Transcriptional profile reveals altered hepatic lipid and cholesterol metabolism in hyposulfatemic NaS1 null mice

Sulfate plays an essential role in human growth and development, and its circulating levels are maintained by the renal Na+-SO42- cotransporter, NaS1. We previously generated a NaS1 knockout ( Nas1(-/-)) mouse, an animal model for hyposulfatemia, that exhibits reduced growth and liver abnormalities including hepatomegaly. In this study, we investigated the hepatic gene expression profile of Nas1(-/-) mice using oligonucleotide microarrays. The mRNA expression levels of 92 genes with known functional roles in metabolism, cell signaling, cell defense, immune response, cell structure, transcription, or protein synthesis were increased ( n = 51) or decreased ( n = 41) in Nas1(-/-) mice when compared with Nas1(-/-) mice. The most upregulated transcript levels in Nas1(-/-) mice were found for the sulfotransferase genes, Sult3a1 ( approximate to 500% increase) and Sult2a2 ( 100% increase), whereas the metallothionein-1 gene, Mt1, was among the most downregulated genes ( 70% decrease). Several genes involved in lipid and cholesterol metabolism, including Scd1, Acly, Gpam, Elov16, Acsl5, Mvd, Insig1, and Apoa4, were found to be upregulated ( >= 30% increase) in Nas1(+/+) mice. In addition, Nas1(+/+) mice exhibited increased levels of hepatic lipid ( approximate to 16% increase), serum cholesterol ( approximate to 20% increase), and low-density lipoprotein ( approximate to 100% increase) and reduced hepatic glycogen ( approximate to 50% decrease) levels. In conclusion, these data suggest an altered lipid and cholesterol metabolism in the hyposulfatemic Nas1(-/-) mouse and provide new insights into the metabolic state of the liver in Nas1(-/-) mice.

Downregulation of IgE antibody and allergic responses in the lung by epidermal biolistic microparticle delivery

Background: Biolistic injections provide a needle-free delivery of antigen-laden microparticles to the epithelium. The precision of the injection preferentially targets the Langerhans cell network, which, although ideal for vaccination, might not be suitable for the downregulation of immune responses in immunotherapy. Objective: We sought to determine the ability of biolistic injection of antigen into the epithelium of sensitized mice to inhibit IgE antibody and lung inflammatory responses produced by further exposure to antigen. Methods: Mice were sensitized by means of a needle injection of ovalbumin (OVA) in alum and given a series of biolistic injections of OVA or vehicle control, followed by a boost of OVA in alum. Serum IgE and IgG antibodies were measured before and after the boost. The mice were then challenged intranasally, and the infiltration of inflammatory cells was measured by means of bronchoalveolar lavage. Airway reactivity of the challenged mice was measured by examining responses to methacholine with forced oscillatory techniques. Results: Biolistic injection of OVA into the dorsal skin of sensitized mice markedly inhibited IgE and IgG1 antibody responses induced by boosting. IgG2a antibody responses were reduced rather than stimulated. The eosinophilic inflammation in the bronchoalveolar lavage fluid induced by intranasal challenge was also markedly inhibited. Lung hyperreactivity showed an initial increase and then a decrease of responsiveness to methacholine, with elastance returning to the level of unsensitized mice. Biolistic injection into the buccal epithelium was also inhibitory. Conclusions: Biolistic injection of allergen inhibited the boosting of IgE antibody and eosinophilic lung inflammatory responses without inducing TO immunity.

