Comprehensive gene expression profiling in lungs of mice infected with Mycobacterium tuberculosis following DNAhsp65 immunotherapy

Background The continued increase in tuberculosis (TB) rates and the appearance of extremely resistant Mycobacterium tuberculosis strains (XDR-TB) worldwide are some of the great problems of public health. In this context, DNA immunotherapy has been proposed as an effective alternative that could circumvent the limitations of conventional drugs. Nonetheless, the molecular events underlying these therapeutic effects are poorly understood. Methods We characterized the transcriptional signature of lungs from mice infected with M. tuberculosis and treated with heat shock protein 65 as a genetic vaccine (DNAhsp65) combining microarray and real-time polymerase chain reaction analysis. The gene expression data were correlated with the histopathological analysis of lungs. Results The differential modulation of a high number of genes allowed us to distinguish DNAhsp65-treated from nontreated animals (saline and vector-injected mice). Functional analysis of this group of genes suggests that DNAhsp65 therapy could not only boost the T helper (Th)1 immune response, but also could inhibit Th2 cytokines and regulate the intensity of inflammation through fine tuning of gene expression of various genes, including those of interleukin-17, lymphotoxin A, tumour necrosis factor-cl, interleukin-6, transforming growth factor-beta, inducible nitric oxide synthase and Foxp3. In addition, a large number of genes and expressed sequence tags previously unrelated to DNA-therapy were identified. All these findings were well correlated with the histopathological lesions presented in the lungs. Conclusions The effects of DNA therapy are reflected in gene expression modulation; therefore, the genes identified as differentially expressed could be considered as transcriptional biomarkers of DNAhsp65 immunotherapy against TB. The data have important implications for achieving a better understanding of gene-based therapies. Copyright (C) 2008 John Wiley & Sons, Ltd.

Effect of growth hormone on in vitro osteogenesis and gene expression of human osteoblastic cells is donor-age-dependent

it has been demonstrated that the effect of GH on bone tissue is reduced with aging. In this study we tested the hypothesis that the action of GH on osteoblastic cells is donor-age-dependent by investigating the effect of GH on the development of osteoblastic phenotype in cultures of cells from adolescents (13-16 years old), young adults (18-35 years old), and adults (36-49 years old). Osteoblastic cells derived from human alveolar bone were cultured with or without GH for periods of up to 21 days, and parameters of in vitro osteogenesis and gene expression of osteoblastic markers were evaluated. GH increased culture growth, collagen content and alkaline phosphatase (ALP) activity in cultures from adolescents and young adults, whereas non-significant effect was observed in cultures from adults. While GH significantly increased the bone-like formation in cultures from adolescents, a slightly effect was observed in cultures from young adults and no alteration was detected in cultures from adults. Results from real-time PCR demonstrated that GH upregulated ALP, osteocalcin, type I collagen, and Cbfa1 mRNA levels in cultures from adolescents. In addition, cultures from young adults showed higher ALP mRNA expression and the expression of all evaluated genes was not affected by GH in cultures from adults. These results indicate that the GH effect on both in vitro osteogenesis and gene expression of osteoblastic markers is donor-age-dependent, being more pronounced on cultures from adolescents.

Patch dynamics and metapopulation theory: the case of successional species

We present a mathematical framework that combines extinction-colonization dynamics with the dynamics of patch succession. We draw an analogy between the epidemiological categorization of individuals (infected, susceptible, latent and resistant) and the patch structure of a spatially heterogeneous landscape (occupied-suitable, empty-suitable, occupied-unsuitable and empty-unsuitable). This approach allows one to consider life-history attributes that influence persistence in patchy environments (e.g., longevity, colonization ability) in concert with extrinsic processes (e.g., disturbances, succession) that lead to spatial heterogeneity in patch suitability. It also allows the incorporation of seed banks and other dormant life forms, thus broadening patch occupancy dynamics to include sink habitats. We use the model to investigate how equilibrium patch occupancy is influenced by four critical parameters: colonization rate? extinction rate, disturbance frequency and the rate of habitat succession. This analysis leads to general predictions about how the temporal scaling of patch succession and extinction-colonization dynamics influences long-term persistence. We apply the model to herbaceous, early-successional species that inhabit open patches created by periodic disturbances. We predict the minimum disturbance frequency required far viable management of such species in the Florida scrub ecosystem. (C) 2001 Academic Press.

Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Glyt-1 expression in cultured human Muller cells and intact retinae

In this study, we demonstrate that Muller cells cultured from human retinas are capable of strongly expressing the glycine transporter Glyt-1 as assessed by immunocytochemistry. By contrast, intact normal and pathological human retinas exhibit Glyt-1 immunoreactivity only in neurons. These data suggest that Glyt-1 expression in cultured Muller cells is an epiphenomenon associated with culturing in vitro, rather than a normal physiological or even pathophysiological phenomenon in vivo. (C) 2001 Wiley-Liss, Inc.

Growth hormone (GH)/GH receptor expression and GH-mediated effects during early bovine embryogenesis

Pituitary growth hormone (GH) stimulates postnatal growth and metabolism. The role of CH and its receptor (GHR) during prenatal development, however, is still controversial. As shown by reverse transcription polymerase chain reaction (RT-PCR), bovine in vitro fertilization embryos synthesized the transcript of GHR from Day 2 of embryonic life onwards. Real time RT-PCR revealed that synthesis of GHR mRNA was increased 5.9-fold in 6-day-old embryos compared with 2-day-old embryos. Using in situ hybridization, the mRNA encoding GHR was predominantly localized to the inner cell mass of blastocysts. The GHR protein was first visualized 3 days after fertilization. GH-specific transcripts were first detected in embryos on Day 8 of in vitro culture. As shown by transmission electron microscopy, GH treatment resulted in elimination of glycogen storage in 6- to 8-day-old embryos and an increase in exocytosis of lipid vesicles. These results suggest that a functional GHR able to modulate carbohydrate and lipid metabolism is synthesized during preimplantation development of the bovine embryo and that this GHR may be subject to activation by embryonic GH after Day 8.

Tissue-specific induction of SOCS gene expression by PRL

The mechanisms whereby tissue sensitivity to PRL is controlled are not well understood. Here we report that expression of mRNA and protein for members of the SOCS/CIS/JAB family of cytokine signaling inhibitors is increased by PRL administration in ovary and adrenal gland of the lactating rat deprived of circulating PRL and pups for 24 h but not in mammary gland. Moreover, suckling increases SOCS mRNA in the ovary but not in the mammary gland of pup-deprived rats. Deprivation of PRL and pups for 48 h allows the mammary gland to induce SOCS genes in response to PRL administration, and this is associated with a decrease in basal SOCS-3 mRNA and protein expression to the level seen in other tissues, suggesting that SOCS-3 induced refractoriness related to filling of the gland. In reporter assays, SOCS-1, SOCS-3, and CIS, but not SOCS-2, are able to inhibit transactivation of the STAT 5-responsive beta -lactoglobulin promoter in transient transfection assays. Moreover, suckling results in loss of ovarian and adrenal responsiveness to PRL administered 2 h after commencement of suckling, as determined by STAT 5 gel shift assay. Immunohistochemistry was used to localize the cellular sites of SOCS-3 and CIS protein expression in the ovary and adrenal gland. We propose that induced SOCS-1, SOCS-3, and CIS are actively involved in the cellular inhibitory feedback response to physiological PRL surges in the corpus luteum and adrenal cortex during lactation, but after pup withdrawal, the mammary gland is rendered unresponsive to PRL by increased levels of SOCS-3.

Expression and function of colonic epithelial K(v)LQT1 K+ channels

1. K(V)LQT1 (KCNQ1) is a voltage-gated K+ channel essential for repolarization of the heart action potential Defects in ion channels have been demonstrated in cardiac arrhythmia. This channel is inhibited potently by the chromanol 293B, The same compound has been shown to block cAMP-dependent electrolyte secretion in rat and human colon, Therefore, it was suggested that a K+ channel similar to K(V)LQT1 is expressed in the colonic epithelium. 2, In the present paper, expression of K(V)LQT1 and its function in colonic epithelial cells is described. Reverse transcription-polymerase chain reaction analysis of rat colonic mucosa demonstrated expression of K(V)LQT1 in both crypt cells and surface epithelium. When expressed in Xenopus oocytes, K(V)LQT1 induced a typical delayed activated K+ current. 3, As demonstrated, the channel activity could be further activated by increases in intracellular cAMP. These and other data support the concept that K(V)LQT1 is forming a component of the basolateral cAMP-activated Kf conductance in the colonic epithelium.

