bluuebunny/crossref_metadata_2025 huggingface dataset:
Growth-rate-dependent regulation of 6-phosphogluconate dehydrogenase level mediated by an anti-Shine-Dalgarno sequence located within the Escherichia coli gnd structural gene.
Authors: Carter-Muenchau P , Wolf R E
Published: May 2006

Previous work has shown that in Escherichia coli K-12 growth-rate-dependent regulation of expression of 6-phosphogluconate dehydrogenase, encoded by the gnd gene, occurs at the posttranscriptional level and is mediated by a negative control element that lies deep in the coding sequence, somewhere between codons 48 and 118. Deletion analysis of a growth-rate-regulated gnd-lacZ translational fusion showed that the element is the segment of gnd mRNA between codons 67 and 78 that is complementary to an extensive portion of the gnd ribosome-binding site, including its Shine-Dalgarno sequence. The boundaries of the element were further defined by the cloning of a synthetic "internal complementary sequence." The core internal complementary sequence element effected growth-rate-dependent regulation when placed at several sites between codon 40 and codon 69, but it severely reduced gene expression when moved to codon 13. The effect on regulation of single and double mutations introduced into the element by site-directed mutagenesis correlated with the ability of the respective mRNAs to fold into secondary structures that sequester the ribosome-binding site. Thus the gnd gene's internal regulatory element appears to function as a cis-acting antisense RNA.
The two subunits of the human asialoglycoprotein receptor have different fates when expressed alone in fibroblasts
Authors: Shia Michael A. , Lodish Harvey F.
Published: May 2006

Two related polypeptides, H1 and H2, comprise the human asialoglycoprotein receptor (ASGP-R). Stable lines of murine NIH 3T3 fibroblasts expressing H1 alone or H2 alone do not bind or internalize the ligand asialoorosomucoid (ASOR), which contains triantennary oligosaccharides. In contrast, cells expressing H1 and H2 together bind and degrade ASOR with properties indistinguishable from those of the ASPG-R in human hepatoma HepG2 cells. Whether or not H2 is coexpressed, H1 is synthesized as a 40-kDa precursor bearing high-mannose oligosaccharides, processed to its mature 46-kDa form, and transported to the cell surface. In cells expressing only H1, homodimers and -trimers of H1 are formed. In contrast, when expressed in 3T3 cells without H1, H2 is synthesized as its 43-kDa precursor, bearing high-mannose oligosaccharides, but is rapidly degraded. When H1 and H2 are coexpressed in the same cell, the H1 polypeptide “rescues” the H2 polypeptide; H2 is processed to its characteristic 50-kDa mature form and is transported to the surface. We conclude that the human ASGP-R is a multichain heterooligomer, probably a trimer of H1 molecules in noncovalent association with one, two, or three H2 molecules, and that the two polypeptides normally interact early in biosynthesis.
UV-induced DNA-binding proteins in human cells.
Authors: Glazer P M , Greggio N A , Metherall J E , Summers W C
Published: May 2006

To investigate the response of human cells to DNA-damaging agents such as UV irradiation, we examined nuclear protein extracts of UV-irradiated HeLa cells for the presence of DNA-binding proteins. Electrophoretically separated proteins were transferred to a nitrocellulose filter that was subsequently immersed in a binding solution containing radioactively labeled DNA probes. Several DNA-binding proteins were induced in HeLa cells after UV irradiation. These included proteins that bind predominantly double-stranded DNA and proteins that bind both double-stranded and single-stranded DNA. The binding proteins were induced in a dose-dependent manner by UV light. Following a dose of 12 J/m2, the binding proteins in the nuclear extracts increased over time to a peak in the range of 18 hr after irradiation. Experiments with metabolic inhibitors (cycloheximide and actinomycin D) revealed that de novo synthesis of these proteins is not required for induction of the binding activities, suggesting that the induction is mediated by protein modification.
Highly attenuated vaccinia virus mutants for the generation of safe recombinant viruses.
Authors: Rodriguez D , Rodriguez J R , Rodriguez J F , Trauber D , Esteban M
Published: May 2006

An attenuated vaccinia virus mutant with specific genetic lesions has been used to develop a vehicle for safer live recombinant virus vaccines. The mutant virus 48-7 has an 8-MDa deletion starting 2.2 MDa from the left end of the viral genome and point mutations in the gene encoding the 14-kDa fusion protein that determines the plaque-size phenotype of the virus. Using the highly sensitive reporter gene luciferase, we have shown that this mutant can generate recombinant viruses that infect cultured cells and animals with normal vaccinia virus tropism. Insertion of the envelope and gag genes of human immunodeficiency virus type 1 into the attenuated vaccinia mutant resulted in their efficient expression and precursor processing in infected cultured cells. Infection of mice with human immunodeficiency virus-vaccinia recombinant viruses elicited human immunodeficiency virus-specific antibodies. Using mice pretreated with cyclophosphamide as a model for immunosuppression, the reduced virulence of the mutant recombinant virus was clearly evident. These findings demonstrate that the highly attenuated vaccinia virus mutant 48-7 can be used to generate effective and safer vaccines.
Immunocytochemical localization of the beta I subspecies of protein kinase C in rat brain.
Authors: Hosoda K , Saito N , Kose A , Ito A , Tsujino T , Ogita K , Kikkawa U , Ono Y , Igarashi K , Nishizuka Y
Published: May 2006

