Induction of abortion with aglepristone significantly changed the expression of progesterone and estrogen receptors in canine endometrial stromal cells

In the present study, resorption/abortion was induced between days 25 and 45 of gestation with aglepristone (group IRA, n=10). The aim was to observe the change in the distribution of progesterone (PR) and estrogen receptors (ER), in comparison to a group of spontaneous resorptions/abortions (group SRA, n=5), and a further group of normal healthy pregnant animals, ovariohysterectomized between days 25 and 45 of gestation (controls, n=7). The receptors were assessed by means of immunohistochemistry (IHC) and RT-PCR, at the placental and interplacental sites of the uterine horn as well as in the corpus uteri. Significant differences were observed between the controls on one side and the groups of resorption/abortion on the other side. The total scores of the progesterone receptors (TPR) in the placental and interplacental part of the uterine horn, was significantly lower in the endometrial stromal cells (ESC) of the control group than in those of the SRA- and IRA-group, respectively (placenta: 5.8 vs. 6.5 and 6.7, p<0.01; interplacental sites: 5.6 vs. 6.6 and 6.6, p<0.05). In contrary, the total scores of the estrogen receptors (TER) at interplacental sites and the corpus uteri, respectively, was significantly higher in the myometrial smooth muscle cells (MSMC) and the ESC (p<0.05) of the controls. We therefore conclude, that the here observed differences between groups point to an up-regulation of TPR- and a down-regulation of TER-scores in endometrial stromal cells at different uterine sites during resorption/abortion, which indicates a special role of these cells.     

Prostaglandin E1 (PGE1), but not prostaglandin E2 (PGE2), alters luteal and endometrial luteinizing hormone (LH) occupied and unoccupied LH receptors and mRNA for LH receptors in ovine luteal tissue to prevent luteolysis

Loss of luteal progesterone secretion at the end of the ovine estrous cycle is via uterine PGF₂α secretion. However, uterine PGF₂α secretion is not decreased during early pregnancy in ewes. Instead, the embryo imparts a resistance to PGF₂α. Prostaglandins E (PGE; PGE₁ + PGE₂) are increased in endometrium and uterine venous blood during early pregnancy in ewes to prevent luteolysis. Chronic intrauterine infusion of PGE₁ or PGE₂ prevents spontaneous or IUD, estradiol-17β, or PGF₂α-induced premature luteolysis in nonbred ewes. The objective was to determine whether chronic intrauterine infusion of PGE₁ or PGE₂ affected mRNA for LH receptors, occupied and unoccupied receptors for LH in luteal and caruncular endometrium, and luteal function. Ewes received Vehicle, PGE₁, or PGE₂ every 4 h from days 10 to 16 of the estrous cycle via a cathether installed in the uterine lumen ipsilateral to the luteal-containing ovary.Jugular venous blood was collected daily for analysis of progesterone and uterine venous blood was collected on day-16 for analysis of PGF₂α and PGE. Corpora lutea and caruncular endometrium were collected from day-10 preluteolytic control ewes and day-16 ewes treated with Vehicle, PGE₁ or PGE₂ for analysis of the mRNA for LH receptors and occupied and unoccupied receptors for LH. Luteal weights on day-16 in ewes treated with PGE₁ or PGE₂ and day-10 control ewes were similar (P ≥ 0.05), but were greater (P ≤ 0.05) than in day-16 Vehicle-treated ewes. Progesterone profiles on days 10–16 differed (P ≤ 0.05) among treatment groups: PGE₁ > PGE₂ > Vehicle-treated ewes. Concentrations of PGF₂α and PGE in uterine venous plasma on day-16 were similar (P ≥ 0.05) in the three treatment groups. Luteal mRNA for LH receptors and unoccupied and occupied LH receptors were similar (P ≥ 0.05) in day-10 control ewes and day-16 ewes treated with PGE₂ and were lower (P ≤ 0.05) in day-16 Vehicle-treated ewes. PGE₂ prevented loss (P ≤ 0.05) of day-16 luteal mRNA for LH receptors and occupied and unoccupied LH receptors. Luteal and caruncular tissue mRNA for LH receptors and occupied and unoccupied LH receptors were greater (P ≤ 0.05) on day-16 of PGE₁-treated ewes than any treatment group. mRNA for LH receptors and occupied and unoccupied receptors for LH in caruncules were greater (P ≤ 0.05) in day-16 Vehicle or PGE₂-treated ewes than in day-10 control ewes. It is concluded that PGE₁ and PGE₂ share some common mechanisms to prevent luteolysis; however, only PGE₁ increased luteal and endometrial mRNA for LH receptors and occupied and unoccupied LH receptors. PGE₂ prevents a decrease in luteal mRNA for LH receptors and occupied and unoccupied receptors for LH without altering endometrial mRNA for LH receptors or occupied and unoccupied receptors for LH.     

