A mass shooting at Port Arthur, Tasmania, Australia: a study of its impact on early pregnancy losses using a conception time-based methodology.

STUDY QUESTION: Does an acute calamity in a community cause early miscarriage and is this association the same for male and female fetuses? SUMMARY ANSWER: Estimated losses of 29.5% of first trimester pregnancies in the affected region could be associated with an acute calamity, with no statistically significant difference in estimated losses by fetal sex. WHAT IS KNOWN ALREADY: There are very few studies on the impact of a calamity on early pregnancy loss and its differential effects on male and female fetuses. A decline in the human sex ratio at birth associated with the events of 9/11 in New York has been documented. STUDY DESIGN, SIZE, DURATION: This is a retrospective descriptive study of birth register data in Tasmania, Australia, from 1991 to 1997, covering the period in which the calamity occurred. The register contains data on all pregnancies that proceeded to >20 weeks gestation. The conception date was calculated by subtracting gestational age from birth date. We estimated that 40 318 pregnancies were conceived in the period 1991-1996 inclusive. These were aggregated to 4-weekly blocks classified by region and sex. PARTICIPANTS/MATERIALS, SETTING, METHODS: The acute calamity was at Port Arthur, Tasmania, Australia. On 28 April 1996, a gunman opened fire on visitors and staff in a tourist cafe. A very stressful 20 h period, ended with 35 people dead and 22 injured. A negative binomial regression model was used to assess the association between this calamity and pregnancy loss. This loss is evidenced by a shortfall in the registration of pregnancies that were in their first trimester at the time of the calamity. MAIN RESULTS AND THE ROLE OF CHANCE: We estimated a shortfall of 29.5% or 229 registered pregnancies among those in the first trimester at the time of the calamity (P < 0.001), in the region surrounding the calamity site. There was no sex effect in this shortfall (P = 0.911). There was no corresponding shortfall in other parts of Tasmania (P = 0.349). LIMITATIONS, REASONS FOR CAUTION: The study is descriptive and cannot produce causal inferences. These first trimester miscarriages are estimated statistically and it is understood that gestational age is an estimate. The use of maternal residential postcodes at birth as a surrogate for geographic area or space assumes that the mother has not moved into the postcode area after the calamity and before the reporting of a birth. WIDER IMPLICATIONS OF THE FINDINGS: The results of this study suggest that calamities bring about significant pregnancy loss affecting both sexes. The methodology presented of inferring conception date from birth date and using this for analysis, provides a more accurate assessment of first trimester pregnancy losses than raw birth data or sex ratio at birth.

Chronic kidney disease: cardiac and renal angiotensin-converting enzyme (ACE) 2 expression in rats after subtotal nephrectomy and the effect of ACE inhibition.

Renin-angiotensin system blockade slows but does not prevent the cardiovascular complications of chronic kidney disease (CKD). Angiotensin-converting enzyme (ACE) 2 is differentially regulated in acute kidney injury, with increased cardiac ACE2 but decreased kidney ACE2 levels. This study investigated the effect of long-term ACE inhibition on cardiac and renal ACE2 in rats with CKD induced by subtotal nephrectomy (STNx). Sprague-Dawley rats had sham (control) or STNx surgery. Control rats received vehicle (n = 9) and STNx rats ramipril (1 mg kg(-1) day(-1); n = 10) or vehicle (n = 10) for 28 days. Subtotal nephrectomy resulted in impaired creatinine clearance (P < 0.05), proteinuria (P < 0.05), renal fibrosis (P < 0.05) and reduced renal cortical ACE2 mRNA (P < 0.05) and activity (P < 0.05). In rats with CKD, ramipril improved creatinine clearance (P < 0.05) and was associated with an increase in cortical but not medullary ACE2 activity (P < 0.05). Compared with control rats, STNx rats were hypertensive (P < 0.01), with increased left ventricular end-diastolic pressure (LVEDP; P < 0.01), left ventricular hypertrophy (LVH; P < 0.05) and interstitial (P < 0.05) and perivascular fibrosis (P < 0.01). In rats with CKD, ramipril decreased blood pressure (P < 0.001) and reduced LVEDP (P < 0.01), LVH (P < 0.01) and perivascular fibrosis (P < 0.05) but did not significantly reduce interstitial fibrosis. There was no change in cardiac ACE2 in rats with CKD compared with control rats. In rats with CKD, ACE inhibition had major benefits to reduce blood pressure and cardiac hypertrophy and to improve creatinine clearance, but did not significantly impact on cardiac ACE2, cardiac interstitial fibrosis, renal fibrosis or proteinuria. Thus, in rats with CKD, renal ACE2 deficiency and lack of activation of cardiac ACE2 may contribute to the progression of cardiac and renal tissue injury. As long-term ACE inhibition only partly ameliorated the adverse cardio-renal effects of CKD, adjunctive therapies that lead to further increases in ACE2 activity may be needed to combat the cardio-renal complications of CKD.

