Subclade of flavin-monooxygenases involved in aliphatic glucosinolate biosynthesis

Glucosinolates (GSLs) are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. We previously identified the flavin-monooxygenase FMO(GS-OX1) as an enzyme in the biosynthesis of aliphatic GSLs in Arabidopsis (Arabidopsis thaliana) that catalyzes the S-oxygenation of methylthioalkyl to methylsulfinylalkyl GSLs. Here, we report the fine mapping of a quantitative trait locus for the S-oxygenating activity in Arabidopsis. In this region, there are three FMOs that, together with FMO(GS-OX1) and a fifth FMO, form what appears to be a crucifer-specific subclade. We report the identification of these four uncharacterized FMOs, designated FMO(GS-OX2) to FMO(GS-OX5). Biochemical characterization of the recombinant protein combined with the analysis of GSL content in knockout mutants and overexpression lines show that FMO(GS-OX2), FMO(GS-OX3), and FMO(GS-OX4) have broad substrate specificity and catalyze the conversion from methylthioalkyl GSL to the corresponding methylsulfinylalkyl GSL independent of chain length. In contrast, FMO(GS-OX5) shows substrate specificity toward the long-chain 8-methylthiooctyl GSL. Identification of the FMO(GS-OX) subclade will generate better understanding of the evolution of biosynthetic activities and specificities in secondary metabolism and provides an important tool for breeding plants with improved cancer prevention characteristics as provided by the methylsulfinylalkyl GSL.     

Cellular and subcellular localization of phototropin 1

Phototropin 1 (phot1) is a Ser/Thr photoreceptor kinase that binds two molecules of flavin mononucleotide as its chromophores and undergoes autophosphorylation in response to blue light. Phot1 is plasma membrane associated and, as with phot2, has been shown to function as a photoreceptor for phototropism, blue light-induced chloroplast movement, and blue light-induced stomatal opening. Phot1 likely also plays a redundant role with phot2 in regulating the rate of leaf expansion. Understanding the mechanism(s) by which phot1 initiates these four different responses requires, at minimum, knowledge of where the photoreceptor is located. Therefore, we transformed a phot1 null mutant of Arabidopsis with a construct encoding translationally fused phot1-green fluorescent protein (GFP) under the control of the endogenous PHOT1 promoter and investigated its cellular and subcellular distribution. This PHOT1-GFP construct complements the mutant phenotype, restoring second positive curvature. Phot1 is expressed strongly in dividing and elongating cortical cells in the apical hook and in the root elongation zone in etiolated seedlings. It is localized evenly to the plasma membrane region in epidermal cells but is confined largely to the plasma membrane region of the transverse cell walls in the cortical cells of both root and hypocotyl. It is found at both apical and basal ends of these cortical cells. In light-grown plants, phot1-GFP is localized largely in the plasma membrane regions adjacent to apical and basal cell end walls in the elongating inflorescence stem, where the photoreceptor is expressed strongly in the vascular parenchyma and leaf vein parenchyma. Phot1 also is localized to the plasma membrane region of leaf epidermal cells, mesophyll cells, and guard cells, where its distribution is uniform. Although phot1 is localized consistently to the plasma membrane region in etiolated seedlings, a fraction becomes released to the cytoplasm in response to blue light. Possible relationships between observed phot1 distribution and the various physiological responses activated by blue light are discussed.     

Cellular and subcellular localization of calcium in gravistimulated corn roots

Summary Antimonate staining procedures and energy dispersive X-ray microanalytical techniques were used to determine the patterns of localization of calcium in nonstimulated and gravistimulated corn roots. In horizontally positioned roots within the region of the developing bend there was a change in the staining from that principally localized within cells of the stele to asymmetric staining within the vacuoles of the cortical cells along the upper root surface. There was little staining in the walls. The pattern observed is quite different from that seen in gravistimulated coleoptiles. Staining of mitochondria, plastids and Golgi stacks was seen in most cell types, but no asymmetry of staining was observed. In the rootcap where graviperception is thought to occur, there was little staining of any cellular organelles.     

