Leupeptazin, a highly modified tripeptide isolated from cultures of a Streptomyces sp. inhibits cathepsin K.

Using a human cathepsin K-targeting inhibitor screen, a new leupeptin analogue, leupeptazin (1), containing an unprecedented piperidinotriazine moiety, was isolated from a liquid culture of soil Streptomyces sp. IS2-4 collected in northern Italy. The structure of leupeptazin was established using HRESIMS as well as 1D and 2D NMR data. The inhibitory activity of the compound towards the collagenase cathepsin K was tested in vitro to reveal moderate activity with an inhibition constant, Ki, of 44µM.

Affinity Crystallography: A New Approach to Extracting High-Affinity Enzyme Inhibitors from Natural Extracts.

Natural products are an important source of novel drug scaffolds. The highly variable and unpredictable timelines associated with isolating novel compounds and elucidating their structures have led to the demise of exploring natural product extract libraries in drug discovery programs. Here we introduce affinity crystallography as a new methodology that significantly shortens the time of the hit to active structure cycle in bioactive natural product discovery research. This affinity crystallography approach is illustrated by using semipure fractions of an actinomycetes culture extract to isolate and identify a cathepsin K inhibitor and to compare the outcome with the traditional assay-guided purification/structural analysis approach. The traditional approach resulted in the identification of the known inhibitor antipain (1) and its new but lower potency dehydration product 2, while the affinity crystallography approach led to the identification of a new high-affinity inhibitor named lichostatinal (3). The structure and potency of lichostatinal (3) was verified by total synthesis and kinetic characterization. To the best of our knowledge, this is the first example of isolating and characterizing a potent enzyme inhibitor from a partially purified crude natural product extract using a protein crystallographic approach.

Herbicidin congeners, undecose nucleosides from an organic extract of Streptomyces sp. L-9-10.

Four new undecose nucleosides (herbicidin congeners), three known herbicidins, and 9-(ß-d-arabinofuranosyl)hypoxanthine (Ara-H) were isolated from the organic extract of a fermentation culture of Streptomyces sp. L-9-10 using proton NMR-guided fractionation. Their structures were elucidated on the basis of comprehensive 1D and 2D NMR and mass spectrometry analyses. These structures included 2'-O-demethylherbicidin F (1), 9'-deoxy-8',8'-dihydroxyherbicidin B (2), 9'-deoxy-8'-oxoherbicidin B (2a), and the 8'-epimer of herbicidin B (3). This is the first detailed assignment of proton and carbon chemical shifts for herbicidins A, B, and F. The isolated compounds were evaluated for cancer chemopreventive potential based on inhibition of tumor necrosis factor alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) activity.

Synthetic Analogs of the Sponge Sesterterpenoid Alotaketal C are Potent Inhibitors of SARS-CoV-2 Omicron BA.1 and BA.5 Infections of Human Lung Cells.

The protein kinase C-activating sponge natural product alotaketal C (1) potently inhibits the infection of human Calu-3 lung cells by SARS-CoV-2 Omicron BA.1 and BA.5 variants. Simplified analogs of 1 have been synthesized and tested for anti-SARS-CoV-2 activity providing SAR data for the antiviral pharmacophore of 1. Analogs 19 and 23, which are missing the C-11 substituents in 1 and have modified C-13 appendages, are ∼2- to 7-fold more potent than 1 and have equal or larger selectivity indices.

A novel class of broad-spectrum active-site-directed 3C-like protease inhibitors with nanomolar antiviral activity against highly immune-evasive SARS-CoV-2 Omicron subvariants.