Patterns of gene expression in pig adipose tissue: insulin-like growth factor system proteins, neuropeptide Y (NPY), NPY receptors, neurotrophic factors and other secreted factors.

Although cDNA microarray studies have examined gene expression in human and rodent adipose tissue, only one microarray study of adipose tissue from growing pigs has been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) from gilts at 90, 150, and 210 d (n=5 age(-1)). Dye labeled cDNA probes were hybridized to custom porcine microarrays (70-mer oligonucleotides). Gene expression of insulin-like growth factor binding proteins (IGFBPs), hormones, growth factors, neuropeptide Y (NPY) receptors (NPYRs) and other receptors in OSQ and MSQ changed little with age in growing pigs. Distinct patterns of relative gene expression were evident within NPYR and IGFBP family members in adipose tissue from growing pigs. Relative gene expression levels of NPY2R, NPY4R and angiopoietin 2 (ANG-2) distinguished OSQ and MSQ depots in growing pigs. We demonstrated, for the first time, the expression of IGFBP-7, IGFBP-5, NPY1R, NPY2R, NPY, connective tissue growth factor (CTGF), brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes in pig adipose tissue with microarray and RT-PCR assays. Furthermore, adipose tissue CTGF gene expression was upregulated while NPY and NPY2R gene expression were significantly down regulated by age. These studies demonstrate that expression of neuropeptides and neurotrophic factors in pig adipose tissue may be involved in regulation of leptin secretion. Many other regulatory factors were not influenced by age in growing pigs but may be influenced by location or depot.

Expression of CCAAT/enhancer binding protein C/EBPalpha, beta and delta in rat adipose stromal-vascular cells in vitro.

Stromal-vascular (S-V) cells from rat inguinal fat depots were isolated and cultured in medium containing fetal bovine serum (FBS) and differentiated in defined medium until lipid accumulation was apparent. C/EBPalpha, beta and delta levels were evaluated for different growth conditions and at different times using Western blots. Immediately after isolation C/EBPalpha, beta and delta could not be detected in S-V cells. After seeding for 24 h in Dulbecco's modified Eagle's medium (DMEM) with FBS, C/EBPalpha, beta and delta could all be detected. Cells at day 1 of culture in insulin, transferrin, triiodothyronine and selenium (ITTS) had increased levels of C/EBPalpha and continued steady high levels to day 6 of culture. Cultures grown in DMEM alone, with no ITTS, showed C/EBPalpha levels similar to ITTS cultures at day 1 and day 3; however, levels diminished after day 3. DMEM cultures also showed lipid accumulation at day 6; however, the number of cells and the amount of lipid cell were reduced from levels observed in ITTS cultures. C/EBPbeta was expressed uniformly throughout the culture period in either DMEM or ITTS cultures while C/EBPdelta expression was higher with DMEM treatment than with ITTS. Treatment of 2 day DMEM cultures with FBS increased levels of C/EBPbeta and delta but significantly reduced levels of C/EBPalpha. Immunocytochemical analysis of S-V cells at day 1 of culture showed a similar percentage of cells stained in DMEM cultures and ITTS cultures. However, by day 6 of culture the percentage of cells staining positively for C/EBPalpha in DMEM had been reduced by one half while in ITTS the percent positive cells remained about the same. Our results indicate that ITTS is not necessary for the induction of C/EBPalpha and accumulation of lipid in S-V cells. However, ITTS is responsible for maintaining C/EBPalpha and enhanced lipid accumulation. Because C/EBPalpha, beta and delta expression occurs very early in cell culture and C/EBPalpha and delta expression continues to increase in DMEM without any apparent inducing agents, our results suggest that these factors may be expressed by the same cells in vivo before being placed in culture. Thus, a large fraction of S-V cells may be further along in the differentiation program than 3T3 cells are when they begin differentiation.

