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Purification and properties of lectin from potato tubers and leaves; interaction with acid phosphatase from potato tuber.

The haemagglutination activity of lectin, (STA) was determined during vegetation of potatoes (Solanum tuberosum var. Uran). Traces of haemagglutination activity were found in young stalks and first leaves. In the next leaves the maximal activity appeared after 6-8 weeks of vegetation and, simultaneously with the decrease in leaves it appeared in young tubers (at the time of their differentiation from stolons), reaching the maximum in the 18th week of vegetation. Lectins from potato tubers and leaves were extracted with acetate buffer, pH 3.6, salted out with ammonium sulphate, and purified alternatively by affinity chromatography on GlcNAc-Spheron or by adsorption on trypsinated and glutaraldehyde-fixed rabbit erythrocytes. Chromatography on Sephadex G-200 was used for preparative separation of isomeric forms. In the tubers four isolectins were found, differeing in molecular mass, pI values, content of sugars and haemagglutination activity. Two of these forms were isolated in homogeneous form: a heavy dimeric form, Mr 130,000-142,000 containing 59.6% of sugars (including 28.8% arabinose, 6.9% galactose, 16.5% glucose, 1.6% xylose and 3.1% mannose), and a light monomeric form. of Mr 21,000-22,000, containing 20% of neutral sugars (2.5% of pentoses and 17.5% of hexoses). From leaves, a single heavy dimeric lectin of Mr 136,000, containing 14.3% of neutral sugars was isolated. The effect of tuber isolectin on the activity, pH optima and heat-stability of the potato acid phosphatase has been described.

Acid Phosphatase↗

Improved detection of cortical and subcortical tubers in tuberous sclerosis by fluid-attenuated inversion recovery MRI.

We carried out fluid-attenuated inversion recovery (FLAIR) pulse sequences with long repetition and echo times in seven children with tuberous sclerosis, and compared them with conventional spin-echo (SE) sequences. FLAIR images exhibited higher sensitivity than conventional SE images to cortical and subcortical tubers. The low signal intensity of cerebrospinal fluid on FLAIR images allowed more accurate delineation of the cortical and subcortical tubers. However, T1-weighted imaging was still superior for delineation of subependymal nodules.

Adolescent↗

Expression of an Escherichia coli phosphoglucomutase in potato (Solanum tuberosum L.) results in minor changes in tuber metabolism and a considerable delay in tuber sprouting.

The aim of this work was to evaluate the influence of elevating the cytosolic activity of phosphoglucomutase (PGM; EC 5.4.2.2) on photosynthesis, growth and heterotrophic metabolism. Here we describe the generation of novel transgenic plants expressing an Escherichia coli phosphoglucomutase (EcPGM) under the control of the 35S promoter. These lines were characterised by an accumulation of leaf sucrose, despite displaying no alterations in photosynthetic carbon partitioning, and a reduced tuber starch content. Determinations of the levels of a wide range of other metabolites revealed dramatic reductions in maltose and other sugars in leaves of the transformants, as well as a modification of the pattern of organic and amino acid content in tubers of these lines. Intriguingly, the transgenics also displayed a dramatically delayed rate of sprouting and significantly enhanced rate of respiration, however, it is important to note that the severity of these traits did not always correlate with the level of transgene expression. These results are discussed in the context of current understanding of the control of respiration and the breaking of tuber dormancy.

Carbohydrate Metabolism↗

Computed tomography in diagnosis of intracranial tumors versus tubers in tuberous sclerosis.

Intraventricular gliomas producing obstruction of the foramen of Monro were observed by computed tomography in 4 of 8 patients with tuberous sclerosis. Bilateral tumors arising from the caudate nuclei were present in one patient. Characteristic subependymal calcified tubers were also seen, but contrast enhancement occurred only in the tumors and not in the tubers. The location of the tumor prevented communication between the 2 lateral ventricles so that surgical placement of a single ventricular shunt resulted in only unilateral relief of hydrocephalus. Shunting in one case was ineffective because of high cerebrospinal fluid viscosity due to elevated protein concentration.