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB₁ and CB₂ receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins. Additionally, we studied the functionality of the cannabinoid receptors in glioblastomas by using WIN 55,212-2 stimulated [³⁵S]GTPγS binding.Western blot analysis showed that CB₁ receptor immunoreactivity was significantly lower in glioblastoma multiforme (?43%, n = 10; p < 0.05) than in normal post-mortem brain tissue (n = 16). No significant differences were found for astrocytoma (n = 6) and meningioma (n = 8) samples. Conversely, CB₂ receptor immunoreactivity was significantly greater in membranes of glioblastoma multiforme (765%, n = 9; p < 0.05) and astrocytoma (471%, n = 4; p < 0.05) than in control brain tissue (n = 10). Finally, the maximal stimulation of [³⁵S]GTPγS binding by WIN 55,212-2 was significantly lower in glioblastomas (134 ± 4%) than in control membranes (183 ± 2%; p < 0.05). The basal [³⁵S]GTPγS binding and the EC₅₀ values were not significantly different between both groups.The present results demonstrate opposite changes in CB₁ and CB₂ receptor protein expression in human gliomas. These changes may be of interest for further research about the therapeutic effects of cannabinoids in glial tumors.     

Identification and evaluation of nutrient limitation on periphyton growth in headwater streams in the Pawnee Nation,Oklahoma

The objective of the study was to identify nutrient impacts, if any, on stream periphyton growth in Black Bear Creek (north central Oklahoma) and its tributaries. Passive diffusion periphytometers were deployed at ten study sites within the Black Bear Creek basin to evaluate periphyton growth in response to nutrient enrichment. These sites were selected to represent a gradient of land uses, from predominantly agricultural to predominantly urban. Periphytometer treatments included phosphorus (P) (1.0 mg/L PO₄-P, n = 10), nitrogen (N) (10.0 mg/L NO₃-N, n = 10), N plus P (n = 10) and control (reverse osmosis-treated water, n = 10). Results indicated that average dissolved inorganic N (DIN, PQL = 0.04 mg/L) concentrations were significantly correlated (R² = 0.63, p < 0.01) with chlorophyll a production on the periphytometer control treatments in the Black Bear Creek basin. Periphytic growth was nutrient-limited (increased chlorophyll a was measured on nutrient-enriched growth media) at four of the ten sites sampled; two sites were limited by N and two sites were co-limited by both N and P. The lotic ecosystem trophic status index (LETSI), the ratio of C to N + P chlorophyll a, was calculated to compare treatment responses across sites. At nutrient-limited sites, LETSI was positively correlated to ambient DIN values (R² = 0.97, p < 0.01). However, some sites that were not nutrient-limited had ambient nutrient concentrations similar to sites with observed nutrient limitation, indicating other factors were limiting periphyton growth at those sites.     

Postpartum ovarian activity and uterine involution in non-seasonal Shiba goats,with or without nursing

