An age difference of two billion years between a metal-rich and a metal-poor globular cluster.

Globular clusters trace the formation history of the spheroidal components of our Galaxy and other galaxies, which represent the bulk of star formation over the history of the Universe. The clusters exhibit a range of metallicities (abundances of elements heavier than helium), with metal-poor clusters dominating the stellar halo of the Galaxy, and higher-metallicity clusters found within the inner Galaxy, associated with the stellar bulge, or the thick disk. Age differences between these clusters can indicate the sequence in which the components of the Galaxy formed, and in particular which clusters were formed outside the Galaxy and were later engulfed along with their original host galaxies, and which were formed within it. Here we report an absolute age of 9.9 ± 0.7 billion years (at 95 per cent confidence) for the metal-rich globular cluster 47 Tucanae, determined by modelling the properties of the cluster's white-dwarf cooling sequence. This is about two billion years younger than has been inferred for the metal-poor cluster NGC 6397 from the same models, and provides quantitative evidence that metal-rich clusters like 47 Tucanae formed later than metal-poor halo clusters like NGC 6397.

Extremely prematurely born adolescents self-report of anxiety symptoms, and the mothers' reports on their offspring.

AIM: To compare anxiety symptoms in adolescents born extremely prematurely to term-born controls. METHODS: We had 96 preterm-born adolescents and 40 term-born controls from Denmark, and their mothers score the adolescents on the Revised Children Anxiety and Depression scale. We analysed group differences, cross-informant correlations and relative risks for elevated anxiety symptoms. RESULTS: Self-reported anxiety symptoms did not significantly differ, although the upper confidence limit (95% CI: -3.3 to 5.1) supported an odds ratio of 2 for the preterm-born participants. Mothers of the preterm-born participants reported higher social anxiety symptoms than did mothers of controls (51.7 versus 46.8, p = 0.001). The relative risk for being above a threshold indicating distressing anxiety was small from self-reports (1.39; p = 0.60). From mother-reports, the relative risk was noticeable but not significant (4.58; p = 0.14). Cross-informant scores correlated significant for total anxiety and social anxiety for the preterm-born (rτ = 0.2, p = 0.001; rτ = 0.3, p ≤ 0.001). CONCLUSIONS: Self-reports did not clearly indicate more anxiety in the preterm group, although confidence intervals supported a possible twofold increase. Mother- and self-reports correlated only for the preterm group, which may indicate increased sensitivity for their children's symptoms.

Increased nuchal translucency, normal karyotype and infant development.

OBJECTIVE: To investigate whether chromosomally normal fetuses with a nuchal translucency (NT) ≥ 99th percentile(3.5 mm) in the first trimester have an increased risk of delayed development at 2 years of age. METHODS: The study included children of women from 10 Danish hospitals who had fetal NT either ≥ 99th percentile (Group 1) or <95th percentile (Group 2) in the first trimester. The groups were matched by gender,gestational age at birth and maternal age. There were twice as many children in Group 2 as in Group 1. Follow-up was conducted at 2 years of age, infant development being assessed by the 'Ages and Stages Questionnaire'. The cutoff value for delayed development was defined as the 5th percentile from the first 100 questionnaires from Group 2. RESULTS: In a 1-year period 202 of 33 266 fetuses (0.6%)had NT ≥ 3.5 mm. Of these, 99 (49.0%) were liveborn with normal karyotype and normal ultrasound findings during pregnancy. The response rate to the ASQ was 83.3% in Group 1 and 71.4% in Group 2. A low ASQ score was found in 1.3% (1/80) and 4.4% (6/137)in Groups 1 and 2, respectively (P = 0.265), but no difference was found in the mean ASQ score between the two groups (P = 0.160). CONCLUSION: Fetuses with NT ≥ 99th percentile, normal karyotype and normal ultrasound findings during pregnancy had no increased risk of developmental delay at 2 years of age compared with fetuses with normal NT(<95th percentile).

Germination and conjugation of Bacillus thuringiensis subsp. israelensis in the intestine of gnotobiotic rats.

