Immature performance linked with exaggeration of a sexually selected trait in an armed beetle.

Exaggerated traits can be costly and are often trade-off against other characters, such as life-history traits. Thus, the evolution of an exaggerated trait is predicted to affect male life-history strategies. However, there has been very little experimental evidence of the impact of the evolution of sexually selected traits on life-history traits. This study investigated whether increased investment in exaggerated traits can generate evolutionary changes in the life-history strategy for armed males. Male flour beetles, Gnatocerus cornutus, have enlarged mandibles that are used in male-male competition, but females lack this character exaggeration completely. We subjected these weapons to 11 generations of bidirectional selection and found a correlated response in pupal survival but not in larval survival or adult longevity in the male. That is, selecting for male mandibles negatively impacted survival during the production of mandibles. There is no correlated response in the life-history traits of the female.

Partial functional overlap of the three ras genes in mouse embryonic development.

In mammals, three ras genes, H-ras, N-ras and K-ras, encode homologous but distinct 21-kDa Ras proteins. We examined the in vivo functional relationship of the three ras genes in mouse embryonic development by investigating the phenotypes of mice deficient in one or multiple ras genes. H-ras-/- mice and N-ras-/- mice as well as a substantial proportion of H-ras-/-/N-ras-/- mice expressing only the K-ras gene were viable, while K-ras-/- mice were embryonically lethal, as have been reported previously. N-ras-/-/K-ras+/- mice died neonatally, while H-ras-/-/K-ras-/- embryos died much earlier than K-ras homozygous mutant fetuses. To further investigate the functional relationship of the ras genes in embryonic development, we introduced a human H-ras transgene into single or multiple ras mutant mice and found that the transgene rescued mice, including triple ras mutants, from embryonic lethality in association with correction of thin ventricular walls of the heart in null K-ras mutant mice. In situ hybridization revealed that the expression of the H-ras transgene on embryonic day E13.5 and E15.5 was more intense in major organs, including the heart, than those of endogenous ras genes. We therefore conclude that the functions of the ras genes are partially overlapping in mouse embryonic development.

Cot/Tpl2 is essential for RANKL induction by lipid A in osteoblasts.

Lipopolysaccharide (LPS) is a pathogenic factor that increases bone resorption in periodontal diseases. LPS treatment of osteoblasts was shown to induce the receptor activator of NF-kappa B ligand (RANKL), an essential secretory or membrane-bound factor for osteoclast function, in a manner dependent on extracellular signal-regulated kinase (ERK) activation. However, the mechanisms regulating this process remained unknown. Here, we show that RANKL mRNA induction and ERK activation, when treated with synthetic lipid A (an active center of LPS), were markedly reduced in mouse osteoblasts lacking Cot/Tpl2, which was recently recognized as an essential kinase for the induction of TNF-alpha by LPS in macrophages. In contrast, c-Jun N-terminal kinase (JNK), p38 kinase, Raf-1, and NF-kappa B were normally activated in cot/tpl2-/- osteoblasts. These findings indicate that Cot/Tpl2 is essential for LPS-induced ERK activation and RANKL induction in osteoblasts.

Tendon grafting for multiple extensor tendon ruptures of fingers in rheumatoid hands.

We assessed the outcome of tendon grafting of multiple finger extensor tendon ruptures in 14 patients with rheumatoid arthritis. Extensor lags improved from a preoperative mean of 33 degrees (range, 20 degrees-65 degrees) to a postoperative mean of 18 degrees (range, 0-60 degrees). However, loss of finger flexion was observed, with a mean postoperative fingertip to palm distance of 1.6 (range: 0-7.5) cm. Patient satisfaction correlated with the fingertip to palm distance, though not with the postoperative extensor lag. Because of the loss of finger flexion which was probably due to muscle contracture, we conclude that the results of tendon grafts in this situation are unsatisfactory.

Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) stimulates primitive and definitive erythropoiesis in mouse embryos expressing hGM-CSF receptors but not erythropoietin receptors.

