Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress.

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

Exploring the mechanism of lung injury induced by lunar dust simulant in rats based on metabolomic analysis.

Inflammatory response and oxidative stress are considered to be important mechanisms of lung injury induced by lunar dust. However, the pulmonary toxicological mechanism remains unclear. In the present study, Wistar rats were exposed to CLDS-i 7 days/week, 4 h/day, for 4 weeks in the mouth and nose. Lung tissue samples were collected for histopathological analysis and ultra-performance liquid chromatography-mass spectrometry analysis. Enzyme activities and expression levels of key metabolic enzymes were detected by biochemical analysis and real-time PCR. The pathological features of lung tissue showed that CLDS-i caused congestion and inflammation in the lungs, and the lung structure was severely damaged. Metabolomics analysis showed that 141 metabolites were significantly changed in the lung tissue of the CLDS-i group compared with the control group. Combined with Kegg pathway analysis, it was found that the changes of amino acid metabolites were involved in these pathways, indicating that the simulated lunar dust exposure had the most obvious effect on amino acid metabolism in the lung tissue of rats. Real-time PCR analysis showed that the mRNA expression of six key enzymes related to amino acid metabolism was changed, and the enzyme activities of these key enzymes were also changed, which were consistent with the results of qPCR. These results suggest that changes in amino acid metabolism may be closely related to the pathogenesis of lung injury induced by lunar dust, and amino acid metabolism may be a potential biomarker of lung diseases related to lunar dust exposure.

Chirality-Selected Chemical Modulation of Amyloid Aggregation.

Due to the composed α-helical/ß-strand structures, ß-amyloid peptide (Aß) is sensitive to chiral environments. The orientation and chirality of the Aß strand strongly influence its aggregation. Aß-formed fibrils have a cascade of chirality. Therefore, for selectively targeting amyloid aggregates, chirality preference can be one key issue. Inspired by the natural stereoselectivity and the ß-sheet structure, herein, we synthesized a series of d- and l-amino acid-modified polyoxometalate (POM) derivatives, including positively charged amino acids (d-His and l-His) and negatively charged (d-Glu and l-Glu) and hydrophobic amino acids (d-Leu, l-Leu, d-Phe, and l-Phe), to modulate Aß aggregation. Intriguingly, Phe-modified POMs showed a stronger inhibition effect than other amino acid-modified POMs, as evidenced by multiple biophysical and spectral assays, including fluorescence, circular dichroism, NMR, molecular dynamic simulations, and isothermal titration calorimetry. More importantly, d-Phe-modified POM had an 8-fold stronger inhibition effect than l-Phe-modified POM, indicating high enantioselectivity. Furthermore, in vivo studies demonstrated that the chiral POM derivatives crossed the blood-brain barrier, extended the life span of AD transgenic Caenorhabditis elegans CL2006 strain, and had low cytotoxicity, even at a high dosage.

Amino acid based prodrugs of a fosmidomycin surrogate as antimalarial and antitubercular agents.

Fosmidomycin is a natural antibiotic with promising IspC (DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase) inhibitory activity. This enzyme catalyzes the first committed step of the non-mevalonate isoprenoid biosynthesis pathway, which is essential in Plasmodium falciparum and Mycobacterium tuberculosis. Mainly as a result of its high polarity, fosmidomycin displays suboptimal pharmacokinetic properties. Furthermore, fosmidomycin is inactive against M. tuberculosis as a result of its inability to penetrate the bacterial cell wall. Temporarily masking the phosphonate moiety as a prodrug has the potential to solve both issues. We report the application of two amino acid based prodrug approaches on a fosmidomycin surrogate. Conversion of the phosphonate moiety into tyrosine-derived esters increases the in vitro activity against asexual blood stages of P. falciparum, while phosphonodiamidate prodrugs display promising antitubercular activities. Selected prodrugs were tested in vivo in a P. berghei malaria mouse model. These results indicate good in vivo antiplasmodial potential.

Changes in amino acid profiles and liver alterations in pregnant rats with a high carbohydrate/low protein diet.

