Effect of steroid hormones on the innate immune response induced by Staphylococcus aureus in the goat mammary gland.

The objective of this study was to compare the dynamics of innate immune components after intramammary infusion of Staphylococcus aureus (SA) under conditions of high oestrogen and high progesterone in goats. In one group ("E-group"), controlled internal drug release (CIDR) devices were inserted intravaginally from days -11 to -4. Prostaglandin F2α was administered immediately after removal of the CIDR device at day -3, and then oestradiol benzoate (E) was injected intramuscularly once a day from days -2 to 3. Heat-inactivated SA was then administered via intramammary infusion to the left udder at day 0, whilst only saline was infused to the right udder as a control. In a second group ("P-group"), CIDR devices were inserted intravaginally from days -3 to 7 and SA was infused at day 0 in the same way as in the E-group. The milk yield and the concentration of innate immune components (somatic cell count (SCC), lactoferrin (LF), S100A7 and goat ß-defensin 1 (GBD-1)) in the milk were measured. Milk yield decreased drastically in both SA and control udders in the E-group, whereas the P-group exhibited increased milk yield in both SA and control udders. SCC increased after SA infusion in both E- and P-groups, although it was higher in the E-group than in the P-group. There was no significant change in LF concentration in the E-group, but a decrease was observed in the P-group. Concentrations of S100A and GBD-1 were significantly increased after SA infusion in the E-group but not in the P-group. These results suggest that E enhances the innate immune response induced by SA in the goat mammary gland. This effect may be due to the reduction in milk yield and upregulation of innate immune components.

Magnetic structure of phase II in U(Ru(0.96)Rh(0.04))2Si2 determined by neutron diffraction under pulsed high magnetic fields.

We report neutron diffraction measurements on U(Ru(0.96)Rh(0.04))(2)Si(2) single crystal under pulsed high magnetic fields up to 30 T applied along the tetragonal c axis. The high-field experiments revealed that the field-induced phase II above 26 T corresponds to a commensurate up-up-down ferrimagnetic structure characterized by the wave vector q=(2/3,0,0) with the magnetic moments parallel to the c axis, which naturally explains the one-third magnetization plateau and the substantially changed Fermi surface in phase II. This a-axis modulated magnetic structure indicates that the phase II near the hidden order phase is closely related to the characteristic incommensurate magnetic fluctuations at Q(1)=(0.6,0,0) in the pure system URu(2)Si(2), in contrast to the pressure-induced antiferromagnetic order at Q(0)=(1,0,0).

Molecular mechanism of immunoglobulin V-region diversification regulated by transcription and RNA metabolism in antigen-driven B cells.

The immune system produces specific antibodies (Ab) against any antigens (Ag) of exogenous and endogenous origins with a diverse repertoire of V-region specificities. The primary V-region repertoire is created by the rearrangement of immunoglobulin (Ig) V-region, D- and J-segments with the insertion of N- and P-sequences during early B cell differentiation. Recent studies revealed that secondary diversification of the IgV-region generated in the peripheral lymphoid organs plays a critical role in the generation of effective Ab production for protection from various pathogens. Naïve B cells that react with Ags initiate proliferation and differentiation in the follicular region and create the germinal centres (GCs), where activation-induced cytidine deaminase (AID)-dependent IgV-region somatic hypermutation (SHM) and class-switch recombination generate high-affinity and class-switched mature Ag-specific B cells. Our studies have discovered a 210-kDa nuclear protein, named GC-associated nuclear protein (GANP) that is up-regulated in GC B cells during the T cell-dependent (TD) immune responses. By studying mice with mutant forms of the ganp gene, we demonstrated that GANP is essential for the generation of high-affinity B cells against TD-Ag by affecting SHM at the IgV-regions. GANP is associated with AID in the cytoplasm and the GANP/AID complex is recruited to the nucleus, specifically, the chromatin, and targeted selectively to the IgV-region gene in B cells. GANP augments the access of AID towards IgV-regions in B cells. Here, we review the role of GANP in acquired immunity through the detailed analysis of the molecular mechanism generating SHM specifically at IgV-regions in B cells.

Angiotensin II type 1 receptor-independent beneficial effects of telmisartan on dietary-induced obesity, insulin resistance and fatty liver in mice.

