Interferon γ but not tumor necrosis factor α decreases susceptibility of human renal cell cancer cell lines to lymphokine-activated killer cells

Summary Human renal cell cancer (RCC) cell lines, ACHN and KRC/Y, with or without exposure to cytokines, were examined for their susceptibility to lymphokine-activated killer (LAK) cells. Flow-cytometric analysis demonstrated constitutional expression of class I antigen on both cell lines, which was enhanced by interferon (IFN), IFN and tumor necrosis factor (TNF). A 4-h⁵¹Cr-release cytotoxicity assay demonstrated that pretreatment of both cell lines with IFN or IFN, but not with TNF, decreased their susceptibility to LAK cells. IFN also decreased susceptibility to natural killer cells in a 16-h⁵¹Cr-release cytotoxicity assay. IFN treatment decreased the susceptibility of ACHN cells in a dose-dependent manner. Cold-target competition assay clearly showed that IFN- but not TNF-pretreated cells compete less effectively than do untreated target cells. Pretreatment with IFN, however, increased expression of intercellular adhesion molecule-1 (ICAM-1) to a degree comparable to that with TNF. Northern blot analyses using a 520-base-pair ICAM-1 cDNA as a probe demonstrated that more 3.3-kb mRNA is expressed in IFN- and TNF-pretreated cells. These results suggest that IFN-treated RCC cell lines may reduce their ability to be recognized by LAK cells, and that IFN-induced protection of RCC cell lines against LAK cells may depend upon a mechanism independent of the expression of class I antigens or ICAM-1 on tumor cells.     

α-Thalassemia and the production of different α chain variants in heterozygotes

The production of five chain variants (Hb G-Georgia, Hb St. Luke's, Hb Lloyd, Hb Montgomery, and Hb G-Philadelphia) in heterozygotes was evaluated through hematological observations, hemoglobin quantification, and biosynthetic studies. All heterozygotes for Hb St. Luke's and Hb Lloyd and most heterozygotes with Hb G-Georgia and Hb Montgomery had normal hematology and average / values of about 1.1. They were assigned a normal genotype (^G/), although the proportions of Hb St. Luke's and Hb G-Georgia were low (10 to 13%) and those of Hb Lloyd and Hb Montgomery twice as high (20%). Data from short-term incubations confirmed this genotype for some of these heterozygotes. Isolated Hb St. Luke's and Hb G-Georgia gave low ^G/ values (0.2 and 0.3) indicating that these Hb variants were defective at the level of Hb assembly. Isolated Hb Montgomery and Hb G-Philadelphia, however, gave higher ^G/ values of 0.6 and 0.8, respectively. A second type of variability existed among Hb G-Georgia (20 vs. 13%), Hb Montgomery (28 vs. 20%), and Hb G-Philadelphia (47 vs. 34%) heterozygotes, in whom the levels of Hb G differed. The occurrence of higher levels of these three chain heterozygosities was associated with hematological or biosynthetic evidence of a mild or moderate chain deficiency due to an -thalassemia-2 heterozygosity (^G/⁰ or ^0G/) or a homozygosity (^0G/⁰), respectively.This study was supported in part by USPHS Research Grants HLB-05168 and HLB-15158.     

Linkage of the β-Like ω-Globin Gene to α-Like Globin Genes in an Australian Marsupial Supports the Chromosome Duplication Model for Separation of Globin Gene Clusters

The structure, function, and evolutionary history of globin genes have been the subject of extensive investigation over a period of more than 40 years, yet new globin genes with highly specialized functions are still being discovered and much remains uncertain about their evolutionary history. Here we investigate the molecular evolution of the -globin gene family in a marsupial species, the tammar wallaby, Macropus eugenii. We report the complete DNA sequences of two -like globin genes and show by phylogenetic analyses that one of these genes is orthologous to embryonically expressed -globin genes of marsupials and eutherians and the other is orthologous to adult expressed -globin genes of marsupials and eutherians. We show that the tammar wallaby contains a third functional -like globin gene, -globin, which forms part of the -globin gene cluster. The position of -globin on the 3 side of the -globin cluster and its ancient phylogenetic history fit the criteria, originally proposed by Jeffreys et al. (1980), of a fossil -globin gene and suggest that an ancient chromosome or genome duplication preceded the evolution of unlinked clusters of - and -globin genes in mammals and avians. In eutherian mammals, such as humans and mice, -globin has been silenced or translocated away from the -globin locus, while in marsupials -globin is coordinately expressed with the adult -globin gene just prior to birth to produce a functional hemoglobin (_{2 2}).     

