Age-related variations of leptin receptor expression in the growth plate of spine and limb: gender- and region-specific changes

Leptin is a potent growth-stimulating factor of bone. The effects of leptin on bone growth differ significantly between axial and appendicular regions. Gender differences of leptin function have also been suggested in normal pubertal development. To explore the mechanisms underlying these effects, we investigated the spatial and temporal expressions of the active form of the leptin receptor (Ob-Rb) in the tibial and spinal growth plates of the female and male rats during postnatal development. The 1-, 4-, 7-, 12- and 16-week age stages are representative for early life, puberty and early adulthood after puberty, respectively. Quantitative real-time PCR was used for Ob-Rb mRNA examination and comparison. The spatial location of Ob-Rb was determined by immunohistochemical analysis. There were gender- and region-specific differences in Ob-Rb mRNA expression in the growth plate. Mainly cytoplasm staining of Ob-Rb immunoreactivity was observed in the spinal and tibial growth plate chondrocytes of both genders. Spatial differences of region- and gender-related Ob-Rb expression were not observed. Ob-Rb immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in early life stage and during puberty. After puberty, staining was mainly located in the late proliferative and hypertrophic chondrocytes. The results of Ob-Rb HSCORE analysis were similar to those obtained from quantitative real-time PCR. Our study indicated direct effects on the chondrocytes of the growth plate in different development stages. The region-specific expression patterns of Ob-Rb gene might be one possible reason for contrasting phenotypes in limb and spine. Different Ob-Rb expression patterns might partly contribute to age- and gender- related differences in trabecular bone mass.     

Cholecystokinin levels in prohormone convertase 2 knock-out mouse brain regions reveal a complex phenotype of region-specific alterations

Prohormone convertase 2 is widely co-localized with cholecystokinin in rodent brain. To examine its role in cholecystokinin processing, cholecystokinin levels were measured in dissected brain regions from prohormone convertase 2 knock-out mice. Cholecystokinin levels were lower in hippocampus, septum, thalamus, mesencephalon, and pons in knock-out mice than wild-type mice. In cerebral cortex, cortex-related structures and olfactory bulb, cholecystokinin levels were higher than wild type. Female mice were more affected by the loss of prohormone convertase 2 than male mice. The decrease in cholecystokinin levels in these brain regions shows that prohormone convertase 2 is important for cholecystokinin processing. Quantitative polymerase chain reaction measurements were performed to examine the relationship between peptide levels and cholecystokinin and enzyme expression. They revealed that cholecystokinin and prohormone convertase 1 mRNA levels in cerebral cortex and olfactory bulb were actually lower in knock-out than wild type, whereas their expression in other brain regions of knock-out mouse brain was the same as wild type. Female mice frequently had higher expression of cholecystokinin and prohormone convertase 1, 2, and 5 mRNA than male mice. The loss of prohormone convertase 2 alters CCK processing in specific brain regions. This loss also appears to trigger compensatory mechanisms in cerebral cortex and olfactory bulb that produce elevated levels of cholecystokinin but do not involve increased expression of cholecystokinin, prohormone convertase 1 or 5 mRNA.     

The effect of aging on glutathione and cysteine levels in different regions of the mouse brain

A general glutathione (GSH) deficiency occurs in many tissues of the aging mouse. However, there is no information on GSH in the aging brain even though it has been involved in a number of neurobiologic reactions. To this end, C57BL/6 mice, 3-31 months old, representing the growth, maturation, and aging periods of the life-span were studied. Brain cortex, hippocampus, and stem samples were dissected, processed, and analyzed specifically for reduced and oxidized glutathione (GSH, GSSG) and cyst(e)ine using high performance liquid chromatography with dual electrochemical detection. The GSH content of each brain region varied in the order brain cortex greater than brain hippocampus greater than brainstem. However, the GSH profiles of all regions were the same through the life-span, namely, high values during growth dropping to a maturation plateau and then decreasing 30% during aging. In contrast to GSH, the order of cysteine levels was brain cortex less than brain hippocampus less than brainstem and no life-span changes occurred in any region. In addition, the brain GSSG and cystine contents of all regions were very low and did not change during the life-span. Thus, the GSH loss was not accountable by oxidation to GSSG or degradation to cyst(e)ine. Altogether these results demonstrated a GSH deficiency in brain tissues of aging mice like that found previously in other tissues. These findings suggest an increased susceptibility of the aging brain to oxidative damage.     

