Carboxy-terminal Extension Research Articles

Role of Structural Plasticity in Signal Transduction by the Cryptochrome Blue-Light Photoreceptor

Cryptochromes are blue-light photoreceptors that regulate a variety of responses such as growth and circadian rhythms in organisms ranging from bacteria to humans. Cryptochromes share a high level of sequence identity with the light-activated DNA repair enzyme photolyase. Photolyase uses energy from blue light to repair UV-induced photoproducts in DNA through cyclic electron transfer between the catalytic flavin adenine dinucleotide cofactor and the damaged DNA. Cryptochromes lack DNA repair activity, and their mechanism of signal transduction is not known. It is hypothesized that a light-dependent signaling state in cryptochromes is created as a result of an intramolecular redox reaction, resulting in conformational rearrangement and effector binding. Plant and animal cryptochromes possess 30-250 amino acid carboxy-terminal extensions beyond the photolyase-homology region that have been shown to mediate phototransduction. We analyzed the structures of C-terminal domains from an animal and a plant cryptochrome by computational, biophysical, and biochemical methods and found these domains to be intrinsically unstructured. We show that the photolyase-homology region interacts with the C-terminal domain, inducing stable tertiary structure in the C-terminal domain. Importantly, we demonstrate a light-dependent conformational change in the C-terminal domain of Arabidopsis Cry1. Collectively, these findings provide the first biochemical evidence for the proposed conformational rearrangement of cryptochromes upon light exposure.

Characterization of a Cloned Temperature-Sensitive Construct of the Diphtheria Toxin A Domain

Our goal was to determine how the DTM mutant construct of the A domain of diphtheria toxin (DTx) causes temperature-sensitive effects in Drosophila and yeast [Bellen, H. J., D'Evelyn, D., Harvey, M., Elledge, S. J. (1992) Development 114, 787-796]. Because DTM fortuitously bears the same point mutation as found in the A chain of CRM197, an ADP-ribosyltransferase (ADPrT)-deficient form of DTx, we hypothesized that the dramatic low-temperature-sensitive effects did not stem from ADP-ribosylation of elongation factor 2 (EF-2). To rule out acquisition of ADPrT activity at low temperatures, we assayed mutant forms of the A domain of DTx produced by in vitro transcription/translation and found that DTM has no ability to ADP-ribosylate EF-2 at 18 or 30 degrees C. Because the DTM gene results in a protein with a 23-amino acid missense carboxy-terminal extension, we also constructed a form without this extension. Assays for nuclease activity revealed that nuclease activity comigrated with the two distinguishable E. coli-cloned mutant proteins DTM and DTM-23, regardless of whether electrophoresis was conducted under denaturing or nondenaturing conditions in gels embedded with DNA. Studies with CRM197 showed that Ca(2+) and Mg(2+) promote single-strand DNA nicks, whereas Mn(2+) promotes double-strand DNA breaks. Evidence that the cation-dependent nuclease and NAD-dependent ADPrT enzymic sites are distinct is that NAD protected only the A domain of DTx from proteolytic cleavage, whereas DNA protected the A domains of both DTx and CRM197. We conclude that the nuclease activity of DTM is responsible for the temperature-sensitive effects associated with its expression in both yeast and Drosophila.

Study of some common biochemical bone turnover markers in postmenopausal women

Markers of bone formation in serum include total and bone specific alkaline phosphatase, osteocalcin and Type 1 collagen carboxy terminal extension peptide. Bone resorption can be assessed by measuring plasma tartarate resistant acid phosphtase and urinary excretion of collagen degradation products: hydroxyproline, hydroxylysine glycosides and more recently the pyridinium crosslinks and associated peptides. We compared the excretion of hydroxyproline in women of reproductive age group to those of menopausal age group and found a significant difference in the two age groups. Urinary hydroxyproline was found to be significantly raised in post menopausal women. Thus hydroxyproline maybe used as the earliest indicator in the prognostic assessment of postmenopausal women of their risk of developing osteoporosis and fracture.