Primary structure and subcellular localization of two fimbrial subunit-like proteins involved in the biosynthesis of K99 fibrillae

Analysis of the nucleotide sequence of the distal part of the fan gene cluster encoding the proteins involved in the biosynthesis of the fibrillar adhesin, K99, revealed the presence of two structural genes, fanG and fanH. The amino acid sequence of the gene products (FanG and FanH) showed significant homology to the amino acid sequence of the fibrillar subunit protein (FanC). Introduction of a site-specific frameshift mutation in fanG or fanH resulted in a simultaneous decrease in fibrillae production and adhesive capacity. Analysis of subcellular fractions showed that, in contrast to the K99 fibrillar subunit (FanC), both the FanH and the FanG protein were loosely associated with the outer membrane, possibly on the periplasmic side, but were not components of the fimbriae themselves.     

Cellular and subcellular localization of heme oxygenase-2 in monkey retina

Carbon monoxide (CO), an activator of soluble guanylate cyclase (SGC) and generated enzymatically by heme oxygenases (HO), is considered to function as an intra- and intercellular neuromodulator or neurotransmitter in the central and peripheral nervous systems. HO-2 is the constitutive isoform of HO and is more prevalent in nervous tissues than in the other peripheral tissues. Because previous studies have demonstrated different distributions of HO-2 in the retina depending on the species of animals, the aim of this study was to identify which cell types of the monkey retina express HO-2. The expression of HO-2 protein was examined in monkey retina by Western blot analysis. Immunoblottings from monkey homogenates revealed a single clear protein band with a molecular mass of 36 kDa that is corresponding to rat HO-2. Immunoreactivity of HO-2 was found in the perikarya of ganglion cells. Density of immunoreactive ganglion cells was higher in the central area of retina than in the peripheral retina, and somata of larger ganglion cells were stained more densely than smaller ones. In electron microscopy, immunoreactivity of HO-2 was localized on the membrane of the endoplasmic reticulum and the nuclear outer membrane of the ganglion cells. By contrast, inner plexiform layer, inner nuclear layer and outer nuclear layer were devoid of HO-2 immunoreactivity. cGMP were strongly localized in all of ganglion cells. Some cells contributed to the relatively faint cGMP staining were seen in the inner nuclear layer. In combination of HO-2 and cGMP immunocytochemistry, the overlap of co-localization of HO-2 and cGMP would suggest that HO-2 in the ganglion cells would serve as a source for CO generation and CO could serve as a gaseous signaling molecule modulator of neural activity in the retina of monkey.     

Cellular and subcellular 3H-estradiol localization in the pituitary by autoradiography

Summary The cellular and subcellular localization of 6.7-³H-estradiol-17 in the pituitaries of five immature and two mature castrated female rats was studied by autoradiography. The autoradiographic technique used minimizes or eliminates translocation. It is based on low temperature tissue preparation of freeze-dried sections which are dry-mounted on dried photographic emulsion, excluding known sources of translocation artifacts such as liquid fixatives, dehydrating and clearing fluids, embedding media and thawing. The animals were given from 0.093 to 0.63 g of ³H-estradiol and were sacrificed at 15 min, 1, 2, or 6 hours after the injection. A large portion of the anterior pituitary cells was found to be labeled; the extent of this labeling varied with dose, time of sacrifice after the injection, and photographic exposure time, but apparently not with the endocrine status of the animal. The portions labeled were 76 and 86 per cent for the immature and mature rats respectively, exceeding single tinctorial light-microscopic groups. Gomori trichrome chromophiles and chromophobes, cells with intense and weak pyronin basophilia, as well as morphologically defined castration cells, were all partially labeled and unlabeled. Acidophiles appeared to be labeled in a somewhat higher proportion. Cells of the intermediate and posterior lobe were generally unlabeled except for occasionally interspersed cell groups or single cells, especially at the border between intermediate and posterior lobe, probably identical with basophilic invaginations in man and other mammals. The subcellular concentration of radioactivity was always nuclear. The findings are interpreted as suggesting a) feedback control on the pituitary level, in addition to the diencephalic level, b) pluripotentiality of anterior pituitary cells, and c) possible positive feedback mechanism of estradiol with secondary negative effect. Dry-mount autoradiography with labeled hormones, as applied in this study, provides a new methodological approach to the elucidation of pituitary physiology and pharmacology.Supported by USPHS Grants 1-ROl-AM-12, 649-01, GRS Grant FR-5367, ACS Grant IN-41-H, and Otho Sprague Memorial Institutional Grant. — The author thanks Dr. N. S. HALMI for consultation.     

