Enterochromaffin-like cells in the rat stomach: effect of α-fluoromethylhistidine-evoked histamine depletion

Summary In the rat, gastric histamine is stored predominantly in the enterochromaffin-like (ECL) cells, which are located basally in the oxyntic mucosa. The functional significance of histamine in the ECL cells is a matter of speculation. In this study the effect of depletion of histamine on the properties and ultrastructure of the ECL cells was examined. Histamine synthesis was inhibited with -fluoromethylhistidine (3 mg·kg^-1·h^-1) given via osmotic minipumps over a period of 24 h. The treatment reduced the histidine decarboxylase activity (approximately 20% remaining) and histamine concentration (less than 20% remaining) in the oxyntic mucosa, as well as the intensity of histamine- and chromogranin A-immunostaining in the ECL cells, compared to control rats. The cytoplasmic (secretory) granules/vesicles were greatly reduced in number and size following -fluoromethylhistidine administration. The histamine immunostaining of the mast cells, which occurs at the mucosal surface and in the submucosa, appeared unaffected. We conclude that ECL cell histamine accounts for at least 80% of the total oxyntic mucosal histamine in the rat and that it represents a more mobile pool than mast cell histamine. The reduction in the number and size of the ECL cell granules/vesicles following histamine depletion is in accord with the idea that they represent the storage site for histamine.     

Regulation of polyphosphate metabolism in Acinetobacter strain 210A grown in carbon- and phosphate-limited continuous cultures

The response of Acinetobacter strain 210A to low phosphate concentrations was investigated in P- or C-limited chemostat cultures. The organism accumulated poly--hydroxybutyric acid under P-deprivation, at phosphate concentrations ranging from 0.1 to 0.7 mM. The amount of biomass was proportional to the phosphate concentration in the medium and no polyphosphate was formed. When shifting a culture from P- to C-limitation phosphate was accumulated as polyphosphate. No poly--hydroxybutyrate could be detected in these cells. The amount of polyphosphate in the cell showed a hysteresis. When cultures were shifted from low to high phosphate concentrations, polyphosphate reached a maximum of about 60 mg P per gram of dry weight at about 3 times excess phosphate (ca. 2.5 mM P_i). It decreased to 45 mg P per gram dry weight at approximately 5 times the phosphate needed for growth (ca. 3.5 mM P_i). In the reverse case (high to low) polyphosphate did never exceed 45 mg P per gram dry weight. The specific activities of alkaline phosphatase and the phosphate uptake system were induced at residual P_i concentrations below the detection limit (<10 M). The specific uptake rate followed also a hysteresis. The specific activities of polyphosphatase and polyphosphate: AMP phosphotransferase increased when polyphosphate formation was possible.Abbreviations HPP High polymeric polyphosphates - PHB Poly--hydroxybutyric acid - PP_n Polyphosphate - PQQ Pyrrolo-quinoline quinone - U 1 mol product formed · min^-1     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund     

The “ON”-bipolar agonist,L-2-amino-4-phosphonobutyrate,blocks light-evoked cone contraction in xenopus eye cups

Rhythmic photoreceptor metabolism in relationship to light-dark cycles is now thought be regulated through a retinal feed-back mechanism with dopamine serving as a principal signal initiating light-evoked events. In order to test the hypothesis that depolarizing ON-bipolar neurons participate in the retinal signalling pathway, we determined the effects of L-2-amino-4-phosphonobutyrate (L-APB) on light-evoked cone contraction in eye cups fromXenopus laevis. L-APB blocked the response stereospecifically when applied over a broad concentration range. The high specificity of L-APB in retina suggests that sign-inverting bipolar neurons which depolarize in light are in the signalling pathway. One possibility is that this pathway conveys signals that regulate dopamine release.Special issue dedicated to Dr. Frederick E. Samson.     

