Structure and variability of mammalian peroxisomal membrane proteins.

Peroxisomal membrane proteins (PMPs) from the Swiss-Webster mouse are analyzed and compared to those of rats and humans using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. A purification procedure for fresh mouse, rat, or human biopsy liver which enriches peroxisomal/mitochondrial marker enzyme ratios over 100-fold is characterized. When analyzed by SDS-PAGE, membranes of purified liver peroxisomes are shown to contain the same complement of 145-, 70-, 55-, 36-, and 22-kDa PMPs in rats, mice, and humans. A rabbit polyclonal antibody raised against mouse peroxisomal membranes demonstrates immunoreactivity to 145- and 70-kDa proteins in fresh liver homogenates from all three species and in control or Zellweger syndrome fibroblasts from humans. Human autopsy or placental tissues which were refrigerated before analysis exhibited 105-, 55-, and 36-kDa peptides which may be derived from the 145- and 70-kDa peptides. Such conversions, if related to degradation, may explain difficulties in purifying peroxisomes from human autopsy specimens. Variable amounts of the 55-kDa peptide also occurred in mouse adrenal and lung, and the conversion of higher to lower molecular weight PMPs could not be demonstrated by in vitro incubation of mouse liver. Further definition of the structure and variability of mammalian PMPs should be helpful in understanding polyenzymopathies such as Zellweger syndrome.     

Evaluation of the role of the endoplasmic reticulum-Golgi transit in the biogenesis of peroxisomal membrane proteins in wild type and peroxisome biogenesis mutant CHO cells

Peroxisomes are thought to be formed by division of pre-existing peroxisomes after the import of newly synthesized proteins. However, it has been recently suggested that the endoplasmic reticulum (ER) provides an alternative de novo mechanism for peroxisome biogenesis in some cells. To test a possible role of the ER-Golgi transit in peroxisome biogenesis in mammalian cells, we evaluated the biogenesis of three peroxisomal membrane proteins (PMPs): ALDRP (adrenoleukodystrophy related protein), PMP70 and Pex3p in CHO cells. We constructed chimeric genes encoding these PMPs and green fluorescent protein (GFP), and transiently transfected them to wild type and mutant CHO cells, in which normal peroxisomes were replaced by peroxisomal membrane ghosts. The expressed proteins were targeted to peroxisomes and peroxisomal ghosts correctly in the presence or absence of Brefeldin A (BFA), a drug known to block the ER-Golgi transit. Furthermore, low temperature did not disturb the targeting of Pex3p-GFP to peroxisomes. We also constructed two chimeric proteins of PMPs containing an ER retention signal "DEKKMP": GFP-ALDRP-DEKKMP and myc- Pex3p-DEKKMP. These proteins were mostly targeted to peroxisomes. No colocalization with an ER maker was found. These results suggest that the classical ER-Golgi pathway does not play a major role in the biogenesis of mammalian PMPs.     

