Ice retreat in Wilkes Basin of East Antarctica during a warm interglacial.

Efforts to improve sea level forecasting on a warming planet have focused on determining the temperature, sea level and extent of polar ice sheets during Earth's past interglacial warm periods1-3. About 400,000 years ago, during the interglacial period known as Marine Isotopic Stage 11 (MIS11), the global temperature was 1 to 2 degrees Celsius greater2 and sea level was 6 to 13 metres higher1,3. Sea level estimates in excess of about 10 metres, however, have been discounted because these require a contribution from the East Antarctic Ice Sheet3, which has been argued to have remained stable for millions of years before and includes MIS114,5. Here we show how the evolution of 234U enrichment within the subglacial waters of East Antarctica recorded the ice sheet's response to MIS11 warming. Within the Wilkes Basin, subglacial chemical precipitates of opal and calcite record accumulation of 234U (the product of rock-water contact within an isolated subglacial reservoir) up to 20 times higher than that found in marine waters. The timescales of 234U enrichment place the inception of this reservoir at MIS11. Informed by the 234U cycling observed in the Laurentide Ice Sheet, where 234U accumulated during periods of ice stability6 and was flushed to global oceans in response to deglaciation7, we interpret our East Antarctic dataset to represent ice loss within the Wilkes Basin at MIS11. The 234U accumulation within the Wilkes Basin is also observed in the McMurdo Dry Valleys brines8-10, indicating11 that the brine originated beneath the adjacent East Antarctic Ice Sheet. The marine origin of brine salts10 and bacteria12 implies that MIS11 ice loss was coupled with marine flooding. Collectively, these data indicate that during one of the warmest Pleistocene interglacials, the ice sheet margin at the Wilkes Basin retreated to near the precipitate location, about 700 kilometres inland from the current position of the ice margin, which-assuming current ice volumes-would have contributed about 3 to 4 metres13 to global sea levels.

Novel Asymmetric Synthesis of Atropisomeric 6-Aryl Pyrazinones via an Unusual Chirality Transfer Process.

Cyclization of (S,S)-alpha-[(1-phenylethyl)amino]-alpha-(2-iodophenyl)acetonitrile with (COCl)(2) in toluene or chlorobenzene afforded the atropisomeric pyrazinone (aS,S) 6-(2-alpha-iodophenyl)-3,5-dichloro-1-(1-phenylethyl)-2(1H)-pyrazinone in 57% yield. With smaller ortho substituents (F, Cl, CH(3), CF(3), OCH(3)) on the aromatic ring, mixtures of atropisomers were obtained from the cyclization reaction. All of the individual atropisomers prepared were stable at room temperature. All but the o-fluoro-substituted atropisomers were stable at elevated temperatures. This paper describes a stereoselective synthesis of pyrazinones and suggests a mechanism for formation via an interesting transfer of chirality.

Effect of diltiazem and a prostaglandin derivative (PGBx) on platelet function during long-term storage.

Calcium is an intermediate messenger between platelet stimuli and platelet response. Published studies have shown that the decreased ability of platelets to control calcium flux during long-term storage leads to platelet senility. Platelet metabolism might be more efficient during storage if pharmacologic agents that limit calcium movement were incorporated into the platelet concentrate storage solution. This hypothesis was tested by storing platelets with the calcium channel blocker, diltiazem, or with a prostaglandin B1 derivative, PGBx. During a 15-day storage period, platelets incubated with either diltiazem or PGBx showed improved function, as measured by aggregation, as compared to control platelets. The PGBx -enhanced platelet function during storage was accompanied by a significant decrease in glucose and an increase in adenosine triphospate concentrations. Platelet function after storage with PGBx improved in spite of significantly lower pH levels of the platelet concentrates at all time points tested. These studies suggest that the maintenance of calcium ion homeostasis during long-term platelet storage is important to in vitro platelet function even if the Ca2+ balance is maintained at the expense of pH and the glucose concentrations.

Calculation of NH...pi hydrogen bond energies in basic pancreatic trypsin inhibitor.

