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Characterization of the Hox gene cluster in the malaria vector mosquito, Anopheles gambiae.

The Hox genes play a central role in regulating development and are involved in the specification of cell fates along the anteroposterior axis. In insects and vertebrates, these genes are clustered and organized in an arrangement that is largely conserved across evolutionary lineages. By exploiting the sequence conservation of the homeobox, orthologues of the Hox genes Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), and abdominal-A (abd-A) have been isolated from the malaria vector mosquito, Anopheles gambiae. These genes were first identified in Drosophila, where they achieve a high level of functional complexity, in part, by the use of alternative promoters, polyadenylation sites, and splicing to generate different protein isoforms. Preliminary analyses of the Anopheles Hox genes suggest that they do not achieve their functional complexity in the same manner. Using a combination of in situ hybridization to polytene chromosomes and chromosome walking, the Anopheles Hox genes have been localized to a single cluster in the region 19D-E on chromosome 2R, a situation distinct from that of Drosophila where the Hox complex is split into two clusters. This study, therefore, provides a framework for future comparative analyses of the structure, organization, and expression of developmental regulatory genes between the lower and higher Diptera. Moreover, the genes that have been isolated enhance the genetic and physical maps of chromosome 2R in this medically important mosquito species.

Amino Acid Sequence↗

Identification of a murine TEF-1-related gene expressed after mitogenic stimulation of quiescent fibroblasts and during myogenic differentiation.

Fibroblast growth factor (FGF)-1 binding to cell surface receptors stimulates an intracellular signaling pathway that ultimately promotes the transcriptional activation of specific genes. We have used a mRNA differential display method to identify FGF-1-inducible genes in mouse NIH 3T3 fibroblasts. Here, we report that one of these genes, FGF-regulated (FR)-19, is predicted to encode a member of the transcriptional enhancer factor (TEF)-1 family of structurally related DNA-binding proteins. Specifically, the deduced FR-19 amino acid sequence has approximately89, 77, and 68% overall identity to chicken TEF-1A, mouse TEF-1, and mouse embryonic TEA domain-containing factor, respectively. Gel mobility shift experiments indicate that FR-19, like TEF-1, can bind the GT-IIC motif found in the SV40 enhancer. The FR-19 gene maps in the distal region of mouse chromosome 6, and analysis of several FR-19 cDNA clones indicates that at least two FR-19 isoforms may be expressed from this locus. FGF-1 induction of FR-19 mRNA expression in mouse fibroblasts is first detectable at 4 h after FGF-1 addition and is dependent on de novo RNA and protein synthesis. FGF-2, calf serum, platelet-derived growth factor-BB, and phorbol 12-myristate 13-acetate can also induce FR-19 mRNA levels. We have also found that FR-19 mRNA expression increases during mouse C2C12 myoblast differentiation in vitro. The FR-19 gene is expressed in vivo in a tissue-specific manner, with a relatively high level detected in lung. These results indicate that increased expression of a TEF-1-related protein may be important for both mitogen-stimulated fibroblast proliferation and skeletal muscle cell differentiation.

3T3 Cells↗

Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton.

Expression of connective tissue growth factor (CTGF) was induced in renal mesangial cells by activation of heptahelical receptors by serotonin (5-HT) and lysophosphatidic acid (LPA). Induction of CTGF mRNA was transient with maximal expression after 1 to 2 h, whereas induction of CTGF by transforming growth factor beta (TGF-beta) increased over time. In contrast to the induction of other early response genes (Egr-1 and cyclooxygenase-2), LPA-mediated induction of CTGF was pertussis toxin-insensitive and independent of p42/44 MAP kinase activation. 5-HT-mediated CTGF induction was due to activation of 5-HT(2A) receptors and likewise independent of p42/44 MAP kinase activation. Upon stimulation, enhanced levels of CTGF protein were detected in cellular homogenates, whereas no protein was detectable in cell culture supernatants. Inhibition of proteins of the Rho family by toxin B abrogated basal as well as CTGF expression stimulated by LPA, 5-HT, and TGF-beta. Inhibition of the downstream mediator of RhoA, the Rho kinase by Y-27632 partially reduced induction of CTGF by LPA and TGF-beta. Toxin B not only affected gene expression, but disrupted the actin cytoskeleton similarly as observed after treatment with cytochalasin D. Disassembly of actin stress fibers by cytochalasin D partially reduced basal and stimulated CTGF expression. These data indicate that an intact actin cytoskeleton is critical for the expression of CTGF. Elimination of the input of Rho proteins by toxin B, however, was significantly more effective and their effect on CTGF expression thus goes beyond disruption of the cytoskeleton. These findings thus establish activation of heptahelical receptors coupled to pertussis toxin-insensitive G proteins as a novel signaling pathway to induce CTGF. Proteins of the Rho family and an intact cytoskeleton were identified as critical determinants of CTGF expression induced by LPA and 5-HT, and also by TGF-beta.