Differentiation-dependent expression of obese (ob) gene by preadipocytes and adipocytes in primary cultures of porcine stromal-vascular cells.

The relationship between obese (ob) gene expression and preadipocyte differentiation was examined in primary cultures of porcine stromal-vascular (S-V) cells by Northern-blot analysis using a pig ob cDNA probe. Isolated adipocytes expressed high levels of ob gene, but S-V cells did not express the ob gene. Cultures were seeded with fetal bovine serum (FBS) plus dexamethasone (Dex) for 3 days followed by ITS (insulin 5 microg/ml, transferrin 5 microg/ml, and selenium 5 ng/ml) treatment for 6 days. Detectable levels of ob mRNA first appeared at day 1 with very low activity of glycerol phosphate dehydrogenase (GPDH). Levels of ob mRNA increased in parallel with preadipocyte number or GPDH activity at the later times in cultures. The depletion of preadipocytes by complement-mediated cytotoxicity at day 3 of culture resulted in markedly decreased ob mRNA expression. Immunocytochemical analysis showed that ob protein was localized in the cytosol of preadipocytes and adipocytes. These data indicated that the ob gene is expressed by preadipocytes and ob gene expression may be correlated with preadipocyte recruitment as well as fat cell size.

Elevated hypothalamic neuropeptide Y levels in rats with dorsomedial hindbrain lesions.

Lesions centered on the area postrema (AP) and adjacent nucleus of the solitary tract (AP/mNTS-lesions) are reported to result in increased consumption of highly palatable diets. Recent studies suggest that neuropeptide Y (NPY) may cause a preference for carbohydrate-rich diets. Thus, it is possible that NPY may play a role in the enhanced intake of highly palatable diets by AP/mNTS-lesioned rats. In the studies reported here, we found that lesions centered on the AP result in increased levels of NPY-immunoreactivity in the paraventricular nucleus of the hypothalamus. Additionally, steady-state NPY mRNA in the basomedial hypothalamus including the arcuate nucleus was elevated. Enhanced NPY was not found throughout the hypothalamus however, as NPY-immunoreactivity was not elevated in the lateral hypothalamus or the tissue bordering the anteroventral third ventricle. These data suggest the possibility that elevated hypothalamic NPY, particularly in the arcuate and paraventricular nuclei, may contribute to the altered food intake and energy balance observed in rats with lesions centered on the AP.

Neuropeptide Y expression in rat brain: effects of adrenalectomy.

Neuropeptide Y (NPY) messenger RNA was measured by hybridization of mRNA from the cortex, hippocampus, hypothalamus and striatum of rat brains. Adrenalectomized rats showed lowered level of NPY message in the striatum. A similar decline was found in the hypothalamus, while the cortex and hippocampus were unchanged. Levels of NPY message per unit total RNA were about the same for hypothalamus, cortex and striatum and about 50% less for hippocampus. Adrenalectomized rats that received replacement corticosterone had levels of NPY message that had returned to the levels found in rats receiving sham operation. Response elements consistent with our findings are reported in the NPY genomic sequence.

Experimentally induced attenuation of neuropeptide-Y gene expression in transgenic mice increases mortality rate following seizures.

Previous experiments have reported increased seizure susceptibility in transgenic mice lacking normal neuropeptide-Y (NPY) gene expression (i.e. NPY 'knock-out' mice). A critical issue inherent in such experiments concerns the confounding of developmental influences of NPY and its neurotransmitter functions in the mature organism. The present experiments directly addressed this issue by studying seizure susceptibility in transgenic mice possessing an inducible antisense transcript that can be experimentally manipulated to attenuate NPY synthesis. NPY-deficient and control mice were injected with kainic acid (40 mg/kg, i.p.) and several seizure-related behaviors were measured. Consistent with previously reported effects in NPY knock-out mice, significantly more NPY-deficient mice died within 24 h than control mice. In situ hybridization analyses confirmed a decrease in prepro-NPY gene expression in transgenic mice. The experiments support the hypothesis that the control of neural excitability is a prominent function of NPY.

