Refined crystal structure of an octanucleotide duplex with G . T mismatched base-pairs

Single crystal X-ray diffraction techniques have been used to determine the structure of the DNA octamer d(G-G-G-G-C-T-C-C) at a resolution of 2.25 A. The asymmetric unit consists of two strands coiled about each other to produce an A-type DNA helix. The double helix contains six G . C Watson-Crick base-pairs and two G . T mismatched base-pairs. The mismatches adopt a "wobble" type structure in which both bases retain their major tautomer forms. The double helix is able to accommodate this G . T pairing with little distortion of the overall helical conformation. Crystals of this octamer melt at a substantially lower temperature than do those of a related octamer also containing two G . T base-pairs. We attribute this destabilization to disruption of the hydration network around the mismatch site combined with changes in intermolecular packing. Full details are given of conformational parameters, base stacking, intermolecular contacts and hydration involving 52 solvent molecules.     

Progesterone pretreatment has a direct effect on GnRH-induced preovulatory follicles to determine their ability to develop into normal corpora lutea in anoestrous ewes

In two experiments carried out during seasonal anoestrus, Romney Marsh ewes were treated with small-dose (250 ng) multiple injections of GnRH at 2-h intervals with and without progesterone pretreatment. In Exp. 1, 8/8 progesterone-primed ewes ovulated and produced functionally normal corpora lutea compared with 2/9 non-primed ewes. Follicles were recovered from similarly treated animals 18 or 28 h after the start of GnRH treatment (at least 14 h before the estimated time of the LH peak) and assessed in terms of diameter, granulosa cell number, oestradiol, testosterone and progesterone concentrations in the follicular fluid, oestradiol production in vitro and binding of 125I-labelled hCG to granulosa and theca. There were no significant differences in any of these measures in 'ovulatory' follicles recovered from the progesterone-pretreated compared to non-pretreated animals. In Exp. 2, follicles were removed from similar treatment groups just before and 2 h after the start of the LH surge. Unlike 'ovulatory' follicles recovered from the non-pretreated ewes, those recovered from progesterone-pretreated ewes responded to the LH surge by significantly increasing oestradiol secretion (P less than 0.01) and binding of 125I-labelled hCG (P less than 0.05) to granulosa cells. Overall there was also more (P less than 0.05) hCG binding to granulosa and theca cells from progesterone-pretreated animals. Non-ovulatory follicles recovered from progesterone-primed ewes had more (P less than 0.05) binding of 125I-labelled hCG to theca and a higher testosterone concentration in follicular fluid (P less than 0.05) than did those from non-primed ewes. These results suggest that inadequate luteal function after repeated injections of GnRH may be due to a poor response to the LH surge indicative of a deficiency in the final maturational stages of the follicle.     

Diglycosyl diacylglycerol of Mycobacterium tuberculosis.

A diglycosyl diacylglycerol was isolated from Mycobacterium tuberculosis, and its structure was established by a combination of methylation analysis, 1H nuclear magnetic resonance, and fast atom bombardment-mass spectrometry. It is a 1,2-diacyl-[beta-D-glucopyranosyl(1"----6')-beta-D-glucopyranosyl(1'---- 3)]- sn-glycerol and exists in at least five molecular species differing in fatty acyl substituents. The major constituent fatty acids were identified as iso- and anteisopentadecanoate, iso- and n-hexadecanoate, and iso- and anteisoheptadecanoate. Although glycosyl diacylglycerols are common membrane components of gram-positive bacteria, this report represents the first substantial evidence for the presence of a glycosyl diacylglycerol within a member of the Mycobacterium genus. Although the glycolipid is not a major component of M. tuberculosis, it reacts readily in enzyme-linked immunosorbent assay against rabbit antibodies raised against whole bacteria and thus may be useful for the serodiagnosis of tuberculosis.     

Position dependence of ankle joint dynamics--II. Active mechanics

System identification techniques were used to examine the position dependence of active ankle joint mechanics. Subjects were required to maintain tonic contractions in either the tibialis anterior (TA) or triceps surae (TS) muscles while the ankle was stochastically displaced about different mean angular positions. The dynamic relation between ankle position and torque was determined for each mean position/tonic torque combination; a non-linear minimization technique was used to estimate the three parameters (inertial, viscous and elastic) of a second-order, underdamped system. Whereas the inertial parameter remained essentially invariant across all test conditions, the viscous and elastic (K) parameters became larger as the level of tonic activity increased and as the joint was rotated toward the extremes of the range of motion. The relation between K and torque was linear at all ankle angles. The slope of this relation remained constant at all mean positions during plantarflexor contractions; during dorsiflexor contractions the slope increased as the ankle was rotated from maximum plantarflexion to maximum dorsiflexion. These findings are discussed in terms of: the physiological correlates of ankle mean position, the relative significance of passive and active joint mechanics and contrasts in joint behaviour during active dorsiflexor and plantarflexor contractions.     

