The plug domain of a neisserial TonB-dependent transporter retains structural integrity in the absence of its transmembrane beta-barrel.

Transferrin binding protein A (TbpA) is a TonB-dependent outer membrane protein expressed by pathogenic bacteria for iron acquisition from human transferrin. The N-terminal 160 residues (plug domain) of TbpA were overexpressed in both the periplasm and cytoplasm of Escherichia coli. We found this domain to be soluble and monodisperse in solution, exhibiting secondary structure elements found in plug domains of structurally characterized TonB-dependent transporters. Although the TbpA plug domain is apparently correctly folded, we were not able to observe an interaction with human transferrin by isothermal titration calorimetry or nitrocellulose binding assays. These experiments suggest that the plug domain may fold independently of the beta-barrel, but extracellular loops of the beta-barrel are required for ligand binding.

Characterization of the G91del CRYBA1/3-crystallin protein: a cause of human inherited cataract.

Congenital cataract is a leading cause of visual disability in children. Inherited isolated (non-syndromic) cataract represents a significant proportion of cases and the identification of genes responsible for inherited cataract will lead to a better understanding of the mechanism of cataract formation at the molecular level both in congenital and age-related cataract. Crystallins are abundantly expressed in the developing human lens and represent excellent candidate genes for inherited cataract. A genome-wide search of a five-generation family with autosomal dominant lamellar cataract demonstrated linkage to the 17p12-q11 region. Screening of the CRYBA1/3 gene showed a 3 bp deletion, which resulted in a G91del mutation within the tyrosine corner, that co-segregated with disease and was not found in 96 normal controls. In order to understand the molecular basis of cataract formation, the mutant protein was expressed in vitro and its unfolding and refolding characteristics assessed using far-UV circular dichroism spectroscopy. Defective folding and a reduction in solubility were found. As the wild-type protein did not refold into the native conformation following unfolding, a corresponding CRYBB2 mutant was genetically engineered and its refolding characteristics analysed and compared with wild-type CRYBB2. Its biophysical properties support the hypothesis that removal of the glycine residue from the tyrosine corner impairs the folding and solubility of beta-crystallin proteins. This study represents the first comprehensive description of the biophysical consequences of a mutant beta-crystallin protein that is associated with human inherited cataract.

The stability of human acidic beta-crystallin oligomers and hetero-oligomers.

Crystallins are bulk structural proteins of the eye lens that have to last a life time. They gradually become modified with age, denature and form light scattering centres. High thermodynamic and kinetic stability of the crystallins enables them to resist unfolding and delay cataract. Here we have made recombinant human betaA1-, betaA3-, and betaA4-crystallins. The betaA3-crystallin formed higher oligomers that lead to precipitation at ambient temperature. Heat-induced precipitation of betaA3-crystallin was compared with human and calf betaB2-crystallins, showing that the human proteins start to precipitate above 50 degrees C while the calf betaB2-crystallin stays in solution even when unfolded. The stabilities of these human acidic beta-crystallin homo-oligomers have been estimated by measuring their unfolding in urea at neutral pH. BetaA3/1/betaB1 and betaA4/betaB1-crystallin hetero-oligomers have been prepared from homo-oligomers by subunit exchange. The resolution of the methodology used was insufficient to detect a stabilization of the betaA4-crystallin subunit in the hetero-oligomer, the betaA1-crystallin subunit was clearly stabilized by its interaction with betaB1-crystallin. Circular dichroism and fluorescence spectroscopies show that homo-dimer surface tryptophans become buried in the betaA3/1/betaB1-crystallin hetero-dimer concomitant with changes in polypeptide chain conformation.

Synchrotron radiation circular dichroism spectroscopy: vacuum ultraviolet irradiation does not damage protein integrity.

Synchrotron radiation circular dichroism (SRCD) spectroscopy is an emerging technique for sensitive determination of protein secondary structures and for monitoring of conformational changes. An important issue for its adoption as a useful technique is whether the high-intensity low-wavelength vacuum ultraviolet radiation in the SRCD chemically damages proteins. In this paper, using horse myoglobin as a test sample, it is shown that extensive irradiation in the SRCD does not produce any change in the chemical nature of the protein as detected by either SDS gel electrophoresis or mass spectrometry. In addition, no changes in the protein secondary structure are detectable from the SRCD spectra after extensive exposure to the SRCD beam.

A temperature-sensitive mutation of Crygs in the murine Opj cataract.

