Mitochondrial proteins, learning and memory: biochemical specialization of a memory system.

The enzyme cytochrome c oxidase is a mitochondrial protein complex that plays a crucial role in oxidative metabolism. In the present study we show that amounts of two of its protein subunits (cytochrome c oxidase subunit I [CO-I] and II [CO-II]) are influenced by both learning-independent and learning-dependent factors. Converging evidence has consistently implicated the left intermediate medial mesopallium (IMM) in the chick brain as a memory store for the learning process of visual imprinting. This form of learning proceeds very shortly after chicks have been hatched. In the left IMM, but not in three other brain regions studied, amounts of CO-I and CO-II co-varied: the correlation between them was highly significant. This relationship did not depend on learning. However, learning influenced the amounts of both proteins, but did so only in the left IMM. In this region, amounts of each protein increased with the strength of learning. These findings raise the possibility that the molecular mechanisms involved in the coordinated assembly of cytochrome c oxidase are precociously developed in the left IMM compared to the other regions studied. This precocious development may enable the region to respond efficiently to the oxidative demands made by the changes in synaptic connectivity that underlie memory formation and would allow the left IMM to function as a storage site within hours after hatching.

Amino acid neurotransmitter release and learning: a study of visual imprinting.

The intermediate and medial part of the hyperstriatum ventrale (IMHV) is an area of the domestic chick forebrain that stores information acquired through the learning process of imprinting. The effects of visual imprinting on the release of the amino acids aspartate, arginine, citrulline, gamma-aminobutyric acid (GABA), glutamate, glycine and taurine from the left and right IMHVs in vitro were measured at 3.5, 10 and 24 h after training. Chicks were exposed to an imprinting stimulus for 1 h, their preferences measured 10 min afterward and a preference score calculated as a measure of the strength of learning. Potassium stimulation was used to evoke amino acid release from the IMHVs of trained and untrained chicks in the presence and absence of extracellular Ca2+. Ca2+-dependent, K+-evoked release of glutamate was significantly (34.4%) higher in trained than in untrained chicks. This effect was not influenced by time after training or by side (left or right IMHV). Training influenced the evoked release of GABA and taurine from the left IMHV at both 3.5 and 10 h. The training effects at the two times were statistically homogeneous so data (< or = 10 h group) were combined for each amino acid respectively. For this < or = 10 h group, evoked release increased significantly with preference score. In contrast, for the 24 h group, evoked release of GABA and taurine was not significantly correlated with preference score. There were no significant correlations between preference score and GABA or taurine release in the right IMHV at any time, nor in the absence of extracellular calcium. No significant effects of training condition, time or side were observed for any other amino acid in the study. The present findings suggest that soon after chicks have been exposed to an imprinting stimulus glutamatergic excitatory transmission in IMHV is enhanced, and remains enhanced for at least 24 h. In contrast, the learning-related elevations in taurine and GABA release are not sustained over this period. The change in GABA release may reflect a transient increase in inhibitory transmission in the left IMHV.

Early stages of memory formation in filial imprinting: Fos-like immunoreactivity and behavior in the domestic chick.

Early stages of memory formation in filial imprinting were studied in domestic chicks. Chicks trained for 15 min showed strong imprinting, demonstrated by a strong preference for their training stimulus, and the time course of this preference over 2 days after training was similar to that of chicks trained for 60 min. The chicks therefore learned characteristics of the training stimulus very early during training. The intermediate and medial hyperstriatum ventrale (IMHV) is a part of the chick forebrain that is crucial for imprinting. Previous experiments have shown a learning-specific increase in Fos-like immunoreactivity, used as a marker of neuronal activity, in the IMHV after training for 60 min. The time course of Fos expression in the IMHV was measured after training for 15 min and 60 min. The same pattern of expression was found for both training times, showing a peak 120 min after the start of training. The time course of expression was stimulus-dependent. Fos expression in the IMHV, but not the hippocampus, was significantly correlated with strength of imprinting. It is concluded that the learning-specific change in Fos expression in the IMHV is associated with very early components of memory formation.

Determination of the biotin content of select foods using accurate and sensitive HPLC/avidin binding.

