Structure-activity relationship, molecular docking, and molecular dynamic studies of diterpenes from marine natural products with anti-HIV activity.

HIV-1 infection is a global epidemic whose treatment is limited majorly by viral resistance and adverse effects. Natural products from algae have been studied for many years, including antiviral, being an alternative to anti-HIV drug design. Since the isolation of natural products can be a hurdle, molecular modeling is an important tool to study these compounds. Herein, structure-activity relationship, molecular docking, and molecular dynamic studies were performed to direct the studies of ten marine natural products with anti-HIV activity. In the structure-activity relationship, descriptors were identified associating the anti-HIV activity of five diterpenes with possible action on the reverse transcriptase allosteric site. These diterpenes were evaluated by molecular docking, and it was identified that only dolabelladienetriol interacted in the allosteric site. Molecular dynamics suggested that the dolabelladienetriol might interfere with the viral RNA binding to HIV-1 RT by inducing a conformational change of the enzyme. Also, in silico ADMET simulations predicts that the dolabelladienetriol present a high potential to be successfully developed as a drug. Thus, applying in silico approaches was possible to suggest potential anti-HIV compounds derived from marine natural products.Communicated by Ramaswamy H. Sarma.

Antiviral activity of diterpenes isolated from the Brazilian marine alga Dictyota menstrualis against human immunodeficiency virus type 1 (HIV-1).

The antiviral effect of the CH(2)Cl(2)/MeOH-soluble fraction from the alga Dictyota menstrualis on HIV-1 replication was evaluated in vitro. The antiretroviral activity was attributed to two diterpenes: (6R)-6-hydroxydichotoma-3,14-diene-1,17-dial, named Da-1, and (6R)-6-acetoxi-dichotoma-3,14-diene-1,17-dial, named AcDa-1. Da-1 or AcDa-1 were added to the culture medium of HIV-1-infected PM-1 cells at different times post-infection or during virus adsorption/penetration. The results indicated that the compounds affected an early step of the virus replicative cycle. Virus binding and entry into the host cells were evaluated in the presence of each diterpene, but no inhibitory effect was observed. To evaluate provirus DNA synthesis/integration into the host genome, the viral protease coding sequence was amplified from total cellular DNA. Proviral DNA was not detected in infected cells incubated with the diterpenes. To investigate the effect of the diterpenes on the reverse transcription of the viral genomic RNA, the recombinant HIV-1 reverse transcriptase (RT) was assayed in vitro in the presence of each diterpene. Da-1 and AcDa-1 inhibited the RNA-dependent DNA-polymerase activity of HIV-1 RT in a dose-dependent manner. Taken together, our results demonstrate that both diterpenes inhibit HIV-1 RT and consequently virus replication.

Ecological roles of natural products of the Brazilian red seaweed Laurencia obtusa

Laboratory and field experiments were performed to assess the ecological roles of natural products produced by the Brazilian red seaweed Laurencia obtusa. Laboratory assays revealed that the natural concentration of the crude organic extract of L. obtusa significantly inhibited feeding by two herbivores: the crab Pachygrapsus transversus and the sea urchin Lytechinus variegatus. It was verified that this chemically defensive action was due to halogenated sesquiterpenoid elatol, found to be the major natural product of this red seaweed. In addition, it was verified that the antifouling property of the chemicals produced by L. obtusa could make this red alga less attractive for fish grazing. Direct protection against two herbivore species and indirect protection against herbivory by fouling inibition constitute evidence that the major natural product from Brazilian L. obtusa plays multiple environmental roles, thereby increasing the adaptive value of these metabolites. On the other hand, the evidence reinforces the idea that marine natural products may have different functions in the sea.

Ecological roles of natural products of the Brazilian red seaweed Laurencia obtusa.

Laboratory and field experiments were performed to assess the ecological roles of natural products produced by the Brazilian red seaweed Laurencia obtusa. Laboratory assays revealed that the natural concentration of the crude organic extract of L. obtusa significantly inhibited feeding by two herbivores: the crab Pachygrapsus transversus and the sea urchin Lytechinus variegatus. It was verified that this chemically defensive action was due to halogenated sesquiterpenoid elatol, found to be the major natural product of this red seaweed. In addition, it was verified that the antifouling property of the chemicals produced by L. obtusa could make this red alga less attractive for fish grazing. Direct protection against two herbivore species and indirect protection against herbivory by fouling inibition constitute evidence that the major natural product from Brazilian L. obtusa plays multiple environmental roles, thereby increasing the adaptive value of these metabolites. On the other hand, the evidence reinforces the idea that marine natural products may have different functions in the sea.