The expression of plasminogen activator system in a rat model of periodontal wound healing

Background: The plasminogen activator system has been proposed to play a role in proteolytic degradation of extracellular matrices in tissue remodeling, including wound healing. The aim of this study was to elucidate the presence of components of the plasminogen activator system during different stages of periodontal wound healing. Methods: Periodontal wounds were created around the molars of adult rats and healing was followed for 28 days. Immunohistochemical analyses of the healing tissues and an analysis of the periodontal wound healing fluid by ELISA were carried out for the detection of tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (u-PA), and 2 plasminogen activator inhibitors (PAI-1 and PAI-2). Results: During the early stages (days 1 to 3) of periodontal wound healing, PAI-1 and PAI-2 were found to be closely associated with the deposition of a fibrin clot in the gingival sulcus. These components were strongly associated with the infiltrating inflammatory cells around the fibrin clot. During days 3 to 7, u-PA, PAI-1, and PAI-2 were associated with cells (particularly monocytes/macrophages, fibroblasts, and endothelial cells) in the newly formed granulation tissue. During days 7 to 14, a new attachment apparatus was formed during which PAI-1, PAI-2, and u-PA were localized in both periodontal ligament fibroblasts (PDL) and epithelial cells at sites where these cells were attaching to the root surface. In the periodontal wound healing fluid, the concentration for t-PA increased and peaked during the first week. PAI-2 had a similar expression to t-PA, but at a lower level over the entire wound-healing period. Conclusions: These findings indicate that the plasminogen activator system is involved in the entire process of periodontal wound healing, in particular with the formation of fibrin matrix on the root surface and its replacement by granulation tissue, as well as the subsequent formation of the attachment of soft tissue to the root surface during the later stages of wound repair.

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear H-1-N-15 NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta -strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. N-15 longitudinal and transverse relaxation rates, and {H-1}-N-15 heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85 +/- 0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides. (C) 2001 Academic Press.

Glucose induced IEG expression in the thiamin-deficient rat brain

Glucose loading of rats made thiamin deficient by dietary deprivation of thiamin and the administration of pyrithiamin (40 mug/100 g, i.p.) precipitates an acute neuropathy, a model of Wernicke's encephalopathy in man (Zimitat and Nixon, Metab. Brain Dis. 1999;14:1-20). Immunohistochemical detection of Fos proteins was used as a marker to identify neuronal populations in the thiamin-deficient rat brain affected by glucose loading. As thiamin deficiency progressed, the extent and intensity of Fos-Like immunoreactivity (FLI) in brain structures typically affected by thiamin deficiency (the thalamus, mammillary bodies, inferior colliculus, vestibular nucleus and inferior olives) were markedly increased when compared to thiamin-replete controls. Glucose loading for 1-3 days further increased the intensity of FLI in these same regions, consistent with a dependence of Fos expression on carbohydrate metabolism as well as on thiamin deficiency. The timed acute changes that follow a bolus glucose load administered to thiamin-deficient animals may provide a sequential account of events in the pathogenesis of brain damage in this model of Wernicke's encephalopathy. (C) 2001 Elsevier Science B.V. All rights reserved.

Enzymology, structure, and dynamics of acetohydroxy acid isomeroreductase

Acetohydroxy acid isomeroreductase is a key enzyme involved in the biosynthetic pathway of the amino acids isoleucine, valine, and leucine. This enzyme is of great interest in agrochemical research because it is present only in plants and microorganisms, making it a potential target for specific herbicides and fungicides. Moreover, it catalyzes an unusual two-step reaction that is of great fundamental interest. With a view to characterizing both the mechanism of inhibition by potential herbicides and the complex reaction mechanism, various techniques of enzymology, molecular biology, mass spectrometry, X-ray crystallography, and theoretical simulation have been used. The results and conclusions of these studies are described briefly in this paper.