Untersuchungen Mit J131-Markiertemβ-Globulin Zur Frage Der Abstammung Der Liquoreiweisskörper

Zusammenfassung Die Abstammung der-Globuline im Liquor wurde bei 20 Fällen mit den verschiedensten neurologischen Erkrankungen untersucht. — Die spezifische Aktivität der-Globuline war bei normalen und pathologischen Liquors ausnahmslos niedriger als im Serum. Es treten demnach nur einzelne Serum--Globuline in den Liquor über, ein verschieden großer Anteil der Liquor--Globuline wird im Liquorraum gebildet. Die-Fraktion im Liquor besitzt einen Serumanteil, von dem die liquoreigenen oder auch cerebrogenen-Globuline unterschieden werden können. Beziehungen zwischen der Höhe des liquoreigenen-Globulinanteils zu einzelnen Krankheitsgruppen waren nicht herzustellen. Es ließ sich aber zeigen, daß eine Erhöhung des elektrophoretisch ermittelten relativen-Globulingehaltes im Liquor bei pathologischen Fällen nicht — wie bisher angenommen wurde — mit einer Zunahme des cerebrogenen Eiweißes einherzugehen braucht. — Die Bedeutung des liquoreigenen-Globulins ist noch unbekannt, auch ist es nicht möglich zu entscheiden, welche einzelnen Proteine innerhalb der-Fraktion im Liquorraum entstanden sind oder aus dem Serum stammen.Mit Unterstützung der Deutschen Forschungsgemeinschaft.     

Calcium binding to an elastic portion of connectin/titin filaments

-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the -connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on -connectin. Purified -connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 10⁷ M^–1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 M. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of -connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of -connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl₂ and 70 M leupeptin to split connectin filaments into -connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while - and -connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 m at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N₂ line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction–relaxation cycle of skeletal muscle.     

Some properties of the UL-FS 49 block of the hyperpolarization-activated current (i f) in sino-atrial node myocytes

Block of the hyperpolarization-activated pacemaker current (i _f) by the bradycardic agent UL-FS 49 was studied in isolated sino-atrial (SA) node myocytes. Using repetitive activation/deactivation protocols, micromolar concentrations of UL-FS 49 blockedi _f in a dose-dependent fashion. Block development was slow, with time constants decreasing with drug concentration and ranging from 25.8 s at 10 M to 75.5 s at 1 M UL-FS 49. Block did not develop in cells held at –35 mV, at which voltagei _f channels are closed, indicating that channels must open before blocking occurs. Apparently in contrast with the requirement of negative voltages for block development, block was relieved by hyperpolarization with a time course slower than current kinetics. Due to the hyperpolarization-induced block relief, current/voltage (I/V) relations in the presence of UL-FS 49 displayed inward-going rectification. Experimental data fitted the hypothesis that UL-FS 49 behaves as an open channel blocker of single-ioni _f channels. Block occurs within the pore, at a distance of about 39% of the membrane thickness from its internal side.     

Role of elevated monocyte transforming growth factorβ (TGFβ) production in posttrauma immunosuppression

We previously reported that increased production of prostaglandin E₂ by monocytes is a pivotal mechanism in posttrauma immunopathology. Here we characterize monocyte levels of transforming growth factor and examine the effects of elevated transforming growth factor on prostaglandin E₂ release by patients' monocytes. Trauma patients' and normals' monocyte supernates (± stimulation with muramyl dipeptide) were acid treated and assayed for transforming growth factor using the mink lung-cell bioassay. Alternatively, human transforming growth factor was added to patients' and normals' monocytes and prostaglandin E₂ production assayed. Significantly elevated transforming growth factor levels (median=181.7 pmol/10⁶ monocytes) were detected in immunosuppressed patients' monocytes but not immuno-competent trauma patients' (median=32.0 pM) or normals' (median=20.4 pM) monocytes. Adding transforming growth factor to monocytes resulted in a significant elevation of prostaglandin E₂ levels. Elevated monocyte transforming growth factor levels in trauma patients could be both suppressing T-lymphocyte functions and maintaining elevated monocyte prostaglandin E₂ synthesis.     