AIMS/HYPOTHESIS: Evidence suggests that telmisartan, an angiotensin II type 1 receptor (AT1) blocker and peroxisome proliferator-activated receptor-gamma partial agonist, has beneficial actions that limit development of the metabolic syndrome and diabetes. However, the role played by AT1 inhibition in metabolic effects elicited by telmisartan remains uncertain. Here we isolated the metabolic effects of telmisartan from AT1 antagonism. METHODS: Male At1a (also known as Agtr1a)-deficient mice were fed a standard diet or 60% high-fat diet; those on high-fat diet were co-administered telmisartan (3 mg kg(-1) day(-1) by oral gavage) or vehicle for 12 weeks. RESULTS: In At1a-null mice, telmisartan prevented high-fat-diet-induced increases in (1) body weight, epididymal and inguinal white adipose tissue weight, adipocyte size and plasma leptin concentration; (2) plasma glucose and insulin concentrations and HOMA index; and (3) liver weight and triacylglycerol content. Insulin tolerance testing also indicated that telmisartan improved the high-fat-diet-induced reduction of glucose-lowering by insulin. CONCLUSIONS/INTERPRETATION: The present findings demonstrate beneficial, AT1-independent effects of the AT1 blocker telmisartan on dietary-induced obesity, insulin resistance and fatty liver in animals.

Excitation spectrum of PrOs(4)Sb(12) under a magnetic field.

The evolution of the magnetic excitation spectrum of the heavy fermion superconductor PrOs(4)Sb(12) was studied by inelastic neutron scattering on crossing the critical field H(c2) for superconductivity at low temperature. The peak positions in energy and the peak intensities of the modes of the triplet split by magnetic field confirm the known crystal field parameters for PrOs(4)Sb(12) in T(h) symmetry. A selective broadening of the lineshape occurs on increasing the magnetic field: the linewidth of the upper mode of the triplet increases while the one of the middle mode does not.

Blockade of the natriuretic peptide receptor guanylyl cyclase-A inhibits NF-kappaB activation and alleviates myocardial ischemia/reperfusion injury.

Acute myocardial infarction (AMI) remains the leading cause of death in developed countries. Although reperfusion of coronary arteries reduces mortality, it is associated with tissue injury. Endothelial P-selectin-mediated infiltration of neutrophils plays a key role in reperfusion injury. However, the mechanism of the P-selectin induction is not known. Here we show that infarct size after ischemia/reperfusion was significantly smaller in mice lacking guanylyl cyclase-A (GC-A), a natriuretic peptide receptor. The decrease was accompanied by decreases in neutrophil infiltration in coronary endothelial P-selectin expression. Pretreatment with HS-142-1, a GC-A antagonist, also decreased infarct size and P-selectin induction in wild-type mice. In cultured endothelial cells, activation of GC-A augmented H2O2-induced P-selectin expression. Furthermore, ischemia/reperfusion-induced activation of NF-kappaB, a transcription factor that is known to promote P-selectin expression, is suppressed in GC-A-deficient mice. These results suggest that inhibition of GC-A alleviates ischemia/reperfusion injury through suppression of NF-kappaB-mediated P-selectin induction. This novel, GC-A-mediated mechanism of ischemia/reperfusion injury may provide the basis for applying GC-A blockade in the clinical treatment of reperfusion injury.

The neuron-restrictive silencer element-neuron-restrictive silencer factor system regulates basal and endothelin 1-inducible atrial natriuretic peptide gene expression in ventricular myocytes.