Characterization and Purification of Flap Endonuclease-1(xiFEN-1) fromXiphophorus maculatus

The cloning, gene structure, and expression of flap endonuclease-1 (xiFEN1) from Xiphophorus maculates are presented. The xiFEN1 gene structure was found to include 8 exons and 7 introns. The Xiphophorus FEN1 cDNA sequence contained an open reading frame that encoded a 380 amino acid protein with a predicted mass of 43 kDa. The intact FEN1 cDNA was subcloned into a bacterial expression vector (pET101-xiFEN1ct) and recombinant xiFEN1 enzyme purified from E. colicell extracts. The pET101-xiFEN1ct translation product was a 3' fusion protein with a ~3 kDa vector-encoded carboxy terminal extension designed to facilitate protein recognition and purification. The xiFEN1 fusion protein was purified and its amino acid sequence verified by Western blot analysis and tryptic peptide mass fingerprinting. The purified recombinant protein was assessed for enzyme specificity using several different oligonucleotide substrates having select flap overhangs. Also reported are Michaelis steady state kinetic values of enzymatic activity for the xiFEN1 directly compared with human FEN1 activity. xiFEN1 displayed a five-fold greater Km and six-fold lower catalytic efficiency (kcat/Km) than observed for the hFEN1.

Activation of LIMK1 by binding to the BMP receptor, BMPRII, regulates BMP-dependent dendritogenesis

The growth and morphological differentiation of dendrites are critical events in the establishment of proper neuronal connectivity and neural function. One extrinsic factor, BMP7, has been shown to specifically affect dendritic morphogenesis; however, the underlying mechanism by which this occurs is unknown. Here we show that LIM kinase 1 (LIMK1), a key downstream effector of Rho GTPases, colocalizes with the BMP receptor, BMPRII, in the tips of neurites and binds to BMPRII. This interaction is required for BMP-dependent induction of the dendritic arbor in cortical neurons. Furthermore, we demonstrate that the physical interaction of LIMK1 with BMPRII synergizes with the Rho GTPase, Cdc42, to activate LIMK1 catalytic activity. These studies thus define a Smad-independent pathway that directly links the BMP receptor to regulation of actin dynamics and provides insights into how extracellular signals modulate LIMK1 activity to permit fine spatial control over cytoskeletal remodelling during dendritogenesis.

CORRELATIONS BETWEEN LEPTIN, BODY COMPOSITION, BONE MINERAL DENSITY AND BONE METABOLISM IN KIDNEY TRANSPLANT RECIPIENTS AND DIALYZED PATIENTS

P301 Aims: In kidney transplant recipients leptin levels are often elevated and BMD decreased, however, so far data about correlations between leptin and BMD in this population is lacking. It has been suggested that leptin is a predictor of BMD in postmenopausal women. Moreover, leptin acts a a marker of fat stores. We examined the relationships between leptinemia, some markers of nutritional status, BMD and bone metabolism in kidney transplant recipients as well as in dialyzed patients (hemodialyzed -HD, peritoneally dialyzed -CAPD). We also assessed whether leptin is significant and independent predictor of BMD in this population. Methods: Methods: BMD and fat content (global, percentage, trunk) was measured using dual energy X-ray absorptiometry (DEXA) in 27 kidney allograft recipients, 25 HD and 23 CAPD patients,. Intact PTH and osteocalcin were studied by radioimmunoassay using commercially available kits from Cis, France. Vitamin 1,25 (OH)2D3 and IGF-1 concentrations were measured RIA using commercially available kits from Biosource, Europe S.A., Belgium. Levels of IGF-BP-1 and 3 were assayed by IRMA (Diagnostic Systems Laboratories, Inc, USA). Levels of PICP (procollagen type I carboxy-terminal extension peptide) and ICTP (procollagen type I cross-linked carboxy-terminal telopeptide) were estimated by radioimmunoassays (Orion Diagnostica, Finland). Bone-specifc alkaline phosphatase-bALP was estimated using ELISA kit from Metra Biosystem, USA. Serum beta2microglobulin was estimated using kits from AlphaDialab, Vienna, Austria. Beta Ctx (degradation products of C-terminal telopeptides of type I collagen), were assayed by ELISA (Osteometer, Denmark). Serum tartrate-resistant acid phosphatase-TRAP was estimated using kit from Suomen Bioanalytiikka Oy, Finland. DPD (deoxypirydynoline) in urine was assayed by immunochemiluminescence (ACS 180 Bayer) and expressed as DPD/creatinine ratios. Serum leptin was assessed by RIA using kits from Linco Research, USA. Results: Leptin correlated with percentage of body fat, trunk fat, lean body mass, serum creatinine and urea. Insulin growth factor binding protein 1 was negatively related to waist-hip ratio, global and trunk fat., whereas BMD of the lumbar spine was related to the dose of prednisone, azathioprine, level of cyclosporine, serum calcium as well as osteoprotegerin level in kidney transplant recipients. In femoral neck BMD was significantly higher in CAPD patients, without significant differences in BMD in lumbar spine. There was a negative correlation between BMD at the femoral neck and time on hemodialysis (r=-0.45, p<0.05). In CAPD patients BMD at the lumbar spine correlated negatively with vitamin D3 (r=-0.54, p<0.05), osteocalcin (r=-0.54, p<0.05), and positively with BMI (r=0.63, p<0.01). BMD at the femoral neck correlated positively with BMI (r=0.59, p<0.01), and negatively with osteocalcin (r=-0.63, p<0.05) and time on CAPD (r=-0.52, p<0.05). Leptin correlate only with cholesterol (r=0.25, p<0.05), TSH (r=0.35, p<0.01), beta2microglobulin (r=0.32, p<0.001) and BMD at the femoral neck (r=-0.23, p<0.05) in all dialyzed (HD and CAPD) patients. Conclusions: Leptin levels are associated with renal function and body fat in kidney allograft recipients. Leptin is not related to nutritional status, BMD and bone metabolism BMD in kidney allograft recipients, but depends on currents dosage of immunosuppressants and serum calcium. Leptin is not a predictor of BMD in dialyzed patients.