Evidence for a nucleotide-dependent topoisomerase activity from yeast mitochondria

Yeast mitochondria were found to contain a novel topoisomerase-like activity which required nucleoside di- or tri-phosphates as a cofactor. ADP supported activity as effectively as ATP and the optimal concentration for each was approximately 20 M. None of the other standard ribo- or deoxyrib-onucleotides could fully substitute for either ADP or ATP. The non-hydrolyzable ATP analogs, adenosine-5-0-(3-thiotriphosphate) (ATP--S), adenylyl (, -methylene) (AMP-PCP), and andenyl-imidodiphosphate (AMP-PNP) also supported activity suggesting that the nucleotide cofactor regulated topoisomerase activity rather than serving as an energy donor in the reaction. The mitochondrial topoisomerase activity relaxed both positively and negatively supercoiled DNA. It was not inhibited by concentrations of ethidium bromide up to 2 g/ml nor by either nalidixic or oxolinic acids; novobiocin, coumermycin, and berenil inhibited the activity. Genetic and biochemical analysis of the mitochondrial topoisomerase activity indicated that it was not encoded by the nuclear TOP1, TOP2, and TOP3 genes.     

TRP channels: a TR(I)P through a world of multifunctional cation channels

The transient receptor potential (TRP) family of ion channels comprises more than 50 cation-permeable channels expressed from yeast to man. On the basis of structural homology, the TRP family can be subdivided in to seven main subfamilies: the TRPC (Canonical) group, the TRPV (Vanilloid) group, the TRPM (Melastatin) group, the TRPP (Polycystin), the TRPML (Mucolipin), the TRPA (Ankyrin) and the TRPN (NOMP) family. The cloning and characterization of members of this cation channel family has exploded during recent years, leading to a plethora of data concerning TRPs in a variety of cell types, tissues and species. This paper briefly reviews the TRP superfamily and the basic properties of its many members as a readers guide in this Special Issue. Hopefully, a better understanding of TRP channel physiology will provide important insight into the relationship between TRP channel dysfunction and human diseases.     

Differential effects of interleukin-1α and β on the arachidonic acid cascade in human synovial cells and chondrocytes in culture

The effects of interleukin-1 and were tested on the [³H]-arachidonic acid release and the prostaglandin synthesis by human cultured synovial cells and chondrocytes. Both forms of interleukin-1 stimulated the arachidonic acid release but interleukin-1 was more potent than IL-1. Human synovial cells and chondrocytes synthesized three types of prostaglandins upon stimulation with interleukin-1 or : prostaglandin E₂, F₂ and 6-keto-prostaglandin F₁. Regarding the synthesis of these prostaglandins, IL-1 was again more potent than IL-1. A comparison between interleukin-1-stimulated synovial cells and chondrocytes revealed neither significant quantitative nor qualitative differences in both the arachidonic acid release and the prostaglandin synthesis.     

Cardiac Purkinje fibres

Summary The influence of intracellular calcium concentration [Ca²⁺] _i on the steady state membrane currentsi was studied in a range of clamp potentials between –20 and –100 mV. Injection of CaCl₂ or Ca-EGTA (pCa 6) increasedi whereas injection of K-EGTA diminished it. The changes i were attributed to a change in steady state potassium conductance, g_K, by four arguments: i was restricted to potentials negative to –20 mV and depended on clamp potential in an inward rectifying manner. i displayed a reversal potential, E_rev, which followed log [K⁺]₀ with 60 mV for a tenfold change. Since E_rev obtained during Ca injection agreed with E_rev observed during EGTA injection the potassium driving force had to be constant. Wheng _K was blocked by superfusion with 20 mM Cesium neither CaCl₂ nor K-EGTA injection modifiedi .Supported by SFB 38, Membranforschung, project G2     

Measurement of β-glucuronidase in effluent of perifused alveolar macrophages challenged with chemically modified chrysotile asbestos

