Immunogenicity of a non-class I MHC expressing murine tumor transfected with the influenza virus hemagglutinin or murine interleukin-2 genes

Summary The transfection of murine SP1 tumor cells with the hemagglutinin (HA) gene of influenza virus results, after fluorescent-activated cell sorting (FACS), in the selection of high-HA-expressing cell lines called H4A and H4B. Both lines fail to grow in syngeneic animals at doses that result in 100% tumor take of non-transfected tumor cells. Both grow in immunosuppressed mice. SP1 and H4A or H4B cells express few class I major histocompatibility complex (MHC) antigens but do express class II IA^k antigens. H4A or H4B cells engender a cytotoxic T lymphocyte (CTL) response but cannot protect against a challenge with SP1 cells. This CTL response is inhibited by anti-CD4 but not anti-CD8 antibodies. Using FACS, we were able to select a population (called H5AK5) with high class-I MHC antigen expression. Like H4A and H4B, H5AK5 cells fail to grow in syngeneic animals but do grow in immunosuppressed mice. However, unlike H4A or H4B, H5AK5 can induce protection against a challenge with 1 × 10⁵ SP1 cells. These studies indicate that the immunogenicity ofHA-transfected SP1 cells may correlate with the cell-surface expression of class II MHC antigens. However, HA-expressing SP1 cells seem able to induce a protective response against a parent SP1 cell challenge only if they also express class I MHC antigens. This view is supported by the observations that SP1 cells expressing murine interleukin-2 do not express class I MHC antigens, fail to grow in syngeneic animals, do grow in immunosuppressed mice but do not protect against a challenge with parental SP1 cells.This work was supported by The Clayton Fund, The Sid W. Richardson Foundation and PHS grants CA 39853 and 41525. Toshiyuki Itaya is a visiting scientist supported by the Smith Education Fund of the Department of Cell Biology. Troy Fiesinger is a summer research investigator sponsored by The University of Texas M. D. Anderson Cancer Center Summer Program for College Students     

Properties of the complexes of riboflavin 3',5'-bisphosphate and the apoflavodoxins from Megasphaera elsdenii and Desulfovibrio vulgaris

Megasphaera elsdenii and Desulfovibrio vulgaris apoflavodoxins have been reconstituted with riboflavin 3',5'-bisphosphate. Several biochemical and biophysical properties of the complexes have been investigated and the results are compared with the properties of the native proteins. The dissociation constant of the modified complex of M. elsdenii flavodoxin is increased by a factor of about 23 by comparison with that of the native protein. The rate constant for the formation of the complex of M. elsdenii flavodoxin is about 26 times lower than that for the native protein. The redox potential of the transition between the oxidized and semiquinone state is similar to that of the native protein. On the other hand, the redox potential of the semiquinone-hydroquinone transition is about 20 mV more negative than that of the native protein. Absorbance and circular dichroic spectra of the protein-bound artificial prosthetic group and the protein-bound natural prosthetic group are very similar. In both the oxidized and in the fully reduced state only minor differences in interaction between the isoalloxazine ring and the apoprotein for the two flavin derivatives are found by 13C and 15N NMR. 31P-NMR studies show that the 5'-phosphate group of the two flavin derivatives is bound in the same way and that it is dianionic in the complex. In contrast, the 3'-phosphate group in riboflavin 3',5'-bisphosphate is monoanionic or even neutral when bound to the protein. The 3'-phosphate group is also close to or on the surface of the protein. Desulfovibrio vulgaris apoflavodoxin has an affinity for riboflavin 3',5'-bisphosphate which is 10 times higher as compared to Megasphaera elsdenii apoflavodoxin (Ka = 10(8) M-1). Also the association rate constant of Desulfovibrio vulgaris apoprotein and riboflavin 3'5'-bisphosphate is found to be 10 times faster than for the Megasphaera elsdenii flavodoxin reaction. The dissociation behaviour of native Desulfovibrio vulgaris flavodoxin measured under identical conditions as for the riboflavin 3',5'-bisphosphate analog gives a value (Kd approximately equal to 0.2 nM) which is considerably lower than reported earlier [Dubourdieu, M., MacKnight, M. L. & Tollin, G. (1974) Biochem. Biophys. Res. Commun. 60, 649-655]. The results are discussed in the light of the existing crystallographic data of flavodoxins and the recently proposed theory on the regulation of the redox potential in flavoproteins [Moonen, C. T. W., Vervoort, J. & Müller, F. (1984) in Flavins and flavoproteins, pp. 493-496, Walter de Gruyter, Berlin].     

Repression of Meiotic Crossing over by a Centromere (CEN3 ) in SACCHAROMYCES CEREVISIAE

The location of the centromere of chromosome III (CEN3) of Saccharomyces cerevisiae has been altered by means of transformation. The frequency of meiotic crossing over in the CEN3-PGK1 and LEU2-CEN3 intervals increases approximately 1.5- and fourfold, respectively, when CEN3 is repositioned at HIS4. The centromere-distal HIS4-LEU2 region experiences a three- to fivefold decrease in the frequency of meiotic exchange when CEN3 is repositioned at HIS4. The inhibition of meiotic crossing over is conferred by a 627-base-pair fragment of CEN3 DNA and is not dependent on the orientation of CEN3 relative to the rest of chromosome III.     

