High capacity homogeneous non-radioactive cortisol detection assays for human 11beta-hydroxysteroid dehydrogenase type 1

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) catalyzes the interconversion of inert glucocorticoid (cortisone) to the active glucocorticoid (cortisol) and is enriched in liver and fat tissues. Increasing evidence suggests that selective inhibition of 11beta-HSD1 may reduce the excess glucocorticoid levels that underlie the etiology of many common disorders that constitute the metabolic syndrome. Measurement of 11beta-HSD1 activity has historically involved the detection of cortisol by methods unfavorable for large-scale screening, such as high performance liquid chromatography or thin layer chromatography. Here we describe the development and validation of novel homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) and electrochemiluminescence assays for the measurement of cortisol. These non-radioactive assays were easy to perform and produced robust results with reference compound values comparable to those obtained by conventional methods. The TR-FRET assay was easily automated and was successfully employed for the high-throughput screening of a large compound library for inhibitors of purified human recombinant 11beta-HSD1.     

Penitentiary population of Mycobacterium tuberculosis in Kyrgyzstan: Exceptionally high prevalence of the Beijing genotype and its Russia-specific subtype

Here, we present results of the first study of the Mycobacterium tuberculosis genotypes circulating in Kyrgyzstan. We focused on the incarcerated population known to be at high-risk for tuberculosis (TB) and with a significant impact on TB incidence in the general population. Beijing genotype was detected in 42 of 56 M. tuberculosis sputum-extracted DNA samples from newly-diagnosed adult pulmonary TB patients. RIF and INH resistance was genotypically detected in 28% and 55% samples; 13 of 15 MDR strains belonged to Beijing genotype. 12-locus MIRU-VNTR typing showed 8 of 56 samples to be mixed cases; 7 of them contained a Beijing strain. MIRU analysis demonstrated a high homogeneity of the studied collection (HGI = 0.66) while 28 of 56 strains had a profile 223325153533 corresponding to Beijing/M2 subtype highly prevalent in different Russian settings. Three hypervariable loci, QUB-3232, VNTR-3820 and VNTR-4120, permitted to further subdivide 28 Beijing/M2 strains into 11 subtypes shared by 1 to 9 strains. To conclude, all markers taken together, the penitentiary population of M. tuberculosis in Kyrgyzstan exhibited a strong genetic affinity to Russia and a weak relatedness to East Asia.     

Colony-stimulating factor-1 in immunity and inflammation

Colony-stimulating factor-1 (CSF-1, also known as macrophage-CSF) is the primary regulator of the survival, proliferation, differentiation and function of mononuclear phagocytes. Studies that involve CSF-1-deficient mice demonstrate that there is a variable requirement for CSF-1 in the development of individual mononuclear phagocyte populations. However, these cells uniformly express the CSF-1 receptor, and their morphology, phagocytosis and responsiveness to infectious and non-infectious stimuli is regulated by CSF-1. CSF-1 plays important roles in innate immunity, cancer and inflammatory diseases, including systemic lupus erythematosus, arthritis, atherosclerosis and obesity. In several conditions, activation of macrophages involves a CSF-1 autocrine loop. In addition, secreted and cell-surface isoforms of CSF-1 can have differential effects in inflammation and immunity.     

Evolution of drug resistance in different sublineages of Mycobacterium tuberculosis Beijing genotype

We compared the population structure and drug resistance patterns of the Mycobacterium tuberculosis strains currently circulating in the Beijing area of China. One hundred thirteen of 123 strains belonged to the Beijing family genotypes defined by spoligotyping. The Beijing genotype strains were further subdivided into old and modern sublineages on the basis of NTF locus analysis. A stronger association with resistance to the more recently introduced antituberculosis drugs has been observed for old versus modern strains of the Beijing genotype, suggesting that its different sublineages may differ in their mechanisms of adaptation to drug selective pressure.     