In Opj, an inherited cataract in mice, opacity is associated with a mutation in Crygs, the gene for gammaS-crystallin, the first mutation to be associated with this gene. A single base change causes replacement of Phe-9, a key hydrophobic residue in the core of the N-terminal domain, by serine. Despite this highly non-conservative change, mutant protein folds normally at low temperature. However, it exhibits a marked, concentration-dependent decrease in solubility, associated with loss of secondary structure, at close to physiological temperatures. This is reminiscent of processes thought to occur in human senile cataracts in which normal proteins become altered and aggregate. The Opj cataract is progressive and more severe in Opj/Opj than in Opj/+. Lens histology shows that whereas fiber cell morphology in Opj/+ mice is essentially normal, in Opj/Opj, cortical fiber cell morphology and the loss of maturing fiber cell nuclei are both severely disrupted from early stages. This may indicate a loss of function of gammaS-crystallin which would be consistent with ideas that members of the betagamma-crystallin superfamily may have roles associated with maintenance of cytoarchitecture.

Deletion of 24 amino acids from the C-terminus of phosphatidylinositol transfer protein causes loss of phospholipase C-mediated inositol lipid signalling.

Phosphatidylinositol transfer protein alpha (PITPalpha) is a 32 kDa protein of 270 amino acids that is essential for phospholipase C-mediated phosphatidylinositol bisphosphate hydrolysis. In addition, it binds and transfers phosphatidylinositol and phosphatidylcholine between membrane compartments in vitro. Here we have used limited proteolysis of PITPalpha by subtilisin to identify the structural requirements for function. Digestion by subtilisin results in the generation of a number of slightly smaller peptide fragments, the major fragment being identified as a 29 kDa protein. The fragments were resolved by size-exclusion chromatography and were found to be totally inactive in both in vivo PLC reconstitution assays and in vitro phosphatidylinositol transfer assays. N-terminal sequencing and MS of the major 29 kDa fragment shows that cleavage occurs at the C-terminus of PITP at Met246, leading to a deletion of 24 amino acid residues. We conclude that the C-terminus plays an important role in mediating PLC signalling in vivo and lipid transfer in vitro, supporting the notion that lipid transfer may be a facet of PITP function in vivo.

Antibacterial activity of peptides homologous to a loop region in human lactoferrin.

Human lactoferrin contains a 46 residue sequence named lactoferricin H thought to be responsible for its antimicrobial properties. Synthetic peptides HLT1, corresponding to the loop region of human lactoferricin (FQWQR-NMRKVRGPPVS) and HLT2, corresponding to its charged portion (FQWQRNMRKVR), exerted significant antibacterial effects against E. coli serotype O111 strains NCTC 8007 and ML35. The corresponding sequences in native human lactoferrin were shown to adopt a charged helix and hydrophobic tail within the N-lobe remote from the iron binding site. Sequence similarities between lactoferricin and dermaseptin and magainins suggest that lactoferricin may act as an amphipathic alpha helix.

[Assessment of residual mediastinal tumor in patients with Hodgkin's lymphoma using computed tomography, magnetic resonance and 67Ga scintigraphy]. / Valutazione del residuo mediastinico nei pazienti affetti da linfoma di Hodgkin con Tomografia Computerizzata, Risonanza Magnetica e oncoscintigrafia con 67Ga.

594 patients with Hodgkin's disease were treated from 1983 to 1993 at the Department of Radiotherapy and Institute of Hematology, "La Sapienza" University, Rome, Italy. 385 patients presented mediastinal involvement; CT and/or chest radiography showed residual mediastinal masses in 96 of them (25%). In this study we included only the patients treated after 1986; they were examined with MRI of the chest (24 patients) and 67Gallium scintigraphy of the mediastinum (44 patients) with or without SPECT, combined with high-dose 67Ga in some cases. Eighteen patients underwent both MRI and 67Gallium scintigraphy. MR accuracy, sensitivity and specificity were respectively 75%, 86% and 86%; gallium scintigraphy had 86%, 77% and 93%. These data were confirmed by the results fo the subgroup of 18 patients submitted to both exams; MRI had higher sensitivity (80% vs. 75%) and lower specificity and accuracy (83% vs. 80% and 72% vs. 67, respectively) than 67Gallium scintigraphy. The predictive value of MR-scintigraphy agreement is high: indeed, no false negatives or false positives were observed when MR and scintigraphy results were in agreement.

The mechanism of iron uptake by transferrins: the structure of an 18 kDa NII-domain fragment from duck ovotransferrin at 2.3 A resolution.

