Binding specificities of cellular retinol-binding protein and cellular retinol-binding protein, type II

Cellular retinol-binding protein (CRBP) and cellular retinol-binding protein, type ii (CRBP(II] are cytoplasmic proteins that bind trans-retinol as an endogenous ligand. These proteins are structurally similar having greater than 50% sequence homology. Employing fluorescence, absorbance, and competition studies, the ability of pure preparations of CRBP(II) and CRBP to bind various members of the vitamin A family has been examined. In addition to trans-retinol, CRBP(II) was able to form high affinity complexes (K'd less than 5 X 10(-8) M) with 13-cis-retinol, 3-dehydroretinol, and all-trans-retinaldehyde. CRBP bound those retinol isomers with similar affinities, but did not bind trans-retinaldehyde. Neither protein bound retinoic acid nor 9-cis- and 11-cis-retinol. The spectra of 13-cis-retinol and 3-dehydroretinol, when bound, were shifted and displayed fine structure compared to their spectra in organic solution. However, the lambda max and fluorescent yield of a particular ligand were different when bound to CRBP(II) versus CRBP. It appears that CRBP(II) and CRBP bind trans-retinol, 13-cis-retinol, and 3-dehydroretinol in a planar configuration. However, the binding sites of CRBP(II) and CRBP are clearly distinct based on the observed spectral differences of the bound ligands and the observations that only CRBP(II) could bind trans-retinaldehyde. The ability of CRBP(II) to bind trans-retinaldehyde suggests a physiological role for the protein in accepting retinaldehyde generated from the cleavage of beta-carotene in the absorptive cell.     

Phenotypic identification of memory cytolytic T lymphocytes in a subset of Lyt-2+ cells

Murine peripheral Lyt-2+ T cells could be subdivided according to surface expression of the Pgp-1 glycoprotein into major (71%) Pgp-1- and minor (29%) Pgp-1+ subsets. A striking correlation was observed between Pgp-1 expression and enrichment for antigen-specific memory cytolytic T lymphocyte precursors (CTLp). After immunization with the male minor transplantation antigen H-Y, virtually all the H-Y-specific CTLp were found in the minor Pgp-1+ subset of Lyt-2+ cells. In addition, after alloimmunization the frequency of allospecific CTLp resistant to inhibition by anti-Lyt-2 antibody was markedly enriched within the Pgp-1+ cells, suggesting an enrichment for CTLp bearing high avidity antigen receptors. Taken together, these data suggest that surface Pgp-1 expression is stably acquired at the time of primary antigenic stimulation by virgin T cells. As such, Pgp-1 represents an important marker for identifying a subset of Lyt-2+ T cells with the quantitative and qualitative properties of memory CTLp.     

Thermodynamics of glycophorin in phospholipid bilayer membranes

We have developed a model of glycophorin in a phospholipid bilayer membrane in order to study the thermodynamics of this system and to understand the detailed behavior of recent calorimetric data. We assume that the larger glycophorin polar group can be considered as either adopting a pancakelike conformation at the bilayer interface (D state) or be directed generally away from the interface (U state) [Ruppel, D., Kapitza, H.G., Galla, H.J., Sixl, F., & Sackmann, E. (1982) Biochim. Biophys. Acta 692, 1-17]. Lipid hydrocarbon chains are described either as excited (e state) with high energy and relatively many gauche conformers or as generally extended (g state) with low energy. We performed a Monte-Carlo simulation using the Glauber and Kawasaki procedures on a triangular lattice which represents the plane of half of the bilayer. Lattice sites can be occupied either by lipid hydrocarbon chains or by model glycophorin alpha-helical hydrophobic cores. The states D and U are represented by hexagons of different sizes in the plane of the lattice, and the hard core repulsion between two such polar groups is accounted for by forbidding hexagon-hexagon overlap. We have studied the effect of having the glycophorin polar group interact in various ways with the lipid bilayer. We find that the protein polar group in its D state interacts, either directly or indirectly, with the lipid bilayer so as to reduce the effective lateral pressure acting on the lipid hydrocarbon chains by about 3 dyn/cm. Polar groups in their U states do not reduce this lateral pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fusion of synaptic vesicle membranes with planar bilayer membranes.

