Allospecific T cell recognition of HLA-A2 antigens: Evidence for group-specific and subgroup-specific epitopes

Interleukin 2-dependent alloreactive cytotoxic T cell lines, with activity predominantly directed against the HLA-A2 antigen, have been generated in vitro by stimulating blood mononuclear cells from donors nonimmune to the Epstein-Barr (EB) virus with appropriate numbers of EB virus-transformed B cells from A2-homozygous individuals. Such effector cells were tested against a panel of EB virus-transformed target cell lines all expressing the serologically defined A2 antigen but typed into common A2 and variant A2 subgroups on the basis of their recognition by A2-restricted EB virus-specific cytotoxic T cells. Variant A2 responder cells cocultivated with common A2-bearing stimulators gave rise to effector T cell lines which recognized only the common A2-bearing subgroup of targets. By contrast, responder cells from A2-negative donors stimulated with common A2-bearing cells produced effector T cell lines in which the strong lysis of common A2-bearing targets was accompanied by a lower, but still significant, lysis directed against all targets within the variant A2 subgroup. In both cases, lysis of the target cells was blocked equally well by the anti-A2-specific monoclonal antibody MA2.1 as by the monoclonal antibody W6/32 specific for HLA-A, -B, and -C determinants. This suggests that HLA-A2 molecules possess at least two distinct sets of epitopes capable of inducing alloreactive T cell cytotoxicity: first, epitopes probably associated with T cell-restricting sites, which generate subgroup-specific responses, and second, epitopes shared by all A2 molecules, and perhaps associated with serologically defined sites, which generate pan A2 group-specific responses.Abbreviations used in this paper EB Epstein-Barr - IL-2 Interleukin 2 - UM unfractionated mononuclear - AET aminoethylisothiouroniumbromide hydrobromide     

Stable production of recombinant pro-urokinase by human lymphoblastoid Namalwa KJM-1 cells: Host-cell dependency of the expressed-protein stability

Human pro-urokinase (pro-UK) gene was engineered for expression in mammalian cells. The stability of recombinant pro-UKs produced by two kinds of cells, Chinese hamster ovary (CHO) and human lymphoblastoid Namalwa KJM-1 cells, were compared. The pro-UK expressed in CHO cells in serum-free medium was degraded by cysteine endopeptidase secreted by CHO cells. This endopeptidase was inhibited by pchloromercuribenzonate (PCMB) and leupeptin more efficiently than by aprotinin. On the other hand, the pro-UK expressed in Namalwa KJM-1 cells was not degraded, resulting in the stable production of pro-UK at a rate of 2–3 g/10⁶ cells/day by use of a gene amplification method with dihydrofolate reductase (DHFR) in serum-free medium. Thus, Namalwa KJM-1 cells showed the desired characteristics as a host cell for the production of recombinant proteins. The stability of recombinant proteins produced in heterologous systems may vary depending on the host cells.Abbreviations ABTS 2,2-azino-di-(3-ethylbenzothiazoline) sulfonic acid diammonium salt - AMC 7-amino-4-methylcoumarin - CHO Chinese hamster ovary - DFP diisopropylfluorophosphate - DHFR dihydrofolate reductase - ELISA enzyme-linked immunosorbent assay - MCA 4-methylcoumaryl-7-amide - MTX methotrexate - NEAA non essential amino acid - NEM N-ethylmaleimide - PCMB p-chloromercuribenzonate - PMSF phenylmethanesulfonyl fluoride - pro-UK pro-urokinase     

The frequency of the γ chain variant AγT in different populations,and its use in evaluating γ gene expression in association with thalassemia

