Improvement of production yield and extraction efficacy of recombinant protein by high endosperm-specific expression along with simultaneous suppression of major seed storage proteins.

Human transforming growth factor-ß1 (hTGF-ß1)　was produced in transgenic rice seeds. To boost its production yield and to extract it simply, it was expressed under the control of seed-specific promoters along with the simultaneous suppression of endogenous seed storage proteins　(SSPs)　through RNA interference (RNAi). When driven by the 26 kDa α-globulin endosperm-specific promoter, it accumulated up to the markedly high level of 452 µg/grain. However, exchange with other seed-specific promoters such as 18 kDa oleosin and AGPase promoters resulted in remarkable reduction to the levels of 62 and 48 µg/grain, respectively, even though endogenous SSPs were reduced to the similar level. These production levels were almost similar to those (42 and 108 µg/grain) produced by the glutelin GluB-1 endosperm-specific promoter and the maize ubiquitin constitutive promoter without reduction of SSPs, respectively. When extracted from these transgenic rice seeds with reduced SSPs with various buffers, it could be solubilized with denaturant solution, which was in remarkable contrast with those without depressed SSPs which required further supplementation of reducing agent for extraction. This difference was associated with the fact that it was mainly deposited to ER-derived structures though self-aggregation or interaction with remaining prolamin via intermolecular disulfide bonds.

Expression and Purification of Recombinant Mouse Interleukin-4 and -6 from Transgenic Rice Seeds.

Transgenic rice seed can be utilized as a bioreactor to produce high-value recombinant proteins. Mouse interleukin 4 (mIL-4) and mIL-6 were specifically expressed as secretory proteins in rice endosperm by ligating the N-terminal glutelin B-1 (GluB-1) signal peptide and the C-terminal KDEL endoplasmic reticulum retention signal under control of the endosperm-specific GluB-1 promoter. In the transgenic rice seed, mIL-4 and mIL-6 accumulated in levels up to 0.43 mg/g grain and 0.16 mg/g grain, respectively. The reducing agents and detergents required for extraction from the transgenic rice seeds differed between the two proteins, indicating differences in their intracellular localization within the endosperm cell. Purified mIL-4 and mIL-6 exhibited high activity and very low endotoxin contamination.

Concentrated protein body product derived from rice endosperm as an oral tolerogen for allergen-specific immunotherapy--a new mucosal vaccine formulation against Japanese cedar pollen allergy.

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.

Endovascular injury induces rapid phenotypic changes in perivascular adipose tissue.

OBJECTIVE: Accumulating evidence suggests that adipose tissue not only stores energy but also secretes various bioactive substances called adipocytokines. Periadventitial fat is distributed ubiquitously around arteries throughout the body. It was reported that inflammatory changes in the periadventitial fat may have a direct role in the pathogenesis of vascular diseases accelerated by obesity. We investigated the effect of endovascular injury on the phenotype of perivascular fat. METHODS AND RESULTS: Endovascular injury significantly upregulated proinflammatory adipocytokines and downregulated adiponectin within periadventitial fat tissue in models of mouse femoral artery wire injury and rat iliac artery balloon injury. Genetic disruption of tumor necrosis factor (TNF)-alpha attenuated upregulation of proinflammatory adipocytokine expression, with reduced neointimal hyperplasia after vascular injury. Local delivery of TNF-alpha to the periadventitial area enhanced inflammatory adipocytokine expression, which was associated with augmented neointimal hyperplasia in TNF-alpha-deficient mice. Conditioned medium from a coculture of 3T3-L1 and RAW264 cells stimulated vascular smooth muscle cell proliferation. An anti-TNF-alpha neutralizing antibody in the coculture abrogated the stimulating effect of the conditioned medium. CONCLUSIONS: Our findings indicate that endovascular injury induces rapid and marked changes in perivascular adipose tissue, mainly mediated by TNF-alpha. It is suggested that the phenotypic changes in perivascular adipose tissue may have a role in the pathogenesis of neointimal hyperplasia after angioplasty.

Extraction and purification of human interleukin-10 from transgenic rice seeds.

Recombinant protein production system using transgenic rice grain offers many advantages in higher accumulation, preservation, lower production cost, ease of scale up and low risk of contamination by toxic materials. We developed a transgenic rice strain whose seeds accumulate human interleukin (IL)-10, a cytokine that suppresses inflammation-related immune responses. We also developed a method of extracting and purifying IL-10 from rice seeds. A biochemical crosslinking method was used to detect the biologically active noncovalent dimer of IL-10. This method was useful for developing efficient methods of refolding and purification. The purified IL-10 comprised only noncovalent dimers and showed higher activity than the commercial IL-10. The purified IL-10 had very low endotoxin contamination and is expected to have broad clinical application.