The Carboxy-Terminal Extension of Yeast Ribosomal Protein S14 Is Necessary for Maturation of 43S Preribosomes

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants. These mutations not only reveal a more precise function for rpS14 in ribosome biogenesis but also uncover a role in ribosome assembly for the extended tails found in many ribosomal proteins. These studies are one of the first to relate the structure of eukaryotic ribosomes to their assembly pathway—the carboxy-terminal extension of rpS14 is located in the 40S subunit near the 3′ end of 18S rRNA, consistent with a role for rpS14 in 3′ end processing of 20S pre-rRNA.

Mutations in the IGF-II pathway that confer resistance to lytic reovirus infection

BackgroundViruses are obligate intracellular parasites and rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.ResultsA gene entrapment approach was used to identify candidate cellular genes that affect reovirus infection or virus induced cell lysis. Four of the 111 genes disrupted in clones selected for resistance to infection by reovirus type 1 involved the insulin growth factor-2 (IGF-II) pathway, including: the mannose-6-phosphate/IGF2 receptor (Igf2r), a protease associated with insulin growth factor binding protein 5 (Prss11), and the CTCF transcriptional regulator (Ctcf). The disruption of Ctcf, which encodes a repressor of Igf2, was associated with enhanced Igf2 gene expression. Plasmids expressing either the IGF-II pro-hormone or IGF-II without the carboxy terminal extension (E)-peptide sequence independently conferred high levels of cellular resistance to reovirus infection. Forced IGF-II expression results in a block in virus disassembly. In addition, Ctcf disruption and forced Igf2 expression both enabled cells to proliferate in soft agar, a phenotype associated with malignant growth in vivo.ConclusionThese results indicate that IGF-II, and by inference other components of the IGF-II signalling pathway, can confer resistance to lytic reovirus infection. This report represents the first use of gene entrapment to identify host factors affecting virus infection. Concomitant transformation observed in some virus resistant cells illustrates a potential mechanism of carcinogenesis associated with chronic virus infection.

Sirtuins: Sir2-related NAD-dependent protein deacetylases.

Silent information regulator 2 (Sir2) proteins, or sirtuins, are protein deacetylases dependent on nicotine adenine dinucleotide (NAD) and are found in organisms ranging from bacteria to humans. In eukaryotes, sirtuins regulate transcriptional repression, recombination, the cell-division cycle, microtubule organization, and cellular responses to DNA-damaging agents. Sirtuins have also been implicated in regulating the molecular mechanisms of aging. The Sir2 catalytic domain, which is shared among all sirtuins, consists of two distinct domains that bind NAD and the acetyl-lysine substrate, respectively. In addition to the catalytic domain, eukaryotic sirtuins contain variable amino- and carboxy-terminal extensions that regulate their subcellular localizations and catalytic activity.

