Genetics and ontogeny of butyryl CoA dehydrogenase in the mouse and linkage of Bcd-1 with Dao-1

A zymogram method has been developed for fatty acyl CoA dehydrogenase and used to examine the electrophoretic properties of butyryl CoA dehydrogenase (BCD) from mouse tissues. A single form of BCD is present in extracts of liver, kidney, heart, and intestine. Ontogenetic, tissue distribution, and subcellular fractionation results are consistent with the mitochondrial origin previously reported for this enzyme. A genetic variant for BCD-1 was used to provide evidence for a locus determining the electrophoretic properties of this enzyme (designated Bcd-1), which is linked to Dao-1 (encoding d-amino acid oxidase).This research was funded in part by the Australian Research Grants Committee.     

Elimination of an immunodominant CD4+ T cell epitope in human IFN-beta does not result in an in vivo response directed at the subdominant epitope

The BALB/cByJ mouse strain displays an immunodominant T cell response directed at the same CD4(+) T cell epitope peptide region in human IFN-beta, as detected in a human population-based assay. BALB/cByJ mice also recognize a second region of the protein with a lesser magnitude proliferative response. Critical residue testing of the immunodominant peptide showed that both BALB/cByJ mice and the human population response were dependent on an isoleucine residue at position 129. A variant IFN-beta molecule was constructed containing the single amino acid modification, I129V, in the immunodominant epitope. The variant displayed 100% of control antiproliferation activity. Mice immunized with unmodified IFN-beta responded weakly in vitro to the I129V variant. However, BALB/cByJ mice immunized with the I129V variant were unable to respond to either the I129V variant or the unmodified IFN-beta molecule by either T cell proliferation or Ag-specific IgG1 Ab production. This demonstrates that a single amino acid change in an immunodominant epitope can eliminate an immune response to an otherwise intact therapeutic protein. The elimination of the immunodominant epitope response also eliminated the response to the subdominant epitope in the protein. Modifying functionally immunodominant T cell epitopes within proteins may obviate the need for additional subdominant epitope modifications.     

Inhibition of nitric oxide synthesis increases mortality in Sindbis virus encephalitis.

Sindbis virus (SV) is an alphavirus that causes acute encephalomyelitis in mice. The outcome is determined by the strain of virus and by the age and genetic background of the host. The mortality rates after infection with NSV, a neurovirulent strain of SV, were as follows v: 81% (17 of 21) in BALB/cJ mice; 20% (4 of 20) in BALB/cByJ mice (P < 0.001); 100% in A/J, C57BL/6J, SJL, and DBA mice; and 79% (11 of 14) in immunodeficient scid/CB17 mice. Treatment with Nomega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthetase (NOS) inhibitor, increased mortality to 100% (P < 0.05) in NSV-infected BALB/cJ mice, to 95% (P < 0.001) in BALB/cByJ mice, and to 100% in scid/CB17 mice. BALB/cJ and BALB/cByJ mice had similar levels of inducible NOS mRNA in their brains, which were not affected by L-NAME or NSV infection. Brain NOS activity was similar in BALB/cJ and BALB/cByJ mice before and after infection and was markedly inhibited by L-NAME. NSV replication in the brains of BALB/cJ mice, BALB/cByJ mice, and mice treated with L-NAME was similar. Treatment of N18 neuroblastoma cells with NO donors S-nitroso-N-acetylpenicillamine or sodium nitroprusside in vitro before infection increased cell viability at 42 to 48 h compared with untreated NSV-infected N18 cells with little effect on virus replication. These data suggest that NO protects mice from fatal encephalitis by a mechanism that does not directly involve the immune response or inhibition of virus growth but rather may enhance survival of the infected neuron until the immune response can control virus replication.     