Mice expressing the E7 oncogene of HPV16 in epithelium show central tolerance, and evidence of peripheral anergising tolerance, to E7-encoded cytotoxic T-lymphocyte epitopes

In order to derive mice which expressed both the E7 open reading frame transgene of human papillomavirus type 16 in skin and MHC class 1 restriction elements for several E7-encoded cytotoxic T-lymphocyte (CTL) epitopes, K14.HPV16E7 mice which express E7 in basal keratinocytes were crossed to the F1 generation with A2.1 K-b transgenic mice which express the MHC binding cleft domains of human HLA A*0201, and murine H-2(b). F1 mice (denoted K14E7xA2.1) expressed E7 in the thymus at least as early as 2-5 days before birth. Immunisation of FVBxA2.1 control mice (transgenic for HLA A*0201 and H-2(b) but not for E7), with two HLA A*0201-restricted epitopes of E7 and one H-2(b)-restricted CTL epitope of E7, gave strong primary CTL responses recognising epitope-pulsed or constitutively E7-expressing syngeneic target cells. In contrast, in immunised K14E7xA2.1 mice, the CTL responses to the H-2(b) epitope and one of the HLA A*0201 CTL epitopes were strongly down-regulated, and to the other HLA A*0201 epitope, completely abolished, as demonstrated by percentage specific killing by bulk splenocyte cultures in cyrotoxicity assays, and by CTL precursor frequency analysis, In thymus-transplanted bone marrow radiation chimeras in which the immune system of K14E7xA2.1 mice was replaced by a FVBxA2.1 immune system, specific immunisation did not result in reemergence of strong E7-directed CTL responses. In agreement with these in vitro findings, specific immunisation failed to significantly alter the course of E7-associated tumour development in K14E7xA2.1 mice. These data are consistent with a model of central deletional CTL tolerance to E7-encoded epitopes recognised in the context of two distinct MHC class 1 restriction elements, and with the possibility of peripheral T-cell anergy maintained by expression of E7 in the skin. (C) 1998 Academic Press.

Improved beef production from supplementation of Hereford, Brahman and crossbred cattle grazing low and medium quality pastures in the subtropics of Australia

Subtropical grasslands are low in organic matter digestibility (OMD) (0.60) and nitrogen (N) (15 g/kg) for much of the year and this limits cattle production which is characterized by low calving rates and low weaning weights. Production has been based on Bos taurus British breeds of cattle but this is changing and now many breeding herds comprise B, indicus cows and their crosses. This change has increased some aspects of production, but low calving rates persist. A 4-year study was undertaken with a view to improve calving rates and weaner output by supplementing cows grazing either native or improved pastures with a high protein oilseed meal (cottonseed meal; CSM) on four sites. These sites were subdivided into a total of 36 paddocks to allow for two replications in a 3 breeds X 3 supplementation rates X 2 pastures factorial design. Selected cows (no. = 216) from Hereford (H), Brahman (B) and Brahman X Hereford (BH) breed types were set to graze either native pastures (0.45 to 0.62 OMD, 8 to 15 g N per kg; low quality) or improved pastures (0.47 to 0.67 OA ID, 10 to 22 g N per kg; medium quality). Cows were given either 0, 750 or 1500 g/day of CSM for 130 days from calving until 4 weeks into a 12- to 13-week mating period. The CSM was given as two meals per week. Live weight at mating of cows on the low quality pasture was increased (P < 0.01) over those not supplemented by feeding either 750 g CSM per day (H and B cows) or 1500 g CSM per day tall cows). There was no significant effect of supplementation on the mating weights of B cows grazing the medium quality sites. Calving rate of B cows was not increased by their supplementation on either low (4-year mean 58.3 %) or medium quality pastures (66.8%) but did tend to be higher in H cows when supplemented at 1500 g CSM per day on the low (66.7 v. 78.0 (s.e. 6.09) %; P < 0.1) and medium quality pastures (70.5 v. 93.5 (s.e. 4.72) %). An increased calving rate (65.8 (s.e. 6.6) % to 83.2 (s.e. 5.82) % in supplemented BH cows grazing low quality pastures approached significance (P < 0.1) when given CSM at 1500 g/day but there was no increased trend in calving rate when this breed type was supplemented on medium quality pastures. Weaning weights of calves from and B and BH cows were increased (P < 0.05) by supplementation of their darns at 750 g/day and for calves weaned from H cows supplemented at 1500 g/day of CSM. Supplementation at 1500 g/day on low quality pastures increased weaner output per cow mated by 120% for H, by 65% for BH cows and by 50% for B cows. Weaner output was increased by 34 and 40%, respectively, for B and H cows when supplemented at 750 g/day and grazing medium quality pastures but there teas no significant effect of supplementation on output from BH cows. Responses in many parameters differed between years. These results were interpreted as a response to the protein in the oilseed meal supplement by B, taurus and B. taurus X B. indicus cross cows grazing on the subtropical pastures. The study also highlighted that responses to the meal differed between breed types, between the quality of the grazed pasture and between the years of supplementation.