Chrysotile asbestos has been implicated with lung disorders, notably fibrotic lesions and cancer. In vitro, chrysotile fibers are cytotoxic to lung macrophages and stimulate the release of inflammatory mediators. Reports to the effect that chemical modifications of asbestos fibers reduce their cytotoxic and inflammatory potential initiated the present study of three fiber modifications. The cytotoxic and inflammatory effects of magnesium-leached chrysotile, POCL₃-treated chrysotile, and CaO-treated chrysotile were studied in a perifused rat alveolar macrophage culture system, relative to untreated fibers. Natural Canadian chrysotile (UICC B) caused dose-dependent cell mortality and clumping. The release of-glucuronidase (-Glu), a lysosomal enzyme, was also dose dependent. Rhodesian chrysotile (UICC A) caused similar cytotoxic and inflammatory effects. However, magnesium-leached chrysotile was less cytotoxic (39% less) and had a lesser clumping capacity (31% less) than untreated chrysotile. Total secretion of-Glu elicited by magnesium-leached chrysotile was reduced by 43% from the untreated sample, but kinetic monitoring indicates that this reduction in inflammatory potential is only significant during the first 12 h of an 18-h culture period. POCl₃ treatment of chrysotile fibers produced differing effects depending on the length of the fibers under study. Treating fibers with a mean length of 8 m produced a secretion pattern similar to that produced by acid leaching. The total output for the treated sample was 44% lower than with untreated chrysotile; the difference was only significant during the first 12 h of perifusion. Cell mortality and aggregation were not reduced in any important way with POCl₃ treatment of these longer fibers. When ultra-short fibers (mean length=0.8 m) were treated with POCl₃, the total decrease in-Glu output was equal to 41%, and the release of enzyme was significantly lower during the whole 18-h experiment. Cell aggregation was not appreciably affected, but cell mortality was significantly lower than for untreated fibers. CaO treatment did not alter the cytotoxic (cell death and aggregation) or inflammatory (-Glu release) effects of chrysotile asbestos. These results suggest that chemical modifications affecting the integrity of the surface magnesium and/or the polarity of the surface charge of chrysotile can reduce to some extent the cytotoxic and inflammatory properties of this type of asbestos.     

Modulation of hematopoietic colony formation of stem cells in peripheral blood by anti-TGF-β in patients with severe immunosuppression

Summary The influence of transforming growth factor- (TGF-) on hematopoiesis has been evaluated by adding blocking antibodies against TGF- to colony forming assays (CFU-c). When optimum concentrations of recombinant growth factors, granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) were added to stem cells from the peripheral blood of healthy individuals and certain patients with tumors or HIV infection, the anti-TGF- capable of blocking 5 ng/ml of active TGF- had no significant influence on erythroid or myeloid colony formation. However, in certain immunosuppressed individuals, anti-TGF- resulted in a significant decrease of erythroid colony formation and slight suppression of myeloid colony formation. The significant inhibition of hematopoiesis by plasma of HIV patients could be due to the presence of active forms of TGF-. The results of the blocking experiments are consistent with the concept that TGF- in low concentrations is essential for erythropoiesis and myelopoiesis but that higher levels of TGF- primarily inhibit erythropoiesis in vitro. TGF- serves as a coordinating factor when efficient recruitment of granulocytes and monocytes is more essential than erythropoiesis and stem cell growth.Abbreviations BFU-E burst forming unit-erythroid - CFC colony forming cells - CFU-GEMM colony forming unit-granulocyte/erythroid/macrophage/megacaryocyte - CFU-GM colony forming unit-granulocyte/macrophage - EPO erythropoietin - GM-CSF granulocyte/macrophage-colony stimulating factor - HIV human immunodeficiency virus - IL-1 interleukin-1 - IL-3 interleukin-3 - IMDM Iscove's Modified Dulbecco's medium - PBS phosphate buffered saline - TGF- transforming growth factor- - TNF- tumor necrosis factor-     

Isolation of the Bα and Bβ mating-type loci of Schizophyllum commune

The B mating type of the basidiomycete fungus, Schizophyllum commune is determined by two, tightly linked, multi-specificity (also called multi-allelic) loci: B and B. A plasmid library was used in DNA-mediated transformation to obtain transformants that displayed B-directed development. Plasmids that conferred B1 and B1 mating-type specificities were rescued from the transformants. Fragments of DNA from each plasmid hybridized to genomic DNA from the strain used to make the plasmid library; however, they did not hybridize, or hybridized only weakly, to genomic DNA from strains with mating-type specificities different from B1 or B1. The cloned fragments are presumed to correspond to active regions of each B mating-type locus.     

A cyclin protein modulates mitosis in the budding yeast Saccharomyces cerevisiae

Summary For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step start. The threshold amount of cell mass (reflected as a critical size) necessary for start is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at start, termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for start, causing the smaller cells, produced in the previous cell cycle, to be delayed at start while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and start, nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product.     