Domains of the TMV movement protein involved in subcellular localization

To identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C-terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild-type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9–11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49–51 may be involved in co-alignment of the protein with microtubules. The region around residues 88–101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.     

COI1参与茉莉酸调控拟南芥吲哚族芥子油苷生物合成过程

芥子油苷是一类具有防御作用的植物次生代谢产物,外源激素茉莉酸对吲哚族芥子油苷的合成具有强烈的诱导作用,但茉莉酸调控吲哚族芥子油苷生物合成的分子机制并不清楚。以模式植物拟南芥(Arabidopsis thaliana)的野生型和coi1-22、coi1-23两种突变体为研究材料,通过茉莉酸甲酯(MeJA)处理,比较了拟南芥野生型和coi1突变体植株吲哚族芥子油苷含量、吲哚族芥子油苷合成前体色氨酸的生物合成基因(ASA1、TSA1和TSB1)、吲哚族芥子油苷生物合成基因(CYP79B2、CYP79B3和CYP83B1)及调控基因(MYB34和MYB51)的表达对MeJA的响应差异,由此确定茉莉酸信号通过COI1蛋白调控吲哚族芥子油苷生物合成,即茉莉酸信号通过信号开关COI1蛋白作用于转录因子MYB34和MYB51,进而调控吲哚族芥子油苷合成基因CYP79B2、CYP79B3、CYP83B1和前体色氨酸的合成基因ASA1、TSA1、TSB1。并且推断,COI1功能缺失后,茉莉酸信号可能通过其他未知调控因子或调控途径激活MYB34转录因子从而调控下游基因表达。     

Cellular and subcellular localization of uncoupling protein 2 in the human kidney

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.     

Cellular and subcellular localization of 3H-diethylstilboestrol in the central nervous system

Summary The cellular and subcellular localization of radioactivity in the brain of immature female rats was determined by dry-mount autoradiography 2 h after iv injection of 1.0 g of (monethyl-³H) diethylstilboestrol per 100 g body weight. A specific topographic pattern of nuclear concentration of the synthetic oestrogen was obtained similar to that for ³H-oestradiol-17 in specific neurons of the basal hypothalamus, preoptic region and amygdala. In competition experiments, the nuclear concentration of radioactivity in all areas studied was inhibited by unlabeled oestradiol, while unlabeled testosterone had no effect. These data suggest that although oestradiol can bind to androgen receptors, the oestrogen receptor itself can account for the localization seen after the injection of ³H-oestradiol.This research was supported in part by US PHS Grant No. NS12933NIH Career Development Awardee No. NS00164The expert technical assistance of Ms. Riki Ison and Ms. Linda Furr is gratefully acknowledged.     

Cellular and subcellular localization of the inhibitory glycine receptor in hippocampal neurons

Inhibitory glycine receptors are most abundant in spinal cord and brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Little is known about the function of glycine receptors in cortex and hippocampus, where GABA plays a dominant role in synaptic inhibition. Therefore, we have investigated tissue and cellular expression of glycine receptor alpha-subunits. Western blot and immunohistochemical analyses reveal the presence of glycine receptors in hippocampal tissue. Immunocytochemical experiments in hippocampal cultures show prominent cellular expression of glycine receptors in pyramidal neurons and GAD-positive interneurons similar to the calcium-binding protein VILIP-1 with widespread hippocampal distribution. On the subcellular level we found co-staining of GlyR and the presynaptic marker synapsin I. Furthermore, co-staining with GAD at synaptic terminals indicated partial co-localization of GABA- and glycine receptors.     

Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity

The peripheral benzodiazepine receptor (PBR) is currently used as a marker of inflammation and gliosis following brain injury. Previous reports suggest that elevated PBR levels in injured brain tissue are specific to activated microglia and infiltrating macrophages. We have produced hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the cellular and subcellular nature of the PBR response. Degenerating, argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14 days after TMT exposure. Reactive microglia were also evident at both times with a maximal response observed at 14 days, subsiding by 6 weeks. Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT administration, suggesting that the onset of the astroglia response is delayed, but more persistent, compared with microgliosis. Morphological evidence from [3H]PK11195 microautoradiography and PBR immunohistochemistry indicates that both astrocytes and microglia are capable of expressing high levels of PBR after injury. This was confirmed by double labeling of either Griffonia simplicifolia isolectin B4, a microglial-specific marker, or glial fibrillary acidic protein, an astrocyte-specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells.     

Immunocytological localization of two enzymes involved in berberine biosynthesis

Using post-embedding immunogold techniques the cytological localization of the two branchpoint enzymes of isoquinoline biosynthesis, berberine bridge enzyme (BBE) and (S)-tetrahydroprotoberberine oxidase (STOX), was demonstrated. Electron-microscopic examination revealed their exclusive compartmentation within vesicles. After these vesicles have fused with the central vacuole, they release their contents, resulting in a characteristic electron-dense precipitate at the tonoplast. Vesicles of similar structure could be identified in young meristematic tissues of roots or shoots from different Berberis species and Papaver somniferum L. The appearance of electron-dense osmiophilic material is strictly correlated with the alkaloid content of the tissue. Immunocytological staining of P. somniferum tissue with antibodies directed against BBE led to a characteristic labeling of electron-dense aggregates in idioblasts that are not connected to the laticifer system. This localization demonstrates the strictly cytological separation of benzophenanthridine and morphine biosyntheses within this plant.     

Farnesylation directs AtIPT3 subcellular localization and modulates cytokinin biosynthesis in Arabidopsis

Cytokinins regulate cell division and differentiation as well as a number of other processes implicated in plant development. The first step of cytokinin biosynthesis in Arabidopsis (Arabidopsis thaliana) is catalyzed by adenosine phosphate-isopentenyltransferases (AtIPT). The enzymes are localized in plastids or the cytoplasm where they utilize the intermediate dimethylallyl-diphosphate from the methylerythritolphosphate or mevalonic acid pathways. However, the regulatory mechanisms linking AtIPT activity and cytokinin biosynthesis with cytokinin homeostasis and isoprenoid synthesis are not well understood. Here, we demonstrate that expression of AtIPT3, one member of the adenosine AtIPT protein family in Arabidopsis, increased the production of specific isopentenyl-type cytokinins. Moreover, AtIPT3 is a substrate of the protein farnesyl transferase, and AtIPT3 farnesylation directed the localization of the protein in the nucleus/cytoplasm, whereas the nonfarnesylated protein was located in the plastids. AtIPT3 gain-of-function mutant analysis indicated that the different subcellular localization of the farnesylated protein and the nonfarnesylated protein was closely correlated with either isopentenyl-type or zeatin-type cytokinin biosynthesis. In addition, mutation of the farnesyl acceptor cysteine-333 of AtIPT3 abolishes cytokinin production, suggesting that cysteine-333 has a dual and essential role for AtIPT3 farnesylation and catalytic activity.     

Cellular and subcellular localization of stathmin during oocyte and preimplantation embryo development