INTRODUCTION AND AIM: Intake of a high-carbohydrate, low-protein diet (HCD/LPD) during pregnancy promotes metabolic disturbances. It has been suggested that liver function during pregnancy contributes to the synthesis of proteins necessary for fetal development during this stage. The liver is a site of response to the synthesis of macronutrients such as proteins. However, it is unknown how HCD/LPD is associated with modifications to the amino acid profiles and hepatic alterations in the maternal environment during pregnancy. MATERIALS AND METHODS: A transverse longitudinal study was done in primiparous mothers during gestation (G) (G1 day 1, G5 day 5, G15 day 15, and G20 day 20). Histological analysis was used to assess hepatic alterations, and amino acid profiles in the liver were analyzed with high performance liquid chromatography (HPLC). Food and water intake was quantified, and peripheral biochemical indicators in serum were measured. RESULTS: Mothers with HCD/LPD had increased micro and macro vesicles of fat, necrosis, and inflammation in the liver on G5. The total concentration of hepatic amino acids increased by 40% on G1, 17% on G5, and 25% on G15; and, there was a 12% decrease on G20. The following increases were observed in the liver on G1: arginine 68%, histidine 75%, alanine 18%, methionine 71%, and phenylalanine 51% (p>0.05); on G5: arginine 12%, methionine 34%, and phenylalanine 83% (p>0.05); on G15: arginine and phenylalanine 66%, tryptophan 81% and histidine 60.4% (p>0.05); and on G20: arginine 32% (p>0.05). No weight loss, changes in food consumption, or hepatomegaly occurred. CONCLUSIONS: HCD/LPD during pregnancy in primiparous mothers may promote development of fat vesicles. Possibly, this condition causes metabolic adaptations and nitrogen management reflected in decreased levels of serum urea and altered amino acid profiles in the liver.

Design and Toxicity Evaluation of Novel Fatty Acid-Amino Acid-Based Biocompatible Surfactants.

Surfactants occupy an important place owing to their wide application, but primarily compromised due to its toxicity issues. This raises the need for exploration of newer surfactants with increased biocompatibility. Novel fatty acid- and amino acid-based surfactants were prepared using standard carbodiimide chemistry. Pyrene assay was implemented to confirm the amphiphilic nature of the surfactants and to calculate their CMC (critical micellar concentration). In vitro hemolytic and cell culture study in MCF-7 and HEK cell line were done to check the in vitro biocompatibility of the developed surfactants in comparison to marketed surfactants Triton X-100 and Tween ® 80. In vivo biocompatibility test in female Swiss albino mice was carried out in comparison to marketed surfactants with respect to serum markers, organ histology, and RBC morphology. Surfactant synthesis provided more than 60% yield in all the conjugates. Pyrene assay concluded the amphiphilic nature of the surfactants with lowest CMC of 0.083% w/v in the case of stearic acid and valine conjugate. In vitro hemolytic and cell culture study depicted highest biocompatibility in vitro as compared to marketed surfactants. Similar results were obtained in in vivo biocompatibility with respect to AST (aspartate transaminase), ALT (alanine transaminase), BUN (blood urea nitrogen), and creatinine serum levels and histology of spleen, liver, and kidney in comparison to marketed surfactants Triton X-100 and Tween ® 80. The developed surfactant also depicted least RBC morphology changes in vivo. Stearic acid valine conjugate thus depicted potential for further application in formulation development replacing the commercially available surfactants.

Safety Assessment of Amino Acid Alkyl Amides as Used in Cosmetics.

The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the product use, formulation, and safety data of 115 amino acid alkyl amides, which function as skin and hair conditioning agents and as surfactants-cleansing agents in personal care products. Safety test data on dermal irritation and sensitization for the ingredients with the highest use concentrations, lauroyl lysine and sodium lauroyl glutamate, were reviewed and determined to adequately support the safe use of the ingredients in this report. The Panel concluded that amino acid alkyl amides are safe in the present practices of use and concentration in cosmetics, when formulated to be nonirritating.

Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability.

OBJECTIVES: The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities. METHODS: Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy. RESULTS: Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all d-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified l-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm. CONCLUSIONS: Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.

Caged xanthones: Potent inhibitors of global predominant MRSA USA300.

Total of 22 caged xanthones were subjected to susceptibility testing of global epidemic MRSA USA300. Natural morellic acid showed the strongest potency (MIC of 12.5µM). However, its potent toxicity diminishes MRSA therapeutic potential. We synthetically modified natural morellic acid to yield 13 derivatives (3a-3m). Synthetically modified 3b retained strong potency in MRSA growth inhibition, yet the toxicity was 20-fold less than natural morellic acid, permitting the possibility of using caged xanthones for MRSA therapeutic.

Acute hyperammonaemia induces a sustained decrease in vigilance, which is modulated by caffeine.

