Expression cloning of a cDNA encoding a fish prolactin receptor.

By using an expression cloning strategy, we isolated a single positive clone encoding a tilapia prolactin (PRL) receptor. Tilapia PRL188 was used to screen a freshwater tilapia kidney expression library transfected in COS cells. The tilapia PRL receptor is a mature protein of 606 amino acids. The extracellular domain is devoid of the tandem repeat units present in birds and has two pairs of cysteine residues, a Trp-Ser-Xaa-Trp-Ser motif, and two potential N-glycosylation sites. The cytoplasmic domain contains 372 amino acids, including box 1, a sequence previously shown to be important for signal transduction in mammalian species. Thus, the general structure is similar to the long form of mammalian PRL receptors; however, amino acid comparisons reveal a rather low identity (approximately 37%). Northern blot analysis shows the existence of a single transcript in osmoregulatory tissues and reproductive organs. This localization is in agreement with known functions of PRL in teleosts.

Distinct regions of c-Mpl cytoplasmic domain are coupled to the JAK-STAT signal transduction pathway and Shc phosphorylation.

c-Mpl, a member of the hematopoietic cytokine receptor family, is the receptor for thrombopoietin. To investigate signal transduction by c-Mpl, a chimeric receptor, composed of the extracellular domain of human growth hormone receptor and the intracellular domain of c-Mpl, was introduced into the interleukin 3-dependent cell line Ba/F3. In response to growth hormone, this chimeric receptor induced growth in the absence of interleukin 3. Deletion analysis of the 123-amino acid intracellular domain indicated that the elements responsible for this effect are present within the 63 amino acids proximal to the transmembrane domain. Mutation of the recently described box 1 motif abrogated the proliferative response. Tyrosine phosphorylation of the tyrosine kinase JAK-2 and activation of STAT proteins were dependent on box 1 and sequences within 63 amino acids of the plasma membrane. STAT proteins activated by thrombopoietin in a megakaryocytic cell line were purified and shown to be STAT1 and STAT3. A separate region located at the C terminus of the c-Mpl intracellular domain was found to be required for induction of Shc phosphorylation and c-fos mRNA accumulation, suggesting involvement of the Ras signal transduction pathway. Thus, at least two distinct regions are involved in signal transduction by the c-Mpl.

The Lutheran blood group glycoprotein, another member of the immunoglobulin superfamily, is widely expressed in human tissues and is developmentally regulated in human liver.

Glycoproteins expressing the Lutheran blood group antigens were isolated from human erythrocyte membranes and from human fetal liver. Amino acid sequence analyses allowed the design of redundant oligonucleotides that were used to generate a 459-bp, sequence-specific probe by PCR. A cDNA clone of 2400 bp was isolated from a human placental lambda gt 11 library and sequenced, and the deduced amino acid sequence was studied. The predicted mature protein is a type I membrane protein of 597 amino acids with five potential N-glycosylation sites. There are five disulfide-bonded, extracellular, immunoglobulin superfamily domains (two variable-region set and three constant-region set), a single hydrophobic, membrane-spanning domain, and a cytoplasmic domain of 59 residues. The overall structure is similar to that of the human tumor marker MUC 18 and the chicken neural adhesion molecule SC1. The extracellular domains and cytoplasmic domain contain consensus motifs for the binding of integrin and Src homology 3 domains, respectively, suggesting possible receptor and signal-transduction function. Immunostaining of human tissues demonstrated a wide distribution and provided evidence that the glycoprotein is under developmental control in liver and may also be regulated during differentiation in other tissues.

Correlation of peptide specificity and IgG subclass with pathogenic and nonpathogenic autoantibodies in pemphigus vulgaris: a model for autoimmunity.

Pemphigus vulgaris (PV) is a rare, potentially fatal, autoimmune disease that affects the skin and mucous membranes. The PV antigen (PVA) has been characterized as desmoglein 3. PV patients carry HLA-DR4- or HLA-DR6-bearing extended haplotypes. We recently demonstrated that patients with active disease have high titers of PV autoantibodies of the IgG1 and IgG4 subclasses. Patients in remission, healthy unaffected relatives, and some MHC-matched normal individuals have low levels of PV autoantibodies, which are IgG1 only. Furthermore, intraperitoneal injection of IgG from patients with active disease caused clinical disease in mice, but IgG from patients in remission, healthy relatives, or MHC-matched normal individuals did not. We prepared 12 peptides of 30 amino acids each (peptides Bos 1-12) spanning the extracellular domain of PVA. Patients with active disease recognize peptides Bos 1 and Bos 6 with high titers of IgG1 and IgG4 autoantibodies. Patients in remission have IgG1 autoantibodies to peptide Bos 1 only, in statistically significantly lower titers (P < 0.01). They no longer have IgG4 subclass autoantibodies to peptide Bos 6. Healthy relatives and normal unrelated individuals have low levels of only IgG1 autoantibodies that recognize only Bos 1. In vitro studies indicate that Bos 6-specific IgG and, to a lesser extent, Bos 1-specific IgG can cause acantholysis. Our data suggest that Bos 6-specific IgG4 is probably the main acantholytic autoantibody, while Bos 1-specific IgG4 may act as a facilitator or enhancer of the process. In this study we illustrate some of the paradigms that demonstrate the interactions between the MHC, subclass of autoantibodies, and peptide specificities of the autoantibodies in the autoimmune process. Thus, PV provides an important model to study the pathogenesis of autoimmunity.

