Purification and characterization of fructan: fructan fructosyl transferase from chicory (Cichorium intybus L.) roots

Fructan: fructan fructosyl transferase (FFT, EC 2.4.1.100) was purified from chicory (Cichorium intybus L. var. foliosum cv. Flash) roots by a combination of ammonium sulfate precipitation, concanavalin A affinity chromatography, and anion- and cation-exchange chromatography. This protocol produced a 60-fold purification and a specific activity of 14.5 mol·(mg protein) ^–1·min^–1. The mass of the enzyme was 69 kDa as estimated by gel filtration. On sodium dodecyl sulfatepolyacrylamide gel electrophoresis and mass spectrometry, 52-kDa and 17-kDa fragments were found, suggesting that the enzyme was a heterodimer. Optimal activity was found between pH 5.5 and 6.5. The enzyme used 1-kestose, 1,1-nystose, oligofructan and commercial chicory root inulin (degree of polymerization 10) as donors and acceptors. Sucrose was the best acceptor but could not be used as a donor. However, at higher concentrations sucrose acted as a competitive inhibitor for donors of FFT. 1-Kestose was the most efficient and 1,1-nystose the least efficient donor. The purified enzyme exhibited -fructosidase activity, specially at higher temperatures and lower substrate concentrations. The synthesis of fructans from 1-kestose decreased at higher temperatures (5–50°C). Therefore enzyme assays were performed at 0°C. The same fructan oligosaccharides, with a distribution similar to that observed in vivo, were obtained upon incubation of the enzyme with sucrose and commercial chicory root inulin.Abbreviations Con A concanavalin A - DP degree of polymerization - FFT fructan: fructan fructosyl transferase - Fru fructose - Glc glucose - Kes 1-kestose - MALDI-TOF MS matrix-assisted laser desorption ionisation time of flight mass spectrometry - Nys 1,1-nystose - pI isoelectric point - SST sucrose: sucrose fructosyl transferase - Suc sucrose The authors would like to thank E. Nackaerts for valuable assistance. W. Van den Ende is also grateful to the National Fund for Scientific Research (NFSR Belgium) for giving a grant for research assistants. P. Verhaert is a research associate of the NFSR. This work was also supported by grant OT/91/18 from the Research Fund K.U. Leuven.     

C-terminal polypeptides are necessary and sufficient for in vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured plant cells

Summary The potential of tobacco BY-2 suspension-cultured cells for examining in vivo targeting and import of proteins into plant peroxisomes was shown recently in our laboratory. In the current study, the necessity and sufficiency of putative C-terminal targeting signals on cottonseed malate synthase and bacterial chloramphenicol acetyl-transferase (CAT) were examined in BY-2 cells. Cotton suspension cells also were evaluated as another in vivo peroxisome targeting system. Ultrastructural views of BY-2 cells showed that the peroxisomes were relatively small (0.1-0.3 m diameter), a characteristic of so-called unspecialized peroxisomes, Peroxisomes in cotton and tobacco cells were identified with anti-cottonseed catalase IgGs as distinct immunofluorescent particles clearly distinguishable from abundant immunofluorescent mitochondria and plastids, marked with antibodies to -ATPase and stearoyl-ACP 9 desaturase, respectively. The C-terminal ser-lys-leu (SKL) motif is a well-established peroxisome targeting signal (PTS 1) for mammals and yeasts, but not for plants. Antiserum raised against SKL peptides recognized proteins only in peroxisomes in cotton and tobacco cells. The necessity of SKL-COOH for targeting of proteins to plant peroxisomes had not been demonstrated; we showed that SKL-COOH was necessary for directing cottonseed malate synthase to BY-2 peroxisomes. KSRM-COOH, a conservative modification of SKL-COOH, was shown by others to be sufficient for redirecting CAT in stably-transformed Arabidopsis plants to the leaf peroxisomes. Here we show with the same CAT constructs (e.g., pMON316CAT-KSRM) that KSRM is sufficient for targeting transiently-expressed passenger proteins to unspecialized BY-2 peroxisomes. These results provide new direct evidence for the necessity of SKL-COOH (a type 1 PTS) and sufficiency of a conservative modification of the PTS 1 (KSRM-COOH) for in vivo, heterologous targeting of proteins to plant peroxisomes.Abbreviations CAT chloramphenicol acetyltransferase - CHO cells Chinese hamster ovary cells - DAB 3,3-diaminobenzidine - GUS -glucuronidase - ICL isocitrate lyase - KSRM lysine-serine-arginine-methionine - MS malate synthase - PBS phosphate-buffered saline - PTS peroxisome targeting signal - SKL serine-lysine-leucine - tobacco BY-2 Bright Yellow-2 Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Hormonal regulation of iron-stress response in sunflower roots: a morphological and cytological investigation