UNLABELLED: Hyperammonaemia is observed after prolonged, intense exercise, or in patients with hepatic failure. In the latter, it is associated with a set of neurological and psychiatric abnormalities termed hepatic encephalopathy. THE AIMS OF OUR STUDY WERE: 1. to measure vigilance in a condition of induced hyperammonaemia; 2. to assess whether caffeine modulates the effects of hyperammonaemia on vigilance, if any. Ten healthy volunteers (28.5 ± 5 years; 5 males) underwent three experimental sessions consisting of two-hourly measurements of capillary ammonia, subjective sleepiness (Karolinska Sleepiness Scale) and vigilance (Psychomotor Vigilance Task, PVT), in relation to the intake of breakfast (+/-coffee), an amino acid mixture which induces hyperammonaemia (amino acid challenge; AAC), and AAC+coffee (only for participants who had coffee with their standard breakfast). The AAC resulted in: 1. the expected increase in capillary ammonia levels, with highest values at approximately 4 h after the administration; 2. a significant increase in subjective sleepiness ratings; 3. a sustained increase in PVT-based reaction times. When caffeine was administered after the AAC, both subjective sleepiness and the slowing in RTs were significantly milder than in the AAC-only condition. In conclusion, acute hyperammonaemia induces an increase in subjective sleepiness and a sustained decrease in vigilance, which are attenuated by the administration of a single espresso coffee.

Safety assessment of animal- and plant-derived amino acids as used in cosmetics.

The Cosmetic Ingredient Review Expert Panel (Panel) reviewed the safety of animal- and plant-derived amino acid mixtures, which function as skin and hair conditioning agents. The safety of α-amino acids as direct food additives has been well established, based on extensive research through acute and chronic dietary exposures and the Panel previously has reviewed the safety of individual α-amino acids in cosmetics. The Panel focused its review on dermal irritation and sensitization data relevant to the use of these ingredients in topical cosmetics. The Panel concluded that these 21 ingredients are safe in the present practices of use and concentration as used in cosmetics.

Persistent fibrosis in the liver of choline-deficient and iron-supplemented L-amino acid-defined diet-induced nonalcoholic steatohepatitis rat due to continuing oxidative stress after choline supplementation.

Nonalcoholic steatohepatitis (NASH) is characterized by combined pathology of steatosis, lobular inflammation, fibrosis, and hepatocellular degeneration, with systemic symptoms of diabetes or hyperlipidemia, all in the absence of alcohol abuse. Given the therapeutic importance and conflicting findings regarding the potential for healing the histopathologic features of NASH in humans, particularly fibrosis, we investigated the reversibility of NASH-related findings in Wistar rats fed a choline-deficient and iron-supplemented l-amino acid-defined (CDAA) diet for 12weeks, with a recovery period of 7weeks, during which the diets were switched to a choline-sufficient and iron-supplemented l-amino acid-defined (CSAA) one. Analysis showed that steatosis and inflammation were significantly resolved by the end of the recovery period, along with decreases in AST and ALT activities within 4weeks. In contrast, fibrosis remained even after the recovery period, to an extent similar to that in continuously CDAA-fed animals. Real-time reverse transcriptase-polymerase chain reaction, Western blot, and immunohistochemical investigations revealed that expression of some factors indicating oxidative stress (CYP2E1, 4-HNE, and iNOS) were elevated, whereas catalase and SOD1 were decreased, and a hypoxic state and CD34-positive neovascularization were evident even after the recovery period, although the fibrogenesis pathway by activated α-SMA-positive hepatic stellate cells via TGF-ß and TIMPs decreased to the CSAA group level. In conclusion, persistent fibrosis was noted after the recovery period of 7weeks, possibly due to sustained hypoxia and oxidative stress supposedly caused by capillarization. Otherwise, histopathological features of steatosis and inflammation, as well as serum AST and ALT activities, were recovered.

Amino acid analog toxicity in primary rat neuronal and astrocyte cultures: implications for protein misfolding and TDP-43 regulation.

Amino acid analogs promote translational errors that result in aberrant protein synthesis and have been used to understand the effects of protein misfolding in a variety of physiological and pathological settings. TDP-43 is a protein that is linked to protein aggregation and toxicity in a variety of neurodegenerative diseases. This study exposed primary rat neurons and astrocyte cultures to established amino acid analogs (canavanine and azetidine-2-carboxylic acid) and showed that both cell types undergo a dose-dependent increase in toxicity, with neurons exhibiting a greater degree of toxicity compared with astrocytes. Neurons and astrocytes exhibited similar increases in ubiquitinated and oxidized protein following analog treatment. Analog treatment increased heat shock protein (Hsp) levels in both neurons and astrocytes. In neurons, and to a lesser extent astrocytes, the levels of TDP-43 increased in response to analog treatment. Taken together, these data indicate that neurons exhibit preferential toxicity and alterations in TDP-43 in response to increased protein misfolding compared with astrocytes.

