The attachment of biotin to apocarboxylases is catalyzed by holocarboxylase synthetase (HCS). An inherited deficiency of HCS results in the disorder 'multiple carboxylase deficiency', which is characterized by reduced activity of all biotin-dependent carboxylases. Here we show that the majority of HCS localizes to the nucleus rather than the cytoplasm based on immunofluorescence studies with antibodies to peptides and full length HCS and on the expression of recombinant HCS. Subnuclear fractionations indicate that HCS is associated with chromatin and the nuclear lamina, the latter in a discontinuous distribution in high salt-extracted nuclear membranes. During mitosis, HCS resolves into ring-like particles which co-localize with lamin B. Nuclear HCS retains its biotinylating activity and was shown to biotinylate purified histones in vitro. Significantly, fibroblasts from patients with HCS deficiency are severely deficient in histone biotinylation in addition to the deficiency of carboxylase activities. We propose that the role of HCS in histone modification may be linked to the participation of biotin in the regulation of gene expression or cell division and that affected patients may have additional disease beyond that due to the effect on carboxylases.

B cell activation requires sustained elevation of cytoplasmic free calcium, achieved by influx through storeoperated calcium (SOC) channels. The molecular identity of these channels is not known. Ectopic expression of the raft-associated tetraspan protein CD20 in Chinese hamster ovary cells introduced a novel SOC entry pathway that was permeable to strontium as well as to calcium. The activity of this SOC pathway was abolished by deletion of a cytoplasmic sequence in CD20 essential for its efficient raft localization. Strontium-permeable SOC channels were detected in B cells, and B cell receptorstimulated influx was significantly reduced by downregulation of CD20 expression using short interfering RNA and also by cholesterol depletion. This is the first evidence that raft-associated CD20 constitutes a component of a SOC entry pathway activated by the B cell receptor.The function of CD20, a tetraspan transmembrane protein expressed in B lymphocytes, is not yet fully elucidated, although electrophysiological evidence of calcium channel activity has been reported. Increased Ca 2ϩ conductance, detected by whole cell patch clamp recordings, was induced by membrane hyperpolarization in a variety of cell types expressing CD20 ectopically and was similar to the native conductance found in B cells (1, 2). The type of calcium channel that is formed or regulated by CD20 and the conditions of its activation are not known.Intracellular calcium is an essential regulator of cell function (3, 4). Receptor-mediated activation of phospholipase C and consequent inositol 1,4,5-trisphosphate production leads to rapid release of Ca 2ϩ from intracellular stores in the endoplasmic reticulum. Store depletion activates Ca 2ϩ influx across the plasma membrane, replenishing empty stores and providing a sustained increase in the concentration of cytoplasmic free Ca 2ϩ ([Ca 2ϩ ] c ). The Ca 2ϩ influx has been termed capacitative or store-operated Ca 2ϩ (SOC) 1 entry and is essential for regulating diverse cellular responses to receptor-mediated stimuli, including enzyme activation, gene expression, secretion, and cell division (5-7). Electrophysiological studies distinguish several classes of SOC channels, including the highly Ca 2ϩ -selective Ca 2ϩ release-activated Ca 2ϩ (CRAC) channel described in mast cells and T lymphocytes and others with distinct characteristics (7,8). Despite intensive research efforts, the molecular identity of SOC channel proteins remains unclear. The mammalian homologues of the Drosophila transient receptor potential (TRP) gene products, now forming a TRP superfamily with more than 20 members in 3 subfamilies (9), provide the only candidate SOC channel proteins currently known. All TRPs apparently form cation channels but with variable selectivity and activation properties (10, 11).In B lymphocytes, expression of specific genes requires sustained elevation in [Ca 2ϩ ] c (12). As in other cell types, B lymphocytes activate Ca 2ϩ entry after store-depletion, but the molecular components and the mechanism of acti...

The migration of leucocytes from blood into the joint is a key feature of human and experimental arthritis. Adhesion molecules on leucocytes and vascular endothelium are important in this process and may be therapeutic targets for intervention in arthritis. We investigated whether monoclonal antibody treatment to block the α4 integrin, very late activation antigen‐4 (VLA‐4), and β2 integrins (CD11/CD18) administered to rats during the preclinical (day 5) or clinical phase (day 10+) would modify disease. When treatment was initiated 5 days after induction of disease, development of arthritis was significantly reduced by either anti‐α4 (TA‐2) or anti‐β2 (WT.3) monoclonal antibodies (mAb) and the combination of both mAb was even more effective (clinical scores: control 11.4; anti‐α4 6.6; anti‐β2 6.8; anti‐α4 + anti‐β2 3.9). When treatment was delayed until arthritis was apparent (day 10), the anti‐α4 + anti‐β2 mAb combination still significantly diminished the arthritis score on day 14 (control 13; anti‐α4 + anti‐β2 7.9). Treatment with anti‐α4 + anti‐β2 mAb decreased the migration to the joints of blood polymorphonuclear leucocytes (PMNL) by 66–79% and of spleen T lymphocytes by 56–75%, depending on the joint. In contrast, PMNL migration was abolished (>98%) and T‐cell migration markedly (87%) inhibited to dermal inflammatory reactions in the same animals. These findings demonstrate: that blocking mAb to α4 and β2 integrins can reduce the severity of adjuvant arthritis, even after joint inflammation has developed; that this treatment can markedly inhibit PMNL and T‐lymphocyte migration to the joints; and that yet to be defined mechanisms distinct from α4 (CD49d) and β2 (CD11/CD18) integrins, also contribute to leucocyte migration to inflamed joints. Identifying these additional adhesion mechanisms may be required to control joint inflammation further.

