Characterization of the spontaneous murine B cell leukemia (BCL1). III. Evidence for monoclonality by using an anti-idiotype antibody.

We have raised an anti-idiotypic antibody against the cell surface IgM of the murine BCL1 tumor cells. This antiserum reacts exclusively with the IgM expressed on the tumor cells and detects a unique population of cells in the spleen and blood of the tumor-bearing mice. When these cells are stimulated in vitro with LPS, they secrete an IgM bearing the same idiotype as the cell surface Ig. These results are discussed in terms of a model for the immunotherapy of a chronic lymphocytic leukemia-like syndrome in mice.     

Catalytic detection principle for high-performance liquid chromatography: Determination of enantiomeric iodinated thyronines in blood serum

A method for the trace determination of iodinated thyronines with differentiation of the optical isomers by high performance liquid chromatography (HPLC) is described. The detection is effected by means of a catalytic principle based on the iodide-catalysed reaction of chloramine-T and N,N′-tetramethyldiaminodiphenylmethane, producing a coloured complex that can be measured spectrophotometrically at 600 nm. Owing to the selectivity of the catalytic reaction, iodine-containing compounds can be easily determined in a complex matrix such as blood plasma. The sensitivity is sufficient for the detection of plasma levels of iodinated thyronines. The limit of detection for thyroxine is in the sub-nanogram range. The enantiomers of thyronines can be separated on commercial reversed phases after pre-column synthesis of diastereomers. For this derivatization the reagent tert.-butyloxy-carbonyl-l-leucine-N-hydroxysuccinimide ester is used. The coupling of the stereospecific HPLC separation with the catalytic detector offers the possibility of determining both d- and l-thyroxine in human plasma.     

Dihydroisocoumarins and phthalide from wood samples infested by fungi

Wood samples, infested by fungi during storage, were shown to contain, besides the known 5-methyl-mellein, additional (3R)-8-hydroxy-3-methyl-3,4-dihydroisocoumarins substituted by 7-methyl, 5-formyl, 5-carboxy, 5-hydroxy, 5-methoxy, 6-methoxy-5-methyl and 6,7-dimethoxy-5-methyl groups, as well as 6-formyl-7-hydroxy-5-methoxy-4-methylphthalide. Several 2-methylchromanones were synthesized in order to show that this class of compounds can be distinguished from 3-methyl-3,4-dihydroisocoumarins by MS.     

Purification and characterization of an (Na++K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain

Highly purified lamb kidney (Na⁺+K⁺)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na⁺+K⁺)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (γ1 and γ2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that γ1 is an aggregated form of γ2. The total yield was 0.8–1.5 mol of γ component per mol of catalytic subunit recovered. This indicates that the γ component is present in stoichiometric amounts in the (Na⁺+K⁺)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.     

Transforming growth factor-β inhibition of interleukin-1 activity involves down-regulation of interleukin-1 receptors on chondrocytes

Interleukin-1 (IL-1) plays an important role in cartilage destruction associated with inflammatory and degenerative arthritis because of its ability to induce matrix degrading enzymes. Previously, we have shown that the IL-1-induced chondrocyte protease activity was inhibited by transforming growth factor-β (TGF-β). In this paper, we show that TGF-β inhibits the IL-1-induced synthesis of collagenase and stromelysin by reducing the steady-state mRNA levels in rabbit articular chondrocytes. We further demonstrate that TGF-β-treated chondrocytes show reduced ¹²⁵I-IL-1 binding that returns to a normal level when TGF-β is removed from the culture medium. The inhibitory effect of TGF-β is observed for both naturally occurring as well as fibroblast growth factor (FGF)-inducible binding sites (receptors). Scatchard analysis of receptor—ligand interactions demonstrate that the reduced binding is due to a reduction in the number of receptors for IL-1 and is not due to changes in affinity. Affinity cross-linking studies suggest that control chondrocytes contain two major cross-linked bands of M_r =116 and 80 kDa and a minor band of M_r =100 kDa. FGF-treated cells show enhanced levels of all the bands, plus an additional 200-kDa band. TGF-β treatment of chondrocytes results in the reduction of all of these bands in both control as well as FGF-induced cells. These observations suggest that the ability of TGF-β to down-regulate the IL-1 receptor may be a mechanism by which it exerts its effects in antagonizing the IL-1 activity on chondrocytes.     

Matrix-assisted laser desorption mass spectrometry of rhodopsin and bacteriorhodopsin.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes.     

Hirudisins. Hirudin-derived thrombin inhibitors with disintegrin activity.

Recombinant hirudin variants have been designed which inhibit alpha-thrombin by the hirudin mechanism and which in addition exhibit disintegrin activity. These proteins, called "hirudisins," have been engineered by replacing the Ser-Asp-Gly-Glu sequence at the tip of hirudin's finger-like structure (residues 32-35) by Arg-Gly-Asp-Ser (RGDS) to yield hirudisin and Lys-Gly-Asp-Ser (KGDS) to obtain hirudisin-1. Comparison of thrombin inhibition activities showed that hirudisin is 2-fold more potent (K(i) = 160 +/- 70 fM) than hirudisin-1 (K(i) = 370 +/- 44 fM) and recombinant (r)-hirudin (K(i) = 270 +/- 50 fM). alpha-Thrombin-stimulated platelet aggregation was effectively inhibited by r-hirudin, hirudisin, and hirudisin-1 with IC50 of 5.7 to 6.8 nM. Unlike r-hirudin, hirudisin inhibits ADP-induced platelet aggregation (IC50 = 65 microM) 3- to 5-fold stronger than the linear GRGDS- and RGDS-peptide. Direct interaction of hirudisin with purified glycoprotein IIb-IIIa demonstrated that antiplatelet aggregation activity is due to the integrin-directed RGD motif. Disintegrin activity of hirudisin relative to that of reduced and carboxymethylated hirudisin suggests that the conformational strain favors binding to integrins. On the basis of these results, hirudisins appear to be interesting molecules for the design of potential antithrombotic agents with antithrombin as well as antiplatelet aggregation activities.     

IgM and IgG but not cytokine secretion is restricted to the CD27+ B lymphocyte subset.

In a recent study we reported that CD27 is expressed on a subpopulation of human B lymphocytes and presented circumstantial phenotypic evidence that CD27 expression may be acquired late during B cell differentiation. Here we present functional data showing that, after in vitro stimulation, CD27+ but not CD27- B cells secrete large amounts of both IgM and IgG. Using double immunofluorescence staining of CD27 and IgD, three functionally different B cell subsets representing distinct stages of B cell differentiation can be isolated: 1) the CD27- IgD+ B cells, which do not secrete appreciable Ig; 2) the CD27+IgD+ B cells, which exclusively secrete IgM; and 3) the CD27+IgD- B cells, which comprise the IgG-producing cells. Furthermore, costimulation of CD27- B cells with low m.w. B cell growth factor, in the presence or in the absence of a CD40 mAb, does not induce these cells to become Ig-secreting cells. Although CD27- B cells hardly secrete Ig of any isotype in response to Staphylococcus aureus+IL-2, these cells proliferate vigorously and express the IL-2R alpha chain (CD25) under these stimulatory conditions. Furthermore, both CD27- and CD27+ B cells are capable of producing similar amounts of IL-6 and TNF-alpha. Taken together, these findings indicate that CD27 is a unique non-Ig surface marker discriminating naive from primed B lymphocytes. Furthermore, the capacity to proliferate and to secrete the B cell differentiation factors IL-6 and TNF-alpha already exists at an early B cell differentiation stage at which the cells lack CD27 expression and are not induced to produce Ig.