Hepatic triglyceride contents are genetically determined in mice: results of a strain survey

To assess whether genetic factor(s) determine liver triglyceride (TG) levels, a 10-mouse strain survey of liver TG contents was performed. Hepatic TG contents were highest in BALB/cByJ, medium in C57BL/6J, and lowest in SWR/J in both genders. Ninety and seventy-six percent of variance in hepatic TG in males and females, respectively, was due to strain (genetic) effects. To understand the physiological/biochemical basis for differences in hepatic TG among the three strains, studies were performed in males of the BALB/cByJ, C57BL/6J, and SWR/J strains. In vivo hepatic fatty acid (FA) synthesis rates and hepatic TG secretion rates ranked BALB/cByJ approximately C57BL/6J > SWR/J. Hepatic 1-(14)C-labeled palmitate oxidation rates and plasma beta-hydroxybutyrate concentrations ranked in reverse order: SWR/J > BALB/cByJ approximately C57BL/6J. After 14 h of fasting, plasma-free FA and hepatic TG contents rose most in BALB/cByJ and least in SWR/J. beta-Hydroxybutyrate concentrations rose least in BALB/cByJ and most in SWR/J. Adaptation to fasting was most effective in SWR/J and least in BALB/cByJ, perhaps because BALB/cByJ are known to be deficient in SCAD, a short-chain FA oxidizing enzyme. To assess the role of insulin action, glucose tolerance test (GTT) was performed. GTT-glucose levels ranked C57BL/6J > BALB/cByJ approximately SWR/J. Thus strain-dependent (genetic) factors play a major role in setting hepatic TG levels in mice. Processes such as FA production and hepatic export in VLDL on the one hand and FA oxidation on the other, explain some of the strain-related differences in hepatic TG contents. Additional factor(s) in the development of fatty liver in BALB/cByJ remain to be demonstrated.     

Improved electrocompetence and metabolic engineering of Clostridium pasteurianum reveals a new regulation pattern of glycerol fermentation

Clostridium pasteurianum produces industrially valuable chemicals such as n‐butanol and 1,3‐propanediol from fermentations of glycerol and glucose. Metabolic engineering for increased yields of selective compounds is not well established in this microorganism. In order to study carbon fluxes and to selectively increase butanol yields, we integrated the latest advances in genome editing to obtain an electrocompetent Clostridium pasteurianum strain for further engineering. Deletion of the glycerol dehydratase large subunit (dhaB) using an adapted S. pyogenes Type II CRISPR/Cas9 nickase system resulted in a 1,3‐propanediol‐deficient mutant producing butanol as the main product. Surprisingly, the mutant was able to grow on glycerol as the sole carbon source. In spite of reduced growth, butanol yields were highly increased. Metabolic flux analysis revealed an important role of the newly identified electron bifurcation pathway for crotonyl‐CoA to butyryl‐CoA conversion in the regulation of redox balance. Compared to the parental strain, the electron bifurcation pathway flux of the dhaB mutant increased from 8 to 46% of the overall flux from crotonyl‐CoA to butyryl‐CoA and butanol, indicating a new, 1,3‐propanediol‐independent pattern of glycerol fermentation in Clostridium pasteurianum.     

The role of acetyl coenzyme A: butyrate coenzyme A in the transferase uptake of butyrate by isolated membrane vesicles of Escherichia coli

The acetyl CoA:butyrate CoA transferase catalyzes the translocation of butyrate in membrane vesicles prepared from a strain of Escherichia coli which is depressed for the acetoacetate degradation operon. Butyrate accumulated in the membranes as butyryl CoA. The role of the transferase in uptake is supported by the following observations: (i) uptake is stimulated by acetyl CoA; (ii) the solubilized CoA transferase and uptake exhibit K_mS for butyrate, pH optima and levels inhibition by N-ethylmaleimide that are virtually identical; (iii) significant amounts of the CoA transferase are found associated with the membranes and uptake is rapidly inhibited by butyryl CoA and acetate, the products of the CoA transferase-catalyzed reaction. The fact that butyrate uptake did not exhibit saturation kinetics with increasing concentrations of acetyl CoA suggested that the transferase is not localized on the outer surface of the membrane. The level of free butyrate in the vesicles, the fact that butyrate uptake exhibited saturation kinetics with increasing concentrations of butyrate, and the observation that radioactivity was not rapidly lost from the vesicles following addition of butyryl CoA or acetate to incubation mixtures indicated that butyrate is translocated rather than trapped by the CoA transferase.     