Feeding preferences of the endemic gastropod Astraea latispina in relation to chemical defenses of Brazilian tropical seaweeds.

Seaweed preference by the Brazilian endemic gastropod Astraea latispina was examined in the laboratory to evaluate the role of secondary metabolites in determining food choice. Of three species of seaweeds examined, Plocamium brasiliense was highly preferred; less so were Sargassum furcatum and Dictyota cervicornis were preferred less. Extracts and/or pure major metabolites of the two potentially chemically-defended seaweeds (P. brasiliense and D. cervicornis) were tested as feeding deterrents against A. latispina. Algal extract assays demonstrated that three concentrations of crude organic extract of the red alga P. brasiliense (50%, 100%: natural concentration, and 200% of dry weight: dw) did not affect feeding of this gastropod. In contrast, the three concentrations of crude organic extract of the brown alga D. cervicornis (50%, 100% and 200% dw) inhibited feeding by A. latispina. The chemical deterrent property of D. cervicornis extract against the gastropod A. latispina occurred due to a mixture of the secodolastane diterpenes isolinearol/linearol (4:1--0.08% dry weight). This is the first report showing that Dictyota cervicornis produces a chemical defense against herbivores using secodolastane diterpenoid. In addition, these results widen the action spectrum of secondary metabolites found in seaweed belonging to this brown algal genus.

Feeding preferences of the endemic gastropod Astraea latispina in relation to chemical defenses of Brazilian tropical seaweeds

Seaweed preference by the Brazilian endemic gastropod Astraea latispina was examined in the laboratory to evaluate the role of secondary metabolites in determining food choice. Of three species of seaweeds examined, Plocamium brasiliense was highly preferred; less so were Sargassum furcatum and Dictyota cervicornis were preferred less. Extracts and/or pure major metabolites of the two potentially chemically-defended seaweeds (P. brasiliense and D. cervicornis) were tested as feeding deterrents against A. latispina. Algal extract assays demonstrated that three concentrations of crude organic extract of the red alga P. brasiliense (50 percent, 100 percent: natural concentration, and 200 percent of dry weight: dw) did not affect feeding of this gastropod. In contrast, the three concentrations of crude organic extract of the brown alga D. cervicornis (50 percent, 100 percent and 200 percent dw) inhibited feeding by A. latispina. The chemical deterrent property of D. cervicornis extract against the gastropod A. latispina occurred due to a mixture of the secodolastane diterpenes isolinearol/linearol (4:1 -- 0.08 percent dry weight). This is the first report showing that Dictyota cervicornis produces a chemical defense against herbivores using secodolastane diterpenoid. In addition, these results widen the action spectrum of secondary metabolites found in seaweed belonging to this brown algal genus

Altered dopaminergic transmission in the anorexic anx/anx mouse striatum.

We demonstrate abnormal dopaminergic neurotransmission in anorexic mice, homozygous for a recessive mutation (anx) causing starvation and motor disturbances. Isolated neurons from anx/anx striatum displayed a markedly increased activity of the Na+,K+-ATPase compared with normal littermates. Dopamine down-regulates Na+,K+-ATPase activity in striatal medium spiny neurons in rat, mouse and guinea pig. However, addition of dopamine in vitro failed to suppress the increased activity in anx/anx striatal neurons. Striatal dopamine and its metabolites, but not norepinephrine, were slightly but significantly lower in anx/anx mice than in normal littermates. We suggest that abnormal dopaminergic transmission may contribute to the anx phenotype.

Chemotaxis and chemical defenses in seaweed susceptibility to herbivory

Recent studies have show that small marine herbivores with limited mobility (mesograzers) often feed on macroalgae chemically defended against fishes or sea-urchins. In order to verify the involved mechanisms of chemotaxis or chemical defense into this process in Brazilian littoral, two species of brown alga Dictyota menstrualis and Dictyota mertensii were studied against the limited mobility herbivores, the amphipod Parhyale hawaiensis and the crab Pachygrapsus transversus. These two species were studied in order to verify the action of their crude extracts in the defense and chemotaxis processes related to limited mobility of these herbivores. Feeding preference assays revealed that P. hawaiensis do not eaten these Dictyota species. P. transversus do not eaten D. menstrualis either, but consumed large amounts of D. mertensii. Chemical deterrence assays showed that extracts of these species act as feeding deterrent to both species of herbivores. In addition, chemotaxis assays demonstrated that both herbivores are significantly negative chemotactic probably due to the presence of complementary metabolites into artificial foods. Considering that both Dictyota species exhibit active extracts against these small herbivores, we suppose that the non-occurrence of these herbivore species in close relationship with the seaweeds D. menstrualis and D. mertensii may explain the defense action of both extracts related to these mesograzers

Chemotaxis and chemical defenses in seaweed susceptibility to herbivory.

