Amelogenins make up a class of proteins associated with the formation of mineralized enamel in vertebrates, possess highly conserved N-and C-terminal sequence regions, and represent an interesting model protein system for understanding biomineralization and protein assembly. Using bioinformatics, we report here the identification of molecular traits that classify 12 amelogenin proteins as members of the intrinsically disordered or unstructured protein family (IDPs), a group of proteins that normally exist as unfolded species but are capable of transformation to a folded state as part of their overall function. Using biophysical techniques (CD and NMR), we follow up on our bioinformatics studies and confirm that one of the amelogenins, recombinant porcine rP172, exists in an extended, unfolded state in the monomeric form. This protein exhibits evidence of conformational exchange between two states, and this exchange may be mediated by Pro residues in the sequence. Although the protein is globally unfolded, we detect the presence of local residual secondary structure [α-helix, extended β-strand, turn/loop, and polyproline type II (PPII)] that may serve several functional roles within the enamel matrix. The extended, labile conformation of rP172 amelogenin is compatible with the known functions of amelogenin in enamel biomineralization, i.e., self-assembly, associations with other enamel matrix proteins and with calcium phosphate biominerals, and interaction with cell receptors. It is likely that the labile structure of this protein facilitates interactions of amelogenin with other macromolecules or with minerals for achievement of internal protein stabilization.The formation of inorganic compounds by organisms (biomineralization) is a substantial scientific puzzle. The ability of cells to employ proteins to control nucleation, crystal morphology, polymorphism, and the material properties of living tissues requires precise molecular control and efficient mechanisms (1). One such protein, amelogenin, is found in mammalian tooth enamel, one of the most highly mineralized materials of vertebrates (1-3). Amelogenin is essential for normal enamel development and is capable of protein selfassociation, forming supramolecular assemblies under defined conditions in the laboratory (4-6). These supramolecular assemblies (nanospheres) are believed to exert control over the † This work was supported by funding from the Department of Energy (DE-FG02-03ER46099) and the National Institute of Dental and Craniofacial Research (DE-013414 morphology, organization, and directionality of hydroxyapatite crystal growth (7,8). Primary sequence analysis of 26 mammalian lineages indicates that the N-terminus (Tyr-rich) and Cterminus (charged) of amelogenin are highly conserved, whereas variations occur in the central regions (9). Amelogenin sequence mutations lead to defective enamel crystal formation and organization (10,11), and deletion of the conserved terminal domains leads to the formation of ill-defined enamel crystals, high...

The use of steroids in treating acute respiratory obstruction is still controversial. In this double-blind controlled trial, we decided to examine the beneficial effects of a single large dose of methylprednisolone (MSSP), using objective criteria. In the emergency setting, methylprednisolone (30 mg/kg) has been shown to decrease the need for hospital admission in patients with acute bronchospasm. No difference in this improvement was seen among patients in the steroid-dependent or non-steroid-dependent populations. Based on our findings, we suggest that the early use of single-dose steroid therapy is appropriate treatment for patients with acute bronchospastic attacks.

An L-cysteinyl-tRNA synthetase (EC 6.1.1.16) from Phaseolus aureus has been purified approximately 200-fold. The enzyme uses selenocysteine as substrate in the ATP-PPi exchange assay; other cysteine analogs were inactive. The molecular weight as determined by Sephadex G-200 column chromatography is about 61,000; sodium dodecyl sulfate and 8 Murea acrylamide gel electrophoresis indicate that the enzyme is a dimer conssting of two identical monomers of molecular weight 30,000.A method for the preparation of selenocysteine from selenocystine is described.digests from several organisms (2, 10, 15, 23), but it was reported absent, despite the presence of selenomethionine, from proteins of other organisms grown with radioactive selenite or selenate (13, 21). Such findings suggest that different organisms vary in their ability to activate selenocysteine and incorporate it into their proteins. Therefore, to examine the activation of selenocysteine, a method for its preparation from selenocystine and its use as substrate by a purified cysteinyl-tRNA synthetase from Phaseolus aureus was undertaken and will be described here. MATERIALS AND METHODSThe ability of methionyl-and cysteinyl-tRNA synthetases to use selenium isologs as substrates has been suggested as the cause of selenium toxicity. According to this hypothesis, both the sulfur and selenium amino acids are activated and incorporated into proteins; these altered proteins, with an excess of selenoamino acids, function abnormally (7,29). On the other hand, tolerance of selenium, notably in selenium-accumulator plants, is thought to have arisen from evolutionary modifications to these aminoacyl-tRNA synthetases so that the selenium isologs, no longer activated, are excluded from proteins; instead, the selenium isologs are converted to and accumulate as nonprotein selenoamino acids (7,24,28,31). Altered aminoacyl-tRNA synthetases have been described in other plants which synthesize large amounts of nonprotein amino acids. Modified prolyl-tRNA synthetases, for example, can be isolated from Polygonatum and Convallaria species that synthesize azetidine-2-carboxylic acid; this nonprotein amino acid analog of proline is excluded from the proteins of these plants but not from the proteins of organisms poisoned by the analog (7,8,22,25 Buffer B, for dissolving the 45 to 65% (NH4)2SO4 fraction, and for equilibration of Sephadex and DE-52 columns, contained 0.05 M tris and the other components of buffer A; the pH was brought to 7.7 with HCl.Buffer C, pH 7.7, for hydroxylapatite and cellulose phosphate columns, contained 50 ml of 0.02 M KH2PO4, 500 ml of 0.02 K2HPO4, 25 mm 2-mercaptoethanol, and 10% (w/v) glycerol.Buffer D, pH 7.7, for gradient elution of hydroxylapatite and cellulose phosphate columns, contained 70 ml of 0.4 M KH2PO4 and 500 ml of 0.4 M K2HPO4, 25 mm 2-mercaptoethanol, andPreparation of Crude Enzyme Extract and Preliminary Fractionation. All operations were carried out at 3 to 4 C. Phaseolus aureus seed, milled to a powder, was homogenized for 1 min in ...