Ruolo di Clusterina nella progressione del tumore prostatico

Clusterina (CLU) è una proteina ubiquitaria, presente nella maggior parte dei fluidi corporei e implicata in svariati processi fisiologici. Dalla sua scoperta fino ad oggi, CLU è risultata essere una proteina enigmatica, la cui funzione non è ancora stata compresa appieno. Il gene codifica per 3 varianti trascrizionali identificate nel database NCBI con i codici: NM_001831 (CLU 1 in questo lavoro di tesi), NR_038335 (CLU 2 in questo lavoro di tesi) e NR_045494 (CLU 3 in questo lavoro di tesi). Tutte le varianti sono trascritte come pre-mRNA contenenti 9 esoni e 8 introni e si differenziano per l’esone 1, la cui sequenza è unica e caratteristica di ogni variante. Sebbene in NCBI sia annotato che le varianti CLU 2 e CLU 3 non sono codificanti, tramite analisi bioinformatica è stato predetto che da tutti e tre i trascritti possono generarsi proteine di differente lunghezza e localizzazione cellulare. Tra tutte le forme proteiche ipotizzate, l’unica a essere stata isolata e sequenziata è quella tradotta dall’AUG presente sull’esone 2 che dà origine a una proteina di 449 aminoacidi. Il processo di maturazione prevede la formazione di un precursore citoplasmatico (psCLU) che subisce modificazioni post-traduzionali tra cui formazione di ponti disolfuro, glicosilazioni, taglio in due catene denominate β e α prima di essere secreta come eterodimero βα (sCLU) nell’ambiente extracellulare, dove esercita la sua funzione di chaperone ATP-indipendente. Oltre alla forma extracellulare, è possibile osservare una forma intracellulare con localizzazione citosolica la cui funzione non è stata ancora completamente chiarita. Questo lavoro di tesi si è prefissato lo scopo di incrementare le conoscenze in merito ai trascritti CLU 1 e CLU 2 e alla loro regolazione, oltre ad approfondire il ruolo della forma citosolica della proteina in relazione al signaling di NF-kB che svolge un ruolo importante nel processo di sviluppo e metastatizzazione del tumore. Nella prima parte, uno screening di differenti linee cellulari, quali cellule epiteliali di prostata e di mammella, sia normali sia tumorali, fibroblasti di origine polmonare e linfociti di tumore non-Hodgkin, ha permesso di caratterizzare i trascritti CLU 1 e CLU 2. Dall’analisi è emerso che la sequenza di CLU 1 è più corta al 5’ rispetto a quella depositata in NCBI con l’identificativo NM_001831 e il primo AUG disponibile per l’inizio della traduzione è localizzato sull’esone 2. È stato dimostrato che CLU 2, al contrario di quanto riportato in NCBI, è tradotto in proteina a partire dall’AUG presente sull’esone 2, allo stesso modo in cui viene tradotto CLU 1. Inoltre, è stato osservato che i livelli d’espressione dei trascritti variano notevolmente tra le diverse linee cellulari e nelle cellule epiteliali CLU 2 è espressa sempre a bassi livelli. In queste cellule, l’espressione di CLU 2 è silenziata per via epigenetica e la somministrazione di farmaci capaci di rendere la cromatina più accessibile, quali tricostatina A e 5-aza-2’-deossicitidina, è in grado di incrementarne l’espressione. Nella seconda parte, un’analisi bioinformatica seguita da saggi di attività in vitro in cellule epiteliali prostatiche trattate con farmaci epigenetici, hanno permesso di identificare, per la prima volta in uomo, una seconda regione regolatrice denominata P2, capace di controllare l’espressione di CLU 2. Rispetto a P1, il classico promotore di CLU già ampiamente studiato da altri gruppi di ricerca, P2 è un promotore debole, privo di TATA box, che nelle cellule epiteliali prostatiche è silente in condizioni basali e la cui attività incrementa in seguito alla somministrazione di farmaci epigenetici capaci di alterare le modificazioni post-traduzionali delle code istoniche nell’intorno di P2. Ne consegue un rilassamento della cromatina e un successivo aumento di trascrizione di CLU 2. La presenza di un’isola CpG differentemente metilata nell’intorno di P1 spiegherebbe, almeno in parte, i differenti livelli di espressione di CLU che si osservano tra le diverse linee cellulari. Nella terza parte, l’analisi del pathway di NF-kB in un modello sperimentale di tumore prostatico in cui CLU è stata silenziata o sovraespressa, ha permesso di capire come la forma citosolica di CLU abbia un ruolo inibitorio nei confronti dell’attività del fattore trascrizionale NF-kB. CLU inibisce la fosforilazione e l’attivazione di p65, il membro più rappresentativo della famiglia NF-kB, con conseguente riduzione della trascrizione di alcuni geni da esso regolati e coinvolti nel rimodellamento della matrice extracellulare, quali l’urochinasi attivatrice del plasminogeno, la catepsina B e la metallo proteinasi 9. È stato dimostrato che tale inibizione non è dovuta a un’interazione fisica diretta tra CLU e p65, per cui si suppone che CLU interagisca con uno dei componenti più a monte della via di segnalazione responsabile della fosforilazione ed attivazione di p65.