Ab initio molecular orbital procedures have been employed to investigate associations of the Tyr35 aromatic ring with Gly37 NH and Asn44 NHz in the X-ray crystal structure of basic pancreatic trypsin inhibitor. Formamide and phenol were used to model the key features in the protein. The calculations provide evidence for energetically favorable NH...pi hydrogen bonding.

Structure-activity relationships for drugs binding to the agonist and antagonist states of the primary morphine receptor.

Thirty-three opiate drugs have been considered in an investigation of the geometric and electronic features required for association with the agonist and antagonist states of the principal morphine receptor. Conformational analysis was carried out by means of molecular mechanics, and electronic properties were calculated with an ab initio SCF-MO procedure using FSGO basis sets. Statistical analysis of receptor binding based on a free-energy model reveals several properties of the molecules under study that affect the stability of the drug-receptor complex. The results suggest that the same drug conformation is involved in binding at both the agonist and antagonist states of the receptor. A single set of drug features serves to rationalize association with both receptor states, but differences in binding-site topography are revealed by the relative importance of the various structural features in the regression equations for the two states.

Effect of sodium ion on the affinity of naloxone for the kappa opioid receptor.

Several investigators have observed that sodium ion enhances the binding of naloxone to opioid receptors. This effect has generally been attributed to allosteric modulation of the state of the mu receptor. However, a recent claim has been made that the enhancement does not involve a change in the mu receptor, but instead occurs because naloxone becomes a more kappa-specific drug when sodium ion is present in high concentration. Since the claim was not based on experimental evidence from binding studies involving known high-affinity kappa ligands, we have investigated the competition of naloxone for the kappa site using [3H]U-69593 as the marker for receptor binding. Assays were carried out in the presence and absence of 100 mM NaCl. The results of the study indicate that sodium ion does not increase the affinity of naloxone or U-69593 for the kappa receptor.

Factors affecting binding of trans-N-[2-(methylamino)cyclohexyl]benzamides at the primary morphine receptor.

In this paper, we describe the synthesis of a series of trans-N-[2-(methylamino)cyclohexyl]benzamides possessing morphine-like pharmacological properties. The affinity of the compounds for the agonist and antagonist states of the mu opioid receptor has been established by means of an in vitro binding assay. We have investigated the geometry and electronic structure of the molecules using molecular mechanics and an ab initio SCF-MO procedure with FSGO basis sets. Comparison to naloxone reveals properties of possible importance in receptor association. We have considered both the S,S and R,R isomers in the binding model. Statistical analyses imply that three factors play a significant role in binding: (1) membrane-water partitioning, (2) the capacity of the aromatic ring and amine N-substituent to act as electron acceptors, (3) the conformational energy required to attain the binding configuration.

Treatment of prehospital refractory ventricular fibrillation with bretylium tosylate.

Among 218 patients treated for prehospital arrest during an eight-month baseline period prior to addition of bretylium tosylate to the paramedic protocol in Columbus, 16 (7.3%) were seen with refractory ventricular fibrillation (RVF). These patients failed to respond to multiple countershocks, lidocaine, bicarbonate and epinephrine, and either were transported in arrest during cardiopulmonary resuscitation (CPR)(14) or were pronounced dead at the scene (2). A single patient was eventually resuscitated in and discharged from the hospital. During the subsequent 16 1/2-month experience with bretylium used only for prehospital RVF, 421 patients with prehospital arrest were seen, 35 of whom (8.3%) had RVF. All but five patients were defibrillated successfully, and 14 (40%) were converted to a rhythm sufficient to obviate CPR during transportation. Eleven patients (31%) survived to be admitted to the hospital, and eight of 35 (23% vs 1/16 or 6.2% above, P less than .05) were discharged and remained well three to 17 months later. Bretylium tosylate may provide life-saving therapy for refractory prehospital ventricular fibrillation so that survival from an almost uniformly fatal condition is improved. While patients with persistent arrest generally should be transported to the hospital, such patients should not be subjected to the difficulties of CPR in transit unless they are first given bretylium if RVF is present.

Characteristics of lympho-myelopoietic stem cells isolated from canine peripheral blood.

