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The stringent response is a form of bacterial response to adverse environmental conditions. Its effectors are guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp], which are synthetized by RelA, SpoT and their homologs (RSH). RelA, a (p)ppGpp synthase, is activated when there is a shortage of amino acids, whereas SpoT, which has the ability to synthetize and hydrolyze (p)ppGpp, responds to fatty acids, iron and carbon limits. Accumulation of (p)ppGpp causes an inhibition of translation, replication, a decrease in the transcription of many genes, e.g. rRNA, tRNA, encoding ribosomal proteins, and an increase in the transcription of genes whose proteins are important in bacterial stress response. The stringent response alarmones are crucial for bacterial resistance to oxidative stress and antibiotics. They also regulate the production of specific molecules, the so-called quorum sensing autoinducers, which help bacteria communicate the density of their own population, which enables them to adjust their metabolism to the prevailing conditions, to form a biofilm – a community of microorganisms attached to a certain surface, ensuring them appropriate conditions to survive in an unfavourable environment, and to colonize new niches. (p)ppGpp has a positive impact on biofilm formation not only via the regulation of quorum sensing, but also by stimulating the synthesis of potential elements of the biofilm. It also appears that the stringent response alarmones decrease the ability of Agrobacterium tumefaciensbacteria to transform plants and thus their potential to cause disease. (p)ppGpp enables the bacteria to perform swarming motility, a movement that increases their resistance to adverse environmental factors.

Objective:To test the hypothesis that multiple courses of antenatal corticosteroids accentuate the decreases in blood-brain barrier permeability observed after a single course of corticosteroids in pretern ovine fetuses.Methods:Chronically instrumented 106-day gestation ovine fetuses were studied after single and multiple courses of dexamethasone or placebo were given to ewes beginning at 104 to 106 or 76 to 78 days of gestation, respectively. In the single-course groups, the ewes received dexamethasone (6 mg, n = 6) or placebo (n = 6) as four intramuscular injections every 12 hours over 48 hours. In the multiple course groups, the ewes received the same treatment (dexamethasone, n = 9, or placebo, n = 8), once per week for 5 weeks starting at 76 to 78 days of gestation. Blood-brain barrier permeability was quantified with the blood-to-brain transfer constant (Ki) for α-aminoisobytyric acid (AIB) in the brain regions of the fetuses 12 hours after the last injection of dexamethasone was given to the ewes at 106 to 107 days of gestation.Results:Both single (analysis of variance [ANOVA]; main effects for dexamethasone treatment, F = 5.92, P <.04) and multiple (ANOVA; main effects for dexamethasone treatment, F = 4.74, P <.04) course of antenatal corticosteriods were associated with decreases in blood-brain barrier permeability in the brain regions of the ovine fetus. However, the multiple course did not accentuate (ANOVA; main effects for single versus multiple courses, F = 1.06, P = .32) the decreases in permeability observed after a single course.Conclusion:Contrary to our hypothesis, antenatal treatment with a 5-week course of corticosteroids did not accentuate the reductions in blood-brain barrier permeability that we observed after a single course of corticosteroids in the fetus.

We investigated the effects of maternal antenatal dexamethasone (Dex) treatment given as a single course (4 doses) or multiple courses (20 doses) on fetal skeletal muscle glucose transporter (GLUT) protein concentrations at 70% of gestation (106 to 107 days with term being 145 to 150 days) in the ovine fetus. Antenatal corticosteroid administration was associated with a decrease in endogenous fetal plasma cortisol concentrations (P< 0.05), fetal hyperglycemia (P< 0.02) and hyperinsulinemia (P< 0.05). These metabolic/hormonal changes were associated with a decrease in fetal body weight (P< 0.05) in the multiple course Dex group compared with the multiple course placebo group. These perturbations were associated with an increase in fetal skeletal muscle GLUT 1 concentrations that mediate basal glucose transport in the extensor digitorum lateralis and extensor digitorum longus muscles (P< 0.05) 18 h after the last dose of Dex was given in the single course group. However, in the multiple course Dex group, a small increase in GLUT 1 was observed only in the biceps femoris. In contrast, both single and multiple courses of antenatal Dex were associated with an increase in the extensor digitorum lateralis and biceps femoris muscle GLUT 4 (insulin-responsive) concentrations (P< 0.05). We conclude that antenatal corticosteroids perturb fetal glucose/insulin homeostasis, which is associated with increases in fetal skeletal muscle glucose transporters to compensate for and attenuate the associated catabolic fetal state. These changes consist of an increase in proteins that mediate basal glucose transport (GLUT 1) to meet immediate energy requirements of the fetal skeletal muscle with an increase in basal insulin sensitivity (GLUT 4) to compensate for the Dex-induced catabolic state after exposure to multiple courses of Dex.