Assessing dietary biotin content, biotin bioavailability, and resulting biotin status are crucial in determining whether biotin deficiency is teratogenic in humans. Accuracy in estimating dietary biotin is limited both by data gaps in food composition tables and by inaccuracies in published data. The present study applied sensitive and specific analytical techniques to determine values for biotin content in a select group of foods. Total biotin content of 87 foods was determined using acid hydrolysis and the HPLC/avidin-binding assay. These values are consistent with published values in that meat, fish, poultry, egg, dairy, and some vegetables are relatively rich sources of biotin. However, these biotin values disagreed substantially with published values for many foods. Assay values varied between 247 times greater than published values for a given food to as much as 36% less than the published biotin value. Among 51 foods assayed for which published values were available, only seven agreed within analytical variability (720%). We conclude that published values for biotin content of foods are likely to be inaccurate.

Analysis of differential gene expression supports a role for amyloid precursor protein and a protein kinase C substrate (MARCKS) in long-term memory.

Previous work has identified the intermediate and medial part of the hyperstriatum ventrale (IMHV) as a region of the chick brain storing information acquired through the learning process of imprinting. We have examined in this brain region changes in expression of candidate genes involved in memory. Chicks were exposed to a rotating red box and the strength of their preference for it, a measure of learning, determined. Brain samples were removed approximately 24 h after training. Candidate genes whose expressions were different in IMHV samples derived from strongly imprinted chicks relative to those from chicks showing little or no learning were identified using subtractive hybridization. The translation products of two candidate genes were investigated further in samples from the left and right IMHV and from two other brain regions not previously implicated in imprinting, the left and right posterior neostriatum. One of the proteins was the amyloid precursor protein (APP), the other was myristoylated alanine rich C kinase substrate (MARCKS). In the left IMHV the levels of the two proteins increased with the strength of learning. The effects in the right IMHV were not significantly different from those in the left. There were no effects of learning in the posterior neostriatum. This is the first study to relate changes in the amounts of MARCKS and APP proteins to the strength of learning in a brain region known to be a memory store and demonstrates that the systematic identification of protein molecules involved in memory formation is possible.

GABA, taurine and learning: release of amino acids from slices of chick brain following filial imprinting.

The intermediate and medial hyperstriatum ventrale (IMHV) is a forebrain region in the domestic chick that is a site of information storage for the learning process of imprinting. We enquired whether imprinting is associated with learning-related increases in calcium-dependent, potassium-stimulated release of neurotransmitter amino acids from the IMHV. Chicks were hatched and reared in darkness until 15-30 h after hatching. They then either remained in darkness or were trained for 2 h by exposure to an imprinting stimulus. One hour later, the chicks were given a preference test and a preference score was calculated from the results of this test, as a measure of imprinting. Chicks were killed 2 h after training. Slices from the left and right IMHV of trained and untrained chicks were superfused with Krebs' solution either with or without calcium and the superfusate assayed for arginine, aspartate, citrulline, GABA, glutamate, glycine and taurine using high-performance liquid chromatography. For calcium-containing superfusates from the left IMHV, preference score was significantly correlated with potassium-stimulated release of (i) GABA (r=0.51, 23 d.f., P=0.008) and (ii) taurine (r=0.77, 23 d.f., P<0.0001). There was no significant difference between the mean values of trained and untrained chicks for either compound. However, examination of the variance of the data indicated that release of both GABA and taurine increased as a result of learning. No significant correlation between preference score and release was found for any of the amino acids from the right IMHV, nor for control tissue from the left IMHV superfused with calcium-free solution. These results demonstrate that the learning process of imprinting is associated with increases in releasable pools of GABA and taurine and/or membrane excitability in the left IMHV.

Neural cell adhesion molecules, CaM kinase II and long-term memory in the chick.

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick brain is a site of recognition memory for filial imprinting. Previous results have demonstrated learning-related changes in the amounts of the three major isoforms of neural cell adhesion molecule (NCAM) in the left IMHV. The increases were present 24 h after training. The present study enquired whether the increases persisted and were present 48 h after training. The brain regions analysed were the left and right IMHV and the left and right hyperstriatum accessorium (HA), a visual projection area. The alpha-subunit of calcium/calmodulin protein kinase II (CaMKIIalpha) was also assayed. There were significant correlations between a measure of the strength of learning and the amount of NCAM 180 in the right IMHV (r = +0.65; p = 0.012) but not in the left, and in the left HA (r = -0.61; p = 0.02), but not in the right. There were no learning-related changes for CaMKIIalpha. We conclude that in IMHV the effects of imprinting on NCAM 180 are expressed mainly in the left IMHV 24 h after training, but 48 h after training are expressed mainly in the right IMHV.