The interaction of synaptic vesicles with horizontal bilayer lipid membranes (BLMs) was investigated as a model system for neurotransmitter release. High concentrations (200 mM) of the fluorescent dye, calcein, were trapped within synaptic vesicles by freezing and thawing. In the presence of divalent ions (usually 15 mM CaCl2), these frozen and thawed synaptic vesicles (FTSVs) adhere to squalene-based phosphatidylserine-phosphatidylethanolamine BLMs whereupon they spontaneously release their contents which is visible by fluorescence microscopy as bright flashes. The highest rate of release was obtained in KCl solutions. Release was virtually eliminated in isotonic glucose, but could be elicited by perfusion with KCl or by addition of urea. The fusion and lysis of adhering FTSVs appears to be the consequence of stress resulting from entry of permeable external solute (KCl, urea) and accompanying water. An analysis of flash diameters in experiments where Co+2, which quenches calcein fluorescence, was present on one or both sides of the BLM, indicates that more than half of the flashes represent fusion events, i.e., release of vesicle contents on the trans side of the BLM. A population of small, barely visible FTSVs bind to BLMs at calcium ion concentrations of 100 microM. Although fusion of these small FTSVs to BLMs could not be demonstrated, fusion with giant lipid vesicles was obvious and dramatic, albeit infrequent. Addition of FTSVs or synaptic vesicles to BLMs in the presence of 100 microM-15 mM Ca2+ produced large increases in BLM conductance. The results presented demonstrate that synaptic vesicles are capable of fusing with model lipid membranes in the presence of Ca+2 ion which, at the lower limit, may begin to approach physiological concentrations.     

Normal mouse strains differ in the site of initiation of closure of the cranial neural tube

The scanning electron microscopic study of day 9 embryos reported here documents differences among normal mouse strains in morphology of cranial neural tube closure. The site of initiation of contact and fusion of the cranial neural folds, previously defined as Closure 2 (Macdonald et al., '89), is located in the region of the junction between the forebrain (prosencephalon) and midbrain (mesencephalon) in three normal strains: LM/Bc, AEJ/RkBc, and ICR/Bc. However in a fourth normal strain, SWV/Bc, Closure 2 is initiated much further rostral, in the prosencephalic region. In addition, the anterior neuropore, rostral to Closure 2, closes late in ICR/Bc embryos, relative to the posterior progress of development of the Closure 2 seam. Initiation of closure from the most rostral end of the neural tube (Closure 3) appears to be relatively delayed in ICR/Bc embryos. We hypothesize that the observed genetic polymorphism in location of the first site of fusion between the cranial neural folds in normal mouse embryos may be one basis for differences among normal strains in liability to exencephaly induced by teratogens.     

Stimulation of insulin release from pancreatic islets by quinones

Coenzyme Q (CoQ₀) and other quinones were shown to be potent insulin secretagogues in the isolated pancreatic islet. The order of potency was CoQ₀benzoquinonehydroquinonemenadione. CoQ₆ and CoQ₁₀ (ubiquinone), duroquinone and durohydroquinone did not stimulate insulin release. CoQ₀'s insulinotropism was enhanced in calcium-free medium and CoQ₀ appeared to stimulate only the second phase of insulin release. CoQ₀ inhibited inositol mono-, bis- and trisphosphate formation. Inhibitors of mitochondrial respiration (rotenone, antimycin A, FCCP and cyanide) and the calcium channel blocker verapamil, did not inhibit CoQ₀-induced insulin release. Dicumarol, an inhibitor of quinone reductase, did not inhibit CoQ₀-induced insulin release, but it did inhibit glucose-induced insulin release suggesting that the enzyme and quinones play a role in glucose-induced insulin release. Quinones may stimulate insulin release by mimicking physiologically-occuring quinones, such as CoQ₁₀, by acting on the plasma membrane or in the cytosol. Exogenous quinones may bypass the quinone reductase reaction, as well as many reactions important for exocytosis.     

Randomised controlled trial of effects of coordinating care for terminally ill cancer patients.

OBJECTIVES--To measure effects on terminally ill cancer patients and their families of coordinating the services available within the NHS and from local authorities and the voluntary sector. DESIGN--Randomised controlled trial. SETTING--Inner London health district. PATIENTS--Cancer patients were routinely notified from 1987 to 1990. 554 patients expected to survive less than one year entered the trial and were randomly allocated to a coordination or a control group. INTERVENTION--All patients received routinely available services. Coordination group patients received the assistance of two nurse coordinators, whose role was to ensure that patients received appropriate and well coordinated services, tailored to their individual needs and circumstances. MAIN OUTCOME MEASURES--Patients and carers were interviewed at home on entry to the trial and at intervals until death. Interviews after bereavement were also conducted. Outcome measures included the presence and severity of physical symptoms, psychiatric morbidity, use of and satisfaction with services, and carers'' problems. Results from the baseline interview, the interview closest to death, and the interview after bereavement were analysed. RESULTS--Few differences between groups were significant. Coordination group patients were less likely to suffer from vomiting, were more likely to report effective treatment for it, and less likely to be concerned about having an itchy skin. Their carers were more likely to report that in the last week of life the patient had had a cough and had had effective treatment for constipation, and they were less likely to rate the patient''s difficulty swallowing as severe or to report effective treatment for anxiety. Coordination group patients were more likely to have seen a chiropodist and their carers were more likely to contact a specialist nurse in a night time emergency. These carers were less likely to feel angry about the death of the patient. CONCLUSIONS--This coordinating service made little difference to patient or family outcomes, perhaps because the service did not have a budget with which it could obtain services or because the professional skills of the nurse-coordinators may have conflicted with the requirements of the coordinating role.     