Induction of the atrial natriuretic peptide (ANP) gene is a common feature of ventricular hypertrophy. A number of cis-acting enhancer elements for several transcriptional activators have been shown to play central roles in the regulation of ANP gene expression, but much less is known about contributions made by transcriptional repressors. The neuron-restrictive silencer element (NRSE), also known as repressor element 1, mediates repression of neuronal gene expression in nonneuronal cells. We found that NRSE, which is located in the 3' untranslated region of the ANP gene, mediated repression of ANP promoter activity in ventricular myocytes and was also involved in the endothelin 1-induced increase in ANP gene transcription. The repression was conferred by a repressor protein, neuron-restrictive silencer factor (NRSF). NRSF associated with the transcriptional corepressor mSin3 and formed a complex with histone deacetylase (HDAC) in ventricular myocytes. Trichostatin A (TSA), a specific HDAC inhibitor, relieved NRSE-mediated repression of ANP promoter activity, and chromatin immunoprecipitation assays revealed the involvement of histone deacetylation in NRSE-mediated repression of ANP gene expression. Furthermore, in myocytes infected with recombinant adenovirus expressing a dominant-negative form of NRSF, the basal level of endogenous ANP gene expression was increased and a TSA-induced increase in ANP gene expression was apparently attenuated, compared with those in myocytes infected with control adenovirus. Our findings show that an NRSE-NRSF system plays a key role in the regulation of ANP gene expression by HDAC in ventricular myocytes and provide a new insight into the role of the NRSE-NRSF system outside the nervous system.

Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization.

Type I secretion system (TISS) of Gram-negative bacteria permits proteins to be secreted directly from the cytoplasm to the external medium by a single, energy-coupled step. To examine whether this system can be used as an extracellular production system of recombinant proteins, Escherichia coli alkaline phosphatase (AP) was fused to a C-terminal region of Pseudomonas sp. MIS38 lipase (PML) and examined for secretion using the E.coli cells carrying the heterologous TISS. PML is one of the passenger proteins of TISS and contains 12 repetitive sequences and a secretion signal at the C-terminal region. The fusion protein was efficiently secreted to the extracellular medium, while AP was not secreted at all, indicating that the secretion of AP is promoted by a secretion signal of PML. The repetitive sequences were not so important for secretion of the fusion protein, because the secretion level of the fusion protein containing entire repeats ( approximately 10 mg/l culture) was only 2-fold higher than that of the fusion protein without repeats. The fusion protein purified from the culture supernatant existed as a homodimer, like AP, and was indistinguishable from AP in enzymatic properties and stability.

Induction of JAB/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3 is involved in gp130 resistance in cardiovascular system in rat treated with cardiotrophin-1 in vivo.

CIS (cytokine-inducible SH2 protein), SOCS (suppressor of cytokine signaling), or SSI (signal transducers and activators of transcription [STAT]-induced STAT inhibitor) proteins are a family of cytokine-inducible negative regulators of cytokine signaling via Janus kinase (JAK)-STAT pathways. Given the evidence that the JAK-STAT pathway plays a critical role in the cardiovascular system, the primary objective of this study was to assess the effects of the CIS family on JAK-STAT signaling in the cardiovascular system in rats treated with cardiotrophin-1 (CT-1), an interleukin-6 family of cytokines. Intravenous injection of 20 microgram/kg body weight of CT-1 induced a transient, marked increase in STAT3 activation in various tissues, including heart and lung, and subsequent upregulation of 2 members of the CIS family, JAK-binding protein (JAB)/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3, in the same tissues. It was also observed that CIS3 was directly associated with JAK2 in vivo. Pretreatment with the same dose of CT-1 60 minutes before significantly attenuated the STAT3 activation induced by a second injection of CT-1. We previously reported that intravenous injection of CT-1 results in the nitric oxide (NO)-dependent hypotension accompanied by the induction of inducible NO synthase mRNA. In rats pretreated with CT-1, the induction of inducible NO synthase mRNA or hypotension by subsequent CT-1 injection was not observed. Forced expression of JAB or CIS3, but not other CISs, directly blocked CT-1-induced STAT3 activation in 293 cells. These results suggest that JAB and CIS3 serve as endogenous inhibitors of CT-1-mediated JAK-STAT signaling in the cardiovascular system in vivo.

Lethal effect of bleomycin and peplomycin on HeLa cells in multicell tumor spheroids.