Adolescent↗

Production of volatile organic compounds (VOCs) by yeasts isolated from the ascocarps of black (Tuber melanosporum Vitt.) and white (Tuber magnatum Pico) truffles.

Twenty-nine yeast strains were isolated from the ascocarps of black and white truffles (Tuber melanosporum Vitt. and Tuber magnatum Pico, respectively), and identified using a polyphasic approach. According to the conventional taxonomic methods, MSP-PCR fingerprinting and sequencing of the D1/D2 domain of 26S rDNA, the strains were identified as Candida saitoana, Debaryomyces hansenii, Cryptococcus sp., Rhodotorula mucilaginosa, and Trichosporon moniliiforme. All isolates assimilated L: -methionine as a sole nitrogen source and produced the volatile organic compounds (VOCs), 2-methyl butanol, 3-methyl butanol, methanethiol, S-methyl thioacetate, dimethyl sulfide, dimethyl disulfide, dimethyl trisulfide, dihydro-2-methyl-3(2H)-thiophenone and 3-(methylthio)-1-propanol (MTP). ANOVA analysis of data showed significant (P<0.01) differences in VOCs produced by different yeasts, with MTP as the major component (produced at concentrations ranging from 19.8 to 225.6 mg/l). In addition, since some molecules produced by the isolates of this study are also characteristic of truffle complex aroma, it is possible to hypothesize a complementary role of yeasts associated with this ecosystem in contributing to final Tuber spp. aroma through the independent synthesis of yeast-specific volatile constituents.

Ascomycota↗

Tuber aestivum and Tuber uncinatum: two morphotypes or two species?

Tuber spp. are ectomycorrhizal fungi that establish symbioses with shrubs and trees. Because of their different smell and taste, Tuber uncinatum and Tuber aestivum are two truffle morphotypes with a different market value, but whether or not T. uncinatum and T. aestivum are different taxa is still an open debate among mycologists. In order to identify molecular keys characterizing both T. aestivum and T. uncinatum morphotypes, ITS/RFLPs analyses were carried out on a large collection of samples from all over Italy and from other European countries, followed by a study of the phylogenesis of ITS, beta-tubulin and EF 1-alpha genes, on representative samples. The present study provides compelling evidence that: (i) T. uncinatum and T. aestivum belong to the same species, (ii) neither morphotype presents a specific molecular fingerprint, but they may even share identical alleles at any of the loci analysed; (iii) T. aestivum is most likely under a selfing reproductive mode. Our findings suggest that ecological, rather than genetic causes may account for differences in sporal morphology, taste and smell between T. aestivum and T. uncinatum truffles.

Ascomycota↗

A calmodulin binding site in the tuberous sclerosis 2 gene product is essential for regulation of transcription events and is altered by mutations linked to tuberous sclerosis and lymphangioleiomyomatosis.

Mutations in the tuberous sclerosis 2 (TSC2) gene product have been genetically linked to the pathology of both tuberous sclerosis (TSC) and the gender-specific lung disease, lymphangioleiomyomatosis (LAM). Both diseases are classified as disorders of cellular migration, proliferation, and differentiation. Earlier studies from our laboratory (1) linked TSC2 with steroid/nuclear receptor signaling. Studies presented here provide evidence for calmodulin (CaM) signaling in the propagation of this TSC2 activity. Far Western screening of a lambda phage human brain cDNA library to identify interacting proteins for the TSC2 gene product (tuberin) yielded multiple clones encoding human CaM. Direct binding with 32P-labeled tuberin demonstrated Ca2+-dependent binding to CaM-Sepharose which was lost upon deletion of the C-terminal 72 residues. The sequence (1740)WIARLRHIKRLRQRIC(1755) was identified as one capable of forming a basic amphipathic helix indicative of CaM binding domains in known calmodulin binding proteins. Studies with a synthetic peptide of this sequence demonstrated very tight Ca2+-dependent binding to CaM as judged by tryptophan fluorescence perturbation studies and phosphodiesterase activation by CaM. Deletion mutagenesis studies further suggested that this CaM binding domain is required for tuberin modulation of steroid receptor function and that mutations in this region may be involved in the pathology of TSC and LAM.