Tumor necrosis factor alpha is not a pathogenic determinant in acute lethal encephalitis induced by a highly neurovirulent strain of mouse hepatitis virus.

To investigate the role of tumor necrosis factor alpha (TNFalpha) in the pathogenesis of acute viral encephalitis, TNFalpha-deficient mice were infected with a highly neurovirulent strain of mouse hepatitis virus, JHM, and compared with JHM-infected C57BL/6 mice as controls. All the JHM-infected mice had succumbed to infection by 6 days postinfection. The virus replication kinetics, histopathological changes and mRNA expression levels of proinflammatory cytokines in the brain did not differ between TNFalpha-deficient and control C57BL/6 mice. These results suggest that TNFalpha is not a pathogenic determinant in JHM-induced acute lethal encephalitis.

Negative feedback regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by heme oxygenase-1 induction in macrophages.

Heme oxygenase-1 (HO-1) is induced under infectious diseases in macrophages. We performed experiments using various gene deficient mouse-derived macrophages to determine a detailed induction mechanism of HO-1 by lipopolysaccharide (LPS) and the functional role of HO-1 induction in macrophages. LPS (1 microg/mL) maximally induced inducible nitric oxide synthase (iNOS) and HO-1 mRNAs in wild-type (WT) macrophages at 6h and 12h after treatment, respectively, and liberated tumor necrosis factor alpha (TNFalpha) from WT macrophages. LPS also induced iNOS and HO-1 in TNFalpha(-/-) macrophages, but not in iNOS(-/-) macrophages. Interestingly, although LPS strongly induced iNOS, it failed to induce HO-1 almost completely in nuclear-factor erythroid 2-related factor 2 (Nrf2)(-/-) macrophages. The LPS-induced iNOS gene expression was suppressed by pretreatment with HO-1 inducers, hemin and Co-protoporphyrin (CoPP), but not with HO-1 inhibitor, Sn-protoporphyrin in WT macrophages. In the Nrf2(-/-) macrophages, the ability of CoPP to induce HO-1 and its inhibitory effect on the LPS-induced iNOS gene expression were lower than seen in WT macrophages. The present findings suggest that HO-1 is induced via NO-induced nuclear translocation of Nrf2, and the enzymatic function of HO-1 inhibits the overproduction of NO in macrophages.

Impaired LPS-induced signaling in microglia overexpressing the Wiskott-Aldrich syndrome protein N-terminal domain.

Wiskott-Aldrich syndrome protein (WASP) plays important roles in TCR signaling, but its roles in signal transduction in innate immune cells have not been well characterized. As microglia are the primary immune effector cells in the brain, WASP may possibly have important roles in microglial activation, such as production of inflammatory and anti-inflammatory cytokines and neurotoxic factors. Here, we established a microglial cell line from WASP dominant-negative transgenic (Tg) mice overexpressing the N-terminal enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domain. WASP Tg microglia were impaired in production of inflammatory cytokines such as tumor necrosis factor-alpha, IL-6 and IL-1beta upon LPS stimulation, whereas anti-inflammatory IL-10 production was significantly enhanced. Also, LPS-induced phosphorylation of nuclear factor kappaB was reduced in WASP Tg microglia. Furthermore, WASP Tg microglia exhibited less cytotoxicity against co-cultured neurons after stimulation by LPS and IFN-gamma, with a concordant decrease in nitric oxide production. These results strongly suggest that WASP may have pivotal roles through the EVH1 domain in the LPS signaling cascade, either directly or indirectly, and modulates inflammatory immune responses in microglia.

Protection of zinc against tumor necrosis factor induced lethal inflammation depends on heat shock protein 70 and allows safe antitumor therapy.