Antivirals with broad coronavirus activity are important for treating high-risk individuals exposed to the constantly evolving SARS-CoV-2 variants of concern (VOCs) as well as emerging drug-resistant variants. We developed and characterized a novel class of active-site-directed 3-chymotrypsin-like protease (3CLpro) inhibitors (C2-C5a). Our lead direct-acting antiviral (DAA), C5a, is a non-covalent, non-peptide with a dissociation constant of 170 nM against recombinant SARS-CoV-2 3CLpro. The compounds C2-C5a exhibit broad-spectrum activity against Omicron subvariants (BA.5, BQ.1.1, and XBB.1.5) and seasonal human coronavirus-229E infection in human cells. Notably, C5a has median effective concentrations of 30-50 nM against BQ.1.1 and XBB.1.5 in two different human cell lines. X-ray crystallography has confirmed the unique binding modes of C2-C5a to the 3CLpro, which can limit virus cross-resistance to emerging Paxlovid-resistant variants. We tested the effect of C5a with two of our newly discovered host-directed antivirals (HDAs): N-0385, a TMPRSS2 inhibitor, and bafilomycin D (BafD), a human vacuolar H+-ATPase [V-ATPase] inhibitor. We demonstrated a synergistic action of C5a in combination with N-0385 and BafD against Omicron BA.5 infection in human Calu-3 lung cells. Our findings underscore that a SARS-CoV-2 multi-targeted treatment for circulating Omicron subvariants based on DAAs (C5a) and HDAs (N-0385 or BafD) can lead to therapeutic benefits by enhancing treatment efficacy. Furthermore, the high-resolution structures of SARS-CoV-2 3CLpro in complex with C2-C5a will facilitate future rational optimization of our novel broad-spectrum active-site-directed 3C-like protease inhibitors.

Discovery of lead natural products for developing pan-SARS-CoV-2 therapeutics.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global public health crisis. The reduced efficacy of therapeutic monoclonal antibodies against emerging SARS-CoV-2 variants of concern (VOCs), such as omicron BA.5 subvariants, has underlined the need to explore a novel spectrum of antivirals that are effective against existing and evolving SARS-CoV-2 VOCs. To address the need for novel therapeutic options, we applied cell-based high-content screening to a library of natural products (NPs) obtained from plants, fungi, bacteria, and marine sponges, which represent a considerable diversity of chemical scaffolds. The antiviral effect of 373 NPs was evaluated using the mNeonGreen (mNG) reporter SARS-CoV-2 virus in a lung epithelial cell line (Calu-3). The screening identified 26 NPs with half-maximal effective concentrations (EC50) below 50 µM against mNG-SARS-CoV-2; 16 of these had EC50 values below 10 µM and three NPs (holyrine A, alotaketal C, and bafilomycin D) had EC50 values in the nanomolar range. We demonstrated the pan-SARS-CoV-2 activity of these three lead antivirals against SARS-CoV-2 highly transmissible Omicron subvariants (BA.5, BA.2 and BA.1) and highly pathogenic Delta VOCs in human Calu-3 lung cells. Notably, holyrine A, alotaketal C, and bafilomycin D, are potent nanomolar inhibitors of SARS-CoV-2 Omicron subvariants BA.5 and BA.2. The pan-SARS-CoV-2 activity of alotaketal C [protein kinase C (PKC) activator] and bafilomycin D (V-ATPase inhibitor) suggest that these two NPs are acting as host-directed antivirals (HDAs). Future research should explore whether PKC regulation impacts human susceptibility to and the severity of SARS-CoV-2 infection, and it should confirm the important role of human V-ATPase in the VOC lifecycle. Interestingly, we observed a synergistic action of bafilomycin D and N-0385 (a highly potent inhibitor of human TMPRSS2 protease) against Omicron subvariant BA.2 in human Calu-3 lung cells, which suggests that these two highly potent HDAs are targeting two different mechanisms of SARS-CoV-2 entry. Overall, our study provides insight into the potential of NPs with highly diverse chemical structures as valuable inspirational starting points for developing pan-SARS-CoV-2 therapeutics and for unravelling potential host factors and pathways regulating SARS-CoV-2 VOC infection including emerging omicron BA.5 subvariants.

The long-term regulation of food intake and body weight depends on the availability of thyroid hormones in the brain.

OBJECTIVES: We evaluated the contribution of the thyroid hormones to the long-term maintenance of feeding behavior and body weight, while distinguishing their direct central effects from those resulting from the metabolic rate in the peripheral tissues. METHODS: We assessed the effect of hypothyroidism on the long-term (6 months) regulation of food intake, body weight, and energy expenditure in rats. We then generated the recovery of a euthyroid condition in the brain while maintaining a low T3 availability for the peripheral organs, i.e. a combined condition of central euthyroidism with peripheral hypothyroidism, with the aid of a pharmacological combination. RESULTS: Hypothyroidism caused a decrease in the daily food intake, body weight, and body temperature. The food intake and body temperature stabilized at a lower value, whereas body weight kept decreasing at a constant rate. The administration of exogenous T4 increased food intake and body-weight gain, but had no effect on body temperature. CONCLUSIONS: The thyroid hormones are necessary for the long-term regulation of energy intake, storage, and expenditure by different mechanisms. The feeding behavior seems to be partially dependent on a direct action of the thyroid hormones on the brain and this effect is independent of the energy expenditure in the peripheral organs. The body weight is closely dependent on the thyroid status and its maintenance seems to involve thyroid action on mechanisms other than feeding and metabolic rate.

