Identification of the prion protein allotypes which accumulate in the brain of sporadic and familial Creutzfeldt-Jakob disease patients.

A characteristic feature of Creutzfeldt-Jakob disease (CJD) is the accumulation in the brain of the amyloid protease-resistant protein PrPres. PrPres derives from a host-encoded, protease-sensitive isoform, PrPsen. Mutations of this protein are linked to familial variants of the disease, and the presence of a methionine or valine residue at the polymorphic position 129 may be critical in sporadic CJD cases. We found that in the brain of patients heterozygous for the mutation in which isoleucine is substituted for valine at codon 210 (Val21Olle), the PrPres is formed by both the wild-type and mutant PrPsen. We also found that in a sporadic CJD patient, who was heterozygous (Met/Val) at position 129, PrPres is also formed by both allotypes. These data associate transmissible spongiform encephalopathies with other amyloidosis, although the nature of the transmissible agent remains unsettled.

Chlamydia pneumoniae induces T cell apoptosis through glutathione redox imbalance and secretion of TNF-alpha.

Chlamydia pneumoniae persistent infection has been implicated in the pathogenesis of several chronic inflammatory diseases including atherosclerosis, and we hypothesized that modulation of the apoptosis of macrophages and/or T cells by C. pneumoniae infection may contribute to the development of such diseases. We therefore evaluated apoptosis, cytokine response, and redox status in human primary T cells and macrophages infected with C. pneumoniae. In addition, co-cultures of T cells and macrophages infected with C. pneumoniae were also carried out. Apoptosis, and levels of glutathione (GSH), glutathione disulfide (GSSG), and tumour necrosis factor (TNF)-alpha were measured by flow cytometry, high performance liquid chromatography and enzyme-linked immunosorbent assay. C. pneumoniae induced apoptosis in T cells as well as in co-cultures of T cells and infected macrophages by marked decrease in GSH/GSSG ratio and increased production of TNF-alpha, respectively. The results demonstrate that interaction of C. pneumoniae with T cells and/or macrophages characterized by interference with redox status, and secretion of tumour necrosis factor-alpha culminates in the induction of T cell apoptosis and survival of infected macrophages. In conclusion, the inappropriate T cell response against C. pneumoniae and survival of infected macrophages could explain the persistence of this intracellular obligate pathogen in the host-organism; it may contribute to the development of chronic inflammatory diseases, although further studies are needed to clarify such a complex mechanism.

Quantitative profiling of the pathological prion protein allotypes in bank voles by liquid chromatography-mass spectrometry.

The conversion of the cellular prion protein (PrP(C)) into a misfolded isoform (PrP(TSE)) that accumulates in the brain of affected individuals is the key feature of transmissible spongiform encephalopaties (TSEs). Susceptibility to TSEs is influenced by polymorphisms of the prion gene suggesting that the presence of certain amino acid residues may facilitate the pathological conversion. In this work, we describe a quantitative, fast and reliable HPLC-MS method that allowed to demonstrate that in the brain of 109(Met/Ile) heterozygous bank voles infected with the mouse adapted scrapie strain 139A, there are comparable amounts of PrP(TSE) with methionine or isoleucine in position 109, suggesting that in this TSE model the two allotypes have similar rates of accumulation. This method can be easily adapted for the quantitative determination of PrP allotypes in the brain of other natural or experimental TSE models.

Purification and characterization of an antifungal thaumatin-like protein from Cassia didymobotrya cell culture.

A 23-kDa antifungal thaumatin-like protein was isolated and purified from Cassia didymobotrya (Fres.) cell cultures for the first time. The protein was secreted in the culture medium, but it could be also isolated after elution of whole cells with a 0.5 M CaCl(2) solution. Treatment of the cells with laminarin oligosaccharides or salicylic acid, but not with NaCl, resulted in enhancement of expression of the protein. A rapid purification protocol was used based on cationic exchange chromatography. The protein, with a highly basic character (pI 10), has an exact molecular mass of 23034 Da, as determined by MALDI-ToF mass spectrometry analysis. N-terminal sequencing of the intact polypeptide and the sequencing of two internal tryptic peptides indicated significant identity with other thaumatin-like proteins (TLP). The protein exerted antifungal activity towards some Candida species showing EC(50) values comparable to those of other antifungal TLPs. The collected data lead to classify this TLP as a new PR-5 protein.