Microcirculatory pathways in normal human spleen, demonstrated by scanning electron microscopy of corrosion casts

Confusion regarding microcirculatory pathways in normal human spleen has arisen due to extrapolation from pathological material and from other mammalian spleens, not to mention difficulties in tracing intricate three-dimensional routes from the study of thin sections or cut surfaces of tissue. We examined microcirculatory pathways in normal human spleens freshly obtained from organ transplant donors. A modified corrosion casting procedure was used to obtain an open view of vessels and their connections. Our results demonstrate: 1) "arteriolar-capillary bundles" within lymphatic nodules and extensive branching of arterioles in the marginal zone (MZ); 2) the marginal sinus around lymphatic nodules; 3) the peri-marginal cavernous sinus (PMCS) outside the MZ or immediately adjacent to the nodule itself; the PMCS receives flow via ellipsoid sheaths and MZ, or directly from arterial capillaries, and drains into venous sinuses; 4) fast pathways for flow into venous sinuses via ellipsoid sheaths; 5) arterial capillary terminations in the reticular meshwork of the red pulp or MZ ("open" circulation); direct connections to venous sinuses also occur ("closed" circulation), although rarely; and 6) numerous open-ended venous sinuses in the MZ, allowing a large proportion of the splenic inflow to bypass the red cell filtration sites in the reticular meshwork and at venous sinus walls.     

A membrane-bound diacylglycerol kinase that selectively phosphorylates arachidonoyl-diacylglycerol. Distinction from cytosolic diacylglycerol kinase and comparison with the membrane-bound enzyme from Escherichia coli

The membrane-bound diacylglycerol kinase from Swiss 3T3 cells (M-DG kinase) was characterized with a mixed micellar assay system, and compared with the cytosolic diacylglycerol kinase from 3T3 cells and with the membrane-bound diacylglycerol kinase from Escherichia coli. M-DG kinase selectively phosphorylated arachidonoyl-diacylglycerols, at a rate 2- to 8-fold higher than that for other naturally occurring long-chain diacylglycerols. In contrast, the cytosolic 3T3 enzyme exhibited little or no selectivity among long-chain diacylglycerols but had higher activity with more soluble substrates such as 1,2-didecanoylglycerol. Comparison of the properties of M-DG kinase with those of the bacterial membrane-bound enzyme revealed that selectivity for arachidonoyl-diacylglycerol was unique to the mammalian enzyme. All three kinases were activated by phosphatidylserine, but activation did not alter the arachidonoyl selectivity of M-DG kinase. Phosphatidylserine activated M-DG kinase by increasing Vm and decreasing the apparent Km for diacylglycerol. High concentrations of diacylglycerol reduced the Ka for phosphatidylserine, but did not abolish the phosphatidylserine requirement for maximum activity. Examination of the thermal lability of M-DG kinase revealed that this enzyme was rapidly and selectively inactivated by preincubation with its preferred substrate. This novel effect may have obscured previous attempts to discern substrate selectivity. Taken together, the results provide evidence that M-DG kinase is an arachidonoyl-diacylglycerol kinase that may participate in the formation of arachidonoyl-enriched species of phosphatidylinositol.     

Regulation of malate dehydrogenase activity by glutamate, citrate, alpha-ketoglutarate, and multienzyme interaction

Binding experiments indicate that mitochondrial aspartate aminotransferase can associate with the alpha-ketoglutarate dehydrogenase complex and that mitochondrial malate dehydrogenase can associate with this binary complex to form a ternary complex. Formation of this ternary complex enables low levels of the alpha-ketoglutarate dehydrogenase complex, in the presence of the aminotransferase, to reverse inhibition of malate oxidation by glutamate. Thus, glutamate can react with the aminotransferase in this complex without glutamate inhibiting production of oxalacetate by the malate dehydrogenase in the complex. The conversion of glutamate to alpha-ketoglutarate could also be facilitated because in the trienzyme complex, oxalacetate might be directly transferred from malate dehydrogenase to the aminotransferase. In addition, association of malate dehydrogenase with these other two enzymes enhances malate dehydrogenase activity due to a marked decrease in the Km of malate. The potential ability of the aminotransferase to transfer directly alpha-ketoglutarate to the alpha-ketoglutarate dehydrogenase complex in this multienzyme system plus the ability of succinyl-CoA, a product of this transfer, to inhibit citrate synthase could play a role in preventing alpha-ketoglutarate and citrate from accumulating in high levels. This would maintain the catalytic activity of the multienzyme system because alpha-ketoglutarate and citrate allosterically inhibit malate dehydrogenase and dissociate this enzyme from the multienzyme system. In addition, citrate also competitively inhibits fumarase. Consequently, when the levels of alpha-ketoglutarate and citrate are high and the multienzyme system is not required to convert glutamate to alpha-ketoglutarate, it is inactive. However, control by citrate would be expected to be absent in rapidly dividing tumors which characteristically have low mitochondrial levels of citrate.