Learning-related fos-like immunoreactivity in the chick brain: time-course and co-localization with GABA and parvalbumin.

Previous work has shown that, after domestic chicks have learned the characteristics of an object (visual imprinting), there is a learning-related increase in the numerical density of Fos-immunopositive neurons in the intermediate and medial part of the hyperstriatum ventrale, a forebrain region that is a site of recognition memory for the imprinted object. The present study describes the time-course of this effect and has used double-labelling immunocytochemistry to identify neuronal types in which the effect occurs. Chicks were trained by exposure for 1 h to an imprinting (training) stimulus and then given a preference test to determine the strength of imprinting (i.e. of learning). Strongly imprinted chicks were killed 2, 2.5, 3, 3.5 or 4 h (12 chicks in each group) after the start of training and a further group of 12 chicks remained untrained. Sections from the chicks' brains were stained for Fos-like immunoreactivity, and the numerical density of Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale was counted. Relative to untrained chicks, there was a 60% increase in the number of Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale 2 h after the start of training (P = 0.02), but not at any other time. Sections from 10 trained chicks, two killed at each of the above times after training, and from two untrained chicks were stained with anti-Fos antibody as before and also with an antibody against GABA. Approximately 95% of the Fos-positive neurons in the intermediate and medial part of the hyperstriatum ventrale were also immunopositive for GABA. In neurons immunopositive for GABA, there were significantly (P = 0.02) more Fos-positive nuclei in the intermediate and medial part of the hyperstriatum ventrale 2 h after the start of training than in untrained chicks. Five chicks killed 2 h after training and five untrained chicks yielded sections for the next experiment; sections were double labelled for (i) Fos and (ii) either Calbindin-D28k or parvalbumin. Training gave rise to a significant (P = 0.017) increase in numerical density of Fos-positive nuclei of neurons that were immunonegative for Calbindin-D28k. This increase occurred in neurons that were immunopositive for parvalbumin. The use of alternative antibodies for GABA, Calbindin-D28k and parvalbumin in trained and untrained chicks confirmed the double-staining pattern observed in the quantitative experiments. The results demonstrate that the learning-related increase in Fos-like immunoreactivity following training is transitory and have localized the increase to a population of neurons immunopositive for GABA and parvalbumin, but not Calbindin-D28k.

The recognition memory of imprinting: biochemistry and electrophysiology.

A restricted part of the intermediate and medial part of the hyperstriatum ventrale (IMHV) of the domestic chick forebrain is pivotal to the learning process of imprinting and is probably the site at which information about an imprinting stimulus is stored. A range of learning-related changes occur in the IMHV between 1 and 24 h after training. The earliest change described is in Fos-like immunoreactivity. There follow changes in phosphorylation of the protein kinase C substrate MARCKS, morphological changes in axospinous synapses, an increase in NMDA receptor number and increases in amounts of the major isoforms of the neural cell adhesion molecule and clathrin heavy chain. All but the change in Fos-immunopositivity occurs in the left, but not the right, IMHV. Insufficient nitric oxide synthase is available in the IMHV to support the hypothesis that nitric oxide is a retrograde messenger contributing to the effect on Fos-like immunoreactivity. In chicks anaesthetised approximately 24 h after imprinting training, the spontaneous mean neuronal firing rate is related to a preference score (a measure of learning). In unanaesthetised chicks 24 h after training, the responsiveness of some IMHV neurons is biassed specifically towards the imprinting stimulus.The responses of other neurons in the IMHV generalise across some features of the training stimulus, such as form or colour. Some neurons in the IMHV of unanaesthetised chicks are responsive to the distance of an imprinting stimulus from the chick; distance-sensitive neurons can be distinguished from distance-insensitive neurones by the action potential shape.

Expression of the GABA(A) receptor gamma 4-subunit gene: anatomical distribution of the corresponding mRNA in the domestic chick forebrain and the effect of imprinting training.

