The effect of repeated administration of insulin on pain threshold and gamma-aminobutyric acid level in mouse brain

In experiments on male Albino-Swiss mice weighing 18-22 g insulin given in doses of 2 i.u./kg caused no change in the time of reaction to pain, while the same dose administered daily for 7 days potentiated the analgesic action of morphine (3 mg/kg s.c.). Glucose caused no change in this effect of insulin. After 14 days of insulin treatment the time of reaction to pain in the animals subjected to the action of morphine returned to its initial value. Twenty-four hours after the last administration of morphine the level of gamma-aminobutyric acid (GABA) was found to be decreased in the animals receiving insulin with glucose. These results suggest that the central action of insulin is dependent not only on hypoglycaemia produced by it, but may be due also to its direct action on the central structures and an indirect action mediated by its effect on other neurotransmitter systems.     

Degradation of heparin proteoglycan in cultured mouse mastocytoma cells.

Pulse-labelling of mouse mastocytoma cell cultures, established from ascites fluid, with inorganic [35S]sulphate for 1 h yielded labelled heparin proteoglycan containing polysaccharide chains of Mr 60,000-100,000. After chase incubation for 24 h most of the 35S appeared in intracellular polysaccharide fragments similar in size to commercially available heparin, Mr 5000-25,000, as indicated by gel chromatography. Products isolated from cultures after 6 h of chase incubation consisted of partially degraded free polysaccharide chains and, in addition, residual proteoglycans that were of smaller size than the proteoglycans initially pulse-labelled. The polysaccharide chains released by alkali treatment from the residual chase-incubated proteoglycans were of the same size as the chains derived from proteoglycans after 1 h of pulse labelling. These results suggest that the intracellular degradation of heparin proteoglycan to polysaccharide fragments is initiated by release of intact polysaccharide chains, probably by action of a peptidase, and is pursued through cleavage of these chains by an endoglycosidase. An endoglucuronidase with stringent substrate specificity [Thunberg, Bäckström, Wasteson, Ogren & Lindahl (1982) J. Biol. Chem. 257, 10278-10282] has previously been implicated in the latter step. Cultures of more purified mastocytoma cells (essentially devoid of macrophages) did not metabolize [35S]heparin proteoglycan to polysaccharide fragments, but instead accumulated free intact polysaccharide chains, i.e. the postulated intermediate of the complete degradation pathway. When such purified cells were co-cultured with adherent mouse peritoneal cells, presumably macrophages, formation of polysaccharide fragments was observed. It is tentatively proposed that the expression of endoglucuronidase activity by the mast cells depends on collaboration between these cells and macrophages.     

Subcellular location of enzymes involved in the N-glycosylation and processing of asparagine-linked oligosaccharides in Saccharomyces cerevisiae

A particulate translation system isolated from the yeast Saccharomyces cerevisiae was shown to translate faithfully in-vitro-transcribed mRNA coding for a mating hormone precursor (prepro-alpha-factor mRNA) and to N-glycosylate the primary translation product after its translocation into the lumen of the microsomal vesicles. Glycosylation of its three potential sugar attachment sites was found to be competitively inhibited by acceptor peptides containing the consensus sequence Asn-Xaa-Thr, supporting the view that the glycan chains are N-glycosidically attached to the prepro-alpha-factor polypeptide. The accumulation in the presence of acceptor peptides of a membrane-specific, unglycosylated translation product (pp-alpha-F0) differing in molecular mass from a cytosolically located, protease-K-sensitive alpha-factor polypeptide (pp-alpha-Fcyt) by about 1.3 kDa, suggests that, in contrast to previous reports, a signal sequence is cleaved from the mating hormone precursor on/after translocation. This conclusion is supported by the observation that the multiply glycosylated alpha-factor precursor is cleaved by endoglucosaminidase H to a product with a molecular mass smaller than the primary translation product pp-alpha-Fcyt but larger than the membrane-specific pp-alpha-F0. Translation and glycosylation experiments carried out in the presence of various glycosidase inhibitors (e.g. 1-deoxynojirimycin, N-methyl-1-deoxynojirimyin and 1-deoxymannojirimycin) indicate that the N-linked oligosaccharide chains of the glycosylated prepro-alpha-factor species are extensively processed under the in vitro conditions of translation. From the specificity of the glycosidase inhibitors applied and the differences in the molecular mass of the glycosylated translation products generated in their presence, we conclude that the glycosylation-competent microsomes contain trimming enzymes, most likely glucosidase I, glucosidase II and a trimming mannosidase, which process the prepro-alpha-factor glycans down to the (Man)8(GlcNAc)2 stage. Furthermore, several arguments strongly suggest that these three enzymes, which apparently represent the full array of trimming activities in yeast, are exclusively located in the lumen of microsomal vesicles derived from endoplasmic reticulum membranes.     

