bluuebunny/crossref_metadata_2025 huggingface dataset:
Evolutionary origin of autoreactive determinants (autogens).
Authors: Kieber-Emmons T , Kohler H
Published: May 2006

The question addressed in this report focuses on the autoantigenicity of self antigens, principally cytochrome c and lysozyme. Of interest is whether the immune system produces autoantibodies to its host proteins reacting randomly with all potential antigen sites or is autoreactively selective for certain determinants. Based on experimental evidence from autoantibodies against cytochromes c, Jemmerson and Margoliash [Jemmerson, R. & Margoliash, E. (1979) Nature (London) 282, 468-471] have described a striking correlation between autoreactive sequence regions and evolutionary instability. While their analysis of evolutionary variation was based on simple sequence variability plots, we present here a refined approach that takes into account the distinction between evolutionary substitutions that induce a change in the protein surface from those that do not (surface-neutral substitutions). A quantitative aspect of surface variation (surface consensus) is included in the algorithm that produces a ranked order for autoantigenic determinants. The final plot, called surface variability, indicates sequence regions having a preference for autoimmune reaction. We propose the term "autogen" to designate such protein determinants.
Evidence for an X-linked modifier gene affecting the expression of Tourette syndrome and its relevance to the increased frequency of speech, cognitive, and behavioral disorders in males.
Authors: Comings D E , Comings B G
Published: May 2006

A number of disorders affecting speech, learning, and behavior have a 3:1 or greater male:female ratio. This has usually been explained on the basis of a developmental model postulating a difference in the young male versus female brain. Tourette syndrome is a hereditary neurobehavioral disorder due to a single major autosomal gene. It is associated with learning disorders and attention deficit disorder with hyperactivity and shows a similar predilection for males. Because of the more obvious nature of the tics and vocal noises, it is uniquely suited for examining an alternative hypothesis of an X-linked modifying gene to account for the increased incidence in males. Two alternative models tested were an autosomal modifying gene and the developmental model. Family pedigrees on a series of 430 consecutive cases of Tourette syndrome with 966 age-corrected offspring were used to compare the observed affected and unaffected sons and daughters for nine different phenotype matings with the expected ratios for three different models. The X-linked modifier model provided a better fit to the observed data and accounted for marked differences in the percentage of affected sons and daughters when the father versus the mother transmitted the Tourette syndrome gene. A similar model may account for the male predominance in other genetically influenced disorders of speech, learning, and behavior.
Site-specific covalent modification of monoclonal antibodies: in vitro and in vivo evaluations.
Authors: Rodwell J D , Alvarez V L , Lee C , Lopes A D , Goers J W , King H D , Powsner H J , McKearn T J
Published: May 2006

A strategy for covalent modification of monoclonal antibodies utilizing the oxidized oligosaccharide moieties on the molecule was evaluated and compared to more conventional methods. As judged by quantitative in vitro measurements, a monoclonal antibody conjugate prepared via the oligosaccharides retained the homogeneous antigen binding property and affinity of the unmodified antibody. In contrast, conjugates of the same antibody, modified to the same degree on either lysines or aspartic and glutamic acid side chains, were heterogeneous in their antigen binding and had lowered affinity. In vivo biodistribution and nuclear-imaging experiments were also performed with a second monoclonal antibody and a tumor xenograft model. Antibodies modified on the oligosaccharides with either a peptide labeled with iodine-125 or a diethylenetriaminepentaacetic acid chelate with indium-111 localize into target tumors more efficiently than the same antibody radiolabeled on either tyrosines or lysines. These in vivo results, when compared to those reported in the literature for conventionally modified antibodies, suggest that oligosaccharide modification of monoclonal antibodies is a preferred method of preparing conjugates.
Metabolic activation and detoxication of nephrotoxic cysteine and homocysteine S-conjugates.
Authors: Elfarra A A , Lash L H , Anders M W
Published: May 2006

S-(1,2-Dichlorovinyl)-L-homocysteine (DCVHcy), an analogue of the nephrotoxin S-(1,2-dichlorovinyl)-L-cysteine (DCVCys), is a much more potent nephrotoxin than DCVCys both in vivo and in isolated renal proximal tubular cells. S-(1,2-Dichlorovinyl)-DL-alpha-methylhomocysteine, at equimolar doses relative to DCVHcy, is not nephrotoxic. Agents that inhibit pyridoxal phosphate-dependent enzymes (DL-propargylglycine and aminooxyacetic acid) or renal organic anion transport (probenecid) protect against DCVHcy-induced nephrotoxicity. With kidney cytosol, DCVHcy or the analogue S-(2-benzothiazolyl)-L-homocysteine (BTHcy) is not metabolized to 2-ketobutyrate, but 2-mercaptobenzothiazole is a metabolite of BTHcy and the Vmax for its formation is enhanced by addition of 2-ketobutyrate. These results are consistent with a bioactivation mechanism for DCVHcy that involves enzymatic deamination followed by a nonenzymatic beta-elimination to produce two reactive intermediates--i.e., S-(1,2-dichlorovinyl)thiol and 2-keto-3-butenoic acid. The Km values for the N-acetylation of DCVCys and DCVHcy by kidney microsomal N-acetyltransferase are similar, but the rate of DCVCys N-acetylation is 4-fold greater than the rate measured with DCVHcy as the substrate. Thus, the remarkable nephrotoxic potency of DCVHcy compared with DCVCys may be attributable to intrarenal differences in activation and detoxication.
L-glutamic acid, a neurotrophic factor for maintenance of acetylcholinesterase and butyrylcholinesterase in the preganglionically denervated superior cervical ganglion of the cat.
Authors: Koelle G B , Sanville U J , Thampi N S , Wall S J
Published: May 2006