Polyclonal antibodies were raised against an oligopeptide containing an amino acid sequence specific for beta I protein kinase C (PKC) in order to localize this subspecies of PKC in the rat brain. beta I PKC immunoreactivity was widely distributed but discretely localized in particular brain regions. The immunoreactivity was associated with neurons but not glial cells and was generally weak in the neutrophils with the exception of areas such as the triangular septal nucleus, pontine nuclei, superficial layer of the superior colliculus, and gray matter of the spinal cord. The largest number of beta I PKC-immunoreactive cells was seen in the triangular septal nucleus and pontine nuclei. In these areas of dense beta I immunoreactivity, there was no or little gamma PKC immunoreactivity. The beta I PKC immunoreactivity was observed in perikarya, dendrites, and axons but not in nuclei or nucleoli. There were various patterns of cytoplasmic immunoreactivity, but the immunoreactivity was seen frequently in the periphery of the perikarya and dendrites. The intraneuronal localization of beta I PKC immunoreactivity differs from that of the subspecies that is coded by the gamma gene sequence. The results suggest that beta I PKC may play a role in the regulation of cell membrane responsiveness in particular neurons.
Molecular structure of fructose-1,6-bisphosphatase at 2.8-A resolution.
Authors: Ke H , Thorpe C M , Seaton B A , Marcus F , Lipscomb W N
Published: May 2006

Fructose-1,6-bisphosphatase (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) from the cortex of pig kidney and its complexes with either fructose 2,6-bisphosphate (Fru-2,6-P2) or adenosine monophosphate (AMP) have been crystallized in the space group P3(2)21. The three-dimensional structure of the native enzyme has been solved at 3.0-A resolution by the multiple isomorphous replacement method and refined at 2.8-A resolution to a crystallographic R factor of 0.194. A total of 316 of 335 residues, omitting disordered regions 1-5 and 54-67, have been built into the monomer, which has average dimensions of about 30 A by 50 A by 35 A. Four monomeric units aggregate into a molecular tetramer with D2 symmetry, which approximates a disk about 35 A thick. Each monomer consists of about 33% alpha-helix, 23% beta-strand, and 6% beta-turn. Four sites for Fru-2,6-P2 and two major sites for AMP binding per tetramer have been identified by difference Fourier techniques. The binding site for Fru-2,6-P2 is shared by two neighboring monomers and consists of side-chain atoms of Asn-212, Tyr-244, Tyr-264, and Lys-274; backbone atoms of Gly-246 through Met-248; and only Arg-243 from the adjacent subunit. In addition, Asn-125, Tyr-215, and Lys-269 are located within a distance of about 5 A of Fru-2,6-P2. A negatively charged pocket near this binding site includes Asp-118, Asp-121, Glu-280, Glu-97, and Glu-98. The AMP binding site is located near Val-17, Gln-20, Gly-21, Ala-24 through Met-30, Lys-112, Tyr-113, Arg-140, and Met-177.
Why are blue visual pigments blue? A resonance Raman microprobe study.
Authors: Loppnow G R , Barry B A , Mathies R A
Published: May 2006

A resonance Raman microscope has been developed to study the structure of the retinal prosthetic group in the visual pigments of individual photoreceptor cells. Raman vibrational spectra are obtained by focusing the probe laser on intact photoreceptors frozen on a 77 K cold stage. To elucidate the mechanism of wavelength regulation in blue visual pigments, we have used this apparatus to study the structure of the chromophore in the 440-nm absorbing pigment found in "green rods" of the toad (Bufo marinus). The 9-cis isorhodopsin form of the green rod pigment exhibits a 1662-cm-1 C = NH+ Schiff base stretching mode that shifts to 1636 cm-1 in deuterium-substituted H2O. This demonstrates that the Schiff base linkage to the protein is protonated. Protonation of the Schiff base is sufficient to explain the 440-nm absorption maximum of this pigment without invoking any additional protein-chromophore interactions. The absence of additional perturbations is supported by the observation that the ethylenic band and the perturbation-sensitive C-10-C-11 and C-14-C-15 stretching modes have the same frequency as those of the 9-cis protonated retinal Schiff base in solution. Our demonstration that a blue visual pigment contains an unperturbed protonated Schiff base provides experimental evidence that the protein charge perturbation responsible for the opsin shift in the 500-nm absorbing pigments is removed in the opsins of blue pigments, as suggested by the sequence data.
mRNA from NCB-20 cells encodes the N-methyl-D-aspartate/phencyclidine receptor: a Xenopus oocyte expression study.
Authors: Lerma J , Kushner L , Spray D C , Bennett M V , Zukin R S
Published: May 2006

The mouse neuroblastoma--Chinese hamster brain hybrid cell line NCB-20 is the only clonal cell line in which binding studies indicate the presence of phencyclidine (PCP) receptors. We report here that Xenopus oocytes injected with NCB-20 cell poly(A)+ RNA express N-methyl-D-aspartate (NMDA)-activated channels and that these channels include the PCP receptor site. In injected oocytes, NMDA application evoked a partially desensitizing inward current that was potentiated by glycine, blocked by the competitive antagonist D-2-amino-5-phosphonovaleric acid, blocked by Mg2+ and by Zn2+, and blocked in a use-dependent manner by the PCP receptor ligands PCP and MK-801. There was little or no response to kainate or quisqualate (agonists of the other excitatory amino acid receptors), to gamma-aminobutyric acid (an inhibitory transmitter), or to glycine (an inhibitory transmitter as well as an allosteric potentiator of NMDA channels). Thus, NMDA/PCP receptors expressed from NCB-20 cell mRNA exhibit properties similar to those of the neuronal receptors. The absence of expression of other excitatory amino acid receptors in this system makes it particularly useful for study of NMDA-evoked responses without interference from responses mediated by other receptors. Moreover, NCB-20 mRNA may be an appropriate starting material for cloning the cDNA(s) encoding the NMDA/PCP-receptor complex.
Reconstitution of Golgi vesicle CMP-sialic acid and adenosine 3'-phosphate 5'-phosphosulfate transport into proteoliposomes.
Authors: Milla M E , Hirschberg C B
Published: May 2006