Tumor necrosis factor (TNF)-induced inflammation prevents its broad application as an antitumor agent. We here report that addition of ZnSO(4) to the drinking water of mice induces expression of heat shock protein 70 (HSP70) in several organs, notably the gastrointestinal track. Zinc conferred dose-responsive protection against TNF-induced hypothermia, systemic induction of interleukin-6 and NO(x), as well as against TNF-induced bowel cell death and death of the organism. The protective effect of zinc was completely absent in mice deficient in the major HSP70-inducible gene, hsp70.1, whereas transgenic mice constitutively expressing the human HSP70.A gene, under control of a beta-actin promoter, was also protected against TNF, indicating that an increase in HSP70 is necessary and sufficient to confer protection. The therapeutic potential of the protection induced by ZnSO(4) was clearly shown in a TNF/IFNgamma-based antitumor therapy using three different tumor models. In hsp70.1 wild-type mice, but not in hsp70.1-deficient mice, zinc very significantly protected against lethality but left the antitumor effect intact. We conclude that zinc protects against TNF in a HSP70-dependent way and that protection by zinc could be helpful in developing a safer anticancer therapy with TNF/IFNgamma.

Tumor-necrosis-factor-alpha-gene-deficient mice have improved cardiac function through reduction of intercellular adhesion molecule-1 in myocardial infarction.

BACKGROUND: Tumor necrosis factor (TNF)-alpha is linked to the pathogenesis of cardiovascular diseases, but how it affects myocardial infarction (MI), so the present study examined the effects of TNF-alpha and the involvement of intercellular adhesion molecule (ICAM)-1 on MI. METHODS AND RESULTS: Left coronary arteries of C57BL/6 wild type (WT) and TNF-alpha knockout (KO) mice were ligated and the mice were killed 1, 3, and 7 days later. Fractional shortening on echocardiography of the KO mice was significantly higher than that of the WT mice from day 1 to 7 (p<0.01). The ICAM-1 mRNA in the infarcted area of the KO mice was significantly lower than that of the WT from day 1 (p<0.01) to 7. In immunohistochemistry, the expression of ICAM-1 was weaker in the KO than in the WT mice. The number of neutrophils in the KO mice peaked at day 1, but even this peak level failed to reach the levels in the infarcted (p<0.01) and peri-infarcted areas (p<0.05) in the WT. The number of macrophages in the KO mice peaked at day 7, but this peak level failed to reach the levels in the infarcted (p<0.01) and peri-infarcted areas (p<0.05) in the WT. CONCLUSION: In a permanent occlusion model of MI TNF-alpha decreased cardiac function and ameliorated myocardial remodeling through the induction of ICAM-1.

Protective effects of antimicrobial peptides derived from the beetle Allomyrina dichotoma defensin on endotoxic shock in mice.

Synthetic peptides, Arg-Leu-Tyr-Leu-Arg-Ile-Gly-Arg-Arg-NH2 (peptide A) and Arg-Leu-Arg-Leu-Arg-Ile-Gly-Arg-Arg-NH2 (peptide B), derived from the beetle Allomyrina dichotoma defensin, have not only antimicrobial activities but also anti-inflammatory effects by inhibiting tumour necrosis factor-alpha(TNF-alpha) production. In the present study, we evaluated the lipopolysaccharide (LPS)-binding activities and the protective effects of these peptides on LPS-induced lethal shock in d-galactosamine (GalN)-sensitized mice. These peptides were shown to bind to erythrocytes coated with LPS and the binding activity of peptide A to LPS was significantly higher than those of peptide B and polymyxin B. Mice were injected intraperitoneally with peptide A or B at doses of 25, 50, 100 and 150 mg/kg before an injection of Salmonella abortusequi LPS (5 microg/kg) and GalN (1 g/kg) (LPS+GalN). All of wild-type mice died within 24 h after challenged with LPS+GalN. All of TNF-alpha-deficient mice challenged with LPS+GalN survived. An injection of peptide A immediately after challenge with LPS+GalN resulted in significantly improved survival rates in a dose dependent manner. Peptide B showed only minor protection. The levels of TNF-alpha in the ameliorated mice by peptide A were significantly lower than those of challenge control, suggesting a suppressive effect of peptide A on TNF-alpha production. Furthermore, peptide A-treated mice showed significantly lower levels of asparate aminotransferase and alanine aminotransferase when compared to challenge control. Concordantly, hemorrhage and necrosis in the liver of peptide A-treated mice were less apparent than those of untreated control mice. These results suggest that peptide A has a protective effect on LPS-induced mortality in this mouse model.

Impaired contact hypersensitivity reaction and reduced production of vascular endothelial growth factor in tumor necrosis factor-alpha gene-deficient mice.

