Genetic evidence for α2-adrenergic receptor subtypes in rat brain,heart and adrenal gland

Summary A complementary DNA (cDNA) clone - cA₂-47 - corresponding to a new ₂-adrenergic receptor subtype has been isolated from a rat brain cDNA library and used as a hybridization probe to scrutinize the ₂-receptor poly(A⁺) RNAs in rat brain, heart and adrenal gland. Hybridization of the 5 half of the coding region of this cDNA at 37°C to rat brain poly(A+) RNA revealed a single band at 5.8 kb as the size of its corresponding mRNA. Under identical hybridization conditions, a human platelet ₂-receptor genomic probe failed to hybridize to any rat brain mRNAs.Under lower stringency conditions, hybridization of the full-length cDNA, cA₂-47, to selected rat tissue poly(A⁺) RNA showed the presence of four different sized mRNAs in brain and three in both heart and adrenal gland. Messages of 1.3 kb and 2.1 kb were common in all three tissues (although the band at 2.1 kb was slightly higher in the heart and adrenal gland). A 5.8 kb mRNA was unique to the brain and a slightly higher band at 6.0 kb was consistently present in heart and adrenal gland but was absent in the brain. A fourth message at 3.4 kb was found predominantly in the brain and was either absent or present at very low levels in the other tissues examined. Under the same conditions, a human platelet ₂-receptor probe hybridized to similar sized messages of 2.1 and 5.8 kb in rat brain and 2.2 and 6.0 kb in rat heart and adrenal gland. This probe, however, failed to detect the abundant 1.3 kb mRNA common to all tissues or the 3.4 kb message in rat brain. The extent of homology of these messages with cA₂-47 is not confined to limited regions of the cDNA since similar hybridization patterns were observed using either 5-noncoding or 5-coding regions of the probe.These results provide the first direct evidence of a surprisingly large range of mRNA sizes for members of the ₂-receptor family in brain, heart, and adrenal gland. The unique nature of certain members of the family in each of the tissues examined raises the curious possibility that these members might contribute to some of the individualized functions of the brain, cardiovasculature and adrenal gland.     

Catecholamine receptor in the seawater eel intestine

Dose-response relationships of catecholamines on seawater eel intestinal ion transport were obtained; the potency order being (-)adrenalin>(-)noradrenalin=clonidine>(±)noradrenalin (racemic form of noradrenalin)>(-)phenylephrine>dopamine>(±)isoproterenol, indicating that ₂ are more potent than ₁- or -agonists. In addition, the effects of adrenalin were completely blocked by yohimbine (₂-antagonists) but not by prazosin (₁-antagonists) or propranolol (-antagonist). These results indicate the existence of an ₂-receptor in the seawater eel intestine. Adrenalin may activate the ₂-receptor physiologically, since adrenalin is the most potent stimulant and is the predominant catecholamine in American eel plasma (Hathaway and Epple 1989). Presumably ion and water absorption across the seawater eel intestine will be maintained by adrenalin. From the structure and the action of various agents used in the present study, structure-activity relationships of catecholamines are considered: hydroxyl groups on the benzene ring (catechol) seem to be essential for the ₂-action in the seawater eel intestine and the presence of OH and CH₃ on -carbon and amide, respectively, seems to potentiate the ₂-action.Abbreviations ACh acetylcholine - AD adrenalin - CONH 2-cyclooctyl-2-hydroxyethylamine; n-methyltransferase - DA dopamine - 5-HT serotonin - IBMX 3-isobutyl-1-methylxanthine - I _sc short-circuit current - MCh methacholine - NA noradrenalin - PD transepithelial potential difference - PNMT phenylethanolamine N-methyltransferase - R _t tissue resistance     

Pyridoxal isonicotinoyl hydrazone (PIH) prevents copper-mediated in vitro free radical formation

Since it was reported in 1991 by Schaffer et al. that myocardial contractile responsiveness was altered in NIDDM in the absence of alterations in the -adrenergic receptor population, researchers have been seeking a post-receptor defect to account for this. The present study addresses this issue by comparing alterations occurring in the myocardial -receptor signalling pathway in two different models of rat NIDDM, as well as the response of the pathway after stimulation with isoproterenol in the presence or absence of insulin. The characteristics of the -receptor population, adenylyl cyclase activity and cAMP levels were determined at three different ages. The main results demonstrate that: (i) the two models of NIDDM myocardium differ biochemically; (ii) the -adrenergic signalling system of the insulin deficient model was altered more than the hyperinsulinemic model and (iii) the observed exaggerated cAMP response of NIDDM hearts after stimulation with a -adrenergic agonist is in contrast with lower responsivity.     