A Multilead Scheme Based on Periodic Component Analysis for T-Wave Alternans Analysis in the ECG

T-wave alternans (TWA) is a cardiac phenomenon that appears in the electrocardiogram (ECG) and is associated with the mechanisms leading to sudden cardiac death (SCD). In this study, we propose the use of a multilead TWA analysis scheme that combines the Laplacian likelihood ratio (LLR) method and periodic component analysis (πCA), an eigenvalue decomposition technique whose aim is to extract the most periodic sources of the signal. The proposed scheme is evaluated in different scenarios—from synthetic signals to stress test ECGs—and is compared to other reported schemes based on the LLR method. Results demonstrate that the πCA-based scheme provides a superior ability to detect TWA than previously reported schemes, and has the potential to improve the prognostic value of testing for TWA.     

Epigenetic modifications induced by RGC-32 in colon cancer

First described as a cell cycle activator, RGC-32 is both an activator and a substrate for CDC2. Deregulation of RGC-32 expression has been detected in a wide variety of human cancers. We have now shown that RGC-32 is expressed in precancerous states, and its expression is significantly higher in adenomas than in normal colon tissue. The expression of RGC-32 was higher in advanced stages of colon cancer than in precancerous states or the initial stages of colon cancer. In order to identify the genes that are regulated by RGC-32, we used gene array analysis to investigate the effect of RGC-32 knockdown on gene expression in the SW480 colon cancer cell line. Of the 230 genes that were differentially regulated after RGC-32 knockdown, a group of genes involved in chromatin assembly were the most significantly regulated in these cells: RGC-32 knockdown induced an increase in acetylation of histones H2B lysine 5 (H2BK5), H2BK15, H3K9, H3K18, and H4K8. RGC-32 silencing was also associated with decreased expression of SIRT1 and decreased trimethylation of histone H3K27 (H3K27me3). In addition, RGC-32 knockdown caused a significantly higher percentage of SW480 cells to enter S phase and subsequently G2/M. These data suggest that RGC-32 may contribute to the development of colon cancer by regulating chromatin assembly.     

Comparative analysis of lesion development and intraspinal inflammation in four strains of mice following spinal contusion injury

Susceptibility to neuroinflammatory disease is influenced in part by genetics. Recent data indicate that survival of traumatized neurons is strain dependent and influenced by polygenic loci that control resistance/susceptibility to experimental autoimmune encephalomyelitis (EAE), a model of CNS autoimmune disease. Here, we describe patterns of neurodegeneration and intraparenchymal inflammation after traumatic spinal cord injury (SCI) in mice known to exhibit varying degrees of EAE susceptibility [EAE-resistant (r) or EAE-susceptible (s) mice]. Spinal cords from C57BL/6 (EAE-s), C57BL/10 (EAE-r), BALB/c (EAE-r), and B10.PL (EAE-s) mice were prepared for stereological and immunohistochemical analysis at 6 hours or 3, 7, 14, 28, or 42 days following midthoracic (T9) spinal contusion injury. In general, genetic predisposition to EAE predicted the magnitude of intraparenchymal inflammation but not lesion size/length or locomotor recovery. Specifically, microglia/macrophage activation, recruitment of neutrophils and lymphocytes, and de novo synthesis of MHC class II were greatest in C57BL/6 mice and least in BALB/c mice at all times examined. However, lesion volume and axial spread of neurodegeneration were similar in C57BL/6 and BALB/c mice and were significantly greater than in C57BL/10 or B10.PL mice. Strains with marked intraspinal inflammation also developed the most intense lesion fibrosis. Thus, strain-dependent neuroinflammation was observed after SCI, but without a consistent relationship to EAE susceptibility or lesion progression. Only in C57BL/6 mice was the magnitude of intraspinal inflammation predictive of secondary neurodegeneration, functional recovery, or fibrosis.     