Down-regulation of tumor necrosis factor α activity by acute ethanol treatment in human peripheral blood monocytes

As the most commonly used drug that can modulate both metabolic and immune pathways, ethanol is evaluated in this report as a regulator of tumor necrosis factor (TNF) production in human peripheral blood monocytes (M) in combination with a variety of stimuli. While acute ethanol treatment did not induce TNF in M, it was a potent down-regulator of M TNF production whether induced by the combination of interferon- plus muramyl dipeptide (MDP) (P<0.001), lipopolysaccharide (LPS) alone (P<0.01), or interferon- plus LPS. Down-regulation of M TNF by ethanol was dose dependent and statistically significant in the biologically relevant, 25–150 mM, ethanol concentration range. We also demonstrate that these ethanol concentrations did not affect M viability. TNF down-regulation by ethanol was most effective when ethanol was administered 4 hr prior to MDP stimulation; however, it was also effective—though to a lesser extent—if it was added at the time of MDP stimulation. Furthermore, ethanol also down-regulated TNF production of thein vivo preactivated M of trauma patients, which produce hyperelevated levels of TNF. We have previously shown that the majority of posttrauma elevated M TNF is produced by the M subpopulation expressing high-affinity type I Fc receptors (FcRI). When the FcRI cross-linking-stimulated M subpopulation was treated with acute ethanol, TNF production was suppressed again both inin vivo preactivated M of trauma patients and in M of normal controls. In experiments utilizing cyclooxygenase inhibitor, we also demonstrate that ethanol has a direct, prostaglandin E₂-independent, effect on M TNF production. These results demonstrate that acute ethanol exposure has the potential to down-regulate M production of TNF significantly regardless of the TNF-inducing stimulus. Decreased capacity of M to produce TNF might, therefore, contribute to the immunological and metabolic abnormalities described after ethanol uptake.     

Isoproterenol and GTPγS inhibit L-type calcium channels of differentiating rat skeletal muscle cells

Summary In adult skeletal muscle, G-proteins have been shown to modulate the calcium channels both directly and through a cAMP-dependent phosphorylating mechanism. We have investigated the action of G-proteins on the L-type calcium current in cultured rat muscle cells (myoballs) under voltage clamp in whole cell or perforated patch modes. Intracellular photolytic release of 200 M GTPS inhibited the L-type calcium current. Inclusion of 500 M uncaged GTPS in the patch pipette in the whole cell configuration reduced the calcium current by a similar amount. Under perforated patch conditions external application of 10 M of the -adrenergic agonist isoproterenol also reduced the calcium current. Pretreatment of the cells with pertussis toxin reversed the effect of GTPS and removed that of isoproterenol. We conclude that rat myoballs contain -adrenergic receptors that inhibit the L-type calcium current, and that this inhibition is mediated by a pertussis toxinsensitive G-protein.     

Immunoglobulin and T cell receptor gene rearrangements and in situ immunophenotyping in lymphoproliferative disorders

Summary We investigated for rearrangements of the immunoglobulin (Ig) heavy and light chain genes and of the T cell receptor (TCRT) and (TCr) genes 45 biopsy samples from a variety of lymphoproliferative disorders. They were diagnosed histopathologically and immunophenotypically as non-Hodgkin's lymphomas (NHLs) of the B cell type (19 cases), NHLs of the T cell type (3 cases), NHLs of undetermined cell type (3 cases), atypical lymphoid proliferation (1 case) and AIDS-related lymphadenopathies with florid polyclonal follicular hyperplasia (19 cases). A monoclonal proliferation of B cells was shown by DNA analysis in all 19 B cell NHLs. In two immunohistologically determined T cell NHLs (both diagnosed as mycosis fungoides) the cells had rearrangements of TCr gene, whereas in the third case (lymphoblastic NHL) the cells had rearrangements of Ig heavy chain and TCr and TCr genes. None of the B cell NHLs exhibited TCrand TCr gene rearrangement bands. All the undetermined cell NHLs demonstrated rearrangements of Ig heavy chain gene associated with the germ line TCrand TCr genes; in two cases light chain gene rearrangements were also found. The atypical lymphoid proliferation, in which the differential diagnosis was between a reactive or malignant process, and two out of 19 cases of florid polyclonal follicular hyperplasia showed a clonal B cell population by DNA analysis. This study indicates that there was a strong correlation between the rearrangements of specific genes and the immunophenotype of the NHL; moreover, DNA analysis of tissue biopsy specimens from phenotypically undetermined cell NHLs and from equivocal lymphoid proliferation using Ig and TCR gene probes yelded an answer in the cases analyzed. The significance of clonal B cell expansions found in two AIDS-related lymphadenopathies should be interpreted with caution.This work was supported in part by a Grant No 86.00644.44 from the Consiglio Nazionale delle Ricerche, Progetto Finalizzato Oncologia, Rome, and by the Associazione Italiana per la Ricerca sul Cancro, Milan, Italy     