Postpartum ovarian activity, uterine involution, and the relationship with hormonal profiles were studied in non-seasonal Shiba goats, with or without nursing of their kids. After parturition, does were allocated to one of two groups, with either weaning on the day of parturition (n = 3; non-nursing group) or weaning at 7–10 weeks after parturition (n = 4; nursing group). Blood sampling (starting 7 days prior to expected day of parturition) and transrectal ultrasound evaluations (starting 2 days after parturition) were conducted every other day or daily to monitor the follicular dynamics, uterus size, and the levels of plasma progesterone, blood glucose, and insulin concentration until at 3 weeks following the first postpartum ovulation. In the nursing group, blood samples were also collected every 10 min for an 8 h period on the day before weaning, 2 and 6 days after weaning, for the analysis of the pulsatile patterns of LH release. In all animals, the blood glucose concentrations increased transiently on the day before parturition and were significantly (p < 0.05) higher than those on the day of parturition (mean of 144 ± 48.8 mg/dl vs 63 ± 11.2 mg/dl). In the non-nursing group, the first postpartum ovulation was observed 9.3 ± 3.2 days after parturition, while in the nursing group, no ovulation occurred before weaning in any of the goats. Here ovulation was observed 18.8 ± 5.0 days after weaning, which was significantly (p < 0.05) later than in the non-nursing group. After first ovulation, all animals in both groups showed early luteal regression. No significant difference in the time required for the completion of uterine involution was recorded between the non-nursing and nursing groups (18.3 ± 4.2 days vs 19.3 ± 3.6 days, respectively), nor in the plasma LH pulse frequency obtained before (1.3 ± 0.5 pulses/8 h), 2 (1.3 ± 1.0 pulses/8 h) and 6 days (2.0 ± 0.8 pulses/8 h) after parturition, in the nursing group. It can be concluded that no ovulation occurs during nursing postpartum, suggesting that nursing is a determinant of the resumption of ovulation in these non-seasonal goats. Ultrasonic observations of the ovary suggest that uterine involution was not influenced by nursing or suckling.     

Early liraglutide treatment is better in glucose control, β-cell function improvement and mass preservation in db/db mice

Glucagon-like peptide-1 (GLP-1) has been proved to have effects of anti-hyperglycemia and β-cell preservation. However, it is still unclear whether there are differences between early and late GLP-1 intervention in type 2 diabetes mellitus (T2DM). We divided the mice into 5 groups: early treated group (n = 7, 8-week old, fasting glucose > 10 mmol/l), late treated group (n = 7, 10-week old, fasting glucose > 20 mmol/l), early control group (n = 7), late control group (n = 7) and wild type group (n = 7). Treated group was injected with liraglutide (a GLP-1 analog) 300 μg/kg bid for 4 weeks, while control group was given saline at the same time. The results showed that compared with control group, food intake and body weight gain were reduced in both early and late treated group (p < 0.05), and there was no significance between the two treated groups. Early liraglutide intervention showed better improvements in glucose control, acute insulin response to glucose (AIRg) and disposition index (before vs. after treatment, AIRg 1.01 ± 0.53 vs. 2.98 ± 0.63, disposition index 10.81 ± 0.89 vs. 27.4 ± 2.15) than late intervention (AIRg 0.99 ± 0.02 vs. 1.41 ± 0.32, disposition index 3.47 ± 0.38 vs. 6.43 ± 1.62, p = 0.001). The histopathology of the pancreas showed the estimated β-cell mass (BCM) was increased more in early treated group than that in late one (0.03 vs. 0.01 g). Expressions of the proliferation related genes PDX-1, MafA and GLP-1 receptor (GLP-1R) in early treated group were 1.81, 2.57 and 1.59 times as much as that in late treated group. In conclusion, early liraglutide intervention was better in glucose control, β-cell function improvement and β-cell mass preservation.     

Valeur de la TEP-FDG dans la détection précoce des métastases osseuses : étude d’une série de 20 cas

FDG-PET is useful for metabolic analysis of metastatic cells. Based on a retrospective study relating to 20 cases of osseous metastases (MO), and a literature review, we will try to specify the main characteristics of bone metastases detected with FDG-PET. In our study, the primary malignant findings included lymphoma (n = 7), lung cancer (n = 5), breast cancer (n = 4), colon cancer (n = 2), melanoma (n = 1), and nasopharyngeal cancer (n = 1). On FDG-PET, 4 patients had a solitary metastatic focus (20%), 1 patient had two metastatic focuses (5%) and 15 patients had multiple lesions (75%). Most of the lesions showed intense abnormal uptake with SUVmax > 4.0 (14 patients, 70%); 6 patients had both intense and moderate FDG uptake with SUVmax > 2.1. Only 6 patients had MO diagnosed with standard diagnostic procedures (SDP) before FDG-PET. However, FDG-PET consistently revealed more metastatic foci missed by SDP. We conclude that FDG-PET is better than SDP in detecting all bone metastases at all sites of the skeleton according to the data of the literature.     