AIMS: To study the ability of Bacillus thuringiensis subsp. israelensis spores to germinate and subsequently transfer a conjugative plasmid in the intestinal tract of gnotobiotic rats. METHODS AND RESULTS: Germination was studied by feeding germ-free rats with spores of a B. thuringiensis strain harbouring a plasmid encoding green fluorescent protein (GFP), which enabled quantification of germinated bacteria by flow cytometry. To study in vivo conjugation, germ-free rats were first associated with a B. thuringiensis recipient strain and after 1 week an isogenic donor strain harbouring the conjugative plasmid pXO16 was introduced. Both strains were given as spores and transfer of pXO16 was observed from the donor to the recipient strain. CONCLUSIONS: Bacillus thuringiensis is able to have a full life cycle in the intestine of gnotobiotic rats including germination of spores, several cycles of growth and sporulation of vegetative cells. For the first time conjugative plasmid transfer in a mammalian intestinal tract was shown between two B. thuringiensis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of B. thuringiensis are used worldwide to combat insect pests, and this study brings new insights into the nature of B. thuringiensis showing the potential of the bacteria to germinate and transfer DNA in the mammalian intestinal tract.

The members of the Bacillus cereus group are commonly present contaminants of fresh and heat-treated milk.

To determine the level of milk contamination by Bacillus cereus sensu lato, 44 samples were collected from a dairy farm and two independent dairies in northeastern Poland. A total of 680 B. cereus sensu lato isolates were recovered. Based on spore counts, their highest level in milk was found during the spring and summer months. Although significant variations in chromosomal DNA polymorphisms among B. cereus sensu lato isolates were noted based on repetitive element sequence polymorphism (rep-PCR) and pulse-field gel electrophoresis (PFGE), indistinguishable B. cereus isolates were observed in all sampling points and locations. Both B. cereus sensu stricto/Bacillus weihenstephanensis and Bacillus thuringiensis cultured from milk harbored nheA, hblA, and cytK in, respectively, 80%, 55%, and 60% of the isolates. With respect to Bacillus mycoides/Bacillus pseudomycoides, 30% and 70% of theses isolates harbored, respectively, nheA and hblA. The presence of cytK was not detected in any isolate. Our data show the occurrence of potentially toxic B. cereus s.l. in both raw and heat-treated milk, thus emphasizing the requirement for precautions that prevent spore germination and vegetative proliferation by keeping the milk at low temperatures during all steps of production and dispensation to the consumers.

Double versus single intensive phototherapy with LEDs in treatment of neonatal hyperbilirubinemia.

OBJECTIVE: We investigate whether double phototherapy reduces total serum bilirubin concentration faster than single light during intensive phototherapy with high levels of irradiance using light-emitting diodes. STUDY DESIGN: Eighty-three infants with gestational age ⩾33 weeks and uncomplicated hyperbilirubinemia were randomized to either double (n=41) or single phototherapy (n=42) for 24 h. The mean irradiance was 64.8 µW cm-2 nm-1 from above and 39 µW cm-2 nm-1 from below. RESULTS: The percentage decreases of total serum bilirubin after 12 h of double vs single phototherapy were (mean (95% confidence interval (CI))) 39% (37 to 42) vs 30% (27 to 32), respectively (P<0.001). After 24 h, the decreases were 58% (56 to 61) vs 47% (44 to 50), respectively (P<0.001). The results were still significant after adjustment for confounding. The only side effect was loose stools. CONCLUSION: Even with intensive phototherapy increasing spectral power by increasing the irradiated body surface area, the efficacy of phototherapy is improved.

Developmental changes in intracellular calcium regulation in rat cerebral cortex during hypoxia.