Characterization of C. elegans RING finger protein 1, a binding partner of ubiquitin-conjugating enzyme 1

In a yeast two-hybrid screen, RING finger protein 1 (RFP-1) and UBR1 were identified as potential binding partners of C. elegans UBC-1, a ubiquitin-conjugating enzyme with a high degree of identity to S. cerevisiae UBC2/RAD6. The interaction of RFP-1 and UBC-1 was confirmed by co-immunoprecipitation experiments. Yeast interaction trap experiments mapped the region of interaction to the basic N-terminal 313 residues of RFP-1. The acidic carboxy-terminal extension of UBC-1 was not required for the interaction with RFP-1. Western blot analysis and indirect immunohistochemical staining show that RFP-1 is present in embryos, larvae, and adults, where it is found in intestinal, nerve ring, pharyngeal, gonadal, and oocyte cell nuclei. Double-stranded RNA interference experiments against rfp-1 indicate that this gene is required for L1 development, vulval development, and for egg laying. By contrast, RNA interference against ubc-1 gave no obvious phenotype, suggesting that ubc-1 is nonessential or is functionally redundant.

Identification of mitogen-activated protein kinase homologues from Leishmania mexicana

Mitogen-activated protein kinases are key-regulatory elements in the differentiation, proliferation, apoptosis and stress response of eukaryotic cells. Our recent identification of a mitogen-activated protein kinase homologue in Leishmania mexicana which is essential for the proliferation of the amastigote stage of the parasite living in the parasitophorous vacuole of the infected macrophage prompted us to screen the genome of L. mexicana for additional mitogen-activated protein kinase homologues using degenerate oligonucleotide primers in a polymerase chain reaction amplification approach. We cloned and sequenced the genes for eight new mitogen-activated protein kinase homologues which were subsequently shown to be present in one copy per haploid genome. The mRNA levels of the kinases varied significantly in pro- and amastigote life stages of the parasite. We used the structural information of the p38 stress-activated protein kinase, which belongs to the family of mitogen-activated protein kinases, for the alignment of the deduced proteins and the verification of the predicted secondary structure elements. All new mitogen-activated protein kinases reveal the typical 12 subdomain primary structure, the conserved residues characterising serine/threonine protein kinases and the characteristic TXY motif in the phosphorylation lip. Typical features of some of the molecules are amino acid insertions between the subdomains and long carboxy-terminal amino acid extensions carrying putative src-homology 3-binding motifs.

Purification and characterization of hexahistidine-tagged elongation factor SelB

The cotranslational incorporation of selenocysteine into proteins is mediated by a specialized elongation factor, named SelB. Its amino-terminal three domains show homology to elongation factor EF-Tu and accordingly bind GTP and selenocysteyl-tRNA Sec. In addition, SelB exhibits a long carboxy-terminal extension that interacts with a secondary structure of selenoprotein mRNAs (SECIS element) positioned immediately downstream of the in-frame UGA codons specifying the sites of selenocysteine insertion. In this report, a fast and efficient method for the purification of large amounts of hexahistidine-tagged SelB is presented. After two chromatographic steps, 10 mg pure protein was isolated from 12 g wet cell pellet. Biochemical analysis of the purified protein showed that the tag does not influence the interaction of SelB with guanine nucleotides, SECIS elements, and selenocysteyl-tRNA Sec. In addition, the fusion protein is fully functional in mediating UGA read-through in vivo. It therefore represents an excellent model for studying the function of SelB and the mechanisms of selenocysteine incorporation.

Correlations of new markers of bone formation and resorption in kidney transplant recipients

Renal osteodystrophy is a common complication of chronic renal failure and renal replacement therapy. Successful kidney transplantation reverses many of these abnormalities, but the improvement is often incomplete. The evaluation of renal osteodystrophy in everyday practice is based on noninvasive measurements. Taking this into consideration the aim of the present study was to assess new markers of bone metabolism: serum CrossLaps degradation products of C-terminal telopeptides of type I collagen tartrate-resistant acid phosphatase (TRAP) and bone-specific alkaline phosphatase (bALP), as well as their correlations with bone mineral disease (BMD) in kidney transplant recipients. Twenty-six patients (aged 26 to 54 years) receiving a triple immunosuppressive regimen with stable graft function were enrolled in the study. Serum parathormone (PTH) osteocalcin type collagen C-terminal peptides (ICTP), and procollagen type I carboxyterminal extension peptide (PICP) concentrations were measured by radioimmunoassay (RIA), Serum CrossLaps, bALP, β2-microglobulin, TRAP 5b by enzyme-linked immunoassay (ELISA), and deoxypyridinoline (DPD) in urine immunochemiluminescence. BMD, as measured by dual-energy X-ray absorptiometry (DEXA), correlated negatively with markers of bone formation (bALP, osteoclacin, and PICP) and resorption (TRAP, ICTP, and β2-microglobulin). The only positive correlation was between urine DPD and BMD at the femoral neck. Interestingly, BMD correlated negatively with CsA concentration. TRAP 5b correlated positively with serum creatinine, ALP, bALP, osteocalcin, iPTH, ICTP, and serum β2-microglobulin, and negatively with CsA concentration, and azathioprine and prednisone dose. DPD did not correlate with any parameters. Serum CrossLaps correlated with markers of both bone formation and resorption. Because TRAP and serum CrossLaps correlated with markers of both bone formation and or resorption, additional studies are needed to establish the value of these markers of bone resorption to assess renal osteodystrophy.