A virus-induced gene silencing screen identifies a role for Thylakoid Formation1 in Pseudomonas syringae pv tomato symptom development in tomato and Arabidopsis.

Pseudomonas syringae pv tomato DC3000 (Pst DC3000), which causes disease in tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana), produces coronatine (COR), a non-host-specific phytotoxin. COR, which functions as a jasmonate mimic, is required for full virulence of Pst DC3000 and for the induction of chlorosis in host plants. Previous genetic screens based on insensitivity to COR and/or methyl jasmonate identified several potential targets for COR and methyl jasmonate. In this study, we utilized Nicotiana benthamiana and virus-induced gene silencing to individually reduce the expression of over 4,000 genes. The silenced lines of N. benthamiana were then screened for altered responses to purified COR. Using this forward genetics approach, several genes were identified with altered responses to COR. These were designated as ALC (for altered COR response) genes. When silenced, one of the identified genes, ALC1, produced a hypersensitive/necrosis-like phenotype upon COR application in a Coronatine-Insensitive1 (COI1)-dependent manner. To understand the involvement of ALC1 during the Pst DC3000-host interaction, we used the nucleotide sequence of ALC1 and identified its ortholog in Arabidopsis (Thylakoid Formation1 [THF1]) and tomato (SlALC1). In pathogenicity assays performed on Arabidopsis thf1 mutant and SlALC1-silenced tomato plants, Pst DC3000 induced accelerated coalescing necrotic lesions. Furthermore, we showed that COR affects ALC1 localization in chloroplasts in a COI1-dependent manner. In conclusion, our results show that the virus-induced gene silencing-based forward genetic screen has the potential to identify new players in COR signaling and disease-associated necrotic cell death.

Synthesis of a new trifluoromethylketone analogue of l-arginine and contrasting inhibitory activity against human arginase I and histone deacetylase 8.

As part of our continuing search for new amino acid inhibitors of metalloenzymes, we now report the synthesis and biological evaluation of the trifluoromethylketone analogue of L-arginine, (S)-2-amino-8,8,8-trifluoro-7-oxo-octanoic acid (10). While this novel amino acid was initially designed as a potential inhibitor of human arginase I, it exhibits no measurable inhibitory activity against this enzyme. Surprisingly, however, 10 is a potent inhibitor of human histone deacetylase 8, with IC(50)=1.5 ± 0.2 µM. Additionally, 10 weakly inhibits the related bacterial enzyme, acetylpolyamine amidohydrolase, with IC(50)=110 ± 30 µM. The lack of inhibitory activity against human arginase I may result from unfavorable interactions of the bulky trifluoromethyl group of 10 in the constricted active site. Since the active site of histone deacetylase 8 is less constricted, we hypothesize that it accommodates 10 as the gem-diol, which mimics the tetrahedral intermediate and its flanking transition states in catalysis. Therefore, we suggest that 10 represents a new lead in the design of an amino acid or peptide-based inhibitor of histone deacetylases with simpler structure than previously studied trifluoromethylketones.

Amino Acid and Biogenic Amine Adductions Derived from Reactive Metabolites.

Reactive metabolites (RMs) are products generated from the metabolism of endogenous and exogenous substances. RMs are characterized as electrophilic species chemically reactive to nucleophiles. Those nucleophilic species may be nitrogen-containing bio-molecules, including macro-biomolecules, such as protein and DNA, and small biomolecules, i.e., amino acids (AAs) and biogenic amines (BAs). AAs and BAs are essential endogenous nitrogen-containing compounds required for normal development, metabolism, and physiological functions in organisms, through participating in the intracellular replication, transcription, translation, division and proliferation, DNA and protein synthesis, regulation of apoptosis, and intercellular communication activities. These biological amines containing an active lone pair of electrons on the electronegative nitrogen atom would be the proper N-nucleophiles to be attacked by the abovementioned RMs. This review covers an overview of adductions of AAs and BAs with varieties of RMs. These RMs are formed from metabolic activation of furans, naphthalene, benzene, and products of lipid peroxidation. This article is designed to provide readers with a better understanding of biochemical mechanisms of toxic action.

Fullerene-Based Mimics of Biocatalysts Show Remarkable Activity and Modularity.

