Transduction of the IFN-gamma signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.     

Modification of γ-Aminobutyric AcidA Receptor Binding and Function by N-Ethoxycarbonyl-2-Ethoxy-1,2-Dihydroquinoline In Vitro and In Vivo: Effects of Aging

The irreversible protein-modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) was used to investigate binding site characteristics on the gamma-aminobutyric acidA (GABAA) receptor complex. In vitro, preincubation with EEDQ led to a concentration-dependent decrease in receptor number for benzodiazepine, t-butylbicyclophosphorothionate (TBPS), and GABA binding sites in cerebral cortex. The effect was maximal at the highest concentration of EEDQ used (10(-4) M) and was greatest for the benzodiazepine site. Pretreatment of membranes with the benzodiazepine antagonist Ro 15-1788, 1 or 10 microM, or the agonist lorazepam, 10 microM, largely prevented the effects of EEDQ. Scatchard analysis indicated no effect of EEDQ, 10(-4) M, on apparent affinity, but a decrease in receptor density for each site. Administration of EEDQ to mice, 12.5 mg/kg i.p., led to a substantial (55-65%) decrease in number of benzodiazepine binding sites in cortex after 4 h. Slightly smaller changes were observed for TBPS and GABA binding. No changes were observed in apparent affinity at any site. Prior administration of Ro 15-1788, 5 mg/kg, prevented the effect of EEDQ on benzodiazepine binding. Density of benzodiazepine binding sites gradually recovered over time, and receptor density returned to control values by 96 h after EEDQ injection. Number of binding sites in cortex for TBPS and GABA also increased over time after EEDQ. Benzodiazepine sites in cerebellum were decreased proportionally to cortex after EEDQ, and increased over a similar time course. Function of the GABAA receptor in chloride uptake in cortex was markedly reduced (65%) by EEDQ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alterations in Cortical [3H]Kainate and α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding in a Spontaneous Canine Model of Chronic Hepatic Encephalopathy

Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L-[3H]Glutamate, (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK-801), [3H]kainate, and alpha-[3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L-[3H]-glutamate or [3H]MK-801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low-affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non-N-methyl-D-aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.     

Glutamine Synthetase in Oligodendrocytes and Astrocytes: New Biochemical and Immunocytochemical Evidence

The results of recent immunocytochemical experiments suggest that glutamine synthetase (GS) in the rat CNS may not be confined to astrocytes. In the present study, GS activity was assayed in oligodendrocytes isolated from bovine brain and in oligodendrocytes, astrocytes, and neurons isolated from rat forebrain, and the results were compared with new immunochemical data. Among the cells isolated from rat brain, astrocytes had the highest specific activities of GS, followed by oligodendrocytes. Oligodendrocytes isolated from white matter of bovine brain had GS specific activities almost fivefold higher than those in white matter homogenates. Immunocytochemical staining also showed the presence of GS in both oligodendrocytes and astrocytes in bovine forebrain, in three white-matter regions of rat brain, and in Vibratome sections as well as paraffin sections.     

Aboriginal preparation ofCycas seeds in Australia1

The seeds of cycad plants are a toxic food used by many Aboriginal groups in northern Australia. Acute symptoms produced after consumption of untreated Cycas seeds are due to azoxyglycosides, especially cycasin, although the toxic dose depends on the animal species tested. There are three traditional methods used to treat these seeds: brief leaching in water; prolonged leaching in water; and aging. Aboriginal people living at Donydji outstation in northeast Arnhem Land, most regularly consume aged seeds ofCycas angulata R.Br. Analyses of fresh seeds and seeds prepared at Donydji and in the laboratory indicate that cycasin is effectively removed by all the traditional preparation techniques, although each technique has an end product with different storage and handling properties. The social implications of processing need further elaboration, but these techniques have a long history and archaeological remains of seeds in Australia may date back to the Pleistocene.     

Synthesis of 2-chloro-2′-deoxyadenosine by washed cells ofE. coli

Summary Washed cells ofE. coli ATCC 5275, a thymine auxotroph, catalysed formation of 2-chloro-2-deoxyadenosine when incubated with 2-chloroadenosine and a variety of deoxynucleosides. This transdeoxyribosylation reaction was complete after 4 h of shaking at 37°C. The equilibrium reaction mixture favoured product formation when purine rather than pyrimidine deoxyribonucleosides were used as cosubstrates, and when the ratio of deoxysugar donor to 2-chloroadenosine was high. Using deoxyadenosine as cosubstrate, chlorodeoxyadenosine was purified from larger scale reaction mixtures by treatment with Dowex-1 (OH-form) or by high performance liquid chromatography.     

Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate

We have used the technique of phosphate: water oxygen exchange to measure the rate of ATP and Pi release and Pi binding to myosin subfragment 1 and actomyosin subfragment 1 from rabbit skeletal muscle. The oxygen exchange distributions for ATP and Pi release fit a simple kinetic model with a single set of rate constants for each step. For actomyosin subfragment 1 (20 degrees C, pH 7.0, I = 50 mM), the rate constant governing ATP release is approximately 8 s-1, Pi release is at approximately 60 s-1 and Pi rebinds to an ADP state at greater than 120 M-1 s-1. These rate constants are similar to those that may occur for undistorted cross-bridges within glycerinated rabbit psoas fibers (Bowater, R., Webb, M. R., and Ferenczi, M. A. (1989) J. Biol. Chem. 264, 7193-7201.     

A labile,Ca2+-dependent cytoskeleton in rhabdomeral microvilli of blowflies

Summary Rhabdomeral microvilli of photoreceptors of the blowfly Lucilia are shown to contain a cytoskeleton. An axial filament ( 6–11 nm) in each microvillus is inserted into a terminal cap distally, and into a plug filling the narrow neck of the microvillus proximally. In some states, the axial filament projects beyond the neck; within the microvillus it is surrounded by amorphous material. Together, they form an axial complex, which supports side-arms linking it to the plasma membrane. Conventional fixation for examination with the electron microscope destroys the cytoskeleton. To preserve it, retinae are pre-treated with a Ringer's solution buffered with 20 mM imidazole and containing, minimally, the following components: (i) a protease inhibitor, usually phenylmethylsulphonyl-fluoride (PMSF); (ii) either the Ca²⁺-chelator EGTA, or the calmodulin-blocking agent trifluoperazine (TFP); and (iii) a source of divalent cations to preserve the side-arms. When EGTA is used, Mg²⁺, Sr²⁺, Ba²⁺, Mn²⁺ and Co²⁺ are effective, Ba²⁺ giving the most satisfactory contrast, and Mg²⁺ and Co²⁺ the best preservation. It is inferred that the cytoskeletal complex includes at least one Ca²⁺-activated protease, and possibly calmodulin. Microvilli are bonded together by intermicrovillar bridges with a periodicity of 11–17nm. The cytoskeleton is destroyed by pretreatment with 1 mM dithiothreitol (DTT), possibly by the activation of a thiol protease. It does not survive osmication unless treated with low molecular weight tannic acid (LMWT). The evidence does not discriminate between actin and intermediate filaments as the basis of the cytoskeleton. Attention is drawn to similarities and differences between the rhabdomeral cytoskeleton and that of vertebrate intestinal brush-borders. The extreme lability of the rhabdomeral cytoskeleton to conventional methods of fixation is attributed in part to the Ca²⁺ fluxes experienced by invertebrate photoreceptors, and in part to the effects of osmication.The authors thank Dr. Lindsay Barton-Brown and Tom Van Gerwen for supplying flies from CSIRO cultures: Smith Kline and French Laboratories Ltd., French's Forest, N.S.W. for a generous gift of trifluoperazine, and Mallinckrodt, Inc., St. Louis, Missouri, USA, for a gift of low molecular weight tannic acid. Many colleagues, especially Richard Payne, Steve Shaw and Gert De Couet helped by discussing the results. George Weston and the staff of the Electron Microscope Unit provided support and advice. Sandy Smith prepared Table 1     

Translocation(X;Y)(p22.33;p11.2) in XX males: Etiology of male phenotype

Summary Analysis of G-banded prometaphase chromosomes from three XX males revealed extra bands on the distal end of one X short arm. These bands were similar both in size and staining properties to the distal Y short arm of their fathers (in the two cases examined) and also to other chromosomally normal males. The extra material on the abnormal X chromosomes was not C-or G-11 positive in the two cases examined, suggesting that the proximal Y long arm was not present.Previous karyotype-phenotype correlations with structurally altered Y chromosomes provided evidence for localization of male determinants on the Y short arm. The present findings in XX males provide support for more precise localization, to bands p11.2pter of Y short arm.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.