Antibody levels to the class I and II epitopes of the M protein and myosin are related to group A streptococcal exposure in endemic populations

Rheumatic fever (RF)/rheumatic heart disease (RHD) and post-streptococcal glomerulonephritis are thought to be autoimmune diseases, and follow group A streptococcal (GAS) infection. Different GAS M types have been associated with rheumatogenicity or nephritogenicity and categorized into either of two distinct classes (I or II) based on amino acid sequences present within the repeat region ('C' repeats) of the M protein. Sera from ARF patients have previously been shown to contain elevated levels of antibodies to the class I-specific epitope and myosin with the class I-specific antibodies also being cross-reactive to myosin, suggesting a disease association. This study shows that immunoreactivity of the class I-specific peptide and myosin does not differ between controls and acute RF (ARF)/RHD in populations that are highly endemic for GAS, raising the possibility that the association is related to GAS exposure, not the presence of ARF/RHD. Peptide inhibition studies suggest that the class I epitope may be conformational and residue 10 of the peptide is critical for antibody binding. We demonstrate that correlation of antibody levels between the class I and II epitope is due to class II-specific antibodies recognizing a common epitope with class I which is contained within the sequence RDL-ASRE. Our results suggest that antibody prevalence to class I and II epitopes and myosin is associated with GAS exposure, and that antibodies to these epitopes are not an indicator of disease nor a pathogenic factor in endemic populations.

A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression

The c fins gene encodes the receptor for macrophage colony-stimulating factor-1. This gene is expressed selectively in the macrophage cell lineage. Previous studies have implicated sequences in intron 2 that control transcript elongation in tissue-specific and regulated expression of c -fms. Four macrophage-specific deoxyribonuclease I (DNase I)-hypersensitive sites (DHSS) were identified within mouse intron 2. Sequences of these DHSS were found to be highly conserved compared with those in the human gene. A 250-bp region we refer to as the fins intronic regulatory element (FIRE), which is even more highly conserved than the c-fins proximal promoter, contains many consensus binding sites for macrophage-expressed transcription factors including Spl, PU.1, and C/EBP. FIRE was found to act as a macrophage-specific enhancer and as a promoter with an antisense orientation preference in transient transfections. In stable transfections of the macrophage line RAW264, as well as in clones selected for high and low-level c -fms mRNA expression, the presence of intron 2 increased the frequency and level of expression of reporter genes compared with those attained using the promoter alone. Removal of FIRE abolished reporter gene expression, revealing a suppressive activity in the remaining intronic sequences. Hence, FIRE is shown to be a key regulatory element in the fins gene.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2(d)), C57BL6 (H-2(b)), DBA/2J (H-2(d)) and CBA/CaH (H-2(k)) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4(+) and CD8(+) T-cell subsets, CD14(+) macrophages and CD19(+) B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8(+) T cells and CD19(+) B cells were found in any of the lesions. The percentages of CD4(+) cells, CD14(+) cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14(+) cells in sham-immunized mice. The percentage of CD14(+) cells was higher than that of CD4(+) cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4(+) and CD14(+) cells predominated in immunized CBA/CaH mice and CD4(+) cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14(+) cells and CD4(+) cells in sham-immunized mice. IgG1(+) plasma cells were more dominant than IgG2a(+) cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a(+) plasma cells were more obvious in sham-immunized mice. IgG2a(+) plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