Adrenalectomy decreases neuropeptide Y mRNA levels in the arcuate nucleus.

Recent studies suggest that glucocorticoids may increase NPY and NPY mRNA levels. To determine if endogenous corticosterone affects the level of NPY mRNA in areas that control NPY levels in the paraventricular nucleus, we examined the effects of adrenalectomy and corticosterone replacement on NPY mRNA levels in the arcuate nucleus and brainstem. Rats were either adrenalectomized, adrenalectomized and corticosterone replaced, or sham-operated. The arcuate nucleus, hypothalamus (excluding arcuate nucleus), and brainstem were collected and the RNA isolated. Dot blots were made of each tissue and the NPY mRNA quantitated by densitometry. Adrenalectomy significantly reduced NPY mRNA levels in the arcuate nucleus, while corticosterone replacement restored the NPY mRNA levels. NPY mRNA levels in the remainder of the hypothalamus were not affected by adrenalectomy. Adrenalectomy also had no affect on NPY mRNA levels in the brainstem. These data suggest that the paraventricular nucleus may be affected by glucocorticoids via an NPY pathway and that the two major afferent pathways of NPY-containing neurons to the paraventricular nucleus may be regulated by different mechanisms.

Activation of polycyclic hydrocarbons in Reuber H4-II-E hepatoma cells. An in vitro system for the induction of SCEs.

Activation of polycyclic aromatic hydrocarbons (PAHs) was examined in the Reuber H4-II-E established cell line without the use of exogenous enzyme preparations. Metabolism of PAHs to genotoxic products was determined by the induction of sister-chromatid exchanges (SCEs). The induction of SCEs followed a dose-response pattern with plateaus at high doses of PAH. The effects of metabolic enzyme inducers (3-methylcholanthrene, phenobarbital, Aroclor 1254) and the epoxide hydrase inhibitor 1,1,1-trichloropropylene oxide were assessed as changes in SCE induction and enhanced production of water-soluble metabolites. Results indicate that Reuber H4-II-E cells can be employed in the testing of carcinogens activated by the P1-450 monooxygenase system and would be a useful in vitro system for the study of mechanisms of metabolic induction and their effect on genetic toxicity.

Expression of leptin mRNA and CCAAT-enhancer binding proteins in response to insulin deprivation during preadipocyte differentiation in primary cultures of porcine stromal-vascular cells.

The aim of this study was to examine the correlation between CCAAT-enhancer binding proteins (C/EBPs) and leptin gene expression in response to insulin deprivation in preadipocytes and adipocytes. Adipose tissue from 7 d-old pigs was digested enzymatically and stromal-vascular (S-V) cells were seeded and plated for 3 d in fetal bovine serum (FBS) with dexamethasone (DEX) followed by 6 d (Days 3-9) in serum-free medium with insulin (850 nM or 10 nM), transferrin, and selenium. During FBS+DEX treatment (Days 0-3) a large number of preadipocytes develop with no lipid accretion. In contrast, preadipocyte number does not change with lipid accretion during insulin treatment (Days 3-9). Total RNA and cells were harvested from S-V cultures after periods with and without insulin after FBS+DEX. Northern-blotting and Western blot analysis were used to study leptin mRNA and C/EBP protein expression in cultures, respectively. Insulin deprivation from Days 3-4 reduced leptin mRNA and C/EBP-alpha protein expression. Treatment with 850 nM or 10 nM insulin from Days 3-9 induced leptin mRNA and C/EBP-alpha expression at a similar level. In cultures treated with 10 nM insulin from Days 3-7, leptin and C/EBP-alpha expression were reduced markedly by insulin deprivation from Days 7-9, but were restored by insulin treatment for 6 hr before harvesting. The restoration of leptin expression by insulin was blocked by cycloheximide treatment. However, C/EBP-beta protein levels did not change regardless of insulin deprivation. Insulin deprivation from Days 7-9 in cultures treatedwith 850 nM insulin from Days 3-7 did not influence C/EBP-alpha or leptin mRNA expression, whereas C/EBP-alpha and leptin expression were reduced after treating these cultures with 1.5 uM okadaic acid for 45 min before harvesting on Day 9. However, cycloheximide treatment for 6 hr before harvesting did not reduce leptin mRNA expression. These results suggest that 1) leptin expression is positively correlated with C/EBP-alpha expression, and 2) the maintenance of leptin expression after insulin deprivation in 850 nM insulin-treated cultures on Day 9 may be associated with the presence of C/EBP-alpha expression and/or activation.