Septic arthritis caused by Neisseria meningitidis has been demonstrated in approximately 2% of patients with systemic meningococcal infection (1).Meningococcal arthritis in the absence of documented meningococcemia or meningitis, however, is extremely uncommon. In a recent review, Schaad (1) identified only 25 patients from both personal experience and the literature. The incidence of this entity, termed primary meningococcal arthritis, follows the characteristic agespecific attack rate seen in other forms of N meningifidis infection (1). Most cases have been described in children less than 2 years old: a rapid decline in incidence occurs with increasing age. To our knowledge, the disease has not been reported in patients over the age of 50. We report here an elderly patient with chronic lymphocytic leukemia who presented with primary acute meningococcal arthritis and pseudogout in the same joint.Case history. A 64-year-old white woman with a 3-year history of chronic lymphocytic leukemia (B cell type) and a history of chronic obstructive pulmonary disease presented to the Montefiore Hospital emergency room with 12 hours of progressive tenderness, swelling, and redness of the right knee. She denied rigors or recent trauma to the joint. There was no history of acute arthritis, rash, dysuria, frequency, vaginal discharge, headache, or stiff neck. She did, however, note increased sputum production and cough. Three months earlier she had had acute pneumococcal pneumonia (type 19), despite previous immunization with pneumococcal vaccine. She was allergic to penicillin.On physical examination her respiratory rate was 24/minute and temperature was 101.1"F orally. There was no rash. The pharynx was clear and the neck supple without signs of meningeal irritation. Generalized lymphadenopathy in the neck, axillary, femoral, and epitrochlear regions was present. Auscultation of the lungs revealed bilateral coarse airway sounds with occasional scattered ronchi. Heart sounds were normal and the spleen was not enlarged. Her right knee was warm, swollen. tender, and painful on passive and active motion. The suprapatellar area was boggy and a joint effusion was present. Homan's sign and calf swelling were absent.Significant laboratory data included: a peripheral leukocyte count of 64,7OO/pl (18% neutrophils, 6% band forms, 60% lymphocytes with 16% atypical lymphocytes), hemoglobin 1 1.8 gm/dl, platelet count 230,00O/pl, serum glucose 156 mg/dl, calcium 9.9 mg/dl, phosphorous 2.9 mg/dl, uric acid 4.3 mg/dl, alkaline phosphatase 142 IU/liter, serum albumin 2.8 gm/dl, and total protein 4.7 gm/dl. Serum protein electrophoresis revealed a total gamma globulin fraction of 200 mg/dl With a normal range of 600-1,800 gm/dl. After the acute illness, quantitative immunoglobulin tests were performed by immunodiffusion and showed IgG of 319 mg/dl (normal 800-1,800 mg/dl), IgA of 31 mg/dl (normal >90 mg/dl), and IgM of 0 (normal >90 mg/dl). Complement determinations showed a CH5O of I : 64 (control 1 : 64). A chest roentgenogram showed no inf...

Hospital records were reviewed on 78 consecutive patients (33 homosexuals, 37 Haitians and 8 IV drug abusers) who had AIDS and who underwent fiberoptic bronchoscopy over a two year period. Pneumocystis carinii pneumonia (PCP) was the most common bronchoscopic finding (53.8% of patients) followed by tuberculosis (14.1%), cytomegalovirus infection (12.8%) and atypical mycobacteriosis (3.8%). In 15.4% of patients multiple organisms were found. Twenty-seven percent of patients had non-diagnostic bronchoscopies. In 8 patients with non-diagnostic bronchoscopies, open-lung biopsy or autopsy revealed PCP (2 patients), Kaposi's sarcoma (3 patients) and non-specific lymphocytic-plasmacytic infiltrates (3 patients). Histologic studies of transbronchial biopsy specimens were more sensitive for the diagnosis of PCP than touch imprints. PCP was diagnosed in 15 patients who had received trimethoprim-sulfamethoxazole for up to 4 days. Mean duration of respiratory symptoms prior to bronchoscopy was 4.2 weeks in patients with PCP and was not significantly different in patients with other bronchoscopic diagnoses. Historical, laboratory and chest radiographic parameters were not helpful in establishing a diagnosis or assessing prognosis in these AIDS patients with pulmonary infiltrates. Fiberoptic bronchoscopy with transbronchial biopsy is safe (only one of the patients had a self limited hemorrhagic episode) and sensitive for the diagnosis of pulmonary infiltrates in patients with AIDS.