On the endogenous peroxidase in the spleen of swine (author's transl)]

We studied the distribution of endogenous peroxidase in the spleen of swine by modifications of the Graham and Karnovsky diaminobenzidine procedure. There is a peroxidatic activity in the majority of the ellipsoid cells (cells of the sheathed capillaries of Schweigger-Seidel), which is localized in the endoplasmic reticulum and the perinuclear cisterna. This staining is inhibited completely by aminotriazole and is rapidly destroyed even by low concentratoins of glutaraldehyde. Furthermore, the reaction is abolished after boiling of tissue sections or in the absence of H2O2. The macrophages of the red pulp and a minority of the ellipsoid cells are peroxidase negative. Our results are discussed in respect to some recent studies on the system of mononuclear phagocytes. It is suggested, that the enzyme active ellipsoid cells represent a special form of macrophages, enzyme histochemically related to Kupffer cells and resident peritoneal macrophages. The enzyme negative cells of the ellipsoids are probably fibroblasts.     

The Effects of Neuraminidase and Gangliosides on Ovarian LH/hCG Receptors During Rat Development

Ovaries of neonatal rats are not endowed with specific LH/hCG receptors up to 6–8 days of age. Treatment of ovarian membranes of the neonatal rat with neuraminidase results in a specific binding of radioactively labeled hCG, while an increase of hormone binding is observed after neuraminidase treatment of ovarian membranes of the 21-day-old rat. These changes in hormone receptor sites in the ovary are dependent on the neuraminidase concentration used and are due to a receptor with a dissociation constant (K_D) of about 10⁻⁹ M. The K_D of the receptor in the LH/hCG sensitive ovary without neuraminidase treatment is about 10⁻¹⁰ M. These results indicate the presence of two different LH/hCG receptors in the ovarian membrane. The unmasking effect of neuraminidase onto LH/hCG receptors indicate that ganglioside-like structures are responsible for the masking of receptors in the neonatal, insensitive rat ovary and also in the 21-day-old sensitive ovary. Ganglioside preparations are able to inhibit the binding, and the fractionation of ovary gangliosides results in a fraction with a rather high inhibition potency of LH/hCG binding to the receptor. It is hypothesized that the masked receptors in the sensitive period represent a store of receptors for the reconstitution of the ovarian cells with active receptors after internalization of the hormone-receptor complex. Thus the masking of the receptors in the early postnatal rat ovary could be a prerequisite for the female differentiation of hypothalamic centers. The observed neuraminidase effect in vitro could reflect a physiologic situation. Neuraminidase was found in the ovary, and during early postnatal development the neuraminidase activity pattern coincides with that of the ovarian LH/hCG receptor changes.     

Toward optimal clearance: A universal affinity-based mass spectrometry approach for comprehensive ELISA reagent coverage evaluation and HCP hitchhiker analysis

In the control strategy for process related impurities in biopharmaceuticals, the enzyme linked immunosorbent assay (ELISA) is the method of choice for the quantification of host cell proteins (HCPs). Besides two dimensional-western blots (2D-WB), the coverage of ELISA antibodies is increasingly evaluated by affinity purification-based liquid chromatography–tandem mass spectrometry (AP-MS) methods. However, all these methods face the problem of unspecific binding issues between antibodies and the matrix, involving the application of arbitrarily defined thresholds during data evaluation. To solve this, a new approach (optimized AP-MS) was developed in this study, for which a cleavable linker was conjugated to the ELISA antibodies enabling the subsequent isolation of specifically interacting HCPs. By comparing both approaches in terms of method variability and the number of false positive or negative hits, we could demonstrate that the optimized AP-MS method is very reproducible and superior in the identification of antibody detection gaps, while previously described strategies suffered from over- or underestimating the coverage. As only antibody associated HCPs were identified, we demonstrated that the method is beneficial for hitchhiker analysis. Overall, the method described herein has proven as a powerful tool for reliable coverage determination of ELISA antibodies, without the need to arbitrarily exclude HCPs during the coverage evaluation.     