α-Globin gene deletion causes α-thalassemia syndromes in two German families

Summary Restriction endonuclease mapping of chromosomal DNA has been used to determine whether the -globin gene deletion or non-deletion form of -thalassemia is the underlying molecular defect in individuals of two unrelated German families with -thalassemia syndromes. The obtained DNA pattern in all cases indicated loss of -globin genes resulting in-/,--/, and--/- genotypes in thalassemia-2, -thalassemia-1, and Hb H individuals respectively. The chromosomes showing loss of one -globin gene in -thalassemia-2 and Hb H disease were characterized by the so-called rightward deletion form exhibiting loss of a 3.7 kb DNA fragment in the -gene cluster.     

Carbohydrates of influenza virus. Structure of the oligosaccharides linked to asparagines 406 and 478 in the hemagglutinin of fowl plague virus,strain dutch

Fowl plague virus, strain Dutch, was metabolically labeled withd-[2-³H]mannose, or withd-[6-³H]glucosamine, and the small subunit (HA₂; 0.8 mg in total) of the viral hemagglutinin was isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis. After proteolytic digestion, the radioactive oligosaccharides were sequentially liberated from the glycopeptides by treatment with different endo--N-acetylglucosaminidases and with peptide:N-glycosidase or, finally, by hydrazinolysis. In this manner, four groups of glycans could be obtained by consecutive gel filtrations and were subfractionated by HPLC. The structures of the individual oligosaccharides were analyzed by micromethylation, by acetolysis or by digestion with exoglycosidases. The major species amongst the high mannose glycans at Ans-406 of the viral glycopolypeptide were found to be Man1-2Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNac1-4GlcNAc and Man1-3(Man1-2Man1-6)Man1-6(Man1-2Man1-2Man1-3)Man1-4GlcNAc1-4GlcNAc, while the complex glycans at Asn-478 are predominantly GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc (lacking, in part, one of the outerN-acetylglucosamine residues) and GlcNAc1-2Man1-3(Gal1-4GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc.Abbreviation BSA bovine serum albumin - endo D (F,H) endo--N-acetyl-d-glucosaminidase D (F,H) - HA hemagglutinin (HA₁, large subunit of HA - HA₂ small subunit - FPV fowl plague virus - PNGase F peptide:N-glycosidase F - SDS sodium dodecylsulfate     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

The effects of interleukin-1 therapy on peripheral blood granulocyte function in humans

During a phase I trial of interleukin-1 (IL-1) in patients with ovarian carcinomas, the effects of this treatment on blood granulocyte respiratory burst and locomotive responses were examined. Differences in baseline granulocyte function in patients as well as dose-related effects of IL-1 treatment were observed. Patients enrolled early in the trial (low-dose patients) had significantly lower locomotive responses before treatment than their paired controls; these low responses normalized after 5 days of continuous-infusion IL-1 treatment. Patients enrolled later (high-dose patients) had normal locomotive responses before treatment and IL-1 treatment was associated with suppression of responses to selected stimuli at the end of treatment. Pretreatment respiratory burst responses in both low-and high-dose patient groups were essentially normal, but the rates of granulocyte H₂O₂ production following phorbol myristate acetate stimulation became significantly less than control values at the end of treatment. In vitro exposure of either patient or control cells to 150 U/ml IL-1 did not alter their locomotive or respiratory burst responses, suggesting the observed in vivo effects were not mediated directly by IL-1. Treatment with IL-1 is associated with changes in ex vivo granulocyte function that are related to the IL-1 dose. Treatment with low doses of IL-1 may provide a means of normalizing abnormal polymorphonuclear leukocyte function in some patients with ovarian malignancies.     