Human protein paucimannosylation: cues from the eukaryotic kingdoms

Paucimannosidic proteins (PMPs) are bioactive glycoproteins carrying truncated α‐ or β‐mannosyl‐terminating asparagine (N)‐linked glycans widely reported across the eukaryotic domain. Our understanding of human PMPs remains limited, despite findings documenting their existence and association with human disease glycobiology. This review comprehensively surveys the structures, biosynthetic routes and functions of PMPs across the eukaryotic kingdoms with the aim of synthesising an improved understanding on the role of protein paucimannosylation in human health and diseases. Convincing biochemical, glycoanalytical and biological data detail a vast structural heterogeneity and fascinating tissue‐ and subcellular‐specific expression of PMPs within invertebrates and plants, often comprising multi‐α1,3/6‐fucosylation and β1,2‐xylosylation amongst other glycan modifications and non‐glycan substitutions e.g. O‐methylation. Vertebrates and protists express less‐heterogeneous PMPs typically only comprising variable core fucosylation of bi‐ and trimannosylchitobiose core glycans. In particular, the Manα1,6Manβ1,4GlcNAc(α1,6Fuc)β1,4GlcNAcβAsn glycan (M2F) decorates various human neutrophil proteins reportedly displaying bioactivity and structural integrity demonstrating that they are not degradation products. Less‐truncated paucimannosidic glycans (e.g. M3F) are characteristic glycosylation features of proteins expressed by human cancer and stem cells. Concertedly, these observations suggest the involvement of human PMPs in processes related to innate immunity, tumorigenesis and cellular differentiation. The absence of human PMPs in diverse bodily fluids studied under many (patho)physiological conditions suggests extravascular residence and points to localised functions of PMPs in peripheral tissues. Absence of PMPs in Fungi indicates that paucimannosylation is common, but not universally conserved, in eukaryotes. Relative to human PMPs, the expression of PMPs in plants, invertebrates and protists is more tissue‐wide and constitutive yet, similar to their human counterparts, PMP expression remains regulated by the physiology of the producing organism and PMPs evidently serve essential functions in development, cell–cell communication and host–pathogen/symbiont interactions. In most PMP‐producing organisms, including humans, the N‐acetyl‐β‐hexosaminidase isoenzymes and linkage‐specific α‐mannosidases are glycoside hydrolases critical for generating PMPs via N‐acetylglucosaminyltransferase I (GnT‐I)‐dependent and GnT‐I‐independent truncation pathways. However, the identity and structure of many species‐specific PMPs in eukaryotes, their biosynthetic routes, strong tissue‐ and development‐specific expression, and diverse functions are still elusive. Deep exploration of these PMP features involving, for example, the characterisation of endogenous PMP‐recognising lectins across a variety of healthy and N‐acetyl‐β‐hexosaminidase‐deficient human tissue types and identification of microbial adhesins reactive to human PMPs, are amongst the many tasks required for enhanced insight into the glycobiology of human PMPs. In conclusion, the literature supports the notion that PMPs are significant, yet still heavily under‐studied biomolecules in human glycobiology that serve essential functions and create structural heterogeneity not dissimilar to other human N‐glycoprotein types. Human PMPs should therefore be recognised as bioactive glycoproteins that are distinctly different from the canonical N‐glycoprotein classes and which warrant a more dedicated focus in glycobiological research.     

Novel approaches to plant drug discovery based on high throughput pharmacological screening and genetic manipulation

Plants are potentially important for novel therapeutic drug leads, but the slowness of conventional methods for investigation of plants limits enthusiasm in the pharmaceutical industry. To overcome some of the drawbacks, we have applied high throughput pharmacological screening (HTPS) to crude plant extracts. Using a "differential smart screen", (DSS) the spectrum of activity contained in a crude extract is measured at several closely related receptor subtypes. This spectrum is then compared to that of known compounds. A unique spectrum suggests that the extract merits further investigation. Evaluation of species and environmental libraries of whole plants has demonstrated the value of this approach for rapid prioritization of plants for investigation. In addition, genomic and genetic manipulation of plants and plant cell cultures can increase the value of DSS. For example, the whole genomic potential of a plant species for biodiversity can be accessed by using gain of function mutations to generate a "functional genomics library" of mutant clonal cultures, and the bioactivity of these cultures tested by DSS. Clones that overproduce activity differing from the wild-type plant can be identified in this way. This "Natural Products Genomics" (NPG) strategy is limited by the massive numbers of clonal cultures that are required to cover all possible gain-of-function mutations. The rapidity and efficiency of this process can be improved by using transgenic plants expressing appropriate mammalian proteins. These may be designed to make the plant cell resemble a human cell for a specific form of toxicity. Now, "unnatural selection" of resistant mutant clones can be used to provide a sub-population potentially enriched in useful compounds. Alternatively, transgenic plant cells can be used for "in situ screening" in which a mammalian receptor protein, linked to a reporter construct, such as green fluorescent protein, is expressed. Clonal cultures that produce ligands for this receptor can now be rapidly identified visually in an ultra-HTPS. Overall, our aim is to use pharmacological screening, together with functional genomic approaches, to make plant drug discovery competitive with combinatorial chemistry.     

Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce

Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for the generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties owing to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that are effective, safe, low cost, and amenable to large-scale manufacturing.     

Immunoelectron microscopic evidence for organ differences in the composition of peroxisome-specific membrane polypeptides among three rat organs: liver, kidney, and small intestine

We examined the distribution of peroxisome-specific membrane polypeptides (PMPs) among peroxisomes of the liver, renal cortex, and jejunal mucosa, using antibodies for 70 KD, 26 KD and 22 KD PMPs. Immunoblot analysis showed signals for 70 KD polypeptide in all three kinds of tissue, but for the other two only in the liver and renal cortex, with neither being detected in jejunal mucosa. The total amounts of PMPs increased in all three organs with DEHP (di-(2-ethylhexyl)phthalate) administration. By immunoelectron microscopic analysis using protein A-gold, the three PMPs were localized along the peroxisomal membrane. Quantitation of the gold particles associated with the peroxisomal membrane showed an increase in the density of 70 KD and 26 KD PMPs but a decrease in 22 KD PMP with the administration of DEHP. The presence of tissue-specific localizations of PMPs suggest the 70 KD PMP is a common constituent of peroxisomes of these three tissues, whereas 26 KD and 22 KD PMPs are absent in microperoxisomes of jejunal mucosal epithelium.     

The ER-peroxisome connection in plants: development of the "ER semi-autonomous peroxisome maturation and replication" model for plant peroxisome biogenesis

The perceived role of the ER in the biogenesis of plant peroxisomes has evolved significantly from the original "ER vesiculation" model, which portrayed co-translational import of proteins into peroxisomes originating from the ER, to the "ER semi-autonomous peroxisome" model wherein membrane lipids and post-translationally acquired peroxisomal membrane proteins (PMPs) were derived from the ER. Results from more recent studies of various plant PMPs including ascorbate peroxidase, PEX10 and PEX16, as well as a viral replication protein, have since led to the formulation of a more elaborate "ER semi-autonomous peroxisome maturation and replication" model. Herein we review these results in the context of this newly proposed model and its predecessor models. We discuss also key distinct features of the new model pertaining to its central premise that the ER defines the semi-autonomous maturation (maintenance/assembly/differentiation) and duplication (division) features of specialized classes of pre-existing plant peroxisomes. This model also includes a novel peroxisome-to-ER retrograde sorting pathway that may serve as a constitutive protein retrieval/regulatory system. In addition, new plant peroxisomes are envisaged to arise primarily by duplication of the pre-existing peroxisomes that receive essential membrane components from the ER.     

Multiple Domains in PEX16 Mediate Its Trafficking and Recruitment of Peroxisomal Proteins to the ER

Peroxisomes rely on a diverse array of mechanisms to ensure the specific targeting of their protein constituents. Peroxisomal membrane proteins (PMPs), for instance, are targeted by at least two distinct pathways: directly to peroxisomes from their sites of synthesis in the cytosol or indirectly via the endoplasmic reticulum (ER). However, the extent to which each PMP targeting pathway is involved in the maintenance of pre‐existing peroxisomes is unclear. Recently, we showed that human PEX16 plays a critical role in the ER‐dependent targeting of PMPs by mediating the recruitment of two other PMPs, PEX3 and PMP34, to the ER. Here, we extend these results by carrying out a comprehensive mutational analysis of PEX16 aimed at gaining insights into the molecular targeting signals responsible for its ER‐to‐peroxisome trafficking and the domain(s) involved in PMP recruitment function at the ER. We also show that the recruitment of PMPs to the ER by PEX16 is conserved in plants. The implications of these results in terms of the function of PEX16 and the role of the ER in peroxisome maintenance in general are discussed.      