If hematopoietic stem cells (HSC) could be separated from peripheral blood, it might be possible to harvest these stem cells for potential clinical use. By leukapheresis techniques, we harvested mononuclear cells (MNC) from peripheral blood and then placed these cells over discontinuous stractan gradients of three densities (1.077 gm/ml, 1.071 gm/ml and 1.066 gm/ml). These separated cells were submitted to colony culture to identify colony-forming-unit activity for granulocyte-macrophage (CFU-C) and T-cell lymphocyte (CFU-L) cell lines. The lightest cells (1.066) contained most of the CFU-C and no CFU-L activity. Heavier cells (greater than 1.071) contained CFU-L and very little CFU-C activity. CFU-L colonies could be distinguished from CFU-C by their density and distinct morphological appearance. In addition, the amount of CFU-C could be increased in the animal by increasing the amount of blood processed (from 3.9 +/- .76 CFU-C/10(6) MNC to 6.7 +/- .35 CFU-C/10(6) MNC). This resulted in an increase of CFU-C collected from 7.6 +/- 2.1 CFU-C/10(6) MNC after the first equivalent blood volume to 22.5 +/- 3.4 CFU-C/10(6) MNC after the third equivalent blood volume processed. These results suggest that leukapheresis and gradient density separation may be useful procedures to obtain HSC.

Structure-activity correlations for a series of antiallergy agents. 2. Geometric and electronic characterization of some oxamic and dioxamic acids.

Hartree-Fock self-consistent field calculations using the ab initio molecular fragment technique have been performed on some phenyloxamic and m-phenylenedioxamic acids, which exhibit markedly different activities in the rat passive cutaneous anaphylaxis (PCA) assay. Attention is focused upon structural features that are most likely to affect the drug-receptor interactions, such as the preferred molecular geometry, the electronic charge distribution, and the nature of the higher occupied (HOMO) and lower unoccupied (LUMO) molecular orbitals. Judging from the regions of high density in HOMOs and LUMOs, the benzene ring would preferably act as an electron acceptor, while the oxamic acid moiety would serve best as an electron donor. Factors affecting the relative PCA activities of oxamic and dioxamic acids are discussed.

Structure-activity correlations for a series of antiallergy agents. 3. Development of a quantitative model.

A nonlinear regression model has been employed to investigate the activity of a series of 51 drugs in the rat passive cutaneous anaphylaxis assay. Although several classes of molecules are represented in the sample, there are certain common structural features that enable detailed geometric and electronic comparisons to be made. Ab initio Hartree-Fock SCF calculations were performed by using the molecular fragment approach to characterize the electronic structure and preferred conformations of the molecules. The statistical results establish the importance of nine structural factors in determining the potency as inhibitors of histamine release. Both the conformation of a drug and its capacity to act effectively as an electron acceptor in charge-transfer interactions are shown to be critical for high activity.

Glycerol-glucose cryopreservation of platelets. In vivo and in vitro observations.

Extended storage of platelets can be achieved by cryopreservation. However, most cryopreservation techniques require extensive manipulation prior to administration, limiting their practicality. A simple cryopreservative system using glycerol and glucose as cryoprotectants would eliminate the need to wash the platelets after freezing, since neither of these agents is toxic. We evaluated such a system in vivo and compared the results to 72-hour liquid-stored platelets. The percentage of in vivo recovery was significantly less (p less than 0.01) for cryopreserved (21.1 +/- 3.4% [chi +/- 1 SD]) than liquid-stored (43.8 +/- 7.4%) platelets, but those frozen-thawed cells that were viable had normal survivals (8.4 +/- 1.7 days). Liquid-stored cell appeared to be less viable (5.9 +/- 1.8 days). These results indicate that cryopreservation with the glycerol-glucose system produces significant injury to the majority of platelets and therefore, is inadequate for general blood bank use.

Granulocyte progenitor cell (CFUC) harvest by continuous apheresis in dogs. Effects of blood volume and lithium on yields.

