Human neutrophil interactions of a bispecific monoclonal antibody targeting tumor and human Fcγ RIII

 2B1 is a bispecific murine monoclonal antibody (bsmAb) targeting the c-erbB-2 and CD16 (FcγRIII) antigens. c-erbB-2 is over-expressed by a variety of adenocarcinomas, and CD16, the low-affinity Fcγ receptor for aggregated immunoglobulins, is expressed by polymorphonuclear leukocytes (PMN), natural killer (NK) cells and differentiated mononuclear phagocytes. 2B1 potentiates the in vitro lysis of c-erbB-2 over-expressing tumors by NK cells and macrophages. In this report, the interactions between 2B1 and PMN were investigated to assess the impact of these associations on in vitro 2B1-promoted tumor cytotoxicity by human NK cells. The peak binding of 2B1 to PMN was observed at a concentration of 10 μg/ml 2B1. However, 2B1 rapidly dissociated from PMN in vitro at 37°C in non-equilibrium conditions. This dissociation was not caused by CD16 shedding. When PMN were labeled with ¹²⁵I-2B1 and incubated at 37°C and the supernatants examined by HPLC analysis, the Fab regions of dissociated 2B1 were not complexed with shed CD16 extracellular domain. While most of the binding of 2B1 to PMN was solely attributable to Fab-directed binding to FcγRIII, PMN-associated 2B1 also bound through Fcγ-domain/FcγRII interactions. 2B1 did not promote in vitro PMN cytotoxicity against c-erbB-2-expressing SK-OV-3 tumor cells. When PMN were coincubated with peripheral blood lymphocytes, SK-OV-3 tumor and 2B1, the concentration of 2B1 required for maximal tumor lysis was lowered. Although PMN may serve as a significant competitive binding pool of systemically administered 2B1 in vivo, the therapeutic potential of the targeted cytotoxicity properties of this bsmAb should not be compromised. Received: 3 May 1995 / Accepted: 6 February 1996     

A SALL4 zinc finger missense mutation predicted to result in increased DNA binding affinity is associated with cranial midline defects and mild features of Okihiro syndrome

Truncating mutations of the gene SALL4 on chromosome 20q13.13–13.2 cause Okihiro and acro-renal-ocular syndromes. Pathogenic missense mutations within the SALL4 or SALL1 genes have not yet been reported, raising the question which phenotypic features would be associated with them. Here we describe the first missense mutation within the SALL4 gene. The mutation results in an exchange of a highly conserved zinc-coordinating Histidine crucial for zinc finger (ZF) structure within a C2H2 double ZF domain to an Arginine. Molecular modeling predicts that this exchange does not result in a loss of zinc ion binding but leads to an increased DNA-binding affinity of the domain. The index patient shows mild features of Okihiro syndrome, but in addition cranial midline defects (pituitary hypoplasia and single central incisor). This finding illustrates that the phenotypic and functional effects of SALL4 missense mutations are difficult to predict, and that other SALL4 missense mutations might lead to phenotypes not overlapping with Okihiro syndrome. This study has received approval by the institutional review board (Ethics committee) of the Medical Faculty, University of Freiburg, Germany. Jan Miertus and Wiktor Borozdin have equally contributed to this work.     

Specific dechlorinase activity in lindane degradation by Streptomyces sp. M7

Synthesis of dechlorinase in Streptomyces sp. M7 was induced when the microorganism was grown in the presence of lindane (γ-hexachlorocyclohexane) as the only carbon source. Activity of cells grown with lindane was about four and half times higher compared to cells grown with glucose. Maximum dechlorinase activity was observed at 30°C in alkaline conditions pH (7.9) and the enzyme did not show cation dependency. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed one differential band with a molecular weight similar to serum albumin (M _r 66,200), which corresponded to polynucleotide phosphorylase, an enzyme that plays an important role in the regulation system and could be involved in the regulation of the dechlorinase gene. Detected in cell-free extracts were γ-pentachlorocyclohexene and 1,3,4,6-tetrachloro-1,4-cyclohexadiene, both being products of the dechlorinase activity. This is the first time that the presence of an enzyme with dechlorinase activity has been demonstrated in an actinomycete strain isolated in Tucumán, Argentina. Characteristics of this enzyme revealed that Streptomyces sp. M7 could be useful in the future in bioremediation of soil or as a biosensor.     

The influence of seat height on the mechanical function of the triceps surae muscles during steady-rate cycling

The purpose of this study was to manipulate bicycle seat height in order to perturbate muscle length, contraction velocity and excitation of soleus and medial gastrocnemius muscles. One group of female riders (n = 13) rode a stationary ergometer at 200 W and a cadence of 80 rpm. Individuals rode at a self-selected seat height, a 10% lowered and 5% raised seat position. It was hypothesized that because the two muscles would operate at decreased contraction velocities at the low seat, the integrated EMG would be less for the lowest seat position. The soleus and medial gastrocnemius muscles showed a significant decrease in integrated EMG value with decreased seat height (soleus F_2,24 = 5.4, p < 0.01, gastrocnemius F_2,24 = 51.6, p < 0.0001). The combined effect of the movement at the ankle and knee joints resulted in increased length of gastrocnemius rather than shortening at the lowered seat-height position as anticipated. This suggested that there was a greater role of knee-joint angle in determining the muscle excitation for medial gastrocnemius. The original hypothesis was accepted, confirming the importance of setting proper seat height.     

Electrogenic plasma membrane H<Superscript>+</Superscript>-ATPase activity using voltage sensitive dyes

Fast responding voltage sensitive dyes, RH421 and di-4-ASPBS, were used to study the electrogenic properties of plant plasma membrane proton pumps on sealed plasma membrane vesicles extracted by two-phase partitioning from Beta vulgaris and Avena sativa cv Swan root material. Fluorescence spectroscopy in the presence of the dye RH421 (10.8 nM) was sufficiently sensitive to detect electrogenic activity of the extracted plant vesicles. The dye detection system could detect inhibition of electrogenic activity of vesicles by vanadate (75 μM) and stimulation by nigericin (0.5 μM). The newly developed dye di-4-ASPBS was less sensitive to detecting the electrogenic proton pump activity. This study represents an important innovation in plant biophysics as this class of fast responding voltage sensitive dyes have never to our knowledge been used to study electrogenic proton pump activity derived from plant membranes and represents a novel approach for carrying out such studies.     

N-Substituted acetamidines and 2-methylimidazole derivatives as selective inhibitors of neuronal nitric oxide synthase

A series of N-substituted acetamidines and 2-methylimidazole derivatives structurally related to W1400 were synthesized and evaluated as Nitric Oxide Synthase (NOS) inhibitors. Analogs with sterically hindering isopropyl and phenyl substituents on the benzylic carbon connecting the aromatic core of W1400 to the acetamidine nitrogen, showed good inhibitory potency for nNOS (IC(50)=0.2 and 0.3 μM) and selectivity over eNOS (500 and 1166) and to a lesser extent over iNOS (50 and 100). A molecular modeling study allowed to shed light on the effects of the structural modifications on the selectivity of the designed inhibitors toward the different NOS isoforms.