Development and application of different methods for the detection of Toxoplasma gondii in water

Two methods, centrifugation and flocculation, were evaluated to determine their efficiencies of recovery of Toxoplasma gondii oocysts from contaminated water samples. Demineralized and tap water replicates were inoculated with high numbers of sporulated or unsporulated T. gondii oocysts (1 x 10(5) and 1 x 10(4) oocysts). The strain, age, and concentration of the seeded oocysts were recorded. Oocysts were recovered either by centrifugation of the contaminated samples at various g values or by flocculation with two coagulants, Fe(2)(SO(4))(3) and Al(2)(SO(4))(3). The recovery rates were determined with the final pellets by phase-contrast microscopy. Sporulated oocysts were recovered more effectively by flocculation with Al(2)(SO(4))(3) (96.5% +/- 21.7%) than by flocculation with Fe(2)(SO(4))(3) (93.1% +/- 8.1%) or by centrifugation at 2,073 x g (82.5% +/- 6.8%). For the unsporulated oocysts, flocculation with Fe(2)(SO(4))(3) was more successful (100.3% +/- 26.9%) than flocculation with Al(2)(SO(4))(3) (90.4% +/- 19.1%) or centrifugation at 2,565 x g (97.2% +/- 12.5%). The infectivity of the sporulated oocysts recovered by centrifugation was confirmed by seroconversion of all inoculated mice 77 days postinfection. These data suggest that sporulated Toxoplasma oocysts purified by methods commonly used for waterborne pathogens retain their infectivity after mechanical treatment and are able to induce infections in mammals. This is the first step in developing a systematic approach for the detection of Toxoplasma oocysts in water.     

Oxytocin regulates changes of extracellular glutamate and GABA levels induced by methamphetamine in the mouse brain

Oxytocin (OT), a neurohypophyseal neuropeptide, affects adaptive processes of the central nervous system. In the present study, we investigated the effects of OT on extracellular levels of glutamate (Glu) and γ-aminobutyric acid (GABA) induced by methamphetamine (MAP) in the medial prefrontal cortex (mPFC) and dorsal hippocampus (DHC) of freely moving mice, using in vivo microdialysis coupled to high-performance liquid chromatography and fluorescence detection. The results showed that OT had no effect on basal Glu levels, but attenuated MAP-induced Glu increase in the mPFC and decrease in the DHC. OT increased the basal levels of extracellular GABA in mPFC and DHC of mice, and inhibited the MAP-induced GABA decrease in DHC. Western blot results indicated that OT significantly inhibited the increased glutamatergic receptor (NR1 subunit) levels in the PFC after acute MAP administration, whereas OT further enhanced the elevated levels of glutamatergic transporter (GLT1) induced by MAP in the hippocampus of mice. Atosiban, a selective inhibitor of OT receptor, antagonized the effects of OT. The results provided the first neurochemical evidence that OT, which exerted its action via its receptor, decreased Glu release induced by MAP, and attenuated the changes in glutamatergic neurotransmission partially via regulation of NR1 and GLT1 expression. OT-induced extracellular GABA increase also suggests that OT acts potentially as an inhibitory neuromodulator in mPFC and DHC of mice.     

Two validated HPLC methods for the quantification of alizarin and other anthraquinones in Rubia tinctorum cultivars

Direct and indirect HPLC-UV methods for the quantitative determination of anthraquinones in dried madder root have been developed, validated and compared. In the direct method, madder root was extracted twice with refluxing ethanol-water. This method allowed the determination of the two major native anthraquinone glycosides lucidin primeveroside and ruberythric acid. In the indirect extraction method, the anthraquinone glycosides were first converted into aglycones by endogenous enzymes and the aglycones were subsequently extracted with tetrahydrofuran-water and then analysed. In this case the anthraquinones alizarin, purpurin and nordamnacanthal may be determined. The content of nordamnacanthal is proportional to the amount of lucidin primeveroside originally present. The indirect extraction method is easier to apply. Different madder cultivars were screened for their anthraquinone content.     

Androgen-metabolizing enzymes show region-specific changes across the breeding season in the brain of a wild songbird.

The Lapland longspur (Calcarius lapponicus) is an arctic-breeding songbird that shows rapid behavioral changes during a short breeding season. Changes in plasma testosterone (T) in the spring are correlated with singing but not territorial aggression in males. Also, T treatment increases song but not aggression in this species. In contrast, in temperate-zone breeders, song and aggression are highly correlated, and both increase after T treatment. We asked whether regional or temporal differences in androgen-metabolizing enzymes in the longspur brain explain hormone-behavior patterns in this species. We measured the activities of aromatase, 5alpha-reductase and 5beta-reductase in free-living longspur males. Aromatase and 5alpha-reductase convert T into the active steroids 17beta-estradiol (E(2)) and 5alpha-dihydrotestosterone (5alpha-DHT), respectively. 5beta-Reductase deactivates T via conversion to 5beta-DHT, an inactive steroid. We examined seven brain regions at three stages in the breeding season. Overall, aromatase activity was high in the hypothalamus, hippocampus, and ventromedial telencephalon (containing nucleus taeniae, the avian homologue to the amygdala). 5beta-Reductase activity was high throughout the telencephalon. Activities of all three enzymes changed over time in a region-specific manner. In particular, aromatase activity in the rostral hypothalamus was decreased late in the breeding season, which may explain why T treatment at this time does not increase aggression. Changes in 5beta-reductase do not explain the effects of plasma T on aggressive behavior.     