In continuation of previous studies, the intraarterial fusion of L-glutamic acid for 24 hr was found to oppose the decrease in acetylcholinesterase and butyrylcholinesterase in the superior cervical ganglion of the cat that otherwise occurs 48 hr after preganglionic denervation. The combination of glutamic acid and gamma-aminobutyric acid, in concentrations that were inactive individually, likewise produced the same neurotrophic effect. Inactive in this respect were glycine plus L-glutamine, pyroglutamic acid, gamma-aminobutyric acid, and L-aspartic acid. The possible mechanisms and implications of these findings are discussed.
The ciliate
Euplotes raikovi
heterozygous at the
mat
genetic locus coreleases two individual species of mating pheromone: Genetic and biochemical evidence
Authors: Luporini Pierangelo , Raffioni Simona , Concetti Antonio , Miceli Cristina
Published: May 2006

Euplotes raikovi
of the wild-type strain 13 was found to be heterozygous (
mat-1/mat-2
) for the genetic locus
mat
, which is supposed to control the mating-type specificity of freely released mating pheromones (“euplomones”), and capable of yielding the two types of corresponding homozygotes (
mat-1/mat-1, mat-2/mat-2
). The results of euplomone purification, performed in parallel on
Euplotes
of the three different genotypes, showed that the heterozygous cells corelease two
mat
-specific euplomones (namely, euplomone
r
1 associated with
mat-1
and euplomone
r
2 associated with
mat-2
), while the homozygous cells release either one of the two types according to their genotype. The two euplomones coreleased by the
mat
heterozygous cells were resolved as separate molecular species present in different relative amounts (euplomone
r
1, ≈70%; euplomone
r
2, ≈30%). A closely similar degree of eccentricity in the euplomone
r
1/euplomone
r
2 production was again found between the two homozygous cell types. It was concluded that the alleles
mat-1
and
mat-2
exhibit a relationship of nondominance: the heterozygote apparently behaves as the simple combination of the two corresponding homozygotes. It was inferred that the observed quantitative variations in the production of the different euplomones may be the result of a differential
mat
gene-type amplification occurring during the development of the cell somatic macronucleus.
Relationship between transmitter release and presynaptic calcium influx when calcium enters through discrete channels.
Authors: Zucker R S , Fogelson A L
Published: May 2006

We have used a three-dimensional diffusion model of calcium entering the presynaptic nerve terminal through discrete channels to simulate experiments relating transmitter release to presynaptic calcium current. The relationship will be less than linear, or will curve downward, if calcium channels are well separated. It will resemble a power-law function with exponent less than the cooperativity of calcium action if channels are clustered closer together. Large presynaptic depolarizations elicit more release than small depolarizations admitting the same calcium influx. This occurs because large pulses open more channels near each other, with the result that the calcium concentration near release sites is greater, due to overlap of calcium diffusing from adjacent channels.
Phase separation in lens cytoplasm is genetically linked to cataract formation in the Philly mouse.
Authors: Clark J I , Carper D
Published: May 2006

The variation of the phase-separation temperature, Tc, in lenses was studied during the postnatal development of three genetically different mouse strains: Swiss-Webster, Philly, and the (Swiss-Webster x Philly)F1 hybrid. The general behavior of Tc during early postnatal development has two stages: in stage I, Tc increased to a maximum and then, in stage II, Tc decreased. Philly mice are a strain that develops hereditary cataracts about 36 days following birth. In F1 hybrids of Philly and Swiss-Webster mice, cataracts appeared about 49 days following birth, approximately equal to 13 days later in development than in the Philly mice. In the Philly and hybrid mice, stage I and stage II were followed by stage III in which Tc reached a minimum value and then increased toward body temperature. The values of Tc at birth, the slope of the increase during stage I, and the maximum Tc were characteristic for each mouse strain. These results establish that the behavior of the temperature of the phase separation Tc in mouse lens is linked to the genetic strain of the mice and that the value of Tc at birth is an early indicator of lenses that will develop cataracts and lenses that will develop normally.
Evolutionarily conserved regions of the human c-myc protein can be uncoupled from transforming activity.
Authors: Sarid J , Halazonetis T D , Murphy W , Leder P
Published: May 2006

The myc family of oncogenes contains coding sequences that have been preserved in different species for over 400 million years. This conservation (which implies functional selection) is broadly represented throughout the C-terminal portion of the human c-myc protein but is largely restricted to three clusters of amino acid sequences in the N-terminal region. We have examined the role that the latter three regions of the c-myc protein might play in the transforming function of the c-myc gene. Several mutations, deletions and frameshifts, were introduced into the c-myc gene, and these mutant genes were tested for their ability to collaborate with the EJ-ras oncogene to transform rat embryo fibroblasts. Complete elimination of the first two N-terminal conserved segments abolished transforming activity. In contrast, genes altered in a portion of the second or the entire third conserved segment retained their transforming activity. Thus, the latter two segments are not required for the transformation process, suggesting that they serve another function related only to the normal expression of the c-myc gene.
Cloning of cDNA for maize superoxide dismutase 2 (SOD2).
Authors: Cannon R E , White J A , Scandalios J G
Published: May 2006