Preparation and characterization of cellulose fibers from Meghatyrsus maximus: Applications in its chemical derivatives.

Non-wood lignocellulosic fibers have emerged and are becoming increasingly important as an alternative source of cellulose for derivatives, functional materials, and biofuels. This work aimed, to obtain cellulose from Meghatyrsus maximus grass with adequate properties through an alkaline delignification and alkaline hydrogen peroxide bleaching. Meghatyrsus maximus was chemically characterized as lignocellulosic biomass, which consisted of 45.0 %, cellulose, 35.0 % hemicellulose, and 20.0 % lignin. The obtained cellulose was characterized by Fourier transform infrared spectroscopy, X-ray diffraction analysis, thermogravimetric analysis, and scanning electron microscopy. The alpha-cellulose content was 98.50 % with a crystallinity of 61.0 %. The morphological study by scanning electron microscopy images indicates a clean surface and removal of non-cellulosic components present in the initial raw fibers. These results showed that high-quality cellulose was obtained and is comparable to a commercial alpha-cellulose, highlighting Meghatyrsus maximus as an alternative source of lignocellulosic fibers.

Moderate exercise prevents the cell atrophy caused by hypothyroidism in rats.

Adult­onset hypothyroidism is associated with an increase in cell atrophy of the hippocampal pyramidal neurons. Physical exercise implies diverse actions on the neural tissue that promote neuron proliferation and survival. The beneficial effects of exercise seem to be inversely linked to its intensity, so that strenuous exercise has reduced protective effects. In this study we evaluated the capacity of a moderate forced­exercise routine to counteract the neurodegenerative effects of a hypothyroid condition induced during adulthood. Simultaneously with a chronic anti­thyroid chemical treatment, a group of rats was forced to walk in a motorized wheel for 30 min daily five times a week. In four weeks of treatment the rats developed a plain hypothyroid condition that in non­exercised rats was accompanied by a marked increase in the number of atrophic cells in all CA regions of the hippocampus. The forced­exercise treatment did not counter the development of hypothyroidism and its signs, but it did prevent almost completely the associated neuronal damage in all CA regions. The forced exercise also improved the cognitive function in a spatial­learning test. These results indicate that moderate exercise has the potential to prevent the structural and functional deficits associated with a hypothyroid condition.

Enhancing antibiotic activity: a strategy to control Acinetobacter infections.

OBJECTIVES: The emergence of antibiotic resistance has seriously diminished antibiotic efficacy and an increasing number of infections are becoming difficult to treat. One approach to the restoration of antibiotic activity is to administer them in conjunction with non-antibiotic compounds that depress resistance mechanisms. We describe the activity of ellagic and tannic acids as adjuvants that enhance the activity of aminocoumarin antibiotics against multidrug-resistant (MDR) Acinetobacter baumannii. METHODS: Adjuvant activity of plant phenolics was tested using growth inhibition assays in combination with subinhibitory concentrations of novobiocin. The antibacterial susceptibilities of susceptible and MDR A. baumannii to a variety of antibiotics were determined in the absence and presence of ellagic and tannic acids. The effect of the adjuvants on bacterial outer membrane function was examined by using the fluorescence dye 1-N-phenylnaphthylamine (NPN). The efflux pump inhibition was measured by the intracellular accumulation of ethidium bromide (EtBr) and pyronin Y. RESULTS: At 40 microM, ellagic and tannic acids enhanced the activity of novobiocin, coumermycin, chlorobiocin, rifampicin and fusidic acid against A. baumannii. There were no increases in the uptake of NPN or in the accumulation of EtBr after strains were treated with these adjuvants; however, the intracellular accumulation of pyronin Y by the treated cells was significantly increased, suggesting that ellagic and tannic acids act as efflux pump inhibitors. CONCLUSIONS: Susceptibility of MDR A. baumannii to a variety of antibiotics was enhanced in the presence of ellagic and tannic acids. The use of such plant compounds might provide effective treatments for resistant Gram-negative bacterial infections.