During the first weeks of life, injury to the central nervous system caused by brief periods of oxygen deprivation greatly increases. To investigate possible causes for this change, the effects of hypoxia or application of the excitatory neurotransmitter glutamate on intracellular calcium ([Ca2+]i) and ATP were studied in rat cerebrocortical brain slices. [Ca2+]i was measured fluorometrically with the indicator Fura-2. Hypoxia (95% N2/5% CO2) or 100 microM sodium cyanide produced gradual elevations in [Ca2+]i and ATP depletion in slices from rats < 2 weeks old, but rapid changes in older rats. After 20 min, [Ca2+]i in adult slices exposed to cyanide was 1,980 +/- 310 nM; in day 1-14 animals, it was 796 +/- 181 nM (p < 0.05). Combination of cyanide and a glycolytic inhibitor (iodoacetate) rapidly elevated [Ca2+]i and depleted ATP in all age groups. Energy utilization during anoxia, assessed by measuring ATP fall in cyanide/iodoacetate-treated brain slices, increased with age. Elevations in [Ca2+]i caused by application of 500 microM glutamate increased 240% from days 1-2 to day 28, but ATP loss caused by glutamate did not change with age. The N-methyl-D-aspartate antagonist MK-801 delayed calcium entry during the initial 5-7 min of hypoxia or cyanide in rats < 2 weeks old. We conclude that anaerobic ATP production, conservation of energy by reduced ATP consumption, and reduced sensitivity to glutamate contribute to delaying elevation in [Ca2+]i in neonatal rat brain during hypoxia.

Alpha 2-adrenergic agonists reduce glutamate release and glutamate receptor-mediated calcium changes in hippocampal slices during hypoxia.

The mechanisms by which alpha 2-adrenergic agonists reduce ischemic brain damage are not clear. In ischemia-vulnerable hippocampal neurons we tested whether alpha 2-agonists reduce glutamate efflux and glutamate receptor-mediated increase of cytosolic free calcium. Brain slices (300 microns thick) from rat hippocampal were located with fura-2 for measurements of cytosolic free calcium with a microscope fluorometer. Change of cytosolic calcium in CA1 neurons during application of N-methyl-D-aspartate (NMDA) was measured, as were calcium changes during simulated ischemia (hypoxia, NaCN, iodoacetate) of hypoxia plus high glutamate concentration (pO2 = 25 mmHg, 3 mM glutamate). In order slices, glutamate efflux evoked by anoxia (pO2 = 25 mmHg, 100 microM NaCN) was measured. The selective alpha 2-agonist mivazerol (1 microM) decreased NMDA receptor-mediated calcium changes in hippocampal CA1 neurons by 28% (p = 0.0079). With hypoxia and 3 mM glutamate, 1 microM mivazerol reduced early peak calcium changes in CA1 neurons by 57% (p = 0.0007). An alpha 2-antagonist (rauwolscine, 1 microM) blocked this. Mivazerol did not reduce the rate of calcium change during simulated ischemia. Clonidine (0.1 microM), a partial alpha 2-agonist, decrease glutamate/hypoxia-mediated calcium changes in CA1 (p = 0.01), but 1 microM clonidine, which stimulates alpha 1-receptors, did not. Mivazerol decreased hypoxia and KCl1-evoked glutamate release by 50% and 75% (p < 0.01), respectively. In addition, 1 microM mivazerol reduced lactate dehydrogenase leakage rate from brain slices during anoxia by 61% (p = 0.018). Thus, alpha 2-receptors influence glutamate release, calcium changes, and cell damage in ischemia-vulnerable hippocampal neurons. These effects may contribute to the cerebroprotective actions of alpha 2-agonists.

Polymerase chain reaction assay for the detection of Bacillus cereus group cells.

Recent investigations have shown that members of the Bacillus cereus group carry genes which have the potential to cause gastrointestinal and somatic diseases. Although most cases of diseases caused by the B. cereus group bacteria are relatively mild, it is desirable to be able to detect members of the B. cereus group in food and in the environment. Using 16S rDNA as target, a PCR assay for the detection of B. cereus group cells has been developed. Primers specific for the 16S rDNA of the B. cereus group bacteria were selected and used in combination with consensus primers for 16S rDNA as internal PCR procedure control. The PCR procedure was optimized with respect to annealing temperature. When DNA from the B. cereus group bacteria was present, the PCR assay yielded a B. cereus specific fragment, while when non-B. cereus prokaryotic DNA was present, the consensus 16S rDNA primers directed synthesis of the PCR products. The PCR analyses with DNA from a number of non-B. cereus confirmed the specificity of the PCR assay.

Causes of calcium accumulation in rat cortical brain slices during hypoxia and ischemia: role of ion channels and membrane damage.