Summary The occurrence of the ^AT chain (i.e. ^A75 Ile Thr) in different populations was evaluated through a study of 4250 cord blood samples and blood samples from more than 350 SS¹ patients. High frequencies were observed in Italy, Yugoslavia, Turkey, Holland, but also in Japan, Vietnam, and India. The chain is (nearly) absent in the Black population of Ghana and Kenya, and low frequencies were observed in China and Australian aborigines. Only a few adult SS patients (18 out of 357) were ^AT heterozygotes. The chromosomes with the ^AT globin gene were mapped through an evaluation of the presence of 10 different restriction sites. The ^AT chromosomes from different populations were closely related and had the same subhaplotypes of [--++-+] (Hinc II 5 to ; Xmn I 5 to ^G; Hind III in ^G and ^A; Hinc II in and 3 to ), quite different from the subhaplotypes seen for ^AT negative chromosomes.² This suggests a common ancestor which may have originated in Southern Europe. An evaluation of the chain production by both chromosomes in SS patients and -thalassemia heterozygotes was possible for subjects with an ^AT heterozygosity. It was concluded that in -thalassemia trait, the chain synthesis is directed for about two-thirds by the thalassemic chromosome and for about onethird by the normal chromosome; the contribution by the normal chromosome decreases with a decrease in total chain production.This is contribution #0890 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA     

Hydrogen isotope discrimination in higher plants: Correlations with photosynthetic pathway and environment

Summary The ratio of deuterium to hydrogen (expressed as D) in hydrogen released as water during the combustion of dried plant material was examined. The D value (metabolic hydrogen) determined on plant materials grown under controlled conditions is correlated with pathways of photosynthetic carbon metabolism. C₃ plants show mean D values of-132 for shoots and -117 for roots; C₄ plants show mean D values of -91 for shoots and-77 for roots and CAM plants a D value of-75 for roots and shoots. The difference between the D value of shoot material from C₃ and C₄ plants was confirmed in species growing under a range of glasshouse conditions. This difference in D value between C₃ and C₄ species does not appear to be due to differences in the D value (tissue water) in the plants as a result of physical fractionation of hydrogen isotopes during transpiration. In C₃ and C₄ plants the hydrogen isotope discrimination is in the same direction as the carbon isotope discrimination and factors contributing to the difference in D values are discussed. In CAM plants grown in the laboratory or collected from the field D values range from-75 to +50 and are correlated with ¹³C values. When deprived of water, the D value (metabolic hydrogen) in both soluble and insoluble material in leaves of Kalanchoe daigremontiana Hamet et Perr., becomes less negative. These changes may reflect the deuterium enrichment of tissue water during transpiration, or in field conditions, may reflect the different D value of available water in areas of increasing aridity. Whatever the origin of the variable D value in CAM plants, this parameter may be a useful index of the water relations of these plants under natural conditions.     

Heterogeneity of the α-globin gene defects in German α-thalassemia affected families

Summary Analysis of -thalassemia syndromes in several German families revealed DNA deletion as well as nondeletion forms as the molecular basis for the defects. Thus, the -thalassemia haplotype was identified as the (–)^3.7 rightward deletion form, and the region of the putative recombination process generating such a deletion was further characterized. In addition three different ° haplotypes, (--)^MED, (--)^>26, and ()^T, could be detected using -and -globin gene-specific probes.     

Biometeorological comfort index

The paper aims to represent and scale biometeorological sensations through a thermal index which comprises the effects of temperature, humidity and wind. The cooling power of a measured combination of wind speed and air temperature is equated with that due to the combination of a reference wind speed (related to the air movement experienced by a person walking in still air), and a derived aerodynamic temperature. A biometeorological temperature which is the average of such an aerodynamic temperature and a conventionally measured wet bulb temperature is shown to afford a better measure of comfort in Spain than some other, alternative indices.     

Retinoic acid receptor isoform RARγ 1: an antagonist of the transactivation of the RARβ RARE in epithelial cell lines and normal human keratinocytes