Experimental allergic orchitis in mice

Inbred strains of mice were studied for their susceptibility to the induction of experimental allergic orchitis after sensitization with mouse testicular homogenate in complete Freund's adjuvant accompanied by injections of extract from Bordetella pertussis. Susceptibility to autoimmune orchitis was found to be linked to the major histocompatibility complex in BALB/c and C57BL/10 mice and mapped to genes encoded within the H-2D ^dregion. In five of six groups of bidirectional (susceptible × resistant) F₁ hybrids, H-2D ^d-linked susceptibility was inherited as a dominant autosomal trait. However, in (BALB/cByJ × DBA/2J)F₁ and (DBA/2J × BALB/cByJ)F₁ hybrids, dominant autosomal resistance to the induction of autoimmune orchitis was observed. Backcross analysis between the resistant F₁ hybrid and the susceptible BALB/cByJ parent suggests that a single independently segregating DBA/2J locus is capable of negating H-2D ^d-linked susceptibility, and controls resistance to the induction of autoimmune orchitis.Abbreviations used in this paper BP extract Bordetella pertussis extract - CFA complete Freund's adjuvant - EAO experimental allergic orchitis - Ir immune response - MHC major histocompatibility complex - MLH mouse liver homogenate - MTH mouse testis homogenate - PI pathology index     

A new mouse model reveals a critical role for host innate immunity in resistance to Rift Valley fever

Rift Valley fever (RVF) is an arthropod-borne viral disease repeatedly reported in many African countries and, more recently, in Saudi Arabia and Yemen. RVF virus (RVFV) primarily infects domesticated ruminants, resulting in miscarriage in pregnant females and death for newborns and young animals. It also has the ability to infect humans, causing a feverish syndrome, meningoencephalitis, or hemorrhagic fever. The various outcomes of RVFV infection in animals and humans argue for the existence of host genetic determinants controlling the disease. We investigated the susceptibility of inbred mouse strains to infection with the virulent RVFV ZH548 strain. Compared with classical BALB/cByJ mice, wild-derived Mus m. musculus MBT/Pas mice exhibited earlier and greater viremia and died sooner, a result in sharp contrast with their resistance to infection with West Nile virus and influenza A. Infection of mouse embryonic fibroblasts (MEFs) from MBT/Pas mice with RVFV also resulted in higher viral production. Microarray and quantitative RT-PCR experiments showed that BALB/cByJ MEFs displayed a significant activation of the type I IFN pathway. In contrast, MBT/Pas MEFs elicited a delayed and partial type I IFN response to RVFV infection. RNA interference-mediated inhibition of genes that were not induced by RVFV in MBT/Pas MEFs increased viral production in BALB/cByJ MEFs, thus demonstrating their functional importance in limiting viral replication. We conclude that the failure of MBT/Pas murine strain to induce, in due course, a complete innate immune response is instrumental in the selective susceptibility to RVF.     

BALB/c mice genetically susceptible to proteoglycan-induced arthritis and spondylitis show colony-dependent differences in disease penetrance

Introduction

The major histocompatibility complex (H-2d) and non-major histocompatibility complex genetic backgrounds make the BALB/c strain highly susceptible to inflammatory arthritis and spondylitis. Although different BALB/c colonies develop proteoglycan-induced arthritis and proteoglycan-induced spondylitis in response to immunization with human cartilage proteoglycan, they show significant differences in disease penetrance despite being maintained by the same vendor at either the same or a different location.

Methods

BALB/c female mice (24 to 26 weeks old after 4 weeks of acclimatization) were immunized with a suboptimal dose of cartilage proteoglycan to explore even minute differences among 11 subcolonies purchased from five different vendors. In vitro-measured T-cell responses, and serum cytokines and (auto)antibodies were correlated with arthritis (and spondylitis) phenotypic scores. cDNA microarrays were also performed using spleen cells of naïve and immunized BALB/cJ and BALB/cByJ mice (both colonies from The Jackson Laboratory, Bar Harbor, ME, USA), which represent the two major BALB/c sublines.