Actins↗

Genetic and molecular analysis of wings apart-like (wapl), a gene controlling heterochromatin organization in Drosophila melanogaster.

Mutations in the X-linked gene wings apart-like (wapl) result in late larval lethality associated with an unusual chromosome morphology. In brain cell metaphases of wapl mutants, sister chromatids of all chromosomes are aligned parallel to each other instead of assuming the typical morphology observed in wild type. This effect is due to a loosening of the adhesion between sister chromatids in the heterochromatic regions of the chromosomes. Despite this aberrant chromosome morphology, mutant brains exhibit normal mitotic parameters, suggesting that heterochromatin cohesion is not essential for proper centromere function. On the basis of these observations, we examined the role of wapl in meiotic chromosome segregation in females. wapl exhibits a clear dominant effect on achiasmate segregation, giving further support to the hypothesis that proximal heterochromatin is involved in chromosome pairing during female meiosis. We also examined whether wapl modulates position-effect variegation (PEV). Our analyses showed that wapl is a dominant suppressor of both white and Stubble variegation, while it is a weak enhancer of brown variegation. wapl maps to region 2D of the X chromosome between Pgd and pn. We identified the wapl gene within a previously conducted chromosomal walk in this region. The wapl transcriptional unit gives rise to two alternatively spliced transcripts 6.5- and 5-kb long. The protein encoded by the larger of these transcripts appears to be conserved among higher eukaryotes and contains a tract of acidic amino acids reminiscent of many chromatin-associated proteins, including two [HP1 and SU(VAR)3-7] encoded by other genes that act as suppressors of PEV.

Animals↗

Transient periodicity and episodic predictability in biological dynamics.

Biological time series often contain passing episodes of nearly periodic dynamics. In chaotic systems, such transient periodicity can reflect the existence of semiperiodic saddles--nonstable invariant sets--contained in the attractor. Motion in the vicinity of such objects has a prominent periodic component. In addition, trajectories can become temporarily trapped in these neighbourhoods before exiting. The immediate dynamical antecedents (low-order preimages) of transient periodicity are well defined and, along with the saddles to which they map, correspond to regions of enhanced predictability under nonlinear forecasting. This suggests that it may be possible to forecast the onset of transient periodicity in systems for which overall predictability is low. The present paper reviews these concepts and applies them to biological phenomena at different levels of organization.

Child↗

Cytokinin receptors are required for normal development of auxin-transporting vascular tissues in the hypocotyl but not in adventitious roots.

Plants alter the architecture of their root systems to adapt to the environment by modulating post-embryonic (lateral and adventitious) root formation and growth. To understand better the genetic basis of this regulation, we screened ethylmethane sulfonate-mutagenized lines of Arabidopsis thaliana for adventitious rooting mutants. One mutant showed retardation of the primary root growth, no production of lateral roots and enhanced formation of adventitious roots. Mapping and genetic complementation revealed that this mutant named wooden leg-3 (wol-3) was an allele of ARABIDOPSIS HISTIDINE KINASE 4 (AHK4), a locus known to encode a cytokinin receptor. Although the vascular system of the primary root and hypocotyl in the wol-3 mutant was aborted, that of the adventitious roots was normally developed. In the hypocotyl of the wol-3 mutant, auxin signals accumulated around the aborted vascular system. The application of auxin to primary roots induced lateral root formation in the wol-3 mutant. Transport of radiolabeled auxin from the top of the hypocotyl to the primary root was inhibited in wol-3. Although only a single amino acid alteration had occurred in AHK4, the root morphology in the wol-3 mutant was quite similar to that in the ahk2 ahk3 ahk4 triple mutant, which is a loss-of-function mutant of the three cytokinin receptors. This implies that the functional disturbance of AHK4 affects the function of the other receptors. Our results suggest that cytokinin receptors are necessary for the formation of auxin-transporting vascular tissues in the hypocotyl, but not in adventitious roots.