Tumor necrosis factor (TNF)-alpha is an important proinflammatory cytokine in contact hypersensitivity (CHS) reactions. Previous efforts to assay CHS in TNF-alpha gene-deficient (-/-) mice have demonstrated a significant reduction in ear skin weight at 24 h following challenge by oxazolone, although the mechanisms of this suppression have not been examined. To further characterize the impaired CHS during evolution of the elicitation phase in TNF-alpha -/- mice and to clarify its mechanisms, focusing on the roles of TNF-alpha and vascular endothelial growth factor (VEGF), we used an established method of CHS assay-sensitization and challenge by trinitrochlorobenzene (TNCB)- in TNF-alpha -/- and wild-type mice. We compared the histopathology of the sequential evolution of CHS between the two groups of mice and assessed both the extent of inflammatory cell infiltration and the degree of dilatation in dermal vessels labeled with CD31. We quantified the production of VEGF in the epidermis at specific time points by using a murine VEGF ELISA kit. The CHS reaction was markedly suppressed in TNF-alpha -/- mice at all time points of the elicitation phase. Histologically, in TNF-alpha -/- mice we observed diminished vascular permeability, reduced numbers of infiltrating inflammatory cells, neutrophils at 12 h, mononuclear cells and eosinophils at 24 h, and a decreased area of dilatation of vessels labeled with CD31. The level of epidermal VEGF in wild type mice increased rapidly after challenge and peaked at 24 h, paralleling the peak of ear swelling. In contrast, the peak level of epidermal VEGF in TNF-alpha -/- mice was significantly reduced. These results suggest that TNF-alpha plays an enhancing role in the elicitation phase of the CHS reaction. Diminished degrees of vascular permeability, dilatation of CD31+ vessels, and inflammatory cell infiltration in TNF-alpha -/- mice are likely to be the result of a lack of TNF-alpha and reduced production of epidermal VEGF.

Intrabodies against the EVH1 domain of Wiskott-Aldrich syndrome protein inhibit T cell receptor signaling in transgenic mice T cells.

Intracellularly expressed antibodies (intrabodies) have been used to inhibit the function of various kinds of protein inside cells. However, problems with stability and functional expression of intrabodies in the cytosol remain unsolved. In this study, we show that single-chain variable fragment (scFv) intrabodies constructed with a heavy chain variable (V(H)) leader signal sequence at the N-terminus were translocated from the endoplasmic reticulum into the cytosol of T lymphocytes and inhibited the function of the target molecule, Wiskott-Aldrich syndrome protein (WASP). WASP resides in the cytosol as a multifunctional adaptor molecule and mediates actin polymerization and interleukin (IL)-2 synthesis in the T-cell receptor (TCR) signaling pathway. It has been suggested that an EVH1 domain in the N-terminal region of WASP may participate in IL-2 synthesis. In transgenic mice expressing anti-EVH1 scFvs derived from hybridoma cells producing WASP-EVH1 mAbs, a large number of scFvs in the cytosol and binding between anti-EVH1 scFvs and native WASP in T cells were detected by immunoprecipitation analysis. Furthermore, impairment of the proliferative response and IL-2 production induced by TCR stimulation which did not affect TCR capping was demonstrated in the scFv transgenic T cells. We previously described the same T-cell defects in WASP transgenic mice overexpressing the EVH1 domain. These results indicate that the EVH1 intrabodies inhibit only the EVH1 domain function that regulates IL-2 synthesis signaling without affecting the overall domain structure of WASP. The novel procedure presented here is a valuable tool for in vivo functional analysis of cytosolic proteins.

Tumor necrosis factor-alpha-independent downregulation of hepatic cholesterol 7alpha-hydroxylase gene in mice treated with lead nitrate.

We previously reported that lead nitrate (LN), an inducer of hepatic tumor necrosis factor-alpha (TNF-alpha), downregulated gene expression of cholesterol 7alpha-hydroxylase. Herein, to clarify the role of TNF-alpha in LN-induced downregulation of cholesterol 7alpha-hydroxylase, effects of LN on gene expression of hepatic cholesterol 7alpha-hydroxylase (Cyp7a1) in TNF-alpha-knockout (KO) and TNF-alpha-wild-type (WT) mice were comparatively examined. Gene expression of hepatic Cyp7a1 in both WT and KO mice decreased to less than 5% of the corresponding controls at 6-12 h after treatment with LN (100 mumol/kg body weight, iv). Levels of hepatic TNF-alpha protein in either WT or KO mice were below the detection limit, although expression levels of the TNF-alpha gene markedly increased at 6 h in WT mice by LN treatment, but not in KO mice. In contrast, in both WT and KO mice, levels of hepatic IL-1beta protein, which is known to be a suppressor of the cholesterol 7alpha-hydroxylase gene in hamsters, were significantly increased 3-6 h after LN treatment. Furthermore, LN-induced downregulation of the Cyp7a1 gene did not necessarily result from altered gene expression of hepatic transcription factors, including positive regulators (liver X receptor alpha, retinoid X receptor alpha, fetoprotein transcription factor, and hepatocyte nuclear factor 4alpha) and a negative regulator small heterodimer partner responsible for expression of the Cyp7a1 gene. The present findings indicated that LN-induced downregulation of the Cyp7a1 gene in mice did not necessarily occur through a TNF-alpha-dependent pathway and might occur mainly through an IL-1beta-dependent pathway.