The molecular structure of an iron-containing 18 kDa fragment of duck ovotransferrin, obtained by proteolysis of the intact protein, has been elucidated by protein crystallographic techniques at 2.3 A resolution. This structure supports a mechanism of iron uptake in the intact protein whereby the binding of the synergistic (bi)carbonate anion is followed by binding of the metal with the lobe in the open configuration. These stages are then followed by domain closure in which the aspartic acid residue plays a further key role, by forming an interdomain hydrogen-bond interaction in addition to serving as a ligand to the iron. This essential dual role is highlighted by model building studies on the C-terminal lobe of a known human variant. In this variant a mutation of a glycine by an arginine residue enables the aspartic acid to form an ion pair and reduce its effectiveness for both metal binding and domain closure. The X-ray structure of the 18 kDa fragment strongly suggests that the histidine residue present at the iron binding site of the intact protein and arising from the second interdomain connecting strand has been removed during the preparative proteolysis.

X.a.f.s. studies of chicken dicupric ovotransferrin.

A comparison of Cu K-edge x.a.f.s. spectra with that of the equivalent Fe K-edge for chicken ovotransferrin (COT) indicates that the metal ions occupy essentially the same binding sites in the protein. However, in the case of the Cu2+ complex the metal appears to have reduced co-ordination. Changes are observed in the x.a.f.s. of 90%-saturated COT (Cu1.8COT) on freeze-drying. The three-dimensional X-ray structures of rabbit serum transferrin and human lactoferrin have shown that the ferric cations are co-ordinated by four protein ligands and a bidentate carbonate anion in a distorted octahedral arrangement [Anderson, Baker, Dodson, Norris, Rumball, Waters & Baker (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1768-1774; Anderson, Baker, Norris, Rice and Baker (1989) J. Mol. Biol. 209, 711-734; Bailey, Evans, Garratt, Gorinsky, Hasnain, Horsburgh, Jhoti, Lindley, Mydin, Sarra & Watson (1988) Biochemistry 27, 5804-5812]. This structural information, together with the differences in e.x.a.f.s. spectra for solution and freeze-dried samples of diferric COT [Hasnain, Evans, Garratt & Lindley (1987) Biochem. J. 247, 369-375] suggests that the synergistic carbonate anion may be capable of behaving as a unidentate linkage to the Cu2+ in the dicupric complex. Data for Cu1.8COT are consistent with only three protein ligands bound to Cu2+, monodentate binding of the synergistic anion in one lobe and its bidentate binding in the other lobe. Such flexibility in the anion co-ordination may be a requirement for the uptake and release of metals by the transferrins.

[Role of radiotherapy in the treatment of seminoma of the testis]. / Ruolo della radioterapia nel trattamento del seminoma del testicolo.

From 1965 through 1988, 113 patients affected with testicular seminoma were treated at the Dept. of Radiotherapy, University "La Sapienza", Rome, Italy. Mean age of the patients was 38 years; in 70 cases tumor developed in the right testis and in 43 in the left one. In 9 patients underlying cryptorchidism was observed. All cases underwent radical orchiectomy. Histology diagnosed anaplastic seminoma in 5 cases and pure seminoma in all the other patients. Structures were involved in 7 cases. Eighty-four patients were in stage I, 20 in stage IIA, 4 in IIB, 4 in IIIA, and 1 in stage IIIB. All patients staged as I and IIA were treated with exclusive radiotherapy on paraaortic lymph nodes and inguinal and iliac lymph nodes of the involved sites (total doses: 28-35 Gy in stage I and 34-40 Gy in stage IIA). Before 1970 these patients underwent prophylactic irradiation of mediastinum and of left supraclavicular lymph nodes (total dose: 25-28 Gy). Patients in stage IIB were administered subdiaphragmatic lymph nodes irradiation with inverted-Y field (total dose: 36-45 Gy). Two cases were irradiated also on mediastinum and left supraclavicular lymph nodes, and 2 received two cycles of polychemotherapy (PVB) before irradiation. Patients in stage IIIA underwent sub-/supra-diaphragmatic irradiation (total dose: 40-45 Gy, and 40-42 Gy). The case in stage IIIB underwent palliation chemotherapy and local irradiation. All cases in stages I, IIA and IIB obtained complete remission. Three cases of the 4 in stage IIIA obtained complete remission (75%), while 1 (25%) progressed and died 8 months after diagnosis; the only case in stage IIIB progressed and died after 7 months of follow-up. Two cases in stage I recurred (2.4%), 1 in the mediastinum and 1 in the left supraclavicular lymph nodes. Both were cured with salvage radiation therapy. Toxicity related to treatment was low. Two cases in stage I developed secondary malignant neoplasms, at 4 and 34 months of follow-up, respectively.

[Evolution of the diagnostic accuracy of CT in clinical staging of patients with Hodgkin's disease]. / Evoluzione dell'accuratezza diagnostica della TC nella stadiazione clinica dei pazienti affetti da morbo di Hodgkin.