The design of catalysts with greater control over catalytic activity and stability is a major challenge with substantial impact on fundamental chemistry and industrial applications. Due to their unparalleled diversity, selectivity, and efficiency, enzymes are promising models for next-generation catalysts, and considerable efforts have been devoted to incorporating the principles of their mechanisms of action into artificial systems. We report a heretofore undocumented catalyst design that introduces fullerenes to the field of biocatalysis, which we refer to as fullerene nanocatalysts, and that emulates enzymatic active sites through multifunctional self-assembled nanostructures. As a proof-of-concept, we mimicked the reactivity of hydrolases using fullerene nanocatalysts functionalized with the basic components of the parent enzyme with remarkable activity. Owing to the versatile amino acid-based functionalization repertoire of fullerene nanocatalysts, these next-generation carbon/biomolecule hybrids have potential to mimic the activity of other families of enzymes and, therefore, offer new perspectives for the design of biocompatible, high-efficiency artificial nanocatalysts.

Suppression of MYC transcription activators by the immune cofactor NPR1 fine-tunes plant immune responses.

Plants tailor immune responses to defend against pathogens with different lifestyles. In this process, antagonism between the immune hormones salicylic acid (SA) and jasmonic acid (JA) optimizes transcriptional signatures specifically to the attacker encountered. Antagonism is controlled by the transcription cofactor NPR1. The indispensable role of NPR1 in activating SA-responsive genes is well understood, but how it functions as a repressor of JA-responsive genes remains unclear. Here, we demonstrate that SA-induced NPR1 is recruited to JA-responsive promoter regions that are co-occupied by a JA-induced transcription complex consisting of the MYC2 activator and MED25 Mediator subunit. In the presence of SA, NPR1 physically associates with JA-induced MYC2 and inhibits transcriptional activation by disrupting its interaction with MED25. Importantly, NPR1-mediated inhibition of MYC2 is a major immune mechanism for suppressing pathogen virulence. Thus, NPR1 orchestrates the immune transcriptome not only by activating SA-responsive genes but also by acting as a corepressor of JA-responsive MYC2.

Amino acid-functionalized dendrimers with heterobifunctional chemoselective peripheral groups for drug delivery applications.

Dendrimers have emerged as multifunctional carriers for targeted drug delivery, gene delivery and imaging. Improving the functional versatility at the surface for carrying multiple conjugation reactions is becoming vital. Typically, generation four polyamidoamine (G4-PAMAM) dendrimers bear approximately 64 symmetrical end groups, often requiring different spacers to conjugate various functional groups (drugs and targeting moities), increasing the synthetic steps. In the present study, a simple one-step synthesis to convert each symmetrical end group of G4-PAMAM dendrimers into two reactive, distinct orthogonal and chemoselective groups is described. A near-complete end-capping of the dendrimers (87-93%) with amino acids results in heterobifunctional G4-PAMAM dendrimers bearing a very high (> or = 110) diverse peripheral end groups (OH+NHBoc, OH+COOMe, SH+NHBoc, and COOH+NHBoc). Postfunctionalization ability of these dendrimers was evaluated. The heterobifunctional groups at the dendrimer periphery could be chemoselectively conjugated to multiple moities such as drugs (indomethacin and dexamethasone) and drugs and imaging agents (dexamethasone and FITC). These conjugations could be achieved in immediate succession without functional group conversions, eliminating the additional elaborate synthetic steps traditionally required to append specific linkers. Furthermore, one of the two functional handles at periphery was used to develop in situ forming hydrogels, whereas the other handle could be used for conjugating the drugs (e.g., dexamethasone). The heterobifunctional dendrimers with either "NH(2) or SH (thiopyridyl protected form)" terminations showed in situ hydrogel formation by cross-linking with N-hydroxysuccinimide or thiol-terminated multiarm polyethylene glycol (20 kDa). The choice of amino acids as versatile linkers would enable biocompatible dendrimer scaffolds for use in drug delivery. Zeta-potential measurements showed drastic lowering of the charge on G4-PAMAM-NH(2) dendrimers by end-capping with amino acids, whereas in the case of neutral G4-PAMAM-OH dendrimers, the charge did not increase or decrease substantially. The in vitro cytotoxicity and hemolysis assay showed that the heterobifunctional dendrimers were noncytotoxic in the 100 ng/mL to 1 mg/mL concentration range. With this study, we demonstrate the development of biocompatible dendrimers bearing multiple orthogonal surface groups, enabling the attachment of drugs, imaging agents, and gel formation using minimal synthetic steps.

Plant poisons: their occurrence, biochemistry and physiological properties.

Plants produce poisons as a defence against predators. Many of these substances are biosynthesised from non-protein amino acids by biosynthetic pathways which have been deduced from the results of isotopic tracer analysis. These secondary metabolites have been used by humans over thousands of years, both as drugs and as agents to kill animals and commit homicide.