Stathmin is a 19 kDa cytosolic phosphoprotein, proposed to act as a relay integrating diverse intracellular signaling pathways involved in regulation of cell proliferation, differentiation, and function. To gain further information about its significance during early development, we analyzed stathmin expression and subcellular localization in mouse oocytes and preimplantation embryos. RT‐PCR analysis revealed a low expression of stathmin mRNA in unfertilized oocytes and a higher expression at the blastocyst stage. A fine cytoplasmic punctuate fluorescent immunoreactive stathmin pattern was detected in the oocyte, while it evolved toward an increasingly speckled pattern in the two‐cell and later four‐ to eight‐cell embryo, with even larger speckles at the morula stage. In blastocysts, stathmin immunoreactivity was fine and intense in inner cell mass cells, whereas it was low and variable in trophectodermal cells. Electron microscopic analysis allowed visualization with more detail of two types of stathmin immunolocalization: small clusters in the cytoplasm of oocytes and blastocyst cells, together with loosely arranged clusters around the outer membrane of cytoplasmic vesicles, corresponding to the immunofluorescent speckles in embryos until the morula stage. In conclusion, it appears from our results that maternal stathmin is accumulated in the oocyte and is relocalized within the oocyte and early preimplantation embryonic cell cytoplasm to interact with specific cytoplasmic membrane formations. Probably newly synthesized, embryonic stathmin is expressed in the blastocyst, where it is localized more uniformly in the cytoplasm mostly of inner cell mass (ICM) cells. These expression and localization patterns are probably related to the particular roles of stathmin at the successive steps of oocyte maturation and early embryonic development. They further support the proposed physiologic importance of stathmin in essential biologic regulation. Mol. Reprod. Dev. 53:306–317, 1999. © 1999 Wiley‐Liss, Inc.     

Cellular and subcellular localization of catalase in the heart of transgenic mice.

Previous studies have described a cardiac-specific, catalase-overexpressing transgenic mouse model that was used to study myocardial oxidative injury. This study was undertaken to demonstrate cellular and subcellular localization of catalase in the hearts of transgenic mice. By the light microscopic immunoperoxidase method, we found that the overexpressed catalase was exclusively localized in cardiomyocytes. The ratios of immunoreactive cardiomyocytes in the heart were quite different among three transgenic lines examined but agreed with the elevated levels of catalase activity. In the cardiac blood vessels, positive cells were found in the walls of pulmonary veins and the vena cava, which consist of cardiomyocytes, but not in the pulmonary arteries, aorta, or cardiac valves. The electron microscopic immunogold method revealed that the elevated catalase was in sarcoplasm, nucleus, and peroxisomes, but not in mitochondria. In contrast to these distributions, catalase in the non-transgenic cardiomyocytes was in peroxisomes only. In addition, the number and size of peroxisomes in the transgenic cardiomyocytes were markedly increased, but no other ultrastructural changes were observed in comparison with those of non-transgenic mice. These results demonstrated that the elevated catalase in transgenic mouse heart is localized in cardiomyocytes and is distributed to peroxisomal and extraperoxisomal, but not mitochondrial, compartments.     

Cellular and subcellular localization of an Mr 64,000 protein autoantigen in insulin-dependent diabetes

Antibodies to an Mr 64,000 protein from human or rat islets have been detected at high frequency in newly diagnosed insulin-dependent diabetic patients. In this study, we show that the antigenic and amphiphilic properties of the rat islet Mr 64,000 protein resemble that of the human protein. We have analyzed the expression of the Mr 64,000 protein in populations of pancreatic beta and non-beta cells and in selected rat tissues by immunoprecipitation of [35S]methionine-radiolabeled proteins with sera from diabetic patients or from healthy control individuals. When islet cell populations enriched in beta or non-beta cells were tested for the expression of the Mr 64,000 antigen, the protein was primarily observed in the beta cells. On analyzing preparations of islets, liver, kidney, thyroid, adrenal, pituitary, spleen, and thymus, the protein could only be detected in islets. The protein was also characterized in terms of its subcellular localization by Percoll density gradient centrifugation and was recovered in a fraction enriched in the plasma membrane marker, 5'-nucleotidase. These results are consistent with a beta cell-restricted plasma membrane expression of the protein and support the hypothesis that this protein is a target antigen of beta cell-specific autoimmunity in insulin-dependent diabetes.     

Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants

The cellular and subcellular localization of endogenous nitric oxide (NO.) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO. was mainly present in vascular tissues (xylem and phloem). Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. In senescent leaves, NO. generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)(2) and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO. was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO., with g = 2.05 and a(N) = 12.8 G, was detected in peroxisomes. By fluorometry, NO. was also found in these organelles, and the level measured of NO. was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO. from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO. mg(-1) protein min(-1); was strictly dependent on NADPH, calmodulin, and BH(4); and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO. could be involved in the process of senescence of pea leaves.