Blood-gas,acid-base and catecholamine responses to lactic acid infusion in larval and adult Ambystoma tigrinum

Larval and adult Ambystoma tigrinum were subjected to acidosis by infusing lactic acid (2 M·g^-1) into the peritoneal cavity. Blood was sampled at intervals to establish the time-course of the ensuing acidosis. Both larvae and adults became significantly acidotic after 1 h. The larval acidosis was more pronounced (-4 pH units versus-2 pH units) than adults due to greater extracellular buffering capacity (higher [HCO₃ ^-]) in adults. Both forms recovered in about 8 h. Larvae showed a typical increase in circulating norepinephrine during the initial stages of the acidosis; adults did not, having significantly lower norepinephrine titer than larvae during the acidosis. Both larvae and adults showed transient increases in PO₂ during the acidosis. The ₁ and ₂ antagonists, timolol and butoxamine respectively, (0.2 g·g^-1) were administered to separate groups of larvae. Butoxamine (₂) delayed the recovery from the acidosis by prolonging the increase in arterial PCO₂ and reversing the recovery of [HCO₃ ^-]. Timolol (₁) did not delay recovery. We conclude that ₂ receptors are involved in the catecholamine responses to acidosis in larvae. Catecholamines appear not to play the same role in adult acid-base disturbances as they seem to in larvae.Abbreviations RBC red blood cell     

The pharmacodynamic action of the cyclic AMP phosphodiesterase inhibitor rolipram on prolactin producing rat pituitary adenoma (GH4C1) cells

Rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) represents a new class of specific low K_m cAMP phosphodiesterase (PDE) inhibitors. This compound enhances basal, hormone- and forskolin-elicited cAMP accumulation in prolactin (PRL) producing rat pituitary adenoma (GH₄C₁) cells in culture (ED₅₀=5·10^–8 M). This effect is due to a selective inhibition of the low K_m cAMP PDE (type III), since neither basal nor hormone-stimulated adenylate cyclase (AC) nor the Ca²⁺/calmodulin-dependent PDE were affected by rolipram. The drug enhanced vasoactive intestinal polypeptide (VIP)-stimulated PRL-secretion, while thyroliberin (TRH)- and 12-0-tetradecanoyl phorbol-13-acetate (TPA)-elicited PRL egress were slightly reduced indicating a cAMP-mediated reduction of protein kinase C (PK-C) mediated PRL release. Interestingly, inhibition of PRL secretion by somatostatin (SRIH) was completely suppressed suggesting cAMP-mediated inactivation of some GTP-binding protein(s) of the _i family (G _i2 orG _k). Rolipram did not affect phosphoinositide metabolism (i.e. IP₃ accumulation), neither acutely nor after long term administration. Rolipram, like the cAMP PDE inhibitor Ro 20–1724, did not influence AC and PDE I, but dose-dependently inhibited PDE III activity.Long term incubation of GH₄C₁ cells with rolipram in the presence of noradrenaline (NA) exerted a marginal decrease of -receptor number, AC activation and cAMP accumulation, while Ro 20–1724 brought about a marked down-regulation and desensitization of the AC complex.In summary, rolipram selectively interacts with PDE III in rat pituitary adenoma cells in culture and does not result in -adrenoceptor AC downregulation. These features are not shared by the other drugs tested.     

δ and κ Opiate receptors in primary astroglial cultures from rat cerebral cortex

The effects of , , and receptor-agonists on forskolin stimulated cyclic adenosine-3, 5-monophosphate (cAMP) formation were examined in astroglial enriched primary cultures from the cerebral cortex of newborn rats. Intracellular cAMP accumulation was quantified by radioimmunoassay. Morphine was used as a -receptor agonist, D-Ala-D-Leu-Enkephalin (DADLE) as a -receptor agonist and dynorphine 1–13 (Dyn) as a -receptor agonist. Basal cAMP levels were unaffected by either the opiate agonists or the antagonists used. In the presence of the cAMP stimulator forskolin, morphine had no significant effect on the cytoplasmic cAMP levels. DADLE caused a dose related inhibition of the forskolin stimulated cAMP accumulation. The effects of this receptor stimulation was blocked with the selective antagonist ICI 174.864. In the presence of Dyn, the forskolin stimulated cAMP accumulation was inhibited in a dose related manner. This receptor stimulation was blocked with the selective antagonist MR 2266. Co-administration of DADLE and Dyn resulted in a non additive inhibition of the forskolin stimulated accumulation of cAMP. These findings indicate that astroglial enriched cultures from the cerebral cortex of rats express and -receptors co-localized ont he same population of cells, and that these receptors are inhibitory coupled to adenylate cyclase.     