Zur Früherkennung der diabetischen Nephropathie: Beta2-Mikroglobulin im Serum

Summary Detection of early diabetic nephropathy is necessary to postpone or even prevent progression of irreversible kidney damage by therapeutic measures. Beta₂-microglobulin (₂-MG) as a parameter of the glomerular filtration rate has been measured by immunoassay in the serum of 100 diabetic subjects, 50 insulin-dependent (IDD), and 50 noninsulin-dependent (NIDD) patients. The results are compared with endogenous creatinine-clearance, serum creatinine concentration, and proteinuria and are related to different stages of diabetic retinopathy (RD). Normal values were obtained from 50 healthy age- and sex-matched subjects.A close correlation was found between ₂-MG levels and endogenous creatinine clearance. Thirty-nine diabetics revealed an elevated ₂-MG (2.5 mg/l or higher), only 16 of whom had increased serum creatinine levels (1.4 mg/dl or higher). Significant differences of ₂-MG were obtained between each group of patients with different stages of RD. A relevant difference of serum creatinine was found only between patients with normal eye fundus and advanced proliferative retinopathy, respectively. Without RD 26% of the patients revealed elevated ₂-MG but normal creatinine values demonstrating a latent nephropathy, just 8% showed an increase of both parameters. Of the diabetics with proliferative retinopathy 40% suffered from impaired kidney function proven by reduced creatinine clearance and by elevation of ₂-MG and creatinine as well, 15% just revealed an increase of ₂-MG with normal creatinine levels. The incidence and extent of nephropathy demonstrated by pathologic values of both ₂-MG and serum creatinine were significantly higher in IDD patients with a smaller proportion of latent nephropathy as compared to NIDD patients (p<0.02). This is also true for the markedly increased proteinuria in IDD subjects. In both groups, measurement of ₂-MG disclosed more often decrease of renal function in diabetics than did concentrations of serum creatinine.The determination of serum ₂-MG appears to be a reliable and sensitive method for the early detection of minor impairment of kidney function in diabetes mellitus.

Unterstützt durch die Deutsche Forschungsgemeinschaft (Go 299/2)     

Intratypic antigenic heterogeneity of Coxsackie B viruses

Summary Neutralization tests, employing the cytopathogenic effect in tissue culture tubes, with a variety of homotypic antisera and strains of Coxsackie B viruses often yielded high titers in early readings and low titers in late readings — the break-through phenomenon — and occasionally also low, early-reading titers with heterologous, homotypic sera, which gave high titers with the homologous strains. Of 27 strains of Goxsackie B 1 to B 5, that were tested, 10 showed no break-through tendency while others showed varying degrees of break-through, without reference to any evidence of intratypic antigenic variation. There was a positive correlation between a small number of tissue culture passages away from man or mouse brain and the break-through tendency. Moreover, strains without break-through tendency yielded viral populations with marked break-through properties after a single intracerebral passage in newborn mice, and even after two subsequent tissue culture passages. Plaque-purified progeny exhibited the break-through phenomenon to the same extent as the original, unpurified cultures.The early readings yielded reproducible titers, which could be used for analysis of antigenic variation. Prime antigenic variants, of broader antigenic constitution than their non-prime relatives, were found among the Coxsackie B 2, B 3, and B 4 strains that were tested. These prime strains (e. g., B. V. A. 96- B2; Stevens - B 3, and Burrier or J. V. B. - B 4) were found to be antigenically broader than the prototype strains (Ohio 1 - B 2, Nancy - B 3, Powers or Texas 13 - B 4) generally used for the preparation of diagnostic antisera. The broader antigenicity of the prime variant was also present in plaque-purified progeny.Aided by grants from The National Foundation, Inc.The work reported here was carried out in 1957–1958 during Dr.Wigand's tenure of a fellowship in the Cincinnati Laboratory.     

Electron microscopic study of the formation of African horse-sickness virus

Summary BHK 21/13 cells infected with African horse-sickness virus (strain 13/63, type 3) for varying periods of time have been studied with the electron microscope. Evidence is presented to show that the virus does not multiply in the nucleus, but that replication occurs in the cytoplasmic matrix. It is also indicated that the particle often leaves the host cell with an envelope derived from the cell membrane. The diameter of the particle is roughly estimated to be 70 m., excluding the envelope.     