The learning process of imprinting involves morphological, electrophysiological and biochemical changes in a region of the chick (Gallus gallus domesticus) forebrain known as the intermediate and medial part of the hyperstriatum ventrale (IMHV). The alterations include increases in the mean length of postsynaptic density profiles of axospinous synapses and the number of N-methyl-D-aspartate (NMDA) receptor binding sites, and changes in spontaneous and evoked electrical activity. Recent immunocytochemical and behavioural studies have suggested that inhibitory GABAergic neurotransmission plays a role in learning. In this context, it has previously been reported that a novel avian gamma-aminobutyric acid (GABA) type A (GABA(A)) receptor gene, encoding the gamma4 subunit, is highly expressed in the hyperstriatum ventrale. In this study, we have used in situ hybridization to map, in detail, the expression of the gamma4-subunit gene in the chick brain, and to assess the effect of imprinting training on the level of the corresponding transcript. Our results reveal that the gamma4-subunit mRNA has a restricted distribution, and demonstrate a highly significant, time-dependent effect of training on its steady-state level. At 10 h but not at 5 h after training there is a decrease (25-32%) in the amount of this transcript in parts of the medial hyperstriatum ventrale, including the IMHV. A decrease (28-39%) is also seen in certain visual and auditory pathway areas but no effect was observed in other forebrain regions such as the hyperstriatum intercalatus superior (HIS). These results suggest that imprinting training leads to a time-dependent down-regulation of GABAergic transmission, and raise the possibility that this down-regulation plays a role in learning.

Neural cell adhesion molecules, learning, and memory in the domestic chick.

The intermediate and medial hyperstriatum ventrale (IMHV) of the chick forebrain is a site of recognition memory for the learning process of imprinting. The results reported here demonstrate that neural cell adhesion molecules (NCAMs) play a time-dependent role in this recognition memory. Dark-reared chicks were trained, tested, and assigned a preference score as a measure of learning. Chicks with high preference scores were designated good learners and those with lower preference scores, poor learners. Controls were untrained. Tissue was removed, 9.5 hr or 24 hr after training, from the left and right IMHV, hyperstriatum accessorium, and posterior neostriatum. Three major NCAM isoforms (180, 140, and 120 kDa) were assayed. At 24 hr only, there was in left IMHV significantly more NCAM (for each isoform) in good learners than in the other 2 groups, and also a significant correlation between the amounts of NCAM and preference scores for all isoforms; the amount predicted by each regression line at preference score 50 (no learning) did not differ significantly from the mean value for untrained controls. There were no learning-related effects in either the hyperstriatum accessorium or the posterior neostriatum.

Clathrin proteins and recognition memory.

Strong converging evidence indicates that the intermediate and medial part of the hyperstriatum ventrale (IMHV) of the chick forebrain is a site of recognition memory for the learning process of imprinting. Clathrin proteins have been implicated in synaptic plasticity. In the present study we demonstrate for the first time that they are involved in vertebrate learning. Chicks were trained by exposure to a conspicuous object and their preference for it versus a novel object subsequently measured as a preference score (an index of learning). Trained chicks with low preference scores were classed as "poor learners" and those with high preference scores as "good learners". An additional group of chicks was untrained ("dark-reared"). Tissue was removed from the left and right IMHV, hyperstriatum accessorium and posterior neostriatum 9.5 h or 24 h after training. Clathrin heavy chain and clathrin light chains a and b were assayed using sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting. In the IMHV, and only for clathrin heavy chain, was there a significant effect of training. The effect occurred 24 h but not 9.5 h after training, and was significant only in the left IMHV. In this region at 24 h, there was (i) significantly more clathrin heavy chain in good learners than in dark-reared chicks, and (ii) a significant positive correlation between the amount of clathrin heavy chain and preference score; the amount of protein present in the dark-reared chicks did not differ significantly from the amount predicted from the regression line for trained chicks performing at chance (preference score 50). These findings imply that for the left IMHV, visual experience per se, locomotor activity and other side effects of training did not affect the amount of clathrin heavy chain. Rather, the increase observed was a function of the amounts chick learned and, because it was delayed, is likely to be involved in long-term memory. The results for clathrin heavy chain taken together suggest that enhanced presynaptic events in the IMHV, possibly associated with an increase in synaptic vesicle release/uptake, are important in the recognition memory underlying imprinting.

Differential activation of the rat hippocampus and perirhinal cortex by novel visual stimuli and a novel environment.

Two groups of rats were shown individual novel visual objects. One group had been familiarised to the environmental context within which the objects were shown, the other experienced the situation for the first time. The activation of neurones in perirhinal cortex and the hippocampal formation was determined by counts of nuclei stained for products of the immediate early gene c-fos. The ratio of counts in the hippocampal formation to that in perirhinal cortex was compared for the two groups: the ratio was significantly higher (4.2:1) in the group experiencing the environment for the first time. Thus, whereas perirhinal neurones are activated by novel rather than familiar objects, hippocampal neurones are preferentially activated by a novel rather than a familiar environment.