Enhanced Responsiveness of the Myocardial β-Adrenoceptor-Adenylate Cyclase System in the Perfused Rat Heart (I)

Crude myocardial sarcolemmal membrane fractions were prepared from rat hearts subjected to total global ischemia with and without normoxic reperfusion, or global anoxic (N₂) perfusion with and without normoxic reperfusion. The direct effects on -adrenenoceptor number, G-protein levels and stimulation of the adenylate cyclase (AC) complex were assessed.In terms of AC activation, ischemia led to a marked increase (4-fold) in sensitivity to terbutaline (₂-agonist) and phorbol ester (tetradecanoyl phorbal acetate = TPA) stimulation, whereas the dobutamine (₁) responsiveness and Gpp(NH)p activation through G_S/G_i2 remained unaltered. However, forskolin-elicited holoenzyme activity fell markedly during normoxic reperfusion. Ischemia did not change the ₁-adrenoceptor number, while ₂-receptor population increased by approximately 45%. Western blots of myocardial G_S A and G_i2 contents revealed that ischemia selectively diminished G_i2 levels only by some 50–70%.Contrastingly, anoxia selectively increased the AC sensitivity (2-fold) to ₁-adrenergic stimulation. As subsequent to ischemia, anoxia also increased the sensitivy to TPA stimulation, however, only 2-fold. Gpp(NH)p activation was unchanged, while forskolin-enhanced activity gradually declined, also during ensuing normoxic reperfusion. Anoxia brought about a 75% enhancement in ₁-receptor number, while ₂-receptors remained unaffected. However, altered receptor number normalized on termination of normoxic reperfusion. Finally, anoxia led to a 50–60% decimation of myocardial G_i2 levels, while G_S was only marginally reduced.Despite the fact that the ischemia and anoxia effectuated a similar deterioration of physiological heart parameters, myocardial contents of energy rich phosphate moieties and loss of G_i2, ischemia rendered the most profound increase in responsiveness of the sarcolemmal AC system.     

Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies?

The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408–417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu–MT studies. The Zn/Ag replacement in recombinant mouse Zn₇–MT 1 and corresponding Zn₄-MT 1 and Zn₃-MT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV–vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn₇–MT 1, Zn₄–MT 1 and Zn₃-MT 1 with AgClO₄ at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn₇–MT 1 at pH 7.5 has revealed the subsequent formation of Ag₄Zn₅–MT, Ag₇Zn₃–MT, Ag₈Zn₃–MT, Ag₁₀Zn₂–MT, Ag₁₂Zn₁–MT, Ag_x–MT, x=14–19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Ag_x–MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the _xZn_y–MT (0y<7) species found, only M^I₄Zn₅–MT [(Zn₄)(₄Zn₁)] and M^I₇Zn₃–MT [(₃Zn₂)(₄Zn₁)], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Role of the pituitary gland in experimental hormonal induction and prevention of benign prostatic hyperplasia in the dog

Summary The antiandrogen, cyproterone acetate (CPA), prevents development of prostatic hyperplasia, induced in castrated dogs by a 6 month-treatment with 5-androstane-3,l 7-diol (A)alone or in combination with 17-oestradiol (E ₂). The immunoperoxidase technique was used to study functional cell types in the pars distalis of the pituitary gland and to detect growth hormone (GH) and prolactin (PRL) target sites in the prostate gland. Homologous radioimmunoassays for estimation of serum canine GH and PRL concentrations were also performed. Treatment with the combinations A + E ₂ and A + E ₂ + CPA resulted in morphological indications of stimulated GH and PRL cells and depressed gonadotrophs. This correlates well with an increase in PRL-dependent staining in glandular epithelium and fibromuscular tissue of the prostate gland. However, basal serum PRL and GH levels were not significantly affected. Treatment with A and A + E₂ stimulated, while additional treatment with CPA clearly suppressed adrenocorticotrophin/melanotrophin (ACTH/MSH) cells. These findings indicate that an endocrine imbalance in hypothalamic-pituitary-adrenal function may be involved in induction and prevention of prostatic hyperplasia in the dog.     

