bluuebunny/crossref_metadata_2025 huggingface dataset:
Beta 3-adrenergic receptor stimulation restores message and expression of brown-fat mitochondrial uncoupling protein in adult dogs.
Authors: Champigny O , Ricquier D , Blondel O , Mayers R M , Briscoe M G , Holloway B R
Published: May 2006

Brown adipose tissue (BAT) is present throughout life in rodents and plays an important role in energy balance. However, whereas BAT is clearly recognizable in the neonates of larger mammals (including dogs, cats, sheep, cattle, and humans), it is undetectable or present in only small quantities in adults of these species and is replaced by a tissue with the gross characteristics of white adipose tissue. Here we provide evidence that treatment of adult dogs with a beta 3-adrenergic receptor agonist (ICI D7114) that has thermogenic and antiobesity properties leads to the appearance of BAT at several anatomical sites. The presence of BAT was primarily demonstrated by monitoring the inner mitochondrial membrane uncoupling protein and its mRNA, which are unique to the tissue. Neither message nor protein was detected in adipose tissue samples from control dogs but both were detected in samples from dogs treated with ICI D7114. The data suggest that stimulation of beta 3-adrenergic receptors can reactivate nascent BAT (which has the appearance of white adipose tissue) by increasing expression of the gene coding for uncoupling protein or lead to the recruitment of fully differentiated BAT from preadipocyte precursor cells.
Structure of the human villin gene.
Authors: Pringault E , Robine S , Louvard D
Published: May 2006

We have isolated and characterized the complete human villin gene. The villin gene is located on chromosome 2q35-36 in humans and on chromosome 1 in mice. Villin belongs to a family of calcium-regulated actin-binding proteins that share structural and functional homologies. The villin gene is expressed mainly in cells that develop a brush border, such as mucosal cells of the small and large intestine and epithelial cells of the kidney proximal tubules. Villin gene expression is strictly regulated during adult life and embryonic development in the digestive and urogenital tracts and, thus, may be used as a marker of the digestive and renal cell lineages. The human villin gene has one copy per haploid genome, encompasses about 25 kilobases, and contains 19 exons. Analysis of the structural organization of this gene shows that the two mRNAs that encode villin in humans arise by alternative choice of one of the two polyadenylylation signals located within the last exon. The overall organization of the exons reflects the gene duplication event from which this family of actin-binding proteins originated.
Evolution of behavior by density-dependent natural selection.
Authors: Guo P Z , Mueller L D , Ayala F J
Published: May 2006

Theories of density-dependent natural selection predict that evolution should favor those genotypes with the highest per capita rates of population growth under the current density conditions. These theories are silent about the mechanisms that may give rise to these increases in density-dependent growth rates. We have observed the evolution of six populations of Drosophila melanogaster recently placed in crowded environments after nearly 200 generations at low-population density in the laboratory. After 25 generations in these crowded cultures all six populations showed the predicted increase in population growth rates at high-population density with the concomitant decrease in their growth rates at low densities. These changes in rates of population growth are accompanied by changes in the feeding and pupation behavior of the larvae: those populations that have evolved at high-population densities have higher feeding rates and are less likely to pupate on or near the food surface than populations maintained at low densities. These changes in behavior serve to increase the competitive ability of larvae for limited food and reduce mortality under crowded conditions during the pupal stage of development. A detailed understanding of the mechanisms by which populations evolve under density-dependent natural selection will provide a framework for understanding the nature of trade-offs in life history evolution.
Molecular cloning of a putative plant endomembrane protein resembling vertebrate protein disulfide-isomerase and a phosphatidylinositol-specific phospholipase C.
Authors: Shorrosh B S , Dixon R A
Published: May 2006

cDNA clones containing sequence similarity to the multifunctional vertebrate protein disulfide-isomerase (PDI, EC 5.3.4.1) were isolated from an alfalfa (Medicago sativa L.) cDNA library by screening with a cDNA sequence encoding human PDI. The polypeptide encoded by a clone designated B2 consisted of 512 amino acids and was characterized by a 24-amino acid hydrophobic leader sequence, two regions with absolute identity to the vertebrate PDI active site (Ala-Pro-Trp-Cys-Gly-His-Cys-Lys), and a C-terminal endoplasmic reticulum retention signal (Lys-Asp-Glu-Leu). The overall identity of the B2 sequence to that of human PDI was 35% at the amino acid level (79% when conservative substitutions were included) and 39% at the nucleotide level; this included homology between B2 and the region of human PDI believed to be involved in binding estrogens. The deduced amino acid sequence of B2 was also 35% identical to that of a rat form I phosphatidylinositol-specific phospholipase C. Lysates from Escherichia coli cells harboring an expression plasmid bearing the B2 sequence contained significantly elevated levels of PDI activity. Southern analysis indicated the presence of a small PDI-related gene family in alfalfa, of which B2 appeared to correspond to a single gene. An approximately 2-kilobase B2 transcript was expressed in all alfalfa organs tested. In alfalfa cell suspension cultures, B2 transcripts were strongly induced by tunicamycin but not by exposure to fungal elicitor.
Demographic predisposition to the evolution of eusociality: a hierarchy of models.
Authors: Gadagkar R
Published: May 2006

