Nocturnal pain correlates with effusions in diseased hips.

Thirty-five of 50 patients with different hip joint disease had sonographic evidence of joint effusion. Arthrocentesis confirmed effusions in 30 of these 35 patients. Thirty-two of the 35 patients had nocturnal pain. Both nocturnal pain and sonographic evidence of effusion decreased after aspiration (15 patients) and aspiration and injection of corticosteroids (15 patients). In a further group of 61 patients who subsequently had Charnley arthroplasties, 35 had positive sonograms before operation. Of these, 25 had effusions confirmed at operation, the remaining 10 having synovitis and capsule thickening. Again a correlation was found with nocturnal pain. The sensitivity of sonography in detecting hip joint effusion was 92% with a specificity of 70%. Nocturnal pain had a lower sensitivity, 85%, but higher specificity, 94%.     

The relative importance of thrombin inhibition and factor Xa inhibition to the antithrombotic effects of heparin

The relative importance of antithrombin and anti-factor Xa activities of heparin fractions required to achieve optimal antithrombotic effects is unknown. To study this, we measured the effects of standard heparin, an octasaccharide heparin fraction (anti-factor Xa activity only), and dermatan sulfate (antithrombin activity only) on the prevention of thrombosis and related this to their anticoagulant effects in vivo in rabbits. Thrombosis was measured as the incorporation of 125I- fibrinogen into tissue thromboplastin-induced thrombi using a Wessler- type model. Ex vivo changes in thrombin clotting time (TCT) were used as an index of antithrombin activity, and a chromogenic anti-factor Xa assay was used to measure anti-factor Xa activity. In addition, the ability of the three sulfated polysaccharides to simultaneously inhibit the generation of thrombin activity and to enhance the inactivation of the factor Xa added to initiate thrombin generation in plasma was determined. Standard heparin, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 90%, prolonged the TCT by two seconds, and resulted in an anti-factor Xa level of 0.32 U/mL. The octasaccharide heparin fraction, in a dose of 10 anti-factor Xa U/kg, inhibited thrombus formation by 41%, had no effect on the TCT, and resulted in an anti-factor Xa level of 0.28 U/mL. Higher doses of the octasaccharide resulted in a further increase in the anti-factor Xa levels but had no further effect on thrombus formation. Dermatan sulfate, in a dose of 500 micrograms/kg, inhibited thrombus formation by 95%, but had no affect on the TCT. These results indicate that the antithrombotic effect achieved by inhibiting factor Xa is limited and that better antithrombotic effects are achieved by heparin or heparin- like substances capable of influencing the inactivation and/or the generation of thrombin.     

Clinical features and outcome of T-cell acute lymphoblastic leukemia in childhood with respect to alterations at the TAL1 locus: a Pediatric Oncology Group study

Alteration of the TAL1 locus is the most common nonrandom genetic defect in childhood T-cell acute lymphoblastic leukemia (T-ALL). To determine if rearrangements of the TAL1 proto-oncogene confer a distinct leukemic phenotype, we studied leukemic peripheral blood or bone marrow samples from 182 children with newly diagnosed T-ALL enrolled on Pediatric Oncology Group treatment protocols. Forty-eight (26%) of the samples had a local rearrangement of the TAL1 locus. Demographic and clinical features were compared for patient subgroups with and without TAL1 rearrangements. The only clinical correlates that were significantly associated with TAL1 gene rearrangements were higher white blood cell count (P = .017) and higher hemoglobin (P = .007) at diagnosis. Immunophenotypically, samples with altered TAL1 were more likely to be CD2+ (P = .001) and lack CD10 (cALLa) expression (P = .007) than those without the rearrangement. There was a trend toward improved event-free survival (EFS) in patients with TAL1 rearrangements (4-year EFS was 44% +/- 7% for patients without the rearrangements v 59% +/- 11% for those with rearrangements), but the difference was not significant (P = .34). The role of TAL1 in leukemogenesis has yet to be clearly defined, and the prognostic significance of TAL1 gene rearrangements in T-ALL deserves further study.     

Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly ¹³C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent β-strands to each β-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 μg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.Cataracts are a protein misfolding disease caused by the aggregation of lens crystallin proteins into insoluble deposits that blur vision (1, 2). Because these proteins are not regenerated, damage from UV radiation, oxidative stress, and other chemical modifications accumulates with time (1, 2). As a result, over 50% of the population over 55 develops age-related cataracts (2). Additionally, numerous mutations that destabilize crystallin protein folds are linked to inherited and juvenile-onset cataracts (1). Although the causative factors associated with this disease are known, the structures of the aggregates and the mechanisms by which they form are unknown.Like other protein aggregation diseases such as type II diabetes mellitus and Alzheimer’s disease, the molecular structures of proteins in cataracts are difficult to determine. Atomic-level structures have been obtained for some amyloid aggregates of peptides using NMR spectroscopy (3, 4) and X-ray crystallography (5). However, the most widely used techniques for studying aggregate structures and aggregation mechanisms are circular dichroism spectroscopy, fluorescence spectroscopy, and transmission electron microscopy, which provide little detailed structural information. Two-dimensional (2D) IR spectroscopy is emerging as an important tool for studying protein aggregates such as amyloid fibrils (6–8) because it provides bond-by-bond structural resolution on kinetically evolving samples (6, 8–10). Two-dimensional IR spectroscopy probes secondary structure through cross peak couplings and solvent exposure through 2D lineshapes. Its bond-specific structural resolution comes from isotope labeling. Mechanistic information is obtained from rapid-scan methods that track the kinetics of aggregate formation (6). Spectra can be calculated from molecular dynamics simulations allowing structural models to be tested (11–15). Isotope-edited 2D IR spectroscopy using ¹³C = ¹⁸O labeled backbone amides has been used to study the fibril structure and mechanism of fibril formation for amyloid beta (Aβ) (8) and the human islet amyloid polypeptide (hIAPP) (6, 7), as well as many membrane-bound (13, 16) and soluble polypeptides (9, 10).Thus far, isotope-edited infrared spectroscopy has been limited to peptides that can be made by solid phase synthesis, which is how the ¹³C = ¹⁸O labels are incorporated (17). However, solid phase synthesis is limited to peptides of about 45–70 residues. Even if longer sequences could be synthesized, a single ¹³C = ¹⁸O label would eventually be obscured by Glu, Asp, and Arg side chains, as well as natural abundance ¹³C = ¹⁶O bonds, all of which absorb in the same frequency range (18). Nonnatural amino acids and deuterium labels are well-resolved in protein IR spectra, but only report on local environment not structure (19–21). In this article, we describe an alternative approach in which we isotope label an entire section of a protein rather than a single C═O bond. To do so, we express labeled and unlabeled segments of the protein separately, and then reconstitute the full-length protein using expressed protein ligation (22), which is a variant of native chemical ligation (23). We show that the unlabeled and ¹³C labeled regions are spectroscopically distinguishable, enabling the acquisition of structural and kinetic information on large proteins. We apply our technique to study amyloid fibril formation in the 173 amino acid eye lens protein γD-crystallin and learn that only one of its two structurally homologous domains forms the β-sheet core of the fibrils.γD-crystallin is a member of the γ-crystallin family of monomeric, soluble structural eye lens proteins which constitute about 20% of the total lens proteins and are a major component of insoluble cataract deposits (24). Native γD-crystallin consists of two Greek key domains that are each 80–90 residues long, connected by a short loop (Fig. 1) (25). Numerous modes of aggregation have been proposed for the two-domain structure, including amorphous precipitation, domain-swap oligomerization, and amyloid fibril formation (1, 26, 27). In vitro, fibrillar deposits of γ-crystallins are created by UV damage, low pH, and chemical and thermal denaturation, although it is unclear to what extent each form (or others) contribute to in vivo cataracts (1, 27–29). We utilize acid initiated aggregation because it reproducibly generates fibrillar aggregates of γ-crystallins and because low pH conditions encountered during lens development may be relevant to juvenile cataract formation (27). γD-crystallin, and each of its isolated domains, forms amyloid fibrils in vitro at pH 3 and 37 °C (27). Although there is no domain-specific structural information in the fibril state, fibrils formed from the isolated N-terminal domain (NTD) more closely resemble fibrils from full-length γD-crystallin than do fibrils from the C-terminal domain (CTD), as characterized by FTIR spectroscopy and transmission electron microscopy (TEM) imaging (27). Moreover, the NTD is less stable (30, 31) and has partially unfolded states at pH 7. Although the relationship between domain stability, partially unfolded states, and aggregation propensity is unclear (32), these results have led to the suggestion that the NTD is most likely to form the β-sheets of the amyloid fibril core (33). We show that it is actually the CTD of full-length human γD-crystallin that forms the β-sheets of the amyloid fibril core in low pH aggregates.Open in a separate windowFig. 1.Two-dimensional IR spectroscopy of native human γD-crystallin. (Top) Crystal structure of human γD-crystallin indicating the division of domains in the segmental labeling scheme. (Bottom, A–D) Two-dimensional IR spectra of full-length γD-crystallin at pD = 7. Frequencies and FWHM node slopes are summarized in Table S1.