Fibroblast growth factor increases TNFα-mediated prostaglandin E2 production and TNFα receptor expression in human fibroblasts

To examine the possible role of basic fibroblast growth factor (FGF) in regulating the effects of TNF, we tested the effect of FGF on TNF-mediated PGE₂ production and TNF receptor expression in human fibroblasts. We found that, while FGF alone had no effect on PGE₂ production, it enhanced the amount of PGE₂ produced in response to TNF between 3 and 11-fold. FGF stimulated TNF-induced PGE₂ production independent of potential TNF-mediated IL-1 production, as neither anti-IL-1 mAbs nor IL-1 receptor antagonist protein (IRAP) inhibited TNF induced-PGE₂ production or the stimulatory effect of FGF. A one minute exposure of cells to FGF prior to removal was sufficient to significantly enhance TNF-induced PGE₂ production; the maximal FGF effect was reached after a 6 h preincubation. We also found that FGF significantly enhanced TNF receptor expression. Untreated fibroblasts expressed 3,900 receptors/cell, while cells treated with FGF for 6h expressed 9,500 receptors/cell, a 2.4-fold increase in receptor number; there was no apparent change in affinity for TNF (K_d 3.8×10^–11 M). The FGF-mediated increase in TNF receptor expression and TNF-mediated PGE₂ production could be abolished by FGF mAbs, indicating a specific FGF effect. These results show that FGF increases TNF receptor expression and suggest that this may account, at least in part, for the ability of FGF to enhance TNF-mediated PGE₂ production in human fibroblasts.     

Comparative Analysis of the Mechanisms Underlying Nifedipine-Induced Blockade of Three Subtypes of T-Type Ca2+ Channels

We analyzed the effects of nifedipine on a family of recombinant low-threshold Ca²⁺ channels functionally expressed in Xenopus oocytes and formed by three different subunits (1G, 1H, and 1I). The 1G and 1I channels demonstrated a low sensitivity to nifedipine even in high concentrations (IC₅₀ = 98 and 243 M, maximum blocking intensity A_max = 25 and 47%, respectively). At the same time, the above agent effectively blocked channels formed by the 1H-subunit (IC₅₀ = 5 M and A_max = 41%). The nifedipine-caused effects were voltage-dependent, and their changes depended on the initial state of the channel. In the case of 1G-subunits, the blockade was determined mostly by binding of nifedipine with closed channels, whereas in the cases of 1H- and 1I-subunits this resulted from binding of nifedipine with channels in the activated and inactivated states. The obtained data allow us to obtain estimates of the pharmacological properties of the above three subtypes of recombinant channels and, in the future, to compare these characteristics with the properties of low-threshold Ca²⁺ channels in native cells.     

C-Terminal exchange between Gα o and Gα i3 proteins differently modulates α2A-adrenoceptor activation

Chimeric G proteins, obtained by exchanging their C-terminal portion for that of a G protein from an unrelated class, drive the receptor selectivity to that corresponding to the introduced G protein domain. The _2A-adrenoceptor (_2AAR), which yielded an efficacious and weak [³⁵S]GTPS binding response by respectively G _o and G _i3 protein, was investigated in CHO-K1 cells co-expressing chimeric G proteins for which the six last C-terminal amino acids between G _o and G _i3 proteins, and reciprocally, were permuted. Activation of the chimeric G _{o / i3} protein was highly efficient whereas the G _{i3 / o} protein yielded a weak stimulation. These [³⁵S]GTPS binding responses were not different from their parental wild-type G _o and G _i3 proteins. Similar results were obtained with an _2AAR carrying a facilitating Thr³⁷³Lys mutation in a putative G protein interaction domain. These data indicate that the six terminal G _o protein amino acids do not constitute a major _2AAR interaction domain for G protein activation.     

