The richness and abundance of total sand flies and the abundance of male and female sand flies in the aphotic zone of the caves did not differ between the photophase and scotophase, but differed between photoperiods at the entrance and at sites surrounding the caves. From our study of the daily activity of these insects in this ecotope, it will be possible to know which period of the day is of greatest risk of exposure of vertebrates who visit or live in these environments, including the human population.

Pre-clinical toxicology of garcinielliptone FC, a tautomeric pair of polyprenylated benzophenone, isolated from Platonia insignis Mart seeds.

BACKGROUND: Garcinielliptone FC (GFC) is a tautomeric pair of polyprenylated benzophenone, which has proven to have antiepileptic, cytotoxic and antioxidant activity. PURPOSE: The aim of this study was to investigate the biochemical, hematological and pathological effects of the acute toxicity study as well as to assess the locomotor activity and motor coordination in mice treated with GFC. METHODS: Swiss mice of both sexes weighing 25-30 g divided into three separate groups of five animals matched by weight and size. GFC was aseptically suspended in 0.05% Tween 80, dissolved in 0.9% saline (vehicle) and administered orally (p.o.) and intraperitoneally (i.p.) (500, 1000 and 2000 mg/kg). The acute toxicity study was performed in compliance with the Anvisa regulations. RESULTS: Behavioral manifestations of toxicity, such as state of consciousness, coordination, muscle tone, reflexes, the activity on the central nervous system (shake, seizures, Straub tail reaction and anesthesia) and the activity of the autonomic nervous system (lacrimation, ptosis, urination, piloerection, hypothermia, breathing and hyperemia) were not seen in any of the animals treated with doses of 500, 1000 and 2000 mg/kg. Additionally, no significant difference in body weight, food and water intake, excreta production or macroscopic changes in the organs of treated animals were detected in comparison with control group. GFC did not affect the locomotor activity and motor coordination of the animals. CONCLUSION: The acute toxicity study indicated that GFC treatment, at selected doses given orally and intraperitoneally, showed relatively low risk of toxicity in all test animals, suggesting that it is safe for further investigation.

Exercise on a vibratory platform increases blood perfusion on the stifle joint and rectal temperature in healthy dogs / Exercício em plataforma vibratória aumenta a perfusão sanguínea da articulação do joelho e a temperatura retal de cães saudáveis

The effects of two vibration platform (VP) exercise protocols on stifle and rectal temperatures were evaluated. Eleven animals participated in two exercise protocols, different in duration in each exercise. Exercise protocol 1 (EP1) took 30 seconds and EP2, 60 seconds, with different vibratory levels in both cases (L1 = acceleration ≅ 1g, L4 = acceleration ≅ 2.5g, and L7 = acceleration ≅ 5g). The animals were evaluated before and 1 minute after the exercise, using infrared thermography to obtain stifle temperatures. The rectal temperature (RT) was also checked at each moment. The dogs had higher stifle temperatures in EP1 at all vibratory levels compared to the time before the exercise; EP2 resulted in higher temperature only at maximum vibration intensity (L7). Increase in TR was observed only in EP2. The results suggested that the short duration protocol (EP1) increased the muscular and peripheral vascular activities of the joint, regardless of the vibration intensity. The long duration protocol (EP2) with maximum vibration intensity increased the RT, demonstrating activity beyond the stifle muscle group. It is concluded that exercises on the VP can be used as complementary therapy for low-impact muscle activity in dogs and may be adequate for efficient energy consumption.(AU)

Foram avaliados os efeitos de dois protocolos de exercício em plataforma vibratória (PV) sobre as temperaturas dos joelhos e retais. Onze animais participaram de dois protocolos de exercício, diferentes na duração de cada exercício. O protocolo de exercício 1 (PE1) foi de 30 segundos e o PE2 foi de 60 segundos, com diferentes níveis vibratórios em ambos (L1 = aceleração ≅ 1g; L4 = aceleração ≅ 2,5g; e L7 = aceleração ≅ 5g). Os animais foram avaliados antes e um minuto após os exercícios, por meio de termografia infravermelha para a obtenção das temperaturas dos joelhos. A temperatura retal (TR) foi aferida em cada momento. Os cães apresentaram temperaturas dos joelhos mais elevadas em PE1, em todos os níveis vibratórios, em comparação a antes do exercício; o PE2 resultou em temperatura mais alta apenas na intensidade máxima de vibração (L7). Aumento na TR foi observado apenas no PE2. Os resultados sugerem que o protocolo de curta duração (PE1) aumentou as atividades muscular e vascular periférica da articulação, independentemente da intensidade da vibração; o protocolo de longa duração (PE2) com intensidade máxima de vibração aumentou a TR demonstrando atividade além do grupo muscular do joelho. Conclui-se que os exercícios na PV podem ser utilizados como terapia complementar para atividade muscular de baixo impacto em cães e pode ser adequada para o consumo eficiente de energia.(AU)