From 1983 through 1989, 141 untreated patients with Hodgkin's disease underwent CT of the abdomen. They subsequently underwent staging laparotomy plus splenectomy and multiple biopsies of liver and lymph nodes, at the Institute of Radiology and Hematology, University "La Sapienza", Rome. CT results were compared with surgical findings to evaluate CT sensitivity, specificity, and overall accuracy. The cases from this series were divided into two groups depending on the characteristics of the CT scanners employed. From 1983 to 1985, 78 patients were examined with 2nd-generation CT units; from 1986 to 1989, 63 patients underwent CT performed with 3rd-generation scanners. The results from the two groups were analyzed according to these parameters. A total number of 622 biopsies were performed, of spleen, liver, and lymph nodes. CT sensitivity, specificity, and overall accuracy were: 22.9% (group I) vs 43.7% (group II), 83.1% vs 92%, and 68.4% vs 81.2% for lymph nodes; 28.1% vs 36.3%, 93.5% vs 98%, and 66.7% vs 87.3% for the spleen, and 12.5% vs 42.8%, 97.1% vs 98.2%, and 88.5% vs 92.1% for the liver. Our results demonstrate an obvious increase in reliability with newer units, even though a high percentage of false-negatives were still observed in our series, which caused understaging in 19.4% of cases vs 24.4% in group I.

Molecular structure of serum transferrin at 3.3-A resolution.

Serum transferrin is a metal-binding glycoprotein, molecular weight ca. 80,000, whose primary function is the transport of iron in the plasma of vertebrates. The X-ray crystallographic structure of diferric rabbit serum transferrin has been determined to a resolution of 3.3 A. The molecule has a beta alpha structure of similar topology to human lactoferrin and is composed of two homologous lobes that each bind a single ferric ion. Each lobe is further divided into two dissimilar domains, and the iron-binding site is located within the interdomain cleft. The iron is bound by two tyrosines, a histidine, and an aspartic acid residue. The location of the 19 disulfide bridges is described, and their possible structural roles are discussed in relation to the transferrin family of proteins. Mapping of the intron/exon splice junctions onto the molecule provides some topological evidence in support of the putative secondary role for transferrin in stimulating cell proliferation.

Secondary structure prediction of human SAA1. Presumptive identification of calcium and lipid binding sites.

Serum amyloid A protein (SAA), an apolipoprotein of high density lipoprotein (HDL), is an acute phase protein thought to be the precursor of amyloid fibrils in reactive systemic (AA) amyloidosis. A prediction of the secondary structure of the human serum amyloid protein SAA1(alpha) is presented. The prediction was based upon one-dimensional Fourier analysis of the amino acid sequence together with sequence matching to known structural motifs. The results were compared with those from prediction algorithms based upon statistical techniques. Our findings are consistent with available experimental data. They include the putative identification of the amino-terminal 11 residues as the functionally important lipid-binding site of SAA and of a likely, neutral, calcium-binding sequence: Gly48-Pro49-Gly50-Gly51. Sequence comparisons between SAA and protein tyrosine kinases, phospholipases A2 and delta-crystallin, all of which bind both calcium and phospholipid, revealed significant homologies that support our proposals concerning structure-function relationships in SAA.

X-ray scattering and diffraction by wet gels of AA amyloid fibrils.

Systemic amyloidosis is characterized by the extracellular accumulation of protein fibrils with typical ultrastructural morphology. The persistence in vivo of amyloid fibrils, which is responsible for their serious clinical effects, has been thought to reflect the particular, specific conformation of peptide chains constituting the fibrils. On the basis of earlier structural studies this conformation is generally considered to be almost exclusively anti-parallel beta-sheets. We have re-examined X-ray scattering by human amyloid A protein (AA) amyloid fibrils, with careful attention to the state of hydration of the preparations. We show that a stack of anti-parallel sheets is not consistent with the details of the X-ray pattern, which contains diffracted intensities that can be indexed on a 33A X 33A lattice. A structural model for the AA fibre consistent with the X-ray data is presented. The model takes account of the prediction of the secondary structure of the AA precursor SAA1(alpha) presented in our accompanying paper, and has the AA monomers arranged on a primitive lattice, with two unique molecules per unit cell.

Preliminary X-ray data for an N-terminal fragment of rabbit serum transferrin.

An iron-containing fragment (Mr approximately 39,000) of rabbit serum transferrin has been crystallized from a solution of 25% (w/v) polyethylene glycol 6000, 50 mM-disodium piperazine-N,N'bis(2-ethanesulphonate) adjusted to pH 6.0 at 4 degrees C. The space group is P3(1)21 (or the enantiomorph) with a = b = 66.8(1) A, c = 137.5(3) A and Z = 6. The crystals appear as hexagonal plates, with the unique axis perpendicular to the plate. The crystals, kept at 4 degrees C, are stable in the X-ray beam for at least 130 hours and diffract to better than 1.8 A resolution.