Distinguishing between Nitrification and Denitrification as Sources of Gaseous Nitrogen Production in Soil

The source of N₂O produced in soil is often uncertain because denitrification and nitrification can occur simultaneously in the same soil aggregate. A technique which exploits the differential sensitivity of these processes to C₂H₂ inhibition is proposed for distinguishing among gaseous N losses from soils. Denitrification N₂O was estimated from 24-h laboratory incubations in which nitrification was inhibited by 10-Pa C₂H₂. Nitrification N₂O was estimated from the difference between N₂O production under no C₂H₂ and that determined for denitrification. Denitrification N₂ was estimated from the difference between N₂O production under 10-kPa C₂H₂ and that under 10 Pa. Laboratory estimates of N₂O production were significantly correlated with in situ N₂O diffusion measurements made during a 10-month period in two forested watersheds. Nitrous oxide production from nitrification was most important on well-drained sites of a disturbed watershed where ambient NO₃⁻ was high. In contrast, denitrification N₂O was most important on poorly drained sites near the stream of the same watershed. Distinction between N₂O production from nitrification and denitrification was corroborated by correlations between denitrification N₂O and water-filled pore space and between nitrification N₂O and ambient NO₃⁻. This technique permits qualitative study of environmental parameters that regulate gaseous N losses via denitrification and nitrification.     

Wounding Inhibits Protein Synthesis yet Stimulates Polysome Formation in Aged, Excised Pea Epicotyls

Wounding of aged, previously-excised pea epicotyl segments byremoval of the basal 1–2 mm resulted in a rapid (beginningwithin 15 min) recruitment of monosomes on to polysomes andan even more rapid (maximal between 6–12 min) inhibitionof protein synthesis in the remaining tissue. This inhibitionof protein synthesis in vivo did not appear to be an artefactcaused by the removal of highly active tissue (e.g., callus,contaminating bacteria), since wounds inflicted at a site distantfrom the region analyzed still elicited the response, and proteinsynthesis in the 1–2 mm slices (normally discarded) wasinhibited even more strongly than it was in the remaining tissue.The proportion of radioactive methionine in nascent chains (boundto polysomes) increased, while the production of completed polypeptidesdecreased, after wounding. Cycloheximide, a known inhibitorof the ribosome translocation/release process mimicked someof the effects of wounding. We interpret the results to indicatethat the initial effect of wounding is to inhibit translationby inhibiting the ribosome translocation/release process, whereasthe subsequent recovery in protein synthesis is brought aboutpartly by a recovery in ribosome translocation/release and partlyby enhanced initiation. ¹ Present address: Harvard-MIT Division of Health Science andTechnology, MIT, Cambridge, Massachusetts 02139, U.S.A. ² Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received May 26, 1986; Accepted August 4, 1986)     

Rapid Isolation of the 7S-Nerve Growth Factor Complex and Its Subunits from Murine Submaxillary Glands and Saliva

7S-Nerve growth factor (NGF) and its alpha, beta-NGF, and gamma subunits have been purified from murine submaxillary glands and saliva by a combination of gel filtration on rigid polyvinyl gels, reversed-phase liquid chromatography on short alkyl chain supports (C4 columns), and ion-exchange chromatography on silica-based carboxymethyl columns. This technique is superior to previously used methods in that it is much more rapid and allows the purification of larger quantities of polypeptide from the same amount of starting material. Beta-NGF prepared with this method elicits the outgrowth of fibers of cells of a pheochromocytoma cell line (PC 12) in vitro, indicating that the biological activity is not impaired by the organic solvents and strong acids utilized for its isolation.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Quantitative Isolation of Undegraded Polysomes from Aged Pea Tissue in the Absence of Contaminants and Artefacts

Undegraded polysomes were isolated successfully from aged peaepicotyls by grinding frozen tissue in at least 10 volumes ofbuffer A (0.2 M Tris-HCl, pH 8.5; 0.2 M sucrose; 60 mM KCl;30 mM MgCl₂), taking care to prevent the tissue from thawingprior to homogenization. Supposedly pure polysomes, derivedfrom the membrane-bound polysome fraction, were apparently contaminatedwith membranes, and contained polysomes clumped together vianascent chains. Problems with contaminants and artefacts werepartially alleviated by the use of polyoxyethylene tridecylether as a detergent replacing Triton X-100; further alleviatedby the use of large volumes of detergent-containing buffer toresuspend the membrane-bound polysome; and almost completelyeliminated by brief treatment of resuspended polysomes withprotease K. Optimal conditions for isolating polysomes fromaged tissue are given. ¹ Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received April 24, 1985; Accepted September 2, 1985)     

Phylogenetic Survey of Proteins Related to Synapsin I and Biochemical Analysis of Four Such Proteins from Fish Brain

A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle-associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two-dimensional peptide-mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are wide-spread in the animal kingdom.     

Substitution of selenocysteine for cysteine in a reticulocyte lysate protein synthesis system

Selenocysteine occurs in the peptide backbone of several selenoenzymes. The mechanism, of selenocysteine incorporation has not been well characterized. The incorporation of selenocysteine into protein in a rabbit reticulocyte lysate (RRL) was studied at high levels of selenocysteine. [⁷⁵Se]Selenocysteine incorporation was inhibited by cycloheximide and by nuclease treatment. Random RNA copolymers were tested for protein synthesis activity in the messenger RNA-dependent RRL system. Of the active polymers, poly CIU and GU most strongly stimulated the incorporation of selenocysteine. In a series of four polymers with different ratios of U to G, incorporation of selenocysteine and cysteine increased with increasing percentages of U, suggesting that selenocysteine and cysteine responded to the same codon, presumably UGU. Of the 20 protein amino acids, only cysteine and cystine competed with selenocysteine incorporation. Selenocysteine was charged to cysteine-accepting tRNA in RRL. These results show that at supraphysiological concentrations selenocysteine can substitute for cysteine in RRL protein synthesis. Misincorporation of selenocysteine could be important when animal tissues contain high levels of selenium.