The homeostatic feeding response to fasting is under chronostatic control.

Eating behavior is controlled by the energy needs of the organism. The need to provide a constant supply of energy to tissues is a homeostatic drive that adjusts feeding behavior to the energetic condition of the organism. On the other hand, food intake also shows a circadian variation synchronized to the light-dark cycle and food availability. Thus, feeding is subjected to both homeostatic and circadian regulation mechanisms that determine the amount and timing of spontaneous food intake in normal conditions. In the present study we contrasted the influence of the homeostatic versus the chronostatic mechanisms on food intake in normal conditions and in response to fasting. A group of rats was subjected to food deprivation under two different temporal schemes. A constant-length 24-h food deprivation started at different times of day resulted in an increased compensatory intake. This compensatory response showed a circadian variation that resembled the rhythm of intake in non-deprived animals. When subjected to fasting periods of increasing length (24-66 h), the amount of compensatory feeding varied according to the time of day in which food was made available, being significantly less when the fast ended in the middle of the light phase or beginning of the dark phase. These oscillatory changes did not have a correlation with variations in the level of glucose or ß-hydroxybutyrate in the blood. The results suggest that the mechanism of homeostatic compensation is modulated chronostatically, presumably as part of the alternation of catabolic and anabolic states matching the daily cycles of activity.

Effect of γ-ethyl-γ-phenyl-butyrolactone (EFBL), anticonvulsant and hypnotic drug, on mouse brain catecholamine levels.

γ-Ethyl-γ-phenyl-butyrolactone (EFBL) is a structural combination of the anticonvulsant γ-hydroxy-γ-ethyl-γ-phenylbutyramide (HEPB) and the hypnotic γ-butyrolactone (GBL), which inherits both properties. To clarify its mechanism of action, the effects of EFBL, GBL and HEPB on dopamine (DA) and noradrenaline (NA) brain levels were investigated. Influences of chlorpromazine, phenelzine and aminooxyacetic acid were also studied. EFBL increased DA in a dose-dependent manner, remaining enhanced by 80 % over a period of 24 h and augmented NA by 54 % one hour after treatment. HEPB increased DA and NA approximately 2-fold after the first hour. GBL raised DA and NA after three and 24 h, resp. EFBL reversed chlorpromazine effects but potentiated those of phenelzine on DA. Amino-oxyacetic modified neither DA nor NA brain levels, not even in the presence of EFBL. The anticonvulsant and hypnotic properties of EFBL are attributed to its effect on presynaptic dopaminergic receptors and its lasting effect on ethyl and phenyl radicals that hinder its degradation. The results support the role of DA and NA in regulating seizure activity in the brain and indicate that EFBL offers a potential treatment for refractory epilepsy without complementary drugs and Parkinson's disease, without the drawbacks of oral therapies.

Depletion and recovery of catecholamines in several organs of rats treated with reserpine.

Chemical sympathectomy with reserpine depletes catecholamines in every neuronal or nonneuronal cell producing a nonspecific temporal sympathectomy. After reserpine administration, most of the drug is distributed to tissues based on their blood flow and would then either be metabolized or be reversibly bound in lipid depots from where it might be released. Consequently, reserpine concentration and the catecholamine-depleting effect in the various tissues are expected to differ according to the route of administration. This study was designed to compare the effects of intraperitoneal (i.p.) and subcutaneous (s.c.) administration of reserpine on catecholamine depletion and recovery in the liver, portal vein, and adrenal gland on days 1, 4, and 10 after reserpine dosage. Catecholamine determinations were extended to 25 days after the treatment only in s.c. reserpine-treated rats and adding samples of heart and brown adipose tissue to the testing. I.p. and s.c. reserpine administration had the same norepinephrine-depleting effect in the portal vein and liver but full recovery was present in both tissues only in i.p. reserpine-treated rats. In the adrenal gland, both routes of administration produced the same depleting and recovery effect of norepinephrine and epinephrine concentrations. A significant temporary overshoot in epinephrine levels was observed several days after s.c. reserpine treatment. Except for the liver, reserpine injected s.c. depleted norepinephrine concentrations significantly in all other tissues up to the end of the experiment. Our results suggest that chemical sympathectomy caused by reserpine administered s.c. produces a generalized and prolonged decrease in peripheral sympathetic activity that could be compensated by an increase in activity of the adrenal gland.