Summary Sunflowers are known to respond to Fe deficiency (-Fe) with a typical root tip swelling and the formation of root hairs and transfer cells in the rhizodermis. The possible regulation of this process was examined by a comparative study of root morphology and cytology of intact seedlings (Helianthus annuus L. cv. Giganteus) under -Fe and hormonal treatment in nutrient solution. Longitudinal sections of -Fe roots showed root tip swelling is due to cessation of cell elongation and isodiarnetric volume increase of the cortical cells. Enhanced cell division in the pericycle leads to the formation of lateral root primordia in the swollen zone. Xylem vessel differentiation is markedly accelerated and accompanied by early differentiation of the casparian band in the endodermis. Exogenous application of IAA (10^–8-10^–7 M) via the nutrient solution to Fe sufficient plants causes symptoms which closely mimick the characteristics of Fe deficiency including root hair development. Moreover, rhizodermal cells produce peripheral protuberances reminiscent of -Fe transfer cells. Ethylene-releasing ethephon (10^–4M) also causes subapical swelling and root hair formation. However, wall protuberance development is less pronounced. ABA (10^–5 M) leads to similar root thickening and root hair formation but without any comparable transfer cell differentiation. From the striking similarities between -Fe and IAA treatment it is concluded that this hormone (possibly in cooperation with ethylene) is involved in the Fe stress response of sunflower roots. The importance of a continuous polar IAA transport for this process is discussed.Abbreviations ABA abscisic acid - ACC 1-aminocyclopropane-1-carboxylic acid - Ethephone 2-chloro-ethylphosphonic acid - Fe(III)-EDTA ethylenediaminetetraacetic ferric-sodium salt - IAA indole-acetic acid - TIBA triiodobenzoic acid     

Detection and characterization of a glutenin subunit with unusual high Mr at the Glu-A1 locus in hexaploid wheat

A hexaploid wheat landrace collected from the Baluchistan province of Pakistan was found to possess a novel high-molecular-weight glutenin subunit (HMW-GS). The subunit has a very slow electrophoretic mobility as revealed by SDS-PAGE, and its molecular weight is comparable to that of the highest molecular weight glutenin subunit (2.2 encoded in the D-genome) reported so far in hexaploid wheat varieties and landraces of Japanese origin. Evidence obtained from (PCR) gene amplification studies using the primers specific for Glu-1 loci proved that the gene coding for this novel subunit belongs to the Glu-A1 locus located on the long arm of chromosome 1A. Digestion of the amplified gene (PCR product) with restriction enzymes indicated that the novel gene differs from prevailing Glu-A1 alleles (null, 1 and 2^*) by an extra DNA fragment of approximately 600 base pairs. The results also indicated that the novel subunit is most probably a derivative of subunit 2^* that has very likely incorporated the 600-bp fragment following a process of unequal crossing over. The present findings were further substantiated by reserved phase high performance liquid chromatography (RP-HPLC) analysis.     

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha-1-antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule-associated protein-2 (MAP-2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.     

Modulation of catecholamine storage and release by the pituitary-interrenal axis in the rainbow trout,Oncorhynchus mykiss

This study examined the effects of pituitary-interrenal hormones on catecholamine storage and release in the rainbow trout Oncorhynchus mykiss. An extract of trout pituitary elicited the release of adrenaline, but not noradrenaline, using an in situ perfusion preparation. A variety of doses of adrenocorticotropic hormone (2–2000 mU) caused the release of both catecholamines in situ which was unaffected by pre-treatment with the ganglion blocker, hexamethonium, or the serotonergic receptor antagonist, methysergide, but was abolished in calcium-free media. Intra-arterial injections of adrenocorticotrophic hormone in vivo caused an elevation of plasma adrenaline but not noradrenaline levels. Injections of cortisol in situ did not elicit catecholamine release. Trout given an intraperitoneal implant of cortisol (50 mg·kg^-1 body weight) had significantly higher plasma cortisol concentrations when compared to controls after 7 days of implantation. Increases in the levels of stored catecholamines were observed in various regions of the kidney and posterior cardinal vein following 3 and 7 days of cortisol treatment. The ability of the chromaffin cells to release catecholamines in response to cholinergic stimulation was assessed in situ after 7 days of treatment. Basal (non-stimulated) adrenaline outflowing perfusate levels were greater in the cortisol-treated fish. Cortisol treatment increased the responsiveness of the catecholamine release process to low doses of the cholinoceptor agonist carbachol. Three or 7 days of cortisol treatment did not alter the in vitro activity of the enzyme phenylethanolamine-N-methyl-transferase. The results of this study demonstrate that interactions within the pituitary-adrenal axis can influence both catecholamine storage and release in the rainbow trout.Abbreviations ACTH adrenocorticotropic hormone - AK anterior third of the kidney - APCV anterior third of the PCV - HPLC high performance liquid chromatography - MK middle third of the kidney - M1 maximum value - MPCV middle third of the PCV - MS222 ethyl-aminobenzoate - P1 pre value - PCA perchloric acid - PCV posterior cardinal vein - PK posterior third of the kidney - PNMT phenylethanolamine-N-methyltransferase - PPCV posterior third of the PCV - rbc red blood cells - SEM standard error of the mean - TK total kidney (i.e. the sum of the AK, MK, and PK) - TPCV total PCV (i.e. the sum of the APCV, MPCV and PPCV)     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