Recent studies have show that small marine herbivores with limited mobility (mesograzers) often feed on macroalgae chemically defended against fishes or sea-urchins. In order to verify the involved mechanisms of chemotaxis or chemical defense into this process in Brazilian littoral, two species of brown alga Dictyota menstrualis and Dictyota mertensii were studied against the limited mobility herbivores, the amphipod Parhyale hawaiensis and the crab Pachygrapsus transversus. These two species were studied in order to verify the action of their crude extracts in the defense and chemotaxis processes related to limited mobility of these herbivores. Feeding preference assays revealed that P. hawaiensis do not eaten these Dictyota species. P. transversus do not eaten D. menstrualis either, but consumed large amounts of D. mertensii. Chemical deterrent assays showed that extracts of these species act as feeding deterrent to both species of herbivores. In addition, chemotaxis assays demonstrated that both herbivores are significantly negative chemotactic probably due to the presence of complementary metabolites into artificial foods. Considering that both Dictyota species exhibit active extracts against these small herbivores, we suppose that the non-occurrence of these herbivore species in close relationship with the seaweeds D. menstrualis and D. mertensii may explain the defense action of both extracts related to these mesograzers.

Caco-2 cells in culture synthesize and degrade dopamine and 5-hydroxytryptamine: a comparison with rat jejunal epithelial cells.

To explore the usefulness of Caco-2 cells in the study of intestinal dopaminergic and 5-hydroxytryptaminergic physiology, we have undertaken the study of aromatic L-amino acid decarboxylase (AADC), catechol-O-methyltransferase (COMT) and type A and B monoamine oxidase (MAO-A and MAO-B) activities in these cells using specific substrates. The activity of these enzymes was also evaluated in isolated rat jejunal epithelial cells. The results showed that Vmax values (in nmol mg protein(-1) h(-1)) for AADC, using L-DOPA as the substrate, in rat jejunal epithelial cells (127.3+/-11.4) were found to be 6-fold higher than in Caco-2 cells (22.5+/-2.6). However, Km values in Caco-2 cells (1.24+/-0.37 mM) were similar to those observed in rat jejunal epithelial cells (1.30+/-0.29 mM). Similar results were obtained when AADC activity was evaluated using L-5HTP as substrate; in rat jejunal epithelial cells Vmax values (in nmol mg prot(-1) h(-1)) were found to be 5-fold that in Caco-2 cells (16.3+/-1.0 and 3.0+/-0.2, respectively), and Km values in Caco-2 cells (0.23+/-0.08 mM) were again similar to those observed in rat intestinal epithelial cells (0.09+/-0.03 mM). Caco-2 cells were not able to O-methylate dopamine, in contrast to rat jejunal epithelial cells (Vmax = 8.6+/-0.4 nmol mg protein(-1)(h-1); Km = 516+/-57 microM). Vmax values (in nmol mg protein(-1)(h-1)) for type A and B MAO in Caco-2 cells (19.0+/-0.6 and 5.4+/-0.6, respectively) were found to be significantly lower (P<0.05) than those in rat jejunal epithelial cells (46.9+/-3.1 and 9.6+/-1.2, respectively); however, no significant differences in the Km values were observed between Caco-2 and rat jejunal epithelial cells for both type A and B MAO. In conclusion, Caco-2 cells in culture are endowed with the synthetic and metabolic machinery needed to form and degrade DA and 5-HT, though, no COMT activity could be detected in these cells.

Diterpenes of marine brown algae of the family Dictyotaceae: their possible role as defense compounds and their use in chemotaxonomy.

Marine brown algae of the family Dictyotaceae are protected against predation. Their survival strategy is, at least in part, based on the production of chemical defenses. These compounds are diterpenes that seem to establish a specific predator-prey relationship, and which are found as food-chain markers. The use of Dictyotaceae diterpenes in chemotaxonomy is briefly discussed.

Graves' disease and Hashimoto's thyroiditis: effects of high doses of antithyroid drugs on thyroid autoantibody levels.