Outgrowth morphology and intracellular calcium of crustacean neurons displaying distinct morphologies in primary culture

Peptidesecreting neurons from crustacean X-organ regenerating in defined culture possess different ionic current profiles correlated with two distinct morphological types, veiling and branching; voltage-dependent Ca²⁺ current is prominent in neurons consistently extending large veils, but is small in neurons that repetitively branch. Intracellular free calcium ([Ca²⁺]_i) have been implicated in regulation of neurite outgrowth underlying the establishment of distinct morphologies. Here, basal [Ca²⁺]_i was measured by fura-2 fluorescence ratio imaging from these morphologically distinct neurons and compared. Both morphological tapes can extend out processes over a [Ca²⁺]_i range (approximately 50 to 300 nM) that is much greater than that reported for neurons of other phyla. Application of high k⁺ saline led to increases in [Ca²⁺]_i in soma, neurite, and lamellipodium of veiling neurons. Increase were great for veiling than branching neurons. These observations were consistent with the previous voltage clamp data for calcium currents. Media altered to perturb [Ca²⁺]_i were used to assess the role of [Ca²⁺]_i in veiling or branching outgrowth programs. Outgrowth of veiling cells was arrested addition of 100 μMCD²⁺, a calcium channel blocker. Outgrowth resumed following brief exposures to Cd²⁺. Branching neurons were unaffected by Cd²⁺. Cd²⁺ at lower levels (10 μM) had no effect on outgrowth of either neuronal type, whereas at higher levels (1 mM), outgrowth of both types was arrested. Reduction of extracellular sodium to 0.001 of normal concentration stopped veiling outgrowth, but branching outgrowth continued, although it was less robust. Addition of tetrodotoxin (1 μM) did not alter outgrowth of either neuronal type relative to controls. Thus, peptidergic neurons of differing intrinsic morphologies maintain similar basal [Ca²⁺]_i levels under identical culture conditions, yet show differing sensitivities to manipulations influencing [Ca²⁺]_i with respect to regenerative outgrowth, but not its form. 1994 John Wiley & Sons, Inc.     

A novel site of DNA amplification on chromosome 1p32-33 in a rhabdomyosarcoma revealed by comparative genomic hybridization

Cytogenetic analysis of a human embryonal rhabdomyosarcoma revealed a near-diploid karyotype with structural chromosome aberrations not involving the typical rearrangements of rhabdomyosarcomas, plus a large number of double minutes. Comparative genomic hybridization revealed a previously undescribed site of DNA amplification on the short arm of chromosome 1 (band 1p32-33).     

Detection of avian hematopoietic cell surface antigens with monoclonal antibodies to myeloid cells : Their distribution on normal and leukemic cells of various lineages

The isolation and characterization of monoclonal antibodies reacting with cell surface antigenic determinants of normal and leukemic avian hematopoietic cells is described. The antibodies were produced by immunizing mice with normal macrophages, as well as with myeloid cells transformed with the avian acute leukemia viruses MC29, AMV and E26. Eleven antibodies were characterized for their reactivity with a variety of normal and leukemic cells of the myeloid, B- and T-lymphoid and of the erythroid cell lineage. Using several methods, they could be subdivided into five distinct types: I. Four antibodies were specific for the myeloid lineage, predominantly reacting with immature myeloid cells. II. One antibody reacted with mature and immature myeloid cells as well as with T-lymphoid cells. III. Four antibodies reacted with myeloid, erythroid and T-lymphoid cells. IV. One antibody reacted with myeloid as well as with T- and B-lymphoid cells. V. One antibody reacted with all kinds of chicken hematopoietic cells except erythrocytes. The first type of antibodies detected glycoproteins with MWs of 170 and 130 kD. The pattern of antigens precipitated varied with the different monoclonal antibodies of this group. The antibody of the fourth type precipitated a 30 kD polypeptide from extracts of myeloid and lymphoid cells. None of the other antibodies precipitated any detectable proteins.     

Antibodies against interspecies erythrokaryocyte antigen in patients with partial red cell aplasia of the bone marrow

Immunofluorescense and cytotoxicity test in vitro were used to demonstrate specific antibodies in sera of 11 out of 19 patients with partial red cell aplasia (PRCA). The antibodies reacted with erythroblast cells from embryos and adult men, with bone marrow cells from a female patient suffering from acute erythroleukemia, with erythrokaryocytes of mouse embryos and cells of Rauscher's viral erythroleukemia. The results of cross adsorption and blockade of the immunofluorescent reaction of the sera of PRCA patients with antibodies against mouse erythroblast antigen bearing an interspecies determinant suggest that in the pathogenesis of PRCA there takes part an autoimmune reaction against specific interspecies antigen to erythrokaryocytes. This antigen is apparently similar to antigen against mouse erythroblast cells.