Synthesis and CB1 cannabinoid receptor affinity of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles

A series of 4-alkoxycarbonyl-1,5-diaryl-1,2,3-triazoles were synthesized regioselectively using click chemistry and evaluated at CB1 cannabinoid receptors. The n-propyl ester 11 (K_i = 4.6 nM) and phenyl ester 14 (K_i = 11 nM) exhibited the most potent affinity of the series.     

High serum osteopontin levels in pediatric patients with high risk Langerhans cell histiocytosis

Osteopontin (OPN) acts as an osteoclast activator, a proinflammatory cytokine, and a chemokine attracting histiocytes/monocytes and is abundantly expressed in Langerhans cell histiocytosis (LCH). We investigated whether serum OPN levels are related to disease types in LCH. Fifty-eight newly diagnosed LCH patients were studied; eight with risk organ (liver, spleen and/or hematopoietic) involvements positive multisystem (MS+) disease, 27 with risk organ involvement negative multisystem (MS−) disease and 23 with single system (SS) disease. Pediatric patients with non-inflammatory disease (n = 27) were used as controls. All of patients with MS+ disease were younger than 3 years. Serum OPN levels and 44 kinds of humoral factors were measured by ELISA and Bio-Plex suspension array system, respectively. In the patients younger than 3 years, the median serum OPN level (interquartile range) was 240.3 ng/ml (137.6–456.0) in MS+ (n = 8); 92.7 ng/ml (62.0–213.8) in MS− (n = 14) and 72.5 ng/ml (55.6–94.0) in SS (n = 9) and 74.4 ng/ml (42.2–100.0) in control (n = 12). The OPN values were significantly higher in the MS+ group than the MS−, SS and control groups (p = 0.044, p = 0.001 and p = 0.002, respectively), but not different between the MS−, SS and control groups. In the patients older than 3 years, the median level of serum OPN (IQR) was 56.2 ng/ml (22.9–77.5) in MS− (n = 13), 58.9 ng/ml (31.0–78.7) in SS (n = 14) and 41.9 (28.9–54.1) in control (n = 15). These values did not differ significantly between each group. The serum OPN levels were positively correlated with the serum IL-6, CCL2, IL-18, IL-8 and IL-2 receptor concentration. OPN may be involved in risk organ dissemination and poor prognosis of LCH through the function as inflammatory cytokine/chemokine.     

Sexual performance of Awassi ram lambs reared in different sex composition groups

The objective of this study was to evaluate the effect of sex composition of the group of lambs before puberty on later sexual performance in Awassi ram lambs. Thirteen Awassi ram lambs of the same age were raised in either all-male group (n = 7) or in a group mixed with females (n = 6) from weaning to puberty. Blood samples, body weights (BW) and scrotal circumferences (SC) of ram lambs were recorded between eight and 10 months of age. Sexual performance testing was performed at 9 months of age by individually exposing ram lambs to oestrous females on five occasions. Body weight and SC were greater (P < 0.01) in the all-male compared with the mixed group. Plasma testosterone concentrations were not influenced (P > 0.05) by treatment. Plasma testosterone concentrations were significantly correlated (P ≤ 0.05) with BW (r = 0.3) and SC (r = 0.4). No treatment effects were detected regarding bouts of leg-kicking. Bouts of anogenital sniffing, the frequency of mount attempts and mounting frequency were greater (P < 0.05) in the all-male group. Even though the frequency of raising the fat tail of females was similar between the two treatments, the mixed group tended to be (P = 0.08) more efficient in doing so than the all-male group (higher tail raising/mount). Results of the current study indicate that mixing groups of ram and ewe lambs before puberty may be insufficient to improve later sexual performance of ram lambs.     

Depot-specific modulation of adipokine levels in rat adipose tissue by diet-induced obesity: The effect of aerobic training and energy restriction