Selective age-related changes in the PKC-sensitive, calmodulin-binding protein, neurogranin, in the mouse brain

Brain ageing is associated with a dysregulation of intracellular calcium (Ca(2+)) homeostasis which leads to deficits in Ca(2+)-dependent signalling pathways and altered neuronal functions. Given the crucial role of neurogranin/RC3 (Ng) in the post-synaptic regulation of Ca(2+) and calmodulin levels, age-dependent changes in the levels of Ng mRNA and protein expression were analysed in 3, 12, 24 and 31-month-old mouse brains. Ageing produced significant decreases in Ng mRNA expression in the dorsal hippocampal subfields, retrosplenial and primary motor cortices, whereas no reliable changes were seen in any other cortical regions examined. Western blot indicated that Ng protein expression was also down-regulated in the ageing mouse brain. Analysis of Ng immunoreactivity in both hippocampal CA1 and retrosplenial areas indicated that Ng protein in aged mice decreased predominantly in the dendritic segments of pyramidal neurones. These data suggest that age-related changes of post-synaptic Ng in selected brain areas, and particularly in hippocampus, may contribute to altered Ca(2+)/calmodulin-signalling pathways and to region-specific impairments of synaptic plasticity and cognitive decline.     

Dynamics of the regulation of histamine levels in mouse brain

Abstract— The intraperitoneal administration of L-histidine in a dose of 1000 mg/kg increased threefold the whole brain levels of histamine in the mouse. This increase was evident in all brain regions except the medulla oblongata-pons. The subcellular localization of histamine and histidine was the same in mice administered bhistidine as in salinetreated animals. Cold exposure and restraint further augmented the elevation of histamine elicited by histidine treatment. a-Hydrazino-histidine and 4-bromo-3-hydroxybenzyloxyamine (NSD-1055) but not a-methyl-DOPA inhibited histidine decarboxylase [EC 4.1.1.221 activity in mouse brain homogenates and prevented the increase in brain histamine after histidine administration. NSD-1055 and a-hydrazino-histidine also lowered brain levels of histamine by 50 per cent. NSD-1055 lowered whole brain levels of histamine rapidly, with a half-life for the depletable histamine pool of about 5 min. Assuming that inhibition of histidine decarboxylase accounted for the reduction in histamine, then the rate of histamine decline reflects the rate of histamine turnover, and our results suggest that a portion of mouse brain histamine turns over quite rapidly. Reserpine lowered brain levels of histamine by about 50 per cent, whereas the antihistaminic agent, dexbrompheniramine, and sodium pentobarbital elevated histamine levels.     

Iodine levels in different regions of the human brain

BackgroundIodine is a key component of the thyroid hormones thyroxine (T4) and triiodothyronine (T3), which are crucial for proper growth and development of the human body. In particular, a great body of literature has been published on the link between thyroid hormones and brain development and functioning. However, there is a lack of knowledge on the iodine levels in the human brain. The aim of this work was to determine the brain iodine levels and to contribute to the establishment of “reference” levels for iodine in the different anatomical and functional regions of normal (i.e., subjects without neurological or psychiatric diseases) human brain.MethodsThe iodine levels were determined in 14 brain regions of 52 dead subjects without evidence of neurological or psychiatric disease (n = 728 samples). Iodine was extracted from brain samples using a standard procedure and determined by inductively coupled plasma – mass spectrometry (ICP-MS).ResultsFour subjects presented abnormally high brain iodine levels (26.0 ± 14.2 μg/g) and were excluded from the overall data analysis. The average brain iodine levels for the remaining 48 subjects was 0.14 ± 0.13 μg/g dry weight. Iodine showed very heterogeneous distribution across the different brain regions, with the frontal cortex, caudate nucleus and putamen showing the highest levels. Interestingly, these brain regions are closely related to cognitive function. Iodine levels also showed a tendency to increase with age. The high levels observed in four subjects seemed to be related to previous exposure to iodine-based contrast agents widely used in radiology and computed tomography exams.ConclusionsThis paper provides important data on iodine levels at different brain regions in “normal” people, which can be used to interpret eventual imbalances in subjects with mental disorders and neurodegenerative diseases.     