Disruption of tumor necrosis factor-alpha gene diminishes the development of atherosclerosis in ApoE-deficient mice.

Inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha) have been implicated in atherogenesis. However, the precise role of TNF-alpha in atherogenesis is still unclear. To examine the effect of TNF-alpha on atherogenesis, we generated compound-deficient mice in apolipoprotein E (apoE) and TNF-alpha (apoE-/-/TNF-alpha-/-) and compared them with apoE-/- mice. Although serum total cholesterol levels were markedly elevated in both apoE-/-/TNF-alpha-/- and apoE-/- mice compared to wild-type mice, no differences were observed between apoE-/-/TNF-alpha-/- and apoE-/- mice. The atherosclerotic plaque area in the aortic luminal surface of apoE-/-/TNF-alpha-/- mice (n=8, 3.1+/-0.4%) was significantly smaller than that of apoE-/- mice (n=7, 4.7+/-0.4%, p<0.001) despite the lack of difference in serum cholesterol levels. The atherosclerotic lesion size in the aortic sinus of apoE-/-/TNF-alpha-/- mice (n=10, 5.1+/-0.3 x 10(5)microm(2)) was also significantly smaller than that of apoE-/- mice (n=11, 7.0+/-0.3 x 10(5)microm(2), p<0.0001). RT-PCR analysis indicated that the expression levels of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) were significantly higher in apoE-/- than apoE-/-/TNF-alpha-/- mice. Macrophages from apoE(-/-) mice showed higher uptake level of oxidized LDL and increased expression level of scavenger receptor class A (SRA) compared to those from apoE-/-/TNF-alpha-/- mice. These results indicate that TNF-alpha plays an atherogenic role by upregulating the expressions of ICAM-1, VCAM-1 and MCP-1 in the vascular wall, and by inducing SRA expression and oxidized LDL uptake in macrophages.

IFN-gamma and TNF-alpha are involved in urushiol-induced contact hypersensitivity in mice.

Contact hypersensitivity (CHS) is a cutaneous T-cell-mediated immunological reaction to applied haptens. Activated antigen-specific T cells release several cytokines and chemokines followed by the recruitment of inflammatory cells and skin damage. CD8+ T cells and CD4+ T cells have been involved in the establishment of previously described CHS. In this study, we investigated the induction of CHS by urushiol in mice. Maximum swelling in mouse ears was elicited 24 h after challenge with urushiol on day 9 of sensitization. IFN-gamma, TNF-alpha and IFN-gamma-inducible protein 10 (IP-10) mRNA were expressed after challenge of the antigen in urushiol-sensitized mice, but not in unsensitized mice. IFN-gamma knockout (KO) mice and TNF-alpha KO mice failed to elicit CHS with urushiol. Contact hypersensitivity and expressions of IFN-gamma, TNF-alpha and IP-10 mRNA were markedly suppressed in CD4+ and CD8+ cell-depleted mice. These results suggest that IFN-gamma, TNF-alpha, and possibly IP-10, play a critical role in CHS induced by urushiol, depending on both CD4+ T cells and CD8+ T cells.

Quantitative trait locus analysis of plasma cholesterol and triglyceride levels in C57BL/6J x RR F2 mice.