Different susceptibility of the two dimers of ribonuclease A to subtilisin. Implications for their structure.

RNase A and its minor and major dimers were digested with subtilisin under controlled conditions. The major dimer was found to be slightly more resistant, the minor dimer markedly less resistant to subtilisin than monomeric RNase A. Two S-proteins formed for each RNase A species, one starting with Ser-21, the other with Ser-22. Their relative proportions indicate that the structure of the minor dimer, whose identity with that of a RNase A dimer shown to be 3D domain-swapped is strongly suggested by recent work [S. Sorrentino et al. (2000) FEBS Lett. 466, 35-39], makes its peptide bond between Ser-21 and Ser-22 more accessible to subtilisin than it is in RNase A and its major dimer. Moreover, (i) both subunits constituting the minor dimer are more susceptible to subtilisin than monomeric RNase A, and (ii) the susceptible bonds in one of its two exchanging N-terminal arms are more accessible to the protease than in the other. The properties of the major dimer suggest that its structure could be different.

Proteinase isoinhibitors from bovine spleen: primary structure of an intermediate in the processing of the precursor.

The complete amino acid sequence of the proteinase inhibitor III from bovine spleen is reported. It consists of 62 amino acid residues and is identical to that of spleen inhibitor II (an isoinhibitor of the bovine pancreatic trypsin inhibitor, which shares with the latter 89% of sequence identity), except for four extra residues at the C-terminal side. Inhibitor III appears to be an intermediate in the processing of the putative 100-residue primary expression product, which leads to the mature inhibitor II. These results and those previously obtained for another intermediate, isoinhibitor I, are indicative of the following order for the last steps of the precursor processing inhibitor I----inhibitor III----inhibitor II. The mature protein and the two intermediates isolated have a very similar antiproteolytic activity. However, their in vivo target enzyme(s) are not yet known, as also the target enzyme of the bovine pancreatic trypsin inhibitor is not known. Thus, the available data would indicate that either the three isoinhibitors have a distinct functional role, by inhibiting different target enzymes, or inhibitors I and III are obligatory intermediates for directing the final targeting of the mature, functionally relevant inhibitor II.

Structural and immunological similarities between high molecular weight zinc ion-dependent p-nitrophenylphosphatase and fructose-1,6-bisphosphate aldolase from bovine liver.

High molecular weight zinc ion-dependent acid p-nitrophenylphosphatase (HMW-ZnAPase) was purified from bovine liver to homogeneity as judged by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The partial sequence of the purified enzyme electroblotted on PVDF membrane reveals a 95% sequence homology with human and bovine liver fructose-1,6-bisphosphate aldolase isozyme B (FALD B). FALD B was isolated from bovine liver using an affinity elution from phosphocellulose column. FALD B from bovine liver shows a native and subunit molecular weight that is indistinguishable from that of HMW-ZnAPase. In addition, an affinity purified antiserum raised in rabbits against purified HMW-ZnAPase cross-reacts with bovine liver FALD B and rabbit muscle isozymes. Despite these similarities, HMW-ZnAPase does not show FALD activity and bovine liver FALD does not display any zinc ion-p-nitrophenylphosphatase activity. These results suggested the existence of structural and immunological similarities between bovine liver HMW-ZnAPase and FALD B. Differences in some amino acid residues in enzyme activity indicate that they may be involved in different biochemical functions.

The primary structure of mitochondrial aspartate aminotransferase from human heart.

The complete amino acid sequence of the mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) from human heart has been determined based mainly on analysis of peptides obtained by digestion with trypsin and by chemical cleavage with cyanogen bromide. Comparison of the sequence with those of the isotopic isoenzymes from pig, rat and chicken showed 27, 29 and 55 differences, respectively, out of a total of 401 amino acid residues. Evidence for structural microheterogeneity at position 317 has also been obtained.

Purification of a 76-kDa iron-binding protein from human seminal plasma by affinity chromatography specific for ribonuclease: structural and functional identity with milk lactoferrin.