Although erythropoietin (EPO) and its receptor (EPOR) are crucial for the proliferation, survival, and terminal differentiation of erythroid progenitors, it remains to be elucidated whether EPOR-unique signaling is required for erythropoiesis. To address this issue, human granulocyte-macrophage colony-stimulating factor (hGM-CSF) receptor (hGMR)-transgenic mice and heterozygous EPOR mutant mice were crossed by in vitro fertilization. In methylcellulose clonal culture of fetal liver (FL) cells of generated hGMR-expressing EPOR(-/-) embryos at embryonic day (E) 12.5 of gestation, hGM-CSF stimulated erythroid colony formation under serum-containing and serum-free conditions. Analysis of globin expression in individual erythrocyte-containing colonies formed from E12.5 FL cells showed that hGM-CSF supports primitive and definitive erythropoiesis even in EPOR(-/-) embryos. In comparison of activities between hGM-CSF and EPO in hGMR-expressing EPOR(+/+) embryos, the 2 substances supported the formation of similar numbers of erythroid colonies in clonal culture of E12.5 FL cells; enhanced adult, but not embryonic, globin synthesis; and induced increase of GATA-1 expression and decrease of erythroid Kruppel-like factor and cMyb expression in the FL cells. On the other hand, in E8.0 yolk sac erythropoiesis, both substances had a similar effect on erythroid colony formation, but hGM-CSF induced an increase of beta-major globin expression, while EPO did not. All together, the results of the present study demonstrated that hGM-CSF can stimulate the proliferation and differentiation of primitive and definitive erythroid cells independently of EPOR signal if they express hGMR, and the activity is comparable to that of EPO in definitive, but not primitive, erythropoiesis.

Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development.

SALL1 is a mammalian homolog of the Drosophila region-specific homeotic gene spalt (sal); heterozygous mutations in SALL1 in humans lead to Townes-Brocks syndrome. We have isolated a mouse homolog of SALL1 (Sall1) and found that mice deficient in Sall1 die in the perinatal period and that kidney agenesis or severe dysgenesis are present. Sall1 is expressed in the metanephric mesenchyme surrounding ureteric bud; homozygous deletion of Sall1 results in an incomplete ureteric bud outgrowth, a failure of tubule formation in the mesenchyme and an apoptosis of the mesenchyme. This phenotype is likely to be primarily caused by the absence of the inductive signal from the ureter, as the Sall1-deficient mesenchyme is competent with respect to epithelial differentiation. Sall1 is therefore essential for ureteric bud invasion, the initial key step for metanephros development.

Impaired delay but normal trace eyeblink conditioning in PLCbeta4 mutant mice.

To elucidate the functional role of phospholipase Cbeta4 (PLCbeta4), which is highly expressed in the Purkinje cells of the rostral cerebellum, cerebellar long-term depression (LTD) and delay and trace eyeblink conditioning were investigated in PLCbeta4-deficient mice. Rostral cerebellar LTD and delay eyeblink conditioning were severely impaired, whereas trace eyeblink conditioning was not. These results indicate that PLCbeta4 is essential for LTD in the rostral cerebellum and delay conditioning, but not trace conditioning. Rostral cerebellar LTD may be required as a neural substrate for delay conditioning, but is not required for trace conditioning.

Structural fragility of blood vessels and peritoneum in calponin h1-deficient mice, resulting in an increase in hematogenous metastasis and peritoneal dissemination of malignant tumor cells.

We have observed weak expression of calponin h1, which stabilizes the actin filament system, in blood vessels within human malignant tumors. This observation suggested that because of a deficiency in stabilization by calponin h1, the structure of blood vessels in malignant tumors is fragile compared with blood vessels in normal tissues. We therefore generated calponin h1-deficient (CN(-/-)) mice to examine the effect of calponin h1 on the integrity of the barrier system in blood vessels against cancer metastasis. The CN(-/-) mice exhibited morphological fragility of the tissues, including the uterus and blood vessels. In particular, we frequently observed bleeding into the surrounding tissue from blood vessels of the ocular fundus in CN(-/-) mice. In addition, mesothelial cells, which usually express calponin h1 in normal (CN(+/+)) mice, were retracted in the CN(-/-) mice. When fluorescein was injected i.v. into mice, the CN(-/-) mice exhibited a greater and more rapid leakage of fluorescein from the blood vessels of the ocular fundus compared with the CN(+/+) mice. In the CN(-/-) mice receiving i.v. inoculations of B16 melanoma cells, significantly more metastatic nodules were formed in the lung than in the CN(+/+) mice. When B16 melanoma cells were injected i.p., the severity of peritonitis carcinomatosa was greater in CN(-/-) than in CN(+/+) mice. These results indicate that calponin h1 plays an important role in the regulation of the integrity of the blood vessels and peritoneum, which in turn is an important factor influencing the frequency of cancer metastasis. The CN(-/-) mice, which exhibit fragile blood vessels and peritoneum, could serve as sensitive and useful host models to investigate cancer metastasis.

Phospholipase Cbeta4 and protein kinase Calpha and/or protein kinase CbetaI are involved in the induction of long term depression in cerebellar Purkinje cells.