A cDNA clone encoding maize cytosolic superoxide dismutase 2 (SOD2) was isolated from a cDNA library constructed in pUC9 plasmids from size-selected poly(A)+ RNA. The library was screened with mixed synthetic oligode-oxynucleotide probes. The sequence of the probes was derived from the amino acid sequence from a region of the protein near the NH2 terminus. One positive clone contained an insertion of a 612-base-pair fragment. The amino acid sequence, deduced from the nucleotide sequence of the cDNA, revealed that the clone contained the coding region for all but the first of the 151 amino acids of the SOD2 protein. Additional 5' and 3' flanking sequences, absent on the pUC9 Sod2 clone, were obtained from a lambda gt11 clone isolated from a maize leaf library probed with the pUC9 Sod2 insert. Hybrid-selection translation assays also demonstrate that the cDNA clone contains Sod2 sequences.
Topographical localization of the C-terminal region of the voltage-dependent sodium channel from Electrophorus electricus using antibodies raised against a synthetic peptide.
Authors: Gordon R D , Fieles W E , Schotland D L , Hogue-Angeletti R , Barchi R L
Published: May 2006

A peptide corresponding to amino acid residues 1783-1794 near the C terminus of the electric eel sodium channel primary sequence of the eel (Electrophorus electricus) sodium channel has been synthesized and used to raise an antiserum in rabbits. This antiserum specifically recognized the peptide in a solid-phase radioimmunoassay. Specificity of the antiserum for the native channel protein was shown by its specific binding to a 280-kDa protein in immunoblots of eel electroplax membrane proteins. The antiserum also specifically labeled the innervated membrane of the eel electroplax in immunofluorescent studies; noninnervated membrane was not labeled, consistent with the known distribution of sodium channels in this tissue. The membrane topology of the peptide recognized by this antiserum was probed in binding studies using oriented electroplax membrane vesicles. These vesicles were 98% "right-side-out" as determined by [3H]saxitoxin binding. Binding of the antipeptide antiserum to this fraction was measured before and after permeabilization with 0.01% saponin. Specific binding to intact vesicles was low, but this binding increased 10-fold after permeabilization, implying a cytoplasmic orientation for the peptide. Confirmation for this orientation was then sought by localizing the antibody bound to intact electroplax cells with immunogold electron microscopy. Gold particles identifying the antibody were found almost exclusively associated with the cytoplasmic surface of the innervated membrane. Our data imply that the region of the sodium channel primary sequence near the C terminus that is recognized by our antiserum is localized on the cytoplasmic side of the membrane; this localization provides some further constraints on models of sodium channel tertiary structure.
Oxytocin and cAMP stimulate monovalent cation movements through a Ca2+-sensitive, amiloride-insensitive channel in the apical membrane of toad urinary bladder.
Authors: Van Driessche W , Aelvoet I , Erlij D
Published: May 2006

The effects of oxytocin and cAMP on ion transport were investigated in toad urinary bladders incubated with Ca2+-free solutions on the apical side. Under these conditions both oxytocin and cAMP markedly stimulated the movements of Na+, K+, Rb+, Cs+, Li+, and NH4+ through a pathway that is insensitive to amiloride. The amiloride-insensitive currents were inhibited by the addition of Ca2+, Sr2+, or Mg2+ to the apical solution. The movement of the monovalent cations was associated with a spontaneous Lorentzian component in the power spectrum of the fluctuation in short-circuit current. The plateau of the Lorentzian component was enhanced by oxytocin and cAMP and was depressed by divalent cations. Methohexital inhibited the stimulation of monovalent cation movements caused by oxytocin. These findings suggest that oxytocin and cAMP activate at least two kinds of ionic channels in the apical membrane of toad urinary bladder: the well-known amiloride-sensitive channel and an amiloride-insensitive channel that allows the movement of several monovalent cations and is blocked by Ca2+ and other divalent cations.
Insulin stimulates phosphorylation of a 120-kDa glycoprotein substrate (pp120) for the receptor-associated protein kinase in intact H-35 hepatoma cells.
Authors: Perrotti N , Accili D , Marcus-Samuels B , Rees-Jones R W , Taylor S I
Published: May 2006

The insulin receptor possesses protein kinase activity, which may play a role in mediating insulin action. Recently, we have identified a glycoprotein (pp120) in rat liver plasma membranes that is phosphorylated by the solubilized insulin receptor in a cell-free system. We now report that insulin stimulates phosphorylation of pp120 in intact H-35 cells. H-35 cells were preloaded with [32P]orthophosphate to label the intracellular ATP pool. Insulin caused a 10-fold increase in the phosphorylation of its receptor and a 2-fold increase in phosphorylation of pp120 (P less than 0.001). The time course of insulin's stimulation of pp120 closely paralleled that of insulin receptor phosphorylation over the time period investigated (15-45 min). This effect had the specificity corresponding to the insulin receptor. Epidermal growth factor was inactive, and insulin-like growth factor I had approximately equal to 1% the potency of insulin in this regard. Insulin increased 32P incorporation into pp120 in a linkage that was stable to alkaline hydrolysis, as would be expected for tyrosine-specific phosphorylation. Direct phosphoamino acid analysis confirmed that insulin increased 32P incorporation into phosphotyrosine residues in pp120.
Molecular cloning and characterization of esterase-6, a serine hydrolase of Drosophila.
Authors: Oakeshott J G , Collet C , Phillis R W , Nielsen K M , Russell R J , Chambers G K , Ross V , Richmond R C
Published: May 2006

