The role of the arginine-B22 residue in insulin action.

We describe the modification of the side chain of the arginine-B22 residue of insulin by the N8N9-(1, 2-dihydroxycyclohex-1,2-ylene) group and by the adipoyl group. These are the first insulin derivatives described that contain a modified arginine residue in an otherwise unaltered molecule. When tested for their ability to lower blood sugar concentration, both modified insulins showed a specific activity indistinguishable from that of insulin. In view of the fact that the substituent groups involved are very bulky and in one case of opposite charge to that of the side chain, the retention of biological activity casts doubt on the idea, previously generally accepted, that the arginine-B22 residue is essential to the activity of the hormone.     

Interspecific play between free ranging guerezas (Colobus guereza) and vervet monkeys (Cercopithecus aethiops)

Interspecific play, like other interspecific interactions, seems to be rare among free-living primates, despite the fact that polyspecific associations occur quite frequently, especially among forest-living species. A number of instances of play between young guerezas and vervet monkeys are described. This play may be dyadic, individual-group, or group-group and usually involves non-contact types of play. It is suggested that interspecific play is most likely to occur if the ranging patterns of groups of sympatric species remains the same over considerable periods of time, if animals of similar ages are of similar sizes, if social signals associated with play are similar, if play activities are performed in a similar way, and if the temporal organization of play bouts is the same.     

A ubiquitin C-terminal isopeptidase that acts on polyubiquitin chains. Role in protein degradation.

In the ubiquitin (Ub) pathway, proteins are ligated with polyUb chains and then are degraded by a 26 S protease complex. We describe an enzyme, called isopeptidase T, that acts on polyUb chains. It is a monomeric Ub-binding protein abundant in erythrocytes and reticulocytes. The activity of the isopeptidase is inhibited by iodoacetamide and Ub aldehyde. Treatment of the enzyme with Ub aldehyde increased its affinity for free Ub, indicating the existence of two different Ub-binding sites and cooperativity between the two sites. Isopeptidase T acts on polyUb-protein conjugates, but not on conjugates in which the formation of polyUb chains was prevented by the use of reductively methylated Ub or on abnormal polyUb chains formed with a mutant Ub that contains a Lys----Arg substitution at residue 48. The enzyme converts high molecular mass polyUb-protein conjugates to lower molecular mass forms with the release of free Ub, but not of free protein substrate. The lower molecular mass Ub-protein conjugate products are resistant to further action of the enzyme. Isopeptidase T stimulates protein degradation in a system reconstituted from purified enzyme components. The enzyme also stimulates the degradation of proteins ligated to polyUb chains by the 26 S protease complex. Preincubation of polyUb-protein conjugates with the isopeptidase did not much increase their susceptibility to proteolysis by the 26 S complex. On the other hand, preincubation of conjugates with the 26 S protease complex and ATP increased the release of free Ub upon further incubation with the isopeptidase. It thus seems that a role of this isopeptidase in protein breakdown is to remove polyUb chain remnants following the degradation of the protein substrate moiety by the 26 S complex.     

Characterisation and Regional Localisation of Pre- and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training

To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1-day-old chicks on a one-trial passive avoidance learning task. To increase the reliability of the comparison, a double-labelling method was used. Tissue samples from intermediate medial hyperstriatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region-specific changes, both increases and decreases, in incorporated radioactivity into pre- and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150-180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass approximately 50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150-180, 100-120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2-deoxygalactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre- and postsynaptic neurons of the LPO by growth or modulation of pre- and postsynaptic structures.     

Evolutionarily conserved regions in Caenorhabditis transposable elements deduced by sequence comparison.

In this paper we present the sequence of an intact Caenorhabditis briggsae transposable element, Tcb2. Tcb2 is 1606 base pairs in length and contains 80 base pair imperfect terminal repeats and a single open reading frame. We have identified blocks of T-rich repeats in the regions 150-200 and 1421-1476 of this element which are conserved in the Caenorhabditis elegans element Tc1. The sequence conservation of these regions in elements from different Caenorhabditis species suggests that they are of functional importance. A single open reading frame corresponding to the major open reading frame of Tc1 is conserved among Tc1, Tcb1, and Tcb2. Comparison of the first 550 nucleotides of the sequence among the three elements has allowed the evaluation of a model proposing an extension of the major open reading frame. Our data support the suggestion that Tc1 is capable of producing a 335 amino acid protein. A comparison of the sequence coding for the amino and carboxy termini of the 273 amino acid transposase from Caenorhabditis Tc1-like elements and Drosophila HB1 showed different amounts of divergence for each of these regions, indicating that the two functional domains have undergone different amounts of selection. Our data are not compatible with the proposal that Tc1-related sequences have been acquired via horizontal transmission. The divergence of Tc1 from the two C. briggsae elements, Tcb1 and Tcb2, indicated that all three elements have been diverging from each other for approximately the same amount of time as the genomes of the two species.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Vanadium-containing tunicate blood cells are not highly acidic

The intracellular pH of intact blood cells of the tunicate Ascidia nigra was measured by transmembrane equilibration of [¹⁴C]methylamine. The pH of unfractionated blood cells is 7.39±0.10. The pH of vanadocytes, determined in a fractionation study, is 7.2. Previously used methods, in which pH values less than 3.0 are inferred from cell lysis or vital staining experiments, are shown to be unsuitable for intracellular pH determination due to the chemical composition of these vanadium-containing cells.     

Brain glucose bisphosphatase requires inosine monophosphate

Glucose bisphosphate phosphatase has been partially purified from the cytosol of mouse brain. Enzyme activity required Mg2+ and a heat-stable cofactor. The activator was present in boiled extracts of mouse brain mitochondrial-nuclear fraction, of red blood cells, or of rabbit muscle. The chemical properties of the activator are consistent with its identification as inosine monophosphate (IMP), including its mobility in a high pressure liquid chromatography (HPLC) system capable of resolving all of the biologically important mononucleotides. A large number of other biologically important compounds were not effective, including AMP, cAMP, cGMP, and UMP, GMP, purified by HPLC, (50 or 74 microM), gave a rate about 35% of that obtained with IMP (5 microM). The enzyme was separated completely from phosphoglucomutase and significantly from glucose bisphosphate synthase. The products of the reaction are glucose-P and Pi. Fructose bisphosphate at 500 microM inhibited only 40% in the presence of 20 microM glucose bisphosphate. The activation by IMP follows hyperbolic kinetics with an apparent Ka of 5 microM in the presence of 12 microM glucose bisphosphate. The apparent Km of glucose bisphosphate was 10 microM in the presence of 50 microM IMP. There was no inhibition by 5 or 50 microM AMP or ADP. The possible regulatory importance of glucose bisphosphate in carbohydrate metabolism and the significance of the regulation of the phosphatase by the nucleotide are discussed.     

Genetic factors controlling the susceptibility to experimental autoimmune thyroiditis in inbred rat strains

CDF (high responder) rats were crossed with SHR (low responder) rats to study genetic factors influencing susceptibility to experimental autoimmune thyroiditis (EAT). F1 hybrid rats showed intermediate susceptibility to thyroiditis. Gene segregation patterns of F2 and backcross rats demonstrated that the immune response to rat thyroglobulin (RaTg) is under polygenic control. A main factor controlling immune response to RaTg is associated with the X chromosome. Females of CDF and SHR rats produced higher levels of antibody, suggesting that antibody production to RaTg is a sex-influenced trait. Different genetic control mechanisms are evident in autoantibody production and thyroid lesions. No evidence of linkage of an immune response gene to RaTg to the major histocompatibility complex of the rat (RT1) was observed.