Non-animal origin of animal thioredoxin reductases: implications for selenocysteine evolution and evolution of protein function through carboxy-terminal extensions.

Thioredoxin reductase (TR) and thioredoxin constitute a major cellular redox system present in all organisms. In contrast to a single form of thioredoxin, there are two TR types: One (bacterial type or small TR) is present in bacteria, archaea, plants, and most unicellular eukaryotes, whereas the second (animal or large TR) is only found in animals and typically contains a carboxy-terminal penultimate selenocysteine encoded by TGA. Surprisingly, we detected sequences of large TRs in various unicellular eukaryotes. Moreover, green algae Chlamydomonas reinhardtii had both small and large TRs, with the latter being a selenoprotein, but no examples of horizontal gene transfer from animals to the green algae could be detected. In addition, phylogenetic analyses revealed that large TRs formed a subgroup of lower eukaryotic glutathione reductases (GRs). The data suggest that the large TR evolved in a lower eukaryote capable of selenocysteine insertion rather than in an animal. The enzyme appeared to evolve by a carboxy-terminal extension of GR such that the resulting carboxy-terminal glutathionelike peptide became an intramolecular substrate for GR and a reductant for thioredoxin. Subsequently, small TRs were lost in an organism that gave rise to animals, large TRs were lost in plants and fungi, and selenocysteine/cysteine replacements took place in some large TRs. Our data implicate carboxy-terminal extension of proteins as a general mechanism of evolution of new protein function.

DmMyD88 controls dorsoventral patterning of the Drosophila embryo.

MyD88 is an adapter protein in the signal transduction pathway mediated by interleukin-1 (IL-1) and Toll-like receptors. A Drosophila homologue of MyD88 (DmMyD88) was recently shown to be required for the Toll-mediated immune response. In Drosophila, the Toll pathway was originally characterized for its role in the dorsoventral patterning of the embryo. We found that, like Toll, DmMyD88 messenger RNA is maternally supplied to the embryo. Here we report the identification of a new mutant allele of DmMyD88, which generates a protein lacking the carboxy-terminal extension, normally located downstream of the Toll/IL-1 receptor domain. Homozygous mutant female flies lay dorsalized embryos that are rescued by expression of a transgenic DmMyD88 complementary DNA. The DmMyD88 mutation blocks the ventralizing activity of a gain-of-function Toll mutation. These results show that DmMyD88 encodes an essential component of the Toll pathway in dorsoventral pattern formation.

Gene organization and expression of a neuropeptide Y homolog from the land planarian Arthurdendyus triangulatus.

Neuropeptide Y is one of the most widespread regulatory peptides within the vertebrate nervous system and shares the C-terminal motif [FY]-x(3)-[LIVM]-x(2)-Y-x(3)-[LIVMFY]-x-R-x-R-[YF] with pancreatic polypeptide, peptide YY, and fish pancreatic peptide Y. All four peptides are believed to have arisen from a single ancestral gene through successive gene duplication events in vertebrates. The origin of this peptide family may date back further still; similarly sized peptide transmitters with an identical C-terminal motif have been identified in molluscs and flatworms and designated neuropeptide F (NPF). Cloning of the npf gene from the parasitic flatworm Moniezia expansa identified some unusual features within the peptide precursor organization but, at the same time, provided support for an evolutionary relationship of npf and npy genes through the presence of a single intron at a conserved position. To extend the analysis of the evolutionary relationships between invertebrate NPF and vertebrate NPY family peptides, the NPF precursor from the turbellarian Arthurdendyus triangulatus was characterized. Sequence analysis revealed the npf transcript to be 362 base pairs in length encoding a single open reading frame of 81 amino acids. The precursor comprises a signal peptide followed by the mature peptide of 36 amino acids in length, terminating in the typical invertebrate GRPRF motif, followed by a carboxyterminal glycyl extension. The NPF precursor of A. triangulatus shows significant similarities to the vertebrate NPY peptides. Indeed, the N-terminus of A. triangulatus prepro-NPF corresponds more closely to that of the vertebrate peptide homologs than to that of other invertebrate NPFs isolated to date. Immunocytochemical localization studies have demonstrated NPF immunoreactivity throughout the nervous system of A. triangulatus, particularly in association with muscular structures. The data support an early evolutionary origin for this peptide transmitter family within the nervous system of basal bilaterians.