We have previously shown that Golgi apparatus vesicles transport nucleotide sugars and nucleotide sulfate into their lumen. These transport activities are organelle and substrate specific and are characterized by apparent Km for nucleotide derivatives in the low micromolar range. As part of our goal of purifying and characterizing the above transport proteins, we have reconstituted a protein extract from rat liver Golgi membranes into phosphatidylcholine liposomes. The resulting proteoliposomes transport CMP-N-acetylneuraminic acid (CMP-AcNeu) and adenosine 3'-phosphate 5'-phosphosulfate with very similar affinity and inhibition characteristics as intact Golgi vesicles. Sialic acid and sodium sulfate, which are transported only very slowly into the lumen of Golgi vesicles, are transported at low rates by the reconstituted proteoliposomes. Neither rough endoplasmic reticulum-derived vesicles nor proteoliposomes made from proteins of the rough endoplasmic reticulum transport CMP-AcNeu. The above results demonstrate that this reconstituted system can be used for further purification and characterization of nucleotide sugar and nucleotide sulfate translocator proteins. This approach should also be useful to study membrane transport proteins of lysosomes and endosomes.
Biological photocathodes.
Authors: Griffith O H , Habliston D L , Birrell G B , Skoczylas W P , Hedberg K K
Published: May 2006

Biological surfaces emit electrons when subjected to UV light. This emission is increased greatly after exposure to cesium vapor. Increases from 2 to 3 orders of magnitude are observed, depending on the biochemicals present. Heme and chlorophyll exhibit unusually high photoemission currents, which are increased further after cesiation. Photoemission from proteins and lipids is much less but also is increased by exposure to cesium. The formation of photocathodes with cesium greatly increases the practical magnifications attainable in photoelectron microscopy of organic and biological specimens. Photoelectron micrographs taken at magnifications greater than or equal to X 100,000 of chlorophyll-rich thylakoid membranes and of colloidal gold-labeled cytoskeleton preparations of cultured epithelial cells demonstrate the improvement in magnification. The selectivity and stability of the photocathodes suggest the possibility of detecting chromophore binding proteins in membranes and the design of photoelectron labels for tagging specific sites on biological surfaces.
Human hepatitis delta virus RNA subfragments contain an autocleavage activity.
Authors: Wu H N , Lin Y J , Lin F P , Makino S , Chang M F , Lai M M
Published: May 2006

Hepatitis delta virus (HDV) contains a single-stranded circular RNA genome of 1.7 kilobases. In this report we demonstrate that subfragments of HDV RNA can undergo autocatalytic cleavage. This cleavage requires at least 500 microM of Mg2+ or Ca2+, is not affected by varying the pH from 5.0 to 9.1, and occurs with RNA fragments as small as 133 nucleotides. The larger RNA fragments containing additional HDV sequences have a lower efficiency of cleavage. Deletion analysis at both ends of RNA subfragments suggested that the catalytic ability of HDV RNA resides in a stretch of no more than 117 nucleotides around the cleavage site. The cleavage occurs at the phosphodiester bond between nucleotides 688 and 689 on the HDV genomic map, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3' fragment with a guanosyl residue with a 5'-hydroxyl group. The smallest autocleaving RNA does not contain the "hammerhead" sequence required for the autocleavage of other known self-cleaving RNA. The cleavage of HDV RNA occurs at a much faster rate, even at a very low Mg2+ concentration, than that of other "ribozymes." Thus, HDV RNA represents a distinct class of ribozyme.
Formation of pilin in Pseudomonas aeruginosa requires the alternative sigma factor (RpoN) of RNA polymerase.
Authors: Ishimoto K S , Lory S
Published: May 2006

The promoter region of the Pseudomonas aeruginosa pilin gene has a high degree of similarity to the nitrogen-regulated promoters of enteric bacteria. These promoters are recognized by the alternative sigma factor of RNA polymerase, termed RpoN (NtrA or GlnF). This observation suggested that the P. aeruginosa pilin gene may be transcribed by the RpoN-containing RNA polymerase. We, therefore, cloned the RpoN gene from P. aeruginosa into Escherichia coli (where it formed a functional product) and used that cloned gene to construct a mutant of P. aeruginosa that was insertionally inactivated in its RpoN gene. This mutant failed to synthesize pilin, indicating that the RpoN sigma factor is required for transcription of the pilin gene.
Gangliosides potentiate in vivo and in vitro effects of nerve growth factor on central cholinergic neurons.
Authors: Cuello A C , Garofalo L , Kenigsberg R L , Maysinger D
Published: May 2006