转基因植物表达药用蛋白的研究进展

基因工程技术的进步使得转基因植物广泛应用于工业、农业各个领域,尤其在医药制造领域。研究成果表明,转基因植物作为生物反应器在制备药用蛋白,如重组疫苗、重组动物抗体、细胞因子等方面较其他表达系统,如微生物及动物表达系统具有成本低、应用安全等优势,但在工业化技术方面仍存在障碍。     

Moss bioreactors producing improved biopharmaceuticals

Plants may serve as superior production systems for complex recombinant pharmaceuticals. Current strategies for improving plant-based systems include the development of large-scale production facilities as well as the optimisation of protein modifications. While post-translational modifications of plant proteins generally resemble those of mammalian proteins, certain plant-specific protein-linked sugars are immunogenic in humans, a fact that restricts the use of plants in biopharmaceutical production so far. The moss Physcomitrella patens was developed as a contained tissue culture system for recombinant protein production in photo-bioreactors. By targeted gene replacements, moss strains were created with non-immunogenic humanised glycan patterns. These were proven to be superior to currently used mammalian cell lines in producing antibodies with enhanced effectiveness.     

Human Peroxin PEX3 Is Co‐translationally Integrated into the ER and Exits the ER in Budding Vesicles

The long‐standing paradigm that all peroxisomal proteins are imported post‐translationally into pre‐existing peroxisomes has been challenged by the detection of peroxisomal membrane proteins (PMPs) inside the endoplasmic reticulum (ER). In mammals, the mechanisms of ER entry and exit of PMPs are completely unknown. We show that the human PMP PEX3 inserts co‐translationally into the mammalian ER via the Sec61 translocon. Photocrosslinking and fluorescence spectroscopy studies demonstrate that the N‐terminal transmembrane segment (TMS) of ribosome‐bound PEX3 is recognized by the signal recognition particle (SRP). Binding to SRP is a prerequisite for targeting of the PEX3‐containing ribosome?nascent chain complex (RNC) to the translocon, where an ordered multistep pathway integrates the nascent chain into the membrane adjacent to translocon proteins Sec61α and TRAM. This insertion of PEX3 into the ER is physiologically relevant because PEX3 then exits the ER via budding vesicles in an ATP‐dependent process. This study identifies early steps in human peroxisomal biogenesis by demonstrating sequential stages of PMP passage through the mammalian ER.      

The adult mouse and human pancreas contain rare multipotent stem cells that express insulin

The search for putative precursor cells within the pancreas has been the focus of extensive research. Previously, we identified rare pancreas-derived multipotent precursor (PMP) cells in the mouse with the intriguing capacity to generate progeny in the pancreatic and neural lineages. Here, we establish the embryonic pancreas as the developmental source of PMPs through lineage-labeling experiments. We also show that PMPs express insulin and can contribute to multiple pancreatic and neural cell types in vivo. In addition, we have isolated PMPs from adult human islet tissue that are also capable of extensive proliferation, self-renewal, and generation of multiple differentiated pancreatic and neural cell types. Finally, both mouse and human PMP-derived cells ameliorated diabetes in transplanted mice. These findings demonstrate that the adult mammalian pancreas contains a population of insulin(+) multipotent stem cells and suggest that these cells may provide a promising line of investigation toward potential therapeutic benefit.     

N-Glycosylation in the Moss Physcomitrella patens is Organized Similarly to that in Higher Plants

Abstract: Allergenicity of plant glycoproteins in humans may prevent the use of plants as production factories for pharmaceutically important proteins. The major difference between plant and mammalian N-glycans is the presence of xylosyl and α1,3-fucosyl residues in the former. In a first step towards "humanization" of the N-glycosylation pathway in the moss Physcomitrella patens, which could be an excellent system for industrial production of therapeutic proteins, we isolated the cDNAs and genes for N-acetylglucosaminyltransferase I (GNTI), α1,3-fucosyltransferase, and β1,2-xylosyltransferase. Sequence analysis revealed that all three proteins are homologous to their counterparts from higher plants, however, the conservation of the primary structure was only 35 - 45 %. The gene encoding the key enzyme of the pathway, gntI, was disrupted in P. patens by homologous recombination. Although the mutation of this gene in mouse or A. thaliana led to a significantly altered pattern of N-glycans, the glycosylation pattern in the gntI knockouts did not differ from that in wild-type moss and was identical to that in higher plants. Protein secretion, analysed in assays with recombinant human VEGF₁₂₁ protein, was not affected in the knockouts. We conclude from our findings that the N-glycosylation pathway in P. patens is identically organized to that in higher plants. However, P. patens probably possesses more than one isoform of GNTI which complicates a straightforward knockout. Therefore, and since complex type structures appear more desirable than oligomannosidic N-glycans, future modifications of the pathway should target α1,3-fucosyltransferase and/or β1,2-xylosyltransferase.     