Islet activating protein inhibits physiological responses evoked by cardiac muscarinic acetylcholine receptors. Role of guanosine triphosphate binding proteins in regulation of potassium permeability

The involvement of GTP binding proteins in muscarinic acetylcholine receptor (mAChR) mediated responses of cultured chick embryonic cardiac muscle cells was studied by using islet activating protein (IAP) from Bordetella pertussis. Incubation of cells for 24 h with IAP resulted in inhibition of subsequent IAP-catalyzed incorporation of [alpha-32P]ADP-ribose into membrane proteins of Mr 39 000 (No alpha) and 41 000 (Ni alpha); treatment of cultures with 5 ng/mL IAP was sufficient to ADP-ribosylate all available No alpha and Ni alpha. Inhibition of forskolin-stimulated cAMP accumulation by the muscarinic agonist carbachol was abolished in cultures pretreated with IAP. The affinity of carbachol for the mAChR in membranes from IAP-treated cells was considerably decreased compared to control membranes and was not further decreased by addition of guanyl-5'-yl imidodiphosphate. In contrast, the affinity of carbachol for the mAChR on intact cells was not affected by pretreatment with IAP. To investigate the involvement of No and/or Ni in mAChR-mediated increases in K+ permeability, the effect of IAP treatment on mAChR stimulation of 86Rb+ efflux was determined. Treatment of cultures with 5 ng/mL IAP for 24 h completely blocked the stimulation of 86Rb+ efflux evoked by carbachol. Because previous work has shown that mAChR regulation of K+ permeability is independent of changes in cAMP levels, these results suggest a role for No and/or Ni in coupling the mAChR directly to K+ channels in the heart.     

Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.     

The thermodynamics of bovine and porcine insulin and proinsulin association determined by concentration difference spectroscopy

Difference spectroscopy was used to determine the equilibrium constants and thermodynamic parameters for the monomer-dimer association of bovine and porcine insulin and bovine proinsulin at pH 2.0 and 7.0. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -6.6 kcal/mol, -18 cal/mol-deg, and -12 kcal/mol, respectively. Porcine insulin behaved similarly to bovine insulin in its dimerization properties in that delta G degree 25, delta S degree, and delta H degree were found to be -6.8 kcal/mol, -14 cal/mol-deg, and -11 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -7.2 kcal/mol, -16 cal/mol/deg, and -12 kcal/mol, respectively. At pH 7.0 delta G degree 25, delta S degree, and delta H degree for dimerization of porcine insulin were -6.7 kcal/mol, -11.6 cal/mol-deg, and -10 kcal/mol, respectively. The similarity in the thermodynamic parameters of both insulin species at the different pH's suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range. The dimerization of both insulin species is under enthalpic control. This may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine proinsulin were found to be -5.3 kcal/mol, -26 cal/mol-deg, and -13 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of proinsulin were -5.9 kcal/mol, -4.2 cal/mol-deg, and -7.2 kcal/mol, respectively. Although the presence of the C-peptide on proinsulin does not drastically affect the overall free energy change of dimer formation (as compared to insulin), the other thermodynamic parameters are rather drastically altered. This may be because of electrostatic interactions of groups on the C-peptide with groups on the B-chain which are near the subunit contact site in the insulin dimer.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.     

Evidence for two distinct intracellular pools of inorganic sulfate in Penicillium notatum.

A strain of Penicillium notatum unable to metabolize inorganic sulfate can accumulate sulfate internally to an apparent equilibrium concentration 10(5) greater than that remaining in the medium. The apparent Keq is near constant at all initial external sulfate concentrations below that which would eventually exceed the internal capacity of the cells. Under equilibrium conditions of zero net flux, external 35SO42- exchanges with internal, unlabeled SO42- at a rate consistent with the kinetic constants with the sulfate transport system. Efflux experiments demonstrated that sulfate occupies two distinct intracellular pools. Pool 1 is characterized by the rapid release of 35SO42- when the suspension of preloaded cells is adjusted to 10 mM azide at pH 8.4 (t 1/2, 0.38 min). 35SO42- in pool 1 also rapidly exchanges with unlabeled medium sulfate. Pool 2 is characterized by the slow release of 35SO42- induced by azide at pH 8.4 or unlabeled sulfate (t 1/2, 32 to 49 min). Early in the 35SO42- accumulation process, up to 78% of the total transported substrate is found in pool 1. At equilibrium, pool 1 accounts for only about 2% of the total accumulated 35SO42-. The kinetics of 35SO42- accumulation is consistent with the following sequential process: medium----pool 1----pool 2. Monensin (33 microns) accelerates the transfer of 35SO42- from pool 1 to pool 2. Valinomycin (0.2 microM) and tetraphenylboron- (1 mM) retard the transfer of 35SO42- from pool 1 to pool 2. At the concentrations used, neither of the ionophores nor tetraphenylboron- affect total 35SO42- uptake. Pool 2 may reside in a vacuole or other intracellular organelle. A model for the transfer of sulfate from pool 1 to pool 2 is presented.