The diversity of isoforms of retinoic acid (RA) receptors (RARs) and of DNA sequences of retinoic acid-responsive elements (RAREs) suggests the existence of selectivities in the RAR/RARE recognition or in the subsequent gene modulation. Such selectivities might be particularly important for RAREs involved in positive feedback, eg. the RAR RARE. In the present work we found that in several epithelial cell lines, reporter constructs containing the RAR RARE linked to the HSV-tk promoter were transactivated in the presence of RA by endogenous RARs and co-transfected RAR₁ and RAR₂ isoforms, but not by RAR₁. On the contrary, this latter isoform behaved towards the RAR RARE as an inhibitor of the transactivation produced by endogenous RARs and by cotransfected RAR₁ and RAR₂. RAR₁ also behaved as an antagonist of the transactivation produced by cotransfected RXR. The natural RAR gene promoter or RAR RARE tk constructs were not activated by the endogenous receptors of normal human keratinocytes (NHK), which are known to contain predominantly RAR₁. It was, however, possible to activate to a certain extent RAR RARE-reporter constructs in NHK by co-transfecting RAR₁, RAR₂ or RXR. The antagonist behavior of RAR₁ towards the RAR RARE may explain why in certain cell types such as keratinocytes, RAR is neither expressed nor induced by RA.Abbreviations DMEM Dulbecco's modified Eagle medium - DMSO dimethyl sulfoxide - FCS fetal calf serum - MEM minimal Eagle medium - NHK normal human keratinocyte - RA retinoic acid - RAR retinoic acid receptor - RARE retinoic acid responsive element - TRE thyroid responsive element - VDRE vitamin D response element - RXR retinoid X receptor     

Effect of Intercalators on the Properties of Liquid-Crystalline Dispersions of Nucleic Acid–Chitosan Complex

Molecules of deoxyribonucleic acid and synthetic polydeoxyribonucleotides (NA) in the particles of liquid-crystalline dispersions resulting from interaction with chitosan are accessible to interaction with intercalators. The intercalation is accompanied by alteration in the direction of spatial twist of cholesterics of NA–chitosan complexes. This effect is absent in the case of classical cholesterics produced from NA molecules via phase exclusion, i.e., the cholesteric structure of NA–chitosan complex is very labile as distinct from classical cholesteric NA.     

Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli

Bifidobacterium infantis HL96 produces three -galactosidases (-gal I, II and III). A genomic bank of B. infantis was constructed in E. coli by using pBR322 as a cloning vector. Two E. coli transformants, BIG1 and BIG4, possessing -galactosidase activity, were selected from X-gal plates. They contained two different recombinant plasmids with insert DNA fragments of approx. 4.6 and 4.4 kb, respectively. The restriction maps of pBIG1 and pBIG4 were constructed. -Galactosidases from crude cell-free extracts of B. infantis and of two E. coli recombinants were analyzed by native PAGE and characterized by activity staining. pBIG1 and pBIG4 were shown to carry the genes for -gal I and -gal III, respectively. Optimal pH and temperature for hydrolytic activity of the native enzyme were 7.5 and 40°C, while those for recombinant BIG1 and BIG4 were 7.5, 50°C and 8.0, 40°C, respectively. © Rapid Science Ltd. 1998     

Enzymatic basis for sialyl-Tn expression in human colon cancer cells

Sialyl-Tn antigen (SA2-6 GalNAc-Ser/Thr) is expressed as a cancer-associated antigen on the surface of cancer cells and its expression correlates with a poor prognosis in patients with colorectal and other adenocarcinomas. To understand the enzymatic basis of sialyl-Tn (STn) antigen expression, we used two clonal cell lines, LSB and LSC, derived from LS174T human colonic cancer cells. LSC cells express only the truncated carbohydrate antigen Tn (GalNAc-Ser/Thr) and sialyl-Tn on their mucin molecules, whereas LSB cells express elongated oligosaccharide chains. Both cell lines demonstrated similar activities of glycosyltransferases involved in the biosynthesis of elongated and terminal structures of complex O-glycans. However, LSC cells were unable to synthesize core 1 (Gal1-3GalNAc-) because the ubiquitous enzyme activity of UDP-Gal:GalNAc-R 3-Gal-transferase (core 1 3-Gal-transferase) was lacking. Core 1 3-Gal-transferase could not be reactivated in LSC cells by treatment with sodium butyrate or by in vivo growth of LSC cells in nude mice. In contrast, LSB cells were able to synthesize and process core 1 and core 2 (GlcNAc1-6 (Gal1-3) GalNAc-). LSC cells represent the first example of a non-hematopoietic cell line which lacks core 1 3-Gal-transferase activity. The lack of core 1 3-Gal-transferase in LSC cells explains why they are incapable of forming the common mucin O-glycan core structures and are committed to synthesizing the short Tn and STn oligosaccharides. These findings suggest that the activity of core 1 3-Gal-transferase is an important determinant of the STn phenotype of colon cancer cells.     