The effects of nerve growth factor beta (beta-NGF) and ganglioside GM1 on forebrain cholinergic neurons were examined in vivo and in vitro. Following unilateral decortication of rats, GM1 (5 mg/kg per day) administered intracerebroventricularly could protect forebrain cholinergic neurons of the nucleus basalis magnocellularis from retrograde degeneration in a manner comparable to beta-NGF. Administered in combination with beta-NGF, GM1 produced a significant increase in choline acetyltransferase activity in the nucleus basalis magnocellularis and remaining cortex ipsilateral to the lesion. Concentrations of GM1 that were ineffective when administered alone in this lesion model, when given with beta-NGF, potentiated beta-NGF effects in both of the above brain areas. In dissociated septal cells in vitro, an increase in choline acetyltransferase activity was noted at beta-NGF concentrations as low as 0.1 pM and reached a plateau at 1 nM. A moderate (up to 35%) stimulation of choline acetyltransferase activity was observed with 10 microM GM1. The application of beta-NGF in combination with 10 microM GM1 or 0.1 microM GM1, a concentration that is ineffective in these cultures, produced a much greater increase in choline acetyltransferase activity than did beta-NGF alone. These observations support the idea that exogenously applied gangliosides can elicit trophic responses in cholinergic neurons of the central nervous system. That GM1 increases and even potentiates beta-NGF effects suggests that some of the trophic actions of this compound may be mediated through endogenous trophic factors.
Retinal ganglion beta cells project transiently to the superior colliculus during development.
Authors: Ramoa A S , Campbell G , Shatz C J
Published: May 2006

In adult cats, retinal ganglion cells of the beta class project almost exclusively to the lateral geniculate nucleus rather than to the superior colliculus (SC). We have examined whether this target specificity is present during early development. To identify ganglion cells that send axons to the SC in development, rhodamine-labeled microspheres were deposited in the SC at embryonic day (E) 38, E43, or postnatal day (P) 4. Retinae were then removed between E56 and P32 and kept alive in a tissue-slice chamber so that ganglion cells that had been retrogradely labeled with microspheres could be injected intracellularly with Lucifer yellow to reveal their morphological class. Many beta cells could be retrogradely labeled by microspheres injected into the SC at E38 or E43. They were indistinguishable from beta cells projecting to the lateral geniculate nucleus and were found even when a single injection was restricted to the caudal portion of the SC. In contrast, beta cells could not be retrogradely labeled by microspheres injected into the SC at P4. The disappearance of a beta-cell projection to the SC cannot be explained entirely by cell death since as late as P32, well after the major period of ganglion cell death, many beta ganglion cells labeled with microspheres at E38 were still present. These observations suggest that many beta cells initially extend an axon collateral to the SC that is subsequently lost some time after E43. Thus, to achieve the remarkable specificity present in the adult visual system, beta cells must withdraw axon collaterals from an entire target nucleus. Similar collateral elimination may give rise to the specificity of afferent connections in other sensory systems.
Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation.
Authors: Hendrickson W A , Pähler A , Smith J L , Satow Y , Merritt E A , Phizackerley R P
Published: May 2006

A three-dimensional crystal structure of the biotin-binding core of streptavidin has been determined at 3.1-A resolution. The structure was analyzed from diffraction data measured at three wavelengths from a single crystal of the selenobiotinyl complex with streptavidin. Streptavidin is a tetramer with subunits arrayed in D2 symmetry. Each protomer is an 8-stranded beta-barrel with simple up-down topology. Biotin molecules are bound at one end of each barrel. This study demonstrates the effectiveness of multiwavelength anomalous diffraction (MAD) procedures for macromolecular crystallography and provides a basis for detailed study of biotin-avidin interactions.
Proline isomerism leads to multiple folded conformations of calbindin D9k: direct evidence from two-dimensional 1H NMR spectroscopy.
Authors: Chazin W J , Kördel J , Drakenberg T , Thulin E , Brodin P , Grundström T , Forsén S
Published: May 2006

A complete analysis of calbindin D9k by two-dimensional 1H nuclear magnetic resonance spectroscopy has established the existence of two conformations for the folded protein in solution. Well-resolved major and minor resonances in a ratio of 3:1 are observed throughout the 1H NMR spectrum. Two-dimensional exchange experiments show that the major and minor species are related by an equilibrium process. Analysis of short proton-proton distances along the peptide backbone, identified by two-dimensional nuclear Overhauser effect spectroscopy, provides unambiguous evidence that the two forms of the folded protein differ only in the isomerization state of the peptide bond between Gly-42 and Pro-43. Cis-trans isomerism of Pro-43 is thereby directly identified as the cause of multiple conformations for the folded protein in solution. In addition, when Pro-43 is mutated to a glycine residue there is no indication of multiple conformations. These results provide evidence for the possibility of conformational heterogeneity in the native state of globular proteins.
Regulation of heat production in the inflorescences of an
Arum
lily by endogenous salicylic acid
Authors: Raskin Ilya , Turner Ivan M. , Melander Wayne R.
Published: May 2006

We have recently purified calorigen, the natural trigger for heat production in the inflorescences of
Sauromatum guttatum
Schott (voodoo lily), a thermogenic plant, and identified it as salicylic acid. Since then an analytical assay was developed that allows the quantitation of salicylic acid in plant tissues. This assay was used to demonstrate that on the day preceding the day of blooming the levels of salicylic acid in the thermogenic organs (appendix and lower spadix) of the voodoo lily increased almost 100-fold, reaching a level of 1 μg/g of fresh weight. The level of salicylic acid in the appendix started to rise in the afternoon and reached its maximum in the late evening, whereas the maximum accumulation of salicylic acid in the lower spadix occurred late at night. The increase in salicylic acid level in the appendix was followed the next morning by a spectacular metabolic burst that lasted for about 7 hr and at its peak increased the appendix temperature by over 12°C. The second, 14-hr-long, thermogenic episode in the lower spadix started late at night and ended on the following morning, after maximum temperature increases of more than 10°C. The concentration of salicylic acid in both thermogenic tissues promptly returned to basal, preblooming levels at the end of the thermogenic periods. The thermogenic response was under strong photoperiodic and developmental control, with salicylic acid eliciting much stronger thermogenic responses in light than in darkness. Similar surges in salicylic acid occurred in nonthermogenic male and female flowers, while the concentration of salicylic acid in the spathe remained consistently below 20 ng/g of fresh weight. Of 33 analogs of salicylic acid tested, only 2,6-dihydroxybenzoic acid and acetylsalicylic acid (aspirin) were thermogenic. The activity of 2,6-dihydroxybenzoic acid exceeded that of salicylic acid.
Transforming growth factor beta reverses the glucocorticoid-induced wound-healing deficit in rats: possible regulation in macrophages by platelet-derived growth factor.
Authors: Pierce G F , Mustoe T A , Lingelbach J , Masakowski V R , Gramates P , Deuel T F
Published: May 2006