I present a hierarchy of models that illustrate, within the framework of inclusive fitness theory, how demographic factors can predispose a species to the evolution of eusociality. Delayed reproductive maturation lowers the inclusive fitness of a solitary foundress relative to that of a worker. Variation in age at reproductive maturity makes the worker strategy more profitable to some individuals than to others and thus predicts the coexistence of single-foundress and multiple-foundress nesting associations. Delayed reproductive maturation and variation in age at reproductive maturity also select for mixed reproductive strategies so that some individuals whose reproductive maturation is expected to be delayed can first act as workers and later switch over to the role of a queen or foundress. Assured fitness returns shows how identical mortality rates can have different consequences for workers and solitary nest foundresses because a solitary foundress will have to necessarily survive for the entire duration of development of her brood, whereas a worker can hope to get proportional fitness returns for short periods of work. In concert with assured fitness returns, delayed reproductive maturation and variation in age at reproductive maturity become more powerful in selecting for worker behavior, and mixed reproductive strategies become available to a wider range of individuals. These phenomena provide a consistently more powerful selective advantage for the worker strategy than do genetic asymmetries created by haplodiploidy.
Rapid de novo generation of defective interfering RNA by cucumber necrosis virus mutants that do not express the 20-kDa nonstructural protein.
Authors: Rochon D M
Published: May 2006

It is generally believed that serial passage at high multiplicity of infection (moi) is required for the generation of defective interfering (DI) particles. High levels of DI RNAs are found associated with persistent infections initiated with laboratory cultures of cucumber necrosis virus (CNV). Two synthetic CNV transcripts that were derived through site-directed mutagenesis of a highly infectious CNV cDNA clone and that do not express the CNV 20-kDa nonstructural protein were found to generate high levels of symptom-attenuating DI RNAs de novo without serial high-moi passage in transcript-inoculated plants. Such de novo generation of DI RNAs did not occur in infections initiated with wild-type transcript until at least eight serial high-moi passages. The observation that a CNV nonstructural protein mutant rapidly generates DI RNA de novo may provide insight into mechanisms that underly DI particle formation in RNA viruses in general.
Mechanism of the initial charge separation in bacterial photosynthetic reaction centers.
Authors: Chan C K , DiMagno T J , Chen L X , Norris J R , Fleming G R
Published: May 2006

The initial electron transfer in reaction centers from Rhodobacter sphaeroides R26 was studied by a subpicosecond transient pump-probe technique. The measured kinetics at various wavelengths were analyzed and compared with several mechanisms for electron transfer. An unambiguous determination of the initial electron transfer mechanism in reaction centers cannot be made by studying the anion absorption region (640-690 nm), due to the spectral congestion in this region. However, correlations between the stimulated emission decay of the excited state of the special pair, P*, at 926 nm and bleaching of the bacteriopheophytin Qx absorption at 545 nm suggest that the electron transfer at 283 K is dominated by a two-step sequential mechanism, whereas one-step superexchange and the two-step sequential mechanism have about equal contributions at 22 K.
Human immunodeficiency virus glycoprotein (gp120) infused into rat brain induces interleukin 1 to elevate pituitary-adrenal activity and decrease peripheral cellular immune responses.
Authors: Sundar S K , Cierpial M A , Kamaraju L S , Long S , Hsieh S , Lorenz C , Aaron M , Ritchie J C , Weiss J M
Published: May 2006

Intracerebroventricular (i.c.v.) infusion of glycosylated recombinant gp120, the envelope protein of human immunodeficiency virus, in various doses (100 ng to 4 micrograms) resulted in detection of interleukin 1 (IL-1) activity in a high percentage (61%; 33 of 54) of rat brains, whereas IL-1 was very rarely detected in brains of animals infused with several control substances (4%; 1 of 28). To detect IL-1, clarified glial lysate of diencephalon plus brainstem was subjected to gel exclusion chromatography and fractions were assessed for thymocyte stimulation. IL-1 was seen 2, 6, and 24 hr postinfusion. i.c.v. gp120 also produced known effects of IL-1 in brain, elevating steroid concentration in plasma and decreasing cellular immune responses [natural killer (NK) cell activity and mitogenic response to Con A] of blood and splenic lymphocytes. When gp120 was infused together with alpha-melanocyte-stimulating hormone (20 ng), which blocks many biological actions of IL-1, gp120 no longer elevated steroids or decreased NK cell activity. After intravenous gp120, IL-1 was not found in brain or plasma, indicating that stimulation of IL-1 in brain by i.c.v. gp120 was not due to gp120 affecting infiltrating cells from blood or to elevated circulating IL-1. That induction of IL-1 in brain might have resulted from lipopolysaccharide (LPS) in the gp120 solution was ruled out by studies showing that (i) heating of the infusion solution, which does not affect the capacity of LPS to induce IL-1, eliminated the ability of gp120 infusion to induce brain IL-1, and (ii) gp120 induced IL-1 in brains of LPS-resistant C3H/HeJ mice. Injection of gp120 directly into the hippocampus stimulated IL-1 more readily than i.c.v. infusion. Thymocyte stimulation produced by active fractions of gp120-infused brains was blocked by monoclonal antibody to IL-1 receptors. These findings indicate that elevation of IL-1 in brain can result from infection with human immunodeficiency virus and may be responsible for certain abnormalities (e.g., elevated activity of pituitary-adrenal axis) seen in AIDS patients.
A damage-responsive DNA binding protein regulates transcription of the yeast DNA repair gene PHR1.
Authors: Sebastian J , Sancar G B
Published: May 2006