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Lex determinant

A defined set of oligosaccharides and glycopeptides containing -linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Lex) Gal1-4[Fuc1-3]GlcNAc1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc1-4[Fuc1-3]GlcNAc. The lectin did not bind glycans containing either sialylLex or VIM-2 determinants, nor did it bind the isomeric Lea, Gal1-3[Fuc1-4]GlcNAc-R. Although 2-fucosyllactose Fuc1-2Gal1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc1-2Gal1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Lex antigen and is useful in analyzing specific fucosylation of glycoconjugates. Abbreviations: LTA, Lotus tetragonolobus agglutinin; UEA-1, Ulex europaeus agglutinin-I; LNT, AAL, Aleuria aurantia agglutinin; Gal1-3GlcNAc1-3Gal1-3Glc; LNnT, Gal1-4GlcNAc1-3Gal1-3Glc; Lex, Lewis x antigen; Lea, Lewis a antigen; GDPFuc, guanosine 5-diphosphate--L-fucose     

Binding of the galactose-specificPseudomonas aeruginosa lectin,PA-I,to glycosphingolipids and other glycoconjugates

The carbohydrate-binding specificity ofPseudomonas aeruginosa lectin I (PA-I) in iodinated or biotinylated form was studied. A large number of glycosphingolipids, as well as some glycoproteins and neoglycoproteins were used as ligands. Also, inhibition by free saccharides of PA-I binding to glycosphingolipids was tested. It was found that the lectin binds most strongly to terminal and nonsubstituted Gal3Gal- or Gal4Gal-structures.Abbreviations PA-I Pseudomonas aeruginosa lectin I - Cer ceramide - lactosylceramide Gal4GlcCer - iso globotriaosylcerami Gal3Gal4GlcCer - globotriaosylceramide Gal4Gal4GlcCer - globoside or globotetraosylceramide GalNAc3Gal4Gal4GlcCer - Forssman glycolipid GalNAc3GalNAc3Gal4Gal4GlcCer - P1 glycolipid Gal4Gal4GlcNAc3Gal4GlcCer - lactoneotetraosylceramide Gal4GlcNAc3Gal4GlcCer - B5 glycolipid Gal3Gal4GlcNAc3Gal4GlcCer - gangliotetraosylceramide Gal3GalNAc4Gal4GlcCer - GM1 Gal3GalNAc4(NeuAc3)Gal4GlcCer - RBC red blood cells - BSA bovine serum albumin - PBS phosphate-buffered saline - SDS sodium dodecyl sulfate - TLC thin-layer chromatography - HPLC high pressure liquid chromatography - MS mass spectrometry - FAB fast-atom bombardment - EI electron impact     

11 S seed storage proteins fromHelianthus species (Compositae): Biochemical,size and charge heterogeneity

The seeds of 19 sunflower species were compared on the basis of their protein contents and the relative proportions of their protein fractions. The globulin content varied from 50% to about 70% and the albumin content from 18% to 35% according to the species. The level of intermediateMr polypeptides showed a great variability (9.6 to 24.3%). Comparative studies onMr polymorphism were carried out by means of sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of non reduced and/or reduced samples using both mono- and bidimensional procedures. Polypeptide constituents of helianthinin were compared including both number and molecular size (cultivatedH. annuus was used as a standard). Studies focused on differences observed between the major two (Mr 38 000), (Mr 32 000) and (Mr 25 500), (21 000) polypeptides families constituting the main A, B, and C subunits. and polypeptides analyses permit to discriminate easilyH. petiolaris from the other species. Charge polymorphism was studied using isoelectric focusing (IEF) and IEF-PAGE in mono and bidimensional procedures in the presence or absence of 2-mercaptoethanol (2-ME). Only a specific ₄ polypeptide enables an easy discrimination betweenH. petiolaris and all the other species. Detailed nomenclature of the , and , polypeptides constituting the different helianthinin globulin subunits is given via the results of pI andMr analyses. Monodimensional IEF patterns of the more basic albumins (pI > 8.0) appear to provide a more valuable approach to identifying specific protein markers.     