Transforming growth factor beta (TGF-beta) and the platelet-derived growth factor (PDGF) are potent mitogenic polypeptides which enhance rates of wound healing in experimental animals; in contrast, glucocorticoids inhibit wound repair. The potential of TGF-beta and PDGF to reverse this inhibition in healing was tested in methylprednisolone-treated rats with deficits in skin wound strength of 50%. Single applications of TGF-beta (10-40 pmol per wound, 0.25-1 micrograms) applied locally at the time of wounding fully reversed this deficit in a concentration-dependent and highly reproducible manner. Wounds in glucocorticoid-treated animals were characterized by a near total absence of neutrophils and macrophages and by a delayed influx and reduced density of fibroblasts; however, such wounds treated with TGF-beta showed significant increases in wound fibroblasts and in intracellular procollagen type I. PDGF did not reverse the deficit in wound breaking strength in glucocorticoid-treated rats; there were more fibroblasts in the PDGF-treated wounds, but these fibroblasts lacked the enhanced expression of procollagen type I found in TGF-beta-treated wounds. The wound macrophages, required for normal tissue repair, remained absent from both PDGF- and TGF-beta-treated wounds in glucocorticoid-treated animals. This result suggested that macrophages might normally act as an intermediate in the induction of procollagen synthesis in fibroblasts of PDGF-treated wounds and that TGF-beta might bypass the macrophage through its capacity to stimulate directly new synthesis of procollagen type I in fibroblasts. Whereas PDGF does not stimulate procollagen synthesis, in a rodent macrophage cell line, PDGF induced a highly significant, time-dependent enhancement of expression of TGF-beta.
TCR3: a third T-cell receptor in the chicken.
Authors: Chen C H , Sowder J T , Lahti J M , Cihak J , Lösch U , Cooper M D
Published: May 2006

Avian homologues of mammalian gamma delta and alpha beta T-cell antigen receptors, TCR1 and TCR2, have been identified with monoclonal antibodies. These TCR isotypes are associated with the avian CD3 proteins on the T-cell surface. During chick development, T-cell subpopulations bearing CD3/TCR1 or CD3/TCR2 receptor complexes are generated sequentially in the thymus and seeded to the periphery in the same order. In this study, we used two-color immunofluorescence to identify the subsequent development of a third subpopulation of T cells, provisionally named TCR3. These CD3+ cells, which expressed neither TCR1 nor TCR2, were first detected in the blood 1 week after hatching and increased numerically as a function of age to account for approximately 15% of the circulating T-cell pool in adults. Most (greater than 80%) of the TCR3 cells expressed the CD4 accessory molecule. The relative incidence of the TCR3 subpopulation increased dramatically as a consequence of embryonic treatment with anti-TCR2 antibody and thymectomy after hatching. Two disulfide-linked polypeptides, of Mr 48,000 and 40,000, were associated with the CD3 complex on the TCR3 cells. Examination of the TCR protein backbones and peptide mapping of the TCR chains after partial proteolysis indicated that the TCR3 heterodimer differs from both TCR1 and TCR2. These results suggest the existence of a third class of T-cell receptors in birds.
Molecular cloning of two west African human immunodeficiency virus type 2 isolates that replicate well in macrophages: a Gambian isolate, from a patient with neurologic acquired immunodeficiency syndrome, and a highly divergent Ghanian isolate.
Authors: Kühnel H , von Briesen H , Dietrich U , Adamski M , Mix D , Biesert L , Kreutz R , Immelmann A , Henco K , Meichsner C
Published: May 2006

Human immunodeficiency virus type 2 (HIV-2)-related viruses were isolated from a Gambian dying of exclusively neurological disease (HIV-2D194) and from an asymptomatic Ghanian (HIV-2D205). Both strains exhibited properties of HIV-1 biological subtype c: they grew slowly and induced few or no syncytia but eventually produced high levels of particle-associated reverse transcriptase in cultures of fresh peripheral blood lymphocytes, and they established stable infection of T-lymphoma (HUT-78) and monocytic (U937) cell lines. Each produced even higher levels of reverse transcriptase when fresh human monocytes/macrophages were used as target cells. The viruses were molecularly cloned after a single passage in culture, in order to minimize in vitro selection of subtypes present in vivo. Restriction-site analysis showed heterogeneity within each isolate. Nucleotide sequence analysis of a portion of the HIV-2D194 genome revealed that it is a member of the prototypic HIV-2 family, displaying 13% divergence versus HIV-2ROD and HIV-2NIHZ, as compared to 9% divergence between HIV-2ROD and HIV-2NIHZ. In contrast, HIV-2D205 is the most highly divergent HIV-2 strain yet described: it is equidistant in relation between the known HIV-2 strains and the simian immunodeficiency virus isolates from rhesus macaque monkeys (23-25% divergence).
Neural regulation of sex pheromone biosynthesis in
Heliothis
moths
Authors: Teal P. E. A. , Tumlinson J. H. , Oberlander H.
Published: May 2006