Intravascular granuloma induced by intravenous inoculation ofCryptococcus neoformans

In rodents an intravenous administration of viableCryptococcus (C.) neoformans cells frequently resulted in attachment of intravascular cryptococcal granulomas to inner walls of the large to medium-sized veins of various organs, including the lungs, liver and spleen. In order to elucidate the pathogenesis of granulomatous changes, the cells composing the intravascular granulomas were observed by electron microscopic peroxidase (PO) cytochemistry. The granuloma composing cells could be divided into the following four types according to the pattern of endogenous peroxidase activity: exudate macrophage (Mφ, type I), PO-negative Mφ (type II), resident Mφ (type III) and other inflammatory cells (type IV). In the intravenous granulomas of the lung, the percentages of composed cells were 39.0% for type I, 57.9% for type II, 0% for type III and 3.1% for type IV. By contrast, in the interstitial granulomas in the lung, type III Mφs, possibly derived from alveolar Mφs, played a significant role in granuloma formation. This may indicate that the intravascular granuloma is almost composed of macrophages derived from monocytes rather than alveolar macrophages. The expression of ICAM-1 on endothelia of the pulmonary veins was examined by immunoelectron microscopy. An immunogold labeling index was significantly augmented on the surface of endothelia in response to intravenous challenge ofC. neoformans. The intravascular granuloma demonstrates that the monocytes develop into the granuloma-composing macrophages and suppress the cryptococcal activities even hi the peripheral blood resulting in an assistance of endothelial functions.     

Mixed connective tissue disease. A clinico-serological study of 17 cases

Mixed connective tissue disease (MCTD) was described as a distinct clinical syndrome in 1972. Since then many cases have been reported in the literature worldwide. In this study we present our experience with a group of 17 Mexican patients with this syndrome, and we analyze their clinical and serological features, as well as the causes of death in these patients. The patients are Mexican mestizos living in Guadalajara and most of them have been followed-up at Hospital General de Occidente for a period of 1–10 years. The female/male ratio was 16:1, and their age ranged from 14–55 years with a mean of 29 years. The disease duration has ranged from 1–17 years, with a mean of 6 years. Among the clinical manifestations we have found a high frequency of lymphadenopathy when compared with published series (13/17 or 76%), and the laboratory findings in our patients included a very high polyclonal increase of gammaglobulins (93%), lymphopenia (76%), direct immunofluorescence speckled nuclear epidermal deposits in skin biopsies (75%) and positive rheumatoid factor (65%). Other clinical and serological features were similar to those reported in other series of patients with MCTD. Six of the 17 patients have died (35%), and in 3 of them (17.5%) the cause of death was due to an infectious disease that suddenly presented, and apparently was not related to a concomitant high dose of steroids or malnutrition in the patients. It seems that in addition to the already well known autoimmune abnormalities that occur in MCTD, there are other features like the presence of lymphadenopathy, the high polyclonal increase of gammaglobulins, and the lymphopenia, that reflect the profound disturbance of the immune system in this syndrome, possibly contributing to the sudden appearance of a severe infectious disease in some of our patients.Abbreviations ANA antinuclear antibody - CIE counterimmunoelectrophoresis - MCTD mixed connective tissue disease - PHA passive hemagglutination - PM polymyositis - RF rheumatoid factor - SLE systemic lupus erythematosus - SS systemic sclerosis (SS)     

Isolation of a gene essential for biosynthesis of the lipopeptide antibiotics plipastatin B1 and surfactin in Bacillus subtilis YB8

Bacillus subtilis YB8 was found to produce the lipopeptide antibiotics surfactin and plipastatin B1. A gene, lpa-8, required for the production of both lipopeptides was cloned from strain YB8. When this gene was inactivated in strain YB8, neither surfactin nor plipastatin B1 was produced. However, the defective strain transformed with an intact lpa-8 gene had restored ability to produce both peptides. Nucleotide sequence analysis of the region essential for the production of the peptides revealed the presence of a large open reading frame. The deduced amino acid sequence of lpa-8 (224 amino acid residues) showed sequence similarity to that of sfp (from surfactin-producing B. subtilis), lpa-14 (from iturin A- and surfactin-producing B. subtilis), psf-1 (from surfactin-producing Bacillus pumilus), gsp (from gramicidin-S-producing Bacillus brevis), and entD (from siderophore-enterobactin-producing Escherichia coli), which are able to complement a defect in the sfp gene and promote production of the lipopeptide antibiotic surfactin. The sequence similarity among these proteins and the product similarity of cyclic peptides suggests that they might be involved in the biosynthesis or secretion of the peptides. Received: 14 July 1995 / Accepted: 22 December 1995