Activation of the type-1 metabotropic glutamate receptor (mGluR1) signaling pathway in the cerebellum involves activation of phospholipase C (PLC) and protein kinase C (PKC) for the induction of cerebellar long term depression (LTD). The PLC and PKC isoforms that are involved in LTD remain unclear, however. One previous study found no change in LTD in PKCgamma-deficient mice, thus, in the present study, we examined cerebellar LTD in PLCbeta4-deficient mice. Immunohistochemical and Western blot analyses of cerebellum from wild-type mice revealed that PLCbeta1 was expressed weakly and uniformly, PLCbeta2 was not detected, PLCbeta3 was expressed predominantly in caudal cerebellum (lobes 7-10), and PLCbeta4 was expressed uniformly throughout. In PLCbeta4-deficient mice, expression of total PLCbeta, the mGluR1-mediated Ca(2+) response, and LTD induction were greatly reduced in rostral cerebellum (lobes 1-6). Furthermore, we used immunohistochemistry to localize PKCalpha, -betaI, -betaII, and -gamma in mouse cerebellar Purkinje cells during LTD induction. Both PKCalpha and PKCbetaI were found to be translocated to the plasmamembrane under these conditions. Taken together, these results suggest that mGluR1-mediated activation of PLCbeta4 in rostral cerebellar Purkinje cells induced LTD via PKCalpha and/or PKCbetaI.

Spontaneous tumorigenesis in mice defective in the MTH1 gene encoding 8-oxo-dGTPase.

Oxygen radicals, which can be produced through normal cellular metabolism, are thought to play an important role in mutagenesis and tumorigenesis. Among various classes of oxidative DNA damage, 8-oxo-7,8-dihydroguanine (8-oxoG) is most important because of its abundance and mutagenicity. The MTH1 gene encodes an enzyme that hydrolyzes 8-oxo-dGTP to monophosphate in the nucleotide pool, thereby preventing occurrence of transversion mutations. By means of gene targeting, we have established MTH1 gene-knockout cell lines and mice. When examined 18 months after birth, a greater number of tumors were formed in the lungs, livers, and stomachs of MTH1-deficient mice, as compared with wild-type mice. The MTH1-deficient mouse will provide a useful model for investigating the role of the MTH1 protein in normal conditions and under oxidative stress.

Requirement of phospholipase Cdelta4 for the zona pellucida-induced acrosome reaction.

Several phospholipase C (PLC) isoforms have been found in male and female mammalian gametes, and splicing isoforms of PLCdelta4 are predominantly expressed in testis. Here we report that male mice in which the PLCdelta4 gene had been disrupted either produced few small litters or were sterile. In vitro fertilization studies showed that insemination with PLCdelta4-/- sperm resulted in significantly fewer eggs becoming activated and that the calcium transients associated with fertilization were absent or delayed. PLCdelta4-/- sperm were unable to initiate the acrosome reaction, an exocytotic event required for fertilization and induced by interaction with the egg coat, the zona pellucida. These data demonstrate that PLCdelta4 functions in the acrosome reaction that is induced by the zona pellucida during mammalian fertilization.

Functional dissection of the cytoplasmic subregions of the interleukin-5 receptor alpha chain in growth and immunoglobulin G1 switch recombination of B cells.

The interleukin-5 receptor alpha chain (IL-5Ralpha) is known to regulate the development and function of B cells and eosinophils. Although the functions of IL-5Ralpha cytoplasmic domain subregions have been studied extensively using cultured cell lines, this approach has limitations when studying the functions of distinct primary B-cell subpopulations and their responsiveness to IL-5. In the present study, we generated mice on an IL-5Ralpha null background, each expressing a mutant form of an IL-5Ralpha transgene ligated to a mu enhancer and VH promoter, either lacking the cytoplasmic DC3 region or substituting two proline residues for alanine (ApvA) in the membrane-proximal ppvp motif of the cytoplasmic domain. The ppvp motif, which mediates activation of JAK2/STAT5 and Btk, also contributes to c-fos, c-jun and c-myc expression. IL-5Ralpha null mutant mice showed impaired B-1-cell development, reduced serum immunoglobulin G3 (IgG3) and IgM, no IL-5-induced enhancement of B-cell proliferation and IL-5-induced switch recombination from the mu gene to gamma1 gene; these were not recovered following the expression of the ApvA mutant. In contrast, absence of the DC3 region affected the IL-5-induced switch recombination from the mu to the gamma1 gene and B-1-cell development, while IL-5-induced proliferation and IgM production were at levels similar to those of B cells expressing wild-type IL-5Ralpha transgene. The results clearly indicated that the ppvp motif and the DC3 region of IL-5Ralpha played distinct roles in B-cell proliferation and differentiation. Thus, this present approach offers new insights into the functions of the cytoplasmic subregions of IL-5Ralpha, in particular its carboxy-terminal region.