The PHR1 gene of Saccharomyces cerevisiae encodes the DNA repair enzyme photolyase. Transcription of PHR1 increases in response to treatment of cells with 254-nm radiation and chemical agents that damage DNA. We report here the identification of a damage-responsive DNA binding protein, termed photolyase regulatory protein (PRP), and its cognate binding site, termed the PHR1 upstream repression sequence, that together regulate induction of PHR1 transcription after DNA damage. PRP activity, monitored by electrophoretic-mobility-shift assay, was detected in cells during normal growth but disappeared within 30 min after irradiation. Copper-phenanthroline footprinting of PRP-DNA complexes revealed that PRP protects a 39-base-pair region of PHR1 5' flanking sequence beginning 40 base pairs upstream from the coding sequence. A prominent feature of the foot-printed region is a 22-base-pair palindrome. Deletion of the PHR1 upstream repression sequence increased the basal level expression of PHR1 in vivo and decreased induction after exposure of cells to UV radiation or methyl methanesulfonate, whereas insertion of the PRP binding site between the CYC1 upstream activation sequence and "TATA" sequence reduced basal level expression and conferred damage responsiveness upon a reporter gene. Thus these observations establish that PRP is a damage-responsive repressor of PHR1 transcription.
Differential distribution of AT1 and AT2 angiotensin II receptor subtypes in the rat brain during development.
Authors: Millan M A , Jacobowitz D M , Aguilera G , Catt K J
Published: May 2006

Angiotensin II (AII) receptor subtypes were analyzed in the brains of adult and 2-week-old rats by in vitro autoradiography with 125I-labeled [Sar1,Ile8]AII and competition studies with three AII antagonists: the nonpeptide antagonist, DuP 753, which is specific for AT1 receptors that mediate the calcium-inositol phospholipid signaling actions of AII; and nonpeptide (PD 123177) and peptide (CGP 42112A) antagonists that are selective for AT2 receptors of yet unknown function. In the adult rat brain, DuP 753 inhibited radioligand binding to the circumventricular organs and paraventricular nucleus but not to the lateral septum, subthalamic nucleus, and inferior olive. However, binding of 125I-labeled [Sar1,Ile8]AII in the latter regions was inhibited by the AT2 receptor antagonists PD 123177 and CGP 42112A. These areas showed similar displacement by the AT2 receptor subtype-specific antagonists in 2-week-old rats. In addition, radioligand binding at multiple sites of transient expression of AII receptors in 2-week-old rats, including several thalamic nuclei, the nuclei of the 3rd and 12th cranial nerves, geniculate bodies, cerebellum, and cingulate cortex, was displaced by the AT2 antagonists but not by DuP 753. These studies have demonstrated the presence of two AII receptor subtypes in the brain, one (AT1) in areas related to regulation of blood pressure, water intake, and pituitary hormone secretion, and one (AT2) whose function is not yet defined. The abundance and location of brain AT2 receptors in young animals, and the age-related changes in relative expression of the receptor subtypes, suggest that AII exerts specific actions according to the developmental stage of the central nervous system.
Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: effects of insulin, adenosine, and isoproterenol.
Authors: Nishimura H , Saltis J , Habberfield A D , Garty N B , Greenberg A S , Cushman S W , Londos C , Simpson I A
Published: May 2006

The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulin-stimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.
Genes for two calcium-dependent cell adhesion molecules have similar structures and are arranged in tandem in the chicken genome.
Authors: Sorkin B C , Gallin W J , Edelman G M , Cunningham B A
Published: May 2006

Genomic sequences immediately upstream of the translational start site for the chicken liver cell adhesion molecule (L-CAM) gene contain a second closely related gene, which, because of its location, we have designated the K-CAM gene. Less than 700 base pairs separate the presumed poly(A) site in the K-CAM gene from the translation initiation site for L-CAM. The sizes of exons 4-15 of the K-CAM gene are almost identical to those in the L-CAM gene and the exon/intron junctions occur at exactly equivalent positions in both genes. Exon 16, which includes the 3' untranslated region, is much shorter in the K-CAM gene and intron sizes and sequences are not generally conserved between the two genes. Probes from the K-CAM gene hybridized to a 3-kilobase mRNA that was present at high levels in embryonic skin, at lower levels in kidney, heart, and gizzard, and at still lower levels in brain and liver, as determined by Northern blotting. The sequence of the predicted gene product was nearly identical to that of the chicken B-cadherin cDNA, although the distribution of the K-CAM gene transcript differed from that reported for the cadherin. The proximity and identical overall structure of the K-CAM and L-CAM genes strongly suggest that they arose by gene duplication and raise the possibility that genes for other calcium-dependent CAMs may be located in clusters. Moreover, the tandem arrangement of the genes may have important implications for the regulation of their expression.
Precerebellin is a cerebellum-specific protein with similarity to the globular domain of complement C1q B chain.
Authors: Urade Y , Oberdick J , Molinar-Rode R , Morgan J I
Published: May 2006

