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.

[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.

[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.

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.