We studied the effects of high doses of methimazole (MMI) or propylthiouracil (PTU) on thyroid-stimulating antibody (TSAb), antithyroid microsomal (MCHA) and antithyroglobulin (TGHA) levels in Graves' disease and Hashimoto's thyroiditis. Thirty Graves' hyperthyroid patients were treated for 14 +/- 8 months (mean +/- SD) with MMI, 60-80 mg daily or PTU, 900-1200 mg daily plus T3, 50-75 micrograms daily. Fifteen Hashimoto's thyroiditis patients (4 of whom hypothyroid) received 100-200 micrograms of T4 daily for 4-8 weeks prior to MMI, 60-90 mg daily or PTU, 900 mg daily for 12-16 weeks. In Graves' disease a decrease (p less than 0.001) in TSAb activity (20/25 patients) was observed: before therapy, 0.424 +/- 0.506 pmoles/mg wet wt and at the end of treatment, 0.189 +/- 0.23 pmoles/mg wet wt. The MCHA titers also fell (18/26 patients) from 1:10,403 +/- 20,197 to 1:3,476 +/- 5,252 (p less than 0.01) and was associated with a decrease in free T4 values (1.23 +/- 0.69 vs. 0.51 +/- 0.36 ng/dl; p less than 0.01). A fall of MCHA titers in T4-treated Hashimoto's thyroiditis patients (1:10,416 +/- 25,576) was found when compared with the value before T4 (1:25,920 +/- 39,973; p less than 0.001). However, the titers of MCHA (1:13,280 +/- 25,992) did not change on MMI or PTU plus T4 treatment. The TGHA titers fell in a single patient. No alterations were observed in serum immunoglobulins. Serum concentrations of the complement factor C'3 remained higher (p less than 0.01) than normal values in both Graves' disease and Hashimoto's thyroiditis.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyroid function during the spontaneous course of subacute thyroiditis.

A study of changes in serum T4, T3, and Tg as well as of serum TSH response to TRH was done in ten patients with subacute thyroiditis, from the acute phase up to 56 mo. All patients had symptoms of thyrotoxicosis. The mean +/- s.e.m. serum T4 (21.6 +/- 8.2 micrograms/dl), T3 (315 +/- 191 ng/dl) and Tg (149 +/- 52 ng/ml) concentrations were significantly higher than in normal subjects (8.5 +/- 1.7 micrograms/dl, 136 +/- 34 ng/dl, and 10.5 +/- 1.0 ng/ml, respectively). The basal TSH concentrations failed to increase in response to TRH. Mean serum T3 and serum Tg levels remained higher than in normal subjects until 4 to 5 mo after the acute phase. However, normalization of clinical status and serum thyroid hormone levels did not coincide with the normalization of serum Tg levels. Thyroid autoantibodies were absent during the whole period of study. An exaggerated response of TSH to TRH in six out of seven patients was observed from a 2 to 3 mo period until the end of follow-up. All patients with T3 to T4 ratio above the normal range (7-24 ng/micrograms) showed also an exaggerated response of TSH to TRH. These data suggest that the spontaneous course of subacute thyroiditis may lead to a low thyroid reserve detectable even 5 yr following the acute phase of the disease.

Evidence for centers in the central nervous system that selectively regulate fat mobilization in the rat.

The blood sugar and plasma free fatty acid responses to administration of 2-deoxyglucose were determined in normal rats and in rats subjected to adrenodemedullation and/or hypothalamic deafferentation, as well as in rats with bilateral hypothalamic lesions. Adrenodemedullation of both intact and deafferentated rats reduced the 2-deoxyglucose-induced increase of blood sugar but did not affect the plasma free fatty acid response to 2-deoxyglucose in normal rats. The increases in blood sugar levels induced by the drug in intact rats were not significantly affected by deafferentation, but, in marked contrast, plasma free fatty acid mobilization after 2-deoxyglucose administration was completely suppressed in deafferentated rats, both in the presence and in the absence of the adrenal medulla. These results confirm previous observations indicating that the sympathetic nervous system and adrenalin release from the adrenal medulla participate in the production of hyperglycemia by 2-deoxyglucose. They provide, in addition, evidence for the existence, in the anterior hypothalamus or in limbic structures, of centers that can specifically influence mobilization of free fatty acids through a direct activation of the sympathetic fibers of adipose tissue without intervening in glucose homeostasis. The experiments in animals with bilateral hypothalamic lesions, although small in number, seem to support the above conclusions.