Nucellar callus of ‘Femminello’ lemon,selected for tolerance toPhoma tracheiphila toxin,shows enhanced release of chitinase and glucanase into the culture medium

Phoma tracheiphila is the causative agent of the disease mal secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogenP. tracheiphila and its toxin. Some cultivars (e.g., Femminello lemon) are inherently sensitive while others (e.g., Tarocco orange) are tolerant. Cell lines derived from nucellar tissue of Femminello, Tarocco and a cell line selected for tolerance to the fungal toxin (Femminello-S) were used to study host-pathogen interaction. Our results showed that calli or conditioned media of Tarocco and Femminello-S inhibited the size of co-cultivated fungal colonies when compared to Femminello. In addition, conditioned medium of Tarocco as well as FemminelloS, but not Femminello, promoted bursting of hyphal tips. A ten-fold increase in chitinase and glucanase enzymatic activity, as evaluated by radiometric assay and laminarin hydrolysis respectively, was detected in Femminello-S extracellular extracts as compared to Femminello. An increase in chitinase was also shown by immunoblot analysis. Our findings suggest a positive correlation between the presence of chitinase and glucanase in the conditioned media of the cultured cells and the tolerance of those cells toP. tracheiphila toxin.     

Expression of human lymphotoxin in Namalwa KJM-1 cells adapted to serum-free medium

A Namalwa cell line, KJM-1, which was adapted to serum-free medium is thought to be a good host cell line for recombinant DNA technology. We previously reported the expression of human -interferon (-IFN) in Namalwa KJM-1 (Miyaji, 1989a). The utility of Namalwa KJM-1 for expression of foreign genes was further examined. As a target gene to be expressed, human lymphotoxin (hLT) cDNA was used. It was engineered for expression in Namalwa KJM-1 using a simian virus 40 (SV40)-based expression vector pAGE107 (Miyaji, 1989a). It contains all components necessary for the expression of cDNA in mammalian cells. The expression vector was introduced into Namalwa KJM-1 by electroporation. Among the transformants, clone 7 was further examined for the expression of hLT in serum-free medium. The production level of hLT was augmented with the increase of the cell density. Thus it was further indicated that Namalwa KJM-1 is useful for production of foreign gene products.Abbreviation HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid     

„Kolbenbildungen“ der Muskelfasern des menschlichen Kehlkopfs

Zusammenfassung Fasern der menschlichen Kehlkopfmuskulatur weisen seitliche Aussackungen auf, die unter dem Sarkolemm in lockerer Anordnung Kerne, Lysosomen, Lipofuscingranula und Mitochondrien mit oft konzentrisch angeordneten Cristae enthalten. Sie sind mit den lichtmikroskopisch bekannten End- bzw. Seitenkolben identisch. Diese Aussackungen werden von den gefäßnahen subsarkolemmalen Mitochondrienanhäufungen eines bestimmten, auch in der übrigen Skelettmuskulatur weitverbreiteten Muskelfasertyps abgeleitet. Es wurden keine Verbindungen mit Nervenfasern gefunden. Im Gegensatz zu Goerttler können wir diese Bildungen deshalb nicht als Mechanorezeptoren deuten. Die Endkolben werden als sekantial getroffene Seitenkolben aufgefaßt. Da die Aussackungen der Fasern auch bei gesunden jungen Männern in der Kehlkopfmuskulatur regelmäßig auftreten, handelt es sich hier nicht um eine krankhafte Bildung, wenn auch die Faser im Bereich der Aussackungen einen degenerativen Prozeß durchzumachen scheint.

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Summary Fibers of human larynx muscles show lateral dilatations of the sarcolemma containing loosely disposed mitochondria, lysosomes and eventually one or more nuclei. The cristae of the mitochondria often are arranged concentrically. Corresponding fiber dilatations have already been described by light microscopists as side- resp. end-bulbs.Our electron-microscopical observations lead to the conclusion that the dilatations are derived from accumulations of mitochondria beneath the sarcolemma which in similar form — but smaller — are characteristic of one type of the different muscle fibers, frequently to be found in other muscles of the body. Both subsarcolemma accumulations of mitochondria are close to the muscle capillaries.The fiber dilatations have no connections with nerve axons, and therefore cannot be interpreted as mechanoreceptors (Goerttler, 1960). The end-bulbs are supposed to be secantially (or obliquely) cut side-bulbs. We do not consider them pathologic since they are regularly found in fibers of healthy young men. On the other hand, they may represent a circumscribed degenerative alteration the origin of which remains to be explored.