Modulation of natural killer activity by thymosin alpha 1 and interferon

Summary A single injection of -interferon (-IFN) (30 000 units/mouse), a major biological modifier of natural killer (NK) cytolytic activity, strongly stimulated NK activity in normal mice, as expected, while the same treatment did not statistically alter the NK response in cyclophosphamide (CY)-suppressed animals.We investigated the possibility of thymosin ₁ cooperating with -IFN in boosting NK activity in CY-suppressed animals.The results show that treatment with thymosin ₁ (200 g/kg) for 4 days, followed by a single injection of -IFN 24 h before testing, strongly restored NK activity in CY-suppressed mice. Thymosin ₁ was, moreover, able to accelerate the recovery rate of NK activity in bone marrow reconstituted murine chimeras.Taken together the data support the concept that the synergic effect between thymosin ₁ and -IFN could be the result of effects on differentiation of the NK lineage at different levels.     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

ααααanti-3.7 type II: a new α-globin gene rearrangement suggesting that the α-globin gene duplication could be caused by intrachromosomal recombination

Summary We report here a new human -globin gene rearrangement carrying the two normal, 2 and 1, and two hybrid, 1/2, globin genes in the order 5-2-1/2-1/2-1-3. Both the hybrid genes, subtyped with ApaI and RsaI restriction enzymes, were found to be of the uncommon anti 3.7 type II. The hybrid genes were expressed at the biosynthetic level and their interaction with the -thalassaemia IVS 1 nt 1 GA mutation caused thalassaemia intermedia. We also report a case of an -globin gene rearrangement in the twin of one of the -globin gene carriers; the duplicated gene was of the anti 4.2 type and was associated with the absence of RsaI polymorphism. The singular finding of an -anti 3.7 cluster with two identical rare hybrid genes suggests that the reciprocal unequal recombination causing the -globin gene rearrangements could be of the intra-chromosomal rather than the interchromosomal type.     

α-Thalassemia in Saudi Arabia: deletion pattern

Summary -Thalassemia exists at a high prevalence in several regions of Saudi Arabia. The restriction endonucleases Bam HI and BglII were used to investigate the molecular basis of deletion type of -thalassemia in 226 subjects from the eastern and 61 subjects from the northwestern regions of the country. The arrangements-/ and-/- were common. BglII digestion revealed the existence of rightward deletion in a majority of the cases. Leftward deletions, both homozygous and heterozygous, were also identified. Triple -gene arrangements -/ and -/- were observed at a low frequency in both regions.     

α-Subunit Composition of Nicotinic Cholinoreceptors of Neurons of the Inferior Mesenteric Ganglion of the Guinea Pig: an Electrophysiological Study

We studied the subunit composition of nicotinic cholinoreceptors (nChR) of neurons of the inferior mesenteric ganglion (IMG) of the guinea pig using antibodies against the ₃, ₄, ₅, and ₇ nChR subunits and a standard technique of intracellular recording. Application of the ₃ subunit-specific antibodies resulted in a decrease in the EPSP amplitude by 29.7 ± 1.8%, on average, in 17 of 20 studied neurons. The ₄ subunit-specific antibodies evoked no changes in the amplitude of EPSP recorded from IMG neurons (n = 10). Effects of the ₅ subunit-specific antibodies were studied in 20 neurons, where the EPSP amplitude dropped after application of these antibodies by 40.0 ± 1.8%, on average. Superfusion of the neurons with a solution containing the ₃ and ₅ subunit-specific antibodies completely suppressed synaptic responses (n = 3). The ₇ subunit-specific antibodies provided an increase in the EPSP amplitude in 13 of 15 studied IMG neurons (by 37 ± 6%, on average); this fact allows us to suppose that ₇-containing nChR are present in the IMG neurons and are indirectly involved in the processes of synaptic transmission. Application of the antibodies evoked no significant shifts in the membrane potential in IMG neurons. Our findings demonstrate that synaptic transmission through the guinea pig IMG is realized mostly with the involvement of the ₃- and ₅-containing nChR; the ₄-containing receptors are not engaged in this process.     