Na+/K+-ATPase is a membrane-bound enzyme responsible for Na+/K+ translocation across cell membranes. It is essential for the generation of electrochemical gradients, which control the ionic environment necessary for electrical activity and water and electrolyte balance. Newborn infants who are at risk of developing bronchopulmonary dysplasia (BPD) are frequently treated with corticosteroids. Although these infants are at risk for neurological, water and electrolyte abnormalities, there is little information regarding the effects of clinically relevant doses of corticosteroids on Na+/K+-ATPase activity and protein isoform expression in the brain and kidney of newborns. In the present study, we examined the effects of dexamethasone on cerebral cortical and renal cortical Na+/K+-ATPase activity and ?1- and ?1-protein isoform expression in newborn lambs. Lambs were given four injections of a placebo (n = 11) or one of three different doses of dexamethasone (0.01 mg kg-1, n = 9; 0.25 mg kg-1, n = 11; or 0.50 mg kg-1, n = 9) 12 h apart on Postnatal Days 3 and 4 up to 18 h before harvest of the cerebral cortex and renal cortex. We selected doses in a range to approximate those used to treat infants with BPD. Na+/K+-ATPase activity was measured in membrane preparations as ouabain-sensitive inorganic phosphate liberation from ATP and ?1- and ?1-subunit abundance by Western immunoblot. Postnatal treatment of lambs with dexamethasone resulted in a 21.4% increase in Na+/K+-ATPase activity and a 30.4% increase in catalytic ?1-protein expression in the cerebral cortex at a dose of 0.50 mg kg-1 dexamethasone, but not at the lower doses. Dexamethasone treatment was not associated with changes in ?1-isoform expression in the cerebral cortex. In the kidney, dexamethasone treatment was not associated with significant changes in Na+/K+-ATPase activity or ?1- or ?1-isoform expression for the doses we examined. Therefore, clinically relevant corticosteroid treatment exerts dose-related, differential organ-specific effects on Na+/K+-ATPase activity and protein isoform expression in newborn lambs.

The aim of the present study was to determine the ontogeny and effects of corticosteroid pretreatment on aquaporin 4 (AQP4) channel mRNA and protein expression in the cerebral cortex of sheep during development. A portion of the cerebral cortex was snap-frozen from fetuses of dexamethasone- and placebo-treated ewes at 60%, 80% and 90% of gestation, dexamethasone- and placebo-treated newborn lambs and adult sheep. Cerebral cortical samples were obtained 18 h after the last of four 6 mg dexamethasone or placebo injections were given over 48 h to the ewes and adult sheep. Lambs were treated with 0.01 mg kg-1 dexamethasone or placebo in the same schedule as the ewes and adult sheep. Amplification of an ovine AQP4 cDNA fragment was accomplished by reverse transcription?polymerase chain reaction using primers based on a homologous bovine sequence. The resulting cDNA was used to determine AQP4 channel mRNA expression by Northern hybridisation using phosphorimaging. The relative abundance of AQP4 mRNA was normalised to the ovine ribosomal gene L32. A portion of the frontal cortex was also analysed for AQP4 protein expression by Western immunoblot. Densitometry was performed and the results expressed as a ratio to an adult brain pool. Aquaporin 4 channel mRNA and protein were detectable as early as at 60% gestation. There were no changes in AQP4 mRNA expression among the fetal, newborn and adult groups or after dexamethasone pretreatment in any age group. The expression of the AQP4 protein was higher (P < 0.05) in fetuses at 80% and 90% of gestation (2.9- and 3.3-fold, respectively), in lambs (3.2-fold) and in adult sheep (3.8-fold) compared with fetuses at 60% of gestation, as well as in adult sheep (1.3-fold) compared with fetuses at 80% of gestation. Dexamethasone pretreatment resulted in decreases (P < 0.05) in AQP4 protein expression in the lambs and adult sheep, but not in the fetal groups. We conclude that: (1) AQP4 mRNA and protein were expressed early in fetal and throughout ovine development; (2) protein, but not mRNA, expression increased between 60% and 80% of gestation and did not differ from adult levels by 90% of gestation; and (3) dexamethasone pretreatment resulted in decreases in AQP4 protein expression in lambs and adult sheep, but not in fetuses. The maturational increases in AQP4 protein expression and dexamethasone-related decreases in expression were post-transcriptional, because changes in AQP4 mRNA expression were not observed.