Amino Acid Sequence↗

Reduction of expression of tuberin, the tuberous-sclerosis-complex-gene-2 product in tuberous sclerosis complex associated connective tissue nevi and sporadic squamous and basal cell carcinomas.

BACKGROUND: Patients affected with tuberous sclerosis complex (TSC) are prone to the development of multiple benign tumors of the skin and other organs. Tuberin, the protein product of the tuberous-sclerosis-complex-2 tumor suppressor gene (TSC2) has been shown to inhibit cell proliferation. In TSC associated kidney tumors and sporadic brain tumors the loss/reduction of tuberin has been shown. METHODS: Specimens of nine squamous cell carcinomas (SCC) and five basal cell carcinomas (BCC) from patients without TSC and six biopsies of connective tissue nevi (CTN) of patients with TSC were obtained. Specimens were analyzed by immunoblotting for the expression of tuberin. RESULTS: Absent or reduced levels of tuberin were detected in the dermal parts of three of six shagreen patches, two of five BCC, and four of nine SCC. CONCLUSIONS: In tumors/hamartomas of patients with TSC the complete loss of TSC2 and tuberin is a mechanism which could be shown for CTN, thereby excluding the possibility of haploinsufficiency of TSC2. In a substantial number of cutaneous BCC and SCC the loss or downregulation of tuberin seems to be epigenetic, as alterations of TSC2 are not known in these tumors. The absence or reduction of tuberin might contribute to their proliferation.

Basal Cell Carcinoma↗

Pathogenesis of multifocal micronodular pneumocyte hyperplasia and lymphangioleiomyomatosis in tuberous sclerosis and association with tuberous sclerosis genes TSC1 and TSC2.

Tuberous sclerosis (TSC) is a rare, genetically determined disorder / familial tumor syndrome, currently diagnosed using specific clinical criteria proposed by Gomez, including the presence of multiorgan hamartomas. Pulmonary involvement in TSC is well known as pulmonary lymphangioleiomyomatosis (LAM), which has an incidence of 1-2.3% in TSC patients. LAM has immunohistochemical expression of both smooth-muscle actin and a monoclonal antibody specific for human melanoma, HMB-45. It has recently been reported that multifocal micronodular pneumocyte hyperplasia (MMPH) associated with TSC should be considered as a distinct type of lung lesion, whether it occurs with or without LAM. Two predisposing genes have been found in families affected by TSC; approximately half of the families show linkage to TSC1 at 9q34.3, and the other half show linkage to TSC2 at 16p13.3. TSC genes are considered to be tumor suppressor genes, and mutations in them may lead to abnormal differentiation and proliferation of cells. Tuberin, the TSC2 gene product, has recently been found to be expressed in LAM and MMPH. In this article we discuss the histogenesis and genetic abnormalities of neoplastic lesions associated with TSC, and we review the current understanding of the pathogenesis of pulmonary hamartomatous lesions such as LAM and MMPH in TSC.

Adolescent↗

Tuberous sclerosis: from tubers to mTOR.

Tuberous sclerosis (TSC) is an autosomal dominant hamartoma syndrome whose causative genes (TSC1 and TSC2) were identified 5 and 9 years ago respectively. Their encoded proteins are large, and apart from a strong binding interaction with each other, relatively little was known about their biochemical function. Recent studies in Drosophila have pinpointed a critical function for the DrosophilaTSC1/TSC2 homologues in the regulation of cell size. Epistasis experiments and a variety of biochemical studies that followed have indicated a critical function for these proteins in the highly conserved PI-3-kinase-Akt-mTOR signalling pathway.

Animals↗

Cloning and characterization of PKC-homologous genes in the truffle species Tuber borchii and Tuber magnatum.

The protein kinases C (PKCs) define a growing family of ubiquitous signal transducting serine/threonine kinases that control ion conductance channels, release of hormones and cell growth and proliferation. Degenerated oligonucleotides were used as primers for polymerase chain reactions to amplify PKC-related sequences from the white truffle species Tuber magnatum and Tuber borchii. The deduced amino acid sequences of cloned sequences reveal domains homologous to the regulatory and kinase domains of PKC-related proteins, but lack typical Ca(2+)-binding domain and therefore should be classified as nPKCs. Both contain a large extended N-terminus which is found exclusively in fungi PKCs. Phylogenetic analysis of the kinase domain demonstrates high homology with known filamentous fungi isoenzymes.