Conformational transitions in closed circular DNA molecules I. Topological and energetical considerations

A theory of conformational transitions in closed circular DNA as a function of topological linking number of the molecule () is elaborated taking into account topological and energetical considerations. The theory predicts a step-like dependence of a number of superhelical turns in DNA molecules () on . Thus, the number of superhelical turns = for small values of . For a large (when conformational transitions begin to occur) =–_ij, where _ij is the total angle of conformational transitions for a given . This prediction is in good agreement with published data on the dependence of the sedimentation coefficient of circular DNA molecules on their topological linking number. The results also allow to explain the disagreement between a number of titratable superhelical turns in circular DNA molecules and a number of supercoiles seen on electron micrographs for molecules with sufficiently large .     

Proteolytic digestion of α-lactalbumin: Physiological implications

The kinetics of the partial digestion of bovine -lactalbumin (-LA) by trypsin, -chymotrypsin, and pepsin was monitored by lactose synthase activity, HPLC, and difference spectrophotometry. The relative stabilities of the various metal-bound states of -LA to trypsin and chymotrypsin at 37 and 5°C decrease in the following order: Ca(II)--LA>Zn(II), Ca(II)--LA>apo--LA. The HPLC digestion patterns of Ca(II)--LA and Zn(II), Ca(II)--LA at 5 and 37°C were similar, while the corresponding digestion patterns for apo--LA were quite different, reflecting the existence of the thermally induced denaturation states of apo--LA within this temperature region. Occupation of the first Zn(II)-binding site in Ca(II)-loaded -LA slightly alters the HPLC digestion patterns at both temperatures and accelerates the digestion at 37°C due to Zn(II)-induced shift of the thermal transition of -LA, exposing some portion of thermally denatured protein. The results suggest that the binding of Zn(II) to the first Zn(II)- (or Cu(II))-specific site does not cause any drastic changes in the overall structure of -LA. The acidic form of -LA (atpH 2.2 and 37°C) was digested by pepsin at rates similar to that for the apo- or Cu(II), Ca(II)-loaded forms by trypsin or -chymotrypsin at neutralpH. Complexation of -LA with bis-ANS affords protection against pepsin cleavage. It is suggested that the protective effects of similar small lipophilic compounds to -LA may have physiological significance (e.g., for nutritional transport).On leave from the Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region, 142292, USSR.     

Spectral position control of dye absorption band maximum in an orienting matrix

The continuous control of the maximum position of the dye absorption band (the zero of the derivative dD ()/d of the cell's optical density D ()) in a nematic matrix is demonstrated experimentally, as a result of changing the angle between the optical axis of a planar-oriented sample and the plane of polarization of absorbed light incident normal to the optical axis. The theory proposed describes quantitatively the experimental dependence (). The rotation of the polarizer with given frequency results in the spectral position modulation of the solute band maximum () within (=0°)–(90°)=700 cm^–1.     

Haemoglobin synthesis in 28 obligatory cases for α-thalassemia traits

Summary In the Far East two types of -thalassemia genes, namely -thalassemia₁ (-thal₁) and -thalassemia₂ (-thal₂) exist. Definite diagnosis of the -thal₁ and -thal₂ traits is very difficult because their hematological findings are minimally abnormal or normal. This study attempts to characterize the heterozygotes by hemoglobin chain synthesis in reticulocytes from obligatory cases of the -thal₁ and -thal₂ traits. Twelve parents of babies with hemoglobin Bart's hydrops fetalis (obligatory -thal₁ trait) had the mean total radioactivity / ratio of 0.76±SD 0.04, while that of 7 normal controls was 1.06±SD 0.04. The / globin chain ratios of 16 cases, who were either parents or offspring of patients with hemoglobin H disease, were found to segregate into 2 groups, i.e. 0.78±SD 0.03 (10 cases) and 0.92±SD 0.03 (6 cases), probably representing the -thal₁ and -thal₂ traits respectively. The hematological data of the first group showed definite hypochromic microcytic red cells, similar to thoseof the parents of the hydrops. The second group had significantly higher mean corpuscular hemoglobin than the first group, compatible with -thal₂ trait. Our globin chain synthesis study thus appears to be capable of discriminating normal, -thal₁ and -thal₂ traits.A preliminary report of the results was presented at the XV Congress of the International Society of Haematology, Israel, September, 1974.