Plant-Derived Small Molecule Inhibitors of Neuronal NO-Synthase: Potential Effects on Protein Degradation

Cigarette smoking is known to cause a decrease in NO production in man resulting in a variety of pathological effects, including vascular dysfunction. Aqueous extracts of cigarette and cigarette smoke contain chemical inhibitors to NO-synthases, a heme-containing cytochrome P450 enzymes. More recently, it was shown that freshly harvested leaves from the tobacco plant (Nicotiana tabacum, Solanaceae) also contain chemical inhibitors to neuronal NO-synthase (nNOS). Examination of leaves from 32 other plants representing diverse members of the plant kingdom showed that 17 other plants, besides tobacco, contain these chemical inhibitors. Of all these plants, 16 are members of the core asterids flowering plant group and 6 are members of the Solanaceae family. Although the identity of the chemicals is not known, perhaps the closely related plants contain the same or similar compounds that inhibit nNOS. The inhibitory effects are not attributable to nicotine. The discovery of these chemicals and their further characterization may help to explain the loss of nNOS in smokers. In this addendum, we discuss these results in light of the effect of tobacco-derived chemicals in inhibiting P450 cytochromes, as well as our thoughts on how the inactivation of nNOS leads to its selective downregulation through proteolytic degradation.Key Words: nitric oxide, inhibitors, tobacco, natural products, cytochrome P450, nitric oxide synthase, heat shock proteinsNO-synthase (NOS) is a cytochrome P450 like hemeprotein enzyme that catalyzes the production of NO from L-arginine.^1,2 NO is very important cellular signaling molecule, which plays a role in a variety of physiological processes including blood pressure regulation, neurotransmission, and penile erection. Like other P450 cytochromes, the catalytic site of NOS contains a heme prosthetic group that upon reduction and complexation with CO forms a chromophore that absorbs in the 450 nm region. Thus, although not closely related with respect to amino acid sequence to other P450 enzymes, the catalytic core of NOS and the chemistry involved in catalysis by NOS are highly similar to other P450 cytochromes.The cytochrome P450 enzymes comprise a very large family of enzymes that are responsible for metabolism of drugs and other xenobiotics as well as for synthesis of many hormones. There are over 2,000 P450 genes characterized in animals with over 50 genes in humans and over 1,900 genes in plants. The diversity of the P450 family is thought to reflect a complex “battle” between plants and animals.³ That is, plants are thought to have developed new alkaloids with new enzymes to protect themselves against animals whereas the animals developed new enzymes to metabolize these newly created plant toxins. This is thought to explain why the diversity in CYP genes increased dramatically approximately 400 millions years ago when organisms moved from the oceans to land.^3,4 Consistent with this hypothesis, chemicals in plants have been documented to inhibit mammalian P450 cytochromes. More importantly for humans, a variety of plant products have been documented to interact clinically with P450 cytochromes.⁵ For example, furanocoumarins found in grapefruit juice are known to enhance the oral bioavailability of felodipine, a calcium channel blocker, by inhibiting intestinal P450 metabolism of the drug.⁶In the case of tobacco-based cigarettes, hydrophobic chemicals in cigarettes have been found to inhibit P450 cytochromes. In particular, aromatase, the terminal P450 enzyme responsible for estrogen synthesis in humans, has been shown to be inhibited by tobacco smoke.⁷ Acyl derivatives of noricotine and anabasine were found to be active inhibitors of aromatase. In the case of NOS, studies by Heitzer et al.