Results

The 11 BALB/c colonies could be separated into high (n = 3), average (n = 6), and low (n = 2) responder groups based upon their arthritis scores. While the clinical phenotypes showed significant differences, only a few immune parameters correlated with clinical or histopathological abnormalities, and seemingly none of them affected differences found in altered clinical phenotypes (onset time, severity or incidence of arthritis, or severity and progression of spondylitis). Affymetrix assay (Affymetrix, Santa Clara, CA, USA) explored 77 differentially expressed genes (at a significant level, P < 0.05) between The Jackson Laboratory's BALB/cJ (original) and BALB/cByJ (transferred from the National Institutes of Health, Bethesda, MD, USA). Fourteen of the 77 differentially expressed genes had unknown function; 24 of 77 genes showed over twofold differences, and only 8 genes were induced by immunization, some in both colonies.

Conclusions

Using different subcolonies of the BALB/c strain, we can detect significant differences in arthritis phenotypes, single-nucleotide polymorphisms (SNPs), and a large number of differentially expressed genes, even in non-immunized animals. A number of the known genes (and SNPs) are associated with immune responses and/or arthritis in this genetically arthritis-prone murine strain, and a number of genes of as-yet-unknown function may affect or modify clinical phenotypes of arthritis and/or spondylitis.     

LC-MS/MS quantification of short-chain acyl-CoA's in Escherichia coli demonstrates versatile propionyl-CoA synthetase substrate specificity

Aims: This paper utilized quantitative LC‐MS/MS to profile the short‐chain acyl‐CoA levels of several strains of Escherichia coli engineered for heterologous polyketide production. To further compare and potentially expand the levels of available acyl‐CoA molecules, a propionyl‐CoA synthetase gene from Ralstonia solanacearum (prpE‐RS) was synthesized and expressed in the engineered strain BAP1. Methods and Results: Upon feeding propionate, the engineered E. coli strains had increased the levels of both propionyl‐ and methylmalonyl‐CoA of 6‐ to 30‐fold and 3·7‐ to 6·8‐fold, respectively. Expression of prpE‐RS resulted in no significant increases in acetyl‐, butyryl‐ and propionyl‐CoA when fed the corresponding substrates (sodium acetate, butyrate or propionate). More interesting, however, were the results from strain BAP1 engineered for native prpE overexpression, which indicated increases in the same range of acyl‐CoA formation. Conclusions: The increased acyl‐CoA levels across the strains profiled in this study reflect the genetic modifications implemented for improved polyketide production and also indicate flexibility of the native PrpE. Significance and Impact of the Study: The results provide direct evidence of enhanced acyl‐CoA levels correlating to those strains engineered for polyketide biosynthesis. This information and the inherent flexibility of the native PrpE enzyme support future efforts to characterize, engineer and extend acyl‐CoA precursor supply for additional heterologous biosynthetic attempts.     

Francisella tularensis Vaccines Elicit Concurrent Protective T- and B-Cell Immune Responses in BALB/cByJ Mice

In the last decade several new vaccines against Francisella tularensis, which causes tularemia, have been characterized in animal models. Whereas many of these vaccine candidates showed promise, it remains critical to bridge the preclinical studies to human subjects, ideally by taking advantage of correlates of protection. By combining in vitro intramacrophage LVS replication with gene expression data through multivariate analysis, we previously identified and quantified correlative T cell immune responses that discriminate vaccines of different efficacy. Further, using C57BL/6J mice, we demonstrated that the relative levels of gene expression vary according to vaccination route and between cell types from different organs. Here, we extended our studies to the analysis of T cell functions of BALB/cByJ mice to evaluate whether our approach to identify correlates of protection also applies to a Th2 dominant mouse strain. BALB/cByJ mice had higher survival rates than C57BL/6J mice when they were immunized with suboptimal vaccines and challenged. However, splenocytes derived from differentially vaccinated BALB/cByJ mice controlled LVS intramacrophage replication in vitro in a pattern that reflected the hierarchy of protection observed in C57BL/6J mice. In addition, gene expression of selected potential correlates revealed similar patterns in splenocytes of BALB/cByJ and C57BL/6J mice. The different survival patterns were related to B cell functions, not necessarily to specific antibody production, which played an important protective role in BALB/cByJ mice when vaccinated with suboptimal vaccines. Our studies therefore demonstrate the range of mechanisms that operate in the most common mouse strains used for characterization of vaccines against F. tularensis, and illustrate the complexity necessary to define a comprehensive set of correlates.     