Transition to raloxifene with and without low-dose estrogen therapy in postmenopausal women: effects on serum lipids and fibrinogen - a pilot study

Objective To compare the effects of transferring from low-dose transdermal estrogen to raloxifene (RLX), with a phase of alternate-day RLX therapy with or without low-dose transdermal estrogen, on serum lipids and fibrinogen in postmenopausal women previously administered estrogen plus progestogen therapy. Methods Sixty postmenopausal women (mean age 55 years) were randomized to one of two treatment groups: RLX + low-dose transdermal estrogen (RLX + E) or RLX + placebo. The study consisted of four 8-week phases: phase I (all subjects low-dose transdermal estrogen 25 mug/day), phase II (double-blind RLX 60 mg every 2nd day in combination with either low-dose transdermal estrogen or placebo), phase III (all subjects RLX 60 mg every 2nd day + placebo) and phase IV (all subjects RLX 60 mg/day + placebo). Results No significant differences existed between groups for baseline measurements prior to phase I. In phase I, for all subjects combined, total cholesterol and low-density lipoprotem cholesterol both showed a significant increase (median increase of 0.2 mmol/l, p = 0.008 and 0.4 mmol/l, p < 0.001, respectively), while triglycerides decreased significantly (median decrease of 0.2 mmol/l, p < 0.001). For the primary analysis (phase II to phase IV), the mean change from baseline observations showed no significant differences between the therapy groups for serum lipids, fibrinogen, vital signs or weight. In the comparison phase (phase II), changes in serum lipids, fibrinogen, vital signs and weight were not significantly different between groups. Conclusion Gradual conversion to RLX from low-dose transdermal estrogen, with a phase of alternate-day RLX therapy with or without low-dose transdermal estrogen, does not have any effect on the serum lipid profile or fibrinogen level.

Low-temperature single-crystal Raman and neutron-diffraction study of the hydrogenous ammonium copper(II) tutton salt and the deuterated analogue in the metastable state

Low-temperature (15 K) single-crystal neutron-diffraction structures and Raman spectra of the salts (NX4)(2)[CU(OX2)(6)](SO4)(2), where X = H or D, are reported. This study is concerned with the origin of the structural phase change that is known to occur upon deuteration. Data for the deuterated salt were measured in the metastable state, achieved by application of 500 bar of hydrostatic pressure at similar to303 K followed by cooling to 281 K and the subsequent release of pressure. This allows for the direct comparison between the hydrogenous and deuterated salts, in the same modification, at ambient pressure and low temperature. The Raman spectra provide no intimation of any significant change in the intermolecular bonding. Furthermore, structural differences are few, the largest being for the long Cu-O bond, which is 2.2834(5) and 2.2802(4) Angstrom for the hydrogenous and the deuterated salts, respectively. Calorimetric data for the deuterated salt are also presented, providing an estimate of 0.17(2) kJ/mol for the enthalpy difference between the two structural forms at 295.8(5) K. The structural data suggest that substitution of hydrogen for deuterium gives rise to changes in the hydrogen-bonding interactions that result in a slightly reduced force field about the copper(II) center. The small structural differences suggest different relative stabilities for the hydrogenous and deuterated salts, which may be sufficient to stabilize the hydrogenous salt in the anomalous structural form.