⁸ showed the amelioration of the vascular dysfunction in smokers by tetrahydrobiopterin and the authors concluded that there was a loss of endothelial NO synthesis caused by smoking. Moreover, Xie et al.⁹ showed that exposing rats to cigarette smoke led to the loss of both neuronal NO-synthase (nNOS) activity and protein. This observation reminded us of our earlier studies showing that guanabenz is a time-dependent irreversible inhibitor of nNOS that causes a loss of the penile nNOS activity and protein when given to rats.¹⁰ Thus, based on these findings, we wondered if chemicals in cigarettes could interact with NOS and explain the loss of NOS. We discovered that aqueous extracts of cigarettes and cigarette smoke could inhibit nNOS¹¹ and endothelial NOS.¹² More recently, extracts from freshly harvested tobacco leaves and leaves of various other plants from diverse phylogeny were also examined.¹³Tobacco leaves contained inhibitors to nNOS, indicating that these compounds did not necessarily arise from the curing and processing of the tobacco leaves to make cigarettes or from burning of the cigarettes. Moreover, other extracts made from other plants including 6 from the Solanacea family were found to have inhibitory activity. Thus, we believe that some common chemical or related chemicals exist that inhibits nNOS. The inhibitors are small molecules with hydrophilic and cationic qualities. In comparison, the inhibitors to aromatase are more hydrophobic than those of nNOS, likely reflecting the hydrophobic nature of the active site of aromatase, which accepts steroidal androgens. On the other hand, nNOS accepts L-arginine, a cationic water-soluble compound. The water-soluble nature of the inhibitors has so far hindered the isolation and characterization of the nNOS inhibitors, and thus we do not know the identity of these chemicals.The irreversible nature of the interaction of tobacco inhibitors with nNOS found in our studies may be important in understanding the long-term consequences of smoking. The irreversible inactivation of nNOS by various guanidine-based compounds is known to cause the enhanced proteasomal degradation of nNOS.¹⁴ The dysfunctional nNOS is known to be selectively ubiquitinated¹⁵ by a process involving Hsp70 and CHIP¹⁶ (Fig. 1). The degradation is also accelerated by inhibition of Hsp90, indicating that NOS is also regulated by the Hsp90-based chaperones.¹⁴ The loss of nNOS protein due to cigarette smoke suggests a similar process may be occurring with smokers.Open in a separate windowFigure 1Ubiquitination and Degradation of Inactivated nNOS. The nNOS is inactivated by chemicals found in some plants by alterations directed at the heme-containing active site of nNOS. This dysfunctional form of nNOS is directed for ubiquitination by an E3 ligase, such as CHIP (C-terminal Hsp-interacting protein), hsp70, and E2 ligase enzyme. The ubiquitinated nNOS is then recognized for proteasomal degradation. Closed circles represent ubiquitin molecules.The discovery of nNOS inhibitors in plants could be adequately interpreted in the context of plant-animal warfare. However, in light of the recent discovery of plant NOS,¹⁷ and in particular NOS in tobacco cells,¹⁸ it is possible that these inhibitors are endogenous modulators of the plant NOS. In this respect, an endogenous inhibitor has been identified for mammalian NOS.¹⁹ Furthermore, considering that the Hsp90-and Hsp70-based chaperones, ubiquitin, CHIP, and proteasome are found in plants and serve similar functions,^20–23 the NOS in plants may be similarly regulated. Thus, the mechanism outlined in Figure 1 for nNOS may also be pertinent to regulation of plant NOS. We believe this process reflects a fundamental biological process of protein quality control that is greatly affected by the binding of small molecules to the target protein.     