Diversity of human colonic butyrate‐producing bacteria revealed by analysis of the butyryl‐CoA:acetate CoA‐transferase gene

Butyrate‐producing bacteria play an important role in the human colon, supplying energy to the gut epithelium and regulating host cell responses. In order to explore the diversity and culturability of this functional group, we designed degenerate primers to amplify butyryl‐CoA:acetate CoA‐transferase sequences from faecal samples provided by 10 healthy volunteers. Eighty‐eight per cent of amplified sequences showed > 98% DNA sequence identity to CoA‐transferases from cultured butyrate‐producing bacteria, and these fell into 12 operational taxonomic units (OTUs). The four most prevalent OTUs corresponded to Eubacterium rectale, Roseburia faecis, Eubacterium hallii and an unnamed cultured species SS2/1. The remaining 12% of sequences, however, belonged to 20 OTUs that are assumed to come from uncultured butyrate‐producing strains. Samples taken after ingestion of inulin showed significant (P = 0.019) increases in Faecalibacterium prausnitzii. Because several of the dominant butyrate producers differ in their DNA % G+C content, analysis of thermal melt curves obtained for PCR amplicons of the butyryl‐CoA:acetate CoA‐transferase gene provides a convenient and rapid qualitative assessment of the major butyrate producing groups present in a given sample. This type of analysis therefore provides an excellent source of information on functionally important groups within the colonic microbial community.     

Taenia taeniaeformis: evasion of complement-mediated lysis by early larval stages following activation of the alternative pathway

Activation of the alternative pathway of complement by T. taeniaeformis oncospheres and early stage metacestodes, although a factor in host defense against primary infection, does not directly lead to the killing of the parasite larvae observed prior to day 6 post-infection in innately resistant BALB/cByJ inbred mice. Immunogold labelling techniques clearly demonstrated tegument-associated C3 on in vitro-activated oncospheres incubated with non-immune mouse sera. However, C5, a protease necessary for the assembly of the membrane attack complex, was not detected. Early stage larvae cultured from in vitro-activated oncospheres escaped membrane damage and survived incubation in non-immune sera from both BALB/cByJ and taeniid-susceptible C3H/HeDub mice. Comparisons of cobra venom factor-treated and untreated C5-deficient B10.D2osn mice revealed no significant differences in parasite burden and local eosinophil infiltration at 6 days post-infection, suggesting that the terminal arm of the complement system is necessary for the previously reported role of complement in resistance to primary infection in BALB/cByJ and C3H/HeDub mice. An in vivo test of chemotaxis indicated that although both complement-intact mouse strains examined responded to intraperitoneal injections of inulin, there were lower numbers of eosinophils in C3H/HeDub mice than in BALB/cByJ mice, perhaps pointing to possible mouse strain differences in C5a generation/catabolism or eosinophil ability to respond to C5a. Lectin-binding studies showed an affinity of PNA for the exposed surface of taeniid oncospheres and 4-day post-infection metacestodes; however, binding of lectin to the carbohydrate moiety did not inhibit complement activation.     

Genetic Factors Modulate the Impact of Pubertal Androgen Excess on Insulin Sensitivity and Fertility

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. The syndrome is caused by a combination of environmental influences and genetic predisposition. Despite extensive efforts, the heritable factors contributing to PCOS development are not fully understood. The objective of this study was to test the hypothesis that genetic background contributes to the development of a PCOS-like reproductive and metabolic phenotype in mice exposed to excess DHEA during the pubertal transition. We tested whether the PCOS phenotype would be more pronounced on the diabetes-prone C57BL/6 background than the previously used strain, BALB/cByJ. In addition, we examined strain-dependent upregulation of the expression of ovarian and extra-ovarian candidate genes implicated in human PCOS, genes containing known strain variants, and genes involved with steroidogenesis or insulin sensitivity. These studies show that there are significant strain-related differences in metabolic response to excess androgen exposure during puberty. Additionally, our results suggest the C57BL/6J strain provides a more robust and uniform experimental platform for PCOS research than the BALB/cByJ strain.     