Mapping visual recognition memory through expression of the immediate early gene c-fos.

To study brain regions involved in familiarity discrimination, rats were shown sets of novel and familiar objects. On each trial two objects were shown simultaneously to a rat so that one eye saw a novel object while the other saw a familiar object. Thus novel and familiar objects were seen with the same conditions of alertness and eye movements. Activated neurones were revealed by staining for products of the immediate early gene c-fos. Familiar stimuli activated significantly fewer neurones than novel stimuli in perirhinal cortex and area TE of temporal cortex, and the ventral lateral geniculate nucleus of the thalamus, but not in the hippocampus or other areas sampled. These findings are discussed in relation to recognition memory.

MARCKS and protein F1/GAP-43 mRNA in chick brain: effects of imprinting.

The phosphorylation of MARCKS, but not protein F1/GAP-43, is increased in the intermediate and medial portion of the hyperstriatum ventrale (IMHV) after chick imprinting. Here we investigated if MARCKS, but not F1/GAP-43, gene expression would also be altered after imprinting. We first investigated the constitutive mRNA distribution of MARCKS and F1/GAP-43 in chick brain. MARCKS mRNA was expressed in most cells and exhibited a relatively homogeneous distribution. In contrast, F1/GAP-43 mRNA levels were elevated in discrete brain regions, as we had observed in mammals. The highest F1/GAP-43 mRNA levels in the chick brain were in sensory and associational structures such as the hyperstriatal complex and neostriatum, and lower levels were in structures involved in motor control, such as paleostriatum. These results in chick are consistent with the previously drawn generalization that F1/GAP-43 mRNA is expressed in those brain regions which exhibit synaptic plasticity. After imprinting, MARCKS mRNA levels in IMHV were higher in good learners than poor learners. In contrast, analysis of F1/GAP-43 mRNA levels revealed no differences related to training in any brain region sampled. These selective results for MARCKS but not F1/GAP-43 parallel the prior findings on their phosphorylation, and are consistent with our hypothesis that the very same proteins that are post-translationally modified in association with learning and memory also undergo alterations in their gene expression.

Effects of the novelty or familiarity of visual stimuli on the expression of the immediate early gene c-fos in rat brain.

To investigate substrates of recognition memory, the cellular expression of Fos protein in rat brain has been studied after groups of rats were either shown sets of novel or highly familiar objects, or were exposed to the same pattern of illumination without objects being shown. Counts of stained nuclei were made in eight brain regions, where information about novel or familiar visual stimuli is likely to be processed or stored. The counts were relatively high in occipital visual association cortex and area TE of temporal cortex, intermediate in perirhinal cortex, entorhinal cortex, anterior cingulate cortex and the diagonal band of Broca, and low in the hippocampal formation and mediodorsal nucleus of the thalamus. The number of Fos-stained cells was significantly higher for the rats shown novel objects than for those shown familiar objects in perirhinal cortex, area TE, occipital cortex and anterior cingulate cortex. Arguments are advanced that these differences in counts indicate areas involved in the processing and/or storage of information about the novelty or familiarity of visual stimuli, information important to recognition memory.

Reading skills of dietetic interns and readability of dietetics literature.

OBJECTIVE: To assess and compare reading skills of dietetic interns with reading levels of internship references. DESIGN: A standardized reading test, the Nelson-Denny Reading Test, measured reading skills of entering dietetic interns over 7 years. A computerized readability program assessed the readability of references. SETTING: Dietetic internships in university and Veterans Affairs hospitals. SUBJECTS: Of 194 entering interns, 178 (92%) were included and 16 (89%) were omitted. MAIN OUTCOME MEASURES: Nelson-Denny percentile and grade equivalent scores for vocabulary, comprehension, and total. The Fog Index identified reference reading-grade levels. STATISTICAL ANALYSES PERFORMED: Descriptive statistics and analysis of variance. RESULTS: Interns from the two programs did not differ significantly on Nelson-Denny Reading Test scores or in application grade point average. Percentile means and standard deviations were 54.7 +/- 23.8 for vocabulary, 51.2 +/- 25.0 for comprehension, 52.9 +/- 23.9 for total, and 41.6 +/- 24.7 for reading rate. Nearly 20% (33 of 178) of interns read significantly below expected grade level. The fog Index assigned reference grade levels from 6.98 to 21.63 years. CONCLUSIONS: The majority of dietetic interns have strong reading skills and read within the references' reading levels. A minority may experience difficulties reading assignments. Preinternship reading skills assessment could lead to greater success in reading professional literature.