A pink-colored iron-binding protein has been found in large amount in human seminal plasma and identified as a lactoferrin isoform. Its purification, by a modification of a three-step chromatography procedure developed in an attempt to purify a ribonuclease from the same fluid, provided about 15-18 mg of pure protein from 100 ml of seminal plasma. Despite its ability to bind a ribonuclease ligand during the affinity step, the iron-binding protein did not display any detectable RNase activity in a standard assay with yeast RNA as substrate. It showed an apparent molecular weight of 76 kDa and resulted to be quite similar, if not identical, to human milk lactoferrin in many respects. Its N-terminal sequence (31 amino acid residues) starting with Arg-3 was identical to that of one of the N-terminally truncated lactoferrin variants isolated from human milk. Moreover, the amino acid sequence of a number of peptides, which represented about 23% of the entire sequence, has been also shown to be identical to that of the corresponding peptides of human milk lactoferrin. Double diffusion analysis revealed full recognition by antibodies anti-human milk lactoferrin of the human seminal plasma protein. Using immunoblotting analysis, both human milk lactoferrin and human seminal protein were recognized by antibodies anti-milk lactoferrin. When tested for its iron binding capacity, with Fe-NTA as iron donor, the protein purified was able to bind iron up to 100% saturation, as judged by absorbance at 465 nm.

Cellular nucleic acid binding protein binds a conserved region of the 5' UTR of Xenopus laevis ribosomal protein mRNAs.

Vertebrate ribosomal protein mRNAs share structural features in the 5' untranslated region implicated in the control of their translation. A pyrimidine tract, at the 5' end, is considered the common cis-acting element, but the control requires also the integrity of the conserved downstream region. These sequences interact in vitro with proteins, which may represent the trans-acting factors for a common regulation. The protein that binds the pyrimidine tract has been identified as La and its binding in vitro depends on interaction with a protein factor. In the present study, by purification, microsequencing and immunoprecipitation analysis we have identified the protein that interacts with the region downstream of the pyrimidine tract as the Xenopus laevis cellular nucleic acid binding protein (CNBP). The interaction of this protein with the conserved region of various ribosomal protein (rp)-mRNAs suggests a class-specific recognition. The binding of CNBP to the target region requires the assistance of a protease-sensitive factor, that dissociates after complex formation. Some evidence suggests that this may be the same factor that assists the binding of La to the 5' untranslated region (UTR) of the rp-mRNAs. Considering that CNBP and La come in contact with two typical regions of the 5' UTR, essential for regulation, their interaction with the assisting factor may exert a modulating activity on the translational control of ribosomal protein mRNAs.

Streptomyces griseus aminopeptidase: X-ray crystallographic structure at 1.75 A resolution.

The X-ray crystal structure of the enzyme Streptomyces griseus aminopeptidase (SGAP) has been determined in its double zinc form to 1.75 A resolution, in its apo-enzyme from (zinc removed) to 2.1 A resolution, and as a mercury replaced derivative to 2.1 A resolution. The structure solution was achieved by single isomorphous replacement with phasing from anomalous scattering (SIRAS), followed by density modification with histogram matching. The protein consists of a central beta-sheet made up of eight parallel and antiparallel strands, surrounded by helices on either side. The active site is located at the carbonyl ends of two middle strands of the beta-sheet region. Two sections of the chain that could not be traced were Glu196 to Arg202, which borders the active site, and the final seven C-terminal residues starting with Gly278. The active site contains two zinc cations, each with similar ligands, at a distance of 3.6 A from each other. An unknown molecule appears to be bound to both zinc ions in the active site at partial occupancy and has been modelled as a phosphate ion. A calcium binding site has also been identified, consistent with the observations that calcium modulates the activity of the enzyme, and increases its heat stability. The mechanism by which the calcium cation modulates enzyme activity is not apparent, since the location of the calcium binding site is approximately 25 A distant from the active site zinc ions. Comparison of the structure of SGAP to other known aminopeptidases shows that the enzyme is most similar to Aeromonas proteolytica aminopeptidase (AAP). Both enzymes share a similar topology, although the overall sequence identity is very low (24% in aligned regions). The coordination of the two active site zinc cations in SGAP resembles that of AAP. These two microbial enzymes differ from bovine lens leucine aminopeptidase (LAP) in both overall structure and in coordination of the two zinc ions.