Metastatic thyroid carcinoma arising from congenital goiter due to mutation in the thyroperoxidase gene.

A very large cervical tumor that extended to the upper mediastinum was seen in a newborn after an uneventful pregnancy. The computed axial tomography scan confirmed the presence of a solid mass with precise limits and scattered foci of calcifications situated in the anterolateral region of the neck. The infant underwent thyroidectomy on the seventh day after birth. Pathological examination revealed a follicular carcinoma of the thyroid and probable dyshormonogenetic hyperplastic goiter. At 5 months of age, whole body scans indicated the presence of lung and bone metastases, which were treated with therapeutic doses of radioiodine. Genomic DNA was obtained from the newborn, her parents, her paternal aunt, and her paternal grandparents. Denaturing gradient gel electrophoresis analysis of PCR fragments corresponding to exon 14 of the thyroid peroxidase (TPO) gene indicated the presence of a mutant TPO allele present in the propositus, her father, and her paternal grandmother. Sequencing of the TPO gene demonstrated a mutation resulting from an insertion of a single extra cytosine in a stretch of seven cytosines at positions 2505-2511. The insertion caused a frame shift and a stop signal in exon 16. This sequence would translate into a structurally modified and probably inactive TPO protein. We conclude that the aggressive thyroid metastatic carcinoma arose from a dyshormonogenetic goiter caused by a defective TPO protein.

Phenocopies for deafness and goiter development in a large inbred Brazilian kindred with Pendred's syndrome associated with a novel mutation in the PDS gene.

Pendred's syndrome is an autosomal recessive disease characterized by goiter, impaired iodide organification, and congenital sensorineural deafness. The gene mutated in Pendred's syndrome, PDS (Pendred's syndrome gene), was cloned very recently and encodes the putative sulfate transporter pendrin. Pendred's syndrome may account for up to 10% of the cases with hereditary hearing loss, and pendrin mutations have also been found in a kindred with non-syndromic deafness. In this study, 41 individuals from a large, highly inbred pedigree from Northeastern Brazil were examined for features of Pendred's syndrome. Linkage studies and sequence analysis of the coding region of the PDS gene were performed with DNA from 36 individuals. The index patient, with the classical triad of deafness, positive perchlorate test, and goiter, was found to be homozygous for a deletion of thymidine 279 in exon 3, resulting in a frameshift and a premature stop codon at amino acid 96. This alteration resulted in truncation of the protein in the first transmembrane domain. Two other patients with deafness were found to be homozygous for this mutation; 19 were heterozygous and 14 were homozygous for the wild type allele. Surprisingly, 6 deaf individuals in this kindred were not homozygous for the PDS gene mutation; 3 were heterozygous and 3 were homozygous for the wild type allele, suggesting a probable distinct genetic cause for their deafness. All 3 homozygous individuals for the PDS mutation had goiters. However, goiters were also found in 10 heterozygous individuals and in 6 individuals without the PDS mutation and are most likely caused by iodine deficiency. In conclusion, we identified a novel mutation in the PDS gene causing Pendred's syndrome. The comparison of phenotype and genotype reveals, however, that phenocopies generated by distinct environmental and/or genetic causes are present in this kindred and that the diagnosis of Pendred's syndrome may be difficult without molecular analysis.

Sequential cytotoxicity: a theory evaluated using novel 2-[4-(3-aryl-2-propenoyloxy)phenylmethylene]cyclohexanones and related compounds.