Low temperature induced spikelet sterility in rice. I. Nitrogen fertilisation and sensitive reproductive period

Low temperature during panicle development in rice increases spikelet sterility. This effect is exacerbated by high rates of nitrogen (N) application in the field. Spikelet sterility induced by low temperature and N fertilisation was examined in glasshouse experiments to clarify the mechanisms involved. In two glasshouse experiments, 12-h periods of low (18/13degreesC) and high (28/23degreesC) day/night temperatures were imposed over periods of 5-7 days during panicle development, to determine the effects of low temperature and N fertilisation on spikelet sterility. In one experiment, 50% sunlight was imposed together with low temperature to investigate the additive effects of reduced solar radiation and low temperature. The effect of increased tillering due to N fertilisation was examined by a tiller removal treatment in the same experiment. Pollen grain number and spikelet sterility were recorded at heading and harvest, respectively. Although there was no significant effect of low temperature on spikelet sterility in the absence of applied N, low temperature greatly increased spikelet sterility as a result of a reduction in the number of engorged pollen grains per anther in the presence of applied N. Spikelet sterility was strongly correlated with the number of engorged pollen grains per anther. Low temperature during very early ( late stage of spikelet differentiation-pollen mother cell stage) and peak ( second meiotic division stage-early stage of extine formation) microspore development caused a severe reduction in engorged pollen production mainly as a result of reduced total pollen production. Unlike low temperature, the effect of shading was rather small. The increased tillering due to application of high rates of N, increased both spikelet number per plant and spikelet sterility under low temperature conditions. The removal of tillers as they appeared reduced the number of total spikelets per plant and maintained a large number of engorged pollen grains per anther which, in turn, reduced spikelet sterility. The number of engorged pollen grains per anther determined the numbers of intercepted and germinated pollen grains on the stigma. It is concluded that N increased tillering and spikelet number per plant and this, in turn, reduced the number of engorged pollen grains per anther, leading into increased spikelet sterility under low temperature condition.

Effects of pravastatin on coronary events in 2073 patients with low levels of both low-density lipoprotein cholesterol and high-density lipoprotein cholesterol: results from the LIPID study

Aims Fibrates or nicotinic acid are usually recommended for secondary prevention of coronary heart disease in patients with low plasma levels of both low-density tipoprotein cholesterol (LDL-C) less than or equal to140 mg/dL (less than or equal to3.6 mmol/L) and high-density lipoprotein cholesterol (HDL-C) less than or equal to40 mg/dL (less than or equal to1.03 mmol/L). The LIPID trial, a randomised, placebo-controlled trial in 9014 patients at 87 centres in Australia and New Zealand, provided an opportunity to investigate the effects of an HMG-CoA reductase inhibitor in patients with tow LDL-C and tow HDL-C. Methods and results Participants in this post hoc substudy were 2073 patients aged 31-75 years with baseline LDL-C less than or equal to140 mg/dL (less than or equal to3.6 mmoL/L), HDL-C less than or equal to40 mg/dL (less than or equal to1.03 mmol/L), and triglyceride less than or equal to300 mg/dL (less than or equal to3.4 mmol/L). The relative risk reduction with pravastatin treatment was 27% for major coronary events (95% Cl 8-42%), 27% for coronary heart disease mortality (95% CI 0-47%), 21% for all-cause mortality (95% Cl 0-38%), and 51% for stroke (95% CI 24-69%). The number needed to treat to prevent a major coronary event over 6 years was 22. Conclusions Treatment with pravastatin in patients with both low LDL-C and low HDL-C significantly reduced major coronary events, stroke, and all-cause mortality. The level of HDL-C is crucial to the risk of recurrent CHD events and, consequently, the benefit of lowering LDL-C. (C) 2004 Published by Elsevier Ltd on behalf of The European Society of Cardiology.

Desire to drink in low- and high-risk social drinkers: An experimental analogue of impaired control

This study used a novel cue exposure paradigm to investigate the differences between high- and low-risk drinkers in their desire to drink during a drinking session. Fifty-three self-selected participants were assigned to high- or low-risk drinking groups based on their self-reported consumption of alcohol, then compared on their desire to drink over a 90 min paced drinking session. High-risk drinkers showed increasing desire over the session, while low-risk drinkers' desire began to decrease after only a short drinking period. The perceived and actual effects of the alcohol did not appear to be able to account for the difference. Results are discussed with reference to issues of impaired control. Suggestions for future research directions are also offered.

