Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.     

Cathepsin B from human renal cortex.

Cysteine-proteinase activity was observed in homogenates of human-cadaver renal cortex. This activity co-purified with renin enzymic activity until separation by aminohexyl-Sepharose--pepstatin affinity chromatography. The cysteine proteinase was purified 1780-fold after the following successive chromatographic procedures: Sephadex G-75, DEAE-cellulose DE-52, and an organomercurial affinity resin. The proteinase activity was dependent upon activation by thiol-containing compounds such as dithiothreitol, as well as by EDTA, and was inhibited by the thiol-group-specific alkylating reagents iodoacetic acid and N-ethylmaleimide. DE-52 cellulose chromatography resolved the cysteine proteinase into two components. On the basis of molecular size (26 000 daltons), activity as a function of pH, stability as a function of pH, substrate specificity and thermal lability, the major component (95%) has been identified as cathepsin B. The DE-52 cellulose elution pattern of the minor component (5%) is suggestive of cathepsin H [Schwartz & Barrett (1980) Biochem. J. 191, 487-497] Enzymic activity was determined with synthetic substrates, in particular alpha-N-benzoyl-DL-arginine 2-naphthylamide (Bz-Arg-NNap), thus precluding the detection of cathepsin L [Kirschke, Langner, Wiederanders, Ansorge, Bohley & Broghammer (1976) Acta Biol. Med. Germ. 35, 285-299]. Inhibition by dimethyl sulphoxide was observed in the determination of Km = 7.0 +/- 0.4 mM for the substrate Bz-Arg-NNap, and care must therefore be taken in the preparation of substrate solutions.     

IgG production kinetics in serum free media

Summary A murine hybridoma (455) was cultured in four different serum free media formulations, and a newborn calf serum supplemented medium was used as a basis of comparison. The serum supplemented medium supports a higher cell growth rate and results in a higher IgG titer. However, the antibody secretion rate on a per cell basis is higher in the serum free media, indicating that serum could be inhibitory to antibody secretion. The results identify the possibility of a least eliminating serum during the monoclonal antibody production phase.     

Respiration rates of some greenhouse cultivated tropical and subtropical species

A comparative study of dark respiration and effects of high and low temperature on dark respiration in five greenhouse cultivated tropical and sub-tropical species was made. The respiration rates determined manometrically were low in all species. Respiration rates increased with increasing temperature. Low temperature treatment accelerated the rate of respiration at 25 °G except in two species. An attempt has been made to determine the critical and optimum temperature for these species. The respiratory behaviour of these species has been discussed with respect to their original habitats and prevailing environmental conditions.     

Involvement of the galactosyl-1-phosphate transferase encoded by the Salmonella enterica rfbP gene in O-antigen subunit processing.

rfbT of Salmonella enterica LT2 was previously thought, together with rfaL, to be involved in the ligation of polymerized O antigen to core-lipid A, and three mutants were known. We report the mapping of the mutations to rfbP, the galactosyl-1-phosphate transferase gene, which is now shown to encode a bifunctional protein. The mutations which have the former rfbT phenotype are referred to as rfbP(T). We also show that rfbP(T) mutants are not blocked in the ligation step as previously believed but in an earlier step, possibly in flipping the O-antigen subunit on undecaprenyl pyrophosphate from the cytoplasmic to periplasmic face of the cytoplasmic membrane.     

Increased Expression of Apolipoprotein D Following Experimental Traumatic Brain Injury

Increasing evidence suggests that apolipoprotein D (apoD) could play a major role in mediating neuronal degeneration and regeneration in the CNS and the PNS. To investigate further the temporal pattern of apoD expression after experimental traumatic brain injury in the rat, male Sprague-Dawley rats were subjected to unilateral cortical impact injury. The animals were killed and examined for apoD mRNA and protein expression and for immunohistological analysis at intervals from 15 min to 14 days after injury. Increased apoD mRNA and protein levels were seen in the cortex and hippocampus ipsilateral to the injury site from 48 h to 14 days after the trauma. Immunohistological investigation demonstrated a differential pattern of apoD expression in the cortex and hippocampus, respectively: Increased apoD immunoreactivity in glial cells was detected from 2 to 3 days after the injury in cortex and hippocampus. In contrast, increased expression of apoD was seen in cortical and hippocampal neurons at later time points following impact injury. Concurrent histopathological examination using hematoxylin and eosin demonstrated dark, shrunken neurons in the cortex ipsilateral to the injury site. In contrast, no evidence of cell death was observed in the hippocampus ipsilateral to the injury site up to 14 days after the trauma. No evidence of increased apoD mRNA or protein expression or neuronal pathology by hematoxylin and eosin staining was detected in the contralateral cortex and hippocampus. Our results reveal induction of apoD expression in the cortex and hippocampus following traumatic brain injury in the rat. Our data also suggest that increased apoD expression may play an important role in cortical neuronal degeneration after brain injury in vivo. However, increased expression of apoD in the hippocampus may not necessarily be indicative of neuronal death.     

Atrial natriuretic peptide detected by immunocytochemistry in peripheral organs of Tupaia belangeri

ANP (atrial natriuretic peptide), a peptide found in granules of mammalian atrial cardiac myocytes, has been shown to be active in regulation of blood pressure and body water homeostasis. The existence of ANP in atrium, pituitary, adrenal gland, and kidney of the rat had been immunocytochemically demonstrated with an antibody against rat ANP (102-126). We used the same antibody in immunocytochemical studies for the detection of ANP in peripheral organs of the tree shrew (Tupaia belangeri). The antibody stained granules in myocytes of cardiac atria which indicated that it reacted with tree shrew ANP. In contrast to the rat, no immunoreactive cells were found in pituitaries and adrenal glands. However, in the kidneys distal tubules in outer medulla and cortex were labeled. Ascending limbs of distal tubules were intensely stained when either the peroxidase-antiperoxidase (PAP) or the indirect immunofluorescence method were used. Collecting ducts and convoluted distal tubules in the outer cortex showed a granular type of staining when the immunofluorescence method was used. These data indicate that ANP is present in epithelial cells of distal tubules and collecting ducts, where it may be involved in the regulation of renal salt excretion.     

Purification and characterization of a gelatinase produced by fibroblasts from human gingiva

An endopeptidase which digests denatured collagen to small, dialysable fragments was purified 2675-fold from medium that had been conditioned by the culture of fibroblasts grown from explants of human gingiva. This enzyme was inhibited by chelating agents, but not by phenylmethylsulphonyl fluoride nor by N-ethylmaleimide, and is therefore probably a metalloproteinase. It showed no demonstrable activity against native collagen or ovalbumin, while alpha-casein was digested slowly, if at all. It therefore belongs to the group of enzymes which have been called tissue gelatinases. This gelatinase was secreted in a latent form or forms and could be activated by proteolysis with trypsin. The active enzyme had an apparent molecular weight of 69 000 (gel chromatography) or 72 000 (gel electrophoresis in sodium dodecyl sulphate) and an apparent isoelectric point of 4.15.