Chromosomal changes: induction, detection methods and applicability in human biomonitoring

The objective of this state of the art paper is to review the mechanisms of induction, the fate, the methodology, the sensitivity/specificity and predictivity of two major cytogenetic endpoints applied for genotoxicity studies and biomonitoring purposes: chromosome aberrations and micronuclei. Chromosomal aberrations (CAs) are changes in normal chromosome structure or number that can occur spontaneously or as a result of chemical/radiation treatment. Structural CAs in peripheral blood lymphocytes (PBLs), as assessed by the chromosome aberration (CA) assay, have been used for over 30 years in occupational and environmental settings as a biomarker of early effects of genotoxic carcinogens. A high frequency of structural CAs in lymphocytes (reporter tissue) is predictive of increased cancer risk, irrespective of the cause of the initial CA increase. Micronuclei (MN) are small, extranuclear bodies that arise in dividing cells from acentric chromosome/chromatid fragments or whole chromosomes/chromatids that lag behind in anaphase and are not included in the daughter nuclei in telophase. The cytokinesis-block micronucleus (CBMN) assay is the most extensively used method for measuring MN in human lymphocytes, and can be considered as a "cytome" assay covering cell proliferation, cell death and chromosomal changes. The key advantages of the CBMN assay lie in its ability to detect both clastogenic and aneugenic events and to identify cells which divided once in culture. Evaluation of the mechanistic origin of individual MN by centromere and kinetochore identification contributes to the high sensitivity of the method. A number of findings support the hypothesis of a predictive association between the frequency of MN in cytokinesis-blocked lymphocytes and cancer development. Recent advances in fluorescence in situ hybridization (FISH) and microarray technologies are modifying the nature of cytogenetics, allowing chromosome and gene identification on metaphase as well as in interphase. Automated scoring by flow cytometry and/or image analysis will enhance their applicability.     

HPLC methods for detection of uniconazole-P in soils and plant tissues

High-performance liquid chromatographic (HPLC) methods have been developed for the detection of uniconazole-P [(E)-1-(4-chlorophenyl)-4,4,-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol; XE-1019; the active ingredient in Prunit and Sumagic] in soil and plant tissue samples. Methanolic extracts of soil and plant samples were dried to the aqueous phase, the pH adjusted to 11, and partitioned against methylene chloride. The methylene chloride phases were washed with pH 11 water and then passed through C-18 solid phase extraction (SPE) columns. The soil extracts were then dried and the residues taken up in 1 ml acetonitrile of which 20 l were injected directly onto a C-18 reverse phase analytical column for HPLC analysis. Plant tissue extracts were purified by partitioning and passing through a sequence of Florisil/C-18/Florisil SPE columns before HPLC analysis. Recovery of uniconazole-P was 70% from soils and 40% from plant tissues. Quantitative detection of 10 parts per billion (ppb) uniconazole-P in plant tissues and soil samples was feasible following these procedures. The soil cleanup procedures were also used to detect uniconazole-P in leachates collected from container-grown plants.     

Uridine-induced ultrastructural changes in different formations of the brain

In experiments on the rats there were identified two types of neurons, which age differentiated by their ability to form the coated vesicles in response to the administration of uridine. Presence or absence of the uridine receptors on the neuronal plasmalemmae causes different reaction of the neurons on the action of uridine. Coated vesicles transfer uridine to the lysosomes, where it degrades. Appearance of subsurface cisternae is a compensatory reaction on the deficit of neuronal plasmalemmae, which is necessary for the formation of coated vesicles. The satellite glia is the most resistant for the action of uridine.     

The brain selenoproteome: priorities in the hierarchy and different levels of selenium homeostasis in the brain of selenium-deficient rats

The application of radionuclides for the localization of essential trace elements in vivo and the characterization of their binding proteins is a story of intermittently made improvements of the techniques used for their detection. In this study we present the use of neutron activation analysis and different autoradiographic imaging methods including real-time digital autoradiography to reveal new insights in the hierarchy of selenium homeostasis. Selenoproteins containing the essential trace element selenium play important roles in the CNS. Although the CNS does not show the highest selenium concentration in the case of selenium-sufficient supply in comparison with other organs, it shows a high priority for selenium uptake and retention in the case of dietary selenium deficiency. To characterize the hierarchy of selenium supply in the brain, in vivo radiotracer labeling with ⁷⁵Se in rats with different selenium status was combined with autoradiographic detection of ⁷⁵Se in brain tissue sections and ⁷⁵Se-labeled selenoproteins after protein separation by two-dimensional gel electrophoresis. This study demonstrates significant differences in the uptake of ⁷⁵Se into the brain of rats with different selenium status. A brain region-specific uptake pattern of the radiotracer ⁷⁵Se in selenium-deficient rats could be revealed and the CSF was identified as a key part of the brain selenium homeostasis.