Opioid receptor antagonist affinity ligands: 6β-bromoacetamido-6-desoxynaltrexone and 6β-thioglycolamido-6-desoxynaltrexone

The present study, utilizing thioglycolamido as the reactive group, describes the synthesis and pharmacology of a new opioid antagonist affinity ligand, 6-thioglycolamido-6-desoxynaltrexone (TAN) and compares TAN with a related known compound, 6-bromoacetamido-6-desoxynaltrexone (BAN). Both compounds were tested for their reversible and irreversible inhibition of [³H]naloxone binding to calf brain membranes. Reversible binding of BAN and TAN had K_i values of 1×10^–9 and 1×10^–10 M, respectively as determined by log probit plots. Irreversible binding was determined after extensive washing to remove all non-covalently bound ligand. At a concentration of 5×10^–8 and 1×10^–8 M for BAN and TAN irreversible binding was inhibited 50% of the maximum value. A study of the time course of irreversible inhibition of [³H]naloxone binding revealed that maximal inhibition occurred within 5 min with a concentration of 1×10^–7 M of either agent. TAN but not BAN when administered systematically to mice produced an antinociceptive effect as measured by the writhing test. When administered intracerebraventricularly BAN did not block morphine-induced analgesia for more than 2 hr; whereas, with a single ED₅₀ dose of 20 nmoles of TAN i.c.v. morphine-induced analgesia was almost completely blocked for a period of over 24 hr, as determined by the tail flick test. Although the SH group of TAN were required for the covalent interaction with opioid receptors, the site of TAN's interaction appears to involve other than protein SH groups.     

A nomenclature for the genes encoding the chlorophylla/b-binding proteins of higher plants

We propose a nomenclature for the genes encoding the chlorophylla/b-binding proteins of the light-harvesting complexes of photosystem I and II. The genes encoding LHC I and LHC II polypeptides are namedLhca1 throughLhca4 andLhcb1 throughLhcb6, respectively. The proposal follows the general format recommended by the Commision on Plant Gene Nomenclature. We also present a table for the conversion of old gene names to the new nomenclature.     

Helix packing of leucine-rich peptides: a parallel leucine ladder in the structure of Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe.

The packing of peptide helices in crystals of the leucine-rich decapeptide Boc-Aib-Leu-Aib-Aib-Leu-Leu-Leu-Aib-Leu-Aib-OMe provides an example of ladder-like leucylleucyl interactions between neighboring molecules. The peptide molecule forms a helix with five 5----1 hydrogen bonds and two 4----1 hydrogen bonds near the C terminus. Three head-to-tail NH ... O = C hydrogen bonds between helices form continuous columns of helices in the crystal. The helicial columns associate in an antiparallel fashion, except for the association of Leu ... Leu side chains, which occurs along the diagonal of the cell where the peptide helices are parallel. The peptide, with formula C56H102N10O13, crystallizes in space group P2(1)2(1)2(1) with Z = 4 and cell parameters a = 16.774(3) A, b = 20.032(3) A and c = 20.117(3) A; overall agreement factor R = 10.7% for 2014 data with magnitude of F(obs) greater than 3 sigma (F); resolution 1.0 A.     

Experimental approaches to guarantee minimal risk of potential virus in purified monoclonal antibodies

Regarding biological products, increasing awareness of potential side effects have placed great importance not only at protein purity regarding other proteins but on the removal of biologicals such as DNA and especially virus the importance of which may not be known. Monoclonal antibodies (Mab) have come to be an important class of molecules obtained from hybridoma cells, i.e., nonrecombinant cells in culture. It has been noted during the last years, that with rare exceptions hybridoma cell lines contain retrovirus like particles. The infectious nature of the EM-visible particles has been tested for, however, in most cases not been substantiated. In order to bring these valuable biological reagents, Mab's, to good use in man for imaging or therapy, the remaining concern about a potential retroviral infection has to be reduced to an acceptable minimum. We describe experimental approaches for the validation of chromatographic and ultrafiltration steps used in the production of monoclonal antibodies to remove and inactivate murine retrovirus. Present day biotechnological manufacturing processes have been devised incorporating a number of strategic preventive measures that have found wide spread acceptance. They permit to answer the question: how can a potentially harmful infection by an unknown virus be excluded. Knowledge of the efficacy of purification steps to clear infectious model virus is fundamental to devise biotechnological manufacturing processes yielding a purified antibody for use in man.     

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage

Summary Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD) that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at 180 copies per uninduced cell and was measured at 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.     

An inflatable minirhizotron system for root observations with improved soil/tube contact

Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images.