Structural characterization and kinetics of nitric-oxide synthase inhibition by novel N5-(iminoalkyl)- and N5-(iminoalkenyl)-ornithines

Isoform-specific nitric-oxide synthase (NOS) inhibitors may prove clinically useful in reducing the pathophysiological effects associated with increased neuronal NOS (nNOS) or inducible NOS (iNOS) activity in a variety of neurological and inflammatory disorders. Analogs of the NOS substrate L-arginine are pharmacologically attractive inhibitors because of their stability, reliable cell uptake, and good selectivity for NOS over other heme proteins. Some inhibitory arginine analogs show significant isoform selectivity although the structural or mechanistic basis of such selectivity is generally poorly understood. In the present studies, we determined by x-ray crystallography the binding interactions between rat nNOS and N5-(1-imino-3-butenyl)-L-ornithine (L-VNIO), a previously identified mechanism-based, irreversible inactivator with moderate nNOS selectivity. We have also synthesized and mechanistically characterized several L-VNIO analogs and find, surprisingly, that even relatively minor structural changes produce inhibitors that are either iNOS-selective or non-selective. Furthermore, derivatives having a methyl group added to the butenyl moiety of L-VNIO and L-VNIO derivatives that are analogs of homoarginine rather than arginine display slow-on, slow-off kinetics rather than irreversible inactivation. These results elucidate some of the structural requirements for isoform-selective inhibition by L-VNIO and its related alkyl- and alkenyl-imino ornithine and lysine derivatives and may provide information useful in the ongoing rational design of isoform-selective inhibitors.     

Innate and acquired components of oligophagy in the herbivorous lepidopteran,Manduca sexta

Innate and acquired aspects of oligophagy were investigated in the tobacco hornworm, Manduca sexta L. (Lepidoptera: Sphingidae), a species normally restricted to members of the family Solanaceae. Larvae were reared in the laboratory on solanaceous species tobacco (Nicotiana tabacum L.), strawberry groundcherry (Physalis pruinosa L.), and potato (Solanum tuberosum L.), as well as the non‐solanaceous foods cowpea [Vigna sinensis (L.) (Fabaceae)], rapeseed [Brassica napus L. (Brassicaceae)], and artificial diet. Feeding assays were conducted using the above plants plus corkscrew vine [Vigna caracalla (L.) Verdc. (Fabaceae)], lettuce [Lactuca sativa L. (Asteraceae)], and moist glass‐fiber filter paper. Larval feeding was characterized using two camera‐monitored assays: (1) a quantitative no‐choice disc test that determines consumption over time, delays in initiation of feeding, and % of larvae rejecting the food, and (2) a novel no‐choice emigration test that measures how soon a larva abandons a whole plant. Experimental results from both assays revealed that larvae displayed high levels of acceptance of Solanaceae regardless of whether they had been reared on solanaceous or non‐solanaceous food. We conclude that solanaceous oligophagy in M. sexta is primarily innate and does not require (but may be strengthened by) previous feeding experience on Solanaceae. In contrast, larvae tested on non‐solanaceous plants or moist filter paper showed large variation in both acceptance of foods and emigration times that were strongly dependent on the food on which they had been reared (analogous to the ‘induction of preference’ of earlier literature). Two types of induction were identified: an increase in acceptability of a plant of (1) the same species as the one on which the larva was reared, or (2) a related species. These discoveries both challenge and expand our current understanding of oligophagy in the tobacco hornworm.     

Increasing nitric oxide content in Arabidopsis thaliana by expressing rat neuronal nitric oxide synthase resulted in enhanced stress tolerance

Nitric oxide (NO) plays essential roles in many physiological and developmental processes in plants, including biotic and abiotic stresses, which have adverse effects on agricultural production. However, due to the lack of findings regarding nitric oxide synthase (NOS), many difficulties arise in investigating the physiological roles of NO in vivo and thus its utilization for genetic engineering. Here, to explore the possibility of manipulating the endogenous NO level, rat neuronal NOS (nNOS) was expressed in Arabidopsis thaliana. The 35S::nNOS plants showed higher NOS activity and accumulation of NO using the fluorescent probe 3-amino, 4-aminomethyl-2', 7'-difluorescein, diacetate (DAF-FM DA) assay and the hemoglobin assay. Compared with the wild type, the 35S::nNOS plants displayed improved salt and drought tolerance, which was further confirmed by changes in physiological parameters including reduced water loss rate, reduced stomatal aperture, and altered proline and malondialdehyde content. Quantitative real-time PCR analyses revealed that the expression of several stress-regulated genes was up-regulated in the transgenic lines. Furthermore, the transgenic lines also showed enhanced disease resistance against Pseudomonas syringae pv. tomato (Pst) DC3000 by activating the expression of defense-related genes. In addition, we found that the 35S::nNOS lines flowered late by regulating the expression of CO, FLC and LFY genes. Together, these results demonstrated that it is a useful strategy to exploit the roles of plant NO in various processes by the expression of rat nNOS. The approach may also be useful for genetic engineering of crops with increased environmental adaptations.     