The quantitation of bovine embryo viability using a bioluminescent assay for lactate dehydrogenase.

Forty-seven bovine embryos, ranging from the four-cell to expanded blastocyst stage, with grades ranging from excellent to poor, were collected non-surgically from superovulated Holstein heifers. A viability assay based on the measurement of bioluminescent emission from the media surrounding an embryo was tested. This assay measured the activity of the enzyme lactate dehydrogenase (LDH) released into the media by the embryos. Lactate dehydrogenase has been reported to be released into media by nonviable embryos. The assay used is simple, rapid and nonsubjective, requiring approximately 5 min to complete. The LDH assay proved to be a practical method for distinguishing between nonviable and viable embryos. Viability was determined by the observation of embryo development in culture following the LDH assay. The activity of LDH in the media of nonviable embryos was consistently higher than for viable embryos (P<0.001), with no overlap between the two groups. Thus, the LDH assay was shown to be a reliable test of embryo viability.

Dysdifferentiative nature of aging: age-dependent expression of mouse mammary tumor virus and casein genes in brain and liver tissues of the C57BL/6J mouse strain.

Many aspects of the aging process could be the result of cells slowly drifting away from their proper state of differentiation. This possibility has been studied by searching for an age-dependent increase in the expression of specific genes in tissues where expression of these genes would not normally be expected. In these studies, cDNA probes of specific genes are used in a DNA X NA hybridization assay to detect possible complementary RNA sequences in tissues of different-aged animals. Using this technique in past experiments, a qualitative increase in the RNA sequence complexity of mouse leukemia virus (MuLV) and a quantitative increase in the amount of alpha- and beta-globin RNA were found with increasing age in the brain and liver of the C57BL/6J mouse strain. We report here a similar age-dependent qualitative increase in the RNA sequence complexity for mouse mammary tumor virus (MMTV) but no quantitative or qualitative age-dependent change in casein RNA sequences for the same tissues and mouse strain.

Gene expression profiling in developing pig adipose tissue: non-secreted regulatory proteins.

The expression of many genes encoding secreted and non-secreted factors have been studied in human and rodent adipose tissue with cDNA microarrays, but few such studies in adipose tissue from growing pigs have been reported. Total RNA was collected at slaughter from outer subcutaneous adipose tissue (OSQ) and middle subcutaneous adipose tissue (MSQ) samples from gilts at 90, 150 and 210 days (n = 5/age). Dye-labeled cDNA probes were hybridized to custom microarrays (70-mer oligonucleotides) representing about 600 pig genes involved in growth and reproduction. Gene expression intensity ratios changed little with age for 100 transcription factors, nuclear receptors, enzymes and other regulatory proteins in OSQ and MSQ from pigs between 90 and 210 days of age. However, the relative expression of 13 genes distinguished OSQ and MSQ depots in growing pigs. The expression of several genes were influenced by age including an increase in CCND3, HSF1 and PTGR1 expression in MSQ and a decrease in UCP2 and REA (prohibitin-2) expression in OSQ. These studies demonstrate for the first time the expression of several key regulatory genes in pig adipose tissue. Simple linear regression analysis showed that leptin gene expression was associated with expression of some of these regulatory genes. Negative associations between expression of some regulatory factors and leptin gene expression indicated that local leptin may decrease or antagonize adipogenesis.

Acute kidney injury in the rat causes cardiac remodelling and increases angiotensin-converting enzyme 2 expression.