The lethal effects of bleomycin and its derivative, peplomycin, were determined for HeLa S3 cells grown in multicell spheroids cocultured with human diploid fibroblasts. Both drugs were less effective for cells in spheroids than for cells grown exponentially in monolayers. However, HeLa cells from spheroids exposed to the drugs in single-cell suspension were more sensitive to the drugs than were cells in monolayers. Sequential trypsinization of spheroids after exposure to both drugs showed that the surviving fraction increased sharply with increasing depth of cell layers in the spheroids. The presence of 0.5 mM misonidazole, a hypoxic radiosensitizer, enhanced the lethal effect of peplomycin only for the cells in the deeper layer. These findings suggest that the drug resistance of cells in spheroids was due, at least in part, to the microenvironment of the deeper layers. When spheroids were incubated in fresh medium following exposure to both drugs, the cells recovered from the potentially lethal damage within 1 hr. The extent of the recovery from a fixed drug concentration was higher in cells of the superficial layers than in cells of the deeper layers. It is suggested that the limitation of the lethal effects of bleomycin and peplomycin in solid tumors may be overcome by improving the state of oxygenation of hypoxic cells and by combining either drug with one which inhibits recovery from potentially lethal damage.

Field-induced spin-density wave beyond hidden order in URu2Si2.

URu2Si2 is one of the most enigmatic strongly correlated electron systems and offers a fertile testing ground for new concepts in condensed matter science. In spite of >30 years of intense research, no consensus on the order parameter of its low-temperature hidden-order phase exists. A strong magnetic field transforms the hidden order into magnetically ordered phases, whose order parameter has also been defying experimental observation. Here, thanks to neutron diffraction under pulsed magnetic fields up to 40 T, we identify the field-induced phases of URu2Si2 as a spin-density-wave state. The transition to the spin-density wave represents a unique touchstone for understanding the hidden-order phase. An intimate relationship between this magnetic structure, the magnetic fluctuations and the Fermi surface is emphasized, calling for dedicated band-structure calculations.

A novel 550-kDa protein in skeletal muscle of chick embryo: purification and localization.

We have found a novel protein with a molecular mass of 550 kDa on SDS-polyacrylamide gels, which is abundant in skeletal muscle tissues at an early stage of chick embryonic development. The 550-kDa protein decreased with the progress of development, and only a slight amount of the protein was present in adult chicken skeletal muscle. The 550-kDa protein was purified from the cytoplasm of 18 day embryos by a procedure including ultracentrifugation and gel filtration. The purified 550-kDa protein was essentially free of contaminants as judged by SDS-PAGE. By immunofluorescence and immunoelectron microscopy using the antibody raised against the 550-kDa protein, this protein was shown to be localized in the peripheries of adult muscle fibers and at the Z-disks of isolated myofibrils. These findings have led us to conclude that the 550-kDa protein is a novel myofibrillar protein in chicken skeletal muscle.

Characterization of a novel 550-kDa protein in skeletal muscle of chick embryo.

Some characteristics of a novel 550-kDa protein which is abundant in skeletal muscle tissues at an early stage of the chick embryo, and localized in the peripheries of adult muscle fibers and at the Z-disks of isolated myofibrils, was investigated. A cosedimentation experiment and solid phase immunoabsorbent assay showed that the 550-kDa protein binds directly to F-actin. Therefore, it is concluded that the 550-kDa protein is a novel actin-binding protein. The 550-kDa protein was also interacted with alpha-actinin, laminin, fibronectin and Type IV collagen. Reactions with several kinds of lectin revealed that the 550-kDa protein is a glycoprotein containing oligosaccharides. Electron microscopic observation of negatively stained 550-kDa protein showed that native 550-kDa protein molecules are particles with an average diameter of 26.5 nm, but those particles treated with ethanol/ether are filamentous structures. These results suggest that the 550-kDa protein in the cytoplasma of unorganized skeletal muscle tissues exists as lipid-protein complex. Consequently, the 550-kDa protein may play an important role in the binding of myofibrils to the basal lamina by interaction with F-actin, alpha-actinin, laminin, fibronectin or Type IV collagen.

Involvement of cardiotrophin-1 in cardiac myocyte-nonmyocyte interactions during hypertrophy of rat cardiac myocytes in vitro.