     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.     

Novel prebiotic systems: Nucleotide oligomerization in surfactant entrapped water pools

Summary Oligomerization of 5-TMP in water pools entrapped by dodecyl-ammonium chloride surfactant aggregates in benzene: hexane in the presence of dicyanodiimide at temperatures ranging from 21°–72° resulted in the formation of linear and cyclic oligonucleotides containing up to pentamers. Effects of temperature, time and surfactants have been examined. Rate constants for the formation of oligomers have been determined at five different temperatures. These data afforded values of H = 11.8 ± 1.9 Kcal mole^–1, S=–53 6 e.u. and G = 27.4 4.0 Kcal mole^–1. Prebiotic significance of these results are discussed.     

In vivo fucosylation of lacto-N-neotetraose and lacto-N-neohexaose by heterologous expression of Helicobacter pylori alpha-1,3 fucosyltransferase in engineered Escherichia coli

We report here the in vivo production of type 2 fucosylated-N-acetyllactosamine oligosaccharides in Escherichia coli. Lacto-N-neofucopentaose Gal1-4GlcNAc1-3Gal1-4(Fuc1-3)Glc, lacto-N-neodifucohexaose Gal1-4(Fuc1-3)Glc-NAc1-3Gal1-4(Fuc1-3)Glc, and lacto-N-neodifucooctaose Gal1-4GlcNAc1-3Gal1-4(Fuc1-3)GlcNAc1-3Gal1-4(Fuc1-3)Glc were produced from lactose added in the culture medium. Two of them carry the Lewis X human antigen. High cell density cultivation allowed obtaining several grams of fucosylated oligosaccharides per liter of culture. The fucosylation reaction was catalyzed by an -1,3 fucosyltransferase of Helicobacter pylori overexpressed in E. coli with the genes lgtAB of N. meningitidis. The strain was genetically engineered in order to provide GDP-fucose to the system, by genomic inactivation of gene wcaJ involved in colanic acid synthesis and overexpression of RcsA, positive regulator of the colanic acid operon.To prevent fucosylation at the glucosyl residue, lactulose Gal1-4Fru was assayed in replacement of lactose. Lactulose-derived oligosaccharides carrying fucose were synthesized and characterized. Fucosylation of the fructosyl residue was observed, indicating a poor acceptor specificity of the fucosyltransferase of H. pylori.     

Comparative study of the antitumor effect of two types of murine recombinant interferons, (β) and (γ), against B16-F10 melanoma

Summary A comparative study of the antitumor effect of murine recombinant interferon() Mu-rIFN() and murine recombinant interferon() Mu-rIFN() on B16-F10 melanoma was conducted. Administration of Mu-rIFN() i.p. into C57BL/6 mice on days 1 to 7 produced a higher suppressive effect than Mu-rIFN() both on the growth of s.c. implanted tumor and on the formation of artificial pulmonary metastasis. Pharmacokinetic study of Mu-rIFN() demonstrated that high plasma levels were retained for a long time. In clonogenic assay, Mu-rIFN() at 1000 units/ml showed about 80% inhibition of colonies of B16-F10 melanoma. However, Mu-rIFN() hardly inhibited the colonies, even at 1000 units/ml. Augmentation of natural killer (NK) cytotoxicity was much greater with Mu-rIFN() than Mu-rIFN(), whereas Mu-rIFN() enhanced the cytotoxicity of peritoneal macrophages more strongly than Mu-rIFN(). Injection of Mu-rIFN() i.p. 1 day before tumor challenge also inhibited the formation of pulmonary metastasis of B16-F10 melanoma. However, pretreatment of mice with carrageenan significantly suppressed the inhibitory effect of Mu-rIFN(). From these results, it is suggested that the inhibitory effect of Mu-rIFN() on the tumor growth and metastases of B16-F10 melanoma is mediated partly by direct antitumor effect and partly by the activation of macrophages, and that the augmentation of NK activity contributes mainly to the antitumor effect of Mu-rIFN().