A rapid-equilibrium model for the control of the Calvin photosynthesis cycle by cytosolic orthophosphate

1. A simple model based on rapid-equilibrium assumptions is derived which relates the steady-state activity of the Calvin cycle for photosynthetic carbohydrate formation in C3 plants to the kinetic properties of a single cycle enzyme (fructose bisphosphatase) and of the phosphate translocator which accounts for the export of photosynthate from the chloroplast. Depending on the kinetic interplay of these two catalysts, the model system may exhibit a single or two distinct modes of steady-state operation, or may be unable to reach a steady state. 2. The predictions of the model are analysed with regard to the effect of external orthophosphate on the steady-state rate of photosynthesis in isolated chloroplasts under conditions of saturating light and CO2. Due to the possible existence of two distinct steady states, the model may account for the stimulatory as well as the inhibitory effects of external phosphate observed in experiments with intact chloroplasts. Stability arguments indicate, however, that only the steady-state case corresponding to phosphate inhibition of the rate of photosynthesis could be of physiological interest. 3. It is concluded that chloroplasts under physiological conditions most likely operate in a high-velocity steady state characterized by a negative Calvin cycle flux control coefficient for the phosphate translocator. This means that any factor enhancing the export capacity of the phosphate translocator can be anticipated to decrease the actual steady-state rate of photosynthate export due to a decreased steady-state rate of cyclic photosynthate production.     

The effect of salt acclimation of the water uptake and osmotic permeability of the skin of the toad (Bufo viridis, L.)

1. Water uptake in vivo, and water fluxes across the isolated skin were studied in salt (NaCl) acclimated toads. 2. Water uptake of acclimated toads maintained in the solution of acclimation, decreased with the environmental salinity. 3. The osmotic water permeability (Pos) of the skin increased upon salt (NaCl) acclimation, both in vivo and in vitro. 4. Pos of the skin of toads acclimated to non-permeant solutes such as sucrose (230 mmol/l) or mannitol (400 nmol/l), was greatly reduced. 5. Oxytocin (syntocinon) increased the Pos both in tap water and salt acclimated toads. In high salt (greater than 200 mmol/l NaCl) acclimated toads however, the increased Pos and water flux at larger osmotic gradients, could not be stimulated further by the hormone. 6. The adaptive nature of the selective changes in the permeability properties of the skin under salt acclimation conditions is discussed.     

Ca2+-binding parvalbumin in rat testis. Characterization, localization, and expression during development

Parvalbumin, a Ca2+-binding protein, was isolated from rat testis. This is the first demonstration of the protein in endocrine glands. By using a rat parvalbumin cDNA probe, parvalbumin mRNA was demonstrated in the testis, indicating that the protein is synthesized in this tissue and that testis parvalbumin is a product of the same gene as the one encoding for muscle parvalbumin. Parvalbumin was localized by immunohistochemical methods in the Leydig cells and in the acrosome region of maturing spermatids (stages 1-15). The expression of parvalbumin during testis development was followed. High parvalbumin protein and mRNA levels were found at stages of highest Leydig cell activity, i.e. at late fetal stages until birth and again around postnatal day 50. This suggests that parvalbumin may be involved in the production of testosterone in Leydig cells, a process which is highly dependent on calcium.     