BACKGROUND: The mechanism responsible for cardiac hypertrophy is currently conceptualized as having 2 components, mediated by cardiac myocytes and nonmyocytes, respectively. The interaction between myocytes and nonmyocytes via growth factors and/or cytokines plays an important role in the development of cardiac hypertrophy. We found that cardiac myocytes showed hypertrophic changes when cocultured with cardiac nonmyocytes. Cardiotrophin-1 (CT-1), a new member of the interleukin-6 family of cytokines, was identified by its ability to induce hypertrophic response in cardiac myocytes. In this study, we used the in vitro coculture system to examine how CT-1 is involved in the interaction between cardiac myocytes and nonmyocytes during the hypertrophy process. METHODS AND RESULTS: RNase protection assay revealed that CT-1 mRNA levels were 3. 5 times higher in cultured cardiac nonmyocytes than in cultured cardiac myocytes. We developed anti-CT-1 antibodies and found that they significantly inhibited the increased atrial and brain natriuretic peptide secretion and protein synthesis characteristic of hypertrophic changes of myocytes in the coculture. In addition, non-myocyte-conditioned medium rapidly elicited tyrosine phosphorylation of STAT3 and induced an increase in natriuretic peptide secretion and protein synthesis in cultured cardiac myocytes; these effects were partially suppressed by anti-CT-1 antibodies. Finally, the hypertrophic effects of CT-1 and endothelin-1, which we had previously implicated in the hypertrophic activity in the coculture, were additive in cardiac myocytes. CONCLUSIONS: These results show that CT-1 secreted from cardiac nonmyocytes is significantly involved in the hypertrophic changes of cardiac myocytes in the coculture and suggest that CT-1 is an important local regulator in the process of cardiac hypertrophy.

Aldosterone induces angiotensin-converting-enzyme gene expression in cultured neonatal rat cardiocytes.

BACKGROUND: The cardiac renin-angiotensin-aldosterone system is activated in failing hearts in proportion to the severity of the disease. We hypothesized that a positive feedback mechanism might exist within this system and contribute to the progression of the heart failure. Methods and Results-- To test this hypothesis, we examined whether angiotensin II or aldosterone induces the expression of angiotensin-converting-enzyme (ACE) mRNA in cultured neonatal rat ventricular cardiocytes. Expression of ACE mRNA was detected and quantified using real-time reverse transcription-polymerase chain reaction. Exposure to angiotensin II (10(-5) mol/L) for 24 hours had no significant effect on the expression of ACE mRNA (0.7+/-0.5-fold versus control, P=NS), but similar treatment with aldosterone (10(-5) mol/L) induced a 23.3+/-7.9-fold increase (P<0.01) in ACE mRNA expression. The effect of aldosterone was both time- (maximal effect, 24 hours) and dose-dependent (EC(50), 4x10(-7) mol/L), and it was significantly (P<0.01) inhibited by spironolactone, a specific mineralocorticoid receptor antagonist. CONCLUSIONS: Aldosterone upregulates ACE mRNA expression, which is blocked by spironolactone in neonatal rat cardiocytes. Thus, spironolactone may suppress the progression of heart failure by blocking the effects of aldosterone and angiotensin II.

Relationship between plasma level of cardiotrophin-1 and left ventricular mass index in patients with dilated cardiomyopathy.

OBJECTIVES: The study evaluated the relationship between plasma cardiotrophin-1 (CT-1) concentration and left ventricular (LV) mass in dilated cardiomyopathy (DCM) patients with congestive heart failure (CHF). BACKGROUND: Cardiotrophin-1 is a newly identified member of the interleukin-6 (IL-6) family of cytokines and one of the endogenous ligands for gp130 signaling pathways in the heart, and it has potent hypertrophic and survival effects on cardiac myocytes. However, the clinical significance of CT-1 is poorly understood. METHODS: We measured the plasma CT-1 level in 51 consecutive patients with DCM. Patients were classified into two groups: small LV mass index group and large LV mass index group, based on the median level of LV mass index. RESULTS: The plasma CT-1 level was increased in DCM patients with the severity of CHF and was significantly higher in the large LV mass group than in the small LV mass group, despite the absence of a difference in LV ejection fraction between the two groups. In addition, there was a significant positive correlation between the plasma CT-1 level and the LV mass index (r = 0.627, p < 0.0001). According to stepwise multivariate analyses among hemodynamic and neurohumoral factors, a high plasma CT-1 level showed an independent and significant positive relationship with a large LV mass index in patients with DCM. CONCLUSIONS: These results indicate that the plasma CT-1 level is increased in patients with DCM and is significantly correlated with the LV mass index, suggesting that CT-1 plays an important role in structural LV remodeling in patients with DCM.