Decreased focal inflammatory response by G-CSF may improve stroke outcome after transient middle cerebral artery occlusion in rats

Recent studies have shown that administration of granulocyte colony-stimulating factor (G-CSF) is neuroprotective. However, the precise mechanisms of the neuroprotective effect of G-CSF are not entirely known. We carried out 90-min transient middle cerebral occlusion (tMCAO) of rats. The rats were injected with vehicle or G-CSF (50 mug/kg) immediately after reperfusion and sacrificed 8, 24, or 72 hr later. 2,3,5-Triphenyltetrazolium chloride (TTC) staining was carried out using brain sections of 72 hr, and immunohistochemistry was carried out with those of 8, 24, and 72 hr. TTC-staining showed a significant reduction of infarct volume in the G-CSF-treated group (**P < 0.01). Immunohistochemistry showed a significant decrease of the number of cells expressing tumor necrosis factor-alpha (TNF-alpha) at 8-72 hr, transforming growth factor-beta (TGF-beta) and inducible nitric oxide synthase (iNOS) at 24 and 72 hr after tMCAO in the peri-ischemic area (*P < 0.05 each). Our data suggest that the suppression of inflammatory cytokines and iNOS expression may be one mechanism of neuroprotection by G-CSF.     

Beta-nicotinamide adenine dinucleotide is an inhibitory neurotransmitter in visceral smooth muscle

Peripheral inhibitory nerves are physiological regulators of the contractile behavior of visceral smooth muscles. One of the transmitters responsible for inhibitory neurotransmission has been reputed to be a purine, possibly ATP. However, the exact identity of this substance has never been verified. Here we show that beta-nicotinamide adenine dinucleotide (beta-NAD), an inhibitory neurotransmitter candidate, is released by stimulation of enteric nerves in gastrointestinal muscles, and the pharmacological profile of beta-NAD mimics the endogenous neurotransmitter better than ATP. Levels of beta-NAD in superfusates of muscles after nerve stimulation exceed ATP by at least 30-fold; unlike ATP, the release of beta-NAD depends on the frequency of nerve stimulation. beta-NAD is released from enteric neurons, and release was blocked by tetrodotoxin or omega-conotoxin GVIA. beta-NAD is an agonist for P2Y1 receptors, as demonstrated by receptor-mediated responses in HEK293 cells expressing P2Y1 receptors. Exogenous beta-NAD mimics the effects of the enteric inhibitory neurotransmitter. Responses to beta-NAD and inhibitory junction potentials are blocked by the P2Y1-selective antagonist, MRS2179, and the nonselective P2 receptor antagonists, pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid and suramin. Responses to ATP are not blocked by these P2Y receptor inhibitors. The expression of CD38 in gastrointestinal muscles, and specifically in interstitial cells of Cajal, provides a means of transmitter disposal after stimulation. beta-NAD meets the traditional criteria for a neurotransmitter that contributes to enteric inhibitory regulation of visceral smooth muscles.     

Breast cyst fluids increase the proliferation of breast cell lines in correlation with their hormone and growth factor concentration

OBJECTIVE AND DESIGN: Gross cystic disease (GCD) of the breast is reported to occur in 7% of women in the developed world and, although not premalignant, is thought to be associated with an increased risk of breast cancer. Hormone and growth factor concentration levels were measured in breast cyst fluid (BCF) to correlate them with their mitogenic activity in tumour (MCF-7) or nontransformed (MCF-10A) cells. RESULTS: Oestradiol (E2), oestrone (E1), E2-sulfate (E2-S), E1-sulfate (E1-S) and epidermal growth factor (EGF) concentrations were, as expected, significantly higher in type I than in type II cysts, while transforming growth factor-beta 2 (TGF-beta2) showed higher levels in type II cysts. Fifty per cent of the BCF samples stimulated [3H]-thymidine incorporation into MCF-7 cells while 34.5% inhibited this parameter. In MCF-10A cells, most BCF samples were stimulatory (85%). E2, E1 and EGF concentrations in BCF samples correlated significantly and positively with cell proliferation in MCF-7 cells, whereas a significant negative correlation was found for TGF-beta2. In MCF-10A cells, only E2-S and E1-S exhibited significant positive correlation, whereas a significant negative correlation was found for TGF-beta2. Progesterone (Pg), E2 and EGF incubated under the same conditions had a stimulatory effect on [3H]-thymidine incorporation into MCF-7 cells, whereas TGF-beta2 inhibited this parameter. Pg, E2, E1 and EGF significantly stimulated this parameter in MCF-10A cells. CONCLUSIONS: The stimulatory action of BCF on cell proliferation in a model of human breast epithelial cells could partly explain the increased incidence of breast cancer in cyst-bearing women.