Construction of a Multiplex Promoter Reporter Platform to Monitor Staphylococcus aureus Virulence Gene Expression and the Identification of Usnic Acid as a Potent Suppressor of psm Gene Expression.

As antibiotic resistance becomes phenomenal, alternative therapeutic strategies for bacterial infections such as anti-virulence treatments have been advocated. We have constructed a total of 20 gfp-luxABCDE dual-reporter plasmids with selected promoters from S. aureus virulence-associated genes. The plasmids were introduced into various S. aureus strains to establish a gfp-lux based multiplex promoter reporter platform for monitoring S. aureus virulence gene expressions in real time to identify factors or compounds that may perturb virulence of S. aureus. The gene expression profiles monitored by luminescence correlated well with qRT-PCR results and extrinsic factors including carbon dioxide and some antibiotics were shown to suppress or induce the expression of virulence factors in this platform. Using this platform, sub-inhibitory ampicillin was shown to be a potent inducer for the expression of many virulence factors in S. aureus. Bacterial adherence and invasion assays using mammalian cells were employed to measure S. aureus virulence induced by ampicillin. The platform was used for screening of natural extracts that perturb the virulence of S. aureus and usnic acid was identified to be a potent repressor for the expression of psm.

Influence of high-calorie (cafeteria) diets on the population of Paneth cells in the small intestine of the rat.

A high-calorie (cafeteria) diet is known to cause changes in the intestinal morphology and functioning that seem to be related to calorie overfeeding. Among the cell lineages found in the small intestine epithelium, the Paneth cell (PC) population is known to be influenced by factors related mainly to the intestinal microbiota. The role of PCs in the intestinal cell concert remains unclear, because experimental evidence suggests PC involvement in local processes other than protection against pathogens. Participation of PC in digestive mechanisms has been proposed on this basis. We have analyzed the effect of high-carbohydrate (HC) and high-fat (HF) cafeteria diets on the PC population in the small intestine of the adult rat. For 8 weeks, both HC and HF diets caused a gain in body weight, but whereas the HC-fed rats showed reduced counts of intestinal crypts per 5-mum section, the HF-fed group showed the opposite. In control rats, the number of crypts per section showed a slight tendency to decrease along the duodenum - ileum axis, whereas the number of PCs per crypt was increased towards the ileum. As a result, the number of PCs per section (calculated from these data) remained constant along the three segments of the intestine. The hypercaloric diets did not modify the general tendencies seen in the crypt and PC counts, but reduced the number of PCs per section in the duodenum by 50%. HC-fed, but not HF-fed, rats showed a similar reduction in jejunum also. These changes do not correlate particularly with any of the predictable effects of diet composition, so that a multifactorial control of PC density is proposed.

Screening of Microbial Extracts for Anticancer Compounds Using Streptomyces Kinase Inhibitor Assay.

Eukaryotic kinases are known to play an important role in signal transduction pathways by phosphorylating their respective substrates. Abnormal phosphorylations by these kinases have resulted in diseases. Hence inhibitors of kinases are of considerable pharmaceutical interest for a wide variety of disease targets, especially cancers. A number of reports have been published which indicate that eukaryotic-like kinases may complement two-component kinase systems in several bacteria. In Streptomyces sp. such kinases have been found to have a role in formation of aerial hyphae, spores, pigmentation & even in antibiotic production in some strains. Eukaryotic kinase inhibitors are seen to inhibit formation of aerial mycelia in Streptomyces without inhibiting vegetative mycelia. This property has been used to design an assay to screen for eukaryotic kinase inhibitors. The assay involves testing of compounds against Streptomyces 85E ATCC 55824 using agar well diffusion method. Inhibitors of kinases give rise to "bald" colonies where aerial mycelia and sporulation inhibition is seen. The assay has been standardized using known eukaryotic protein kinase inhibiting anticancer agents like AG-490, AG-1295, AG-1478, Flavopiridol and Imatinib as positive controls, at a concentration ranging from 10 µg/well to 100 µg/well. Anti-infective compounds which are not reported to inhibit eukaryotic protein kinases were used as negative controls. A number of microbial cultures have been screened for novel eukaryotic protein kinase inhibitors. Further these microbial extracts were tested in various cancer cell lines like Panel, HCT116, Calul, ACHN and H460 at a concentration of 10 µg/mL/ well. The anticancer data was seen correlating well with the Streptomyces kinase assay thus validating the assay.