Behavior of newly hatched potato tuber moth larvae,Phthorimaea operculella Zell. (Lepidoptera: Gelechiidae), in relation to their host plants

A study was made of neonate larvae of Phthorimaea operculella.Host finding from soil-laid eggs and dispersal from hosts and nonhosts were first examined. Of first-instar larvae hatching from soil-laid eggs, 80% found the potato plant while roughly 50% found each of the other three plants (datura, tobacco, and tomato). Dispersal from potato, datura, and tobacco was very low, while on tomato it was higher and a high mortality was observed in the 24 h period. Dispersal from nonhost plants was high. Behavior on leaves of hosts and non-hosts is described and some host-plant specific behaviors are identified. Effects on behavior of some of the physical and chemical factors are described. Methylene dichloride extracts of potato leaf wax reduced locomotion rates and the number of turns during locomotion. Also methylene dichloride extract and, to a lesser extent, methanol extract caused biting behavior.     

Oleosin-like proteins are not present on the surface of tobacco pollen

Pollen-stigma interactions on wet- and dry-type stigmas involve similar processes: the hydration of the pollen, followed by pollen tube growth and penetration of the stigma. Furthermore, in some species, identical molecules, namely lipids, are used to achieve this. In addition to lipids, oleosin-like proteins of the pollen coat of dry-type stigma plants have been shown to be involved in pollen-stigma interactions. However, little information is present about the proteins on the surface of pollen of wet-type stigma plants, in particular that of the Solanaceae. To analyze proteins from the surface of pollen of Nicotiana tabacum (tobacco), a solanaceous plant, we used an antiserum raised against Brassica pollen coat, a dry-type stigma plant of the Brassicaceae. In addition we used a molecular approach to identify tobacco homologues of oleosin-like genes. Our results show that no proteins similar to Brassica oleracea pollen coat proteins are present on the surface of tobacco pollen, and that oleosin-like genes are not expressed in tobacco anthers or stigmas.     

Novel Transposable Elements in Solanaceae: Evolutionary Relationships among Tnt1-related Sequences in Wild Petunia Species

Transposable elements (TEs) are widespread in eukaryotic genomes. The diversity and abundance of TEs are highly variable among species and may correspond to particular relationships between a species and the elements in its genome. There are often many TE families within a single genome; thus, the amplification of one TE family may influence the amplification of other families. LTR retrotransposons (LTR-RTs) are extremely abundant in flowering plants, and Tnt1 is one of the most well known. First characterized in tobacco, Tnt1-related sequences have since been reported in other genera of Solanaceae. In this study, we investigated the profile of Tnt1-related sequences among the species of three Solanaceae genera through genomic amplification and the cloning of partial sequences. The analysis of these sequences revealed high levels of diversity and showed that the sequences are not as closely related to Tnt1 as had been previously hypothesized. The classification of the sequences yielded ten possible families of LTR-RTs, which are, in addition to Tnt1, all members of the Tork clade within the Copia superfamily. However, the sequences did not follow the phylogeny of the species and were not homogeneously distributed. One family includes only sequences of taxa that inhabit dry areas. These findings were consistent with previous suggestions of an early association of Tnt1-related elements with the evolution of several Solanaceae species.     

Conserved epitopes on plant H1 histones recognized by monoclonal antibodies

A series of monoclonal antibodies specific for distinct regions of H1 histone from the plant Nicotiana tabacum were obtained from fusion experiments with spleen cells of mice immunized with tobacco nuclear extracts. These monoclonal antibodies were characterized and the evolutionary conservation of the epitopes in higher plants and animals studied by immunoblotting and enzyme-linked immunosorbent assay (ELISA). Whereas some epitopes appear restricted to the Solanaceae plant family, others are common to all higher eukaryotes tested and even detectable on nuclear proteins of yeast. ELISA experiments performed with isolated tobacco chromatin give some indications of the differential accessibility of the epitopes after interaction of H1 histone with the nucleosome.     