To better understand why neurons accumulate calcium during cerebral ischemia, the influence of specific ion channel inhibitors on the rise in cytosolic free calcium ([Ca2+]c) during hypoxia or ischemia was evaluated in rat cerebrocortical brain slices. [Ca2+]c was measured fluorometrically with the dye fura-2 during hypoxia (95% N2/5% CO2 or 100 microM NaCN), simulated ischemia (100 microM NaCN plus 3.5 mM iodoacetate), or 0.5-1.0 mM glutamate. Hypoxia or ischemia increased [Ca+2]c from 100-250 nM to 1,000-2,500 nM within 3-5 min. Greater than 85% of the calcium accumulation was influx from the extracellular medium. The non-competitive N-methyl-D-aspartate (NMDA) inhibitor MK-801 reduced [Ca2+]c accumulation during hypoxia, but antagonism of alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptors or voltage-gated sodium or calcium channels or Na+/Ca2+ exchangers had no effect. During ischemia, combined antagonism of NMDA, AMPA and voltage-gated sodium channels slowed the rate of calcium accumulation, but not concentration at 5 min. Membrane damage, as indicated by leakage of lactate dehydrogenase into superfusate, occurred coincidentally with calcium influx and ATP loss during both hypoxia and ischemia. We conclude that cytosolic calcium changes during hypoxia or ischemia in cortical brain slices are due to multiple mechanisms, are incompletely inhibited by combined ion channel blockade, and are associated with disruption of cell membrane integrity.

Hypoxia-tolerant neonatal CA1 neurons: relationship of survival to evoked glutamate release and glutamate receptor-mediated calcium changes in hippocampal slices.

Neurons in the neonatal mammalian brain survive greater degrees of hypoxic stress than those in the mature brain. To investigate how developmental changes in glutamate receptor-mediated neurotoxicity contribute to this difference, we measured hypoxia-evoked glutamate release, glutamate receptor contribution to hypoxia-evoked intracellular calcium changes, and survival of hypoxia-/ischemia-sensitive CA1 neurons in rat hippocampus. Glutamate release was measured by a fluorescence assay, calcium changes in CA1 neurons with fura-2, and cell viability using Nissl and fluorescence staining with calcein-AM/ethidium homodimer, all in 300-micron thick hippocampal slices from 3-30 post-natal day (PND) rats. Glutamate released from PND 3-7 slices during hypoxia (PO2 = 5 mmHg) was only one third that of PND 18-22 slices. In PND 3-7 slices, survival of CA1 neurons after 5 min of hypoxia and 6 h of recovery was significantly greater than in PND 18-22 slices (viability indices 0.60 and 0.28, respectively, (p < 0.05). Five min of anoxia significantly altered Nissl staining pattern and morphology of CA1 neurons in PND 18-22 but not PND 3-7 slices. Hypoxia (PO2 = 5 mm Hg) caused three to five times greater increases in [Ca2+]i in PND 18-22 slices than in PND 3-7 slices (p < 0.001). During re-oxygenation, [Ca2+]i returned to baseline in PND 3-7 slices, but remained elevated in PND 18-22 slices. Glutamate receptor-mediated calcium changes in CA1 during hypoxia were 33% and 62% of the total calcium change in PND 3-7 and PND 18-22 CA1, respectively. We conclude that survival of CA1 neurons in PND 3-7 slices following hypoxic stress is associated with smaller increases and enhanced recovery of [Ca2+]i, less accumulation of glutamate, and less glutamate receptor-mediated calcium influx than in PND 18-22 slices.

Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis.