Pheromone biosynthesis in females of
Heliothis zea
is regulated endogenously by a neuropeptide produced in the subesophageal ganglion. We have found that the ventral nerve cord must be intact for normal induction of pheromone biosynthesis and that pheromonotropic activity is associated with extracts of the abdominal nerve cord, but only during the period when pheromone is produced. We did not find evidence of pheromonotropic activity in hemolymph obtained from females that were producing pheromone. Extracts of the brain—subesophageal ganglion complex, which contain pheromone biosynthesis activating neuropeptide (PBAN), induced pheromone biosynthesis when applied to the terminal abdominal ganglion only if nerves from this ganglion to the pheromone gland were intact. Brain—subesophageal ganglion extracts did not induce biosynthesis when applied directly to the pheromone glands
in vitro
. From our results, we conclude that the target site of PBAN is not the pheromone gland but the terminal abdominal ganglion, and we hypothesize that the abdominal nerve cord transports PBAN to the terminal abdominal ganglion.
Steroid hormones: modulators of Na+ absorption and Cl- secretion in cultured tracheal epithelia.
Authors: Zeitlin P L , Wagner M , Markakis D , Loughlin G M , Guggino W B
Published: May 2006

Cultures of tracheal epithelial cells from adult nonpregnant female rabbits maintained a different balance of Na+ absorption and Cl- secretion from cells of pregnant animals and fetal cultures. Cells of nonpregnant rabbits had a large Na+ absorptive current (11 microA/cm2 or 53% of baseline short circuit current) and a small Cl- secretory current (2.4 microA/cm2 or 11%). In contrast, fetal cells had greater Cl- secretion (15.1 microA/cm2 or 61%) and much less Na+ absorption (1.6 microA/cm2 or less than 10%). Cells of pregnant animals had amounts of Cl- secretion (11.4 microA/cm2 or 41%) and Na+ absorption (6.7 microA/cm2 or 22%) that were in between that of fetal and adult nonpregnant animals. In addition to these striking differences in baseline ion transport among the three groups, epinephrine was found to stimulate Cl- secretion in cells from pregnant rabbits, both Na+ absorption and Cl- secretion in cells from nonpregnant animals, and neither in fetal cells. Steroid hormones of pregnancy, progesterone and estradiol, when added to cells cultured from nonpregnant rabbits, altered the magnitudes of the individual transport pathways to mimic cells cultured from pregnant animals. In addition to changing baseline transport, these steroids modified the epinephrine-stimulated currents to resemble those in the fetus and in pregnancy. We conclude that steroid hormones regulate the balance of Na+ absorption and Cl- secretion in tracheal epithelia in utero and in adulthood.
A double-stranded RNA unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and Xenopus eggs.
Authors: Wagner R W , Smith J E , Cooperman B S , Nishikura K
Published: May 2006

Amphibian eggs and embryos as well as mammalian cells have been reported to contain an activity that unwinds double-stranded RNA. We have now found that adenosine residues have been modified in the RNA products of this unwinding activity. Although the modified RNA remains double-stranded, the modification causes the RNA to be susceptible to single-strand-specific RNase and to migrate as a retarded smear on a native polyacrylamide electrophoresis gel. The modification is specific for double-stranded RNA. At least 40% of the adenosine residues can be modified in vitro in a given random sequence RNA molecule. By using standard two-dimensional TLC and HPLC analyses, the modified base has been identified as inosine. Mismatched base-pairing between inosine and uridine appears to be responsible for the observed characteristics of the unwound RNA. The biological significance of this modifying activity and also of the modified double-stranded RNA is discussed.
Mutation and selection in bacterial populations: alternatives to the hypothesis of directed mutation.
Authors: Lenski R E , Slatkin M , Ayala F J
Published: May 2006

Bacterial populations have served as model systems for studying evolutionary processes ever since the classic experiments of Luria and Delbrück, which demonstrated the occurrence of mutations prior to selection for the traits they conferred. However, several authors have recently presented experiments suggesting that bacteria may have mechanisms for directing which mutations occur, such that the rate of adaptive mutations is enhanced. Before the hypothesis of directed mutation is accepted, it is imperative to consider alternative hypotheses that might account for the same observations. To this end, we expand upon existing mathematical theory of the dynamics of mutation and selection in clonal populations for two cases of particular interest. The first case concerns selection against mutants before plating; this selection occurs as the result of differences in growth rate between mutants and nonmutants. We demonstrate that this selection model gives rise to distributions of mutants, obtained by plating from sister cultures, that are very similar to those expected when some mutations are induced by the selective environment. The second case concerns the sequential incorporation of two mutations as the result of selection for an intermediate genotype after plating. We demonstrate that this two-step mutation model also yields distributions that are similar to those expected when some mutations are induced by the selective environment. These two cases therefore provide alternatives to the hypothesis of directed mutation. We suggest experiments that might be used to examine our alternative hypotheses. We also contrast the hypothesis of directed mutation with the notion of inheritance of acquired characteristics.
Gastrin releases a blood calcium-lowering peptide from the acid-producing part of the rat stomach.
Authors: Persson P , Håkanson R , Axelson J , Sundler F
Published: May 2006