The effects of β-2-microglobulin on the infectivity of murine cytomegalovirus

Summary The role of -2-microglobulin (2m) in murine cytomegalovirus (MCMV) infection of susceptible (H-2^d) and resistant (H-2^k) murine embryo fibroblasts (MEF) and peritoneal macrophages was evaluated using serum-free virus (SF-MCMV). The infectivity of SF-MCMV was significantly lower than virus propagated in serum, although the concentrations of virions were similar. Infection of cells with SF-MCMV was assessed by measuring the proportion of cells expressing viral antigens, the sizes of plaques formed in fibroblast monolayers and TCID₅₀ titers. Infection of susceptible fibroblasts was significantly increased 1.6–4.7 fold by the addition of whole FCS, a<20 kDa FCS fraction, or purified human 2m. These supplements also significantly enhanced infection of susceptible macrophages and increased TCID₅₀ titers by 3.5–10 fold in susceptible MEF. In relatively resistant H-2^k cells, the TCID₅₀ titer and the proportion of cells expressing viral antigens after infection with SF-MCMV were not affected by 2m or FCS, but plaque sizes were increased 2.5–3 fold in resistant BALB.K MEF.When human or murine 2m was added to infected cultures, immunogold electron microscopy revealed these proteins to be always associated extracellularly with the tegument material of disrupted multicapsid virions, but rarely with the envelope of intact virions. However, no murine 2m was found in association with the envelope or tegument of SF-MCMV. These relatively modest effects of 2m which were restricted to genetically susceptible cells, may be due to tegument-bound 2m facilitating infection by capsids, or the stabilisation of the conformation of Class 1 molecules by exogenous 2m, promoting MCMV binding to the target cell.     

Arachidonic acid inhibits hormone-stimulated cAMP accumulation in the medullary thick ascending limb of the rat kidney by a mechanism sensitive to pertussis toxin

The possible regulation of adenosine 3,5-cyclic monophosphate (cAMP) accumulation by arachidonic acid (AA) was studied in segments, microdissected from the rat kidney, which are sensitive to arginine vasopressin (AVP). In the presence of 5 M indomethacin, the addition of 5 M AA did not impair AVP-dependent cAMP accumulation (measured during 4 min at 35° C) in the cortical or outer medullary collecting tubule, but decreased this response in the thick ascending limb with an inhibition much more pronounced in the medullary portion (MTAL) than in the cortical portion. In MTAL, the response to 10 nM AVP was inhibited by 34.4±9.6% (SEM) and 65.8±5.4% with 1 M and 5 M AA, respectively, N=5 experiments. AVP-, glucagon- and calcitonin-sensitive cAMP levels in MTAL were inhibited by 5 M AA to a similar extent. AA-induced inhibition was unaffected by the presence of inhibitors of AA metabolism: (1) either 10 M indomethacin or 50 M ibuprofen added to all media; (2) a 10-min pre-incubation and a 4-min incubation of MTAL samples with 10 M eicosa-5,8,11,14-tetrayonic acid, (3) a 1-h preincubation with either 30 M SKF-525A, 20 M ketoconazole, or 20 M nordihydroguariaretic acid. In contrast to AA, 11 other saturated or unsaturated fatty acids had no inhibitory effect on the AVP-dependent cAMP level. In fura-2-loaded MTAL samples, AA induced a slow increase of the intracellular calcium concentration ([Ca²⁺]_i) which reached 21.0±3.8 nM and 92.9 ±21.4 nM over basal values (n=11) at 2 min and 4 min, respectively, after the beginning of the superfusion of 5 M AA. AA-induced inhibition of AVP-dependent cAMP accumulation was due neither to the increase in [Ca²⁺]_i elicited by AA, nor to an activation of protein kinase C because this inhibition: (1) was not blocked when MTAL samples were incubated either in zero Ca²⁺ medium, or in the presence of 1,2-bis(2-aminophenoxy)ethane-N, N, N N-tetraacetic acid (BAPTA) to chelate [Ca²⁺ _i, and (2) it was not reproduced by a pre-treatment of MTAL segments with a phorbol ester. Pre-incubation of MTAL (6 h at 35° C) with 500 ng/ml pertussis toxin (PTX) prevented AA-induced inhibition: in the presence of PTX inhibition was 24.7±6.6% vs 10 nM AVP, as compared to 81.6 ± 4.0% in control groups, i.e in the absence of PTX, N=6. AA had no effect on the cAMP level induced by 5M forskolin. It is concluded that AA inhibits AVP-dependent cAMP accumulation in the rat MTAL by a mechanism which implicates a GTP-dependent protein sensitive to PTX.