Photoaffinity labeling of the DDT1 MF-2 cell α1-adrenergic receptor

Summary In this study, we have used an ₁-adrenergic receptor photoaffinity ligand, 2-[4-(4-azido-3-iodo-benzoyl)-piperazin-1-yl]-4-amino-6, 7-dimethoxyquinazoline (¹²⁵I-APD), to label covalently the ₁-adrenergic receptor in a smooth muscle cell line. Our results indicate that in the absence of light, (¹²⁵I)APD binds reversibly to a site in the DDT₁ MF-2 cell membranes having pharmacological characteristics of an ₁-adrenergic receptor. Following incorporation of (¹²⁵I)ADP into partially purified membranes a single labeled band of protein with a M_r of 81 000 was visualized by autoradiography following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Incorporation of (¹²⁵I)-APD into this band was affected by adrenergic agonists and antagonists in a manner consistent with an ₁-adrenergic interaction. Prazosin (₁-selective) blocked incorporation of the label into the Mr = 81 000 protein while yohimbine (₂-selective) did not. Of the adrenergic agonists, (–)-epinephrine and (–)-norepinephrine but not (–)-isoproterenol blocked labeling of the M_r – 81 000 protein. We conclude that the ligand binding site of the DDT₁ MF-2 cell ₁-adrenergic receptor resides in a M_r = 81 000 protein.     

Control of nitrogenase inAzospirillum sp.

Many N₂-fixing organisms can turn off nitrogenase activity in the presence of NH₄ ⁺ and turn it on again when the NH₄ ⁺ is exhausted. One of the most interesting systems for accomplishing this is by covalent modification of one subunit of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). The system can be reactivated when NH₄ ⁺ is exhausted, by dinitrogenase reductase activating glycohydrolase (DRAG) which removes the inactivating group. It is fascinating that some species of the genusAzospirillum possess the DRAT and DRAG systems (A. lipoferum andA. brasilense), whereasA. amazonense in the same genus lacks DRAT and DRAG.A. amazonense responds to NH₄ ⁺ but does not exhibit modification of dinitrogenase reductase characteristic of the action of DRAT. However, it has been possible to clone DRAT and DRAG and to introduce them intoA. amazonense, whereupon they become functional in this organism. The DRAT and DRAG system does not appear to function inAcetobacter diazotrophicus, an organism isolated from sugar cane, that fixes N₂ at a pH as low as 3.0.A. diazotrophicus does show a rather sluggish response to NH₄ ⁺. A level of about 10 M NH₄ ⁺ is required to switch off the system. The response to NH₄ ⁺ is influenced by the dissolved oxygen concentration (DOC) as has been reported forAzospirillum sp. A DOC in equilibrium with 0.1 to 0.2 kPa O₂ seems optimal for the response inA. diazotrophicus.     

Lack of interaction between α2 and dopamine D2-receptors in mediating their inhibitory effects on [3H]dopamine release from rat nucleus accumbens slices

The ₂-adrenoceptor agonist, UK14304, dose-dependently inhibited the electrically stimulated release of dopamine (DA) from rat nucleus accumbens slices. This effect was antagonized by idazoxan, confirming that it was an ₂-adrenoceptor mediated effect. There was no evidence of endogenous activation of noradrenergic receptors suggesting that the ₂-adrenoceptor agonist was not acting presynaptically to inhibit noradrenaline release. An in vitro superfusion technique was used to investigate wheher there was any interaction between ₂-adrenoceptors and DA D₂-receptors in mediating their inhibitory effects on [³H]DA release from rat nucleus accumbens slices. ₂-Adrenoceptor and DA D₂-receptors interact with similar second messenger systems and it was considered that they may compete for a common pool of G-proteins. The inhibitory effects of the ₂-adrenoceptor agonist, UK14304, and the DA receptor agonists, quinpirole, apomorphine and pergolide were not independent. However, there was no evidence of any interaction between UK14304 and the DA D₂-receptor antagonists, sulpiride or haloperidol, suggesting that the two receptors do not compete for a common pool of G-proteins in mediating their inhibitory effects on DA release.     