The Est-6 gene of Drosophila melanogaster was cloned by screening libraries with synthetic oligonucleotides corresponding to tryptic peptides from purified esterase-6 (Est-6) protein. cDNA clones were isolated that hybridized in situ to the site of Est-6 on chromosome 3 at 69A1. Inserts in putative Est-6 cDNA clones were 1.85 kilobases (kb) long, and blot hybridization analysis of electrophoretically fractionated RNA, using a cDNA clone as a probe, revealed two transcripts, of 1.68 and 1.83 kb. The two transcripts showed the same developmental profile as the Est-6 protein. Neither transcript was detected in an Est-6-null line. The cDNA fragment was homologous to a 2.3-kb EcoRI-BamHI fragment in genomic clones, and this region was interrupted by the 8-kb B104 transposable element in the Est-6-null line. Conceptual translation of the cDNA sequence revealed a protein of 548 residues with 19% sequence similarity to acetylcholinesterase from the Torpedo ray.
Chimeric mouse-human IgG1 antibody that can mediate lysis of cancer cells.
Authors: Liu A Y , Robinson R R , Hellström K E , Murray E D , Chang C P , Hellström I
Published: May 2006

A chimeric mouse-human antibody has been created that recognizes an antigen found on the surface of cells from many carcinomas. Immunoglobulin constant (C) domains of the mouse monoclonal antibody L6, C gamma 2a and C kappa, were substituted by the human C gamma 1 and C kappa by recombining cDNA modules encoding variable or C domains. The cDNA constructs were transfected into lymphoid cells for antibody production. The chimeric antibody and mouse L6 antibody bound to carcinoma cells with equal affinity and mediated complement-dependent cytolysis. In the presence of human effector cells, the chimeric antibody gave antibody-dependent cellular cytotoxicity at 100 times lower concentration than that needed for the mouse L6 antibody. The chimeric antibody, but not the mouse L6 antibody, is effective against a melanoma line expressing small amounts of the L6 antigen. The findings point to the usefulness of the chimeric antibody approach for obtaining agents with strong antitumor activity for possible therapeutic use in man.
Endogenous inhibitor for calcium-dependent cysteine protease contains four internal repeats that could be responsible for its multiple reactive sites.
Authors: Emori Y , Kawasaki H , Imajoh S , Imahori K , Suzuki K
Published: May 2006

A cDNA encoding an endogenous inhibitor, termed calpastatin, for calcium-dependent cysteine protease (calpain, EC 3.4.22.17) was cloned by screening rabbit cDNA libraries with a synthetic oligodeoxynucleotide probe based on the partial amino acid sequence of the purified protein. The deduced amino acid sequence contains 718 amino acid residues (Mr, 76,964), and the mature protein corresponds to the deduced sequence from the 80th residue of the primary translation product (resultant Mr, 68,113). This deduced molecular weight is significantly lower than that determined by NaDodSO4/polyacrylamide gel electrophoresis, suggesting the possibility that the inhibitor is post-translationally modified. The sequence of the mature inhibitor contains four consecutive internal repeats approximately 140 amino acid residues long, each of which might be responsible for the inhibitory activity. Calpastatin is apparently different from a typical cysteine protease inhibitor (cystatin), suggesting that the mechanism of inhibition of calcium-dependent cysteine protease by the inhibitor might be different from that of other cysteine proteases by cystatin.
Protein-nucleotide contacts in the immunoglobulin heavy-chain promoter region.
Authors: Landolfi N F , Capra J D , Tucker P W
Published: May 2006

Immunoglobulin heavy-chain variable-region genes contain the octanucleotide ATGCAAAT upstream from the site of transcription initiation. The complement of this sequence, in the reverse orientation, is found at an identical location in light-chain variable-region genes. This sequence element is thought to be involved in the lymphoid-specific expression of immunoglobulin genes. Analysis of nuclear extracts from both lymphoid and nonlymphoid cells in a gel migration inhibition assay, using an immunoglobulin promoter region fragment containing the octamer, reveals multiple migration-retarded species that represent specific DNA-protein complexes. The number and relative level of these complexes vary with cell type; some complexes are detected with all extracts, whereas one complex is lymphoid-specific and may represent an interaction involved in the lymphoid-restricted expression of immunoglobulin genes. Mitogenic stimulation of a B-lymphoid line can increase the level of the protein responsible for this lymphoid-specific complex. Analysis of the complexes detected in the gel migration inhibition assay by DNase I protection ("footprinting") has revealed that all of these DNA-protein complexes involve contact of the protein with the nucleotides of the octamer. One complex, present in both lymphoid and nonlymphoid cells, displays an additional DNA-protein contact adjacent to the octamer. Our results also indicate that the interaction of proteins with the octameric sequence can cause a local alteration in the structure of the DNA helix.
Lysine-156 and serine-119 are required for LexA repressor cleavage: a possible mechanism.
Authors: Slilaty S N , Little J W
Published: May 2006