The cerebellum contains a hexadecapeptide, termed cerebellin, that is conserved in sequence from human to chicken. Three independent, overlapping cDNA clones have been isolated from a human cerebellum cDNA library that encode the cerebellin sequence. The longest clone codes for a protein of 193 amino acids that we term precerebellin. This protein has a significant similarity (31.3% identity, 52.2% similarity) to the globular (non-collagen-like) region of the B chain of human complement component C1q. The region of relatedness extends over approximately 145 amino acids located in the carboxyl terminus of both proteins. Unlike C1q B chain, no collagen-like motifs are present in the amino-terminal regions of precerebellin. The amino terminus of precerebellin contains three possible N-linked glycosylation sites. Although hydrophobic amino acids are clustered at the amino terminus, they do not conform to the classical signal-peptide motif, and no other obvious membrane-spanning domains are predicted from the cDNA sequence. The cDNA predicts that the cerebellin peptide is flanked by Val-Arg and Glu-Pro residues. Therefore, cerebellin is not liberated from precerebellin by the classical dibasic amino acid proteolytic-cleavage mechanism seen in many neuropeptide precursors. In Northern (RNA) blots, precerebellin transcripts, with four distinct sizes (1.8, 2.3, 2.7, and 3.0 kilobases), are abundant in cerebellum. These transcripts are present at either very low or undetectable levels in other brain areas and extraneural structures. A similar pattern of cerebellin precursor transcripts are seen in rat, mouse, and human cerebellum. Furthermore, a partial genomic fragment from mouse shows the same bands in Northern blots as the human cDNA clone. During rat development, precerebellin transcripts mirror the level of cerebellin peptide. Low levels of precerebellin mRNA are seen at birth. Levels increase modestly from postpartum day 1 to 8, then increase more dramatically between day 5 and 15, and eventually reach peak values between day 21 and 56. Because cerebellin-like immunoreactivity is associated with Purkinje cell postsynaptic structures, these data raise interesting possibilities concerning the function of the cerebellin precursor in synaptic physiology.
Phosphoramidon blocks the pressor activity of porcine big endothelin-1-(1-39) in vivo and conversion of big endothelin-1-(1-39) to endothelin-1-(1-21) in vitro.
Authors: McMahon E G , Palomo M A , Moore W M , McDonald J F , Stern M K
Published: May 2006

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate called "big endothelin-1" (big ET-1) by a putative ET-converting enzyme (ECE) that cleaves the 39-mer at the bond between Trp-21 and Val-22. Since big ET-1 has only 1/100-1/150th the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of ET-1. Big ET-1 injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET-1 into ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET-1 in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were all ineffective at blocking the pressor response to big ET-1. However, the metalloprotease inhibitors phosphoramidon and thiorphan dose-dependently inhibited the pressor response to big ET-1, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on mean arterial pressure when administered alone. In a rabbit lung membrane preparation, ECE activity was identified that was blocked by the metalloprotease inhibitors phosphoramidon and 1,10-phenanthroline in a concentration-dependent manner. This enzyme converted big ET-1 to a species of ET that comigrated on HPLC with ET-1 and produced an ET-like contraction in isolated rat aortic rings. Our results suggest that the physiologically relevant ECE is a metalloprotease.
Identification of a T-cell-specific transcriptional enhancer located 3' of C gamma 1 in the murine T-cell receptor gamma locus.
Authors: Spencer D M , Hsiang Y H , Goldman J P , Raulet D H
Published: May 2006

A transcriptional enhancer element has been localized 3 kilobases 3' of the murine T-cell receptor C gamma 1 locus using a chloramphenicol acetyltransferase reporter gene construct. As a monomer the enhancer functions only in PEER gamma delta cells and Jurkat alpha beta cells of the T-cell lines tested. However, a tetramer of the enhancer functions in virtually all T-cell lines tested, including alpha beta T-cell lines, but not in other cell types. These results suggest that elements other than the enhancer are responsible for the failure of rearranged C gamma 1 genes to be expressed in alpha beta T cells. The enhancer has been localized to a 200-base-pair Rsa I restriction fragment, which contains sequence motifs similar to those found in the other T-cell receptor enhancers but not in the immunoglobulin enhancers.
Single-step selection for Ty1 element retrotransposition.
Authors: Curcio M J , Garfinkel D J
Published: May 2006

The yeast retrotransposon Ty1 has been tagged with a reporter gene that allows selection of RNA-mediated transposition events and is applicable to the study of retroelements in other organisms. The reporter gene is a yeast HIS3 gene interrupted by an artificial intron (AI) in the antisense orientation. The HIS3AI sequences were inserted into a Ty1 element such that the intron is on the sense strand of the Ty1 element; therefore, splicing and retrotransposition of marked Ty1 transcripts can give rise to His+ cells. Fusion of the Ty1-H3mHIS3AI element to the inducible GAL1 promoter resulted in a high frequency of histidine prototrophs upon galactose induction. Moreover, spontaneous His+ revertants derived from strains containing genomic TymHIS3AI elements are a result of retrotransposition. By using this assay, we estimated the Ty1 transposition rate to be between 3 x 10(-7) and 1 x 10(-5) transpositions per Ty1 element per generation. Variations in the transposition rate of individual Ty1 elements are correlated with the relative abundance of their transcripts.
Selective inhibition by a synthetic hirudin peptide of fibrin-dependent thrombosis in baboons.
Authors: Cadroy Y , Maraganore J M , Hanson S R , Harker L A
Published: May 2006