A protein induced by NaCl in suspension cultures ofNicotiana tabacum accumulates in whole plant roots

Summary We have characterized a 26 000 dalton (26 000 D) protein which accumulates inNicotiana tabacum cuspension cells grown in media containing 10–25 g/l NaCl (7, 11, 17). Antibody was prepared against this protein and used to examine protein accumulation in both suspension cells and whole plants. Western blot analysis revealed that the 26 000 D protein also accumulates in suspension cells grown in the absence of NaCl as they approach stationary phase but the accumulation never reaches the level seen in the salt adapted cells. This protein also accumulates after treatment with other agents which lower the water potential, such as PEG and KCl, but no increase is seen after nonosmotic stresses such as heat shock and growth in cadmium chloride. The 26 000 D protein is found not only in whole tobacco plants but also in other members of the Solanaceae that were tested, as well as in alfalfa and green beans. The accumulation of the protein seems to be tissue specific as there is considerably more accumulation in roots than in stems or leaves of greenhouse grown plants. We have been unable to correlate accumlation of the 26 000 D protein with salt in wild tomato species but have demonstrated an increase in the accumulation of this protein with salt stress in hydroponically grown tomato plants. These results lead to speculation as to the role of this protein in responding to lowered water potential in the whole plant.     

Indian medicinal plants as antiradicals and DNA cleavage protectors

Celastrus paniculatus L. (Celastraceae) (CP), Picrorhiza kurroa L. (Scrophulariaceae) (PK) and Withania somnifera L. (Solanaceae) (WS) are Indian medicinal plants having a remarkable reputation, as a factor of health care, among the indigenous medical practitioners. The plants exhibit varying degrees of therapeutic value some of which useful in the treatment of cognitive dysfunction, epilepsy, insomnia, rheumatism, gout, dyspepsia. In this work, we have investigated the free radical scavenging capacity of methanolic extracts from CP, PK, WS and the effect on DNA cleavage induced by H2O2 UV-photholysis. In addition, we investigated whether these plant extracts are capable of reducing the hydrogen peroxide-induced cytotoxicity and DNA damage in human non-immortalized fibroblasts. These extracts showed a dose-dependent free radical scavenging capacity and a protective effect on DNA cleavage; methanolic extracts from PK was more active than extracts from CP and WS. These results were confirmed by a significant protective effect on H2O2-induced cytoxicity and DNA damage in human non-immortalized fibroblasts. These antioxidant effects of active principle of CP, PK and WS may explain, at least in part, the reported anti-stress, immunomodulatory, cognition-facilitating, anti-inflammatory and antiaging effects produced by them in experimental animal and in clinical situations and may justify the further investigation of their other beneficial biological properties.     

一株内生砖红镰刀菌促进烟草生长和增强青枯病抗性

课题组前期报道了一株对马铃薯具有促生防病作用的内生砖红镰刀菌Fusarium lateritium (FL617)。为拓展该菌株的应用范围,本研究以同为茄科作物的烟草为研究对象,探究了砖红镰刀菌对其生长和抗病的影响。结果表明,与对照组相比,处理组叶表面积、主根数、叶片数、叶绿素a和叶绿素b含量分别提高了5.0、3.9、1.4、1.3和1.3倍;该结果表明砖红镰刀菌对烟草具有促生作用。生测结果表明,砖红镰刀菌增强了烟草对青枯病的抗病性,其青枯病病情指数下降约30%。植物激素合成相关基因表达模式分析,发现处理组植物激素合成相关基因表达显著上调(1.6-39.9倍);用青枯病菌Ralstonia solanacearum感染寄主植物后分析其水杨酸(SA)、茉莉酸(JA)和R基因信号相关基因的转录模式,发现与对照组相比,处理组SA、JA相关基因均显著上调(1.2-8.3倍),仅有一个R基因显著下调(50%)。进一步用GFP标记的菌株进行荧光定殖观察,发现植物根系周围簇生着带有绿色荧光信号的真菌菌丝,表明砖红镰刀菌可以定殖于烟草根系。综上所述,推测砖红镰刀菌F. lateritium能够通过定殖于烟草根系介导植物激素、免疫防御相关基因的表达从而影响植株的生长发育和抗病性。     