Gastrin-17 induces hypocalcemia in the rat without stimulating calcitonin release. The gastrin-induced hypocalcemia persisted after thyroparathyroidectomy or parathyroidectomy. In contrast, gastrectomy or extirpation of the acid-producing part of the stomach prevented the hypocalcemic effect, suggesting the involvement of the proximal stomach in the gastrin-evoked lowering of blood calcium. The drop in blood calcium upon injection of gastrin-17 did not reflect a loss of calcium via the gastric juice or via the urine. Extracts of the acid-producing mucosa of the rat stomach had a hypocalcemic effect. The extracts were purified by gel chromatography and reversed-phase high-performance liquid chromatography. Digestion with leucine aminopeptidase destroyed the hypocalcemic activity, while trypsin had no effect, suggesting a peptide (or peptides) with an unprotected NH2 terminus and without basic amino acid residues (or with protected basic amino acids). Both gastrin-17 and the mucosal extract stimulated the uptake of 45Ca into bone (radius and sternum). Gastrin-17 was without effect in rats that had undergone gastrectomy, while the mucosal extract was equally effective in gastrectomized and unoperated rats. We suggest that the effects of gastrin-17 on blood calcium and on calcium transfer into bone are indirect and that gastrin-17 stimulates the release of a peptide hormone, tentatively named gastrocalcin, from the acid-producing mucosa of the stomach. Gastrocalcin stimulates the uptake of 45Ca into bone, thereby causing hypocalcemia.
Protein kinase C is not necessary for peptide-induced suppression of M current or for desensitization of the peptide receptors.
Authors: Bosma M M , Hille B
Published: May 2006

Frog sympathetic ganglion cells were studied under whole-cell voltage clamp to determine whether protein kinase C (PKC) mediates peptide-induced suppression of M current (IM) or desensitization of peptide receptors. Low concentrations (10 mM) of chicken II luteinizing hormone-releasing hormone (LHRH) or substance P (SP) suppressed IM; in addition, higher concentrations (1 microM) desensitized receptors. Desensitization is homologous (specific to the peptide) and lasts at least 25 min. Two stimulators of PKC, phorbol 12-myristate 13-acetate and dioctanoylglycerol, partially depressed IM and occluded the response to SP but not to LHRH. The two actions of PKC stimulators were blocked by PKC inhibitors (staurosporine, a pseudosubstrate peptide, and H-7), but SP- and LHRH-mediated suppression of IM and receptor desensitization were not affected. Thus, we conclude that PKC is not necessary for normal IM suppression or receptor desensitization.
Bone remodeling signaled by a dihydropyridine- and phenylalkylamine-sensitive calcium channel.
Authors: Guggino S E , Lajeunesse D , Wagner J A , Snyder S H
Published: May 2006

An osteoblast calcium channel demonstrated by single channel recordings is associated with calcium antagonist receptor binding sites in osteoblast-like osteosarcoma cells. By using whole cell current recordings we now show that this channel is stimulated by the dihydropyridine calcium agonist drug BAY K 8644. A physiological relevance of these channels is apparent from the stereoselective, potent inhibition of parathyroid hormone-stimulated calcium uptake into osteoblast-like cells in culture by desmethoxyverapamil, a phenylalkylamine calcium antagonist. Secretion by these cells of the bone matrix protein osteocalcin is stimulated by BAY K 8644 and blocked by desmethoxyverapamil and nitrendipine. Evidence for a role of this channel in bone remodeling in intact animals comes from enhanced bone resorption in fetal rat bones observed with BAY K 8644 and stereoselective, potent blockade of resorption by desmethoxyverapamil.
Inhibition of beta-adrenergic receptor kinase prevents rapid homologous desensitization of beta 2-adrenergic receptors.
Authors: Lohse M J , Lefkowitz R J , Caron M G , Benovic J L
Published: May 2006

Homologous (agonist-specific) desensitization of beta-adrenergic receptors (beta ARs) is accompanied by and appears to require phosphorylation of the receptors. We have recently described a novel protein kinase, beta AR kinase, which phosphorylates beta ARs in vitro in an agonist-dependent manner. This kinase is inhibited by two classes of compounds, polyanions and synthetic peptides derived from the beta 2-adrenergic receptor (beta 2AR). In this report we describe the effects of these inhibitors on the process of homologous desensitization induced by the beta-adrenergic agonist isoproterenol. Permeabilization of human epidermoid carcinoma A431 cells with digitonin was used to permit access of the charged inhibitors to the cytosol; this procedure did not interfere with the pattern of isoproterenol-induced homologous desensitization of beta 2AR-stimulated adenylyl cyclase. Inhibitors of beta AR kinase markedly inhibited homologous desensitization of beta 2ARs in the permeabilized cells. Inhibition of desensitization by heparin, the most potent of the polyanion inhibitors of beta AR kinase, occurred over the same concentration range (5-50 nM) as inhibition of purified beta AR kinase assessed in a reconstituted system. Inhibition of desensitization by heparin was accompanied by a marked reduction of receptor phosphorylation in the permeabilized cells. Whereas inhibitors of beta AR kinase inhibited homologous desensitization, inhibitors of protein kinase C and of cyclic-nucleotide-dependent protein kinases were ineffective. These data establish that phosphorylation of beta ARs by beta AR kinase is an essential step in homologous desensitization of the receptors. They further suggest a potential therapeutic value of inhibitors of beta AR kinase in inhibiting agonist-induced desensitization.
Isolation and structural characterization of cDNA clones encoding the mating pheromone Er-1 secreted by the ciliate Euplotes raikovi.
Authors: Miceli C , La Terza A , Melli M
Published: May 2006