The present study examined the effects of aerobic training and energy restriction on adipokines levels in mesenteric (MEAT) and retroperitoneal (RPAT) white adipose tissue from obese rats. Male Wistar rats were fed with standard laboratory diet (Control group) or high fat diet (HFD). After 15 weeks, HFD rats were randomly assigned to the following groups: rats submitted to HFD, which were sedentary (sedentary HFD, n = 8) or trained (trained HFD, n = 8); or submitted to energy-restriction (ER), which were sedentary (sedentary ER, n = 8) or trained (trained ER, n = 8). Trained rats ran on a treadmill at 55% VO_2max for 60 min/day, 5 days/week, for 10 weeks. ER rats were submitted to a reduction of 20% daily caloric ingestion compared to the Control group. ER and aerobic training decreased body weight, MEAT and RPAT absolute weight, and fat mass. IL-6, IL-10 and TNF-α levels were decreased and adiponectin did not change in RPAT in response to ER protocol. On the other hand, ER and the aerobic training protocol decreased IL-6, TNF-α and adiponectin levels in MEAT. Absolute MEAT weight showed a positive correlation with IL-6 (r = 0.464), TNF-α (r = 0.508); and adiponectin (r = 0.342). These results suggest a tissue-specific heterogeneous response in adipokines level. The combination of the protocols (aerobic training and energy restriction) did not induce an enhanced effect.     

Increased whole blood manganese concentrations observed in children with iron deficiency anaemia

A prospective observational study was carried out at Alder Hey Children's Hospital, Liverpool, England, UK on children aged 1–6 years attending the pathology department for routine blood tests (n = 225). Whole blood manganese concentrations were measured plus the following markers of iron status; haemoglobin, MCV, MCH, RBC count, ferritin, transferrin saturation and soluble transferrin receptors. Multiple regression analysis was performed, with blood manganese as the dependent variable and factors of iron status, age and gender as independent variables. A strong relationship between blood manganese and iron deficiency was demonstrated (adjusted R² = 34.3%, p < 0.001) and the primary contributing factors to this relationship were haematological indices and soluble transferrin receptors. Subjects were categorised according to iron status using serum ferritin, transferrin saturation and haemoglobin indices. Children with iron deficiency anaemia had higher median blood manganese concentrations (16.4 μg/L, range 11.7–42.4, n = 20) than children with iron sufficiency (11 μg/L, range 5.9–20.9, n = 59, p < 0.001). This suggests that children with iron deficiency anaemia may be at risk from manganese toxicity (whole blood manganese >20 μg/L), and that this may lead to neurological problems. Treatment of iron deficiency in children is important both to improve iron status and to reduce the risk of manganese toxicity.     

Characterization and localization of the endothelin receptors in human end-stage heart failure: ETA/ETB receptor system—Role in the failing human heart

Endothelin (ET) is a potent vasoconstrictor peptide implicated in numerous human diseases, including ischemic cardiomyopathy (ICM). ET binds to receptors ET_A and ET_B. Specific ET receptors were characterized in left atria of patients with end-stage heart failure due to ischemic cardiomyopathy (ICM) (n = 9) and healthy controls (n = 9). Saturation assays revealed a K_d and B_max of 28 ± 8 pM and 87 ± 22 fmol mg^?1 of protein, respectively, from healthy atria and 50 ± 9 pM and 162 ± 19 fmol mg^?1 of protein, respectively, from diseased atria (p < 0.05). For competition studies, we obtained a percentage of ET_A receptors using BQ123, 55% ± 5 and 66% ± 4 in healthy and diseased atria, respectively (p < 0.05). The percentage of ET_B using BQ3020 was 55% ± 14 in healthy and 59% ± 10 in diseased atria. Microautoradiography studies showed a greater number of ET receptors, predominately ET_A, existed in the myocardial layer of diseased atria compared with healthy atria. The percentage of occupied area was greater in the endocardium than the epicardium in diseased atria. These results showed an increase in the ET_A/ET_B receptor system in the failing human heart compared with the non-failing human heart.     

Optimal whole-body vibration settings for muscle strength and power enhancement in human knee extensors

This study compared the effects of 6-week whole-body vibration (WBV) training programs with different frequency and peak-to-peak displacement settings on knee extensor muscle strength and power. The underlying mechanisms of the expected gains were also investigated. Thirty-two physically active male subjects were randomly assigned to a high-frequency/high peak-to-peak displacement group (HH; n = 12), a low-frequency/low peak-to-peak displacement group (LL; n = 10) or a sham training group (SHAM; n = 10). Maximal voluntary isometric, concentric and eccentric torque of the knee extensors, maximal voluntary isometric torque of the knee flexors, jump performance, voluntary muscle activation, and contractile properties of the knee extensors were assessed before and after the training period. Significant improvement in knee extensor eccentric voluntary torque (P < 0.01), knee flexor isometric voluntary torque (P < 0.05), and jump performance (P < 0.05) was observed only for HH group. Regardless of the group, knee extensor muscle contractile properties (P < 0.05) were enhanced. No modification was observed for voluntary muscle activation or electrical activity of agonist and antagonist muscles. We concluded that high-frequency/high peak-to-peak displacement was the most effective vibration setting to enhance knee extensor muscle strength and jump performance during a 6-week WBV training program and that these improvements were not mediated by central neural adaptations.     