Dwarfism and early death in mice lacking C-type natriuretic peptide.

Longitudinal bone growth is determined by endochondral ossification that occurs as chondrocytes in the cartilaginous growth plate undergo proliferation, hypertrophy, cell death, and osteoblastic replacement. The natriuretic peptide family consists of three structurally related endogenous ligands, atrial, brain, and C-type natriuretic peptides (ANP, BNP, and CNP), and is thought to be involved in a variety of homeostatic processes. To investigate the physiological significance of CNP in vivo, we generated mice with targeted disruption of CNP (Nppc(-/-) mice). The Nppc(-/-) mice show severe dwarfism as a result of impaired endochondral ossification. They are all viable perinatally, but less than half can survive during postnatal development. The skeletal phenotypes are histologically similar to those seen in patients with achondroplasia, the most common genetic form of human dwarfism. Targeted expression of CNP in the growth plate chondrocytes can rescue the skeletal defect of Nppc(-/-) mice and allow their prolonged survival. This study demonstrates that CNP acts locally as a positive regulator of endochondral ossification in vivo and suggests its pathophysiological and therapeutic implication in some forms of skeletal dysplasia.

Sez4 gene encoding an elongation subunit of DNA polymerase zeta is required for normal embryogenesis.

BACKGROUND: Sez4 identified as a seizure-activated gene shows a similarity to the yeast REV3 that encodes a catalytic subunit of the nonessential DNA polymerase zeta which is involved in error-prone translesion synthesis. Although yeast REV3 homologues in mouse and human have recently been identified and characterized, their precise roles remain elusive. RESULTS: Here we investigated the role of mouse pol zeta by targeted inactivation of the Sez4 gene. The homozygous Sez4 mutants died around embryonic day (E) 10.5. This lethal effect was the result of developmental defects and apoptotic cell death within the embryo proper at the gastrulation stage, and it was partially rescued at E12.5 by the expression of a Sez4-transgene. In wild-type embryos, Sez4 transcripts were up-regulated within the embryo proper from E7.5, correlating well with the lethal stage of Sez4-inactivation. CONCLUSION: Our findings indicate that Sez4 is essential for epiblast lineage-specific development and suggests a requirement of mammalian DNA polymerase zeta in the survival of certain subcellular populations which are indispensable to normal embryogenesis.

The actin-depolymerizing factor destrin has an actin-stabilizing domain.

Destrin is a 19 kDa actin-depolymerizing protein of the ADF-cofilin family. Destrin was digested with trypsin to a structurally stable 9.2 kDa fragment that contains the actin-binding sequence. The purified 9.2 kDa fragment has an actin filament stabilizing activity, rather than an actin filament depolymerizing activity. The deleted region is probably essential for the actin filament depolymerizing activity of intact destrin. Surprisingly, the 9.2 kDa fragment also has an assembly-promoting activity in the absence of ATP.

Regulation of shortening velocity by calponin in intact contracting smooth muscles.

To elucidate the function of calponin in intact contracting smooth muscle cells in vivo, we generated mice with a mutated basic calponin (h1) locus (Yoshikawa et al., Genes Cells 3, 685-695, 1998). Crossbridge cycling rates were estimated in aortic smooth muscle by the force redevelopment following an isometric step shortening as a function of time after K(+) depolarization. Evidence is presented that calponin is involved in the inhibition of shortening velocity in the tonic phase of contraction. The phosphorylation levels of myosin regulatory light chain and cytosolic calcium concentrations were not significantly different in paired comparisons between calponin-deficient (-/-) and wild-type (+/+) muscles at any time point after stimulation. The force-velocity relationships in vas deferens smooth muscle showed that the maximum shortening velocity of -/- muscle was significantly faster than that of +/+ muscle. There was no change in the length-force relationships in both -/- and +/+ muscles of aorta and vas deferens. The results suggest that calponin plays a role in regulation of the crossbridge cycling and that it may be responsible for reduced shortening velocity during a maintained contraction of mammalian smooth muscle.

Purification and characterization of a new, ubiquitously distributed class of microtubule-associated protein with molecular mass 250 kDa.