Pyrrolizidine alkaloids in human diet.

Pyrrolizidine alkaloids are the leading plant toxins associated with disease in humans and animals. Upon ingestion, metabolic activation in liver converts the parent compounds into highly reactive electrophiles capable of reacting with cellular macromolecules forming adducts which may initiate acute or chronic toxicity. The pyrrolizidine alkaloids present a serious health risk to human populations that may be exposed to them through contamination of foodstuffs or when plants containing them are consumed as medicinal herbs. Some pyrrolizidine alkaloids (PA) adducts are persistent in animal tissue and the metabolites may be re-released and cause damage long after the initial period of ingestion. PAs are also known to act as teratogens and abortifacients. Chronic ingestion of plants containing PAs has also led to cancer in experimental animals and metabolites of several PAs have been shown to be mutagenic in the Salmonella typhimurium/mammalian microsome system. However, no clinical association has yet been found between human cancer and exposure to PAs. Based on the extensive reports on the outcome of human exposure available in the literature, we conclude that while humans face the risk of veno-occlusive disease and childhood cirrhosis PAs are not carcinogenic to humans.     

Import of peroxisomal membrane proteins: the interplay of Pex3p- and Pex19p-mediated interactions

In contrast to the molecular mechanisms underlying import of peroxisomal matrix proteins, those involving the transport of membrane proteins remain rather elusive. At present, two targeting routes for peroxisomal membrane proteins (PMPs) have been depicted: class I PMPs are targeted from the cytoplasm directly to the peroxisome membrane, and class II PMPs are sorted indirectly to peroxisomes via the endoplasmic reticulum (ER). In addition, three peroxins--Pex3p, Pex16p, and Pex19p - have been identified as essential factors for PMP assembly in several species including humans: Pex19p is a predominantly cytoplasmic protein that shows a broad PMP-binding specificity; Pex3p serves as the membrane-anchoring site for Pex19p; and Pex16p - a protein absent in most yeasts--is thought to provide the initial scaffold for recruiting the protein import machinery required for peroxisome membrane biogenesis. Remarkably, the function of Pex16p does not appear to be conserved between different species. In addition, significant disagreement exists about whether Pex19p has a chaperone-like role in the cytosol or at the peroxisome membrane and/or functions as a cycling import receptor for newly synthesized PMPs. Here we review the recent progress made in our understanding of the role of two key players in PMP biogenesis, Pex3p and Pex19p.     

Oxidative metabolites and genotoxic potential of mammalian and plant lignans in vitro

Certain plant lignans, e.g. secoisolariciresinol and matairesinol, are converted by the intestinal microflora to the mammalian lignans enterodiol and enterolactone, which are associated with beneficial health effects in humans. The metabolism of both mammalian and plant lignans in animals and humans is poorly understood, and most studies so far have focused on the conjugation of these diphenolic compounds. However, recent studies have demonstrated that mammalian and plant lignans are good substrates for cytochrome p450-mediated reactions, leading to numerous products of aliphatic and aromatic hydroxylation with microsomes in vitro. The current knowledge of the oxidative metabolism of food-related lignans is briefly reviewed in this paper, including published as well as unpublished data from our laboratory. Moreover, data on the genotoxic potential of the mammalian and plant lignans, determined at various endpoints in cultured mammalian cells, are included in this review.     