LexA repressor of Escherichia coli is inactivated in vivo by a specific cleavage reaction requiring activated RecA protein. In vitro, cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. These two cleavage reactions have similar specificities, suggesting that RecA acts indirectly to stimulate self-cleavage, rather than directly as a protease. We have studied the chemical mechanism of cleavage by using site-directed mutagenesis to change selected amino acid residues in LexA, chosen on the basis of kinetic data, homology to other cleavable repressors, and potential similarity of the mechanism to that of proteases. Serine-119 and lysine-156 were changed to alanine, a residue with an unreactive side chain, resulting in two mutant proteins that had normal repressor function and apparently normal structure, but were completely deficient in both types of cleavage reaction. Serine-119 was also changed to cysteine, another residue with a nucleophilic side chain, resulting in a protein that was cleaved at a significant rate. These and other observations suggest that hydrolysis of the scissile peptide bond proceeds by a mechanism similar to that of serine proteases, with serine-119 being a nucleophile and lysine-156 being an activator. Possible roles for RecA are discussed.
Multiple genes encode the human Na+,K+-ATPase catalytic subunit.
Authors: Shull M M , Lingrel J B
Published: May 2006

A human genomic library was constructed and screened with hybridization probes derived from sheep and rat cDNAs encoding the alpha and alpha(+) isoforms, respectively, of the Na+,K+-ATPase catalytic subunit. Genomic sequences spanning 150 kilobases were isolated. Four genes, designated alpha A, alpha B, alpha C, and alpha D, each 20-25 kilobases in length, were identified by restriction mapping, Southern blot hybridization analysis, and limited DNA sequencing. We present evidence that two of these genes, alpha A and alpha B, encode the alpha and alpha(+) isoforms, respectively. The other genes, alpha C and alpha D, one of which is physically linked to the alpha(+) gene, exhibit nucleotide and amino acid homology to Na+,K+-ATPase catalytic subunit cDNA sequences but do not correspond to any previously identified isoforms.
Deuterium nuclear magnetic resonance measurements of blood flow and tissue perfusion employing 2H2O as a freely diffusible tracer.
Authors: Ackerman J J , Ewy C S , Becker N N , Shalwitz R A
Published: May 2006

The use of deuterium oxide (2H2O) is proposed as a freely diffusible nuclear magnetic resonance (NMR) blood flow and tissue perfusion tracer of potential clinical utility. Deuterium is a stable, nonradiative isotope commercially available as 2H2O at enrichment levels of essentially 100%--i.e., 110 molar equivalent deuterium. This high concentration, together with the short relaxation time of the spin 1 (quadrupole) deuterium nuclide, provides substantial sensitivity for NMR spectroscopy. As a result, when 2H2O is administered in a bolus fashion to a specific tissue or organ in vivo, the deuterium NMR intensity time course can be analyzed, using mathematical models developed by others for radiolabeled tracers, to measure the rate of blood flow and tissue perfusion. Such an application is demonstrated herein at a static magnetic field of 8.5 tesla. Using single-compartment flow modeling, hepatic blood flow and tissue perfusion in fasted (18 hr) male Sprague-Dawley rats was determined to be 61 +/- 17 (mean +/- SD) ml/100 g per min (n = 5).
Direct gene transfer into human cultured cells facilitated by laser micropuncture of the cell membrane.
Authors: Tao W , Wilkinson J , Stanbridge E J , Berns M W
Published: May 2006

The selective alteration of the cellular genome by laser microbeam irradiation has been extensively applied in cell biology. We report here the use of the third harmonic (355 nm) of an yttrium-aluminum garnet laser to facilitate the direct transfer of the neo gene into cultured human HT1080-6TG cells. The resultant transformants were selected in medium containing an aminoglycoside antibiotic, G418. Integration of the neo gene into individual human chromosomes and expression of the gene were demonstrated by Southern blot analyses, microcell-mediated chromosome transfer, and chromosome analyses. The stability of the integrated neo gene in the transformants was shown by a comparative growth assay in selective and nonselective media. Transformation and incorporation of the neo gene into the host genome occurred at a frequency of 8 X 10(-4)-3 X 10(-3). This method appears to be 100-fold more efficient than the standard calcium phosphate-mediated method of DNA transfer.
Interleukin 5, a T-cell-derived B-cell differentiation factor also induces cytotoxic T lymphocytes.
Authors: Takatsu K , Kikuchi Y , Takahashi T , Honjo T , Matsumoto M , Harada N , Yamaguchi N , Tominaga A
Published: May 2006

We describe an interleukin, termed interleukin 5, that is the recombinant product previously referred to as T-cell-replacing factor (TRF), B-cell growth factor II (BCGF II), or killer-helper factor (KHF). TRF has been defined as a T-cell-derived lymphokine that acts on activated B cells as a B-cell differentiation factor. We have previously demonstrated that TRF is identical to BCGF II and induces expression of receptors for interleukin 2 (IL-2) on activated B cells. We also have reported that KHF can induce not only expression of IL-2 receptors on peanut agglutinin-binding (PNA+) thymocytes but also generation of cytotoxic T lymphocytes (CTL) in PNA+ thymocytes in the presence of IL-2. We show here that culture supernatants of T-cell hybridomas that produce TRF as well as TRF purified by high-pressure liquid chromatography (HPLC-TRF) have KHF activity and generate CTL in PNA+ thymocytes in the presence of stimulator cells and IL-2. Moreover, translation products (recombinant TRF) of Xenopus oocytes injected with cDNA encoding for murine TRF (BCGF II) also exert KHF activity. A rat monoclonal anti-TRF antibody TB13 can block generation of CTL by HPLC-TRF or recombinant TRF. These results indicate that TRF acts not only on B cells as BCGF II but also on PNA+ thymocytes as KHF. In view of the diverse activities and targets of TRF, we propose that TRF refers to a different interleukin, interleukin 5.
Quantitative studies of human cardiac metabolism by 31P rotating-frame NMR.
Authors: Blackledge M J , Rajagopalan B , Oberhaensli R D , Bolas N M , Styles P , Radda G K
Published: May 2006