Expression,purification, characterization,and deletion mutations of phosphorylase kinase γ subunit: identification of an inhibitory domain in the γ subunit

A catalytic fragment, _1-298, derived from limited chymotryptic digestion of phosphorylaseb kinase (Harris, W.R.et al., J. Biol. Chem., 265: 11740–11745, 1990), is reported to have about six-fold greater specific activity than does the subunit-calmodulin complex. To test whether there is an inhibitory domain located outside the catalytic core of the subunit, full-length wild-type and seven truncated forms of were expressed inE. coli. Recombinant proteins accumulate in the inclusion bodies and can be isolated, solubilized, renatured, and purified further by ammonium sulfate precipitation and Q-Sepharose column. Four out of seven truncated mutants show similar ( _1-353 and _1-341) or less ( _1-331 and _1-276) specific activity than does the full-length wild-type , _1-386. Three truncated forms, _1-316, _1-300, and _1-290 have molar specific activities approximately twice as great as those of the full-length wild-type and the nonactivated holoenzyme. All recombinant s exhibit similarK _m values for both substrates, i.e., about 18M for phosphorylaseb and about 75 M for MgATP. Three truncated s, _1-316, _1-300, and _1-290, have a 1.9- to 2.5-fold greater catalytic efficiency (V _max/K _m) than that of the full-length wild-type and a 3.5- to 4.5-fold greater efficiency than that of the truncated _1-331. This evidence suggests that there is at least one inhibitory domain in the C-terminal region of , which is located at _301-331· _1-290, but not _1-276, which contains the highly conserved kinase domain, is the minimum sequence required for the subunit to exhibit phosphotransferase activity. Both _1-290 and _1-300 have several properties similar to full-length wild-type , including metal ion responses (activation by free Mg²⁺ and inhibition by free Mn²⁺) pH dependency, and substrate specificities.     

Intracellular calcium activity in split frog skin epithelium: Effect of cAMP

Summary Measurement of intracellular calcium activity (a _Ca ^c ) by ion-selective microelectrodes has previously been technically limited to relatively large cells (20 m). We now report results obtained with this technique in the small epithelial cells (10 m) of split frog skin using microelectrodes having an outer tip diameter of <0.2 m. The basolateral membrane potential was measured with Ca²⁺-selective microelectrodes (E _Ca ^sc ) and with reference micropipettes ( ^sc ) either sequentially or simultaneously in 15 successful experiments. Under baseline conditions,a _Ca ^c was measured to be 215±39nm (mean±se), in close agreement with the mean values estimated from published data obtained withNecturus proximal tubule. Stimulation of Na⁺ transport across six skins with 1mm serosal 8p-chlorophenylthio-3,5 cyclic AMP (CPTcAMP) increaseda _Ca ^c by a factor of 2.6±0.6. The increase ina _Ca ^c preceded the CPTcAMP-induced increase inI _sc. The results of the present study indicate that electrometric determination of intracellular calcium activity is now feasible in a much wider range of cell systems than heretofore possible. CPT cAMP elevates intracellular Ca²⁺ activity; this phenomenon is an early event, preceding the natriferic effect of CPTcAMP.     

Modulation of osteoclastogenesis in porcine bone marrow cultures by quercetin and rutin

Flavonols, in contrast to soybean isoflavones, are the most abundant phytoestrogens in western diets, being present in onions, beans, fruits, red wine, and tea. They may protect against atherosclerosis, inhibit certain cancer cell types, and reduce bone resorption. The most widely distributed flavonol is quercetin, which occurs mainly as its glycoside, rutin, but data are very scarce regarding the precise mechanism of action of these compounds on bone-resorbing cells at concentrations similar to those detected in human plasma. We have therefore investigated the effects of nanomolar concentrations of quercetin and rutin on the development and activity of osteoclasts in vitro compared with the effects of 17-estradiol. Nonadherent porcine bone marrow cells were cultured on dentine slices in the presence of 10 nM 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), with or without 10 nM quercetin, 10 nM rutin or 10 nM 17-estradiol for 11 days. Multinuclear TRAP+ cells that resorbed dentine (osteoclasts) developed in the presence of 1,25(OH)₂D₃, but their number was significantly reduced by quercetin, rutin, and 17-estradiol (P < 0.05). Like 17-estradiol, both flavonols also significantly reduced resorption (P<0.05) as assessed by the size of pits resorbed on dentine slices. Osteoclasts and osteoclast progenitors contained estrogen receptor (ER), ER, and RANK proteins. Both flavonols increased nuclear ER protein and decreased ER protein of osteoclast progenitors. Moreover, rutin reduced RANK protein, whereas 17-oestradiol and quercetin promoted apoptosis by cleavage of caspase-8 and caspase-3. All the effects of flavonols were reversed by 1 M ICI 182,780, an estrogen antagonist. Thus, the anti-resorbing properties of flavonols are mainly mediated by ER proteins through the inhibition of RANK protein or the activation of caspases.C.M.R. was supported by a grant from CAPES (Department of Education, Brasilia, Brasil).     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Symmetric interspecies hybrids of mouse and human hemoglobin: Molecular basis of their abnormal oxygen affinity