Dependence on host constituents controlling food acceptance by Manduca sexta larvae

The host range of Manduca sexta L. (Lepidoptera: Sphingidae) is restricted in nature to plants in the family Solanaceae. However, naive hatchling larvae often accept and continue their development on foliage from a wide spectrum of unrelated plants. In contrast, solanaceous-experienced larvae refuse to feed on other plants. Experiments were designed to explore the role of constituents of various plants in this behavioral phenomenon. Fourth instar larvae reared on solanaceous hosts: tomato (Lycopersicon esculentum Mill.), potato (Solanum tuberosum L.), or tobacco (Nicotiana tabacum L.), and the leguminous non-host cowpea (Vigna sinensis Savi), or on artificial diet based on wheat germ were tested for their acceptance or preference when offered a new diet in both choice and no-choice situations. Under no-choice conditions, acceptability of cowpea foliage was strongly affected by the larval dietary experience. Most larvae reared on solanaceous foliage did not feed, whereas those larvae reared on non-solanaceous food readily accepted cowpea. Moreover, solanaceous foliage, leaf discs and extracts were readily accepted by larvae regardless of their dietary experience. Larvae reared on any of the solanaceous plants strongly preferred cowpea discs that were treated with solanaceous extract, while larvae reared on non-solanaceous diets did not discriminate between treated and control discs. Assays of cowpea extracts as well as acceptance of cowpea discs treated with solanaceous extract indicated a lack of detectable deterrent in cowpea. Our results suggest that the mechanism for induced host specificity in M. sexta larvae involves development of a dependence on solanaceous chemical constituents. We argue that this dependence on host plant chemistry has adaptive significance.     

Aminoguanidine-mediated inactivation and alteration of neuronal nitric-oxide synthase

It is established that aminoguanidine (AG) is a metabolism-based inactivator of the three major isoforms of nitric-oxide synthase. AG is thought to be of potential use in diseases, such as diabetes, where pathological overproduction of NO is implicated. We show here that during the inactivation of neuronal nitric-oxide synthase (nNOS) by AG that the prosthetic heme is altered, in part, to dissociable and protein-bound adducts. The protein-bound heme adduct is the result of cross-linking of the heme to residues in the oxygenase domain of nNOS. The dissociable heme product is unstable and reverts back to heme upon isolation. The alteration of the heme is concomitant with the loss in the ability to form the ferrous-CO complex of nNOS and accounts for at least two-thirds of the activity loss. Studies with [(14)C]AG indicate that alteration of the protein, in part on the reductase domain of nNOS, also occurs but at low levels. Thus, heme alteration appears to be the major cause of nNOS inactivation. The elucidation of the mechanism of inactivation of nNOS will likely lead to a better understanding of the in vivo effects of NOS inhibitors such as AG.     

Solanaceae XIPs are plasma membrane aquaporins that facilitate the transport of many uncharged substrates