Many strains of Bacillus cereus cause gastrointestinal diseases, and the closely related insect pathogen B. thuringiensis has also been involved in outbreaks of diarrhea. The diarrheal types of diseases are attributed to enterotoxins. Two different enterotoxic protein complexes, hemolysin BL (HBL) and nonhemolytic enterotoxin (NHE), and an enterotoxic protein, enterotoxin T, have been characterized, and the genes have been sequenced. PCR primers for the detection of these genes were deduced and used to detect the genes in 22 B. cereus and 41 B. thuringiensis strains. At least one gene of each of the two protein complexes HBL and NHE was detected in all of the B. thuringiensis strains, while six B. cereus strains were devoid of all three HBL genes, three lacked at least two of the three NHE genes, and one lacked all three. Five different sets of primers were used for detection of the gene (bceT) encoding enterotoxin T. The results obtained with these primer sets indicate that bceT is widely distributed among B. cereus and B. thuringiensis strains and that the gene varies in sequence among different strains. PCR with the two primer sets BCET1-BCET3 and BCET1-BCET4 unambiguously detected the bceT gene, as confirmed by Southern analysis. The occurrence of the genes within the two complexes is significantly associated, while neither the occurrence of the two complexes nor the occurrence of the bceT gene is significantly associated in the 63 strains. We suggest an approach for detection of enterotoxin-encoding genes in B. cereus and B. thuringiensis based on PCR analysis with the six primer sets for the detection of genes in the HBL and NHE operons and with the BCET1, BCET3, and BCET4 primers for the detection of bceT. PCR analysis of the 16S-23S rRNA gene internal transcribed spacer region revealed identical patterns for all strains studied.

DNA sequence and transcription of a DNA minicircle isolated from male-fertile sugar beet mitochondria.

A minicircle consisting of 1440 bp was isolated from male fertile sugar beet mitochondria. The entire nucleotide sequence of the minicircle was determined. There are several stop codons in all three reading frames and it is therefore unlikely that the minicircle codes for a polypeptide product. However, the minicircle is transcribed yielding a major RNA product consisting of 850 bases in addition to two very minor RNA products of 1100 and 950 bases, respectively. All three RNA products are transcribed from the same strand. The starting point for transcription of the major RNA product was determined by S1 nuclease protection analysis.

Effects of isoflurane and hypothermia on glutamate receptor-mediated calcium influx in brain slices.

BACKGROUND: To understand how volatile anesthetics protect neurons during cerebral ischemia, we studied the effects of isoflurane on cerebral glutamate receptor-mediated calcium influx. Calcium influx via these key excitatory receptors may mediate pain transmission, memory, and the pathophysiologic sequelae of cerebral anoxia or ischemia. Because cerebral protection by hypothermia may involve a decrease in glutamate receptor activity, we also examined the interaction of temperature and isoflurane on glutamate receptor inhibition. METHODS: We measured glutamate receptor-mediated changes in cytosolic calcium in 300-microns-thick rat cortical brain slices. Temperature was varied to 28, 34, 37, or 39 degrees C and isoflurane partial pressure to 0.016-0.019 atm (equivalent to 1.16 minimum alveolar concentration [MAC], adjusted for temperature and age). Brain slices were loaded with fura-2 to permit measurement of cytosolic free calcium. Calcium changes due to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) (50 microM), to ischemia levels of L-glutamate (1.0 mM) or to simulated ischemia (1.0 mM glutamate, 100 microM NaCN, and 3.5 mM iodoacetate) was then measured. Slice lactate dehydrogenase leakage and adenosine triphosphate were measured as indices of cellular integrity. RESULTS: Isoflurane reduced both L-glutamate and NMDA-mediated calcium fluxes by approximately 60%. Neither the activity of the NMDA receptor nor its inhibition by isoflurane was altered by temperature. The rate of calcium influx during ischemia was significantly reduced both by temperature and by isoflurane (P < 0.05). Adenosine triphosphate loss and lactate dehydrogenase leakage were reduced by isoflurane during simulated ischemia by 37% and 73% (P < 0.05), respectively. CONCLUSIONS: (1) At 1.16 MAC, isoflurane potently inhibits glutamate receptors and delays cellular injury induced by simulated ischemia, and (2) hypothermia does not reduce the intrinsic activity of cortical glutamate receptors but delays calcium accumulation during simulated ischemia. Isoflurane reduces the severity of key pathophysiologic events in an in vitro model of simulated cerebral ischemia.

Influence of thyroxine treatment on thyroid size and anti-thyroid peroxidase antibodies in Hashimoto's thyroiditis.