Interspecies hybrids of HbA and Hb from mouse C57BL/10 [ ₂ ^M ₂ ^H and ₂ ^H ₂ ^M (H=human, M=mouse)], representing 19 and 27 sequence differences per dimers (as compared with human dimer) have been generatedin vitro. The efficiency of the assembly of the interspecies hybrids by the alloplex intermediate pathway is about twofold higher than the low-pH-mediated subunit approach. The interspecies hybrids exhibit a cooperative O₂ binding. The intrinsic O₂ affinity of mouse Hb is slightly lower than HbA, while the 2,3-diphosphoglycerate (DPG) effect is comparable. Interestingly, the interspecies hybrid ₂ ^M ₂ ^H has high O₂ affinity (compared to either human or mouse Hb), while the interspecies hybrid ₂ ^H ₂ ^M exhibits a very low O₂ affinity. These results suggest that the mouse chain generates a tetramer with very low oxygen affinity. However, the complementarity of the mouse and chains generates a set of unique interactions that compensate for the low-oxygen-affinity propensity of the mouse chain. DPG binds the tetramer in the central cavity formed by the two subunits, hence the DPG effects on the interspecies hybrids should be as in the parent molecule. However, the results of the present study demonstrate that the DPG binding pocket is influenced by the nature of the chain present in the tetramer. The mouse chain reduces considerably the DPG right shift of the O₂ affinity of the human-chain containing hybrid. Sequence analysis suggest that perturbations of the _{1 1} (not the _{1 2}) are communicated to the DPG binding pocket in the presence of the alien subunit, and are the primary determinant of the ligand binding properties. The results have implications for the design of Hb-based blood substitutes and understanding of the inhibitory potential of mouse chains in transgenic mouse expressing human ^S chains.     

In vivo effects of tunicamycin on the secretory processes of rat parotid glands

Summary The morphological and functional effects of tunicamycin were studied in rat parotid glands at the stage of the reformation of secretory granules following secretory stimulation by isoproterenol. Tunicamycin inhibited the incorporation of (³H)-mannose into the acid-insoluble fraction but had no effect on total protein synthesis as determined by the incorporation of (¹⁴C)-leucine. Thus the administration of tunicamycin in vivo inhibits the synthesis of mannose-rich glycoproteins in a manner similar to that in an in vitro system. The ultrastructure of the acinar cell showed little change following treatment with this drug, except that the number of reaccumulated secretory granules was greater than in the control. Amylase secretion stimulated by isoproterenol was inhibited in tunicamycin-treated cells, but did not decrease following treatment with N⁶,2-O-dibutyryladenosine 3-5-cyclic monophosphate, a secretory stimulator bypassing the -receptor. A radio-receptor assay using (³H)-dihydroalprenolol and direct localization using the fluorescent -adrenergic blocker 9-amino-acridin propranolol showed a marked reduction in the binding activity of -receptor following treatment with tunicamycin. Thus the inhibition of N-linked glycosylation appears to produce profound effects on the -adrenergic receptor-adenylate cyclase complex of acinar cells, although the steps of the transport and the exocytotic discharge of secretory materials are not affected.     

β-1-3- and β-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl₂, -1-3-linked glucans are mainly produced. By increasing MgCl₂ concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl₂, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl₂.Abbreviations G.S. I glucan synthetase I - G.S. II glucan synthetase II - Dol. P dolichol phosphate