Identification of the pathological prion protein allotypes in scrapie-infected heterozygous bank voles (Clethrionomys glareolus) by high-performance liquid chromatography-mass spectrometry.

Cerebral formation of the pathological isoform of the prion protein (PrP) is a crucial molecular event in prion diseases. The bank vole (Clethrionomys glareolus) is a rodent species highly susceptible to natural scrapie. The PrP gene of bank vole is polymorphic (Met/Ile) at codon 109. Here we show that homozygous 109Met/Met voles have incubation times shorter than heterozygous 109Met/Ile voles after experimental challenge with three different scrapie isolates. An HPLC-MS/MS method was optimized and applied to investigate whether in heterozygous animals both PrP allotypes are able to undergo pathological conversion. The results demonstrate that both allotypes of the prion protein participate to pathological deposition.

Glucan-associated protein modulations and ultrastructural changes of the cell wall in Candida albicans treated with micafungin, a water-soluble, lipopeptide antimycotic.

The composition of glucan-associated proteins (GAP) in the cell wall of Candida albicans was strongly affected by treatment with a sub-MIC yet beta-glucan synthesis inhibitory concentration (0.01 microg/ml) of FK463 (micafungin). Namely, a decrease in enzymes of glucose metabolism (mostly enolase and a novel 40 kDaltons component, here identified as the enzyme fructose-1,6-biphosphate aldolase) was observed, and this was coupled with an increase in two beta1-3 exo-glucanase isoforms (34 and 44 kDa, respectively). No GAP changes were detected in the same strain of the fungus made resistant to the drug, attesting to the specificity of the observed cell wall protein modulation. In addition, GAP changes were accompanied by marked ultrastructural alterations upon treatment with the sub-MIC dose of the drug, the majority of which was an aberrant cell surface morphology and a derangement of the normal layering of the cell wall. Our data demonstrate that sub-MIC doses of micafungin do critically affect not only the beta-glucan synthetic machinery but also protein composition and the whole cell wall structure of Candida albicans.

A chromophore in glutamate decarboxylase has been wrongly identified as PQQ.

Pyrroloquinoline quinone (PQQ) has been claimed to be a component of glutamate decarboxylase from Escherichia coli on the basis of a frequently used procedure in which the protein is extracted with hexanol. We demonstrate that if pyridoxal phosphate (PLP) is not added during the preparation, the apoenzyme prepared from glutamate decarboxylase contains no chromophore absorbing above 280 nm. Full enzyme activity and the original holoenzyme spectrum are restored by the addition of PLP alone. A 340 nm-absorbing band, similar to that which prompted analysis for PQQ, is produced by exposure of the enzyme to solutions of PLP.

Is pantetheinase the actual identity of mouse and human vanin-1 proteins?

Pantetheinase is an amidohydrolase involved in the dissimilative pathway of CoA, allowing the turnover of the pantothenate moiety. We have determined the N-terminal sequence as well as the sequences of a number of tryptic and chymotryptic peptides of the protein isolated from pig kidney. These sequence stretches were used as probes to search in the SwissProt database and significant similarities were found with a GPI-anchored protein (mouse vanin-1, with a suggested role in lymphocyte migration), with two putative proteins encoded by human cDNAs (VNN1 and VNN2) and with human biotinidase. On the basis of sequence similarity, we propose that vanin-1 and VNN1 should be identified as pantetheinase.

Function of saposin C in the reconstitution of glucosylceramidase by phosphatidylserine liposomes.

The function of saposin C (Sap C), a glucosylceramidase activator protein, in the enzyme stimulation by phosphatidylserine (PS) liposomes has been investigated. Using gel filtration experiments evidence was obtained for Sap C binding to PS large unilamellar vesicles (LUV) but not to glucosylceramidase. PS LUV, which by themselves are unable to tightly bind and stimulate the enzyme, acquire the capacity to also bind the enzyme after interaction with Sap C, making it express its full activity. Our results indicate that the primary step in the Sap C mode of action resides in its association with PS membranes; in turn, this association promotes the interaction between the membranes and glucosylceramidase.