The ability to harvest large amounts of hematopoietic stem cells from blood would eliminate the more difficult approach of bone marrow harvest. Unfortunately, concentration of stem cells in the blood compartment is less than 1% of their concentration in bone marrow. Attempts to increase harvest of blood stem cells, as assayed by granulocyte progenitor cells (CFUC), have been only partially successful. Our study confirms previous reports that CFUC can be mobilized into the blood compartment in dogs, but this mobilization is rate-limited. Unlike platelets and granulocytes that are effectively harvested during the first blood volume processed by continuous apheresis, effective CFUC harvest begins during the second blood volume (606 +/- 97.9 CFUC/ml), peaks by the third (740 +/- 30 CFUC/ml), and remains constant through five blood volumes processed (700 +/- 272 CFUC/ml). Since blood CFUC concentration falls at the end of five blood volumes processed (40% of initial values), further continuous apheresis would not be effective. Treatment of animals with lithium did not improve CFUC harvest. These results show that apheresis procedures can be developed to a limited extent to increase the harvest of hematopoietic progenitor and stem cells.

An in vivo comparison of CPD and CPDA-2 preserved platelet concentrates after an 8-hour preprocess hold of whole blood.

To see if citrate-phosphate-dextrose-adenine-two (CPDA-2) anticoagulant-preservative had an effect on the viability of platelets, we studied autologous in vivo recovery and survival in humans for platelet concentrates prepared from six units of blood drawn into CPDA-2 and compared them to six units drawn into citrate-phosphate-dextrose (CPD). These units were prepared from whole blood held at room temperature for 8 hours after collection and were then stored for 3 days at 22 +/- 2 degrees C. The recovery for platelets preserved in CPD was 39.0 +/0 4.8 percent and for platelets preserved in CPDA-2, 32.5 +/- 4.4 percent. The difference was not significant (p greater than 0.10). In order to estimate population differences, in vitro effects on in vivo viability were also evaluated. Six in vitro variables were studied but only pH at 72 hours (r = 0.77), platelet count (r = 0.64), and morphology score (r = 0.66) correlated to recovery. Only pH at 72 hours significantly influenced recovery (p = 0.007). By adjusting for individual pH differences, mean recovery for platelets stored in CPD was 37.5 percent, and for platelets stored in CPDA-2, 34.0 percent. The mean lifespan was 6.7 +/- 0.7 days for platelets preserved in CPD and 6.1 +/- 1.0 days for those preserved in CPDA-2. Although hemostatic function was not studied, these data support in vitro observations that platelets preserved with CPDA-2 are not different from platelets preserved with CPD, even after 8-hours of storage of whole blood at room temperature prior to platelet concentrate preparation.

An analysis of binding at the opioid receptor based upon an agonist/antagonist two-state model.

Experimental binding data for a number of opioid agonists and antagonists in homogenate of rat brain membranes at 0 degrees have been analyzed in terms of the two-state Snyder-Pert receptor model. By means of iterative nonlinear regression techniques, it was possible to evaluate the affinity constants characterizing the binding of each drug at the agonist and antagonist states of the receptor. In addition, the role of Na+ in determining the state of the receptor has been described quantitatively. The findings of this study are discussed in terms of the index, fr, which provides a theoretical measure of the fraction of receptors existing in the agonists state. The graph of fr as a function of drug concentration may be reasonably interpreted as an ideal dose-response curve which might be obtained if the drug were introduced directly at the receptor. The implications of the findings with respect to receptor operation are discussed. Although the binding constants have not been found under physiological conditions, the results indicate that the receptor mechanism remains essentially intact in the brain membrane preparation.

Drug affinities for the agonist and antagonist states of the opioid receptor.

Traditional methods of analyzing the binding of opiates do not accurately establish the affinities of the drugs for the agonist and antagonist states of the opioid receptor. The poor results obtained by the Scatchard procedure may be attributed to the presence of extraneous drug binding sites in the brain-homogenate medium. In this study a nonlinear regression technique has been employed to analyze receptor binding in terms of the two-state Snyder-Pert model while simultaneously considering drug association with the aggregate of non-receptor binding sites. Results are reported for naloxone, pentazocine, and dihydromorphine, which respectively represent a pure antagonist, a mixed agonist-antagonist, and a pure agonist. the affinity constants characterizing equilibrium binding at the agonist and antagonist states of the receptor at 37 degrees C are given for each of the three drugs. In addition, a theoretical index, f tau, is defined which provides a measure of the fraction of receptors existing in the agonist state. It is found that the graft of f tau vs log [D] exhibits features similar to the in vivo dose-response curves for the drugs.