Alleles↗

The Xenopus noggin promoter drives roof-plate specific transcription.

During vertebrate neural development, inductive signals derived from the roof plate specify dorsal neural cell types of spinal cord as well as adjacent dermomytotome. Noggin has been demonstrated to play a pivotal role in this signaling network but how its expression is regulated remains unclear. To investigate this issue, we identified a 2066 bp Xenopus noggin 5' flanking sequence which recapitulates the roof-plate expression of endogenous gene in transgenic frog tadpoles and we further mapped the roof-plate enhancer to a sequence as short as 79 bp. Our results, therefore, not only shed light on but also provide a powerful tool to the study of neural and somite patterning.

Animals↗

Zero-dose extrapolation as part of macromolecular synchrotron data reduction.

Radiation damage to macromolecular crystals at third-generation synchrotron sites constitutes a major source of systematic error in X-ray data collection. Here, a computational method to partially correct the observed intensities during data reduction is described and investigated. The method consists of a redundancy-based zero-dose extrapolation of a decay function that is fitted to the intensities of all observations of a unique reflection as a function of dose. It is shown in a test case with weak anomalous signal that this conceptually simple correction, when applied to each unique reflection, can significantly improve the accuracy of averaged intensities and single-wavelength anomalous dispersion phases and leads to enhanced experimental electron-density maps. Limitations of and possible improvements to the method are discussed.

Algorithms↗

Molecular mechanisms of pancreatic carcinogenesis.

Pancreatic ductal adenocarcinoma is one of the most fatal malignancies. Intensive investigation of molecular pathogenesis might lead to identifying useful molecules for diagnosis and treatment of the disease. Pancreatic ductal adenocarcinoma harbors complicated aberrations of alleles including losses of 1p, 6q, 9p, 12q, 17p, 18q, and 21q, and gains of 8q and 20q. Pancreatic cancer is usually initiated by mutation of KRAS and aberrant expression of SHH. Overexpression of AURKA mapping on 20q13.2 may significantly enhance overt tumorigenesity. Aberrations of tumor suppressor genes synergistically accelerate progression of the carcinogenic pathway through pancreatic intraepithelial neoplasia (PanIN) to invasive ductal adenocarcinoma. Abrogation of CDKN2A occurs in low-grade/early PanIN, whereas aberrations of TP53 and SMAD4 occur in high-grade/late PanIN. SMAD4 may play suppressive roles in tumorigenesis by inhibition of angiogenesis. Loss of 18q precedes SMAD4 inactivation, and restoration of chromosome 18 in pancreatic cancer cells results in tumor suppressive phenotypes regardless of SMAD4 status, indicating the possible existence of a tumor suppressor gene(s) other than SMAD4 on 18q. DUSP6 at 12q21-q22 is frequently abrogated by loss of expression in invasive ductal adenocarcinomas despite fairly preserved expression in PanIN, which suggests that DUSP6 works as a tumor suppressor in pancreatic carcinogenesis. Restoration of chromosome 12 also suppresses growths of pancreatic cancer cells despite the recovery of expression of DUSP6; the existence of yet another tumor suppressor gene on 12q is strongly suggested. Understanding the molecular mechanisms of pancreatic carcinogenesis will likely provide novel clues for preventing, detecting, and ultimately curing this life-threatening disease.

Animals↗

The gene for enhancer binding proteins E12/E47 lies at the t(1;19) breakpoint in acute leukemias.