We have developed 31P NMR spectroscopic methods to determine quantitatively relative levels of phosphorous-containing metabolites in the human myocardium. We have used localization techniques based on the rotating-frame imaging experiment and carried out with a double-surface coil probe. Information is obtained from selected slices by rotating-frame depth selection and from a complete one-dimensional spectroscopic image using phase-modulated rotating-frame imaging. The methods collect biochemical information from metabolites in human heart, and we use the fact that the phosphocreatine/ATP molar ratio in skeletal muscle at rest is higher than that in working heart to demonstrate that localization has been achieved for each investigation. The phosphocreatine/ATP molar ratio in normal human heart has been measured as 1.55 +/- 0.20 (mean +/- SD) (3.5-sec interpulse delay) in six subjects using depth selection and as 1.53 +/- 0.25 (mean +/- SD) in four subjects using spectroscopic imaging. Measurement of this ratio is expected to give a useful and reproducible index of myocardial energetics in normal and pathological states.
Viruses in fungi: infection of yeast with the K1 and K2 killer viruses.
Authors: el-Sherbeini M , Bostian K A
Published: May 2006

We demonstrate here that yeast killer viruses, previously thought to be transmitted only by cytoplasmic mixing during division, mating, or other induced forms of cell fusion, are capable of extracellular transmission. Viral particles from standard K1 and K2 killer strains were used to inoculate sensitive cells of Saccharomyces cerevisiae, rendered competent by spheroplasting, lithium acetate treatment, or by natural mating. Extracellular transmission of the killer viruses was judged by the following criteria and controls. Filter-sterilized virus inocula were shown to be free of viable yeast cells, and host cells treated in the absence of added virus did not yield killer progeny. Infected clones originating from spheroplasts or lithium acetate-treated cells were shown to possess the genotype of the host strain and the killer phenotype of the infecting virus. Infected clones derived from complementary mating pairs were found to be wild-type diploids, whose meiotic segregants exhibited 2:2 segregation for unlinked nutritional markers and 4:0 segregation for the killer phenotype. This technique is generally applicable to the study of interactions between yeast viruses and different hosts and suggests that extracellular transmission may be a natural route for the inheritance and dissemination of mycoviruses.
Two efficient ribosomal frameshifting events are required for synthesis of mouse mammary tumor virus gag-related polyproteins.
Authors: Jacks T , Townsley K , Varmus H E , Majors J
Published: May 2006

The primary translation products of retroviral pol genes are polyproteins initiated in an upstream gene (gag). To investigate the manner in which the gag-initiated polyproteins of the mouse mammary tumor virus are produced, we determined the nucleotide sequence of a 1.8-kilobase DNA fragment that spans the region between gag and pol in the C3H strain of mouse mammary tumor virus. The sequence reveals three overlapping open reading frames: the first encodes products of gag (p27gag and p14gag); the second encodes a protein domain of unknown function (termed X) that is highly related to a similarly positioned sequence in simian type D retroviruses and the viral protease (pro); and the third encodes the reverse transcriptase. The reading frames are organized to permit uninterrupted readthrough from gag to pol if ribosomal frameshifts occur in the -1 direction within each of the two overlapping regions, one of which is 16 nucleotides in length and the other 13 nucleotides. Cell-free translation of RNA containing these overlap regions shows that fusion of the reading frames by ribosomal frameshifting occurs efficiently: about one-fourth of the ribosomes traversing the gag-X/pro overlap and one-tenth traversing the X/pro-pol overlap shift frames, generating gag-related polyproteins in ratios similar to those observed in vivo. Synthetic oligonucleotides containing either of the overlap regions inserted into novel contexts do not induce frameshifting; hence the overlapping portions of the reading frames are not sufficient to induce a frameshift event, and a larger sequence context or secondary structure may be implicated.
Identification of a herpes simplex virus 1 glycoprotein gene within a gene cluster dispensable for growth in cell culture.
Authors: Longnecker R , Chatterjee S , Whitley R J , Roizman B
Published: May 2006

The genome of herpes simplex virus 1 consists of two components, L and S, each containing unique sequences flanked by inverted repeats. Current and earlier studies have shown that 11 of the 12 open reading frames contained in the unique sequences of the S component can be deleted and are dispensable for growth in cell culture. Analyses of one recombinant virus containing a deletion in the open reading frame US7 permitted the identification of a monoclonal antibody specific for the product of this gene. The protein encoded by this gene has a predicted translated molecular weight of 41,366 and an apparent molecular weight of approximately 65,000 in denaturing polyacrylamide gels. The electrophoretic mobility of the protein synthesized by cells in the presence of inhibitory concentrations of tunicamycin is faster than that of the protein accumulating in lysates of untreated infected cells. We conclude that the product of US7 is glycoprotein subject to N-linked glycosylation, and we have designated it glycoprotein I. These studies indicate that the unique sequences of the S component encode four glycoproteins (G, D, I, and E) of which at least three (G, I, and E) are dispensable for growth in continuous lines of primate cells.
Isolation and characterization of a steroid sulfatase cDNA clone: genomic deletions in patients with X-chromosome-linked ichthyosis.
Authors: Ballabio A , Parenti G , Carrozzo R , Sebastio G , Andria G , Buckle V , Fraser N , Craig I , Rocchi M , Romeo G
Published: May 2006