Molecular cloning and characterization of two new isoforms of the protein kinase A catalytic subunit from the human parasite Leishmania

Leishmania are protozoan parasites that cause extensive morbidity and mortality in humans. Genes for two new isoforms of the protein kinase A catalytic subunit (PKAC) in Leishmania, Lmpkac2a and Lmpkac2b, were cloned and characterized. The predicted open reading frames for these isoforms are 93.4% identical over 338 amino acids (aa). The conserved PK catalytic cores (subdomains I–XI) are identical, while the carboxy-terminal extensions differ by only two aa. However, LmPKAC2 shares only 62% identity over the 255 aa catalytic core region with the previously described LmPKAC1 (c-lpk2). Unlike LmPKAC1, the location of the FXXF motif at the carboxy-terminus is conserved in both LmPKAC2 isoforms; however, the aa sequence, LXXF, in isoform-2a is unusual. The leishmanial isoforms can be distinguished by their NH 2-terminal extensions, which show minimal similarity at the primary sequence level. Structural analysis of the three enzymes based on the crystal structure of mammalian PKAs predicts that both LmPKAC2 isoforms, unlike LmPKAC1, have identical α-helix structures in the NH 2-terminal extension. Lmpkac2 genes are located on chromosome 35 just downstream from the leishmanial prp8 gene. This genomic organization is conserved in two species of Leishmania and Crithidia fasciculata and allowed for the partial analysis of Cfpkac2a. Phylogenetic analysis groups the two LmPKAC2 isoforms together and separately from LmPKAC1, which is more similar to the Euglena gracilis PKAC, EPK2. These findings provide the basis for additional studies on the role of the PKA family in parasite differentiation and virulence.

Insulin-Like Growth Factor System Components in Relation to Erythropoietin Therapy and Bone Metabolism in Dialyzed Patients and Kidney Transplant Recipients

Insulin-like growth factor (IGF) system components appear to be the most important regulators of bone cell function. On the other hand, IGF-1 is shown to be an important regulator for erythropoiesis. The aim of the study was to examine the relationships between IGF system, requirements of erythropoietin, endogeneous erythropoietin levels, bone metabolism assesed by biochemical markers, markers of nutrition such as cholesterol and albumin in recombinant human erythropoietin (rHuEPO)-treated patients maintained on chronic hemodialyses or peritoneal dialyses as well as in kidney transplant recipients. The studies were performed on 79 chronically hemodialyzed patients; 28 of them did not receive rHuEPO, 51 subjects received rHuEPO, 34 patients on continuous ambulatory peritoneal dialysis (CAPD), 16 of them did not receive rHuEPO, 18 were given rHuEPO and 46 kidney allograft recipients. Endogeneous erythropoietin concentration, bone-specific alkaline phosphatase and serum CrossLaps were assayed by ELISA. Intact PTH, osteocalcin, 1,25-(OH)₂ D₃, 25-OH D₃, IGF-1, procollagen type I carboxy-terminal extension peptide (PICP) and procollagen type I cross-linked carboxy-terminal telopeptide (ICTP) were studied by RIA, whereas IGFBP-1 and IGFBP-3 concentrations were assayed by IRMA. We found a significantly higher IGF-1 and IGFBP-3 in rHuEPO-treated HD patients when compared to CAPD subjects given rHuEPO as well as to hemodialysis (HD) patients not treated with rHuEPO. IGF-1 was significantly higher in kidney transplant recipients when compared to dialyzed patients without rHuEPO therapy. IGFBP-1 was similar in all groups of patients (including kidney transplant recipients) studied. In CAPD patients not given rHuEPO concentrations of ICTP and PICP were significantly lower when compared to rHuEPO-treated CAPD subjects and HD patients not receiving rHuEPO therapy. Serum CrossLaps in CAPD patients treated with rHuEPO were significantly higher when compared to CAPD subjects without rHuEPO treatment and to kidney transplant recipients. In rHuEPO-treated CAPD subjects IGF-1 and IGFBP-1 correlated positively with serum CrossLaps (r = 0.61, p < 0.05 and r = 0.64, p < 0.05, respectively), whereas in hemodialyzed patients without rHuEPO a significant negative correlation between IGFBP-3 and serum CrossLaps was found (r = –0.69, p < 0.001) as well as between IGFBP-3 and aluminium (r = 0.51, p < 0.05), IGF-1 and ICTP (r = –0.43, p < 0.05). In conclusion, our data indicate a probable functional relationship between IGF system components, erythropoietin treatment in dialyzed patients and bone metabolism in renal replacement therapy in a form of hemodialyses, peritoneal dialyses and kidney transplantation. Dialyzed patients exhibit more pronounced renal osteodystrophy than kidney allograft recipients. IGF system components are influenced by erythropoietin therapy, but are not related to serum erythropoietin levels and rHuEPO requirements.