Physiological activity of E. coli engineered to produce butyric acid

Faecalibacterium prausnitzii (F. prausnitzii) is one of the most abundant bacteria in the human intestine, with its anti-inflammatory effects establishing it as a major effector in human intestinal health. However, its extreme sensitivity to oxygen makes its cultivation and physiological study difficult. F. prausnitzii produces butyric acid, which is beneficial to human gut health. Butyric acid is a short-chain fatty acid (SCFA) produced by the fermentation of carbohydrates, such as dietary fibre in the large bowel. The genes encoding butyryl-CoA dehydrogenase (BCD) and butyryl-CoA:acetate CoA transferase (BUT) in F. prausnitzii were cloned and expressed in E. coli to determine the effect of butyric acid production on intestinal health using DSS-induced colitis model mice. The results from the E. coli Nissle 1917 strain, expressing BCD, BUT, or both, showed that BCD was essential, while BUT was dispensable for producing butyric acid. The effects of different carbon sources, such as glucose, N-acetylglucosamine (NAG), N-acetylgalactosamine (NAGA), and inulin, were compared with results showing that the optimal carbon sources for butyric acid production were NAG, a major component of mucin in the human intestine, and glucose. Furthermore, the anti-inflammatory effects of butyric acid production were tested by administering these strains to DSS-induced colitis model mice. The oral administration of the E. coli Nissle 1917 strain, carrying the expression vector for BCD and BUT (EcN-BCD-BUT), was found to prevent DSS-induced damage. Introduction of the BCD expression vector into E. coli Nissle 1917 led to increased butyric acid production, which improved the strain’s health-beneficial effects.     

Experimental allergic orchitis in mice. V. Resistance to actively induced disease in BALB/cJ substrain mice is mediated by CD4 + T cells

Previous studies have shown that differential susceptibility to actively induced experimental allergic orchitis (EAO) exists among various BALB/c substrains. Of 13 substrains studied, BALB/cJ mice consistently exhibit greater resistance to disease induction. Such resistance is associated with a single recessive genotypic difference in an immunoregulatory locus which is unlinked to any of the known alleles distinguishing the BALB/cJ substrain. In this study, gene complementation protocols were used to study the genetics of susceptibility and resistance to EAO. The results indicate that resistance in BALB/cJ mice is not due to a mutation in theH-2D ^d linked gene which governs the phenotypic expression of autoimmune orchitis. The mechanistic basis for disease resistance was examined using reciprocal bone marrow radiation chimeras generated between the disease-susceptible BALB/ cByJ (ByJ) substrain and BALB/cJ (Jax) mice. All constructs, including Jax - Jax and Jax - ByJ, developed severe EAO following inoculation with mouse testicular homogenate (MTH) and adjuvants whereas control chimeras immunized with adjuvants alone did not. These results suggest that an active immunoregulatory mechanism rather than a passive one, such as the lack of T cells and/or B cells with receptors for the aspermatogenic autoantigens relevant in the induction of EAO, is responsible for disease resistance in BALB/cJ mice. The role of immunoregulatory cells was examined by pretreating BALB/cJ mice with either cyclophosphamide (20 mg/kg) or low-dose whole body or total lymphoid irradiation (350 rads) 2 days prior to inoculation. BALB/cJ mice immunized with MTH plus adjuvants generate immunoregulatory spleen cells (SpCs) that, when transferred to naive BALB/cByJ recipients, significantly reduce the severity of autoimmune orchitis observed during actively induced EAO. Treatment of such cells with either cytotoxic monoclonal anti-Thy-1.2 or anti-CD4 plus C' before transfer abrogates the ability of BALB/cJ spleen cells to inhibit disease. In contrast, neither SpCs from adjuvantimmunized BALB/cJ nor MTH plus adjuvant-primed BALB/cByJ donors significantly influenced the severity of disease observed in recipients. Taken together, these results suggest that genetically controlled resistance to EAO in BALB/cJ mice is associated with a mutation in an immunoregulatory locus whose effects appear to be mediated through a cyclophosphamide and low-dose radiation-sensitive CD4⁺ T-cell population.     

