Genetic aspects of animal reasoning

This paper reviews the investigations of Prof. L. V. Krushinsky and his colleagues into the genetics of complex behaviors in mammals. The ability of animals to extrapolate the direction of a food stimulus movement was investigated in wild and domesticated foxes (including different fur-color mutants), wild brown rats, and laboratory rats and mice. Wild animals (raised in the laboratory) were shown to be superior to their respective domesticated forms on performance of the extrapolation task, especially in their scores for the first presentation, in which no previous experience could be used. Laboratory rats and mice demonstrated a low level of extrapolation performance. This means that only a few laboratory animals were capable of solving the task, i.e., the percentage of correct solutions was equivalent to chance. The brain weight selection program resulted in two mice strains with a 20% (90-mg) difference in brain weight. Ability to solve the extrapolation task was present in low-brain weight mice in generations 7–11 but declined with further selection. Investigation of extrapolation ability in mice with different chromosomal anomalies demonstrated that animals with Robertsonian translocations Rb(8,17) 1lem and Rb(8,17) 6Sic were capable of solving this task in a statistically significant majority of cases, while mice with fusion of other chromosomes, as well as CBA normal karyotype mice, performed no better than expected by chance. Mice with two types of partial trisomies and animals homo- and heterozygous for translocations were also tested. Although mice with T6 trisomy performed no better than expected by chance, animals with trisomy for a chromosome 17 fragment solved the task successfully. Thus, a genetic component underlying the ability to solve the extrapolation task was demonstrated in three animal species. The extrapolation task in animals is considered to reveal a general capacity for elementary reasoning. The genetic basis of this capacity is very complex.     

Age-related working memory deficits in the allocentric place discrimination task: possible involvement in cholinergic dysfunction

It is well known that learning and memory ability declines with aging. Age-related long-term changes in learning and memory ability in rats were investigated with the place navigation task and the allocentric place discrimination task (APDT) in a water maze using the same animals for each task. In a working memory place navigation task, aged animals could learn the location of the platform as well as when they were young, although strategy shifts were observed. In contrast, accuracy in the APDT significantly declined from 90% to 65% with aging. This impairment was ameliorated by an acetylcholine esterase inhibitor physostigmine at 22–23 months old. No amelioration was, however, detected in the same animals tested when they further aged to 26–27 months old. These results suggest that the APDT performance is sensitive to age-related memory deficits and that this may be due to the cholinergic dysfunction.     

A promiscuous T cell hybridoma restricted to various I-A molecules

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-A^g7) mice, we established a T cell hybridoma, NOE33-1-2, specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-A^g7, but also I-A^{d, s, u and v}. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.     

Codon optimization of the human papillomavirus 11 (HPV 11) L1 gene leads to increased gene expression and formation of virus-like particles in mammalian epithelial cells

The 505 amino acid L1 protein of the human papillomavirus type 11 (HPV 11) is the major capsid polypeptide that has been shown to self-assemble into virus-like particles (VLPs) in vivo and in vitro. While L1 is essential for viral infection, expression studies in mammalian cells have been hampered by different codon preference between the virus and its host. To optimize L1 gene expression in mammalian cells, we converted wild-type HPV 11 L1 (11 L1wt) codons to those more common in human genes. The modified HPV 11 L1 gene (11 L1h) generated protein levels that were at least 100-fold higher than those of wild-type HPV 11 L1, while no obvious differences were seen in the level of mRNA. HPV 11 L1 protein was detected in mammalian epithelial and fibroblast cells, by immunoblotting and indirect immunofluorescence (IIF) techniques. Unlike the situation in situ, IIF revealed the presence of L1 mainly at perinuclear sites. Virus-like particles assembled intranuclearly only to a low extent, as indicated by transmission electron microscopy. DNA vaccination using the HPV 11 L1h gene yielded a drastic increase in L1-specific antibody production in mice as compared to immunization with the wild-type gene.     

HLA-C revisited

During the past 10 years knowledge about the interactions between major histocompatibility complex (MHC) class I molecules and the T-cell receptor (TCR) complex of cytotoxic T-cells (CTL) has developed dramatically. But the primary interest, both with respect to structure as well as function, has concentrated on HLA-A and -B molecules because of their high sequence polymorphism and their dominating presence at the cell surface. In contrast, HLA-C molecules seemed to be of only minor importance in the cascade of immune reactions owing to their more limited polymorphism and reduced levels of surface expression. The inability to define a number of antigen specificities had the result that HLA-C molecules were often neglected in studies of immune response, transplantation, and disease association. More recently a new function has been identified for HLA class I molecules where they act as inhibitors of the lytic capacity of natural killer (NK) cells and non-MHC-restricted T-cells. Moreover, the understanding of this novel mode of negative regulation of cytotoxicity was remarkably influenced by HLA-C since these were the first HLA class I molecules found to have such inhibitory potential. With this new inhibitory function serving as an essential component of the immune system, HLA-C molecules can no longer be neglected.     