OBJECTIVE: It has been postulated that a decrease in thyroid size can be achieved by thyroxine treatment in patients with goitrous Hashimoto's thyroiditis but no objective data are available. We have therefore investigated the influence of thyroxine treatment on ultrasonically determined thyroid size. We also measured serum antithyroid peroxidase antibodies. DESIGN: Consecutive patients with goitrous Hashimoto's thyroiditis was studied. PATIENTS: Thirteen women participated; all had goitrous thyroiditis. TREATMENT: To render them euthyroid thyroxine was given for 24 months. MEASUREMENTS: Thyroid size was measured ultrasonically and antithyroid peroxidase antibodies were measured using a commercial radioimmunological method. RESULT: Concomitant with the gradual increase in serum free thyroxine and free triiodothyronine index values and a fall in serum thyrotrophin level, a gradual decrease in thyroid volume from 50.4 +/- 6.8 ml (mean +/- SEM) to 34.1 +/- 5.7 ml (32%), P less than 0.001 was demonstrated. Antithyroid peroxidase antibodies were present in high concentrations in all subjects but the mean serum level was not significantly changed at 24 months after initiation of treatment. CONCLUSION: A clinically significant reduction in thyroid volume related to normalization of thyroid function but unrelated to changes in antithyroid peroxidase antibody can be achieved during L-thyroxine treatment of hypothyroid goitrous Hashimoto's thyroiditis.

The hidden lifestyles of Bacillus cereus and relatives.

Bacillus cereus sensu lato, the species group comprising Bacillus anthracis, Bacillus thuringiensis and B. cereus (sensu stricto), has previously been scrutinized regarding interspecies genetic correlation and pathogenic characteristics. So far, little attention has been paid to analysing the biological and ecological properties of the three species in their natural environments. In this review, we describe the B. cereus sensu lato living in a world on its own; all B. cereus sensu lato can grow saprophytically under nutrient-rich conditions, which are only occasionally found in the environment, except where nutrients are actively collected. As such, members of the B. cereus group have recently been discovered as common inhabitants of the invertebrate gut. We speculate that all members disclose symbiotic relationships with appropriate invertebrate hosts and only occasionally enter a pathogenic life cycle in which the individual species infects suitable hosts and multiplies almost unrestrained.

Enterotoxin-producing strains of Bacillus thuringiensis isolated from food.

Strains of Bacillus thuringiensis were isolated from various food items (pasta, pitta bread and milk) and were found to belong to either H-serotype kurstaki or neoleonensis. The strains were bioassayed against Pieris brassicae and insecticidal activity of strains was found to correspond to the presence of the cry1A-gene. All strains, except one, were found to express cytotoxic effects on Vero cells as an indicator of enterotoxin activity. Further, the B. thuringiensis strains HD-1 (serotype kurstaki), NB-125 (serotype tenebrionis) and HD-567 (serotype israelensis ) which are used commercially for insect pest management, were also found to have cytotoxic effects on Vero cells.

Natural occurrence of Bacillus thuringiensis on cabbage foliage and in insects associated with cabbage crops.

Bacillus thuringiensis was isolated from the phylloplane of organically grown cabbage in one field during two growth seasons (1992-93). The frequency of B. thuringiensis varied between 0.02 and 0.67 of the total B. cereus/B. thuringiensis population, with an average of 0.11. Characterization of the B. thuringiensis isolates from foliage showed that the majority (64% of 150 isolates) belonged to serovar kurstaki, had bipyramidal crystals and toxicity towards Pieris brassicae and/or Trichoplusia ni. Other serovars were also found on the foliage but occurred at very low frequencies (one to three isolates of each serovar). Bacillus thuringiensis was also isolated from insects associated with the cabbage crop (Pieris rapae (Lep.), Delia radicum (Dip.), Syrphidae ribesii (Dip.) and Aleochara bilineata (Col.)), which were collected alive at different developmental stages in the same field. Serologically these isolates were assigned to the serovars kurstaki, aizawai, tochigiensis, colmeri and indiana/colmeri.

Changes in symptoms and colpo-cystourethrography in 35 patients before and after total abdominal hysterectomy: a prospective study.

35 women, referred for simple total hysterectomy, were evaluated pre- and postoperatively for bladder suspension and urodynamic changes. Although all patients denied urological problems, more than half of the patients had some degree of stress and/or urge incontinence. 40% had radiological signs of bladder suspension defects. At postoperative examination the symptomatology of incontinence had declined but the radiological pathologic findings had increased. On this basis it may be concluded that it is unpredictable what happens to the bladder and its suspension following simple hysterectomy.