The gene (E2A) that codes for proteins with the properties of immunoglobulin enhancer binding factors E12/E47 was mapped to chromosome region 19p13.2-p13.3, a site associated with nonrandom translocations in acute lymphoblastic leukemias. The majority of t(1;19)(q23;p13)-carrying leukemias and cell lines studied contained rearrangements of E2A as determined by DNA blot analyses. The rearrangements altered the E2A transcriptional unit, resulting in the synthesis of a transcript larger than the normal-sized E2A mRNAs in one of the cell lines with this translocation. These observations indicate that the gene for a transcription factor is located at the breakpoint of a consistently recurring chromosomal translocation in many acute leukemias and suggest a direct role for alteration of such factors in the pathogenesis of some malignancies.

Child↗

Direct transcriptional repression by pRB and its reversal by specific cyclins.

It was recently shown that the E2F-pRB complex is a negative transcriptional regulator. However, it was not determined whether the whole complex or pRB alone is required for repression. Here we show that pRB and the related protein p107 are capable of direct transcriptional repression independent of E2F. When fused to the DNA binding domain of GAL4, pRB or p107 represses transcription of promoters with GAL4 binding sites. Thus, E2F acts as a tether for pRB or p107 but is not actively involved in repression of other enhancers. This function of pRB maps to the pocket and is abrogated by mutation of this domain. This result suggests an intriguing model in which the pocket has a dual function, first to bind E2F and second to repress transcription directly, possibly through interaction with other proteins. We also show that direct transcriptional repression by pRB is regulated by phosphorylation. Mutations which render pRB constitutively hypophosphorylated potentiate repression, while phosphorylation induced by cyclin A or E reduces repression ninefold.

Base Sequence↗

Microcomputer stereotactic atlas.

A stereotactic atlas to determine thalamic target sites has been incorporated into a microcomputer. Variability studies of the thalamus with mean and standard deviations of nuclear borders are depicted graphically for overlay onto operative images. Internal landmarks traditionally used to reference target points for functional stereotaxis may be determined by conventional ventriculography or derived from magnetic resonance scans. Modeling of polyline vertices established from gray scale contour mapping and atlas reconstructions further enhance the spatial understanding of relationships to midline structures. Computer integration of anatomic reference points, graphically depicted images and stereotactic atlas data into head frame coordinates can be accomplished. This method is consistent with established stereotactic techniques and allows the visual conceptualization of imaged and graphic data for functional stereotaxis.

Brain↗

Defining the human macula transcriptome and candidate retinal disease genes using EyeSAGE.

PURPOSE: To develop large-scale, high-throughput annotation of the human macula transcriptome and to identify and prioritize candidate genes for inherited retinal dystrophies, based on ocular-expression profiles using serial analysis of gene expression (SAGE). METHODS: Two human retina and two retinal pigment epithelium (RPE)/choroid SAGE libraries made from matched macula or midperipheral retina and adjacent RPE/choroid of morphologically normal 28- to 66-year-old donors and a human central retina longSAGE library made from 41- to 66-year-old donors were generated. Their transcription profiles were entered into a relational database, EyeSAGE, including microarray expression profiles of retina and publicly available normal human tissue SAGE libraries. EyeSAGE was used to identify retina- and RPE-specific and -associated genes, and candidate genes for retina and RPE disease loci. Differential and/or cell-type specific expression was validated by quantitative and single-cell RT-PCR. RESULTS: Cone photoreceptor-associated gene expression was elevated in the macula transcription profiles. Analysis of the longSAGE retina tags enhanced tag-to-gene mapping and revealed alternatively spliced genes. Analysis of candidate gene expression tables for the identified Bardet-Biedl syndrome disease gene (BBS5) in the BBS5 disease region table yielded BBS5 as the top candidate. Compelling candidates for inherited retina diseases were identified. CONCLUSIONS: The EyeSAGE database, combining three different gene-profiling platforms including the authors' multidonor-derived retina/RPE SAGE libraries and existing single-donor retina/RPE libraries, is a powerful resource for definition of the retina and RPE transcriptomes. It can be used to identify retina-specific genes, including alternatively spliced transcripts and to prioritize candidate genes within mapped retinal disease regions.

Adult↗

Allergens.