Major intrinsic proteins (MIPs) transport water and uncharged solutes across membranes in all kingdoms of life. Recently, an uncharacterized MIP subfamily was identified in the genomes of plants and fungi and named X Intrinsic Proteins (XIPs). Here, we describe the genetic features, localization, expression, and functions of a group of Solanaceae XIPs. XIP cDNA and gDNA were cloned from tobacco, potato, tomato, and morning glory. A conserved sequence motif in the first intron of Solanaceae XIPs initiates an RNA-processing mechanism that results in two splice variants (α and β). When transiently or stably expressed in tobacco plants, yellow fluorescent protein-tagged NtXIP1;1α and NtXIP1;1β were both localized in the plasma membrane. Transgenic tobacco lines expressing NtXIP1;1-promoter-GUS constructs and RT-PCR studies showed that NtXIP1;1 was expressed in all organs. The NtXIP1;1 promoter was mainly active in cell layers facing the environment in all above-ground tissues. Heterologous expression of Solanaceae XIPs in Xenopus laevis oocytes and various Saccharomyces cerevisiae mutants demonstrated that these isoforms facilitate the transport of bulky solutes, such as glycerol, urea, and boric acid. In contrast, permeability for water was undetectable. These data suggest that XIPs function in the transport of uncharged solutes across the cell plasma membrane in specific plant tissues, including at the interface between the environment and external cell layers.     

Age Related Changes from Youth to Adulthood in Rat Brain Cortex: Nitric Oxide Synthase and Mitochondrial Respiratory Function

Age related changes in brain cortex NO metabolism were investigated in mitochondria and cytosolic extracts from youth to adulthood. Decreases of 19%, 40% and 71% in NO production were observed in mitochondrial fractions from 3, 7, and 14 months old rats, respectively, as compared with 1-month-old rats. Decreased nNOS protein expression in 14 months old rats was also observed in mitochondria as compared with the nNOS protein expression in 1-month-old rats. Low levels of eNOS protein expression close to the detection limits and no iNOS protein expression were significantly detected in mitochondrial fraction for both groups of age. NO production in the cytosolic extracts also showed a marked decreasing tendency, showing higher levels than those observed in mitochondrial fractions for all groups of age. In the cytosolic extracts, however, the levels were stabilized in adult animals from 7 to 14 months. nNOS protein expression showed a similar age-pattern in cytosolic extracts for both groups of age, while the protein expression pattern for eNOS was higher expressed in adult rats (14 months) than in young animals. As well as in mitochondrial extracts iNOS protein expression was not significantly detected in cytosolic extracts at any age. RT-PCR assays indicated increased levels of nNOS mRNA in 1-month-old rats as compared with 14 months old rats, showing a similar pattern to that one observed for protein nNOS expression. A different aged pattern was observed for eNOS mRNA expression, being lower in 1-month-old rats as compared with 14 months old animals. iNOS mRNA was very low expressed in both groups of age, showing a residual iNOS mRNA that was not significantly detected. State 3 respiration rates were 78% and 85% higher when succinate and malate-glutamate were used as substrates, respectively, in 14 months rats as compared with 1-month-old rats. No changes were observed in state 4 respiration rates. These results could indicate 1 that nNOS and eNOS mRNA and protein expression can be age-dependent, and confirmed the nNOS origin for the mitochondrial NOS. During rat growth, the respiratory function seems to be modulated by NO produced by the different NOS enzymes: nNOS, eNOS and mtNOS present in the cytosol and in the mitochondria.     

Cloning and functional characterization of a fructan 1-exohydrolase (1-FEH) in the cold tolerant Patagonian species <Emphasis Type="Italic">Bromus pictus</Emphasis>

Protein phosphorylation/dephosphorylation plays critical roles in stress responses in plants. This report presents a comparative characterization of the serine/threonine PP2A catalytic subunit family in Solanum tuberosum (potato) and S. lycopersicum (tomato), two important food crops of the Solanaceae family, based on the sequence analysis and expression profiles in response to environmental stress. Sequence homology analysis revealed six isoforms in potato and five in tomato clustered into two subfamilies (I and II). The data presented in this work show that the expression of different PP2Ac genes is regulated in response to environmental stresses in potato and tomato plants and suggest that, in general, mainly members of the subfamily I are involved in stress responses in both species. However, the differences found in the expression profiles between potato and tomato suggest divergent roles of PP2A in the plant defense mechanisms against stress in these closely related species.