Biochemical and molecular biological studies on infection (Ascochyta rabiei)-induced thaumatin-like proteins from chickpea plants (Cicer arietinum L.).

A pathogenesis-related protein induced by infection with Ascochyta rabiei was purified from intercellular washing fluid of chickpea (Cicer arietinum L.) leaves. Amino-terminal sequencing identified the protein, named PR-5a, as a thaumatin-like protein. The isoelectric point was determined with 6.5 and the molecular mass is 16 kDa. Therefore, chickpea PR-5a is the first dicot member of a TLP subgroup containing small TLPs with a molecular weight between 15 and 18 kDa. PR-5a shows no antifungal activity towards A. rabiei. Screening of a chickpea cDNA library led to the isolation of a cDNA clone (p5a-241) for this protein. A second cDNA clone (ELR112) encoding a TLP was isolated using differential hybridisation of cDNA libraries obtained from elicited and water treated cell suspension cultures of chickpea. The deduced protein (PR-5b) has a molecular mass of 22 kDa. PR-5b is postulated to be located in the vacuole due to the presence of a respective N-terminal signal peptide and a carboxy-terminal extension. Southern blot analyses showed that ELR112 and p5a-241 represent single copy genes. During fungal infection of chickpea plants expression of both genes proceeds much faster in an A. rabiei resistant cultivar than in a susceptible one.

Characterization of two forms of cocaine- and amphetamine-regulated transcript (CART) peptide precursors in goldfish: molecular cloning and distribution, modulation of expression by nutritional status, and interactions with leptin.

Complementary DNAs encoding two forms of cocaine- and amphetamine-regulated transcript (CART) peptide precursors were identified from goldfish brain and named CART I and CART II. Each cDNA contains a signal peptide sequence, the putative CART-like peptide, and a carboxy-terminal extension peptide. Form I encodes a 117-amino acid pro-CART, whereas form II encodes a 120-amino acid pro-CART. Both forms resemble mammalian CART peptides. Each goldfish CART precursor is encoded by three exons interrupted by two introns within genomic DNA. RT-PCR, slot blot, and Northern blot analysis showed that the mRNAs for form I and II precursors have a widespread distribution. Form I and II are present in the brain, pituitary, eye, gonads, and kidney. Form I is also present in the gill. In the brain, form I is predominant in the olfactory bulb and hypothalamus, and form II is predominant in the optic tectum. Food deprivation for 96 h induced a decrease in form I mRNA levels in the telencephalon-preoptic region, hypothalamus, and olfactory bulb and in form II mRNA expression in the olfactory bulb. An increase in mRNA levels was observed 2 h following a meal in the olfactory bulbs and hypothalamus for form I whereas no postprandial changes in form II mRNA levels were observed. Intracerebroventricular injections of human CART alone induced a significant decrease in food intake. Injections of leptin reinforced the inhibition of feeding behavior and food intake seen in CART-treated fish. Central injection of leptin induced an increase in CART I mRNA in the optic tectum, hypothalamus, and olfactory bulbs but had no effect on CART II mRNA expression in the brain. These results suggest that CART peptides act as leptin-regulated satiety factors in goldfish and that they might have other physiological roles besides feeding, possibly in sensory information processing.