A highly significant cholesterol quantitative trait locus (QTL) (Cq6) was identified on chromosome 1 in C57BL/6J x RR F2 mice. The Cq6 was located over the gene for apolipoprotein A-Il (Apoa2), and the RR allele was associated with increased plasma cholesterol. C57BL/6J has Apoa2a alleles and RR has Apoa2b alleles. Three different Apoa2 alleles are known on the basis of amino acid substitutions at four residues. Analysis with partial Apoa2 congenic strains possessing Apoa2a, Apoa2b, and Apoa2C alleles revealed that the Apoa2b allele is unique in the ability to increase cholesterol among the three Apoa2 alleles, and that the Ala-to-Val substitution at residue 61 may be crucial as far as cholesterol metabolism is concerned. We also investigated the question of whether the Apoa1 gene is responsible for the cholesterol QTLs (Cq4 and Cq5) that had been identified previously on chromosome 9 in C57BL/6J x KK-Ay/a F2 and in KK x RR F2, but not in C57BL/6J x RR F2 mice. Similar to Apoa2 alleles, three different Apoal alleles with two successive amino acid substitutions were revealed among the strains. However, we could not correlate Apoal polymorphisms with the occurrence of QTLs in these three sets of F2 mice.

Susceptibility of TNF-alpha-deficient mice to Trypanosoma congolense is not due to a defective antibody response.

C57BL/6 mice deficient in one or two copies of the gene for tumor necrosis factor alpha (TNF-alpha) were more susceptible to Trypanosoma congolense infection than their resistant, wild-type counterparts. The number of TNF-alpha genes was correlated with the capacity to control parasitaemia and with survival time. Absence of TNF-alpha resulted in a diminished capacity to form germinal centres in lymph nodes and spleen. Since germinal centres are involved in antibody production and affinity maturation, the susceptibility of the TNF-alpha-deficient mice could have been due to this secondary defect. Despite the lack of the germinal centres, the antibody responses to internal and exposed trypanosome antigens and to non-trypanosome antigens were not significantly different. Also the relative avidities measured in infected sera did not significantly differ between the two mouse strains. These data suggest that the role of TNF-alpha in control of T. congolense was not due to its role in the development of an antibody response.

Further mapping and characterization of Naq1, a quantitative trait locus responsible for maternal inferior nurturing ability in RR mice.

Females of the inbred mouse RR strain have a limited ability to nurture their offspring, and frequently the young die during rearing. We previously identified a significant quantitative trait locus (QTL) responsible for the inferior nurturing ability on chromosome 5 (Naq1), on the basis of litter weight of six pups at days 7, 12, and 21 after parturition. Here, we carried out further mapping of Naq1 to define the confidence interval precisely. At the same time, we analyzed new quantitative trait variables, litter weight gain between days 7 and 12 (WG1), and that between days 12 and 21 (WG2), to characterize further the physiology of inferior nurturing ability. Consequently, a peak LOD score for the Naq1 was identified on D5Mit218 (72 cM), which was located approximately 2 cM distal to our previous expectation, as a significant QTL for WG1 (LOD 5.5), but not for WG2 (LOD 0.9). Because the growth of pups depends purely on milk obtained from the dam up to day 12 after birth, it seems possible to assume that the inferior nurturing ability in RR mice is related to defects in maternal nutritional support (that is, lactation) rather than to defects in pup growth. Naq1 is a novel QTL as far as the QTL results of relevant female reproductive traits in cattle and pigs are concerned.

Confirmation and characterization of murine body weight QTLs, Bwq1 and Bwq2, identified in C57BL/6J x KK-Ay/a F2-Ay/a mice.

Body weight quantitative trait loci (QTLs), Bwq1 and Bwq2, identified previously in C57BL/6J x KK-Ay/a F2-Ay/a mice, were further confirmed and characterized. Body weight measurement was done from 21 days after birth (Day 21) through Day 100, at 10-day intervals. Bwq1 was statistically significant only on Days 40, 50, and 60, whereas Bwq2 was statistically significant on and after Day 40. When body weight gain (WG) between two successive weight measurements was evaluated, both Bwq1 and Bwq2 were statistically significant only for WG between Days 30 and 40. The results suggest that variations in body weight among F2-Ay/a individuals in later life have been determined by variations in WG during the period shortly after weaning. The results also suggest that Bwq1 is related to increased body weight in the KK strain, because the effect of Bwq1 on the body weight is observed not only in F2-Ay/a, but also in F2-a/a. On the other hand, it is suggested that Bwq2 is related to enhanced obesity caused by Ay mutation and therefore is a genetic modifier that specifically interacts with the Ay allele, because the effect of Bwq2 is only observed in F2-A y/a. There are two candidate genes, Pparg and Hrh1, which are located near the 95% confidence interval of Bwq2, and which are expressed in the adipose tissue; however, we could not find any nucleotide differences in both cDNAs between KK and C57BL/6J strains.