Four subclones with distinct immunoglobulin light chain phenotypes. (kappa+lambda+, kappa+, lambda+ and kappa-lambda-) from acute leukemia.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-9, was established from the peripheral blood specimen of a immunoglobin (lg) phenotype, the established BALM-9 cell line expressed both kappa and lambda light (L) chains simultaneously in a range of 30-80%. Two-color flow cytometric analysis demonstrated that there was a distinct population of kappa lambda double positive cells as well as kappa single, lambda single, and double negative populations. Therefore, subclones were obtained from each population by limiting dilution and were designated BALM-9KL (kappa+lambda+), BALM-9K (kappa+lambda-), BALM 9N (kappa-lambda-). Western blotting confirmed the results of the immunofluorescence test at the protein level. In BALM-9N, L chains were absent even in the cytoplasm as demonstrated by Western blotting. Evidence that the subclones have the same ancestry was provided both by cytogenetic analysis and by Southern blotting, which revealed the 14q32 chromosomal rearrangement as a common abnormality and the same IgH gene arrangement among the subclones. The existence of a kappa lambda positive B cell population suggests a transient stage of normal B cell maturation. These subclones might represent such a stage and thus provide a useful means of analyzing the mechanism of this double light chain expression.

A novel ALL-L3 cell line, BALM-16, lacking expression of immunoglobulin chains derived from a patient with hypercalcemia.

A human acute lymphoblastic leukemia (ALL) cell line, BALM-16, was established from the peripheral blood specimen of a patient with B cell ALL L3 type (ALL-L3) in relapse. As with the original leukemia cells, the established line was negative for both cell surface and cytoplasmic immunoglobulin (Ig) chains. Absence of Ig expression was confirmed by Western blotting. Southern blot analysis demonstrated homozygous deletion of the C kappa gene, germ line configuration of the C lambda and rearrangement of IgJH genes. Cytogenetic analysis of both leukemic bone marrow and BALM-16 cells showed the t(8;22)(q24;q11) abnormality which is specifically associated with ALL-L3 and Burkitt lymphoma. The patient's serum showed hypercalcemia, prompting further investigation of the established cell lines which showed parathyroid hormone-related peptide (PTHrP) mRNA detected by reverse-transcriptase polymerase chain reaction. However, PTHrP production was not detected in the culture supernatant. The established cell line, BALM-16, could provide a useful material for analyzing the lack of Ig expression and of clarifying the pathogenesis of this type of B cell malignancy.

Identification of Fabry's disease by the screening of alpha-galactosidase A activity in male and female hemodialysis patients.

BACKGROUND: Although previous studies reported that the prevalence of Fabry's disease was 0.16 - 1.2% in hemodialysis (HD) patients based on measurement of a-galactosidase A (alpha-Gal A) activity, few reports detected female patients by the screening for alpha-Gal A. Here we determined the prevalence of Fabry's disease not only in male but also in female HD patients by measuring alpha-Gal A. METHODS: Plasma alpha-Gal A was measured in 696 consecutive males (n = 401) and females (n = 295) on HD. Patients with low plasma alpha-Gal A were examined for leukocyte alpha-Gal A, and patients with low leukocyte alpha-Gal A underwent alpha-Gal A gene sequence analysis for possible mutations, and family survey. RESULTS: Among 15 patients with low plasma alpha-Gal A activity, 4 male patients with low leukocyte alpha-Gal A and 1 female patient revealing low plasma alpha-Gal A were detected in 696 HD patients (0.7% of total patients). 3 of these 5 patients were already diagnosed to have the classical type of Fabry's disease. The other 2 patients were newly diagnosed as Fabry's disease, and did not have typical manifestations of Fabry's disease other than renal failure and left ventricular hypertrophy. DNA analysis of these 2 newly diagnosed patients revealed that each had an alpha-Gal missense mutation, previously identified (E66Q, M2961). CONCLUSION: Fabry's disease should be considered in the etiology of unexplained end-stage renal disease. Not only affected males but also affected females undergoing HD patients can be readily diagnosed by alpha-Gal A activities and gene analysis. These patients and their family members may benefit from enzyme replacement therapy for Fabry's disease.