Five series of novel compounds were synthesized in order to evaluate the theory of sequential cytotoxicity which seeks to exploit the view that various cancer cells are particularly susceptible to successive attacks by cytotoxic agents. The compounds prepared were various 2-[4-(3-aryl-2-propenoyloxy)phenylmethylene]cyclohexanone s 1 and the related Mannich bases 2. In addition the analogues 3-5 lacking an olefinic bond in the ester group were also synthesized, which were predicted to be less cytotoxic than the compounds of series 1 and 2. The atomic charges at the potential sites for interaction with cellular constituents were determined by molecular modeling calculations. The biodata obtained from murine and human neoplastic cells revealed that the predictions made regarding the viability of the theory were fulfilled in approximately two-thirds of the cases indicating that further investigation of this hypothesis is warranted. In addition, the significant potencies of some of the Mannich bases toward human tumor cell lines, in particular coupled to their selective toxicity toward human leukemic and colon cancer cells, confirms their usefulness in serving as lead molecules for further development. A preliminary investigation into the mode of action of representative compounds revealed their ability to induce apoptosis and inhibit the biosyntheses of ribonucleic acid and proteins.

A conformational and structure-activity relationship study of cytotoxic 3,5-bis(arylidene)-4-piperidones and related N-acryloyl analogues.

A series of 3,5-bis(arylidene)-4-piperidones 1 and related N-acryloyl analogues 2 were prepared as candidate cytotoxic agents with a view to discerning those structural features which contributed to bioactivity. A number of the compounds were markedly cytotoxic toward murine P388 and L1210 leukemic cells and also to human Molt 4/C8 and CEM neoplasms. Approximately 40% of the IC50 values generated were lower than the figures obtained for melphalan. In virtually all cases, the N-acyl compounds were significantly more bioactive than the analogues 1. In general, structure-activity relationships revealed that the cytotoxicity of series 1 was correlated positively with the size of the aryl substituents, while in series 2, a -sigma relationship was established. In particular, various angles and interatomic distances were obtained by molecular modeling, and the presence of an acryloyl group on the piperidyl nitrogen atom in series 2 affected the relative locations of the two aryl rings. This observation, along with some differences in distances between various atoms in series 1 and 2, may have contributed to the disparity in cytotoxicity between 1 and 2. The results obtained by X-ray crystallography of representative compounds were mainly in accordance with the observations noted by molecular modeling. Selected compounds interfered with the biosynthesis of DNA, RNA, and protein in murine L1210 cells, while others were shown to cause apoptosis in the human Jurkat leukemic cell line. This study has revealed the potential of these molecules for development as cytotoxic and anticancer agents.

Conformational and quantitative structure-activity relationship study of cytotoxic 2-arylidenebenzocycloalkanones.

Various 2-arylideneindanones 1, 2-arylidenetetralones 2, and 2-arylidenebenzosuberones 3 were synthesized with the aim of determining the relative orientations of the two aryl rings which favored cytotoxicity. Molecular modeling of the unsubstituted compound in each series revealed differences in the spatial arrangements of the two aryl rings, and evaluation of these compounds against P388, L1210, Molt 4/C8, and CEM cells as well as a panel of human tumor cell lines indicated that in general the order of cytotoxicity was 3 > 2 > 1. In particular 2-(4-methoxyphenylmethylene)-1-benzosuberone (3k) had the greatest cytotoxicity, possessing 11 times the potency of the reference drug melphalan when all five screens were considered. Series 3 was considered in further detail. First, excision of the aryl ring fused to the cycloheptanone moiety in series 3 led to some 2-arylidene-1-cycloheptanones 4 which had approximately one-third of the bioactivity of the analogues 3. Second, in some screens cytotoxicity was correlated negatively with the sigma values and positively with the MR constants of the substituents in the arylidene aryl ring of 3. Third, X-ray crystallography of five representative compounds (3i,k-n) revealed differences in the locations of the aryl rings which may have contributed to the variations in cytotoxicity. Finally three members of series 3 inhibited RNA and protein syntheses and induced apoptosis in human Jurkat T cells. This study has revealed that 2-arylidene-1-benzosuberones are a group of useful cytotoxic agents, and in particular 3k serves as a prototypic molecule for subsequent structural modifications.

Overexpression of epidermal growth factor and epidermal growth factor-receptor mRNAs in dyshormonogenetic goiters.