To determine the importance of the thrombin substrate recognition exosite for fibrinogen binding in the formation of both arterial and venous thrombi, we evaluated the antithrombotic effects of the tyrosine-sulfated dodecapeptide from residues 53-64 of hirudin (H peptide) in a nonhuman primate model. This peptide was studied because it inhibits thrombin cleavages of fibrinogen by simple competition without blocking enzyme catalytic-site function. When an exteriorized arteriovenous access shunt model was used in baboons (Papio anubis), thrombus formation was induced by placing a thrombogenic device made of (i) a segment of tubing coated covalently with type I collagen, which generated platelet-rich thrombi under arterial flow conditions, and (ii) two subsequent annular regions of flow expansion that produced fibrin-rich thrombi typically associated with venous valves and veins. Thrombus formation was quantified by measurements of 111In-labeled platelet and 125I-labeled fibrinogen deposition in both arterial-flow and venous-flow portions of the device. Continuous infusion of H peptide (0.5, 15, and 75 mg/kg) proximal to the device for 40 min interrupted, in a dose-response fashion, formation of fibrin-rich thrombus in the regions of disturbed flow and generation of fibrinopeptide A. In contrast, H peptide did not inhibit the capacity of platelets to deposit on the collagen surface (P greater than 0.2 at all doses) or to form hemostatic plugs (as assessed by measurements of bleeding time; P greater than 0.1 at all doses). These findings suggest that, by competitive inhibition of fibrinogen binding to thrombin, fibrin-rich venous-type thrombus formation may be selectively prevented. This strategy may be therapeutically attractive for preserving normal platelet function when conventional anticoagulant therapy is contraindicated.
Sexually dimorphic distribution of substance P in specific anterior pituitary cell populations.
Authors: Brown E R , Roth K A , Krause J E
Published: May 2006

Substance P (SP) immunoreactivity is detectable in the rat pituitary by RIA; however, immunolocalization has been difficult. We used a sensitive immunogold silver-enhancement staining technique to cytochemically locate SP in the gland. SP-immunoreactive (SP-ir) cells were seen in anterior pituitary (AP), and occasional SP-ir fibers and terminals were seen in both AP and posterior pituitary. Colocalization studies showed the vast majority of SP-ir cells in the male AP to be also immunoreactive for growth hormone (GH). These GH/SP-ir cells represent approximately 23% of the somatotroph population in the male. SP-ir cells did not colocalize with lactotrophs, gonadotrophs, or corticotrophs; however, rare thyroid-stimulating hormone/SP-ir cells were found in the male AP. Comparisons of pituitaries from males and females revealed that females have 70% fewer SP-ir cells and that only approximately 6% of the somatotrophs in the female express SP. This sexual dimorphism is diminished in 6-day ovariectomized rats because this treatment increases the GH/SP-ir cell population 3-fold. This result suggests that the previously reported estrogen-induced decrease in SP gene and peptide expression in the pituitary occurs, at least in part, in a subpopulation of somatotrophs. To test this hypothesis, distribution of SP-ir cells was examined in pituitaries from estrogen- and oil-treated ovariectomized rats. Estrogen reduced the percentage of somatotrophs with SP immunoreactivity by 70% compared with ovariectomized oil-treated controls, indicating that estrogen most likely regulates SP levels in the pituitary by acting on a subpopulation of somatotrophs to suppress SP expression. Estrogen does not appear to alter SP immunoreactivity that is detected in the additional population of SP cells that colocalize with thyroid-stimulating hormone. These SP-expressing thyrotrophs were seen 6-fold more frequently in the female than in the male pituitary, regardless of steroid status. These studies reveal that males have more total SP-ir cells in the AP than do females and that there is a sexually dimorphic pattern of SP distribution in the gland. Males have a higher percentage of SP-ir GH cells, whereas females have more SP-ir thyrotrophs than do males. Identification of independently regulated SP-ir somatotroph and thyrotroph populations provides a basis for investigating the roles of SP in autocrine or paracrine regulation of pituitary hormone secretion.
Paired MyoD-binding sites regulate myosin light chain gene expression.
Authors: Wentworth B M , Donoghue M , Engert J C , Berglund E B , Rosenthal N
Published: May 2006

The muscle-specific enhancer element located downstream of the myosin light chain (MLC) locus encoding MLC1 and MLC3 contains three binding sites (A, B, and C) for the myogenic determination factor MyoD. A 173-base-pair region of the MLC gene enhancer, including these three sites, retains full enhancer function when transfected into muscle cells. Whereas mutation of either upstream MyoD binding site (A or B) has a mild effect on muscle-specific enhancer activity, mutation of the third MyoD binding site (C) substantially weakens the enhancer, both in muscle cells or in nonmuscle cells cotransfected with a MyoD, myogenin, or myf5 expression vector. Site C is necessary but insufficient, since double mutation of two MyoD binding sites (A plus B) abrogates enhancer activity. Thus, site C requires either site A or B for enhancer function. This study shows a hierarchy of function among the three MyoD binding sites in the MLC enhancer. We propose that a protein-DNA complex is formed with at least two of these sites (A and C or B and C) to effect activation of the locus encoding MLC1/3 during myogenesis.
Clustered tRNA genes in Schizosaccharomyces pombe centromeric DNA sequence repeats.
Authors: Kuhn R M , Clarke L , Carbon J
Published: May 2006

The centromere-associated B' and B DNA sequence repeats of Schizosaccharomyces pombe chromosomes I and II have been found to contain clusters of tRNA genes. The centromere II region (cen2) includes at least 22 tRNA genes distributed among five copies of the B sequence repeat containing genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Val). Individual B repeats are variously associated with other tRNA genes, including those specifying tRNA(Lys), tRNA(Arg), and tRNA(Glu2). The centromere I region (cen1) contains at least six tRNA genes in two copies of the B' repeated element, including genes specifying tRNA(Ile), tRNA(Ala), and tRNA(Glu3). Multiple tandemly arranged clusters of tRNA genes are presumably conserved due to restricted recombination frequencies in the centromere regions.
Atypical prodynorphin gene expression in corticosteroid-producing cells of the rat adrenal gland.
Authors: Day R , Schafer M K , Collard M W , Watson S J , Akil H
Published: May 2006

Prodynorphin (proDyn) gene expression was examined in the rat adrenal gland. In situ hybridization revealed a heterogenous proDyn mRNA distribution limited almost exclusively to the adrenal cortex; the inner cortical layers contained the highest amounts. In the adrenal medulla, only scattered single cells were seen. By Northern (RNA) blot analysis, adrenocortical proDyn mRNA levels were highly abundant but of smaller size than proDyn transcripts found in the brain. Low levels of the brain-size proDyn mRNA transcript were detected but restricted to the medulla. A discrepancy was suggested when comparing the high abundance of proDyn mRNA levels with the low levels of proDyn-derived peptide in the adrenal. A hypothesis of nontranslation of the shorter proDyn mRNA by adrenocortical cells was rejected because polysomal loading analysis suggests that the mRNA is translated. We propose that adrenocortical proDyn-derived peptides are not targeted for storage but are released shortly after synthesis, thus accounting for low peptide levels. We also measured proDyn mRNA levels in response to stimuli known to affect adrenocortical cells and their most important function--steroidogenesis. Adrenals from hypophysectomized rats had less proDyn mRNA by a factor of 5 than adrenals from normal sham-operated rats. Normal levels were restored by adrenocorticotrophic hormone administration, indicating a potential importance of adrenal proDyn in the hypothalamic-adrenal-pituitary axis.
Human catechol-O-methyltransferase: cloning and expression of the membrane-associated form.
Authors: Bertocci B , Miggiano V , Da Prada M , Dembic Z , Lahm H W , Malherbe P
Published: May 2006

A cDNA clone for human catechol-O-methyltransferase (hCOMT; S-adenosyl-L-methionine:catechol O-methyltransferase; EC 2.1.1.6) was isolated from a human hepatoma cell line (Hep G2) cDNA library by hybridization screening with a porcine cDNA probe. The cDNA clone was sequenced and found to have an insert of 1226 nucleotides. The deduced primary structure of hCOMT is composed of 271 amino acid residues with the predicted molecular mass of 30 kDa. At its N terminus it has a hydrophobic segment of 21 amino acid residues that may be responsible for insertion of hCOMT into the endoplasmic reticulum membrane. The primary structure of hCOMT exhibits high homology to the porcine partial cDNA sequence (93%). The deduced amino acid sequence contains two tryptic peptide sequences (T-22, T-33) found in porcine liver catechol-O-methyltransferase (COMT). The coding region of hCOMT cDNA was placed under the control of the cytomegalovirus promoter to transfect human kidney 293 cells. The endogenous COMT activity, which was approximately 9.98 units per mg of protein in the untransfected cells, increased to 206 units per mg of protein upon transfection with a plasmid containing the COMT cDNA. The COMT activity of recombinant protein was inhibited competitively (IC50 = 700 nM) by the selective COMT inhibitor Ro 40-7592. An anti-COMT monoclonal antibody recognized, on immunoblots, a major polypeptide with apparent molecular mass of 29 kDa, in reasonable agreement with the predicted molecular mass. The recombinant hCOMT was shown by immunoblot analysis to be mainly associated with the membrane fraction. RNA blot analysis revealed one COMT mRNA transcript of 1.4 kilobases in Hep G2 poly(A)+ RNA.
Stimulation of cAMP and phosphomonoester production by melanotropin in melanoma cells: 31P NMR studies.
Authors: Degani H , DeJordy J O , Salomon Y
Published: May 2006

A major part of the present understanding of the molecular basis of signal transduction has been gained from in vitro studies using classical biochemical methods. In this study, we used 31P NMR spectroscopy to investigate the response of live M2R mouse melanoma cells to stimulation by melanocyte-stimulating hormone (MSH; melanotropin). In the presence of 3-isobutyl-1-methylxanthine and a synergistic dose of forskolin (1.67 microM), MSH induced a transient (approximately 60-min) rise in the cellular concentration of 3',5'-cyclic adenosine monophosphate (cAMP), which coincided in time with an equivalent decrease (approximately 40%) in ATP. However, no detectable change in phosphocreatine concentration was observed. Concomitantly, MSH induced a striking and unexpected increase in the concentration of three phosphomonoester (PME) metabolites (approximately 2-fold increase in total PME signal area); one signal has been assigned to phosphoethanolamine. The levels of the PMEs remained high for 2-4 hr and declined slowly (approximately 10 hr) to basal level, following perfusion with fresh culture medium. The increase in PME was also observed after stimulation with MSH alone. In contrast, stimulation with a high dose of forskolin (50 microM) and isobutylmethylxanthine (0.2 mM), although effective in stimulating the production of cAMP, did not induce the PME response. Evaluation of the cells' energetics indicated that the enhanced production of phosphoethanolamine is probably not due to ethanolamine phosphorylation. Therefore, it is likely to result from hydrolysis of phosphatidylethanolamine by a specific phospholipase C. The response of the PMEs appears to be regulated by a cAMP-independent process, suggesting the existence of an alternative transduction pathway controlled by MSH.
cDNA analysis predicts a cornea-specific collagen.
Authors: Marchant J K , Linsenmayer T F , Gordon M K
Published: May 2006

In the development of chicken corneal stroma, two or more collagens often interact, either as constituents of a single heterotypic fibril or as components of the fibril surface. The latter, fibril-associated collagens, may facilitate interactions between fibrils and the surrounding extracellular matrix or between fibrils themselves. In an effort to isolate putative nonfibrillar collagens that may have such a function, we screened a 13-day embryonic cornea cDNA library under reduced stringency conditions, using a cDNA probe for a collagenous domain of type XII collagen. We isolated a 4.2-kilobase (kb) cDNA that predicts a "collagenous" protein that has three unusual, if not unique, features. (i) The putative polypeptide encoded by this cDNA has a structural arrangement in which numerous stretches of Gly-Xaa-Yaa triplets, typical of collagens, are interrupted by non-Gly-Xaa-Yaa regions. One of the potential triple-helical domains is 246 amino acids long, but most are much smaller, consisting of 15-36 amino acids. Many are very rich in the helix-stabilizing imino acid proline. (ii) Northern blot analyses demonstrated strong cDNA hybridization to a 6.8-kb mRNA whose expression is restricted to the cornea. No hybridization was observed to mRNAs from the nine other tissues used in these analyses, even with extended exposure of the film. (iii) The cDNA contains a short (less than or equal to 425-base-pair) sequence in the 3' untranslated region of the 6.8-kb mRNA that hybridizes to a 7.8-kb mRNA that has a wide tissue distribution.
Photosensitized destruction of human bladder carcinoma cells treated with chlorin e6-conjugated microspheres.
Authors: Bachor R , Shea C R , Gillies R , Hasan T
Published: May 2006

A photosensitizer conjugate, chlorin e6 (Ce6) covalently bound to 1-micron-diameter polystyrene microspheres, has been investigated in the photodynamic destruction of MGH-U1 human bladder carcinoma cells in vitro. The microspheres were taken up avidly by the carcinoma cells; confocal laser scanning fluorescence microscopy showed them to be localized in the cytoplasm, apparently within lysosomes, visualized by labeling with acridine orange. In contrast, fluorescence of unconjugated Ce6 was present within most cellular membranes. Use of Ce6-microsphere conjugates led to a 20-fold-higher mean intracellular concentration, compared with unconjugated Ce6. Cells incubated in the presence of Ce6-microsphere conjugates (0.43 microM equivalent) and subsequently irradiated at 659 nm with a dye laser pumped by an argon-ion laser showed dose-dependent phototoxicity, leading to total inhibition of colony formation at a radiant exposure of 5J/cm2; in contrast, cells incubated with either unconjugated Ce6 (0.43 microM) or unconjugated microspheres before laser irradiation were unaffected. Cells pretreated with Ce6-microsphere conjugates and irradiated in the presence of 90% 2H2O showed significantly increased phototoxicity, an effect consistent with an important role for excited-state singlet oxygen in the mechanism of injury. In solution, however, photosensitized generation of singlet oxygen with Ce6-microsphere conjugates was 9 times less efficient than with unconjugated Ce6. The markedly greater phototoxicity of Ce6-microsphere conjugates compared to unconjugated Ce6 was therefore a consequence of the high intracellular Ce6 concentration attained by phagocytosis of the conjugates and their particular sites of intracellular localization. Thus, these conjugates are an efficient system for the delivery of photosensitizing drugs to carcinoma cells.
A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia.
Authors: Ginsberg H S , Moldawer L L , Sehgal P B , Redington M , Kilian P L , Chanock R M , Prince G A
Published: May 2006

Intranasal inoculation of type 5 adenovirus (Ad5) produced pneumonia in mice even though the virus did not replicate. To induce the pneumonia, however, a large viral infectious dose was required--i.e., 10(10) plaque-forming units. Four strains of inbred mouse were studied (C57BL/6N, C57BL/10ScN, CBA/N, and C3H/N): all showed similar inflammatory responses, although the greatest infiltration occurred in the C57BL/6N mice. The pathological response to Ad5 infection resembled that previously described in cotton rats: it consisted of overlapping early and late phases, and the infiltration contained primarily lymphocytes and monocytes/macrophages with a scattering of polymorphonuclear leukocytes. The prominent early phase and the presence of polymorphonuclear leukocytes suggested that induction of cytokines may play an important role in the pathogenesis of this pneumonia. Assays showed the appearance of tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), and IL-6 in the infected mouse lungs concomitant with the developing early-phase infiltration. Only IL-6 was found in the peripheral blood. IL-6 reached maximum titers 6-24 hr after infection, whereas maximum levels of TNF-alpha and IL-1 were attained 2-3 days after infection. Specific RNAs for each of these cytokines were demonstrated in the infected lungs. To test the hypothesis that a cytotoxic T-cell response was responsible for the second phase, which primarily consisted of a perivascular and peribronchial infiltration of lymphocytes, Ad5 was used to infect C57BL/10ScN Nu/Nu and parent mice. The nude mice showed a normal early-phase response, but essentially no peribronchial and only minimal perivascular infiltrations occurred.
Characterization of a tachykinin peptide NK2 receptor transfected into murine fibroblast B82 cells.
Authors: van Giersbergen P L , Shatzer S A , Henderson A K , Lai J , Nakanishi S , Yamamura H I , Buck S H
Published: May 2006

Membranes isolated from a murine fibroblast B82 cell line (SKLKB82#3) transfected with the bovine stomach cDNA pSKR56S exhibited binding of [His(125I)1]neurokinin A (125I-NKA) to a single population of sites with a Bmax of 147 fmol/mg of protein and a Kd of 0.59 nM. Control cell lines had little or no specific binding. The ligand binding in SKLKB82#3 cells was reversible and was inhibited by peptides in the potency rank of neuropeptide gamma greater than neuropeptide K greater than neurokinin A greater than [10-norleucine]neurokinin A-(4-10) greater than substance P much greater than senktide (succinyl-Asp-Phe-MePhe-Gly-Leu-Met-NH2). Specific binding was enhanced by Mn2+, Mg2+, and Ca2+ and was inhibited by guanine nucleotide analogues. Thus, SKLKB82#3 cells have been transfected with NK2 receptors that have become associated with an endogenous guanine nucleotide-binding protein. In comparison with membranes from the hamster urinary bladder, a tissue enriched in NK2 receptors, NK2 receptor antagonists displayed markedly different potencies, either more or less potent, in inhibiting specific binding in membranes of the transfected cells. Furthermore, inhibition of 125I-NKA binding by nucleotide analogues was markedly different in SKLKB82#3 cells compared with hamster bladder tissue. The different binding profile in the cells is not due to an artefact introduced during cDNA transfection because a similar profile was also observed in bovine stomach membranes. These results may indicate the existence of two distinct NK2 receptors.
Isolation and characterization of yeast artificial chromosome clones linking the HLA-B and HLA-C loci.
Authors: Bronson S K , Pei J , Taillon-Miller P , Chorney M J , Geraghty D E , Chaplin D D
Published: May 2006

A 290-kilobase-pair chromosomal segment containing the genes encoding the human class I major histocompatibility complex molecules HLA-B and HLA-C as well as a class I pseudogene has been isolated on three overlapping yeast artificial chromosome (YAC) clones. One YAC clone contains both the HLA-B and HLA-C genes. These loci are located approximately 85 kilobase pairs apart, each in close association with a CpG island. Southern blotting and nucleotide sequencing showed no evidence of alteration of the structure of the cloned DNA in the YACs. End fragments from the YAC inserts have been isolated and used to confirm the overlaps between clones. These fragments can also serve as polymorphic markers for structural analysis of the major histocompatibility complex. Our data show that YAC cloning offers an attractive alternative for analysis of the structures of large gene complexes such as HLA.
Thermal response of rat fibroblasts stably transfected with the human 70-kDa heat shock protein-encoding gene.
Authors: Li G C , Li L G , Liu Y K , Mak J Y , Chen L L , Lee W M
Published: May 2006

The major heat shock protein hsp70 is synthesized by cells of a wide variety of organisms in response to heat shock or other environmental stresses and is assumed to play an important role in protecting cells from thermal stress. We have tested this hypothesis directly by transfecting a constitutively expressed recombinant human hsp70-encoding gene into rat fibroblasts and examining the relationship between the levels of human hsp70 expressed and thermal resistance of the stably transfected rat cells. Successful transfection and expression of the gene for human hsp70 were characterized by RNA hybridization analysis, two-dimensional gel electrophoresis, and immunoblot analysis. When individual cloned cell lines were exposed to 45 degrees C and their thermal survivals were determined by colony-formation assay, we found that the expression of human hsp70 conferred heat resistance to the rat cells. These results reinforce the hypothesis that hsp70 has a protective function against thermal stress.