Invriant chain peptides enhancing or inhibiting the presentation of antigenic peptides by major histocompatibility complex class II molecules

Two soluble invariant chain (Ii) peptides with overlapping sequences had contrasting effects on the presentation of antigenic peptides by murine A^d, A^k, E^d, and E^k major histocompatibility complex (MHC) class II molecules. Naturally produced class II-associated invariant chain peptides human (h)Ii81–104/murine (m)Ii80–103 inhibited antigen presentation on these MHC class II alleles in a manner consistent with competitive inhibition. The Ii-4 peptides hIi77–92/mIi76–91 enhanced presentation of antigenic peptides on I-E class II alleles by promoting the exchange of peptides at the cell surface. Treatment of antigenpresenting cells (APC) with Ii-4 before the addition of antigenic peptide greatly enhanced subsequent T cell responses, while treatment of APC with Ii–4 after antigenic peptide binding decreased subsequent T cell responses. The hIi81–104 and mIi80–103 peptides inhibited T cell responses in both types of assays. The binding of biotinylated antigenic peptide to MHC class II-transfected L cells, as measured by flow cytometry, was inhibited by mIi80-103 and enhanced by mIi-4. Segments of Ii fragments remaining associated with MHC class II, or released Ii peptides, appear to regulate the formation of stable antigenic peptide/MHC class II complexes either positively or negatively through interactions at or near the antigenic peptide binding site. These findings open a pathway for the design of novel therapeutics based on the structure and function of natural and rationally designed fragments of Ii.     

The growth hormone response to apomorphine at 4 days postpartum in women with a history of major depression

We have examined the responsiveness of dopamine sensitive neurones in the postpartum period in woman with a history of major depression who are at high risk of experiencing a recurrence of illness in the postpartum period. Fourteen women were assessed at 36 weeks of pregnancy and during the 3 months following delivery, using the Schedule for Affective Disorders and Schizophrenia, including its change version. They were not depressed at initial assessment. Five of the 14 women went on to experience a postpartum relapse (2 major depressive disorder, 2 generalised anxiety disorder, 1 panic disorder). On the fourth day postpartum, ie before relapse, the growth hormone response to the dopamine agonist apomorphine was measured as an index of the functional state of hypothalamic dopamine D2 receptors. Women who subsequently relapsed had a significantly greater growth hormone response to apomorphine than those who remained well. This was particularly marked in women with anxiety/panic. The development of increased sensitivity of hypothalamic dopamine D2 receptors in the postpartum period appears to predict the onset of depressive and anxiety disorders.     

Premenstrual tension,expectancy, and mother-child relations

The role of expectancy in enhancing or mitigating premenstrual symptoms was examined in 51 mothers of preschoolers. Expectancy was manipulated by providing information either in support of a biological cause for genuine universal mood changes or in support of a psychological cause arising out of negative societal myths. A third group was given no information. Mood, cognitive function, and mother-child interaction were assessed both at midcycle and premenstrually, and the results suggested that expectancy enhances symptoms. The Psychological group lowered their symptom expectations and reported less negative mood premenstrually as well as fewer symptoms at the end of the test month. The other groups reported no change in symptoms and greater premenstrual negative mood than the Psychological group. All groups demonstrated greater task persistence and more positive motherchild interaction during the premenstrual phase.This study is based upon the doctoral dissertation completed in partial fulfillment of the Ph.D. degree from the University of Ottawa.     

Presentation of viral antigens restricted by H-2Kb,Db or Kd in proteasome subunit LMP2-and LMP7-deficient cells

In the class II region of the major histocompatibility complex (MHC), four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP 1 and TAP 2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP 2 and LMP 7) code for subunits of the proteasome. While TAP 1 and TAP 2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP 2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP 1/2 and LMP 2/7 genes, it was recently shown that expression of TAP 1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP 2/7, we transfected T2 cells with TAP 1, TAP 2 and either of the H-2K^b, D^b or K^d genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP 1/2 gene products, presented all tested viral antigens restricted through either the H-2K^b, D^b or K^d class I molecules. We conclude that the proteasome subunits LMP 2/7 as well as other gene products in the MHC class II region, except from TAP 1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP 2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.     

Genes encoded in the major histocompatibility complex affecting the generation of peptides for TAP transport

The B cell line 721.174 has lost the ability to present intracellular antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). This phenotype results from a homozygous deletion in the MHC that includes the peptide transporter genes TAP1 and TAP2, and the proteasome subunits LMP2 and LMP7. Recent work has shown that such cells transfected with TAP genes load their class I molecules with endogenous peptides, and present several viral epitopes to class I-restricted CTL. These data implied that the LMP2 and LMP7 genes were not required for the presentation of most epitopes through class I molecules. By contrast, while confirming the previous reports, we have identified several epitopes that appear to require genes in the MHC in addition to the TAP for their presentation. Further analysis localizes the defect to proteolysis in the cytosol. In one case, presentation could be partially restored by re-expression of full-length LMP7. Control experiments with LMP7, from which the putative pro-region had been removed, failed to restore presentation, and this lack of effect correlated with failure of the shortened LMP7 to incorporate into the proteasome. These results suggest a role for LMP7 in the generation of a viral epitope, but leave open the possibility that additional genes within the .174 deletion are required for full restoration of antigen presentation.