A heat-stable microtubule-associated protein (MAP) with relative molecular mass 250 000, termed 250-kDa MAP, was purified from bovine adrenal cortex. It is classified as a MAP subspecies distinct from MAP1, MAP2, tau, and MAP4, as judged from its electrophoretic mobility, heat stability and immunoreactivity. Purified 250-kDa MAP was able to bind to taxol-stabilized microtubules, although it lacked the ability to polymerize purified tubulin into microtubules. Western-blot analysis showed that this MAP was expressed ubiquitously in mammalian tissues. Immunofluorescence microscopy revealed that polyclonal antibodies raised against 250-kDa MAP stained many punctate structures in the cytoplasm of cultured cells. Blurry cytosolic staining was also observed. Judging from the result of nocodazole treatment, the punctate structures were associated with the microtubule network throughout the cytoplasm, while cytosolic 250-kDa MAP colocalized with free tubulin. Under electron microscopy, 250-kDa MAP has the appearance of a hollow sphere of about 12 nm diameter.

Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-alpha/beta gene induction.

Induction of the interferon (IFN)-alpha/beta gene transcription in virus-infected cells is an event central to innate immunity. Mice lacking the transcription factor IRF-3 are more vulnerable to virus infection. In embryonic fibroblasts, virus-induced IFN-alpha/beta gene expression levels are reduced and the spectrum of the IFN-alpha mRNA subspecies altered. Furthermore, cells additionally defective in IRF-7 expression totally fail to induce these genes in response to infections by any of the virus types tested. In these cells, a normal profile of IFN-alpha/beta mRNA induction can be achieved by coexpressing both IRF-3 and IRF-7. These results demonstrate the essential and distinct roles of thetwo factors, which together ensure the transcriptional efficiency and diversity of IFN-alpha/beta genes for the antiviral response.

Diversity of T cell repertoire shaped by a single peptide ligand is critically affected by its amino acid residue at a T cell receptor contact.

T cell differentiation in the thymus is driven by positive selection through the interaction of alphabeta T cell receptors (TCRs) with self-peptides bound to self-major histocompatibility complex molecules, yet the influence of the peptide sequence on this process remains unknown. To address this issue, we have compared CD4(+) T cell differentiation between two sets of mouse lines in which MHC class II I-A(b) molecules are occupied with either Ealpha chain-derived peptide ((p)Ealpha) or its variant, (p)60K, with one amino acid substitution from leucine to lysine at P5 residue of TCR contacts. Here, we show that despite the comparable expression of I-A(b)-peptide complex in the thymus, this substitution from leucine to lysine affects efficiency of positive selection, resulting in extremely small numbers of CD4(+) T cells to be selected to mature on I-A(b)-(p)60K complex. Furthermore, we show that, although I-A(b)-(p)Ealpha complex selects diverse T cells, T cell repertoire shaped by I-A(b)-(p)60K complex is markedly constrained. Our findings thus suggest that positive selection is both specific and degenerate, depending on the amino acid residues at TCR contacts of the selecting self-peptides.

Different mutation frequencies and spectra among organs by N-methyl-N-nitrosourea in rpsL (strA) transgenic mice.

The frequencies and spectra of N-methyl-N-nitrosourea (MNU)-induced in vivo somatic mutations were determined in rpsL (strA) transgenic mice. The wild-type rpsL gene, which exhibits a streptomycin-sensitive (Sm(S)) phenotype, was used as the rescue marker gene. Studies of mutation spectra among different organs and tissues were simplified using this system because of the short coding sequence (375 bp) of the rpsL gene. MNU administration to transgenic mice significantly elevated the mutation frequencies in various adult organs. Two distinctive patterns of mutation spectrum were observed, depending on the organs tested. Mutations derived from labile organs (spleen and thymus) were predominantly G:C to A:T transitions, as expected for MNU mutagenesis. Stable organs like the liver and brain, however, carried many fewer G:C to A:T transitions but significantly more single base deletions, of which the spectrum was very similar to that of background mutations in the rpsL transgenic mice. This spectrum difference among more and less proliferating organs was confirmed by the predominant occurrence of G:C to A:T transitions in fetal liver cells exposed to transplacental MNU treatment. In addition, most (approximately 90%) of the G:C to A:T transitions induced by MNU were detected in the first nucleotide of some 5'-G-(C or G)-3' sequences, many of which corresponded to the middle guanine residue of 5'-purine-G-(C or G)-3' sequences. It is thus suggested that at particular sites, the neighboring bases in both the 5' side and 3' side seem to influence either the susceptibility to DNA damage or the ability to repair MNU-induced lesions.