Antigen processing in vivo and the elicitation of primary CTL responses.

CD8+ T lymphocytes (TCD8+) play an important role in cellular immune responses. TCD8+ recognize MHC class I molecules complexed to peptides of 8 to 10 residues derived largely from cytosolic proteins. Proteins are generally thought to be fragmented in the cytoplasm and delivered to nascent class I molecules in the endoplasmic reticulum (ER) by a peptide transporter encoded by the MHC. To explore the extent to which TCD8+ induction in vivo is limited by proteolysis or peptide transport into the ER, mice were immunized with recombinant vaccinia viruses containing mini-genes encoding antigenic peptides (bypassing the need for proteolysis), or these peptides with a NH2-terminal ER insertion sequence (bypassing the requirements for both proteolysis and transport). Additionally, mice were immunized with recombinant vaccinia viruses encoding rapidly degraded fragments of proteins. We report that limitations in induction of TCD8+ responses vary among Ags: for some, full length proteins are as immunogenic as other forms tested; for others, maximal responses are induced by peptides or by peptides targeted to the ER. Most importantly, in every circumstance examined, targeting peptides to the ER never diminished, and in some cases greatly enhanced, the TCD8+ immune response and provide an important alternative strategy in the design of live viral or naked DNA vaccines for the treatment of cancer and infectious diseases.

Synthetic oligonucleotide expressed by a recombinant vaccinia virus elicits therapeutic CTL.

P815A is a naturally occurring tumor rejection Ag of the methylcholanthrene-induced murine mastocytoma P815. The gene encoding the Ag P815A, designated P1A, is identical to that encoded in the normal genome of the DBA/2 mouse. A recombinant vaccinia virus (rVV) was constructed that expressed a synthetic oligonucleotide encoding the minimal determinant peptide of the tumor-associated Ag. Although the rVV recombinant expressing this mini-gene was recognized efficiently in vitro, it was an ineffective immunogen in vivo. The addition of an endoplasmic reticulum insertion signal sequence to the NH2 terminus of the minimal determinant resulted in a rVV that elicited CD8+ T cells that could lyse P815 mastocytoma cells in vitro and that were therapeutic in vivo. Recombinant viruses expressing synthetic oligonucleotide sequences preceded by the insertion signal sequences allow the expression of Ag directly into the endoplasmic reticulum, where binding to MHC class I molecules is most efficient. Vaccines based on synthetic oligonucleotides could be constructed with ease and rapidity but, most importantly, such constructs avoid the dangers associated with the expression of full length genes encoding TAA that are potentially oncogenic.

Minimal determinant expressed by a recombinant vaccinia virus elicits therapeutic antitumor cytolytic T lymphocyte responses.

Anticancer vaccine strategies can now target intracellular antigens that are involved in the process of malignant transformation, such as oncogene products or mutated tumor suppressor genes. Fragments of these antigens, generally 8-10 amino acids in length and complexed with MHC class I molecules, can be recognized by CD8+ T lymphocytes (TCD8+). To explore the possibility of using a genetically encoded, minimally sized fragment of an intracellular antigen as an immunogen, we constructed a recombinant vaccinia virus encoding an 8-residue peptide derived from chicken ovalbumin that is known to associate with the mouse H-2Kb molecule. Compared to standard methods of immunization, recombinant molecule. Compared to standard methods of immunization, recombinant vaccinia virus expressing the minimal determinant as well as full length ovalbumin were the only approaches that elicited specific primary lytic responses in C57BL/6 mice against E.G7OVA, a transfectant of the murine thymoma EL4 containing the ovalbumin gene. Stimulating these effectors in vitro with OVA257-264 peptide induced H-2Kb-restricted TCD8+ that not only lysed but also specifically secreted IFN-gamma in response to an antigen. Furthermore, when transferred adoptively, these anti-OVA257-264 TCD8+ cells significantly reduced the growth of established ovalbumin-transfected tumors in a pulmonary metastasis model system. Synthetic transfected tumors in a pulmonary metastasis model system. Synthetic oligonucleotides encoding minimal antigenic determinants within expression constructs may be a useful approach for treatment of neoplastic disease, thus avoiding the potential hazards of immunizing with full-length cDNAs that are potentially oncogenic.