We have isolated several cDNA clones from a lambda gt11 expression library by screening with antibodies prepared against the microsomal enzyme steroid sulfatase, which is deficient in classical X-chromosome-linked ichthyosis patients. One of these clones (p422) has been assigned by mapping with a somatic cell hybrid panel and by in situ hybridization to Xp22.3. Clone p422 therefore has a coincident localization with the previously identified locus for steroid sulfatase expression in the region of the X chromosome escaping from inactivation. Twelve steroid sulfatase-deficient patients, including eight cases of classical ichthyosis, were found to be deleted for genomic sequences detected by the clone.
K
+
regulates bacteroid-associated functions of
Bradyrhizobium
Authors: Gober James W. , Kashket Eva R.
Published: May 2006

Cowpea
Bradyrhizobium
32H1 cells, when grown under 0.2% O
2
, synthesize nitrogenase, as well as a methylammonium (ammonium) transport system and an electrogenic K
+
/H
+
antiporter. This effect was seen in growth medium containing 8-12 mM K
+
but not with 50 μM K
+
. Addition of K
+
to cells growing under low O
2
tensions in low-K
+
medium led to various phenotypic properties associated with bacteroids, including the ability to reduce acetylene, induction of an ammonium transport carrier and the K
+
/H
+
antiporter, and increased synthesis of two heme-biosynthetic enzymes, δ-aminolevulinate synthase and δ-aminolevulinate dehydratase. K
+
addition caused the repression of glutamine synthetase and of capsular polysaccharide synthesis, functions related to the free-living state. A similar pattern of regulation was observed in
Bradyrhizobium japonicum
. In addition, K
+
-mediated depression in
Bradyrhizobium
32H1 was inhibited by exudate of
Vigna unguiculata
, its host plant. We conclude that K
+
ions, in addition to low O
2
tension, are needed for the expression of several bacteroid-related functions in bradyrhizobia and thus are a major controlling influence in bacteroid development.
Matrix-driven translocation: dependence on interaction of amino-terminal domain of fibronectin with heparin-like surface components of cells or particles.
Authors: Newman S A , Frenz D A , Hasegawa E , Akiyama S K
Published: May 2006

During the process of matrix-driven translocation, certain types of cells or polystyrene latex beads are transported between compositionally different regions of a collagen matrix. Under appropriate conditions this translocation depends on an interaction between the cell or particle surface and fibronectin. We now show that this interaction takes place at a site located within the first 31 kDa of the amino-terminal end of the fibronectin molecule. Using defined fibronectin fragments and monoclonal antibodies directed against specific fibronectin domains, this site is established as both necessary and sufficient for the promotion of matrix-driven translocation. Competition experiments using heparin, heparan sulfate, and other sulfated polysaccharides show that this fibronectin site interacts with heparin-like cell or particle surface components in promoting matrix-driven translocation. Treatment of cells with heparinase renders them unresponsive to the translocational effect. An antibody directed against the amino-terminal domain of fibronectin completely inhibits matrix-driven translocation without interfering with heparin binding, suggesting that a post-binding conformational change in fibronectin may be required for promotion of the effect.
Purification and subunit structure of the [3H]phenamil receptor associated with the renal apical Na+ channel.
Authors: Barbry P , Chassande O , Vigne P , Frelin C , Ellory C , Cragoe E J , Lazdunski M
Published: May 2006

Sodium crosses the apical membrane of tight epithelia through a sodium channel, which is inhibited by the diuretic amiloride and by analogs such as phenamil. Target size analysis indicated that the functional size of the [3H]phenamil binding sites associated with the epithelial Na+ channel from pig kidney is 92 +/- 10 kDa. The [3H]phenamil receptor was solubilized by using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized material displayed the same properties of interaction with amiloride and its derivatives as the membrane-bound receptor. A two-step purification of the epithelial Na+ channel was achieved by using QAE Sephadex chromatography and affinity chromatography on a Bandeiraea simplicifolia lectin column. It results in an 1100-fold purification of the Na+ channel as compared to pig kidney microsomes with a yield of 15% +/- 5%. The maximal specific activity was 3.7 nmol/mg of protein. NaDodSO4/poly-acrylamide gel electrophoresis of the purified Na+ channel under nonreducing conditions showed the presence of a single major polypeptide chain of apparent molecular mass 185 kDa. Under disulfide-reducing conditions, the purified epithelial Na+ channel migrated as a single band of apparent molecular mass 105 kDa. It is suggested that the epithelial Na+ channel from pig kidney has a total molecular mass of 185 kDa and consists of two nearly identical 90- to 105-kDa polypeptide chains crosslinked by disulfide bridges.
Complete cDNA and derived amino acid sequence of human factor V.
Authors: Jenny R J , Pittman D D , Toole J J , Kriz R W , Aldape R A , Hewick R M , Kaufman R J , Mann K G
Published: May 2006

cDNA clones encoding human factor V have been isolated from an oligo(dT)-primed human fetal liver cDNA library prepared with vector Charon 21A. The cDNA sequence of factor V from three overlapping clones includes a 6672-base-pair (bp) coding region, a 90-bp 5' untranslated region, and a 163-bp 3' untranslated region within which is a poly(A) tail. The deduced amino acid sequence consists of 2224 amino acids inclusive of a 28-amino acid leader peptide. Direct comparison with human factor VIII reveals considerable homology between proteins in amino acid sequence and domain structure: a triplicated A domain and duplicated C domain show approximately equal to 40% identity with the corresponding domains in factor VIII. As in factor VIII, the A domains of factor V share approximately 40% amino acid-sequence homology with the three highly conserved domains in ceruloplasmin. The B domain of factor V contains 35 tandem and approximately 9 additional semiconserved repeats of nine amino acids of the form Asp-Leu-Ser-Gln-Thr-Thr/Asn-Leu-Ser-Pro and 2 additional semiconserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues.
Functional regions of the cauliflower mosaic virus 35S RNA promoter determined by use of the firefly luciferase gene as a reporter of promoter activity
Authors: Ow David W. , Jacobs Jerry D. , Howell Stephen H.
Published: May 2006