Amino Acid Sequence↗

Phylogenetic relationships between Tuber pseudoexcavatum, a Chinese truffle, and other Tuber species based on parsimony and distance analysis of four different gene sequences.

Phylogenetic relationships between Tuber pseudoexcavatum and other Tuber species were investigated by studying the sequences of four genes: 5.8S-ITS2, beta-tubulin, protein kinase C and elongation factor 1alpha. The four phylogenetic trees allowed to differentiate the black truffle clade, composed of two subclades, one comprising the Asian black truffles (T. indicum, T. sinense, T. himalayense) and the Perigord black truffle (T. melanosporum), the second comprising T. pseudoexcavatum and T. brumale. These two subclades diverged relatively early. We propose a common ancestor, located between Europe and China, to all the black truffles. The T. brumale/pseudoexcavatum subclade would have started to diverge and migrate first, T. brumale towards Europe through a northern route and T. pseudoexcavatum towards China. Later the T. melanosporum subclade would have started to migrate through the same route, T. melanosporum towards Europe and T. indicum towards China, leading to vicariant species.

Ascomycota↗

Role of the tuberous sclerosis gene-2 product in cell cycle control. Loss of the tuberous sclerosis gene-2 induces quiescent cells to enter S phase.

Tuberous sclerosis is an autosomal dominant disorder characterized by the development of benign growths in many tissues and organs. Linkage analysis revealed two disease-determining genes on chromosome 9 and chromosome 16. The TSC2 gene on chromosome 16 encodes a 1784-amino acid tumor suppressor protein, tuberin, that functions as a GTPase-activating protein for Rap1, a member of the superfamily of Ras-related proteins. By immunoblot analyses, we found TSC2 expression to be high in G0 as well as in early small G1 cells. Analyses after different cell synchronization procedures revealed that TSC2 mRNA and protein expression do not fluctuate throughout the cell cycle. Using inducible expression systems we further demonstrated that TSC2 expression is not affected by overexpression of the mitogenic transcription factor E2F-1 or c-Myc. Nevertheless, antisense inhibition of tuberin expression in logarithmically growing cells markedly decreased the percentage of cells in G1. Furthermore, we found that cells exposed to TSC2 antisense oligonucleotides did not undergo G0 arrest after serum withdrawal. Antisense inhibition of TSC2 expression also induced quiescent G0-arrested fibroblasts to reenter the cell cycle. Our data show for the first time that the absence of tuberin can both induce cells to pass through the G1/S transition of the eukaryotic cell cycle and prevent them from entering a quiescent state. These results have clear implications for the tumor suppressor function of TSC2. We further found that reentry into the cell cycle upon loss of TSC2 is dependent on the activity of the G1 cyclin-dependent kinases (CDKs), Cdk2 or Cdk4. Taken together with our finding that antisense inhibition of TSC2 causes up-regulation of cyclin D1 expression, these results provide the first evidence for a connection between tuberin/Rap1 and the G1 CDK-dependent regulation of the transition from G0/G1 to S phase.

Animals↗

Localization of tuberous sclerosis 2 mRNA and its protein product tuberin in normal human brain and in cerebral lesions of patients with tuberous sclerosis.