Thyroid malignancy has been induced by long-term endogenous thyrotropin (TSH) stimulation in experimental animals, leading to local and distant metastasis. It has been postulated that constant and prolonged endogenous TSH stimulation in dyshormonogenetic thyroid tissues could result in thyroid neoplasia. The possible role of growth factors and oncogenes in goitrogenesis and favoring neoplasia has also been mentioned. Overexpression of certain growth factors and/or their receptors, and of oncogenes implicated in growth promotion may play a significant role in the relatively frequent finding of thyroid malignancy in congenital goiters. In this study the expression of epidermal growth factor (EGF), epidermal growth factor receptor (EGF-R), transforming growth factor-beta (TGF-beta), c-myc, and p53 mRNAs was determined in 14 thyroid tissue samples: 6 from patients with thyroid peroxidase (TPO) gene mutations, 4 with thyroglobulin (Tg) gene defects and 4 normal thyroid tissues. EGF mRNA overexpression was seen in 7 of 10 dyshormonogenetic tissues (3.5 to 12.0 arbitrary optical densitometry units [AODU]) and considered significantly higher (p < 0.01) when compared to normal thyroid tissues (0.25 to 0.32 AODU). Moreover, overexpression of EGF-R mRNA was present in 6 of 10 dyshormonogenetic tissues (2.23 to 13.03 AODU) and considered significantly higher (p < 0.01) when compared to normal thyroid tissues (0.42 to 0.65 AODU). There was no difference in c-myc, p53, and TGF-beta mRNAs expression between dyshormonogenetic and normal tissues. The overexpression of EGF and EGF-R mRNAs found in dyshormonogenetic tissues may suggest that this growth factor may play a role in cellular proliferation and contribute to goiter formation.

Thyrotropin receptor mutations in hyperfunctioning thyroid adenomas from Brazil.

Constitutively activating mutations in the thyrotropin (TSH) receptor have been identified as a major molecular cause of hyperfunctioning thyroid adenomas. A smaller subset of these benign tumors is caused by constitutive activation of the adenylyl cyclase cascade by somatic mutations in the Gsalpha gene. In this study, we analyzed hyperfunctioning thyroid adenomas from seven Brazilian patients for TSH receptor and G(s)alpha gene mutations. Solitary autonomous thyroid adenomas were identified by ultrasound and scintigraphy, and DNA was extracted from adenomatous and periadenomatous tissue. Exons 9 and 10 of the TSH receptor gene, and exons 8 and 9 of the G(s)alpha gene, were amplified by polymerase chain reaction (PCR) and subjected to direct sequence analysis. Six of seven adenomas harbored heterozygous mutations known to confer constitutive activity to the TSH receptor. In one case, aspartate 619 was substituted by glycine (D619G). In four adenomas, alanine 623 was replaced by valine (A623V). Both residues are located in the third intracellular loop. In one instance, aspartate 633 located in the sixth transmembrane domain was replaced by tyrosine (D633Y). In this patient, one allele also contained a change of aspartate 727 to glutamate (D727E). This substitution is thought to be a polymorphic variant of the wild-type but it has also been associated with toxic multinodular goiters. Functional comparison of D727 with E727 did not reveal differences in basal or TSH-stimulated cyclic adenosine monophosphate (cAMP)-dependent luciferase activity in transiently transfected cells. These results demonstrate a high prevalence of activating TSH receptor mutations in toxic adenomas in this small series from Brazil (approximately 86%). These findings are in agreement with reports from other countries with a marginal iodine intake but contrast with studies from regions with a high iodine intake where these mutations appear to be less prevalent.

Congenital aneurysm of the right atrium.

Congenital aneurysm of the right atrium is described in a 1-year-old girl who presented with cardiomegaly and symptoms of paroxysmal supraventricular tachycardia. Echocardiography and cineangiography both established the definitive diagnosis and surgical resection was successful. The rarity of this condition is pointed out and its main features outlined.

A direct method for cDNA cloning employing translation-released mRNA.

1. A new cloning procedure is described for cDNA synthesis from mRNA released by in vitro translation of polysomes in a cell-free amino acid incorporating system. The usefulness of the method lies in the feasibility of employing nanogram amounts of mRNA. 2. Complementary DNA is synthesized directly in the translation mixture simply by adjusting the concentration of some components and removing ribosomes by boiling and centrifugation. 3. As an example, we report here the construction and characterization of a cDNA clone corresponding to chick alpha(I)procollagen starting from a collagen-synthesizing polysome fraction obtained from chick embryos.