The primary structure of human chromogranin A and pancreastatin

A full-length clone encoding human chromogranin A has been isolated from a lambda gt10 cDNA library of a human pheochromocytoma. The nucleotide sequence reveals that human chromogranin A is a 439-residue protein preceded by an 18-residue signal peptide. Comparison of the protein sequence of human chromogranin A with that of bovine chromogranin A shows high conservation of the NH2-terminal and COOH-terminal domains as well as the potential dibasic cleavage sites, whereas the middle portion shows remarkable sequence variation (36%). This part of human chromogranin A contains a sequence homologous to porcine pancreastatin at residues 250-301. The sequence variation in this part of human chromogranin A compared to porcine pancreastatin is 32% and thus of the same magnitude as that between human and bovine chromogranin A. Therefore, the difference between porcine pancreastatin and the corresponding portions of bovine or human chromogranin A can be explained by species variation, suggesting that pancreastatin is derived from chromogranin A itself rather than a protein that is only similar to chromogranin A. Moreover, the pancreastatin sequence contained in human chromogranin A is flanked by sites for proteolytic processing. Together, these observations suggest that human chromogranin A may be the precursor for a human pancreastatin molecule and possibly for other, as yet unidentified, biologically active peptides.     

Membrane glycoproteins involved in neurite fasciculation

Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.     

A mechanical evaluation of an elastic femoral prosthetic stem

Studies are being conducted in our laboratory to test the concept of introducing an elastomer to attenuate and damp forces applied to the bone interface in a major weightbearing joint replacement prosthesis. An analogue of a fully constrained intramedullary stem type prosthesis has been developed in a segmental femoral replacement prosthesis of the dog. The layer of silastic was introduced to damp forces at the bone-prosthesis interface. This paper describes the response of this elastomer prosthesis to torsional and bending loads, and defines the upper limits of elastomer strain. The low modulus silastic displayed surprisingly low strain for applied loads, particularly in bending tests, in this prosthetic configuration. The results of these mechanical studies serve as a bench mark for the eventual design and material selection of an elastomer for human prosthetic use.     

Constitutive and IL 1-regulated murine complement gene expression is strain and tissue specific

To study the molecular mechanisms accounting for strain- and tissue-specific variations in the production of complement proteins, complementary DNA probes were used to assess qualitative and quantitative differences in specific mRNA content of complement proteins C2, factor B, and C3 in extracts of tissues (liver, lung, spleen, kidney, and peritoneal macrophages) isolated from various mouse strains. Northern blot analysis of total hepatic RNA revealed differences in C2, factor B, and C3 mRNA levels in strains that share B10 background but differ in the H-2 region (e.g., H-2k, H-2u, H-2d, H-2f). In each instance, hepatic mRNA specific for the individual gene product corresponded in amount to the serum levels. By contrast, specific mRNA content of C2 and factor B in macrophages differed significantly from those observed in liver for each strain. Modulation of C2, factor B, and C3 expression was studied after in vivo administration of recombinant IL 1 or endotoxin to H-2k (B10.AKM) or H-2u (B10.PL) strain mice. As assessed by Northern blot analysis, neither endotoxin nor IL 1 affected liver C2-specific mRNA but increased specific C2 mRNA levels in kidney and lung. For both strains, IL 1 increased specific factor B mRNA in all tissues examined except for the H-2u strain liver factor B mRNA content, which was not affected by IL 1, whereas that of H-2k mice was increased. The lack of factor B modulation by IL 1 in the H-2u lines was specific to that gene and not a reflection of a generalized IL 1 unresponsiveness. Differences in tissue and strain specific constitutive and IL 1-regulated expression of the C3 gene were also observed in the H-2u and H-2k strains.