Active and passive immunization against oral Candida albicans infection in a murine model

Background/aims: Clinical and laboratory studies are consistent with a major role for cell-mediated immunity in recovery from oral infection with Candida albicans, but the role of humoral immunity remains controversial. The purpose of this study was to establish the relative contributions of cellular and humoral immunity to protection against oral candidiasis in a murine model, and to determine whether host responses could be enhanced by different immunization strategies. Results: Active oral immunization was protective in BALB/c and CBA/CaH mice, reducing both fungal burden and duration of infection after secondary challenge, whereas systemic immunization failed to protect against subsequent oral challenge. Candida-specific IgM was the predominant antibody detected in serum following both primary and secondary oral challenge; however, Candida-specific salivary IgA was not detectable. Immunization by passive transfer of either lymphocytes or immune serum did not confer any significant protection against oral infection in either susceptible or resistant mouse strain. Conclusion: The data demonstrate a possible role for mucosa-associated immunity following active immunization by the oral route, most likely exerted by local T lymphocytes resident in the oral mucosa, but there was no evidence to support a role for humoral immunity in protection against oral candidiasis.

Mice and men: Their promoter properties

Using the two largest collections of Mus musculus and Homo sapiens transcription start sites ( TSSs) determined based on CAGE tags, ditags, full- length cDNAs, and other transcript data, we describe the compositional landscape surrounding TSSs with the aim of gaining better insight into the properties of mammalian promoters. We classified TSSs into four types based on compositional properties of regions immediately surrounding them. These properties highlighted distinctive features in the extended core promoters that helped us delineate boundaries of the transcription initiation domain space for both species. The TSS types were analyzed for associations with initiating dinucleotides, CpG islands, TATA boxes, and an extensive collection of statistically significant cis- elements in mouse and human. We found that different TSS types show preferences for different sets of initiating dinucleotides and ciselements. Through Gene Ontology and eVOC categories and tissue expression libraries we linked TSS characteristics to expression. Moreover, we show a link of TSS characteristics to very specific genomic organization in an example of immune- response- related genes ( GO: 0006955). Our results shed light on the global properties of the two transcriptomes not revealed before and therefore provide the framework for better understanding of the transcriptional mechanisms in the two species, as well as a framework for development of new and more efficient promoter- and gene- finding tools.

Redundant roles of Sox17 and Sox18 in postnatal angiogenesis in mice

Sox7, Sox17 and Sox18 constitute group F of the Sox family of HMG box transcription factor genes. Dominant-negative mutations in Sox18 underlie the cardiovascular defects observed in ragged mutant mice. By contrast, Sox18(-/-) mice are viable and fertile, and display no appreciable anomaly in their vasculature, suggesting functional compensation by the two other SoxF genes. Here, we provide direct evidence for redundant function of Sox17 and Sox18 in postnatal neovascularization by generating Sox17(+/-)-Sox18(-/-) double mutant mice. Whereas Sox18(-/-) and Sox17(+/-)-Sox18(+/)-mice showed no vascular defects, approximately half of the Sox17(+/-)-Sox18(-/-) pups died before postnatal day 21 (P21). They showed reduced neovascularization in the liver sinusoids and kidney outer medulla vasa recta at P7, which most likely caused the ischemic necrosis observed by P14 in hepatocytes and renal tubular epithelia. Those that survived to adulthood showed similar, but milder, vascular anomalies in both liver and kidney, and females were infertile with varying degrees of vascular abnormalities in the reproductive organs. These anomalies corresponded with sites of expression of Sox7 and Sox17 in the developing postnatal vasculature. In vitro angiogenesis assays, using primary endothelial cells isolated from the P7 livers, showed that the Sox17(+/-)-Sox18(-/-)endothelial cells were defective in endothelial sprouting and remodeling of the vasculature in a phenotype-dependent manner. Therefore, our findings indicate that Sox17 and Sox18, and possibly all three SoxF genes, are cooperatively involved in mammalian vascular development.