Evidence for a protein kinase cascade in higher plants. 3-Hydroxy-3-methylglutaryl-CoA reductase kinase.

Protein phosphorylation is well established as a regulatory mechanism in higher plants, but only a handful of plant enzymes are known to be regulated in this manner, and relatively few plant protein kinases have been characterized. AMP-activated protein kinase regulates key enzymes of mammalian fatty acid, sterol and isoprenoid metabolism, including 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. We now show that there is an activity in higher plants which, by functional criteria, is a homologue of the AMP-activated protein kinase, although it is not regulated by AMP. The plant kinase inactivates mammalian HMG-CoA reductase and acetyl-CoA carboxylase, and peptide mapping suggests that it phosphorylates the same sites on these proteins as the mammalian kinase. However, with the target enzymes purified from plant sources, it inactivates HMG-CoA reductase but not acetyl-CoA carboxylase. The kinase is located in the soluble, and not the chloroplast, fraction of leaf cells, consistent with the idea that it regulates HMG-CoA reductase, and hence isoprenoid biosynthesis, in vivo. The plant kinase also appears to be part of a protein kinase cascade which has been highly conserved during evolution, since the kinase is inactivated and reactivated by mammalian protein phosphatases (2A or 2C) and mammalian kinase kinase, respectively. This contrasts with the situation for many other mammalian protein kinases involved in signal transduction, which appear to have no close homologue in higher plants. To our knowledge, this represents the first direct evidence for a protein kinase cascade in higher plants.     

Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose

The plant glycosyltransferases, beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT), are responsible for the transfer of beta1,2-linked xylose and core alpha1,3-linked fucose residues to glycoprotein N-glycans. These glycan epitopes are not present in humans and thus may cause immunological responses, which represent a limitation for the therapeutic use of recombinant mammalian glycoproteins produced in transgenic plants. Here we report the genetic modification of the N-glycosylation pathway in Arabidopsis thaliana plants. Knockout plants were generated with complete deficiency of XylT and FucT. These plants lack antigenic protein-bound N-glycans and instead synthesise predominantly structures with two terminal betaN-acetylglucosamine residues (GlcNAc(2)Man(3)GlcNAc(2)).     

Interactions of target population size,population parameters,and program management on viability of captive populations

When established conservation programs expand and evolve, management practices may become inconsistent with program goals. In the past decade, the American Zoo and Aquarium Association expanded species conservation programs by increasing the number of Species Survival Plans (SSP) and establishing more than 300 new Population Management Plan (PMP) programs. However, limited space in captive breeding facilities forces a competition among SSPs and less intensively managed PMPs. Regional Collection Plans establish priorities and allocate space accordingly by setting target population size for each species; species of high conservation priority (SSPs) are allocated space at the expense of lower priority species (PMPs). Because population size and genetic composition interact to impact population viability, target population size is a significant factor to a population’s prospects for long‐term survival. We examined four population parameters (current population size, target population size, current gene diversity, and mean generation time) for 46 mammalian SSPs and 17 PMPs. Relative to SSPs, PMPs combine smaller current and target population sizes, lower levels of current gene diversity, and shorter mean generation times than SSPs. Thus, the average PMP population can expect to lose gene diversity more rapidly than the average SSP population. PMPs are projected to lose 10% or more of their founding gene diversity, within only 2 years. In contrast, the average SSP population is projected to lose 10% in 40 years. Populations with small current or target population sizes require intensive management to avoid extinction. More intensive genetic management of populations typically designated as PMPs, through recruitment of potential founders and equalization of founder representation, could increase gene diversity and improve viability. Less rigorous population management should be reserved for populations whose long‐term survival is either secure or that can be readily replenished from the wild. Because PMP populations need intense genetic management similar to that currently in effect for SSPs, there should be neither a management‐level distinction between programs nor an arbitrary difference in space allocated to programs. Zoo Biol 20:169–183, 2001. © 2001 Wiley‐Liss, Inc.     

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo.