bluuebunny/crossref_metadata_2025 huggingface dataset:
A mutation in segment IVS6 disrupts fast inactivation of sodium channels.
Authors: McPhee J C , Ragsdale D S , Scheuer T , Catterall W A
Published: May 2006

Na(+)-channel inactivation is proposed to occur by binding of an intracellular inactivation gate to a hydrophobic inactivation gate receptor in the intracellular mouth of the pore. Amino acid residues in transmembrane segment S6 of domain IV (IVS6) that are critical for fast inactivation were identified by alanine-scanning mutagenesis. Mutant VIL1774-6AAA, in which three adjacent residues (Val-Ile-Leu) at the intracellular end of segment IVS6 were converted to alanine, had substantial (> 85%) sustained Na+ currents remaining 15 ms after depolarization, while a nearby mutation of three residues to alanine had no effect. Single-channel analysis revealed continued reopenings late in 40-ms depolarizing pulses indicating that inactivation was substantially impaired compared to wild type. The mean open time for VIL1774-6AAA was longer than wild type, suggesting that this mutation also decreases the rate of entry into the fast inactivated state. These results suggest that residues near the intracellular end of segment IVS6 are critical for fast Na(+)-channel inactivation and may form part of the hydrophobic receptor site for the fast inactivation gate.
The essential light chain is required for full force production by skeletal muscle myosin.
Authors: VanBuren P , Waller G S , Harris D E , Trybus K M , Warshaw D M , Lowey S
Published: May 2006

Myosin, a molecular motor that is responsible for muscle contraction, is composed of two heavy chains each with two light chains. The crystal structure of subfragment 1 indicates that both the regulatory light chains (RLCs) and the essential light chains (ELCs) stabilize an extended alpha-helical segment of the heavy chain. It has recently been shown in a motility assay that removal of either light chain markedly reduces actin filament sliding velocity without a significant loss in actin-activated ATPase activity. Here we demonstrate by single actin filament force measurements that RLC removal has little effect on isometric force, whereas ELC removal reduces isometric force by over 50%. These data are interpreted with a simple mechanical model where subfragment 1 behaves as a torque motor whose leyer arm length is sensitive to light-chain removal. Although the effect of removing RLCs fits within the confines of this model, altered crossbridge kinetics, as reflected in a reduced unloaded duty cycle, probably contributes to the reduced velocity and force production of ELC-deficient myosins.
Eicosanoids mediate insect nodulation responses to bacterial infections.
Authors: Miller J S , Nguyen T , Stanley-Samuelson D W
Published: May 2006

We propose that nodule formation is mediated by eicosanoids in insects. Nodulation is the temporally and quantitatively predominant cellular defense response to bacterial infection in insects and other invertebrates. Inhibition of eicosanoid biosynthesis in larvae of the tobacco hornworm Manduca sexta immediately prior to intrahemocoelic infections with the bacterium Serratia marcescens strongly reduced the nodulation response. Inhibition of eicosanoid biosynthesis also reduced formation of cellular aggregates at 1 hr postinfection, which indicates that eicosanoids mediate early stages of nodulation. Separate treatments with specific inhibitors of phospholipase A2, cyclooxygenase, and lipoxygenase reduced nodulation, which supports the view that nodule formation is a complex process involving prostaglandins and lipoxygenase products. The inhibitory effects of the phospholipase A2 inhibitor dexamethasone on nodulation were apparent by 1 hr after infection, and the effects increased, relative to controls, over 24 hr. The dexamethasone effects were expressed in a dose-dependent manner, and they were reversed by treating infected insects with eicosanoid-precursor polyunsaturated fatty acids. Treatments with the saturated fatty acid 16:0, which is not an eicosanoid precursor, did not reverse the dexamethasone effects on nodulation. These findings strongly support the identification of nodulation as a specific insect cellular defense mechanism that is mediated by eicosanoids.
Galanin antisense oligonucleotides reduce galanin levels in dorsal root ganglia and induce autotomy in rats after axotomy.
Authors: Ji R R , Zhang Q , Bedecs K , Arvidsson J , Zhang X , Xu X J , Wiesenfeld-Hallin Z , Bartfai T , Hökfelt T
Published: May 2006

Antisense (AS) oligonucleotides (ONs) to galanin (GAL) were applied to the proximal end of a transected sciatic nerve, allowing their cellular uptake and transport into injured axons. GAL expression in dorsal root ganglia and self-mutilation behavior (autotomy) were then studied. AS-ONs with phosphorothioate or allyl modifications significantly suppressed the axotomy-induced increase in GAL levels, as demonstrated by immunohistochemistry and exaggerated autotomy behavior, whereas no significant effect on GAL mRNA levels could be demonstrated with in situ hybridization. Allyl-ONs were more effective than phosphorothioate-ONs. An AS-ON with three base mismatches did not induce any of the above effects. These results support the view that the inhibition of axotomy-induced GAL up-regulation is related to autotomy.
Altered striatal function in a mutant mouse lacking D1A dopamine receptors.
Authors: Drago J , Gerfen C R , Lachowicz J E , Steiner H , Hollon T R , Love P E , Ooi G T , Grinberg A , Lee E J , Huang S P
Published: May 2006

Of the five known dopamine receptors, D1A and D2 represent the major subtypes expressed in the striatum of the adult brain. Within the striatum, these two subtypes are differentially distributed in the two main neuronal populations that provide direct and indirect pathways between the striatum and the output nuclei of the basal ganglia. Movement disorders, including Parkinson disease and various dystonias, are thought to result from imbalanced activity in these pathways. Dopamine regulates movement through its differential effects on D1A receptors expressed by direct output neurons and D2 receptors expressed by indirect output neurons. To further examine the interaction of D1A and D2 neuronal pathways in the striatum, we used homologous recombination to generate mutant mice lacking functional D1A receptors (D1A-/-). D1A-/- mutants are growth retarded and die shortly after weaning age unless their diet is supplemented with hydrated food. With such treatment the mice gain weight and survive to adulthood. Neurologically, D1A-/- mice exhibit normal coordination and locomotion, although they display a significant decrease in rearing behavior. Examination of the striatum revealed changes associated with the altered phenotype of these mutants. D1A receptor binding was absent in striatal sections from D1A-/- mice. Striatal neurons normally expressing functional D1A receptors are formed and persist in adult homozygous mutants. Moreover, substance P mRNA, which is colocalized specifically in striatal neurons with D1A receptors, is expressed at a reduced level. In contrast, levels of enkephalin mRNA, which is expressed in striatal neurons with D2 receptors, are unaffected. These findings show that D1A-/- mice exhibit selective functional alterations in the striatal neurons giving rise to the direct striatal output pathway.
Chimeric dopamine-norepinephrine transporters delineate structural domains influencing selectivity for catecholamines and 1-methyl-4-phenylpyridinium.
Authors: Buck K J , Amara S G
Published: May 2006

The dopamine (DA) and norephinephrine (NE) transporters demonstrate important differences in their selectivity for catecholamines and the parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+), yet their primary sequences and predicted topology are strikingly similar. To delineate discrete structural domains contributing to pharmacologic and kinetic differences between the DA and NE transporters, a series of recombinant chimeras was generated by a restriction site-independent method and expressed in mammalian cells. Functional analyses of the chimeras delineate two discrete regions spanning the first through the third transmembrane domains (TM1-3) and TM10-11 that contribute to differences in their apparent affinities for DA, NE, and MPP+. These studies also suggest that TM2-3 of the DA transporter have a role in selectively increasing the rate of DA uptake as compared with NE. TM4-8 of the DA transporter may influence the relative rate with which MPP+ is taken up into cells and could contribute to its selective toxicity in neurons expressing the DA transporter. These structure-function studies using chimeras of members of the superfamily of Na(+)- and Cl(-)-dependent transporters provide a framework for identifying the specific structural or regulatory determinants contributing to substrate recognition and translocation by the DA and NE transporters.
Cationic modulation of rho 1-type gamma-aminobutyrate receptors expressed in Xenopus oocytes.
Authors: Calvo D J , Vazquez A E , Miledi R
Published: May 2006

A study was made of the effects of di- and trivalent cations on homomeric rho 1-type gamma-aminobutyrate (GABA rho 1) receptors expressed in Xenopus oocytes after injection of mRNA coding for the GABA rho 1 subunit. GABA elicited large currents with a Kd approximately 1 microM. The properties of these GABA rho 1 receptors were similar to those of native bicuculline-resistant GABA receptors expressed by retinal mRNA. GABA rho 1 currents showed very little desensitization, were blocked by picrotoxin but not by bicuculline, and were not modulated by barbiturates, benzodiazepines, or beta-carbolines. Zn2+ reversibly decreased GABA rho 1 responses (IC50 = 22 microM). Other divalent cations were also tested and their rank order of potency was: Zn2+ approximately Ni2+ approximately Cu2+ >> Cd2+, whereas Ba2+, Co2+, Sr2+, Mn2+, Mg2+, and Ca2+ showed little or no effect. In contrast, La3+ reversibly potentiated the GABA currents mediated by homomeric GABA rho 1 receptors, with an EC50 = 135 microM and a maximal potentiation of about 100% (GABA, 1 microM; La3+, 1 mM). Other lanthanides showed similar effects (Lu3+ > Eu3+ > Tb3+ > Gd3+ > Er3% > Nd3+ > La3+ > Ce3+). Thus, GABA rho 1 receptors contain sites for cationic recognition, and in particular, Zn2+ may play a role during synaptic transmission in the retina.
Structural basis for chromosome X-linked agammaglobulinemia: a tyrosine kinase disease.
Authors: Vihinen M , Vetrie D , Maniar H S , Ochs H D , Zhu Q , Vorechovský I , Webster A D , Notarangelo L D , Nilsson L , Sowadski J M
Published: May 2006

X-linked agammaglobulinemia (XLA) is a hereditary defect of B-cell differentiation in man caused by deficiency of Bruton tyrosine kinase (BTK). A three-dimensional model for the BTK kinase domain, based on the core structure of cAMP-dependent protein kinase, was used to interpret the structural basis for disease in eight independent point mutations in patients with XLA. As Arg-525 of BTK has been thought to functionally substitute for a critical lysine residue in protein-serine kinases, the mutation Arg-525-->Gln was studied and found to abrogate the tyrosine kinase activity of BTK. All of the eight mutations (Lys-430-->Glu, Arg-520-->Glu, Arg-525-->Gln, Arg-562-->Pro, Ala-582-->Val, Glu-589-->Gly, Gly-594-->Glu, and Gly-613-->Asp) were located on one face of the BTK kinase domain, indicating structural clustering of functionally important residues.
Heterogeneity of genomes: measures and values.
Authors: Karlin S , Ladunga I , Blaisdell B E
Published: May 2006

Genomic homogeneity is investigated for a broad base of DNA sequences in terms of dinucleotide relative abundance distances (abbreviated delta-distances) and of oligonucleotide compositional extremes. It is shown that delta-distances between different genomic sequences in the same species are low, only about 2 or 3 times the distance found in random DNA, and are generally smaller than the between-species delta-distances. Extremes in short oligonucleotides include underrepresentation of TpA and overrepresentation of GpC in most temperate bacteriophage sequences; underrepresentation of CTAG in most eubacterial genomes; underrepresentation of GATC in most bacteriophage; CpG suppression in vertebrates, in all animal mitochondrial genomes, and in many thermophilic bacterial sequences; and overrepresentation of GpG/CpC in all animal mitochondrial sets and chloroplast genomes. Interpretations center on DNA structures (dinucleotide stacking energies, DNA curvature and superhelicity, nucleosome organization), context-dependent mutational events, methylation effects, and processes of replication and repair.
Direct measurement of stiffness of single actin filaments with and without tropomyosin by in vitro nanomanipulation.
Authors: Kojima H , Ishijima A , Yanagida T
Published: May 2006

In order to explain the molecular mechanism of muscle contraction, it is crucial to know the distribution of the sarcomere compliance of active muscle. Here, we directly measure the stiffness of single actin filaments with and without tropomyosin, using a recently developed technique for nanomanipulation of single actin filaments with microneedles. The results show that the stiffness for 1-micron-long actin filaments with and without tropomyosin is 65.3 +/- 6.3 and 43.7 +/- 4.6 pN/nm, respectively. When the distribution of crossbridge forces along the actin filament is taken into account, the elongation of a 1-micron-long thin filament during development of isometric contraction is calculated to be approximately 0.23%. The time constant of force in response to a sudden length change is < 0.2 ms, indicating that the viscoelasticity is negligible in the millisecond time range. These results suggest that approximately 50% of the sarcomere compliance of active muscle is due to extensibility of the thin filaments.
Identification of the MAGE-1 gene product by monoclonal and polyclonal antibodies.
Authors: Chen Y T , Stockert E , Chen Y , Garin-Chesa P , Rettig W J , van der Bruggen P , Boon T , Old L J
Published: May 2006

The human MAGE-1 gene encodes a melanoma peptide antigen recognized by autologous cytotoxic T lymphocytes. To produce antibodies against the MAGE-1 gene product, several approaches were taken. Three oligopeptides were synthesized based on predicted MAGE-1 amino acid sequences and were used to generate rabbit anti-peptide anti-sera. In addition, a truncated MAGE-1 cDNA was cloned into an Escherichia coli expression vector, and recombinant protein was produced and purified. All three rabbit anti-peptide antisera showed reactivity against the immunizing peptide, and one reacted with the recombinant MAGE-1 protein by immunoblotting, but none reacted with cell lysates from MAGE-1 mRNA-positive cells. The recombinant MAGE-1 protein was then used for the generation of mouse monoclonal and rabbit polyclonal antibodies. One IgG1 monoclonal antibody, MA454, as well as rabbit polyclonal antisera recognized a 46-kDa protein in extracts of MAGE-1 mRNA-positive melanoma cell lines. The antibodies showed no apparent cross-reactivity with products of the closely related MAGE-2 and MAGE-3 genes. Serological typing of normal and tumor cell lysates was in full agreement with mRNA analysis, showing expression of MAGE-1 protein in MAGE-1 mRNA-positive testis and a subset of melanomas but not in MAGE-1 mRNA-negative normal or tumor tissues. Transfection of the MAGE-1 gene into a MAGE-1 mRNA-negative melanoma cell line resulted in the expression of the 46-kDa protein, confirming the identity of this protein as the MAGE-1 gene product.
Superoxide and peroxynitrite inatherosclerosis.
Authors: White C R , Brock T A , Chang L Y , Crapo J , Briscoe P , Ku D , Bradley W A , Gianturco S H , Gore J , Freeman B A
Published: May 2006

The role of reactive oxygen species in the
vascular pathology associated with atherosclerosis was examined by testing the
hypothesis that impaired vascular reactivity results from the reaction of nitric
oxide (.NO) with superoxide (O2-), yielding the oxidant peroxynitrite (ONOO-).
Contractility studies were performed on femoral arteries from rabbits fed a
cholesterol-supplemented diet. Cholesterol feeding shifted the EC50 for
acetylcholine (ACh)-induced relaxation and impaired the maximal response to ACh.
We used pH-sensitive liposomes to deliver CuZn superoxide dismutase (SOD;
superoxide:superoxide oxidoreductase, EC 1.15.1.1) to critical sites of .NO
reaction with O2-. Intravenously injected liposomes (3000 units of SOD per ml)
augmented ACh-induced relaxation in the cholesterol-fed group to a greater
extent than in controls. Quantitative immunocytochemistry demonstrated enhanced
distribution of SOD in both endothelial and vascular smooth muscle cells as well
as in the extracellular matrix. SOD activity in vessel homogenates of
liposome-treated rabbits was also increased. Incubation of beta very low density
lipoprotein with ONOO- resulted in the rapid formation of conjugated dienes and
thiobarbituric acid-reactive substances. Our results suggest that the reaction
of O2- with .NO is involved in the development of atherosclerotic disease by
yielding a potent mediator of lipoprotein oxidation, as well as by limiting .NO
stimulation of vascular smooth muscle guanylate cyclase
activity.
Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.
Authors: Wysolmerski J J , Broadus A E , Zhou J , Fuchs E , Milstone L M , Philbrick W M
Published: May 2006

Parathyroid hormone-related peptide (PTHrP) was initially discovered as the cause of the syndrome of humoral hypercalcemia of malignancy. Subsequently, the PTHrP gene has been shown to be expressed in a wide variety of normal tissues, including skin. Because the biological function of PTHrP in skin remains unknown, we used the human keratin 14 promoter to target overexpression of PTHrP to the skin of transgenic mice. We achieved a 10-fold level of overexpression in skin, and human keratin 14 promoter-PTHrP transgenic mice displayed a disturbance in normal hair follicle development. These mice either failed to initiate follicle development or showed a delay in the initiation of follicles. These findings suggest that PTHrP normally plays a role in the early stages of hair follicle development and support previous speculation that the peptide may function in regulating cellular differentiation.
Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade.
Authors: Uhl M A , Miller J F
Published: May 2006

Expression of adhesins, toxins, and other virulence factors of Bordetella pertussis is under control of the BvgA and BvgS proteins, members of a bacterial two-component signal transduction family. BvgA bears sequence similarity to regulator components, whereas BvgS shows similarity to both sensor and regulator components. BvgA and the cytoplasmic portion of BvgS ('BvgS) were overexpressed and purified. 'BvgS autophosphorylated with the gamma-phosphate from [gamma-32P]ATP and phosphorylated BvgA. Kinetic analysis indicated that BvgA receives its phosphate from 'BvgS. Mutations in the transmitter, receiver, and C-terminal domains of BvgS were tested for activation of a BvgAS-dependent fhaB::lacZ reporter fusion in vivo and for autophosphorylation and phosphotransfer to BvgA in vitro. All mutations abolished activation of the fhaB::lacZ fusion. A point mutation in the transmitter (H729Q) prevented autophosphorylation of 'BvgS. In contrast to other characterized sensor proteins, autophosphorylation also required sequences in the 'BvgS receiver and C-terminal domains. A 'BvgS receiver point mutation (D1023N) had the novel phenotype of being able to autophosphorylate but unable to transfer the phosphate to BvgA. Autophosphorylation activity of the D1023N mutant protein was kinetically and chemically indistinguishable from wild-type 'BvgS despite an uncoupling of phosphotransfer from autophosphorylation. 'BvgS was shown to contain primarily amidyl phosphate and BvgA an acyl phosphate linkage. We present a model for a phosphorelay controlling virulence gene expression in B. pertussis.
Identification of active-site residues of the adenovirus endopeptidase.
Authors: Rancourt C , Tihanyi K , Bourbonniere M , Weber J M
Published: May 2006

Multiple sequence alignment of the 12 adenovirus endopeptidases known to date identified a number of conserved residues which might be important for enzyme activity. Eleven mutants were created in the cloned gene by site-directed mutagenesis to identify the active site of this thiol endopeptidase. Analysis of the proteolytic activity in a crude system using viral precursor proteins, as well as in a purified system with activated proteinases using a new chromophoric octapeptide substrate, yielded results consistent with Cys-104 and His-54 being two members of the active site. This result was confirmed by the carboxymethylation of the reactive Cys-104 and its prevention by the active-thiol-specific agent E64. Although Cys-122 and Cys-126 were also reactive cysteines, mutation of these residues did not affect enzyme activity. Replacement of the active-site Cys-104 by serine converted the enzyme into a serine-like proteinase, sensitive to serine proteinase inhibitors. The absence of homology to other proteinases, particularly at the active-site cysteine, coupled with the requirement for activation by a substrate cleavage fragment, indicates that the adenovirus endoproteinase may represent a new subclass of cysteine proteinases.
Analysis of a hyperdeformed band of 152(66)Dy86 on the basis of a structure with two revolving clusters, each with a previously unrecognized two-tiered structure.
Authors: Pauling L
Published: May 2006

Analysis on the basis of the two-revolving-cluster model has been made of a cascade of 11 gamma-rays constituting a hyperdeformed band of 152(66)Dy86 (or possibly 153Dy) reported by Galindo-Uribarri et al. [Galindo-Uribarri, A., et al. (1993) Phys. Rev. Lett. 73, 231-234], leading to the conclusions that the band extends from values K approximately 82-104 for the angular-momentum quantum number, that the moment of inertia is approximately 5650 Da.fm2, that the composition of the central sphere is p40n50 and that of each of the clusters is p13n18, that each of the clusters consists of two tiers of spherons, and that the radii of revolution of the inner and outer tiers have values of about 8.00 and 11.20 fm, respectively.
Identification of a vitamin D-response element in the rat calcidiol (25-hydroxyvitamin D3) 24-hydroxylase gene.
Authors: Zierold C , Darwish H M , DeLuca H F
Published: May 2006

The calcidiol (25-hydroxyvitamin D3) 24-hydroxylase is one of the key enzymes in the metabolism of vitamin D. This enzyme acts on both calcidiol and calcitriol (1,25-dihydroxyvitamin D3) to initiate degradation of these potent vitamin D metabolites and is tightly regulated. Calcitriol itself induces this enzyme and acts at the transcriptional level. Transcriptional regulation of genes by calcitriol has been shown to occur via the vitamin D-receptor binding to a vitamin D-response element located upstream of the transcription start site. We now report a vitamin D-response element located between nt -262 and nt -238 of the rat calcidiol 24-hydroxylase gene. This sequence binds the calcitriol receptor and confers vitamin D-dependent transactivation of transcription to its own, as well as heterologous, promoter.
Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa.
Authors: Portera-Cailliau C , Sung C H , Nathans J , Adler R
Published: May 2006

Retinitis pigmentosa (RP) is a group of inherited human diseases in which photoreceptor degeneration leads to visual loss and eventually to blindness. Although mutations in the rhodopsin, peripherin, and cGMP phosphodiesterase genes have been identified in some forms of RP, it remains to be determined whether these mutations lead to photoreceptor cell death through necrotic or apoptotic mechanisms. In this paper, we report a test of the hypothesis that photoreceptor cell death occurs by an apoptotic mechanism in three mouse models of RP: retinal degeneration slow (rds) caused by a peripherin mutation, retinal degeneration (rd) caused by a defect in cGMP phosphodiesterase, and transgenic mice carrying a rhodopsin Q344ter mutation responsible for autosomal dominant RP. Two complementary techniques were used to detect apoptosis-specific internucleosomal DNA fragmentation: agarose gel electrophoresis and in situ labeling of apoptotic cells by terminal dUTP nick end labeling. Both methods showed extensive apoptosis of photoreceptors in all three mouse models of retinal degeneration. We also show that apoptotic death occurs in the retina during normal development, suggesting that different mechanisms can cause photoreceptor death by activating an intrinsic death program in these cells. These findings raise the possibility that retinal degenerations may be slowed by interfering with the apoptotic mechanism itself.
Spatial interactions in human vision: from near to farvia experience-dependent cascades of connections.
Authors: Polat U , Sagi D
Published: May 2006

Perceptual learning has been shown to affect early
visual processes. Here, we show that learning induces an increase in the spatial
range of lateral interactions. Using a lateral masking/facilitation paradigm and
bandpass-localized stimuli, we measured the interaction range before and after
extensive training on a threshold detection task. For naive observers, target
threshold was found to be facilitated by mask presence at distances up to six
times the target period. However, practice had the effect of increasing the
facilitation range by at least a factor of three. We suggest that the induced
longer-range facilitation is a result of internal response transmission via a
cascade of local connections. The data presented also show that this chain can
be broken. These results suggest a plasticity in early vision governed by
Hebbian-like rules.
Inhibition of gonadotropin hormone-releasing hormonerelease by prolactin from GT1 neuronal cell lines through prolactinreceptors.
Authors: Milenković L , D'Angelo G , Kelly P A , Weiner R I
Published: May 2006

High levels of prolactin (PRL) are associated with
inhibition of luteinizing hormone secretion in several mammalian species. We
asked whether this phenomenon could be explained by a direct inhibitory action
of PRL on hypothalamic gonadotropin hormone-releasing hormone (GnRH) neurons.
The ability of PRL to suppress GnRH release and expression was tested in the
highly differentiated GT1 GnRH cell lines. In static culture, nanomolar
concentrations of either rat or mouse PRL inhibited the release of GnRH in a
dose-dependent fashion. PRL treatment for 24 hr also decreased GnRH mRNA levels
determined by Northern analysis. The cells were shown to express the PRL
receptor gene, and the mRNAs for both the short and long forms were present by
Northern and PCR analysis, although the short form was more abundant. In Western
blots with monoclonal antibody against the rat liver PRL receptor, the short
42-kDa form of the receptor was observed. These results demonstrate that PRL
inhibits GnRH release and possibly gene expression in GnRH neurons. This action
appears to be mediated through prolactin receptors expressed by the
cells.
Free radical adducts induce alterations in DNAcytosine methylation.
Authors: Weitzman S A , Turk P W , Milkowski D H , Kozlowski K
Published: May 2006

Methylation of cytosines in DNA is important for
the regulation of expression of many genes. During carcinogenesis, normal
patterns of gene methylation can be altered. Oxygen radical injury, shown to
damage DNA in a variety of ways associated with cancer development and other
conditions, has been suggested to affect DNA methylation, but a mechanism has
not been demonstrated. Using oligonucleotides containing the common oxygen
radical adduct 8-hydroxyguanine to replace guanine, we found that the enzymatic
methylation of adjacent cytosines is profoundly altered. Furthermore, there is a
high degree of positional specificity with respect to this effect. Thus, free
radical injury may explain some of the altered methylation observed during
carcinogenesis.
Two orphan receptors binding to a common site areinvolved in the regulation of the oxytocin gene in the bovineovary.
Authors: Wehrenberg U , Ivell R , Jansen M , von Goedecke S , Walther N
Published: May 2006

The peptide hormone oxytocin is highly expressed
in the hypothalamus within only a small number of magnocellular neurons.
However, it is also expressed in a much larger number of cells in the bovine
corpus luteum at high levels in an estrous cycle-dependent manner. By using
nuclear extracts from this tissue for in vitro binding studies, two protein
complexes have been shown to bind to a common site in the bovine oxytocin
promoter. One of these proteins has been identified as the bovine homologue of
the chicken ovalbumin upstream promoter transcription factor (COUP-TF). The
second protein is here characterized as the bovine homologue of a
tissue-specific transcription factor, steroidogenic factor 1 (SF-1). The
relative expression of these two factors during luteal development correlates
with the level of luteal oxytocin gene expression, with SF-1 being the factor
binding to the promoter of the oxytocin gene when this promoter is activated.
Cotransfection experiments using the murine testicular cell line TM4 show that
SF-1 can stimulate the expression of a transfected oxytocin gene, suggesting
that SF-1 may be involved in upregulation of the oxytocin gene in vivo, possibly
by transducing a stimulatory signal to the RNA polymerase.
A screen for yeast mutants with defects in thedolichol-mediated pathway for N-glycosylation.
Authors: Roos J , Sternglanz R , Lennarz W J
Published: May 2006

Dolichol in the form of dolichyl phosphate
participates in the synthesis of N- and O-linked glycoproteins and
phosphatidylinositol-linked proteins in the yeast Saccharomyces cerevisiae. In
this organism, as well as in higher eukaryotes, a number of the enzymes in the
polyisoprenoid and glycoprotein biosynthetic pathways have not been identified.
In this study, we have developed a convenient, highly sensitive assay that uses
one of the end products of the dolichylphosphate synthetic pathway,
oligosaccharide-diphosphodolichol, and a 125I-labeled peptide substrate for
N-linked glycosylation to screen a collection of temperature-sensitive yeast
mutants for defects in protein glycosylation. By using a combination of
biochemical and genetic procedures, the defective mutants were grouped into
three categories: those containing defects in dolichyl-phosphate synthesis
(class 1), lipid-linked oligosaccharide assembly (class 2), or oligosaccharide
transferase activity (class 3). Among the mutants identified by this screen were
sec59 (which encodes dolichol kinase) and a mutant that affects the activity of
the ALG1-encoded mannosyltransferase that forms dolichol-PP-(GlcNAc)2Man1. Of
particular interest was a mutant that exhibits a temperature-sensitive defect in
oligosaccharide transferase activity. This mutant, meg1 (microsomal protein
essential for glycosylation 1) assembles a complete oligosaccharide chain and,
therefore, is likely to be a class 3 mutant. We report the cloning of MEG1, the
gene that rescues the oligosaccharide transferase activity defect in this
mutant. A number of criteria distinguish this gene from previously described
genes in this pathway.
Charge replacement near the phosphorylatable serine ofthe myosin regulatory light chain mimics aspects ofphosphorylation.
Authors: Sweeney H L , Yang Z , Zhi G , Stull J T , Trybus K M
Published: May 2006

Phosphorylation of the myosin regulatory light
chains (RLCs) activates contraction in smooth muscle and modulates force
production in striated muscle. RLC phosphorylation changes the net charge in a
critical region of the N terminus and thereby may alter interactions between the
RLC and myosin heavy chain. A series of N-terminal charge mutations in the human
smooth muscle RLC has been engineered, and the mutants have been evaluated for
their ability to mimic the phosphorylated form of the RLC when reconstituted
into scallop striated muscle bundles or into isolated smooth muscle myosin.
Changing the net charge in the region from Arg-13 to Ser-19 potentiates force in
scallop striated muscle and maintains smooth muscle myosin in an unfolded
filamentous state without affecting ATPase activity or motility of smooth muscle
myosin. Thus, the effect of RLC phosphorylation in striated muscle and its
ability to regulate the folded-to-extended conformational transition in smooth
muscle may be due to a simple reduction of net charge at the N terminus of the
light chain. The ability of phosphorylation to regulate smooth muscle
myosin's ATPase activity and motility involves a more complex
mechanism.
Nuclear-mitotic apparatus protein: a structuralprotein interface between the nucleoskeleton and RNA splicing.
Authors: Zeng C , He D , Berget S M , Brinkley B R
Published: May 2006

Vertebrate splicing factors are localized to
discrete domains within the nuclei of somatic cells. The mechanism whereby such
nuclear domains, identified as speckles by immunofluorescence microscopy, are
generated is unclear. Recent studies suggest that the spatial order within the
nucleus is maintained by nuclear matrix factors. Here we show that a protein in
the nuclear matrix and mitotic apparatus [nuclear-mitotic apparatus protein,
NuMA; Lydersen, B. & Pettijohn, D. (1980) Cell 22, 489-499] colocalizes with
splicing factors in interphase nuclei and is associated with small nuclear
ribonucleoproteins in a complex immunoprecipitated from HeLa extract with small
nuclear ribonucleoprotein antibodies. Moreover, NuMA associates with splicing
complexes that are reconstituted in vitro using wild-type pre-mRNA, but not with
nonspecific RNA. Cumulatively, these observations suggest a function of NuMA or
NuMA-like proteins in interphase cells in providing a bridge between RNA
processing and the nucleoskeleton.
Human immunodeficiency virus type 1 Nef associateswith a cellular serine kinase in T lymphocytes.
Authors: Sawai E T , Baur A , Struble H , Peterlin B M , Levy J A , Cheng-Mayer C
Published: May 2006

With T-cell lines constitutively expressing Nef
from the SF2 strain of human immunodeficiency virus type 1 (HIV-1SF2) in the
form of a hybrid CD8-Nef fusion protein or T-cell lines chronically infected
with HIV-1SF2, a cellular serine kinase was found that specifically associates
with Nef. Proteins of 62 kDa and 72 kDa, which coimmunoprecipitated with Nef,
were phosphorylated in in vitro kinase assays. This Nef-associated serine kinase
activity was not blocked by inhibitors of protein kinase C or protein kinase A
and was lost when Nef was truncated at amino acid 94 or 99. These findings
present evidence that a serine kinase activity is associated with Nef expressed
in human T lymphocytes.
Molecular resurrection of an extinct ancestralpromoter for mouse L1.
Authors: Adey N B , Tollefsbol T O , Sparks A B , Edgell M H , Hutchison C A
Published: May 2006

The F-type subfamily of LINE-1 or L1 retroposons
[for long interspersed (repetitive) element 1] was dispersed in the mouse genome
several million years ago. This subfamily appears to be both transcriptionally
and transpositionally inactive today and therefore may be considered
evolutionarily extinct. We hypothesized that these F-type L1s are inactive
because of the accumulation of mutations. To test this idea we used phylogenetic
analysis to deduce the sequence of a transpositionally active ancestral F-type
promoter, resurrected it by chemical synthesis, and showed that it has promoter
activity. In contrast, F-type sequences isolated from the modern genome are
inactive. This approach, in which the automated DNA synthesizer is used as a
"time machine," should have broad application in testing models
derived from evolutionary studies.
Single missense mutation in the tyrosine kinasecatalytic domain of the RET protooncogene is associated with multiple endocrineneoplasia type 2B.
Authors: Carlson K M , Dou S , Chi D , Scavarda N , Toshima K , Jackson C E , Wells S A , Goodfellow P J , Donis-Keller H
Published: May 2006

Multiple endocrine neoplasia type 2B (MEN 2B) is a
human cancer syndrome characterized by medullary thyroid carcinoma (MTC),
pheochromocytomas, mucosal neuromas, ganglioneuromas of the intestinal tract,
and skeletal and ophthalmic abnormalities. It appears both as an inherited
disorder and as de novo disease. Sequence analysis of germ-line DNA from MEN 2B
patients revealed the existence of the same point mutation in the RET
protooncogene in 34 unrelated individuals. This sequence difference was not
observed in 93 unaffected individuals, including the normal parents of 14 de
novo MEN 2B patients. The mutation (ATG-->ACG) results in the replacement of
methionine with threonine within the catalytic core region of the tyrosine
kinase domain. We propose that this amino acid replacement effects substrate
interactions and results in dominant oncogenic activity by the RET protein.
Missense mutations in the extracellular ligand-binding domain of the RET
protooncogene previously have been associated with two other disorders [MEN 2A
and familial MTC (FMTC)] in which MTC is observed. MEN 2B represents the third
form of heritable MTC known to be an allele of RET. Alterations in two different
functional domains of the putative receptor protein tyrosine kinase are
implicated in development of MTC.
Adduct detection by acylation with [35S]methionine:analysis of DNA adducts of 4-aminobiphenyl.
Authors: Sheabar F Z , Morningstar M L , Wogan G N
Published: May 2006

Reaction of synthetic
N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (dGuo-8-ABP) with
t-butoxycarbonyl-L-[35S]methionine, N-hydroxysuccinimidyl ester (35S-labeled
TBM-NHS), under optimized conditions produced mono-, bis-, and tris-TBM-acylated
nucleosides that were separable by HPLC. Reaction of different amounts of
N-(2'-deoxy-1',2'-[3H]guanosin-8-yl)-4-aminobiphenyl
([3H]dGuo-8-ABP) with 35S-labeled TBM-NHS established that total 35S content of
acylated products was linearly related to adduct concentration (r = 0.992) over
the range of 10 fmol to 30.6 pmol. Additionally, the
N-(deoxyguanosin-8-yl)-4-[3H]aminobiphenyl (dGuo-8-[3H]ABP) adduct was isolated
from calf thymus DNA adducted in vitro and from rat liver DNA adducted in vivo
and similarly reacted with 35S-labeled TBM-NHS. Acylation products of dGuo-8-ABP
from all three sources showed HPLC retention times identical to those of
authentic TBM-dGuo-8-ABP, and 35S incorporation into acylated products was
linearly related to amount of adduct reacted. These results indicate that the
procedure, to which we have referred as adduct detection by acylation with
methionine (ADAM), has potential applicability as an analytical procedure for
detection and quantification of DNA adducts in human tissues in the molecular
epidemiology of cancer.
Inhibition of human epithelial ovarian cancer cellgrowth in vitro by agonistic and antagonistic analogues of luteinizinghormone-releasing hormone.
Authors: Yano T , Pinski J , Radulovic S , Schally A V
Published: May 2006

In this study, we investigated the effects of
luteinizing hormone-releasing hormone (LH-RH) agonist [D-Trp6]LH-RH, LH-RH
antagonist [Ac-D-Nal(2)1,D-Phe(pCl)2,D-Pal(3)3,D-Cit6,D-Ala10]LH-RH (SB-75), and
estradiol on the growth of human epithelial ovarian cancer cell line OV-1063.
Cells were cultured under estrogen-deprived conditions. Estradiol inhibited cell
proliferation, as measured by cell number at 10(-9)-10(-7) M and [3H]thymidine
incorporation into DNA at 10(-13)-10(-8) M. Both LH-RH analogs inhibited cell
growth dose dependently in the range 10(-8)-10(-5) M, but SB-75 induced a
greater growth inhibition than [D-Trp6]LH-RH. In OV-1063 cells, 125I-labeled
[D-Trp6]LH-RH was bound to one class of specific, saturable binding sites with
high affinity (Kd = 1.4 +/- 0.3 nM) and low capacity (4000 binding sites per
cell). 125I-labeled [D-Trp6]LH-RH could be displaced by unlabeled [D-Trp6]LH-RH
and SB-75, suggesting that both analogs are bound to the same receptor on
OV-1063 cells. Ligand binding was dependent on time and temperature. Receptor
internalization assay showed that the ligand-receptor complex was internalized
at 37 degrees C, which indicates the presence of biologically active LH-RH
receptors on OV-1063 cells. These results suggest that estradiol and LH-RH
analogs can suppress the growth of OV-1063 human epithelial ovarian cancer cells
by a direct action and that the inhibitory effect of LH-RH analogs is mediated
through the high-affinity LH-RH receptors.
Segmental dynamics of the cytoplasmic domain oferythrocyte band 3 determined by time-resolved fluorescence anisotropy:sensitivity to pH and ligand binding.
Authors: Thevenin B J , Periasamy N , Shohet S B , Verkman A S
Published: May 2006

Interactions between the erythrocyte membrane and
its skeleton are mediated primarily by binding of cytoskeletal components to a
conformationally sensitive structure, the cytoplasmic domain of band 3 (cdb3).
To examine the nanosecond segmental motions of cdb3, band 3 was labeled
selectively by fluorescein maleimide at Cys-201 near the proposed hinge in cdb3
about which pH-dependent conformational changes occur. Time-resolved anisotropy
of labeled cdb3 in isolated form and in stripped erythrocyte membranes was
measured by parallel-acquisition frequency-domain microfluorimetry. Samples had
a single-component fluorescein lifetime of approximately 4 ns. Multifrequency
phase and modulation data (5-200 MHz) fitted well to a segmental motion model
containing two correlation times (tau 1c and tau 2c) and two limiting
anisotropies (r1infinity and r2infinity). Measurements in protease-cleaved and
denatured samples indicated that tau 1c (100-150 ps) corresponded to rapid
rotation of bound fluorescein and tau 2c (2-5 ns) corresponded to segmental
motion of cdb3. Both motions were hindered as quantified by nonzero r1infinity
and r2infinity. The strong pH dependence of segmental motion correlated with
that of cdb3 conformation measured by intrinsic tryptophan fluorescence.
Significant changes in cdb3 segmental motion occurred upon interactions with the
small ligands 2,3-bisphosphoglycerate and calcium and several glycolytic enzymes
known to bind to the N terminus of band 3. These time-resolved fluorescence
measurements of the nanosecond segmental dynamics of a labeled membrane protein
provide evidence for the sensitivity of cdb3 conformation to ligand binding and
suggest long-range structural communication through cdb3.
The formation of mitochondrial ribonucleoproteincomplexes involving guide RNA molecules in Trypanosoma brucei.
Authors: Göringer H U , Koslowsky D J , Morales T H , Stuart K
Published: May 2006

Transcripts from mitochondrial (maxicircle) genes
of kinetoplastid organisms undergo RNA editing characterized by a series of
reactions that insert and delete uridine nucleotides within the sequence of the
pre-mRNAs. Guide RNAs, which complement fully edited mRNAs, provide the
information for the edited sequence by an unknown mechanism. We report here that
guide RNA molecules associate with other mitochondrial components to form four
specific, stable ribonucleoprotein complexes. The complexes form very rapidly at
a low monovalent cation concentration, and their formation is blocked by heparin
or pretreatment of the mitochondrial lysate with SDS. ATP hydrolysis is not
required but slightly stimulates complex association up to concentrations of 5
mM. The results are suggestive of a sequential assembly of the ribonucleoprotein
complexes, and their possible involvement during the kinetoplastid RNA editing
is discussed.
Multiple tyrosine protein kinases in rat hippocampalneurons: isolation of Ptk-3, a receptor expressed in proliferative zones of thedeveloping brain.
Authors: Sánchez M P , Tapley P , Saini S S , He B , Pulido D , Barbacid M
Published: May 2006

Tyrosine protein kinases are likely to play an
important role in the maintenance and/or development of the nervous system. In
this study we have used the PCR cloning technique to isolate sequences derived
from tyrosine kinase genes expressed in cultured hippocampal neurons obtained
from 17.5-day-old rat embryos. Nucleotide sequence analysis of 209 independent
clones revealed sequences derived from 25 tyrosine kinases, of which two
corresponded to previously unreported genes. One of the PCR clones, ptk-2,
belongs to the Jak family of cytoplasmic tyrosine kinases. The second clone,
ptk-3, was derived from a gene encoding an additional class of tyrosine kinase
receptors whose extracellular domains contain regions of homology with
coagulation factors V and VIII and complement component C1. Transcripts encoding
the Ptk-3 receptor are present in a variety of embryonic and adult tissues with
highest levels observed in brain. During development, ptk-3 transcripts are most
abundant in the proliferative neuroepithelial cells of the ventricular zone,
raising the possibility that this receptor may play an important role in the
generation of the mammalian nervous system.
Effect of inducing nocturnal serum melatoninconcentrations in daytime on sleep, mood, body temperature, andperformance.
Authors: Dollins A B , Zhdanova I V , Wurtman R J , Lynch H J , Deng M H
Published: May 2006

We examined effects of very low doses of melatonin
(0.1-10 mg, orally) or placebo, administered at 1145 h, on sleep latency and
duration, mood, performance, oral temperature, and changes in serum melatonin
levels in 20 healthy male volunteers. A repeated-measure double-blind Latin
square design was used. Subjects completed a battery of tests designed to assess
mood and performance between 0930 and 1730 h. The sedative-like effects of
melatonin were assessed by a simple sleep test: at 1330 h subjects were asked to
hold a positive pressure switch in each hand and to relax with eyes closed while
reclining in a quiet darkened room. Latency and duration of switch release,
indicators of sleep, were measured. Areas under the time-melatonin concentration
curve varied in proportion to the different melatonin doses ingested, and the
0.1- and 0.3-mg doses generated peak serum melatonin levels that were within the
normal range of nocturnal melatonin levels in untreated people. All melatonin
doses tested significantly increased sleep duration, as well as self-reported
sleepiness and fatigue, relative to placebo. Moreover, all of the doses
significantly decreased sleep-onset latency, oral temperature, and the number of
correct responses on the Wilkinson auditory vigilance task. These data indicate
that orally administered melatonin can be a highly potent hypnotic agent; they
also suggest that the physiological increase in serum melatonin levels, which
occurs around 2100 h daily, may constitute a signal initiating normal sleep
onset.