LeProT1, a Transporter for Proline, Glycine Betaine, and γ-Amino Butyric Acid in Tomato Pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes.Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70 of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and γ-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.

Amino acid transport in plants

Amino acids are transported between different organs through both xylem and phloem. This redistribution of nitrogen and carbon requires the activity of amino acid transporters in the plasma membrane. In addition, amino acids can be taken up directly by the roots. Amino acid transport has been well characterized in the yeast Saccharomyces cerevisiae, and functional complementation has served as an excellent tool for identifying and characterizing amino acid transporters from plants. The transporters from yeast and plants are related and can be grouped into two large superfamilies. Based on substrate specificity and affinity, as well as expression patterns in plants, different functions have been assigned to some of the individual transporters. Plant mutants for amino acid transporter genes are now being used to study the physiological functions of many of the cloned genes.

Developmental control of H+/amino acid permease gene expression during seed development of Arabidopsis

Long distance transport of amino acids is mediated by several families of differentially expressed amino acid transporters. The two genes AAP1 and AAP2 encode broad specificity H+-amino acid co-transporters and are expressed to high levels in siliques of Arabidopsis, indicating a potential role in supplying the seeds with organic nitrogen. The expression of both genes is developmentally controlled and is strongly induced in siliques at heart stage of embryogenesis, shortly before induction of storage protein genes. Histochemical analysis of transgenic plants expressing promoter-GUS fusions shows that the genes have non-overlapping expression patterns in siliques. AAP1 is expressed in the endosperm and the cotyledons whereas AAP2 is expressed in the vascular strands of siliques and in funiculi. The endosperm expression of AAP1 during early stages of seed development indicates that the endosperm serves as a transient storage tissue for organic nitrogen. Amino acids are transported in both xylem and phloem but during seed filling are imported only via the phloem. AAP2, which is expressed in the phloem of stems and in the veins supplying seeds, may function in uptake of amino acids assimilated in the green silique tissue, in the retrieval of amino acids leaking passively out of the phloem and in xylem-to-phloem transfer along the path. The promoters provide excellent tools to study developmental, hormonal and metabolic control of nitrogen nutrition during development and may help to manipulate the timing and composition of amino acid import into seeds.

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Organic carbon and amino acid concentration of Karelian shungite rocks from the lake Onego area, Russia

The study of amino acids in the Precambrian shungite rocks of Karelia showed that their contents vary within 25-89 µg/g depending on proportions between shungite and mineral components. It was established that the amino acids exhibit an excess of L-enantiomers. In the shungite rocks, they form organomineral complexes with silica and aluminosilicates, being built in the globular structure of shungite matter. There are several sources of amino acids in shungites: secondary synthesis, microbial pollution, and original amino acids of organic matter in shungite rocks.

Humic acids in bottom sediments of the Western Pacific

Elemental composition, functional groups, and molecular mass distribution were determined in humic acids from the Western Pacific abyssal and coastal bottom sediments. Humic acid structure was studied by oxidative degradation with alkaline nitrobenzene and potassium permanganate, p-coumaric, guaiacilic, and syringilic structural units typical for lignin of terrestrial plants were identified in humic acids by chromatographic analysis of oxidation products. Polysubstituted and polycondensed aromatic systems with minor proportion of aliphatic structures were basic structural units of humic acids in abyssal sediments.

(Table T1) Amino acid ratios and concentrations in interstitial waters of seven ODP Leg 201 sites

Proteins and their amino acid building blocks form a major group of biomolecules in all organisms. In the sedimentary environment, proteins and amino acids have two sources: (1) soft tissues and detritus and (2) biotic skeletal structures, dominantly from calcium carbonate-secreting organisms. The focus of this report is on D/L ratios and concentrations of selected amino acids in interstitial waters collected during ODP Leg 201. The Peru margin sites are generally low in carbonates, whereas the open-ocean sites are more carbonate rich. Seifert et al. (1990, doi:10.2973/odp.proc.sr.112.152.1990) reported amino acid concentrations in interstitial waters from Site 681 (ODP Leg 112) comparable to Leg 201 Site 1229.

Amino acid composition of the proteins of the serum lipoproteins /

Unclassified Health and Biology.

Sequences of immunoglobulin chains : tabulation and analysis of amino acid sequences of precursors, V-regions, C-regions, J-chain and -microglobulins /

1976 ed. issued under title: Variable regions of immunoglobulin chains.

Lysine. The unique essential amino acid.

Mode of access: Internet.