We examined the effects of prolonged moderate hyperglycemia with and without an additional rapid glucose injection on ischemic brain injury in the fetus. Twenty-five ewes (117–124 d of gestation) were assigned to one of four groups: 1) glucose-infused fetuses exposed to 30 min of carotid artery occlusion followed by 48 h of reperfusion (I/R-Glu, n8); 2) glucose-infused plus rapid glucose injection given 100 min before 30 min of occlusion followed by 48 h of reperfusion (I/R-GluR, n4); 3) placebo-infused exposed to 30 min of occlusion and 48 h of reperfusion (I/R-PL, n8); and 4) glucose-infused sham occlusion and 48 h of sham reperfusion (control, n5). After baseline measurements, fetuses were infused with glucose (9–16 mg/kg/min) for 48 h before and after carotid occlusion or sham treatment. The I/R-PL group received 0.9% NaCl. Brain pathologic outcome was determined. Serial sections stained with Luxol fast blue-hematoxylin and eosin were scored for white matter, cerebral cortical, and hippocampal lesions. These areas received graded pathologic scores of 0 to 5, reflecting the amount of injury, where 0 0%, 1 1–25%, 2 26–50%, 3 51–75%, 4 76–95%, and 5 96–100% of the area damaged. Comparisons of the pathologic scores for cerebral cortex (CC), white matter (WM), and hippocampus (H) demonstrated that the I/R-GluR (CC: 4.56 ± 0.11, WM: 4.50 ± 0.11, H: 3.44 ± 0.48, mean ± SEM) had more (p< 0.05) damage than the I/R-Glu (CC: 2.46 ± 0.47, WM: 1.97 ± 0.37, H: 1.81 ± 0.36) and control (CC: 1.12 ± 0.13, WM: 0.82 ± 0.34, H: 0.80 ± 0.34) groups. The pathologic scores in the I/R-Glu were (p< 0.05) greater than the control, but not the I/R-PL (CC: 2.12 ± 0.35, WM: 2.20 ± 0.44, H: 1.59 ± 0.41) group. We conclude that exposure to prolonged moderate hyperglycemia before ischemia and during reperfusion does not affect the extent of brain injury, but exposure to an additional acute increase in plasma glucose concentration before ischemia is extremely detrimental to the fetal brain.

Objective:To study the effects of single and multiple courses of antenatal corticosteroids on tissue water content in ovine fetuses.Methods:After chronic catheterization of the ewes and fetuses, the ewes were randomly assigned to receive single or multiple courses of dexamethasone or placebo beginning at 104-106 or 76-78 days' gestation, respectively. In the single course groups, the ewes received dexamethansone (6 mg, n = 6) or placebo (n = 6) as four intramuscular injections every 12 hours over 48 hours. The fetal tissues were harvested for water content determination 66 hours after the first injection of dexamethasone or placebo was given. In the multiple-course groups, the ewes received the same treatment (dexamethasone, n = 10, or placebo, n = 8), once a week for 5 weeks starting at 76-78 days' gestation. In these groups, the tissues were harvested 66 hours after the first the injection of the fifth and last treatment course. In both groups, tissues were harvested at 106-107 days' gestation. Tissue water content was determined by wet-to-dry weight ratio in brain (cerebral cortex, caudate nucleus, cerebellum, midbrain, and medulla) and somatic tissues (kidney, liver, muscle, and skin).Results:Water content in the brain regions (cerebellum and medulla) was lower (P < .05) in fetuses of dexamethasone-treated ewes than placebo-treated ewes than placebo-treated ewes after the multiple courses, but not the single course. Water content of somatic tissue was power (P < .05) in fetuses of dexamethasone-treated ewes than placebo-treated ewes after the multiple courses, and in the liver after a single course.Conclusion:Dexamethasone treatment of ewes at 70% of gestation results in decreased regional brain water content in the fetuses after multiple but not single treatment courses, in somatic tissues (kidney, liver, muscle, and skin) after multiple courses, and in the liver after a single course.