Novel insights into cyclooxygenases,linoleate diol synthases,and lipoxygenases from deuterium kinetic isotope effects and oxidation of substrate analogs

Cyclooxygenases (COX) and 8R-dioxygenase (8R-DOX) activities of linoleate diol synthases (LDS) are homologous heme-dependent enzymes that oxygenate fatty acids by a tyrosyl radical-mediated hydrogen abstraction and antarafacial insertion of O₂. Soybean lipoxygenase-1 (sLOX-1) contains non-heme iron and oxidizes 18:2n ? 6 with a large deuterium kinetic isotope effect (D-KIE). The aim of the present work was to obtain further mechanistic insight into the action of these enzymes by using a series of n ? 6 and n ? 9 fatty acids and by analysis of D-KIE. COX-1 oxidized C₂₀ and C₁₈ fatty acids in the following order of rates: 20:2n ? 6 > 20:1n ? 6 > 20:3n ? 9 > 20:1n ? 9 and 18:3n ? 3 ≥ 18:2n ? 6 > 18:1n ? 6. 18:2n ? 6 and its geometrical isomer (9E,12Z)18:2 were both mainly oxygenated at C-9 by COX-1, but the 9Z,12E isomer was mostly oxygenated at C-13. A cis-configured double bond in the n ? 6 position therefore seems important for substrate positioning. 8R-DOX oxidized (9Z,12E)18:2 at C-8 in analogy with 18:2n ? 6, but the 9E,12Z isomer was mainly subject to hydrogen abstraction at C-11 and oxygen insertion at C-9 by 8R-DOX of 5,8-LDS. sLOX-1 and 13R-MnLOX oxidized [11S-²H]18:2n ? 6 with similar D-KIE (~ 53), which implies that the catalytic metals did not alter the D-KIE. Oxygenation of 18:2n ? 6 by COX-1 and COX-2 took place with a D-KIE of 3–5 as probed by incubations of [11,11-²H₂]- and [11S-²H]18:2n ? 6. In contrast, the more energetically demanding hydrogen abstractions of the allylic carbons of 20:1n ? 6 by COX-1 and 18:1n ? 9 by 8R-DOX were both accompanied by large D-KIE (> 20).     

Modulation of intracellular chloride channels by ATP and Mg2+

We report the effects of ATP and Mg²⁺ on the activity of intracellular chloride channels. Mitochondrial and lysosomal membrane vesicles isolated from rat hearts were incorporated into bilayer lipid membranes, and single chloride channel currents were measured. The observed chloride channels (n = 112) possessed a wide variation in single channel parameters and sensitivities to ATP. ATP (0.5–2 mmol/l) modulated and/or inhibited the chloride channel activities (n = 38/112) in a concentration-dependent manner. The inhibition effect was irreversible (n = 5/93) or reversible (n = 15/93). The non-hydrolysable ATP analogue AMP-PNP had a similar inhibition effect as ATP, indicating that phosphorylation did not play a role in the ATP inhibition effect. ATP modulated the gating properties of the channels (n = 6/93), decreased the channels' open dwell times and increased the gating transition rates. ATP (0.5–2 mmol/l) without the presence of Mg²⁺ decreased the chloride channel current (n = 12/14), whereas Mg²⁺ significantly reversed the effect (n = 4/4). We suggest that ATP-intracellular chloride channel interactions and Mg²⁺ modulation of these interactions may regulate different physiological and pathological processes.     

Liquid chromatography–tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid

BackgroundAnalysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography–tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.MethodsThe method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (¹³C₃-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients’ samples with known differences in SA metabolites like meningitis (n = 6), brain tumour (n = 2), leukaemia (n = 5), and Salla disease (n = 1).ResultsLimit of detection (LOD) was 0.54 μM for FSA and 0.45 μM for TSA. Intra- and inter-assay variation for FSA (21.8 μM) were 4.8% (n = 10) and 10.4% (n = 40) respectively. Intra- and inter-assay variation for TSA (35.6 μM) were 9.7% (n = 10) and 12.8% (n = 40) respectively. Tested patients showed values of TSA above established reference value.ConclusionThe validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.     