What makes an antigen an allergen? This question has been at the core of much research regarding the basic mechanisms of allergy as well as the development of diagnostic and therapeutic allergen products. Great strides have been made since Kjell Aas asked this question in his classic review in Allergy (1), but we appear no closer to the answer. The mechanisms leading to allergic reactions have been well described in recent years and our understanding of it at the molecular level has greatly improved. Conversely, the molecular structures responsible for these allergic reactions are not as well understood. Techniques for the isolation, identification, and epitope mapping of allergens have greatly enhanced our ability to test old and new ideas about what makes an antigen an allergen. We continue to search for "major allergens," and when we identify them, we hope to delineate "allergenic determinants" and "immunodominant epitopes." Even now, unique structural motifs or determinants responsible for allergenicity have not yet been identified, and we will probably never find them.

Allergens↗

Protective effects of apocynin on "two-hit" injury induced by hemorrhagic shock and lipopolysaccharide.

AIM: To evaluate the protective effects of apocynin on "two-hit" injury in rats. METHODS: "Two-hit" injury model of rat was induced by hemorrhagic shock (40 mmHg for 45 min) followed by iv administration of lipopolysaccharide (LPS, 150 microg/kg). Rats were randomized into seven groups: Sham, LPS, hemorrhage, hemorrhage/LPS, and hemorrhage/LPS+apocynin (2.5, 5.0, and 10.0 mg/kg). Apocynin was dissolved in the resuscitation fluid (normal saline, NS) and administered iv for 2 h. After LPS or NS administration, the survival rates at 8 h, 16 h, 24 h, and 48 h were monitored. The content of malondialdehyde (MDA) was measured in lung at 3 h and 6 h after iv LPS and in serum before hemorrhage, after hemorrhage, and at 0, 0.5, 1, 2, 4, and 6 h after iv LPS. Myeloperoxidase (MPO) activity in lung and liver was examined at 3 h and 6 h after iv LPS/NS. RESULTS: After "two-hit" injury, the survival rates of rats at 8 h, 16 h, 24 h, and 48 h were 64.3 %, 35.7 %, 28.6 %, and 14.3 % respectively, there were significant differences as compared to sham group (P<0.05 or P<0.01, respectively), the MDA level in lung and serum were significantly enhanced (P<0.01) as compared to sham group, and MPO activity in lung and liver after "two-hit" injury was also significantly increased (P<0.01). Apocynin treatment enhanced the mean arterial pressure (MAP) of hemorrhagic shock rats dose-dependently (P<0.05), increased the survival rate of "two-hit" injury rats, decreased the serum and lung MDA content, and downregulated MPO activity in lung and liver. CONCLUSION: Apocynin could preventively ameliorate "two-hit" injury in rats induced by hemorrhagic shock and LPS insult.

Acetophenones↗

Do cerebral blood volume and contrast transfer coefficient predict prognosis in human glioma?

INTRODUCTION: Noninvasive measurements of cerebral blood volume (CBV) and contrast transfer coefficient (K(trans)) have potential benefits in the diagnosis and therapeutic management of adult glioma. This study examines the relationship between CBV, K(trans), and overall survival. METHODS AND MATERIALS: Twenty-seven adult patients with glioma underwent T1-weighted dynamic contrast-enhanced MR imaging, and parametric maps of CBV and K(trans) were calculated. The relationship of histologic grade, CBV, K(trans), age, sex, surgical resection, and use of adjuvant therapy to survival were analyzed by using the logrank method and Cox regression analysis. The Kaplan-Meier method for displaying survival curves was used. The relationship of factors such as comorbidity, elevated intracranial pressure, size of nonenhancing tumor, and peritumoral edema were not considered. RESULTS: Both CBV (P < .01) and K(trans) (P < .01) show a significant relationship to histologic grade. CBV (P = .004), K(trans) (P = .008), and histologic grade (P < .001) all demonstrate a significant association with patient survival when analyzed individually. Cox regression analysis identified only histologic grade (P < .01) and K(trans) (P < .05) as independent significant prognostic indicators. Examination of survival data from high-grade (III and IV) tumors demonstrated a linear relationship between K(trans) and patient survival (P < .01). CONCLUSION: This study suggests a direct relationship between K(trans) and length of survival in high-grade gliomas, which could be of clinical importance. CBV relates directly to histologic grade but provides no independent prognostic information over and above that provided by grade. Further large prospective studies should be planned to test whether this observation holds true.

Adult↗

Beneficial effects of tetramethylpyrazine, an active constituent of Chinese herbs, on rats with endotoxemia.

Tetramethylpyrazine, an inhibitor of phosphodiesterase, has been widely used for treatment of cardiovascular diseases in China. Here, we investigate the effects of tetramethylpyrazine on hypotension, vascular hyporeactivity to norepinephrine (NE), release of tumor necrosis factor-alpha (TNF alpha) and nitric oxide (NO) in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide, LPS). Male Wistar-Kyoto rats were anesthetized and instrumented for the measurement of mean arterial pressure (MAP) and heart rate (HR). Injection of LPS (10 mg/kg, i.v.) resulted in a fall in MAP and an increase of HR. In contrast, animals pretreated with tetramethylpyrazine (10 micrograms/kg, i.p. at 30 min prior to LPS) maintained a significantly higher MAP, but tachycardia was further enhanced at 60 min and 120 min when compared to rats given only LPS (LPS-rats). The pressor effect of NE (1 microgram/kg, i.v.) was also significantly reduced after treatment of rats with LPS. Similarly, the thoracic aorta obtained from rats after in vivo studies showed a significant reduction in the contractile responses elicited by NE (1 microM). Pretreatment of LPS-rats with tetramethylpyrazine partially, but significantly, prevented this LPS-induced hyporeactivity to NE in vivo and ex vivo. The injection of LPS resulted in a significant increase in the plasma TNF alpha level at 60 min, whereas the effect of LPS on the plasma nitrate (an indicator of NO formation) level increased in a time-dependent manner. This increment of both TNF alpha and nitrate levels induced by LPS was significantly reduced in LPS-rats pretreated with tetramethylpyrazine. The early hypotension caused by LPS was slightly, but significantly, prevented by pretreatment with tetramethylpyrazine, suggesting that tetramethylpyrazine affects the endothelial constitutive NOS (eNOS). This was examined by the effect of tetramethylpyrazine on acetylcholine (ACh, 1 microM)-induced relaxation in rats treated with tetramethylpyrazine for 4 h. However, tetramethylpyrazine had no significant effects on the ACh-induced relaxation, indicating that tetramethylpyrazine does not affect the activity of eNOS. Thus, tetramethylpyrazine attenuates the early hypotension and the delayed circulatory failure caused by endotoxin in the rat. These effects may be due to inhibition of the release of circulation factors and TNF alpha, which usually reveal synergism upon the induction of iNOS.

Animals↗

Strain differences in the reverse tolerance to methamphetamine and changes in catecholaminergic neurons in mice.

The effect of methamphetamine (MAP) on ambulatory activity and neurochemical changes in catecholaminergic neurons in the brain were investigated in dd and C57BL/6 strains of male mice. The mice were given repeatedly MAP at 2 mg/kg, s.c., 10 times at a fixed interval of 4 days. Single MAP markedly increased the ambulatory activity in both strains of mice. The ambulation-increasing effect was progressively enhanced without accompanying stereotyped behaviors when the drug was repeatedly given. The dd mice showed higher susceptibility not only to single MAP but also to the enhancing effect of the drug (reverse tolerance) that the C57BL/6 mice. On the other hand, non-treated dd mice exhibited lower maximum densities of 3H-spiperone binding sites in the striatum and 3H-WB4101 binding sites in the cortex and hippocampus than non-treated C57BL/6 mice. In contrast, the dd mice exhibited higher noradrenaline turnover than the C57BL/6 mice in the brain regions examined. The repeated administration of MAP produced decrease in the densities of both 3H-spiperone and 3H-WB4101 binding sites in the corresponding regions with increase in catecholamine turnover in dd mice. However, the similar changes were observed only in 3H-WB4101 binding sites and noradrenaline turnover in C57BL/6 mice. These results suggest that the ambulation-increasing effect of MAP is positively correlated with catecholamine turnover, while it was correlated negatively with the densities of catecholamine binding sites. Furthermore, the enhancing effect of MAP is supposed to have been partially elicited by changes in brain catecholaminergic systems, in particular an increase in catecholamine turnover.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