Patients with kidney failure are at high risk of a cardiac death and frequently develop left ventricular hypertrophy (LVH). The mechanisms involved in the cardiac structural changes that occur in kidney failure are yet to be fully delineated. Angiotensin-converting enzyme (ACE) 2 is a newly described enzyme that is expressed in the heart and plays an important role in cardiac function. This study assessed whether ACE2 plays a role in the cardiac remodelling that occurs in experimental acute kidney injury (AKI). Sprague-Dawley rats had sham (control) or subtotal nephrectomy surgery (STNx). Control rats received vehicle (n = 10), and STNx rats received the ACE inhibitor (ACEi) ramipril, 1 mg kg(-1) day(-1) (n = 15) or vehicle (n = 13) orally for 10 days after surgery. Rats with AKI had polyuria (P < 0.001), proteinuria (P < 0.001) and hypertension (P < 0.001). Cardiac structural changes were present and characterized by LVH (P < 0.001), fibrosis (P < 0.001) and increased cardiac brain natriuretic peptide (BNP) mRNA (P < 0.01). These changes occurred in association with a significant increase in cardiac ACE2 gene expression (P < 0.01) and ACE2 activity (P < 0.05). Ramipril decreased blood pressure (P < 0.001), LVH (P < 0.001), fibrosis (P < 0.01) and BNP mRNA (P < 0.01). These changes occurred in association with inhibition of cardiac ACE (P < 0.05) and a reduction in cardiac ACE2 activity (P < 0.01). These data suggest that AKI, even at 10 days, promotes cardiac injury that is characterized by hypertrophy, fibrosis and increased cardiac ACE2. Angiotensin-converting enzyme 2, by promoting the production of the antifibrotic peptide angiotensin(1-7), may have a cardioprotective role in AKI, particularly since amelioration of adverse cardiac effects with ACE inhibition was associated with normalization of cardiac ACE2 activity.

Patterns of gene expression in pig adipose tissue: transforming growth factors, interferons, interleukins, and apolipoproteins.

Although cDNA microarray studies have indicated the expression of unique and unexpected genes and their products in human and rodent adipose tissue, cDNA microarray studies of adipose tissue from growing pigs have not been reported. Total RNA was collected at slaughter from outer s.c. adipose tissue (OSQ), middle s.c. adipose tissue (MSQ), ovary, uterus, hypothalamus, and pituitary tissue samples from gilts at 90, 150, and 210 d (n = 5/age). Dye-labeled cDNA probes were hybridized to custom microarrays (70 mer oligonucleotides) representing approximately 600 pig genes involved in growth and reproduction. Expression intensity ratios revealed little change in expression of 27 cytokines and 4 apolipoproteins with age in OSQ and MSQ from pigs at 90, 150, and 210 d of age. Distinct patterns of relative gene expression were evident within apolipoproteins, IL, interferons, and transforming growth factor beta family members in adipose tissue from growing pigs (90-, 150-, and 210-d-old pigs). Patterns of gene expression within apolipoproteins, IL, interferons, and transforming growth factor beta family members distinguished OSQ and MSQ depots in growing pigs. We also demonstrated, for the first time, the expression of several major cytokine and apolipoprotein genes in pig adipose tissue, including small inducible cytokine A5 (RANTES), IL-1B, IL-1A, IL-12A, IL-1 receptor antagonist, and apolipoproteins A1 and E with microarray and reverse transcription-PCR assays or reverse transcription-PCR assays alone. These studies demonstrate that expression of major cytokine and apolipoprotein genes in pig adipose tissue are not influenced by age in growing pigs but may be influenced by location or depot.

Protein and ribonucleic acid syntheses in heat-damaged and heat-killed Escherichia coli.

Protein and ribonucleic acid (RNA) syntheses were measured in both lethally injured and thermally damaged viable cells after heating at lethal temperatures. Immediately after heating, cells were incubated in growth media containing either [14C]leucine or [3H]uracil. The labeled cells were subsequently treated with penicillin. Viable cells were shown to lyse, and the intact nonviable cells were collected by centrifugation. The results showed that after heating, protein and RNA synthesis were reinitiated in the penicillin-sensitive cell population, but there was no detectable protein or RNA synthesis in the heat-killed cells which were resistant to penicillin. The lack of protein or RNA synthesis in lethally damaged cells during the entire recovery period may be interpreted to reflect the lethal thermal damage.