Estrous cycle effects on behavior of C57BL/6J and BALB/cByJ female mice: implications for phenotyping strategies

Systematic behavioral phenotyping of genetically modified mice is a powerful method with which to identify the molecular factors implicated in control of animal behavior, with potential relevance for research into neuropsychiatric disorders. A number of such disorders display sex differences, yet the use of female mice in phenotyping strategies has been a rare practice because of the potential variability related to the estrous cycle. We have now investigated the behavioral effects of the estrous cycle in a battery of behavioral tests in C57BL/6J and BALB/cByJ inbred strains of mice. Whereas the performance of BALB/cByJ female mice varied significantly depending on the phase of the estrous cycle in the open field, tail flick and tail suspension tests, the behavior of C57BL/6J females, with the exception of the tail suspension performance, remained stable across all four phases of the estrous cycle in all of the tests including open field, rotarod, startle reflex and pre-pulse inhibition, tail flick and hot plate. We also found that irrespective of the estrous cycle, the behavior of C57BL/6J females was different from that of BALB/cByJ groups in all of the behavioral paradigms. Such strain differences were previously reported in male comparisons, suggesting that the same inter-group differences can be revealed by studying female or male mice. In addition, strain differences were evident even for behaviors that were susceptible to estrous cycle modulations, although their detection might necessitate the constitution of large experimental groups.     

Inheritance of retarded forebrain commissure development in fetal mice: results from classical crosses and recombinant inbred strains

Deficiency of the adult corpus callosum in BALB/c mice shows incomplete penetrance and is clearly polygenic, whereas the defect in fetuses shows complete penetrance and a much less complex mode of inheritance. Retardation of the growth of the corpus callosum and the hippocampal commissure in the fetal mouse forebrain was expressed by a standard score (z) derived from body weight, such that a fetus with a score less than -2.0 was held to have commissures abnormally small for the body size. By this index, almost all C57BL/6 fetuses were normal, whereas BALB/c fetuses in the body weight range 0.5 to 1.0 g were often 5 standard deviations below the expected value of 0.0. In classical crosses between C57BL/6J and BALB/cWah, inheritance of the index of abnormality (z) was recessive, and about half of the fetuses in backcrosses to BALB/c were below -2.0. However, the distribution of scores was not bimodal. The results were consistent with a two-locus but not a single-locus difference between parent strains. Among the seven recombinant inbred strains derived from the By strains of C57BL/6 and BALB/c, there were three or possibly four distinct clusters of strains, which also suggested two-locus inheritance and excluded a single-locus difference. Although substantial retardation of commissure growth was evident in fetuses, deficiency or absence of the corpus callosum in weanling and adult By recombinant inbred mice was extremely rare in all strains except BALB/cByJ. These data confirm anatomical results showing that, in all but the most extremely retarded cases, the corpus callosum recovers from an obvious prenatal defect.     

Kinetics of ectromelia virus (mousepox) transmission and clinical response in C57BL/6j, BALB/cByj and AKR/J inbred mice

Research was undertaken to answer basic questions on susceptibility, clinical response and transmission of ectromelia virus in selected strains of inbred mice. C57BL/6J and AKR/J were found to be markedly more resistant to a virulent strain of ectromelia virus (isolated during the 1979-80 outbreak at the National Institutes of Health) than C57LJ, BALB/cByJ, DBA/2J, A.By/SNJ and C3H/HeJ when infected by footpad inoculation. In C57BL/6J and AKR/J the LD50 was about 7 logs higher than the ID50. With one exception, C57LJ, the LD50 and ID50 titers in the other strains were about equal. In C57LJ the LD50 titer was intermediate. Following intragastric inoculation, virus was isolated from feces of C57BL/6J mice for as long as 46 days and up to 29 days from BALB/cByJ mice. Transmission to cage mates from intragastrically infected C57BL/6J and BALB/cByJ occurred up to 36 and 30 days respectively after infection. Virus was isolated from the spleen in 2 of 5 BALB/cByJ mice and 1 of 7 C57BL/6J mice tested 95 days after gastric inoculation. Following footpad inoculation, BALB/cByJ mice consistently transmitted virus to cage mates before death at 10-12 days. C57BL/6J mice transmitted between days 8 and 17, but not beyond. Virus was maintained in C57BL/6J mice by exposure to infected cage mates for seven passages, which was the most attempted. Clinical signs in infected C57BL/6J mice were usually subtle or inapparent.