Epinephrine and dopamine colocalization with norepinephrine in various peripheral tissues: guanethidine effects.

Chemical sympathectomy with guanethidine (Gnt) selectively destroys the postganglionic noradrenergic neurons, whereas dopaminergic fibers and nonneural catecholamine-secreting cells are spared. As a result, the relative proportions of norepinephrine (NE), epinephrine (E), and dopamine (DA) in tissues can be differentially affected. This study was done to show the possible differences in the relative amount of catecholamines in some organs and tissues that might indicate the nature of the secretory cells from which they originate. The contents of NE, E, and DA were assessed in rats neonatally treated with Gnt. Gnt-treated rats showed significantly lower levels of NE (P < 0.01) in all tissues except the adrenal gland and paraganglia. Epinephrine was present in all tissues with mean levels below 25 ng/g, with the exception of the adrenal gland (700 microg/gland) and paraganglia (100 ng/g). Only the heart showed lower values in Gnt-treated rats. Mean DA levels were also very high in paraganglia (530 ng/g). In the Gnt-treated rats, DA levels fell practically to zero except in the duodenum, mesentery, and adrenal, whereas there were high levels in the paraganglia, which were significantly different from controls. The results suggest that the three catecholamines are contained mainly in noradrenergic sympathetic fibers of muscle, white adipose tissue, heart, liver, pancreas, and spleen. The duodenum and mesentery may have dopaminergic fibers or E- and DA-containing nonneural cells. Hepatic-vagus paraganglia contain all the catecholamines in relatively high amounts in nonneural cells, and Gnt treatment raises DA levels without affecting the other amines.

Chemical sympathectomy alters food intake and thermogenic responses to catecholamines in rats.

It has been suggested that the sympathetic nervous system contributes to the short-term control of feeding. The adrenergic innervation of some splanchnic organs seems to be especially involved in such processes, since catecholamines reduce feeding only when injected intraperitoneally or intraportally. In this work, the effects of neonatal sympathetic denervation with guanethidine (Gnt) upon food intake were assessed in adult rats. Gnt-treated male rats had lower body weight gain. The hypophagic response to intraperitoneal (ip) norepinephrine was 70% higher in Gnt-treated animals as compared to controls (P < 0.05); that of epinephrine (E) by 33% (P < 0.05) and that of isoproterenol was not significantly modified. As in normal rats, the hypophagic effect was much stronger after ip than after intramuscular (im) administration (P < 0.05). On the other hand, resting oxygen consumption (VO2) was consistently lower in denervated animals. Ip E administration did not modify VO2, while im E caused increased motor activity and VO2 (P < 0.05). In contrast to control rats, the respiratory exchange ratio in ad libitum fed Gnt rats did not decrease after Ip E administration, suggesting a lack of effect upon lipid mobilization. The lower rate of body weight gain induced by neonatal Gnt sympathectomy might be due to lower daily food intake possibly related, in part, to the sensitization of the alpha-adrenergic porto-hepatic response to endogenous catecholamines. Compared with controls, Gnt-treated rats also showed a limited thermogenic capacity not related to feeding, and a greater degree of carbohydrate oxidation, possibly due to a defect in E-induced lipolysis, which is beta-adrenergic.

The NMDA receptor antagonist MK-801 abolishes the increase in both p53 and Bax/Bcl2 index induced by adult-onset hypothyroidism in rat.

Hypothyroidism affects neuron population dynamics in the hippocampus of the adult rat, with neuronal damage as the main feature of its effect. This effect is prevented by the blockade of NMDA receptors, which suggests that glutamatergic activity mediates cell death in this condition. Glutamate can also stimulate cell proliferation and survival of newborn neurons, indicating that it can affect different stages of the cell cycle. In this work we measured the expression of specific proteins that control cell proliferation (cycline-D1), cell arrest (p21), damage (p53) or apoptosis (Bax and Bcl2) in the hippocampus of hypothyroid rats treated with the NMDA receptor (NMDAR) blocker MK-801 during the induction of hypothyroidism. The results show that hypothyroidism increases the expression of markers of DNA damage, cell arrest, and apoptosis, but does not affect the marker of cell proliferation. NMDAR blockade prevents the increase on markers of DNA damage and apoptosis, but does not influence cell arrest or cell proliferation. This suggests that hypothyroidism promotes cell death mainly by an excitotoxic effect of glutamate.