Objective:We examined the effects of maternal corticosteroid administration on water content in regional tissue in ovine fetuses at 60%, 80%, and 90% of gestation.Methods:After catheters were placed in the fetuses, the ewes were given four 6-mg doses of dexamethasone or placebo injections 12 hours apart over 48 hours. Water conent of fetal tissue was determined 18 hours after the last injection was given to the ewes. Tissue water ws determined by wet-to-dry weight ratio in brain (cerebral cortex, caudate nucleus, cerebellum, midbrain, and medulla) and non-neural tissues (kidney, liver, muscle, and skin) at each gestational age.Results:Water content (P < .05) in brain regions was lower in fetuses from dexamethasone-treated than placebo-treated ewes at 60% but not 80% or 90% of gestation and in non-neural tissues at each gestational age.Conclusion:Maternal treatmet with a contricosteroid regimen similar to that used in the clinical setting was associated with small decreases in brain water content early but not later in gestation. This corticosteroid treatment regimen was also associated with decreased regional non-neural tissue water content at 60%, 80%, and 90% of gestation.

The effects of cerebral ischemia on white matter changes in ovine fetuses were examined after exposure to bilateral carotid artery occlusion. Fetal sheep were exposed to 30 min of ischemia followed by 48 (I/R-48, n = 8) or 72 (I/R-72, n = 10) h of reperfusion or control sham treatment (control, n = 4). Serial coronal sections stained with Luxol fast blue/hematoxylin and eosin were scored for white matter, cerebral cortical, and hippocampal lesions. All areas received graded pathologic scores of 0 to 5, reflecting the degree of injury where 0 = 0%, 1 = 1% to 25%, 2 = 26% to 50%, 3 = 51% to 75%, 4 = 76% to 95%, and 5 = 96% to 100% of the area damaged. Dual-label immunofluorescence using antibodies against glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were used to characterize white matter lesions. Basic fibroblast growth factor (FGF-2) was measured in the frontal cortex by ELISA. Results of the pathologic scores showed that the white matter of the I/R-72 (2.74 ± 0.53, mean ± SEM) was more (p < 0.05) damaged when compared with the control (0.80 ± 0.33) group. Cortical lesions were greater (p < 0.05) in the I/R-48 (2.12 ± 0.35) than the control (0.93 ± 0.09) group. White matter lesions were characterized by reactive GFAP-positive astrocytes and a loss of MBP in oligodendrocytes. The ratio of MBP to GFAP decreased (p < 0.05) as a function of ischemia, indicative of a proportionally greater loss of MBP than GFAP. FGF-2 concentrations were higher (p < 0.05) in the I/R-72 than the control group and there was a direct correlation between the pathologic scores (PS) and FGF-2 concentrations (FGF-2 = e(1.6 PS-0.90)+ 743, n = 17, r = 0.73, p < 0.001). We conclude that carotid artery occlusion results in quantifiable white matter lesions that are associated with a loss of MBP from myelin, and that FGF-2, a purported mediator of recovery from brain injury in adult subjects, increases in concentration in proportion to the severity of brain damage in the fetus.

Background The association between coronary heart disease (CHD) and social status has differed among societies in strength and direction. As years of schooling is a major determinant of socioeconomic status and dyslipidaemia a major CHD determinant, the purpose of this investigation is to estimate the association of years of schooling with plasma lipids and lipoproteins among samples from five countries representing different cultures, socio-political systems and stages of economic development.Methods Men and women from Chinese, Polish, Russian, Israeli and US samples were studied. Years of schooling were analysed both as a multi-category ordinal variable and divided into two strata: less than the equivalent of high school and greater than or equal to high school equivalence. Fasting plasma cholesterol, low density lipoprotein (LDL) cholesterol, high density lipoprotein (HDL) cholesterol and triglycerides were compared across years of schooling strata within each country. Lipid levels were computed unadjusted and then adjusted for age and lipid risk factor variables.Results Total cholesterol, LDL cholesterol, and triglycerides varied directly with years of schooling in Chinese, Polish and Russian men, and in contrast varied inversely with years of schooling among US white men. The HDL cholesterol varied inversely with years of schooling for Chinese, Polish, and Russian men, but varied directly with years of schooling among US white men. The lipid differences between men of high versus low years of schooling were not explained by age, body mass index, smoking, alcohol consumption or blood pressure medication use. Findings were less consistent for women and for Israelis and US blacks of both genders.Conclusions Lipid and lipoprotein levels consistent with atherogenicity varied directly with years of schooling in Chinese, Polish, and Russian samples. Opposite trends were present in US whites. These findings are consistent with a hypothesized influence of social status on CHD risk differing among populations in relation to stages in societal economic development.

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Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV–enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p < 0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.