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     

The ionization and distribution behavior of oleic acid in chylomicrons and chylomicron-like emulsion particles and the influence of serum albumin

A reproducible, fairly narrow-sized population of rat lymph chylomicrons, approximately 100 nm, was isolated by centrifugation and combined with low levels of [1-13C]oleic acid for NMR studies. The carboxyl chemical shift was monitored as a function of aqueous pH to characterize the ionization behavior of the fatty acid in these particles. The titration curves were very similar to those for oleic acid in equivalent-sized emulsion particles composed of egg phosphatidylcholine and triolein. A simple partition-ionization model was fitted to the data to derive values for apparent ionization constant, expressed as pKapp, of 7.4-7.5 and the "true" surface to core partition coefficient of approximately 7 for oleic acid in chylomicrons. The fatty acid in chylomicrons thus appeared to be largely associated with the surface regions of these particles. Addition of bovine serum albumin to the samples showed that near physiologic pH much of the fatty acid was bound to the albumin at fatty acid to albumin-binding stoichiometries as high as 5.1 and with mass ratios of greater than 2 in favor of the lipid or lipoprotein particles. Lowering the pH of the medium shifted the distribution of fatty acid away from albumin so that at pH 5 with the emulsion, virtually all the fatty acid was associated with the lipid. The behavior observed under physiologic conditions is consistent with the rapid clearance and redistribution of fatty acid generated in these particles by lipolytic processes. However, under conditions of severe acidosis, hyperlipidemia, and hypoalbuminemia a significant portion of fatty acids might be retained in triglyceride-rich lipoproteins and their remnants and affect subsequent metabolism.     

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.     

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     

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.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Differential processing of Asn-linked oligosaccharides on pituitary glycoprotein hormones: implications for biologic function

Luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone from pituitary and chorionic gonadotropin from placenta are a family of glycoproteins, each consisting of an and subunit. Within an animal species, the subunit of all four hormones contains the identical amino acid sequence, while each subunit is distinct and confers biologic specificity to the hormone dimer. Despite sharing common subunits, these hormones bear Asn-linked oligosaccharides which differ in structure. Whereas chorionic gonadotropin contains exclusively neutral and sialylated oligosaccharides, the pituitary hormones bear neutral, sialylated, sulfated, and sialylated/sulfated structures. The sulfated oligosaccharides are unique in structure and are more prevalent on certain pituitary hormones, indicating that the synthesis of these unusual oligosaccharides is tightly regulated. The differences in oligosaccharide structures in conjunction with the highly specific endocrine responses elicited by these hormones, suggest an important functional role for the oligosaccharides, such as metabolic clearance, control of hormone response, modulation of hormone potency, and/or intracellular sorting of hormones into separate secretory granules.