Tuberous sclerosis (TSC), an autosomal dominant disorder, is characterized by malformations, hamartomas and tumors in various organs including the brain. TSC is genetically linked to two loci: TSC1 on chromosome 9q34 and TSC2 on 16p13.3. TSC2 has been cloned, sequenced and encodes a protein (tuberin) which functions as a tumor suppressor. We have analyzed the distribution of TSC2 mRNA and tuberin in the brains of TSC patients and non-affected individuals using both autopsy and biopsy material. High levels of transcript and protein expression were observed in choroid plexus epithelium, ependymal cells, most brainstem and spinal cord motor neurons, Purkinje cells and the external granule cell layer of the cerebellum in both TSC and control cases. Individual balloon cells from TSC patients showed very faint expression while other glia showed no expression of either transcript or tuberin. Neocortical and hippocampal neurons expressed high levels of TSC2 transcript, but only modest levels of tuberin. The internal granule cell layer of the cerebellum expressed abundant transcript but low levels of tuberin. These observations suggest either that tuberin expression is controlled at the level of both transcription and translation or the antibody and in-situ hybridization recognize different splice variants of the TSC2 gene. In TSC patients, dysmorphic cytomegalic neurons expressed high levels of tuberin and transcript, particularly when in an 'ectopic' location. Individual cells within subependymal giant cell astrocytomas (SEGAs) and hamartomas from TSC patients expressed moderate to high levels of TSC2 transcript and tuberin. While the TSC2 transcript is widely expressed primarily within neurons, tuberin is demonstrable primarily within dysplastic/cytomegalic cells of the cortex and subependymal hamartomas/SEGAs. CNS expression of tuberin is unique in that primarily non-dividing cells express it in this location, whereas extra-CNS expression of tuberin is mainly found in actively proliferating cell types such as epithelium.

Adolescent↗

Psychopathology in tuberous sclerosis: an overview and findings in a population-based sample of adults with tuberous sclerosis.

BACKGROUND: Tuberous sclerosis (TS) is a multi- system disorder with complex genetics. The neurodevelopmental manifestations of TS are responsible for considerable morbidity. The prevalence of epilepsy and intellectual disabilities among individuals with TS have been well described. Ours is the first study that explores the prevalence and pattern of psychopathology in a population-based sample of adults with TS. METHODS: Sixty subjects were identified through a capture-recapture analysis of TS. Information was gathered as to seizure history, cognitive functioning (WISC-III) and psychopathology (SADS-L, SAPPA). Lifetime psychopathology was categorized according to Research Diagnostic Criteria. The overall pattern of mental illness (MI) was examined as well as how this varied with IQ and seizure history. RESULTS: Twenty-four (40.0%) subjects had a history of MI. The most common diagnosis was that of an affective disorder [18 (30.0%)], the majority of which were major depressive episodes. Alcoholism [4 (6.7%)] and anxiety disorders [3 (5.0%)] were the next most common diagnoses. Two (3.3%) subjects had had a tic disorder. Only one individual had a diagnosis of schizophrenia. MI was found in 75.0% of those with a history of epilepsy and 37.5% of those without epilepsy. MI was significantly more prevalent in those with a full-scale IQ above 70. CONCLUSIONS: A significant proportion of adult with TS experience MI. MI was significantly more [corrected] prevalent in subjects with a full-scale IQ above 70. Reasons for such a finding are explored, and related methodological considerations for future research outlined.

Adult↗

Amino acid sequence of the N-terminal domain of yam (Dioscorea japonica) aerial tuber acidic chitinase. Evidence for the presence of a wheat germ agglutinin domain in matured acidic chitinase from unstressed tuber.

The amino acid sequence of the N-terminal domain of acidic chitinase from unstressed aerial tuber was determined and proved the presence of an N-terminal domain in acidic chitinase. The amino acid sequence was determined on a pyroglutamylaminopeptidase-treated N-terminal fragment of V8 protease and on chymotryptic peptides of this fragment. The sequence determined revealed 8 residues deletion and 2 residues insertion as compared with the N-terminal domain of tobacco basic chitinase. The N-terminal domain determined showed a homology of 40% and 52% with the N-terminal domain of tobacco basic chitinase and wheat germ agglutinin, respectively.

Amino Acid Sequence↗

Detection of potato spindle tuber viroid (PSTV) in dormant potato tubers by concatameric cDNA probe.

The 32P-labelled concatameric insert cut out from a plasmid pSPAv6.2(+), containing 6.2 copies of a full-length PSTV, was used to detect PSTV in dormant potato tubers by dot-blot hybridisation assay. The concatameric insert probe was 4 times more sensitive than the monomeric one. This allowed the detection of 0.5 pg of viroid RNA. The sensitivity makes th eoligomeric cDNA probe a useful alternative to cDNA probes.

DNA Probes↗