The cauliflower mosaic virus (CaMV) 35S RNA promoter has been dissected and examined in a transient expression system using the firefly luciferase gene as a reporter of promoter activity. Deletion analysis has shown that the 35S RNA promoter is composed of at least three regions—distal, medial, and proximal—which are essential for activity. The distal region contains three smaller elements homologous to the simian virus 40 “core” enhancer element, the medial region possesses a CCAAT-like box, and the proximal region contains a TATA box. A DNA segment encompassing the distal region is capable of activating the CaMV 35S core promoter in an orientation-independent, but not position-independent, fashion. The distal region can also activate a heterologous weak promoter, the CaMV 19S RNA promoter, albeit not to the high levels of the 35S RNA promoter. Multimers of the distal region are able to activate the 35S RNA promoter core to even greater levels of expression than the native 35S promoter. These experiments demonstrate that elements outside the boundaries of the core promoter (composed of proximal and medial elements) are recognized in a plant cell transient expression system.
Gap junctional coupling modulates secretion of exocrine pancreas.
Authors: Meda P , Bruzzone R , Chanson M , Bosco D , Orci L
Published: May 2006

Dispersed pancreatic acini were studied to assess the function of junctional coupling between adult secretory cells. Nonstimulated control cells were extensively coupled to their neighbors throughout each acinus. Addition of heptanol caused their uncoupling and increased their basal amylase release. Neurotensin, secretin, and vasoactive intestinal peptide (VIP) stimulated amylase secretion without uncoupling acinar cells. Heptanol rapidly and markedly uncoupled the neurotensin-, secretin-, and VIP-stimulated acinar cells and increased their amylase secretion in an additive manner. By contrast, the secretory response to carbamoylcholine (carbachol), a secretagogue that, alone, uncoupled acinar cells, was not affected by heptanol. Basal as well as neurotensin-, secretin-, and VIP-stimulated output returned to the lower control values following removal of heptanol and recovery of normal coupling. The data provide evidence that blockage of gap junctional coupling increases the basal secretion of exocrine pancreas as well as the response of the gland to a variety of secretagogues.
In vivo-like drug responses of human tumors growing in three-dimensional gel-supported primary culture.
Authors: Vescio R A , Redfern C H , Nelson T J , Ugoretz S , Stern P H , Hoffman R M
Published: May 2006

An in vitro test of cell sensitivity to drugs that indicates in vivo response is an important need in cancer therapy and cancer drug development. Toward this end, we previously developed a collagen gel-supported culture system for growth of human tumors. This three-dimensional culture system is general and grows tumors at high frequency directly from surgery or biopsy that maintain important in vivo properties in vitro, including tissue architecture. We report here that with autoradiographic techniques measuring cellular DNA synthesis the drug responses of individual cells within the tissue structure of in vitro-grown tumors can be determined. Twenty tumor classes, including all the major ones, have been measured in toto at greater than 50% frequency. Quantitative and qualitative results show increasing cell kill with rising cytotoxic drug concentration, differential drug sensitivities of multiple cell types within individual cultured tumors, differential sensitivities of a series of tumors of the same histopathological classification to a single drug, differential sensitivities of individual tumors to a series of drugs, and sensitivity patterns of various tumor types similar to the sensitivities found in vivo. Therefore, the results indicate that potentially important therapeutic data can be obtained from tumor specimens growing in vitro for the individual cancer patient as well as for rational and relevant screening for new agents active against human solid tumors.
Interferon messenger RNA is produced constitutively in the organs of normal individuals.
Authors: Tovey M G , Streuli M , Gresser I , Gugenheim J , Blanchard B , Guymarho J , Vignaux F , Gigou M
Published: May 2006

The use of RNA blot hybridization with DNA or RNA probes of high specific activity has shown that interferon (IFN)-alpha mRNA is present constitutively in the spleen, kidney, liver, and peripheral blood leukocytes of normal individuals. A single band (approximately equal to 1.2 kilobases) was detected in poly(A)+ RNA isolated from human organs. This RNA hybridized specifically to human IFN-alpha 1 DNA and comigrated with mature IFN-alpha mRNA from virus-induced human peripheral blood leukocytes. No IFN-beta RNA transcripts were detected in any of the tissues tested. IFN-gamma mRNA was detected in only one sample of normal human spleen, which also contained an unusually high level of IFN-alpha mRNA. The use of a modified S1 mapping technique revealed the presence of IFN-alpha 1 and -alpha 2 transcripts only. No IFN-alpha 4, -alpha 5, -alpha 6, -alpha 7, -alpha 8, or -alpha 14 transcripts were detected in the same sample. The detection, in all the samples tested, of a characteristic pattern of expression of IFN genes, different from that obtained following induction, together with the low number of transcripts present (less than or equal to 0.03 copy per cell) suggest that specific IFN genes are transcribed constitutively in vivo.