Pantetheinase activity of membrane-bound Vanin-1: lack of free cysteamine in tissues of Vanin-1 deficient mice.

Pantetheinase (EC 3.5.1.-) is an ubiquitous enzyme which in vitro has been shown to recycle pantothenic acid (vitamin B5) and to produce cysteamine, a potent anti-oxidant. We show that the Vanin-1 gene encodes pantetheinase widely expressed in mouse tissues: (1) a pantetheinase activity is specifically expressed by Vanin-1 transfectants and is immunodepleted by specific antibodies; (2) Vanin-1 is a GPI-anchored pantetheinase, and consequently an ectoenzyme; (3) Vanin-1 null mice are deficient in membrane-bound pantetheinase activity in kidney and liver; (4) in these organs, a major metabolic consequence is the absence of detectable free cysteamine; this demonstrates that membrane-bound pantetheinase is the main source of cysteamine in tissues under physiological conditions. Since the Vanin-1 molecule was previously shown to be involved in the control of thymus reconstitution following sublethal irradiation in vivo, this raises the possibility that Vanin/pantetheinase might be involved in the regulation of some immune functions maybe in the context of the response to oxidative stress.

Polymorphism at codon 129 or codon 219 of PRNP and clinical heterogeneity in a previously unreported family with Gerstmann-Sträussler-Scheinker disease (PrP-P102L mutation).

We present a new, large, Italian family affected by Gerstmann-Sträussler-Scheinker syndrome (GSS) associated with the Pro to Leu point mutation at codon 102 of the prion protein gene (PRNP). The affected members of this family show a remarkable phenotypic variability of the disease: three of them had a clinical picture characterized by dementia and a brief illness duration (less than 1 year), while the other five members presented an ataxic, slowly evolving syndrome (a clinical duration of 3 to 4 years) with no evidence of cognitive impairment. Despite these remarkable clinical differences among affected members, we found no correlation between the clinical presentation and the codon 129 or codon 219 genotypes. These data suggest that factors as yet unidentified may influence the clinical expression of the disease.

Isolation and characterization of the d1 domain of Pseudomonas aeruginosa nitrite reductase.

Proteolitic digestion of nitrite reductase from Pseudomonas aeruginosa allows to obtain and purify a domain containing only the d1 heme and constituted by two noncovalently bound peptides. This d1 domain catayzes oxygen consumption, and binds carbon monoxide with a kinetic constant slightly higher than the parental dimeric holoenzyme. The capacity to oxidize the physiological substrate, cytochrome c551, is lost, even when the proteolytic c heme domain is added to this reaction mixture. This finding suggests that the two domains do not have a significant affinity for each other, and are kept together only by being part of the same polypeptide.

123I-Interleukin-2: biochemical characterization and in vivo use for imaging autoimmune diseases.

We describe in detail the labelling of interleukin-2 with I ( I-IL2), its biochemical characterization, the binding assay and its use for the detection of tissues infiltrated with mononuclear cells. Human recombinant IL2 was labelled using an enzymatic method and its biochemical characterization was performed using high performance liquid chromatography (HPLC) analysis of cyanogen bromide-cleaved protein. biological and binding assays were performed on CTLL-2 cell line and on activated peripheral blood lymphocytes. studies were performed 1 h after administration of 2-3 mCi of I-IL2 in 10 newly diagnosed type 1 diabetes patients, five pre-diabetic patients, 10 Hashimoto's thyroiditis patients, 10 coeliac disease patients and 10 normal volunteers. I-IL2 scintigraphy allowed the detection and quantification of activated mononuclear cells in several affected tissues. In detail, I-IL2 accumulation was detected in the thyroid of all patients affected by Hashimoto's thyroiditis, in the bowel of all coeliac disease patients and in the pancreas of all pre-type 1 diabetic patients. By contrast, in newly diagnosed type 1 diabetics, I-IL2 scan was positive in five of the 10 studied patients. I-IL2 scintigraphy may be useful for studying autoimmune phenomena and in diagnostic protocols to evaluate the presence of other tissue involvement in patients with an organ-specific autoimmune disease.