Glycoprotein changes in fresh vs. room temperature-stored platelets and their buoyant density cohorts.

Membranes from platelets obtained from normal human volunteers were isolated for evaluation of their glycoproteins. Values were measured in fresh and stored platelet concentrates at 72 and 96 hr (22 degrees +/- 2 degrees C). Polyacrylamide gels were used to separate the membrane glycoproteins. These were identified as GPI, GPII, GPIII, GPIV, GP77, and GP44. The relative amount of GPI (155,000 daltons) was about 30% less after 72 hr storage than in fresh platelets regardless of the change in the pH of the platelet concentrate. At 96 hr, only an additional 5% loss was seen. The 72 hr value for soluble glycoprotein, glycocalicin, was only 64% of the initial value. GP77 (77,000 daltons) and GP44 (44,000 daltons) became apparent or more prominent with storage. A correlation with pH of the platelet concentrate could be demonstrated for GPI but only with those units in which the pH rose during storage. Density-separated populations of fresh and stored platelets also were studied. They were separated on arabinogalactan (Stractan II) gradients for comparative studies of membrane proteins that might be affected, since platelets become less dense with storage. There was an equal loss of GPI in all populations. However, the change was most striking in the least dense (lightest) fraction because these cells started with 20% less GPI when fresh than did heavier cells. The smallest glycoprotein, GP44, was always present in the lightest platelets after storage, whereas only 40% of the concentrates showed GP77, in small amounts, associated with heavy platelets. Alterations in the membranes of platelets stored as platelet concentrates could result in their functional impairment and loss viability.

Buoyant density of platelets stored at room temperature as platelet concentrates.

Separation of platelets by buoyant density centrifugation was periodically performed on platelet concentrates stored up to 96 hr at room temperature. By 72 hr, platelets were much lighter, depending on pH, platelet concentration, and volume of the bag. The mean proportion of platelets in the light fraction (fraction 1) shifted from 4.3% when the concentrate was fresh to 52.2% at 72 hr and 53.6% at 96 hr. The majority of platelets had densities that ranged from 1.034 to 1.088 gm/ml after storage, whereas densities ranged from 1.054 to 1.088 gm/ml in fresh cells. With storage, the light cells became larger than when they were fresh and were mostly balloon-shaped; the heavy cells became smaller but retained their normal shape. Regression analysis showed that density distribution was highly correlated to pH. Most of the changes occurred after 12 hr; those changes that occurred during the initial 12 hr were not related to pH of the platelet concentrate. The changes were related to storage conditions and may reflect injury to the cells. The use of buoyant density separation may be a useful tool to study storage mechanisms and provide a means of separating cells modified by storage stress.

In vitro evaluation of platelets stored in CDP-adenine formulations.

Little information is available about the effect of adenine and added glucose on stored platelets. Two new formulations, CPDA-2 and CPDA-3, contain 34 mg adenine per 63 ml preservative and extra glucose (1.75 and 2.0 times the glucose in standard CPD). We have studied the in vitro integrity of platelet concentrates stored in CPD, CPDA-1, CPDA-2, and CPDA-3 at 22 C for 72 hours. Morphology score, pH, platelet size, population distribution parameters, and electron microscopic ultrastructure did not show any adverse effects which could be ascribed to the presence of adenine or extra glucose or both. No differences in platelet adenosine triphosphate (ATP) concentration or plasma glucose utilization during storage were found between CPD and CPDA-1 platelets. The results suggest that adenine and added glucose in these preservatives are not detrimental to platelets in vitro by the measures employed.

Structure-activity correlations for a series of antiallergy agents. Oxanilic, quinaldic, and benzopyran-2-carboxylic acids.

Ab initio Hartree-Fock SCF calculations with the molecular fragment technique have been performed on several drugs which exhibit activity in the rat passive cutaneous anaphylaxis (PCA) assay. Representative molecules of the following types were included in the series: oxanilic acids, 1,4-dihydro-4-oxoquinaldic acids, and 4-oxo-4H-1-benzopyran-2-carboxylic acids. A quantitative relationship has been established between the observed biological activity and an electronic index obtained from the calculations. The correlation is rationalized in terms of charge-transfer stabilization of the drug-receptor complex.