cDNA clones comprising the entire coding region for the mating pheromone Er-1 of Euplotes raikovi have been isolated by oligonucleotide screening of two cDNA libraries in the vectors lambda gt10 and pUC12. The cDNA sequence contains an open reading frame of 75 amino acids that constitute pre-pro-Er-1. The amino acid sequence of secreted Er-1 starts at aspartic acid-36 of pre-pro-Er-1 and completely matches that known by direct Er-1 protein sequencing. The coding region of Er-1 cDNA ends with codon TAA, which specifies glutamine in other ciliates. The 5'- and 3'-noncoding regions contain, respectively, two and one inverted repeats. The 3'-noncoding-region inverted repeat, which includes the unusual polyadenylylation signal AACAAA, has been related to RNA 3'-terminus formation.
Tyrosine phosphorylation of the insulin receptor is not required for receptor internalization: studies in 2,4-dinitrophenol-treated cells.
Authors: Backer J M , Kahn C R , White M F
Published: May 2006

The relation between insulin-stimulated autophosphorylation of the insulin receptor and internalization of the receptor was studied in Fao rat hepatoma cells. Treatment of Fao cells with 2,4-dinitrophenol for 45 min depleted cellular ATP by 80% and equally inhibited insulin-stimulated receptor autophosphorylation, as determined by immunoprecipitation of surface-iodinated or [32P]phosphate-labeled cells with anti-phosphotyrosine antibody. In contrast, internalization of the insulin receptor and internalization and degradation of 125I-labeled insulin by 2,4-dinitrophenol-treated cells were normal. These data show that autophosphorylation of the insulin receptor is not required for the receptor-mediated internalization of insulin in Fao cells and suggest that insulin receptor recycling is independent of autophosphorylation.
Intron and upstream sequences regulate expression of the Drosophila beta 3-tubulin gene in the visceral and somatic musculature, respectively.
Authors: Gasch A , Hinz U , Renkawitz-Pohl R
Published: May 2006

The morphogenetic programs involved in the differentiation of internal organs, such as the muscle system, during Drosophila embryogenesis have remained largely obscure. beta 3-tubulin has proved to be a good marker for mesoderm development as this tubulin isotype is detectable soon after mesoderm formation and during the process of mesoderm differentiation. The beta 3-tubulin gene is expressed in the somatic and pharyngeal musculature, the dorsal vessel, and the visceral musculature. To learn more about the programs underlying mesodermal differentiation, we have started to dissect the regulatory elements of the beta 3-tubulin gene by means of P-element-mediated transformation experiments. We show that expression of the beta 3-tubulin gene in the somatic muscles, the pharyngeal muscles, and the dorsal vessel is mediated by far upstream sequences. We also demonstrate that the first intron of the beta 3-tubulin gene bears a tissue-specific enhancer element that is required for expression in the visceral muscles and that also functions efficiently when cloned downstream of an indicator gene. The separability of elements driving beta 3-tubulin expression in the somatic and visceral mesoderm facilitates the investigation of the different programs involved in regulating the early differentiation of this germ layer.
A plant manganese superoxide dismutase is efficiently imported and correctly processed by yeast mitochondria.
Authors: Bowler C , Alliotte T , Van den Bulcke M , Bauw G , Vandekerckhove J , Van Montagu M , Inzé D
Published: May 2006

In the plant Nicotiana plumbaginifolia, manganese superoxide dismutase (MnSOD) is synthesized in the cytoplasm as a preprotein and is subsequently translocated to the mitochondrial matrix with corresponding cleavage of an NH2-terminal leader sequence. To determine whether the plant enzyme could replace the endogenous SOD activities of Escherichia coli and yeast, constructions have been made in appropriate vectors for expression of the preprotein and the mature MnSOD. These were introduced into SOD-deficient strains for complementation studies. In E. coli, both forms of the protein were shown to be active and able to complement SOD deficiency to different degrees. Expression of the preprotein in a yeast strain lacking a mitochondrial MnSOD resulted in a restoration of wild-type growth, only possible if the plant protein was being targeted to the mitochondria. Subsequent studies revealed that the protein was processed and that the leader sequence was cleaved at the identical position as recognized by the mitochondrial peptidase of plants. The components mediating mitochondrial import thus appear to be highly conserved between plants and yeast.
Identification and characterization of a membrane-bound cytotoxin of murine cytolytic lymphocytes that is related to tumor necrosis factor/cachectin.
Authors: Liu C C , Detmers P A , Jiang S B , Young J D
Published: May 2006

Cytotoxic T lymphocytes (CTLs) kill their targets by a contact-dependent mechanism. We investigated the possibility that the CTL membranes themselves could exert direct cytotoxic activity. Murine CTLs that had been fixed with paraformaldehyde retained a slow cytotoxic activity toward various target cells that are also sensitive to another cytokine, tumor necrosis factor (TNF)/cachectin. This cytotoxic activity was neutralized by antibodies specific for TNF. Membrane fractions obtained from CTLs were cytotoxic to TNF-sensitive targets but not to several TNF-resistant cell lines. Immunoblot analysis revealed a membrane protein band of 50-60 kDa from CTLs that reacts with anti-TNF antibodies. The surface localization of this cytokine was further ascertained by flow cytometry, indirect immunofluorescence, and immunoelectron microscopy studies using TNF-specific antibodies. Radioiodination of CTL surface proteins followed by immunoprecipitation with anti-TNF antibodies confirmed the presence of a TNF-related cytokine in the plasma membranes of CTLs that migrated with an apparent molecular mass of 50-60 kDa under disulfide-reducing conditions. This cytokine can be removed from membranes by treatment with detergents but not with high-salt buffers, suggesting that it may be an integral membrane protein.