P-selectin is an important adhesion molecule involved in leukocyte migration. However, to date, no monoclonal antibodies (MAb) generated against rat P-selectin have been identified which block P-selectin mediated leukocyte adhesion. Most studies in the rat have utilized crossreacting antibodies generated against P-selectin in higher species. In a P-selectin deficient mouse we generated an anti-rat/mouse P-selectin MAb, designated RMP-1, by immunization with activated rat platelets. This IgG2a MAb immunoprecipitates a 140 kDa protein under reducing conditions from rat platelet lysate. By ELISA and immunofluorescence flow cytometry, MAb RMP-1 reacts with thrombin-activated but not unactivated rat platelets. In addition, by ELISA MAb RMP-1 binds to activated mouse platelets and recombinant rat and mouse P-selectin. MAb RMP-1 inhibited adhesion of HL-60 myeloid cells to immobilized mouse P-selectin by 97% and to activated rat and mouse platelets by 100% under static conditions, confirming the adhesion function blocking activity of MAb RMP-1. This novel MAb should be useful for studying P-selectin function in vitro and in vivo in both rat and mouse inflammation models.

The prerequisite for the recruitment of circulating leukocytes to sites of inflammation is adhesion to vascular endothelial cells. Selectins play a significant role in the initiation of this multistep process by mediating "rolling" of the leukocytes on the endothelium. Investigation of selectin-dependent cell interactions using function blocking monoclonal antibodies (MAb) provides insights into the mechanisms involved in leukocyte migration into inflammation. Until now most studies in inflammation models in rats have relied on cross-reactive or polyclonal antibodies against rat E-selectin. In an E-selectin knockout mouse, we aimed to generate an adhesion function blocking MAb to rat E-selectin by immunization with rat E-selectin transfected Chinese hamster ovary cells (RESEC). An IgG1 kappa MAb was identified that reacts with RESEC but not with untransfected Chinese hamster ovary cells, as well as with recombinant mouse E-selectin protein as assessed by ELISA. This MAb is designated RME-1. It does not cross-react with rat L-selectin or rat P-selectin or E-selectin expressed on human umbilical vein endothelium. Adhesion of the HL-60 myeloid cells to immobilized mouse E-selectin was completely inhibited by MAb RME-1 under static conditions and adhesion of rat polymorphonuclear leukocytes to recombinant mouse E-selectin was blocked under rotation condition. This novel antibody thus recognizes a function-related epitope on rodent E-selectin.

We measured serum osteocalcin concentrations in 82 pregnant and 21 nonpregnant women. Osteocalcin values declined in the second trimester, but returned to nonpregnant levels late in the third trimester. The mean serum osteocalcin concentration in 36 women during pregnancy (mean gestation, 26 weeks) of 2.8 ng/mL was significantly lower than that in nonpregnant women (6.4 ng/mL; P less than 0.001) or term pregnant women at delivery (6.1 ng/mL; n = 46). Serum immunoreactive PTH (iPTH) levels were significantly higher during pregnancy than in nonpregnant women [97 +/- 5 vs. 56 +/- 4 ng/L (mean +/- SE); P less than 0.001]. No significant correlations were found between maternal osteocalcin concentrations and serum phosphorus, alkaline phosphatase, or iPTH, but significant negative correlations were found between osteocalcin and total calcium or total protein. Osteocalcin concentrations in midtrimester amniotic fluid were very low (mean, 0.3 +/- 0.1 ng/mL; n = 11). In 29 lactating mothers, the mean serum osteocalcin level was 9.5 +/- 1.5 ng/mL, significantly higher than in any of the other groups (P less than 0.05), but their serum calcium and iPTH levels were normal. There was no correlation between serum osteocalcin and calcium or iPTH concentrations in lactating women. These changes are compatible with a sequence in which bone turnover is reduced during early pregnancy, rebounds in the third trimester, and increases in postpartum lactating women.

Objective. To determine the prevalence of autoantibodies to high-mobility group (HMG) proteins in sera from patients with drug-induced lupus (DIL).Methods. Forty-two patients who developed autoantibodies andor lupus after treatment with procainamide or other drugs were tested for HMG autoantibodies by immunoblotting and enzyme-linked immunosorbent assay (ELISA).Results. Twenty-eight of the 42 sera (67%) bound HMG-14 andor HMG-17. In comparison, 9 of 42 (21%) bound HMG-1 and/or HMG-2. There was a good correlation between ELISA results and binding on immunoblots.Conclusion. The high prevalence of antibodies to the nucleosomal core HMGs (HMG-14 and HMG-17) in DIL patients adds evidence implicating nucleosomes as immunogens in drug-induced autoimmunity.

Accurate diagnosis of lymphoid malignancies is essential for appropriate therapeutic intervention. In conjunction with other diagnostic determinants, immunophenotypic analysis of differentially expressed cell surface markers, such as CD5, CD20, CD23 and FMC7, is useful in the subclassification of lymphomas and leukemias arising from the B-cell lineage. Recent evidence suggesting that CD20 predicts FMC7 expression has prompted reappraisal of the utility of monitoring both markers. Here, we report that the FMC7 monoclonal antibody (mAb) specifically and strongly recognized CD20 ectopically expressed in hematopoietic and nonhematopoietic cell lines. The reactivity of FMC7 was abolished by mutations in the extracellular domain of CD20. These data confirm the CD20 specificity of FMC7. Like other CD20 mAbs, FMC7 binding was temperature dependent and induced detergent insolubility of CD20. Of significant interest, the CD20 epitope recognized by FMC7 was unusual in that it was exceptionally sensitive to membrane cholesterol. Cholesterol depletion profoundly reduced expression of the FMC7 epitope, whereas cholesterol enrichment enhanced its expression. FMC7 mAb binding thus appears to be a sensitive indicator of the level of plasma membrane cholesterol and reveals a conformational state of CD20 that is regulated by cholesterol.