Diagnostic performance of salivary cortisol and serum osteocalcin measurements in patients with overt and subclinical Cushing's syndrome

ObjectiveThe cut-off value for salivary cortisol measurement for the diagnosis of Cushing's syndrome (CS) may depend both on the severity of the disease and the composition of control group. Therefore, we examined the utility of midnight salivary cortisol measurements in patients who were evaluated for signs and symptoms of CS or because they had adrenal incidentalomas. Because serum osteocalcin (OC) is considered as a sensitive marker of hypercortisolism, we also investigated whether OC could have a role in the diagnosis of CS.Patients and methodsEach of the 151 patients was included into one of the following groups: (A) overt CS (n = 23), (B) subclinical CS (n = 18), (C) inactive adrenal adenomas (n = 40), (D) patients without HPA disturbances (n = 70). Patients (C + D) were used as controls. Serum, salivary and urinary cortisol, and OC were measured by electrochemiluminescence immunoassay.ResultsGroup A had suppressed OC as compared to both group B and group (C + D). Serum and salivary cortisol concentrations showed strong negative correlations with OC in patients with overt CS. The areas under the curves of salivary and serum cortisol at 24:00 h (0.9790 and 0.9940, respectively) serum cortisol after low dose dexamethasone test (0.9930) and OC (0.9220) obtained from ROC aanalysis for the diagnosis of overt CS were not statistically different.ConclusionThis study confirms the usefulness of midnight salivary cortisol measurements in the diagnosis of overt CS in the everyday endocrinological praxis. Our results suggest that OC may have a role in the diagnosis of overt CS.     

Effects of oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) and the metabolic characterization of Bacillus subtilis NX-2

The effects of different oxygen vectors on the synthesis and molecular weight of poly(γ-glutamic acid) (PGA) were investigated in the batch fermentation of Bacillus subtilis NX-2. n-Hexane, n-heptane, and n-hexadecane enhanced the PGA concentration and molecular weight. The PGA concentration reached a maximum of 39.4 ± 0.19 g L^?1, and the highest molecular weight obtained was (19.0 ± 0.02) × 10⁵ Da with the addition of 0.3% n-heptane. However, n-dodecane decreased the PGA concentration and molecular weight to final values of 20.1 ± 0.10 g L^?1 and (8.4 ± 0.02) × 10⁵ Da, respectively. Analysis of the intracellular nucleotide levels of B. subtilis NX-2 with n-heptane and n-dodecane additives showed that the lowest NADH/NAD⁺ ratio and ATP levels were obtained with the n-dodecane additives, which can explain the decreased PGA yield and molecular weight. The metabolic flux distribution of B. subtilis NX-2 with n-heptane and n-dodecane additives was also investigated. Flux distribution was primarily directed to the EMP and TCA cycles with n-heptane additives. The flux of 2-oxoglutarate to intracellular glutamate and the flux distribution from extracellular to intracellular glutamate both increased to improve PGA production.     

Selection by climatic regime and neutral evolutionary processes in holocentric chromosomes (Carex gr. laevigata: Cyperaceae): A microevolutionary approach

The holocentric structure promotes chromosome rearrangements by fission, fusion, translocation, and inversion, which have been thought to promote differentiation and speciation. The Carex laevigata group (Cyperaceae) comprises four species: two restricted endemics from the western Mediterranean (Carex camposii, 2n = 72, and Carex paulo-vargasii, 2n = 74–75), and two more widespread species, found mostly in Western Europe (C. laevigata, 2n = 69–84, and Carex binervis, 2n = 72–74). We tested the selection for chromosome number by climatic variables by controlling for the non-independence of the data using generalized linear mixed model (GLMM). We obtained chromosome counts as well as DNA sequences for the 5′ trnK intron and the trnV-ndhC intergenic spacer in the chloroplast genome from 181 individuals from 53 populations representing these four species. We also climatically characterized the sites where the 53 populations were found using the WorldClim database. Our results show that the best predictor of chromosome number variation is the climatic environment rather than neutral evolutionary processes like founder events and migration patterns. These results support the adaptive value of the holocentric chromosomes and their role in promoting differentiation and eventually speciation.